The Intratubular and Intracrine Renin-Angiotensin System in the Proximal Tubules of the Kidney and Its Roles in Angiotensin II-Induced Hypertension

*Xiao C. Li, Ana Paula de Oliveira Leite, Xu Chen, Chunling Zhao, Xiaowen Zheng, Jianfeng Zhang and Jia L. Zhuo*

## **Abstract**

The kidney plays a fundamental role in the physiological regulation of basal blood pressure and the development of hypertension. Although the mechanisms underlying hypertension are very complex, the renin-angiotensin system (RAS) in the kidney, especially intratubular and intracellular RAS, undoubtedly plays a critical role in maintaining basal blood pressure homeostasis and the development of angiotensin II (ANG II)-dependent hypertension. In the proximal tubules, ANG II activates two G protein-coupled receptors, AT1 and AT2, to exert powerful effects to regulate proximal tubular sodium and fluid reabsorption by activating cell surface as well as intracellular AT1 receptors. Increased production and actions of ANG II in the proximal tubules may cause salt and fluid retention, impair the pressure-natriuresis response, and consequently increase blood pressure in hypertension. The objectives of this chapter are to critically review and discuss our current understanding of intratubular and intracellular RAS in the kidney, and their contributions to basal blood pressure homeostasis and the development of ANG II-dependent hypertension. The new knowledge will likely help uncover novel renal mechanisms of hypertension, and develop kidney- or proximal tubule-specific strategies or drugs to prevent and treat hypertension in humans.

**Keywords:** angiotensin II, blood pressure, hypertension, kidney, proximal tubule

## **1. Introduction**

According to the most recent American College of Cardiology (ACC)/ American Heart Association (AHA) reports, 46% of U.S. adults now develop hypertension and take antihypertensive drugs in their lifetime [1, 2]. Prevention and treatment of hypertension and its target organ complications cost several hundreds of billion dollars a year to the U.S. economy [3–6]. Although the causes of hypertension are multifactorial, the activation of circulating (endocrine), tissue (paracrine) and intracellular (intracrine) RAS via angiotensin II (ANG II) remains one of most important contributing mechanisms [1–7]. Indeed, angiotensin-converting enzyme (ACE) inhibitors, ANG II receptor blockers (ARBs), and renin inhibitors, which block the RAS at the enzymatic or receptor levels, are widely used to treat hypertension, reduce cardiovascular and renal disease risks, and prevent target organ damage [1–7]. However, clinical trials have shown that not all RAS-targeting drugs have the same efficacy of blocking the actions of ANG II and afford the same degree of cardiovascular, blood pressure and renal protection [1–6]. Some patients continue to develop cardiovascular and renal complications despite being treated with one or more than two of these blockers [7, 8]. The underlying mechanisms responsible for these clinical observations are not well understood. One of the possibilities may be that not all ARBs have the same ability to enter the cells to block intracellular ANG II. Some, but not all, ARB(s) such as telmisartan and losartan may exert therapeutic effects beyond the classic ARBs' properties.

**2. Localization of intratubular and intracellular RAS and its receptors in**

Angiotensinogen, a 60 kDa α2 globulin in the serpin family, is the primary, if

In the kidney, angiotensinogen mRNAs and proteins have been localized in the

kidney, primarily in the proximal tubules [28–30]. Immunohistochemistry, immunoelectron microscopy and non-isotopic hybridization histochemistry have demonstrated the localization of angiotensinogen mRNAs and proteins in the proximal convoluted and straight tubules of the cortex, with glomerular mesangial cells and medullary vascular bundles also being immunopositive in neonatal rat kidney [29, 30]. In the adult rat kidney, however, angiotensinogen mRNA expression was

localized primarily in the proximal convoluted tubules, whereas electron-

suggests that liver-derived angiotensinogen is the primary source of renal

expressed in the kidney proximal tubules.

**2.2 Renin**

**83**

angiotensinogen protein and ANG II under physiological conditions, but during the ANG II-induced hypertension, angiotensinogen mRNAs and proteins are also

Renin, the rate-limiting enzyme first discovered to increase blood pressure in

rabbits by Tigerstedt and Bergman in 1898 [35], is an aspartyl proteinase or angiotensinogenase. Renin plays the most critical role in the initiation of the

cells, or distal nephrons under physiological conditions [29, 30].

microscopic immunohistochemistry localized angiotensinogen immunostaining in the apical membrane of proximal convoluted tubules [29, 30]. By contrast, few if any angiotensinogen mRNAs and proteins are localized in the glomeruli, mesangial

Although most of angiotensinogen in the circulation is derived from the liver, there is evidence showing that angiotensinogen is also expressed and produced in the kidney [28, 31–33]. Kobori et al. have consistently shown that angiotensinogen mRNA expression and proteins are increased in the proximal tubules of the kidney in ANG II-infused rats [28, 31–33]. However, Matsusaka et al. have demonstrated that there were no significant differences in the levels of angiotensinogen and ANG II proteins in the kidney between wildtype mice and mice with kidney-specific angiotensinogen knockout [34]. It was further found that angiotensinogen protein and ANG II levels in the kidney were nearly abolished in mice with liver-specific knockout of angiotensinogen [34]. The studies of Kobori et al. and Matsusaka et al.

angiotensinogen is primarily expressed or produced in the liver under physiological conditions. Human angiotensinogen consists of 452 amino acids, whereas rodent's angiotensinogen may vary in its molecular size slightly from human form [24–27]. Angiotensinogen, not active in itself, is released from the liver and cleaved in the circulation by the rate-limiting enzyme renin to form the still inactive decapeptide ANG I. This is followed by the conversion of inactive ANG I to the active and potent peptide ANG II, initiating important biological and physiological actions. A second enzyme called angiotensin I-converting enzyme (ACE) acts to convert ANG I to form the biologically active ANG II, initiating an important biochemical and physiological angiotensinogen/renin/ANG I/ACE/ANG II cascade (see below section on ACE). Accordingly, the recognized and primary role of angiotensinogen is to serve as a key substrate to the production of ANG II in the circulation and tissues.

not the only, substrate for the RAS super family. It is well-recognized that

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

**the proximal tubules of the kidney**

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

**2.1 Angiotensinogen**

There is accumulating evidence that ANG II acts not only as an endocrine or paracrine hormone activating cell surface ANG II receptors, but also as an intracellular or intracrine peptide activating intracellular ANG II receptors, though the precise roles of the latter remain largely unknown [9–11]. Indeed, in addition to activating cell surface ANG II receptors, circulating and paracrine ANG II can readily enter the cells via AT1 receptor-mediated endocytosis. The ANG II/AT1 receptor complex internalized into endosomes may continue to transmit signals from endosomes or be translocated to the nucleus to induce long-lasting genomic effects [12, 13]. Recently, we and others have used innovative in vitro cell expression system [14–16], in vivo adenoviral gene transfer of an intracellular ANG II protein selectively in proximal tubule cells of the rat and mouse kidneys [17, 18], or genetically modified mouse models to investigate the physiological roles and mechanisms of actions of intratubular and intracellular ANG II in the proximal tubules of the kidney, with a focus on basal blood pressure homeostasis and ANG II-induced hypertension [19, 20]. Specifically, we have determined whether intracellular ANG II is derived from AT1 (AT1a) receptor-mediated uptake by the proximal tubule cells, and whether proximal tubule-selective expression of an intracellular ANG II fusion protein in the rat and mouse kidney increases the expression and activity of NHE3, promotes proximal tubular sodium and fluid reabsorption, and therefore elevates arterial blood pressure [17–23]. These new studies have generated new knowledge to improve, and provided new insights into our understanding of renal mechanisms of hypertension involving both endocrine, paracrine and intracellular ANG II, and perhaps aid the development of new classes of multifunctional drugs to treat ANG II-induced hypertension and its target organ damage by blocking not only extracellular but also intracellular and nuclear actions of ANG II. Accordingly, the objectives of this chapter are to critically review, analyze, and discuss the recent developments and progresses in the studies of novel renal mechanisms of hypertension with a focus on the roles of intratubular and intracellular ANG II in the proximal tubules of the kidney.

## **2. Localization of intratubular and intracellular RAS and its receptors in the proximal tubules of the kidney**

## **2.1 Angiotensinogen**

hypertension and take antihypertensive drugs in their lifetime [1, 2]. Prevention and treatment of hypertension and its target organ complications cost several hundreds of billion dollars a year to the U.S. economy [3–6]. Although the

causes of hypertension are multifactorial, the activation of circulating (endocrine), tissue (paracrine) and intracellular (intracrine) RAS via angiotensin II (ANG II)

receptor levels, are widely used to treat hypertension, reduce cardiovascular and renal disease risks, and prevent target organ damage [1–7]. However, clinical trials have shown that not all RAS-targeting drugs have the same efficacy of blocking the actions of ANG II and afford the same degree of cardiovascular, blood pressure and renal protection [1–6]. Some patients continue to develop cardiovascular and renal complications despite being treated with one or more than two of these blockers [7, 8]. The underlying mechanisms responsible for these clinical observations are not well understood. One of the possibilities may be that not all ARBs have the same ability to enter the cells to block intracellular ANG II. Some, but not all, ARB(s) such as telmisartan and losartan may exert therapeutic effects beyond the classic

There is accumulating evidence that ANG II acts not only as an endocrine or

an intracellular or intracrine peptide activating intracellular ANG II receptors, though the precise roles of the latter remain largely unknown [9–11]. Indeed,

paracrine hormone activating cell surface ANG II receptors, but also as

in addition to activating cell surface ANG II receptors, circulating and paracrine ANG II can readily enter the cells via AT1 receptor-mediated endocytosis. The ANG II/AT1 receptor complex internalized into endosomes may continue to transmit signals from endosomes or be translocated to the nucleus to induce long-lasting genomic effects [12, 13]. Recently, we and others have used innovative in vitro cell expression system [14–16], in vivo adenoviral gene transfer of an intracellular ANG II protein selectively in proximal tubule cells of the rat and mouse kidneys [17, 18], or genetically modified mouse models to investigate the physiological roles and mechanisms of actions of intratubular and intracellular ANG II in the proximal tubules of the kidney, with a focus on basal blood pressure homeostasis and ANG II-induced hypertension [19, 20]. Specifically, we have determined whether intracellular ANG II is derived from AT1 (AT1a) receptor-mediated uptake by the proximal tubule cells, and whether proximal tubule-selective expression of an intracellular ANG II fusion protein in the rat and mouse kidney increases the expression and activity of NHE3, promotes proximal tubular sodium and fluid reabsorption, and therefore elevates arterial blood pressure [17–23]. These new studies have generated new knowledge to

improve, and provided new insights into our understanding of renal mechanisms of hypertension involving both endocrine, paracrine and intracellular ANG II, and perhaps aid the development of new classes of multifunctional drugs to treat ANG II-induced hypertension and its target organ damage by blocking not only extracellular but also intracellular and nuclear actions of ANG II. Accordingly, the objectives of this chapter are to critically review, analyze, and discuss the recent developments and progresses in the studies of novel renal mechanisms of hypertension with a focus on the roles of intratubular and intracellular ANG II in the proximal tubules of

remains one of most important contributing mechanisms [1–7]. Indeed, angiotensin-converting enzyme (ACE) inhibitors, ANG II receptor blockers (ARBs), and renin inhibitors, which block the RAS at the enzymatic or

*Selected Chapters from the Renin-Angiotensin System*

ARBs' properties.

the kidney.

**82**

Angiotensinogen, a 60 kDa α2 globulin in the serpin family, is the primary, if not the only, substrate for the RAS super family. It is well-recognized that angiotensinogen is primarily expressed or produced in the liver under physiological conditions. Human angiotensinogen consists of 452 amino acids, whereas rodent's angiotensinogen may vary in its molecular size slightly from human form [24–27]. Angiotensinogen, not active in itself, is released from the liver and cleaved in the circulation by the rate-limiting enzyme renin to form the still inactive decapeptide ANG I. This is followed by the conversion of inactive ANG I to the active and potent peptide ANG II, initiating important biological and physiological actions. A second enzyme called angiotensin I-converting enzyme (ACE) acts to convert ANG I to form the biologically active ANG II, initiating an important biochemical and physiological angiotensinogen/renin/ANG I/ACE/ANG II cascade (see below section on ACE). Accordingly, the recognized and primary role of angiotensinogen is to serve as a key substrate to the production of ANG II in the circulation and tissues.

In the kidney, angiotensinogen mRNAs and proteins have been localized in the kidney, primarily in the proximal tubules [28–30]. Immunohistochemistry, immunoelectron microscopy and non-isotopic hybridization histochemistry have demonstrated the localization of angiotensinogen mRNAs and proteins in the proximal convoluted and straight tubules of the cortex, with glomerular mesangial cells and medullary vascular bundles also being immunopositive in neonatal rat kidney [29, 30]. In the adult rat kidney, however, angiotensinogen mRNA expression was localized primarily in the proximal convoluted tubules, whereas electronmicroscopic immunohistochemistry localized angiotensinogen immunostaining in the apical membrane of proximal convoluted tubules [29, 30]. By contrast, few if any angiotensinogen mRNAs and proteins are localized in the glomeruli, mesangial cells, or distal nephrons under physiological conditions [29, 30].

Although most of angiotensinogen in the circulation is derived from the liver, there is evidence showing that angiotensinogen is also expressed and produced in the kidney [28, 31–33]. Kobori et al. have consistently shown that angiotensinogen mRNA expression and proteins are increased in the proximal tubules of the kidney in ANG II-infused rats [28, 31–33]. However, Matsusaka et al. have demonstrated that there were no significant differences in the levels of angiotensinogen and ANG II proteins in the kidney between wildtype mice and mice with kidney-specific angiotensinogen knockout [34]. It was further found that angiotensinogen protein and ANG II levels in the kidney were nearly abolished in mice with liver-specific knockout of angiotensinogen [34]. The studies of Kobori et al. and Matsusaka et al. suggests that liver-derived angiotensinogen is the primary source of renal angiotensinogen protein and ANG II under physiological conditions, but during the ANG II-induced hypertension, angiotensinogen mRNAs and proteins are also expressed in the kidney proximal tubules.

## **2.2 Renin**

Renin, the rate-limiting enzyme first discovered to increase blood pressure in rabbits by Tigerstedt and Bergman in 1898 [35], is an aspartyl proteinase or angiotensinogenase. Renin plays the most critical role in the initiation of the

angiotensinogen/renin/ACE/ANG II/AT1 receptor activation in the cardiovascular, kidney, and other major target tissues. Human renin precursor consists of 406 amino acids with a pre- and a pro-segment of 20 and 46 amino acids, respectively [36]. Mature human renin contains 340 amino acids and a molecular wt. of 37 kDa [36]. Renin, renin activity, and its mRNA have been localized in the kidney, submaxillary glands, blood vessels, heart, adrenal glands, and brain tissues by enzymatic assays, immunohistochemistry, in situ hybridization histochemistry etc. [37–39]. In the kidney, active renin is primarily localized in the juxtaglomerular apparatus (JGAs) in the afferent arterioles of the kidney under both physiological and diseased conditions [40–42]. For example, light and electron microscopic immunocytochemistry with an antibody to purified human renal renin localized renin in the secretion granules of the epithelioid cells of the afferent arteriole of the JGAs, in renal artery stenosis, or in Bartter's syndrome [36, 37]. In the dog kidney, we have used an in vitro autoradiographic approach to localize active renin using radiolabeled renin inhibitors [40–42]. High resolution light microscopic autoradiography specifically localized active renin to the vascular pole of the glomeruli, or the JGAs (**Figure 1**) [40–42].

group cloned ACE from the mouse kidney in 1988, respectively [49]. ACE in humans consists of 1306 residues with a signal peptide of 29 amino acids [48], whereas ACE in mice contains 1278 amino acids [49]. Approximately 80% of the amino acid sequences are similar between human and mouse ACE. There are two ACE isozymes, one somatic isozyme in the lung, vascular endothelial cells, renal epithelial cells, and testicular Leydig cells, and the other germinal isoenzyme solely in sperm [50–52]. The key actions of ACE are to convert the biologically inactive ANG I to the active peptide ANG II, and to degrade the vasoactive peptide bradykinin. Thus, ACE is most critical for the generation of ANG II in the circulation

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

Abundant ACE is expressed and localized in the kidney, especially in the proximal tubules and glomerular and vascular endothelial cells of intrarenal blood vessels [53–57]. We and others have localized ACE proteins and its mRNA expression in the kidney using quantitative in vitro autoradiography, immunohistochemistry, and in situ hybridization histochemistry (**Figure 1**). For example, the Mendelsohn's group first localized ACE in the rat kidney using quantitative in vitro autoradiography with the radiolabeled ACE inhibitor lisinopril, 125I-351A [53]. ACE was localized primarily to the inner cortex, corresponding to the proximal tubules and blood vessels [53]. We found that infusion of ANG II for 2 weeks significantly increased, rather than downregulated, ACE in the proximal tubules of the rat kidney [54]. At higher resolutions, Brunevaly et al. and others showed ACE primarily in the microvilli and brush borders of the proximal tubules in the human kidney [55–57]. In the vasculature, ACE was localized to the vascular endothelial cells especially in the peritubular capillaries, but not glomerular capillaries of the kidney [53–57]. ACE was also localized inside the renal

vascular endothelial and proximal tubular cell in endoplasmic reticulum,

endosomes, and nuclear envelope, suggesting the presence of intracellular and/or nuclear ACE [53–57]. However, only very low levels of ACE were detected in the

Angiotensin II (ANG II) is undoubtedly the most powerful peptide in the RAS super family, playing a key role in regulating renal blood flow, glomerular filtration, and proximal tubular reabsorption of sodium and fluid, contributing to normal blood pressure and body salt and fluid homeostasis [58–64]. It is well-recognized that the levels of ANG II in the kidney, especially in the proximal tubules, are higher than in the plasma or other tissues. Indeed, local expression and biosynthesis of angiotensinogen, renin, and ACE in the proximal tubules of the kidney significantly contribute to high levels of ANG II levels in the kidneys under physiological conditions [64–68]. Furthermore, ANG II levels are further increased in the kidney of animal models of ANG II-dependent hypertension, even though the circulating and JGA renin and ACE are suppressed [67–73]. This is likely due to the fact that the proximal tubules express all major components of the RAS necessary for the formation of ANG II [38, 47, 54, 59, 67, 74, 75], the proximal tubules have a greater capacity to take up circulating ANG II via AT1 (AT1a) receptor-mediated mechanisms [14, 19, 20, 67], and to augmentation of the expression or generation of angiotensinogen, ACE and ANG II in ANG II-induced hypertension [54, 67, 70, 73]. Finally, ANG II is not only generated in the intratubular fluid compartment, but also localized in intracellular organelles, such as endosomes, mitochondria, and nuclei [15, 67, 71, 74, 75], where it serves as an important intracellular or intracrine

and tissues.

inner medulla.

peptide.

**85**

**2.4 Angiotensin II (ANG II)**

In the proximal tubule of the kidney, renin mRNAs have been reported [43, 44]. Renin activity and mRNAs were detectable in cultured rabbit proximal tubule cells [45], in isolated proximal convoluted and straight tubules, but not in outer medullary collecting ducts [44]. Tang et al. reported that all major components of the RAS, including angiotensinogen, angiotensin converting enzyme, and renin, were expressed in an immortalized rat proximal tubule cell line [45]. However, there is also evidence that renin localized in the proximal tubules may be due to the uptake of circulating renin after filtration [46, 47]. Taugner et al. demonstrated that the reabsorptive pinocytosis of the filtered renin was the primary source of tubular renin in the kidney [46], whereas Iwao et al. used light and electron microscopic autoradiography to localize 125I-labeled renin accumulated in the apical membranes of the proximal convoluted tubules [47]. Taken together, these studies strongly support the concept that in addition to local biosynthesis and expression, circulating or interstitial renin may be taken up by the proximal convoluted tubules in the kidney.

## **2.3 Angiotensin I-converting enzyme (ACE)**

The 2nd key enzyme for the activation of the RAS is ACE, a dipeptidyl carboxypeptidase I, kininase II and EC 3.4.15.1 [48]. Corvol's group first molecularly cloned ACE from human vascular endothelial cells [48], whereas Bernstein's

#### **Figure 1.**

*Intrarenal localization of renin in the juxtaglomerular apparatus (A: JGA), angiotensin-converting enzyme (B: ACE), and angiotensin II AT1 receptors in the kidney (C: AT1 or AT1a) using quantitative in vitro autoradiography. C, renal cortex; G, glomerulus; IM, inner medulla; ISOM, inner stripe of the outer medulla; PCT, proximal convoluted tubule.*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

group cloned ACE from the mouse kidney in 1988, respectively [49]. ACE in humans consists of 1306 residues with a signal peptide of 29 amino acids [48], whereas ACE in mice contains 1278 amino acids [49]. Approximately 80% of the amino acid sequences are similar between human and mouse ACE. There are two ACE isozymes, one somatic isozyme in the lung, vascular endothelial cells, renal epithelial cells, and testicular Leydig cells, and the other germinal isoenzyme solely in sperm [50–52]. The key actions of ACE are to convert the biologically inactive ANG I to the active peptide ANG II, and to degrade the vasoactive peptide bradykinin. Thus, ACE is most critical for the generation of ANG II in the circulation and tissues.

Abundant ACE is expressed and localized in the kidney, especially in the proximal tubules and glomerular and vascular endothelial cells of intrarenal blood vessels [53–57]. We and others have localized ACE proteins and its mRNA expression in the kidney using quantitative in vitro autoradiography, immunohistochemistry, and in situ hybridization histochemistry (**Figure 1**). For example, the Mendelsohn's group first localized ACE in the rat kidney using quantitative in vitro autoradiography with the radiolabeled ACE inhibitor lisinopril, 125I-351A [53]. ACE was localized primarily to the inner cortex, corresponding to the proximal tubules and blood vessels [53]. We found that infusion of ANG II for 2 weeks significantly increased, rather than downregulated, ACE in the proximal tubules of the rat kidney [54]. At higher resolutions, Brunevaly et al. and others showed ACE primarily in the microvilli and brush borders of the proximal tubules in the human kidney [55–57]. In the vasculature, ACE was localized to the vascular endothelial cells especially in the peritubular capillaries, but not glomerular capillaries of the kidney [53–57]. ACE was also localized inside the renal vascular endothelial and proximal tubular cell in endoplasmic reticulum, endosomes, and nuclear envelope, suggesting the presence of intracellular and/or nuclear ACE [53–57]. However, only very low levels of ACE were detected in the inner medulla.

## **2.4 Angiotensin II (ANG II)**

Angiotensin II (ANG II) is undoubtedly the most powerful peptide in the RAS super family, playing a key role in regulating renal blood flow, glomerular filtration, and proximal tubular reabsorption of sodium and fluid, contributing to normal blood pressure and body salt and fluid homeostasis [58–64]. It is well-recognized that the levels of ANG II in the kidney, especially in the proximal tubules, are higher than in the plasma or other tissues. Indeed, local expression and biosynthesis of angiotensinogen, renin, and ACE in the proximal tubules of the kidney significantly contribute to high levels of ANG II levels in the kidneys under physiological conditions [64–68]. Furthermore, ANG II levels are further increased in the kidney of animal models of ANG II-dependent hypertension, even though the circulating and JGA renin and ACE are suppressed [67–73]. This is likely due to the fact that the proximal tubules express all major components of the RAS necessary for the formation of ANG II [38, 47, 54, 59, 67, 74, 75], the proximal tubules have a greater capacity to take up circulating ANG II via AT1 (AT1a) receptor-mediated mechanisms [14, 19, 20, 67], and to augmentation of the expression or generation of angiotensinogen, ACE and ANG II in ANG II-induced hypertension [54, 67, 70, 73]. Finally, ANG II is not only generated in the intratubular fluid compartment, but also localized in intracellular organelles, such as endosomes, mitochondria, and nuclei [15, 67, 71, 74, 75], where it serves as an important intracellular or intracrine peptide.

angiotensinogen/renin/ACE/ANG II/AT1 receptor activation in the cardiovascular, kidney, and other major target tissues. Human renin precursor consists of 406 amino acids with a pre- and a pro-segment of 20 and 46 amino acids, respectively [36]. Mature human renin contains 340 amino acids and a molecular wt. of 37 kDa [36]. Renin, renin activity, and its mRNA have been localized in the kidney, submaxillary glands, blood vessels, heart, adrenal glands, and brain tissues by enzymatic assays, immunohistochemistry, in situ hybridization histochemistry etc. [37–39]. In the kidney, active renin is primarily localized in the juxtaglomerular apparatus (JGAs) in the afferent arterioles of the kidney under both physiological and diseased conditions [40–42]. For example, light and electron microscopic immunocytochemistry with an antibody to purified human renal renin localized renin in the secretion granules of the epithelioid cells of the afferent arteriole of the JGAs, in renal artery stenosis, or in Bartter's syndrome [36, 37]. In the dog kidney, we have used an in vitro autoradiographic approach to localize active renin using radiolabeled renin inhibitors [40–42]. High resolution light microscopic autoradiography specifically localized active renin to the vascular pole of the glomeruli,

In the proximal tubule of the kidney, renin mRNAs have been reported [43, 44]. Renin activity and mRNAs were detectable in cultured rabbit proximal tubule cells [45], in isolated proximal convoluted and straight tubules, but not in outer medullary collecting ducts [44]. Tang et al. reported that all major components of the RAS, including angiotensinogen, angiotensin converting enzyme, and renin, were expressed in an immortalized rat proximal tubule cell line [45]. However, there is also evidence that renin localized in the proximal tubules may be due to the uptake of circulating renin after filtration [46, 47]. Taugner et al. demonstrated that the reabsorptive pinocytosis of the filtered renin was the primary source of tubular renin in the kidney [46], whereas Iwao et al. used light and electron microscopic autoradiography to localize 125I-labeled renin accumulated in the apical membranes of the proximal convoluted tubules [47]. Taken together, these studies strongly support the concept that in addition to local biosynthesis and expression, circulating

or interstitial renin may be taken up by the proximal convoluted tubules in

The 2nd key enzyme for the activation of the RAS is ACE, a dipeptidyl carboxypeptidase I, kininase II and EC 3.4.15.1 [48]. Corvol's group first molecularly cloned ACE from human vascular endothelial cells [48], whereas Bernstein's

*Intrarenal localization of renin in the juxtaglomerular apparatus (A: JGA), angiotensin-converting enzyme (B: ACE), and angiotensin II AT1 receptors in the kidney (C: AT1 or AT1a) using quantitative in vitro autoradiography. C, renal cortex; G, glomerulus; IM, inner medulla; ISOM, inner stripe of the outer*

**2.3 Angiotensin I-converting enzyme (ACE)**

or the JGAs (**Figure 1**) [40–42].

*Selected Chapters from the Renin-Angiotensin System*

the kidney.

**Figure 1.**

**84**

*medulla; PCT, proximal convoluted tubule.*

## **2.5 AT1 and AT2 receptors**

It is now well-accepted that ANG II binds to and activates two different classes of G protein-coupled receptors (GPCRs) to induce well-recognized cardiovascular, renal and blood pressure responses, following the successful development of nonpeptide ANG II type 1 and type 2 receptor antagonists [76–78]. Molecular cloning of AT1 and AT2 receptors and studies of animal models with genetically knockout of these receptors further confirms their pharmacological characterization. Murphy et al. [79] and Sasaki et al. [80] successfully cloned the AT1 receptor in 1991, showing that the AT1 receptor shares the seven-transmembrane-region motif of the GPCR superfamily. AT1 receptors mediate the well-known actions of ANG II on vasoconstriction, cardiac hypertrophy, hypertensive, renal salt retention, as well as aldosterone biosynthesis [76–78, 81]. The AT2 receptor was cloned by Mukoyama et al. [82], Nakajima et al. [83], and Kambayashi et al. [84], respectively. The AT2 receptor was found to have 34% of the identical sequence to the AT1 receptor, sharing a seven-transmembrane domain topology of GPCRs [82–84]. However, the roles and signal transduction pathways for the AT2 receptor remain incompletely understood.

This disparity in our understanding extracellular versus intracellular ANG II has led many to assume that ANG II only activates cell surface receptors to induce all of its biological and physiological responses, and that all ARBs would only block cell surface receptors to produce the same beneficial effects. Thus, an intracellular ANG II system is thought to be unnecessary in the regulation of cardiovascular, blood

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

However, recent studies strongly suggest that these views may be revised for a number of reasons [96–101]. First, it is well-recognized that extracellular ANG II is continuously internalized with its receptors after it activates cell surface receptors. This has long been interpreted only as required for the desensitization of cell surface receptors to repetitive stimulation by extracellular ANG II by moving the ANG II/ AT1 complex into the lysosomal pathway for degradation. There is evidence, however, that the activated agonist/receptor complex internalized into the endosomes may continue to transmit ras/mitogen-activated protein kinase (MAPK) signaling [12, 13]. Ras and MAPK signaling for AT1a, vasopressin V2, and *β*2 adrenergic receptors (*β*2AR) have been reported in endosomal membranes [12, 13, 15, 16], the endoplasmic reticulum, the Golgi or the nucleus independent of cell surface receptor-initiated signaling [81, 88, 89, 102]. Second, ANG II exerts long-lasting genomic or transcriptional effects, which may be independent from the wellrecognized effects induced by activation of cell surface receptors [97–99, 102, 103]. ANG II induces the expression or transcription of many growth factors and proliferative cytokines including nuclear factor-κB (NF-κB) [104–107], monocyte chemoattractant protein-1 (MCP-1) [106, 108], TNF-α [107], and TGF-β1

[102, 109, 110]. While hemodynamic responses to ANG II often occur in seconds or minutes, cellular growth, mitogenic, proliferative and fibrotic responses to ANG II may last from hours to weeks and months. Since the cell surface AT1 (AT1a) receptors may be desensitized in response to sustained exposure to endocrine and para-

cardiovascular, hypertensive, and renal diseases, are at least in part mediated by intracellular ANG II system. Third, not all ARBs, ACE or renin inhibitors are created equal to block both extracellular and intracellular ANG II systems. ARBs may differ in their lipophilic ability to enter the cells to block intracellular AT1 receptors [111– 113]. Indeed, ARBs show different effects on uric acid metabolism, cell proliferation, oxidative stress, nitric oxide production and PPAR-γ activity [111–113]. We and others have shown that losartan internalized with AT1a and AT1b receptors, albeit at a slower rate than ANG II [19, 20, 67, 103, 114], and to attenuate ANG II-

crine ANG II, the long-term genomic effects of ANG II, as observed in

induced intracellular and nuclear effects [15, 88, 89, 102, 103]. Moreover,

block both extracellular and intracellular ANG II-induced effects.

**87**

**4. Intratubular and intracellular ANG II: AT1a receptor-mediated**

**uptake of circulating and paracrine ANG II in the proximal tubules**

We and others have investigated whether circulating and local paracrine ANG II is taken up by the proximal tubules of the kidney via AT1 (AT1a) receptor-mediated endocytosis [19, 20, 118–121], and whether internalized ANG II and AT1a receptors

telmisartan not only blocks AT1 receptors, but also acts as a partial activator of liverspecific peroxisome proliferator-activated receptor γ (PPAR-γ) [111, 115–157]. Finally, some clinical studies have shown that even treated with renin inhibitors, ACE inhibitors or ARBs, there are some patients who still progress to hypertension and suffer from cardiovascular and renal complications [111, 115–117]. These data suggest that additional mechanisms should be involved and studied accordingly. Thus, the new challenges to the field are to study whether and how intracellular ANG II may contribute to these mechanisms and design multifunctional drugs to

pressure, and renal physiology and diseases.

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

In the kidney, the AT1 receptor is widely expressed and localized in different structures or cell types, most prominent in three anatomical regions, that is, the glomerulus, proximal tubules, and the inner stripe of the outer medulla, corresponding the vasa recta blood vessels and renomedullary interstitial cells (**Figure 1**) [85–87]. We and others have consistently localized the AT1 receptor in the rodent and human kidneys using quantitative in vitro and in vivo autoradiography, with high levels of these receptors in the glomerulus, proximal tubules, and renomedullary interstitial cells (**Figure 1**) [85–87]. Other anatomical regions or renal structures may express low levels of AT1 receptor expression, detectable with RT-PCR or immunohistochemistry. AT1 receptors have also been localized in intracellular organelles, for example, endosomes, mitochondria, and nuclei in the proximal tubule cells, suggesting an important intracellular roles [67, 74, 88–90]. By contrast, the levels of AT2 receptor expression in the kidney are species-related or closely associated with the kidney development. Indeed, high levels of AT2 receptors are expressed extensively in the developing fetal and neonatal tissues, but most of them disappear before reaching the adulthood [87]. Nevertheless, the expression of AT2 receptors appears to persist in the adrenal medulla, proximal tubules, and the adventitia of human kidney blood vessels, suggesting potential roles for these receptors in these target tissues [85–87, 91–93].

## **3. Intratubular and intracellular ANG II: the long-term genomic effects induced by endocrine, paracrine and intracellular ANG II**

In contrast to the classic dogma that ANG II only binds to and activates cell surface GPCRs to initiate downstream signaling responses, ANG II can also bind and activate intracellular GPCRs to induce long-term genomic effects. The RAS includes an extracellular system and an intracellular system. ANG II acts as the principle effector of both extracellular and intracellular RAS. Extracellular ANG II includes circulating (endocrine) and paracrine ANG II, which plays the classical roles of the RAS through activation of cell surface GPCRs [76–78, 81, 94, 95]. Intracellular ANG II includes intracellularly formed ANG II (intracrine) and ANG II internalized through AT1 (AT1a) receptor-mediated endocytosis [96–101]. The roles of circulating and paracrine ANG II and its GPCR-mediated signaling mechanisms via cell surface receptors have been extensively investigated. By contrast, the roles of intracellular ANG II and its mechanisms of actions remain poorly understood.

#### *The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

This disparity in our understanding extracellular versus intracellular ANG II has led many to assume that ANG II only activates cell surface receptors to induce all of its biological and physiological responses, and that all ARBs would only block cell surface receptors to produce the same beneficial effects. Thus, an intracellular ANG II system is thought to be unnecessary in the regulation of cardiovascular, blood pressure, and renal physiology and diseases.

However, recent studies strongly suggest that these views may be revised for a number of reasons [96–101]. First, it is well-recognized that extracellular ANG II is continuously internalized with its receptors after it activates cell surface receptors. This has long been interpreted only as required for the desensitization of cell surface receptors to repetitive stimulation by extracellular ANG II by moving the ANG II/ AT1 complex into the lysosomal pathway for degradation. There is evidence, however, that the activated agonist/receptor complex internalized into the endosomes may continue to transmit ras/mitogen-activated protein kinase (MAPK) signaling [12, 13]. Ras and MAPK signaling for AT1a, vasopressin V2, and *β*2 adrenergic receptors (*β*2AR) have been reported in endosomal membranes [12, 13, 15, 16], the endoplasmic reticulum, the Golgi or the nucleus independent of cell surface receptor-initiated signaling [81, 88, 89, 102]. Second, ANG II exerts long-lasting genomic or transcriptional effects, which may be independent from the wellrecognized effects induced by activation of cell surface receptors [97–99, 102, 103]. ANG II induces the expression or transcription of many growth factors and proliferative cytokines including nuclear factor-κB (NF-κB) [104–107], monocyte chemoattractant protein-1 (MCP-1) [106, 108], TNF-α [107], and TGF-β1 [102, 109, 110]. While hemodynamic responses to ANG II often occur in seconds or minutes, cellular growth, mitogenic, proliferative and fibrotic responses to ANG II may last from hours to weeks and months. Since the cell surface AT1 (AT1a) receptors may be desensitized in response to sustained exposure to endocrine and paracrine ANG II, the long-term genomic effects of ANG II, as observed in cardiovascular, hypertensive, and renal diseases, are at least in part mediated by intracellular ANG II system. Third, not all ARBs, ACE or renin inhibitors are created equal to block both extracellular and intracellular ANG II systems. ARBs may differ in their lipophilic ability to enter the cells to block intracellular AT1 receptors [111– 113]. Indeed, ARBs show different effects on uric acid metabolism, cell proliferation, oxidative stress, nitric oxide production and PPAR-γ activity [111–113]. We and others have shown that losartan internalized with AT1a and AT1b receptors, albeit at a slower rate than ANG II [19, 20, 67, 103, 114], and to attenuate ANG IIinduced intracellular and nuclear effects [15, 88, 89, 102, 103]. Moreover, telmisartan not only blocks AT1 receptors, but also acts as a partial activator of liverspecific peroxisome proliferator-activated receptor γ (PPAR-γ) [111, 115–157]. Finally, some clinical studies have shown that even treated with renin inhibitors, ACE inhibitors or ARBs, there are some patients who still progress to hypertension and suffer from cardiovascular and renal complications [111, 115–117]. These data suggest that additional mechanisms should be involved and studied accordingly. Thus, the new challenges to the field are to study whether and how intracellular ANG II may contribute to these mechanisms and design multifunctional drugs to block both extracellular and intracellular ANG II-induced effects.

## **4. Intratubular and intracellular ANG II: AT1a receptor-mediated uptake of circulating and paracrine ANG II in the proximal tubules**

We and others have investigated whether circulating and local paracrine ANG II is taken up by the proximal tubules of the kidney via AT1 (AT1a) receptor-mediated endocytosis [19, 20, 118–121], and whether internalized ANG II and AT1a receptors

**2.5 AT1 and AT2 receptors**

*Selected Chapters from the Renin-Angiotensin System*

incompletely understood.

**86**

It is now well-accepted that ANG II binds to and activates two different classes of G protein-coupled receptors (GPCRs) to induce well-recognized cardiovascular, renal and blood pressure responses, following the successful development of nonpeptide ANG II type 1 and type 2 receptor antagonists [76–78]. Molecular cloning of AT1 and AT2 receptors and studies of animal models with genetically knockout of these receptors further confirms their pharmacological characterization. Murphy et al. [79] and Sasaki et al. [80] successfully cloned the AT1 receptor in 1991, showing that the AT1 receptor shares the seven-transmembrane-region motif of the GPCR superfamily. AT1 receptors mediate the well-known actions of ANG II on vasoconstriction, cardiac hypertrophy, hypertensive, renal salt retention, as well as aldosterone biosynthesis [76–78, 81]. The AT2 receptor was cloned by Mukoyama et al. [82], Nakajima et al. [83], and Kambayashi et al. [84], respectively. The AT2 receptor was found to have 34% of the identical sequence to the AT1 receptor, sharing a seven-transmembrane domain topology of GPCRs [82–84]. However, the roles and signal transduction pathways for the AT2 receptor remain

In the kidney, the AT1 receptor is widely expressed and localized in different structures or cell types, most prominent in three anatomical regions, that is, the

**3. Intratubular and intracellular ANG II: the long-term genomic effects**

In contrast to the classic dogma that ANG II only binds to and activates cell surface GPCRs to initiate downstream signaling responses, ANG II can also bind and activate intracellular GPCRs to induce long-term genomic effects. The RAS includes an extracellular system and an intracellular system. ANG II acts as the principle effector of both extracellular and intracellular RAS. Extracellular ANG II includes circulating (endocrine) and paracrine ANG II, which plays the classical roles of the RAS through activation of cell surface GPCRs [76–78, 81, 94, 95]. Intracellular ANG II includes intracellularly formed ANG II (intracrine) and ANG II internalized through AT1 (AT1a) receptor-mediated endocytosis [96–101]. The roles of circulating and paracrine ANG II and its GPCR-mediated signaling mechanisms via cell surface receptors have been extensively investigated. By contrast, the roles of intracellular ANG II and its mechanisms of actions remain poorly understood.

**induced by endocrine, paracrine and intracellular ANG II**

glomerulus, proximal tubules, and the inner stripe of the outer medulla, corresponding the vasa recta blood vessels and renomedullary interstitial cells (**Figure 1**) [85–87]. We and others have consistently localized the AT1 receptor in the rodent and human kidneys using quantitative in vitro and in vivo autoradiography, with high levels of these receptors in the glomerulus, proximal tubules, and renomedullary interstitial cells (**Figure 1**) [85–87]. Other anatomical regions or renal structures may express low levels of AT1 receptor expression, detectable with RT-PCR or immunohistochemistry. AT1 receptors have also been localized in intracellular organelles, for example, endosomes, mitochondria, and nuclei in the proximal tubule cells, suggesting an important intracellular roles [67, 74, 88–90]. By contrast, the levels of AT2 receptor expression in the kidney are species-related or closely associated with the kidney development. Indeed, high levels of AT2 receptors are expressed extensively in the developing fetal and neonatal tissues, but most of them disappear before reaching the adulthood [87]. Nevertheless, the expression of AT2 receptors appears to persist in the adrenal medulla, proximal tubules, and the adventitia of human kidney blood vessels, suggesting potential roles for these

receptors in these target tissues [85–87, 91–93].

are co-localized in the endosomal compartment and nucleus (**Figure 2**) [67, 74, 88, 89]. Our studies demonstrated that global deletion of AT1a receptors blocked the uptake of unlabeled Val<sup>5</sup> -ANG II [19] or [125I]Val5 -ANG II in the kidney of AT1a-KO mice [20]. However, these studies focused only on the entire kidney, and what nephron segments involved in taking up unlabeled Val<sup>5</sup> -ANG II or [125I]Val5 -ANG II could not be determined using these approaches [19, 20]. We further used cultured proximal tubules cells to test whether proximal tubule cells take up extracellular ANG II and the mechanisms involved (**Figure 2**) [14, 100, 122–126]. The advantages of using these cells for the proposed studies are that ANG II receptors are abundantly expressed and localized in both apical (AP) and basolateral (BL) membranes [127–131]. However, it has not been determined whether ANG II receptors in AP or BL membranes mediate ANG II uptake in the proximal tubules. In a previous study using a porcine proximal tubule cell line expressing a rabbit AT1 receptor, AT1-mediated uptake of [125I]-ANG II was found to be significantly different between AP and BL membranes [130]. AT1-mediated uptake of [125I]-ANG II was more robust and efficient in AP membranes than in BL membranes [130]. Conversely, ANG II-induced AT1 receptor internalization was reportedly much faster in BL membranes than in AP membranes of OK cells [131]. Thus these differences inAT1-mediated uptake of [125I]-ANG II or ANG II-induced AT1 receptor endocytosis or internalization may underscore the differences in the cell types used or experimental conditions.

extent to which megalin- and caveolin 1-mediate ANG II uptake in proximal tubule cells is significantly smaller than that mediated by AT1 (AT1a) receptor-dependent

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

**5. Intratubular and intracellular ANG II: canonical versus noncanonical endocytic pathways in mediating ANG II uptake in the proximal**

We have mechanistically investigated that AT1 (AT1a) receptor-mediate the uptake of extracellular ANG II by proximal tubule cells in vitro and circulating ANG II in vivo [19, 20, 122–126]. It has been previously shown that in vascular smooth muscle cells (VSMCs), cardiomyocytes, and COS-7 cells, *β*2 adrenergic receptors, AT1a, epidermal growth factor receptors, and insulin receptors are internalized via the canonical clathrin-dependent pathway [137–144]. Clathrin-coated pits play an important role in invaginating and pinching off the plasma membranes to form coated vesicles and targeted to endosomes [138, 140, 142]. GPCR kinases (GRKs),

small GTP-binding proteins, such as Rab5, and β-arrestins are reportedly

involved in clathrin-dependent AT1a endocytosis [145, 146]. However, dominantnegatives, siRNAs or knockout targeting dynamin, GRKs or β-arrestins have little effects on AT1a receptor endocytosis in some studies, suggesting that alternative (non-canonical) pathways may also be involved in AT1a receptor endocytosis

There is evidence to suggest that tyrosine phosphatases may be involved in ANG II-induced AT1 receptor endocytosis in AP and BL membranes, since the endocytic response was inhibited by the tyrosine phosphatases inhibitor, phenylarsine oxide (PAO), rather than by pertussis toxin [147–151]. Colchicine, an inhibitor of cytoskeleton microtubules [148], also appeared to inhibit AT1 receptor-mediated ANG II uptake and its effects in rat proximal tubule cells [150, 151]. The role of clathrincoated pits in mediating AT1 receptor-mediated ANG II uptake was also investigated, but we found that deletion of clathrin-coated pits with sucrose or specific siRNAs to knock down clathrin light (LC) or high chain subunits (HC) failed to


How ANG II and AT1 receptors are internalized into the endosomal compartments and transported to other organelles or the nucleus in proximal tubule cells remains incompletely understood. Intravenous infusion of 125I-labeled ANG II was previously detected in the nuclei of rat vascular smooth muscle cells (VSMCs) and cardiac myocytes [152] or the Golgi of adrenal cells [153]. Cook et al. showed that ANG II and its AT1a receptor were translocated to the nuclei of hepatocytes and VSMCs [154]. In AT1a receptor-expressing HEK 293 cells, internalized AT1a receptors were detected in perinuclear areas as well as in the nuclei [155, 156].

In supporting the above-mentioned studies, we also reported high levels of internalized FITC-labeled ANG II in perinuclear areas and the nucleus, which was inhibited by colchicine and siRNA knockdown of MAP-1A [14, 122, 123, 151]. Taken together, our results strongly suggest that the microtubule-dependent pathway may play an important role in mediating the nuclear translocation of internalized ANG II/AT1 receptor complex in proximal tubule cells. Indeed, a nuclear localization sequence (NLS, KKFKKY, aa307-312) has been identified


mechanism [19, 20, 122, 123].

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

**tubules**

[137–146].

Val<sup>5</sup>

**89**

alter AT1-mediated uptake of Val<sup>5</sup>

mediated uptake of ANG II in proximal tubule cells.

In addition to AT1 (AT1a) receptors, other factors may also regulate the uptake of extracellular ANG II by proximal tubule cells. AP membranes of proximal tubule cells express abundant endocytic receptor megalin, which plays a crucial role in mediating the uptake of low molecular weight (LMW) proteins in proximal tubule cells [132–136]. Deletion of megalin in mice led to the development of LMW proteinuria [135]. Interestingly, megalin also binds and internalizes ANG II in immortalized yolk sac cells (BN-16 cells) [136]. We have demonstrated that siRNA knockdown of megalin expression or caveolin 1 in proximal tubule cells significantly attenuated ANG II uptake by proximal tubule cells [122, 123]. However, the

#### **Figure 2.**

*All major components of the circulating RAS, including angiotensinogen (AGT), renin, angiotensin I (ANG I), and ANG II, may be filtered by the kidney glomerulus and taken up by the proximal tubules. Alternatively, all major components of the RAS may be expressed and localized in the proximal tubules of the kidney. ACE, angiotensin-converting enzyme and APA, aminopeptidase A.*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

extent to which megalin- and caveolin 1-mediate ANG II uptake in proximal tubule cells is significantly smaller than that mediated by AT1 (AT1a) receptor-dependent mechanism [19, 20, 122, 123].

## **5. Intratubular and intracellular ANG II: canonical versus noncanonical endocytic pathways in mediating ANG II uptake in the proximal tubules**

We have mechanistically investigated that AT1 (AT1a) receptor-mediate the uptake of extracellular ANG II by proximal tubule cells in vitro and circulating ANG II in vivo [19, 20, 122–126]. It has been previously shown that in vascular smooth muscle cells (VSMCs), cardiomyocytes, and COS-7 cells, *β*2 adrenergic receptors, AT1a, epidermal growth factor receptors, and insulin receptors are internalized via the canonical clathrin-dependent pathway [137–144]. Clathrin-coated pits play an important role in invaginating and pinching off the plasma membranes to form coated vesicles and targeted to endosomes [138, 140, 142]. GPCR kinases (GRKs), small GTP-binding proteins, such as Rab5, and β-arrestins are reportedly involved in clathrin-dependent AT1a endocytosis [145, 146]. However, dominantnegatives, siRNAs or knockout targeting dynamin, GRKs or β-arrestins have little effects on AT1a receptor endocytosis in some studies, suggesting that alternative (non-canonical) pathways may also be involved in AT1a receptor endocytosis [137–146].

There is evidence to suggest that tyrosine phosphatases may be involved in ANG II-induced AT1 receptor endocytosis in AP and BL membranes, since the endocytic response was inhibited by the tyrosine phosphatases inhibitor, phenylarsine oxide (PAO), rather than by pertussis toxin [147–151]. Colchicine, an inhibitor of cytoskeleton microtubules [148], also appeared to inhibit AT1 receptor-mediated ANG II uptake and its effects in rat proximal tubule cells [150, 151]. The role of clathrincoated pits in mediating AT1 receptor-mediated ANG II uptake was also investigated, but we found that deletion of clathrin-coated pits with sucrose or specific siRNAs to knock down clathrin light (LC) or high chain subunits (HC) failed to alter AT1-mediated uptake of Val<sup>5</sup> -ANG II [151]. However, AT1-mediated uptake of Val<sup>5</sup> -ANG II was significantly inhibited by colchicine or siRNA knocking down of microtubule-associated proteins, MAP-1A or MAP-1B, in proximal tubule cells [151]. Our studies therefore support the scientific premise that the noncanonical microtubule-dependent endocytic pathway may be involved in mediating the AT1 mediated uptake of ANG II in proximal tubule cells.

How ANG II and AT1 receptors are internalized into the endosomal compartments and transported to other organelles or the nucleus in proximal tubule cells remains incompletely understood. Intravenous infusion of 125I-labeled ANG II was previously detected in the nuclei of rat vascular smooth muscle cells (VSMCs) and cardiac myocytes [152] or the Golgi of adrenal cells [153]. Cook et al. showed that ANG II and its AT1a receptor were translocated to the nuclei of hepatocytes and VSMCs [154]. In AT1a receptor-expressing HEK 293 cells, internalized AT1a receptors were detected in perinuclear areas as well as in the nuclei [155, 156]. In supporting the above-mentioned studies, we also reported high levels of internalized FITC-labeled ANG II in perinuclear areas and the nucleus, which was inhibited by colchicine and siRNA knockdown of MAP-1A [14, 122, 123, 151]. Taken together, our results strongly suggest that the microtubule-dependent pathway may play an important role in mediating the nuclear translocation of internalized ANG II/AT1 receptor complex in proximal tubule cells. Indeed, a nuclear localization sequence (NLS, KKFKKY, aa307-312) has been identified

are co-localized in the endosomal compartment and nucleus (**Figure 2**) [67, 74, 88, 89]. Our studies demonstrated that global deletion of AT1a receptors blocked the

mice [20]. However, these studies focused only on the entire kidney, and what

In addition to AT1 (AT1a) receptors, other factors may also regulate the uptake of extracellular ANG II by proximal tubule cells. AP membranes of proximal tubule cells express abundant endocytic receptor megalin, which plays a crucial role in mediating the uptake of low molecular weight (LMW) proteins in proximal tubule cells [132–136]. Deletion of megalin in mice led to the development of LMW proteinuria [135]. Interestingly, megalin also binds and internalizes ANG II in immortalized yolk sac cells (BN-16 cells) [136]. We have demonstrated that siRNA knockdown of megalin expression or caveolin 1 in proximal tubule cells significantly attenuated ANG II uptake by proximal tubule cells [122, 123]. However, the

*All major components of the circulating RAS, including angiotensinogen (AGT), renin, angiotensin I (ANG I), and ANG II, may be filtered by the kidney glomerulus and taken up by the proximal tubules. Alternatively, all major components of the RAS may be expressed and localized in the proximal tubules of the kidney. ACE,*

*angiotensin-converting enzyme and APA, aminopeptidase A.*

II could not be determined using these approaches [19, 20]. We further used cultured proximal tubules cells to test whether proximal tubule cells take up extracellular ANG II and the mechanisms involved (**Figure 2**) [14, 100, 122–126]. The advantages of using these cells for the proposed studies are that ANG II receptors are abundantly expressed and localized in both apical (AP) and basolateral (BL) membranes [127–131]. However, it has not been determined whether ANG II receptors in AP or BL membranes mediate ANG II uptake in the proximal tubules. In a previous study using a porcine proximal tubule cell line expressing a rabbit AT1 receptor, AT1-mediated uptake of [125I]-ANG II was found to be significantly different between AP and BL membranes [130]. AT1-mediated uptake of [125I]-ANG II was more robust and efficient in AP membranes than in BL membranes [130]. Conversely, ANG II-induced AT1 receptor internalization was reportedly much faster in BL membranes than in AP membranes of OK cells [131]. Thus these differences inAT1-mediated uptake of [125I]-ANG II or ANG II-induced AT1 receptor endocytosis or internalization may underscore the differences in the cell types





nephron segments involved in taking up unlabeled Val<sup>5</sup>

*Selected Chapters from the Renin-Angiotensin System*

uptake of unlabeled Val<sup>5</sup>

used or experimental conditions.

**Figure 2.**

**88**

within the AT1a receptor, which may mediate nuclear trafficking and activation of AT1a receptors by ANG II [155, 156].

## **6. Intratubular and intracellular ANG II: intracellular versus extracellular effects and signaling mechanisms in the proximal tubules**

In the proximal tubules of the kidney, extracellular ANG II has been reported to stimulate the expression of Na<sup>+</sup> /H+ exchanger 3 (NHE3) [14, 16, 102, 125], AP insertion of NHE3 [157], Na<sup>+</sup> /H<sup>+</sup> exchanger activity [158–161], or NHE3-induced 22Na+ uptake in cultured or isolated proximal tubule cells [162, 163]. The signaling mechanisms by which extracellular ANG II increases the expression and activity of NHE3 in proximal tubule cells have been well studied and documented [164–169]. The most well-described signal mechanism is that ANG II activates cell surface receptor-coupled G proteins, with subsequent increases in IP3 and [Ca2+]i, generation of DG, and activation of PKC [164–169]. The other well-recognized downstream signaling pathways for extracellular ANG II to induce biological or physiological responses also include activation or inhibition of calcium-dependent calcineurin [170], cAMP-dependent protein kinase A (PKA) [169, 171], Ca2+-independent PLA2 [172], PI 3-kinase [157], c-Src/MAP kinases ERK 1/2 [165], or nuclear factor-κB [173].

According to the principles of the G protein-coupled receptor pharmacology, ANG II must bind to its cell surface receptors to activate intracellular signaling mechanisms in order to induce responses [76–78, 138]. Upon internalization, however, ANG II may act as an intracellular peptide to induce biological or physiological responses. Indeed, blockade of the endocytosis of AT1 receptors is associated with inhibition of PKC, IP3 formation, and Na<sup>+</sup> flux in proximal tubule cells [14, 16, 122– 126, 149, 150]. Furthermore, ANG II-induced AT1 receptor endocytosis is also associated with activation of PLA2 [147, 172], inhibition of adenylyl cyclase [151, 169, 171], and increases in Na<sup>+</sup> uptake from AP membranes [149–151]. We have recently shown that AT1-mediated uptake of extracellular Val<sup>5</sup> -ANG II was indeed associated with inhibition of basal and forskolin-stimulated cAMP accumulation [125, 151], ANG II-stimulated NHE3 expression [14, 16, 122, 123], and ANG II-induced activation of MAP Kinases ERK1/2 and nuclear factor-κB in proximal tubule cells [14, 16, 124, 126, 151].

protein [9, 11, 15, 88–90, 102]. Cook et al. overexpressed a cyan fluorescent, intracellular ANG II construct (ECFP/ANG II) with or without a rat yellow fluorescent AT1a receptor (AT1R/EYFP) in rat VSMCs or hepatocytes [9, 97, 98]. They demonstrated that intracellular ANG II induced the proliferation of VSMC via activation of cAMP response element-binding protein (CREB), p38 MAP kinase, and MAP kinases ERK 1/2 [9, 97, 98]. In another study, an intracellular ANG II (pcDNA/TO-iAng II) was expressed in CHO cells to induce cell proliferation, but none of ARBs was found to attenuate the effect of intracellular ANG II on cell proliferation [178, 179]. Nevertheless, these early proof of concept studies suggest that in vitro or in vivo expression of a cyan fluorescent intracellular ANG II fusion protein (ECFP/ANG II) in the proximal tubule cells of wild-type and AT1a-KO mice may be an innovative approach to distinguish the effects of intracellular

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

*proximal tubule cells. Adapted from Zhuo et al. with permission [15].*

*Intracellular microinjection of angiotensin II induces intracellular calcium mobilization in cultured rabbit*

**7. Intratubular and intracellular ANG II: physiological effects of intracellular versus extracellular ANG II on proximal tubule Na<sup>+</sup>**

The physiological roles of intracellular ANG II in the regulation of proximal tubule Na<sup>+</sup> reabsorption and normal blood pressure homeostasis remain to be determined. Whether intracellular and/or internalized ANG II may physiologically regulate proximal tubule Na<sup>+</sup> transport and blood pressure has not been studied until recently. Indeed, this line of research has been long stymied due to the lack of suitable animal models that express an intracellular ANG II protein, which is not

versus extracellular ANG II.

**Figure 3.**

**91**

**reabsorption and blood pressure**

Nevertheless, these approaches are unlikely able to distinguish the effects of ANG II mediated by cell surface or intracellular receptors. Previous studies have shown that single cell microinjection or microdialysis of ANG II directly into the cells may distinguish between the effects induced by extracellular ANG II from those induced by intracellular ANG II [15, 102, 174–177]. Indeed, we have demonstrated that intracellular microinjection of ANG II directly into single rabbit proximal tubule cells induced intracellular [Ca2+]i responses (**Figure 3**) [10, 15, 16, 81, 177]. We further reported that microinjection of the AT1 blocker losartan abolished the [Ca2+]i response induced by microinjected ANG II, but it only partially blocked the effects of extracellular ANG II [15]. In further proof-of-the concept studies, we showed that ANG II stimulated nuclear AT1a receptors to increase in vitro transcription of mRNAs for TGF1, MCP-1 and NHE3 in isolated rat renal cortical nuclei [102]. These studies provide evidence that intracellular ANG II may activate cytoplasmic and nuclear AT1 receptor to induce important genomic effects in proximal tubule cells [15, 102, 174–177].

Whether intracellular ANG II may alter biological responses in a cell culture model has been determined by directly expressing an intracellular ANG II fusion *The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

#### **Figure 3.**

within the AT1a receptor, which may mediate nuclear trafficking and

**6. Intratubular and intracellular ANG II: intracellular versus**

**extracellular effects and signaling mechanisms in the proximal**

In the proximal tubules of the kidney, extracellular ANG II has been reported to

22Na+ uptake in cultured or isolated proximal tubule cells [162, 163]. The signaling mechanisms by which extracellular ANG II increases the expression and activity of NHE3 in proximal tubule cells have been well studied and documented [164–169]. The most well-described signal mechanism is that ANG II activates cell surface receptor-coupled G proteins, with subsequent increases in IP3 and [Ca2+]i, generation of DG, and activation of PKC [164–169]. The other well-recognized downstream signaling pathways for extracellular ANG II to induce biological or

physiological responses also include activation or inhibition of calcium-dependent calcineurin [170], cAMP-dependent protein kinase A (PKA) [169, 171], Ca2+-independent PLA2 [172], PI 3-kinase [157], c-Src/MAP kinases ERK 1/2 [165], or nuclear

According to the principles of the G protein-coupled receptor pharmacology, ANG II must bind to its cell surface receptors to activate intracellular signaling mechanisms in order to induce responses [76–78, 138]. Upon internalization, however, ANG II may act as an intracellular peptide to induce biological or physiological responses. Indeed, blockade of the endocytosis of AT1 receptors is associated with inhibition of PKC, IP3 formation, and Na<sup>+</sup> flux in proximal tubule cells [14, 16, 122– 126, 149, 150]. Furthermore, ANG II-induced AT1 receptor endocytosis is also associated with activation of PLA2 [147, 172], inhibition of adenylyl cyclase [151, 169, 171], and increases in Na<sup>+</sup> uptake from AP membranes [149–151]. We

indeed associated with inhibition of basal and forskolin-stimulated cAMP accumulation [125, 151], ANG II-stimulated NHE3 expression [14, 16, 122, 123], and ANG II-induced activation of MAP Kinases ERK1/2 and nuclear factor-κB in proximal

Nevertheless, these approaches are unlikely able to distinguish the effects of ANG II mediated by cell surface or intracellular receptors. Previous studies have shown that single cell microinjection or microdialysis of ANG II directly into the cells may distinguish between the effects induced by extracellular ANG II from those induced by intracellular ANG II [15, 102, 174–177]. Indeed, we have demonstrated that intracellular microinjection of ANG II directly into single rabbit proximal tubule cells induced intracellular [Ca2+]i responses (**Figure 3**)

[10, 15, 16, 81, 177]. We further reported that microinjection of the AT1 blocker losartan abolished the [Ca2+]i response induced by microinjected ANG II, but it only partially blocked the effects of extracellular ANG II [15]. In further proof-of-the concept studies, we showed that ANG II stimulated nuclear AT1a receptors to increase in vitro transcription of mRNAs for TGF1, MCP-1 and NHE3 in isolated rat renal cortical nuclei [102]. These studies provide evidence that intracellular ANG II may activate cytoplasmic and nuclear AT1 receptor to induce important genomic

Whether intracellular ANG II may alter biological responses in a cell culture model has been determined by directly expressing an intracellular ANG II fusion

have recently shown that AT1-mediated uptake of extracellular Val<sup>5</sup>

/H+ exchanger 3 (NHE3) [14, 16, 102, 125], AP

/H<sup>+</sup> exchanger activity [158–161], or NHE3-induced


activation of AT1a receptors by ANG II [155, 156].

*Selected Chapters from the Renin-Angiotensin System*

**tubules**

factor-κB [173].

**90**

stimulate the expression of Na<sup>+</sup>

tubule cells [14, 16, 124, 126, 151].

effects in proximal tubule cells [15, 102, 174–177].

insertion of NHE3 [157], Na<sup>+</sup>

*Intracellular microinjection of angiotensin II induces intracellular calcium mobilization in cultured rabbit proximal tubule cells. Adapted from Zhuo et al. with permission [15].*

protein [9, 11, 15, 88–90, 102]. Cook et al. overexpressed a cyan fluorescent, intracellular ANG II construct (ECFP/ANG II) with or without a rat yellow fluorescent AT1a receptor (AT1R/EYFP) in rat VSMCs or hepatocytes [9, 97, 98]. They demonstrated that intracellular ANG II induced the proliferation of VSMC via activation of cAMP response element-binding protein (CREB), p38 MAP kinase, and MAP kinases ERK 1/2 [9, 97, 98]. In another study, an intracellular ANG II (pcDNA/TO-iAng II) was expressed in CHO cells to induce cell proliferation, but none of ARBs was found to attenuate the effect of intracellular ANG II on cell proliferation [178, 179]. Nevertheless, these early proof of concept studies suggest that in vitro or in vivo expression of a cyan fluorescent intracellular ANG II fusion protein (ECFP/ANG II) in the proximal tubule cells of wild-type and AT1a-KO mice may be an innovative approach to distinguish the effects of intracellular versus extracellular ANG II.

## **7. Intratubular and intracellular ANG II: physiological effects of intracellular versus extracellular ANG II on proximal tubule Na<sup>+</sup> reabsorption and blood pressure**

The physiological roles of intracellular ANG II in the regulation of proximal tubule Na<sup>+</sup> reabsorption and normal blood pressure homeostasis remain to be determined. Whether intracellular and/or internalized ANG II may physiologically regulate proximal tubule Na<sup>+</sup> transport and blood pressure has not been studied until recently. Indeed, this line of research has been long stymied due to the lack of suitable animal models that express an intracellular ANG II protein, which is not

secreted outside the cells and only acts intracellularly. Dr. Reudelhuber's group was the first to generate genetically modified mouse model that expresses an ANG IIproducing fusion protein in the cardiomyocytes of the rat heart [180, 181]. They used the α myosin heavy chain promoter to control the expression of ANG IIreleasing fusion protein in the cardiomyocytes. Cardiac specific expression of this ANG II fusion protein led to 10-fold increases in ANG II levels in the heart of these transgenic mice, but it did not elevate ANG II levels in the plasma [180, 181]. This approach is very unique to construct this cardiac-specific ANG II fusion protein with a signal peptide sequence derived from human prorenin and a furin cleavage site. Thus, the expressed ANG II fusion protein will be cleaved by furin, and released into the secretory pathway and the cardiac interstitium [180, 181]. It is expected that this cardiac-specific ANG II fusion protein activates cell surface, but not intracellular receptors. In a different study, Baker et al. expressed an intracellular ANG II peptide in the mouse cardiomyocytes using an adenoviral vector [178]. Cardiac-specific expression of this intracellular ANG II peptide in mice induced cardiac hypertrophy, but not altered blood pressure and plasma ANG II [99, 178]. Furthermore, the AT1 receptor blocker failed to block the cardiac hypertrophic effect of this peptide, suggesting that AT1 receptor may not be involved [99, 178].

(Ad-sglt2-ECFP/ANG II), which encodes a cyan fluorescent intracellular ANG II fusion protein (ECFP/ANG II) [17, 18]. The sodium and glucose cotransporter 2 promoter, sglt2, was used to drive the expression of ECFP/ANG II selectively in the proximal tubule cells of the rat and mouse kidneys. Sglt2 is expressed almost exclusively in S1 and S2 segments of the kidney proximal tubules [186]. Using this approach, we have determined whether intrarenal adenovirus-mediated expression of intracellular ECFP/ANG II selectively in the proximal tubules of the rat and mouse kidneys increases the expression and activity of NHE3, stimulate proximal tubule sodium reabsorption, and increase blood pressure in rats and mice. We demonstrated that expression of intracellular ECFP/ANG II selectively in the proximal tubules of rats and mice significantly increased NHE3 expression, proximal tubule sodium reabsorption, and blood pressure (**Figure 4**) [17, 18]. We further showed that AT1 receptor blocker losartan and deletion of AT1a receptors in mice significantly attenuated intracellular ANG II-induced NHE3 expression, proximal tubule sodium reabsorption, and blood pressure responses, suggesting an AT1

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

**8. Intratubular and intracellular ANG II: role of NHE3 in maintaining**

membranes of the proximal tubules of the kidney [187–190]. NHE3 is directly and indirectly responsible for reabsorbing approximately 50–60% of filtered load of

proximal tubules are mediated by NHE3 [187–190]. The importance of proximal tubule NHE3 in maintaining body salt and fluid balance and blood pressure

homeostasis has not been well studied until recently. Overall, global deletion of the NHE3 gene in all tissues of mice (*Nhe3/*) leads to 50% decreases in fluid, Na+

digestive system, and significantly decreases basal blood pressure [191–194]. One of striking phenotypes is absorptive defects in the small intestines due to intestinal NHE3 deletion [191–194]. Moreover, the transgenic rescue of the NHE3 transgene in small intestines in *Nhe3/* mice, tg*Nhe3/*, failed to rescue the structural and absorptive defects of global NHE3 deletion, with basal blood pressure being similar to those of *Nhe3/* mice [195, 196]. These abnormal phenotypes have been con-

However, these studies using either *Nhe3/* or tg*Nhe3/* mice are unable to determine the roles of NHE3 in the proximal tubules of the kidney, since NHE3 is abundantly expressed not only in the proximal tubules of the kidney, but also in small intestines of the gut. To overcome this limitation, we have generated mutant mice with deletion of NHE3 selectively in the proximal tubules of the kidney, PT-*Nhe3*/, using the state of the art Sglt2-Cre/LoxP approach [23]. We directly tested the hypothesis that deletion of NHE3 selectively in the proximal tubules of the kidney would lower basal blood pressure by inhibiting proximal tubule Na<sup>+</sup> reabsorption and increasing the pressure natriuresis response in mice [23]. We demonstrated that under basal conditions, PT-*Nhe3*/ mice had significantly lower systolic, diastolic, and mean arterial blood pressure than WT mice, accompanied by significantly greater diuretic and natriuretic responses than WT mice, without

more, we demonstrated that the pressure-natriuresis response, as well natriuretic

/H<sup>+</sup> exchanger 3 (NHE3) is the most important Na<sup>+</sup> transporter in AP

absorption in proximal convoluted tubules, causes salt wasting from the

) [187–190]. Indeed,

, and bicarbonate levels. Further-

/H+ exchanger activity in AP membrane vesicles of

**normal blood pressure homeostasis and ANG II-induced**

NaCl and 70–80% of filtered load of bicarbonate (HCO3

(AT1a) receptor-mediated mechanisms.

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

**hypertension**

nearly all of the measured Na<sup>+</sup>

firmed by us recently [21–23].

altering 24 h fecal Na<sup>+</sup> excretion, plasma pH, Na<sup>+</sup>

The Na+

and HCO3

**93**

In the kidney, a proximal tubule cell-specific promoter may be an ideal approach to express an intracellular ANG II protein selectively in the proximal tubules. For example, the kidney androgen-regulated protein gene (KAP) has been used to drive "proximal tubule-specific" expression of human angiotensinogen and renin in the kidney [182, 183]. It has been shown that the KAP gene is widely expressed in the kidney, with its expression reportedly confined to the proximal tubules and regulated by androgen and estrogen [184, 185]. The advantages of this approach are its usefulness for studying the sexual dimorphic regulation of angiotensinogen expression in the proximal tubules of the kidney [182, 183].

We have collaborated with Dr. Julie Cook of Ochsner Clinic and Dr. Isabelle Rubera of University of Nice-Sophia, France to develop an adenoviral construct

#### **Figure 4.**

*Overexpression of an intracellular ECFP/ANG II fusion protein selectively in the proximal tubule of the kidney in C57BL/6J or AT1a-KO mice. ECFP/ANG II increased systolic blood pressure and had a significant antinatriuretic response in C57BL/6J but not in AT1a-KO mice. Green blue represents ECFP/ANG II expression in the proximal tubules, whereas Red represents DAPI-stained nuclei in the cortex after conversion from blue color. G, glomerulus. PT, proximal tubule. \*\*p < 0.01 versus control, whereas ++p < 0.01 versus C57BL/6J mice. Reproduced from Zhuo et al. with permission [15].*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

(Ad-sglt2-ECFP/ANG II), which encodes a cyan fluorescent intracellular ANG II fusion protein (ECFP/ANG II) [17, 18]. The sodium and glucose cotransporter 2 promoter, sglt2, was used to drive the expression of ECFP/ANG II selectively in the proximal tubule cells of the rat and mouse kidneys. Sglt2 is expressed almost exclusively in S1 and S2 segments of the kidney proximal tubules [186]. Using this approach, we have determined whether intrarenal adenovirus-mediated expression of intracellular ECFP/ANG II selectively in the proximal tubules of the rat and mouse kidneys increases the expression and activity of NHE3, stimulate proximal tubule sodium reabsorption, and increase blood pressure in rats and mice. We demonstrated that expression of intracellular ECFP/ANG II selectively in the proximal tubules of rats and mice significantly increased NHE3 expression, proximal tubule sodium reabsorption, and blood pressure (**Figure 4**) [17, 18]. We further showed that AT1 receptor blocker losartan and deletion of AT1a receptors in mice significantly attenuated intracellular ANG II-induced NHE3 expression, proximal tubule sodium reabsorption, and blood pressure responses, suggesting an AT1 (AT1a) receptor-mediated mechanisms.

## **8. Intratubular and intracellular ANG II: role of NHE3 in maintaining normal blood pressure homeostasis and ANG II-induced hypertension**

The Na+ /H<sup>+</sup> exchanger 3 (NHE3) is the most important Na<sup>+</sup> transporter in AP membranes of the proximal tubules of the kidney [187–190]. NHE3 is directly and indirectly responsible for reabsorbing approximately 50–60% of filtered load of NaCl and 70–80% of filtered load of bicarbonate (HCO3 ) [187–190]. Indeed, nearly all of the measured Na<sup>+</sup> /H+ exchanger activity in AP membrane vesicles of proximal tubules are mediated by NHE3 [187–190]. The importance of proximal tubule NHE3 in maintaining body salt and fluid balance and blood pressure homeostasis has not been well studied until recently. Overall, global deletion of the NHE3 gene in all tissues of mice (*Nhe3/*) leads to 50% decreases in fluid, Na+ and HCO3 absorption in proximal convoluted tubules, causes salt wasting from the digestive system, and significantly decreases basal blood pressure [191–194]. One of striking phenotypes is absorptive defects in the small intestines due to intestinal NHE3 deletion [191–194]. Moreover, the transgenic rescue of the NHE3 transgene in small intestines in *Nhe3/* mice, tg*Nhe3/*, failed to rescue the structural and absorptive defects of global NHE3 deletion, with basal blood pressure being similar to those of *Nhe3/* mice [195, 196]. These abnormal phenotypes have been confirmed by us recently [21–23].

However, these studies using either *Nhe3/* or tg*Nhe3/* mice are unable to determine the roles of NHE3 in the proximal tubules of the kidney, since NHE3 is abundantly expressed not only in the proximal tubules of the kidney, but also in small intestines of the gut. To overcome this limitation, we have generated mutant mice with deletion of NHE3 selectively in the proximal tubules of the kidney, PT-*Nhe3*/, using the state of the art Sglt2-Cre/LoxP approach [23]. We directly tested the hypothesis that deletion of NHE3 selectively in the proximal tubules of the kidney would lower basal blood pressure by inhibiting proximal tubule Na<sup>+</sup> reabsorption and increasing the pressure natriuresis response in mice [23]. We demonstrated that under basal conditions, PT-*Nhe3*/ mice had significantly lower systolic, diastolic, and mean arterial blood pressure than WT mice, accompanied by significantly greater diuretic and natriuretic responses than WT mice, without altering 24 h fecal Na<sup>+</sup> excretion, plasma pH, Na<sup>+</sup> , and bicarbonate levels. Furthermore, we demonstrated that the pressure-natriuresis response, as well natriuretic

secreted outside the cells and only acts intracellularly. Dr. Reudelhuber's group was the first to generate genetically modified mouse model that expresses an ANG IIproducing fusion protein in the cardiomyocytes of the rat heart [180, 181]. They used the α myosin heavy chain promoter to control the expression of ANG IIreleasing fusion protein in the cardiomyocytes. Cardiac specific expression of this ANG II fusion protein led to 10-fold increases in ANG II levels in the heart of these transgenic mice, but it did not elevate ANG II levels in the plasma [180, 181]. This approach is very unique to construct this cardiac-specific ANG II fusion protein with a signal peptide sequence derived from human prorenin and a furin cleavage site. Thus, the expressed ANG II fusion protein will be cleaved by furin, and released into the secretory pathway and the cardiac interstitium [180, 181]. It is expected that this cardiac-specific ANG II fusion protein activates cell surface, but not intracellular receptors. In a different study, Baker et al. expressed an intracellular ANG II peptide in the mouse cardiomyocytes using an adenoviral vector [178]. Cardiac-specific expression of this intracellular ANG II peptide in mice induced cardiac hypertrophy, but not altered blood pressure and plasma ANG II [99, 178]. Furthermore, the AT1 receptor blocker failed to block the cardiac hypertrophic effect of this peptide, suggesting that AT1 receptor may not be

In the kidney, a proximal tubule cell-specific promoter may be an ideal approach to express an intracellular ANG II protein selectively in the proximal tubules. For example, the kidney androgen-regulated protein gene (KAP) has been used to drive "proximal tubule-specific" expression of human angiotensinogen and renin in the kidney [182, 183]. It has been shown that the KAP gene is widely expressed in the kidney, with its expression reportedly confined to the proximal tubules and regulated by androgen and estrogen [184, 185]. The advantages of this approach are its usefulness for studying the sexual dimorphic regulation of angiotensinogen expres-

We have collaborated with Dr. Julie Cook of Ochsner Clinic and Dr. Isabelle Rubera of University of Nice-Sophia, France to develop an adenoviral construct

*Overexpression of an intracellular ECFP/ANG II fusion protein selectively in the proximal tubule of the kidney in C57BL/6J or AT1a-KO mice. ECFP/ANG II increased systolic blood pressure and had a significant antinatriuretic response in C57BL/6J but not in AT1a-KO mice. Green blue represents ECFP/ANG II expression in the proximal tubules, whereas Red represents DAPI-stained nuclei in the cortex after conversion from blue color. G, glomerulus. PT, proximal tubule. \*\*p < 0.01 versus control, whereas ++p < 0.01 versus*

sion in the proximal tubules of the kidney [182, 183].

*Selected Chapters from the Renin-Angiotensin System*

*C57BL/6J mice. Reproduced from Zhuo et al. with permission [15].*

involved [99, 178].

**Figure 4.**

**92**

**Acknowledgements**

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

Guangxi, China.

**Author details**

**95**

Jianfeng Zhang and Jia L. Zhuo\*

Medical Center, Jackson, Mississippi, USA

† These authors contributed equally.

provided the original work is properly cited.

\*Address all correspondence to: jzhuo@umc.edu

**Conflict of interest**

The authors declare no conflict of interest.

This work was supported in part by NIH grants, 2R01DK102429-03A1, 2R01DK067299-10A1, and 1R56HL130988-01 to Dr. Zhuo. Ana Paula de Oliveira Leite was supported by scholarships from the Ministry of Education, Brazilian Federal Agency for Support and Evaluation of Graduate Education—CAPES, and Hospital do Rim, Sao Paulo, Brazil, respectively. Drs. Chunling Zhao, Xiaowen Zheng, Jianfeng Zhang were visiting scholars from the Department of Emergency Medicine, The 2nd Affiliated Hospital, Guangxi Medical University, Nanning,

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

Xiao C. Li†, Ana Paula de Oliveira Leite†, Xu Chen, Chunling Zhao, Xiaowen Zheng,

Laboratory of Receptor and Signal Transduction, Department of Pharmacology and Toxicology, Division of Nephrology, Internal Medicine, University of Mississippi

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**Figure 5.**

*Global (*Nhe3/*) or "kidney-selective" deletion of the Na+ /H+ exchanger 3 (NHE3) (tg*Nhe3/*) in mice significantly attenuates systolic blood pressure response to angiotensin II infusion for 2 weeks (ANG II), 1.5 mg/kg/day, i.p. \*\*p < 0.01 versus their control or basal; ++p < 0.01 versus wildtype; ##p < 0.01 versus ANG II.*

responses to acute volume expansion and a high salt diet, were significantly augmented in PT-*Nhe3*/ mice [23]. Thus, our data support the scientific premise and physiological relevance that NHE3 in the proximal tubules plays an important role in maintaining basal blood pressure homeostasis, and genetic deletion of NHE3 selectively in the proximal tubules of the kidney lowers blood pressure by increasing the pressure-natriuretic response.

Recently, we further investigated whether NHE3 in small intestines and proximal tubules of the kidney plays a key role in ANG II-induced hypertension using *Nhe3*/, tg*Nhe3*/, and PT-*Nhe3*/ mice [21, 22]. As expected, infusion of a pressor dose of ANG II, 1.5 mg/kg/day, i.p., via an osmotic minipump for 2 weeks markedly increased blood pressure and caused hypertension in C57BL/6J mice (**Figure 5**) [21, 22]. These hypertensive responses were significantly attenuated in conscious and anesthetized *Nhe3*/, tg*Nhe3*/, and PT-*Nhe3*/ mice [21, 22, 197]. These results strongly support an important role of NHE3 not only in small intestines, but also in the proximal tubules of the kidney in maintaining basal blood pressure homeostasis and in the development of ANG II-induced hypertension.

## **9. Future perspectives and conclusions**

Taken together, there is accumulating evidence to support the existence of the circulating (endocrine), local intratubular (paracrine), and intracellular RAS system in the kidney, especially in the proximal tubules. All major components of the RAS, including the substrate angiotensinogen, renin, ACE, ANG II, AT1 and AT2 receptors, have been localized in the circulation, the kidney, and in the proximal tubule. The roles of the circulating and intratubular RAS in the cardiovascular and kidney, and blood pressure regulation have been extensively studied using molecular, cellular, genetic and pharmacological approaches. It is now well-understood that AGT, prorenin, renin, ACE, ANG II and AT1 and AT2 receptors are not only expressed and localized in the proximal tubules under physiological conditions, but the levels of intratubular angiotensinogen, renin, ACE, and ANG II proteins are also significantly increased in the kidney in response to ANG II infusion in spite of suppression of the circulating RAS. Furthermore, there is also increasing evidence supporting the genomic roles of intracellular and nuclear ANG II in the regulation of proximal tubule reabsorption, blood pressure and the development of hypertension. Future studies should focus more on the long-term genomic and hypertensive roles of intracellular, mitochondrial and nuclear ANG II and the underlying signaling mechanisms in ANG II-dependent hypertension and target organ injury.

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

## **Acknowledgements**

This work was supported in part by NIH grants, 2R01DK102429-03A1, 2R01DK067299-10A1, and 1R56HL130988-01 to Dr. Zhuo. Ana Paula de Oliveira Leite was supported by scholarships from the Ministry of Education, Brazilian Federal Agency for Support and Evaluation of Graduate Education—CAPES, and Hospital do Rim, Sao Paulo, Brazil, respectively. Drs. Chunling Zhao, Xiaowen Zheng, Jianfeng Zhang were visiting scholars from the Department of Emergency Medicine, The 2nd Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi, China.

## **Conflict of interest**

responses to acute volume expansion and a high salt diet, were significantly augmented in PT-*Nhe3*/ mice [23]. Thus, our data support the scientific premise and physiological relevance that NHE3 in the proximal tubules plays an important role in maintaining basal blood pressure homeostasis, and genetic deletion of NHE3 selectively in the proximal tubules of the kidney lowers blood pressure by increas-

*i.p. \*\*p < 0.01 versus their control or basal; ++p < 0.01 versus wildtype; ##p < 0.01 versus ANG II.*

*significantly attenuates systolic blood pressure response to angiotensin II infusion for 2 weeks (ANG II), 1.5 mg/kg/day,*

*/H+ exchanger 3 (NHE3) (tg*Nhe3/*) in mice*

Recently, we further investigated whether NHE3 in small intestines and proximal tubules of the kidney plays a key role in ANG II-induced hypertension using *Nhe3*/, tg*Nhe3*/, and PT-*Nhe3*/ mice [21, 22]. As expected, infusion of a pressor dose of ANG II, 1.5 mg/kg/day, i.p., via an osmotic minipump for 2 weeks markedly increased blood pressure and caused hypertension in C57BL/6J mice (**Figure 5**) [21, 22]. These hypertensive responses were significantly attenuated in conscious and anesthetized *Nhe3*/, tg*Nhe3*/, and PT-*Nhe3*/ mice [21, 22, 197]. These results strongly support an important role of NHE3 not only in small intestines, but also in the proximal tubules of the kidney in maintaining basal blood pressure homeostasis and in the development of ANG II-induced hypertension.

Taken together, there is accumulating evidence to support the existence of the circulating (endocrine), local intratubular (paracrine), and intracellular RAS system in the kidney, especially in the proximal tubules. All major components of the RAS, including the substrate angiotensinogen, renin, ACE, ANG II, AT1 and AT2 receptors, have been localized in the circulation, the kidney, and in the proximal tubule. The roles of the circulating and intratubular RAS in the cardiovascular and kidney, and blood pressure regulation have been extensively studied using molecular, cellular, genetic and pharmacological approaches. It is now well-understood that AGT, prorenin, renin, ACE, ANG II and AT1 and AT2 receptors are not only expressed and localized in the proximal tubules under physiological conditions, but the levels of intratubular angiotensinogen, renin, ACE, and ANG II proteins are also significantly increased in the kidney in response to ANG II infusion in spite of suppression of the circulating RAS. Furthermore, there is also increasing evidence supporting the genomic roles of intracellular and nuclear ANG II in the regulation of proximal tubule reabsorption, blood pressure and the development of hypertension. Future studies should focus more on the long-term genomic and hypertensive roles of intracellular, mitochondrial and nuclear ANG II and the underlying signaling mechanisms in ANG II-dependent hypertension and target

ing the pressure-natriuretic response.

*Global (*Nhe3/*) or "kidney-selective" deletion of the Na+*

*Selected Chapters from the Renin-Angiotensin System*

**Figure 5.**

**9. Future perspectives and conclusions**

organ injury.

**94**

The authors declare no conflict of interest.

## **Author details**

Xiao C. Li†, Ana Paula de Oliveira Leite†, Xu Chen, Chunling Zhao, Xiaowen Zheng, Jianfeng Zhang and Jia L. Zhuo\*

Laboratory of Receptor and Signal Transduction, Department of Pharmacology and Toxicology, Division of Nephrology, Internal Medicine, University of Mississippi Medical Center, Jackson, Mississippi, USA

\*Address all correspondence to: jzhuo@umc.edu

† These authors contributed equally.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Whelton PK, Carey RM, Aronow WS, Casey DE Jr, Collins KJ, Dennison HC, et al. 2017ACC/AHA/AAPA/ABC/ ACPM/AGS/APhA/ASH/ASPC/NMA/ PCNA guideline for the prevention, detection, evaluation, and management of high blood pressure in adults: Executive summary: A report of the American College Of Cardiology/ American Heart Association task force on clinical practice guidelines. Circulation. 2018;**138**(17):e426-e483

[2] Carey RM, Whelton PK. Prevention, detection, evaluation, and management of high blood pressure in adults: Synopsis of the 2017 American College of Cardiology/American Heart Association Hypertension Guideline. Annals of Internal Medicine. 2018;**168**(5):351-358

[3] Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, et al. The seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003;**42**(6):1206-1252

[4] Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De SG, et al. Heart disease and stroke statistics— 2010 update: A report from the American Heart Association. Circulation. 2010;**121**(7):e46-e215

[5] Calhoun DA, Jones D, Textor S, Goff DC, Murphy TP, Toto RD, et al. Resistant hypertension: Diagnosis, evaluation, and treatment. A scientific statement from the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research. Hypertension. 2008;**51**(6):1403-1419

[6] Sacks FM, Svetkey LP, Vollmer WM, Appel LJ, Bray GA, Harsha D, et al. Effects on blood pressure of reduced dietary sodium and the Dietary Approaches to Stop Hypertension (DASH) diet.

DASH-Sodium Collaborative Research Group. The New England Journal of Medicine. 2001;**344**(1):3-10

of Biological Chemistry. 2009;**284**(33):

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

[20] Li XC, Zhuo JL. In vivo regulation of AT1a receptor-mediated intracellular

the kidneys and adrenal glands of AT1a receptor-deficient mice. American Journal of Physiology. Renal Physiology.

[21] Li XC, Shull GE, Miguel-Qin E,

[22] Li XC, Shull GE, Miguel-Qin E, Chen F, Zhuo JL. Role of the Na<sup>+</sup>

exchanger 3 in angiotensin II-induced hypertension in NHE3-deficient mice with transgenic rescue of NHE3 in small intestines. Physiological Reports. 2015;

[23] Li XC, Soleimani M, Zhu D, Rubera I, Tauc M, Zheng X, et al. Proximal tubule-specific deletion of the NHE3

/H<sup>+</sup> exchanger 3) promotes the

[24] Tewksbury DA, Frome WL, Dumas

[25] Tewksbury DA, Dart RA, Travis J. The amino terminal amino acid sequence of human angiotensinogen.

Research Communications. 1981;**99**(4):

[26] Bouhnik J, Clauser E, Strosberg D, Frenoy JP, Menard J, Corvol P. Rat angiotensinogen and des(angiotensin I)

sequencing. Biochemistry. 1981;**20**(24):

Haralambidis J, Richards RI. Molecular

pressure-natriuresis response and lowers blood pressure in mice. Hypertension. 2018;**72**(6):1328-1336

ML. Characterization of human angiotensinogen. The Journal of Biological Chemistry. 1978;**253**(11):

Biochemical and Biophysical

angiotensinogen: Purification, characterization, and partial

[27] Clouston WM, Evans BA,

in angiotensin II-induced hypertension. Physiological Genomics. 2015;**47**(10):


/H<sup>+</sup> exchanger 3

/H+

uptake of [125I]-Val5

2008;**294**:F293-F302

479-487

**3**(11):e12605

(Na+

3817-3820

1311-1315

7010-7015

Zhuo JL. Role of the Na<sup>+</sup>

[14] Li XC, Carretero OA, Zhuo JL. Angiotensin II AT1a receptor siRNA inhibits receptor-mediated angiotensin II endocytosis and NHE3 expression in proximal tubule cells. Journal of the American Society of Nephrology. 2005;

[15] Zhuo JL, Li XC, Garvin JL,

mobilization by stimulating

**290**:F1382-F1390

**303**(12):F1617-F1628

F1076-F1088

Navar LG, Carretero OA. Intracellular angiotensin II induces cytosolic Ca2+

intracellular AT1 receptors in proximal tubule cells. American Journal of Physiology. Renal Physiology. 2006;

[16] Li XC, Hopfer U, Zhuo JL. Novel signaling mechanisms of intracellular

[17] Li XC, Cook JL, Rubera I, Tauc M, Zhang F, Zhuo JL. Intrarenal transfer of an intracellular cyan fluorescent fusion of angiotensin II selectively in proximal tubules increases blood pressure in rats

Physiology. Renal Physiology. 2011;**300**:

[18] Li XC, Zhuo JL. Proximal tubuledominant transfer of AT1a receptors induces blood pressure responses to intracellular angiotensin II in AT1a receptor-deficient mice. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2013;**304**:R588-R598

[19] Li XC, Navar LG, Shao Y, Zhuo JL. Genetic deletion of AT1a receptors attenuates intracellular accumulation of angiotensin II in the kidney of AT1a receptor-deficient mice. American Journal of Physiology. Renal Physiology.

2007;**293**:F586-F593

**97**

and mice. American Journal of

angiotensin II-induced NHE3 expression and activation in mouse proximal tubule cells. American Journal of Physiology. Renal Physiology. 2012;

22411-22425

**16**:573A

[7] Bomback AS, Toto R. Dual blockade of the renin-angiotensin-aldosterone system: Beyond the ACE inhibitor and angiotensin-II receptor blocker combination. American Journal of Hypertension. 2009;**22**(10):1032-1040

[8] Jorde UP, Ennezat PV, Lisker J, Suryadevara V, Infeld J, Cukon S, et al. Maximally recommended doses of angiotensin-converting enzyme (ACE) inhibitors do not completely prevent ACE-mediated formation of angiotensin II in chronic heart failure. Circulation. 2000;**101**(8):844-846

[9] Cook JL, Zhang Z, Re RN. In vitro evidence for an intracellular site of angiotensin action. Circulation Research. 2001;**89**(12):1138-1146

[10] Li XC, Zhu D, Zheng X, Zhang J, Zhuo JL. Intratubular and intracellular renin-angiotensin system in the kidney: A unifying perspective in blood pressure control. Clinical Science (London, England). 2018;**132**(13):1383-1401

[11] Kumar R, Singh VP, Baker KM. The intracellular renin-angiotensin system: Implications in cardiovascular remodeling. Current Opinion in Nephrology and Hypertension. 2008; **17**(2):168-173

[12] Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett NW. Endosomes: A legitimate platform for the signaling train. Proceedings of the National Academy of Sciences of the United States of America. 2009;**0906541106**:1-8

[13] Cottrell GS, Padilla BE, Amadesi S, Poole DP, Murphy JE, Hardt M, et al. Endosomal endothelin-converting enzyme-1: A regulator of beta-arrestindependent ERK signaling. The Journal

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

of Biological Chemistry. 2009;**284**(33): 22411-22425

**References**

[1] Whelton PK, Carey RM, Aronow WS, Casey DE Jr, Collins KJ, Dennison HC, et al. 2017ACC/AHA/AAPA/ABC/ ACPM/AGS/APhA/ASH/ASPC/NMA/ PCNA guideline for the prevention, detection, evaluation, and management

*Selected Chapters from the Renin-Angiotensin System*

DASH-Sodium Collaborative Research Group. The New England Journal of

[7] Bomback AS, Toto R. Dual blockade of the renin-angiotensin-aldosterone system: Beyond the ACE inhibitor and angiotensin-II receptor blocker combination. American Journal of Hypertension. 2009;**22**(10):1032-1040

[8] Jorde UP, Ennezat PV, Lisker J, Suryadevara V, Infeld J, Cukon S, et al. Maximally recommended doses of angiotensin-converting enzyme (ACE) inhibitors do not completely prevent ACE-mediated formation of angiotensin II in chronic heart failure. Circulation.

[9] Cook JL, Zhang Z, Re RN. In vitro evidence for an intracellular site of angiotensin action. Circulation Research. 2001;**89**(12):1138-1146

[10] Li XC, Zhu D, Zheng X, Zhang J, Zhuo JL. Intratubular and intracellular renin-angiotensin system in the kidney: A unifying perspective in blood pressure control. Clinical Science (London, England). 2018;**132**(13):1383-1401

[11] Kumar R, Singh VP, Baker KM. The intracellular renin-angiotensin system:

[12] Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett NW. Endosomes: A legitimate platform for the signaling train. Proceedings of the National Academy of Sciences of the United States of America.

[13] Cottrell GS, Padilla BE, Amadesi S, Poole DP, Murphy JE, Hardt M, et al. Endosomal endothelin-converting enzyme-1: A regulator of beta-arrestindependent ERK signaling. The Journal

Implications in cardiovascular remodeling. Current Opinion in Nephrology and Hypertension. 2008;

**17**(2):168-173

2009;**0906541106**:1-8

2000;**101**(8):844-846

Medicine. 2001;**344**(1):3-10

of high blood pressure in adults: Executive summary: A report of the American College Of Cardiology/ American Heart Association task force

on clinical practice guidelines. Circulation. 2018;**138**(17):e426-e483

of the 2017 American College of

2003;**42**(6):1206-1252

[2] Carey RM, Whelton PK. Prevention, detection, evaluation, and management of high blood pressure in adults: Synopsis

Cardiology/American Heart Association Hypertension Guideline. Annals of Internal Medicine. 2018;**168**(5):351-358

[3] Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, et al. The seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension.

[4] Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De SG, et al. Heart disease and stroke statistics— 2010 update: A report from the American Heart Association. Circulation. 2010;**121**(7):e46-e215

[5] Calhoun DA, Jones D, Textor S, Goff

[6] Sacks FM, Svetkey LP, Vollmer WM, Appel LJ, Bray GA, Harsha D, et al. Effects on blood pressure of reduced dietary sodium and the Dietary Approaches to Stop Hypertension (DASH) diet.

DC, Murphy TP, Toto RD, et al. Resistant hypertension: Diagnosis, evaluation, and treatment. A scientific statement from the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research. Hypertension.

2008;**51**(6):1403-1419

**96**

[14] Li XC, Carretero OA, Zhuo JL. Angiotensin II AT1a receptor siRNA inhibits receptor-mediated angiotensin II endocytosis and NHE3 expression in proximal tubule cells. Journal of the American Society of Nephrology. 2005; **16**:573A

[15] Zhuo JL, Li XC, Garvin JL, Navar LG, Carretero OA. Intracellular angiotensin II induces cytosolic Ca2+ mobilization by stimulating intracellular AT1 receptors in proximal tubule cells. American Journal of Physiology. Renal Physiology. 2006; **290**:F1382-F1390

[16] Li XC, Hopfer U, Zhuo JL. Novel signaling mechanisms of intracellular angiotensin II-induced NHE3 expression and activation in mouse proximal tubule cells. American Journal of Physiology. Renal Physiology. 2012; **303**(12):F1617-F1628

[17] Li XC, Cook JL, Rubera I, Tauc M, Zhang F, Zhuo JL. Intrarenal transfer of an intracellular cyan fluorescent fusion of angiotensin II selectively in proximal tubules increases blood pressure in rats and mice. American Journal of Physiology. Renal Physiology. 2011;**300**: F1076-F1088

[18] Li XC, Zhuo JL. Proximal tubuledominant transfer of AT1a receptors induces blood pressure responses to intracellular angiotensin II in AT1a receptor-deficient mice. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2013;**304**:R588-R598

[19] Li XC, Navar LG, Shao Y, Zhuo JL. Genetic deletion of AT1a receptors attenuates intracellular accumulation of angiotensin II in the kidney of AT1a receptor-deficient mice. American Journal of Physiology. Renal Physiology. 2007;**293**:F586-F593

[20] Li XC, Zhuo JL. In vivo regulation of AT1a receptor-mediated intracellular uptake of [125I]-Val5 -angiotensin II in the kidneys and adrenal glands of AT1a receptor-deficient mice. American Journal of Physiology. Renal Physiology. 2008;**294**:F293-F302

[21] Li XC, Shull GE, Miguel-Qin E, Zhuo JL. Role of the Na<sup>+</sup> /H<sup>+</sup> exchanger 3 in angiotensin II-induced hypertension. Physiological Genomics. 2015;**47**(10): 479-487

[22] Li XC, Shull GE, Miguel-Qin E, Chen F, Zhuo JL. Role of the Na<sup>+</sup> /H+ exchanger 3 in angiotensin II-induced hypertension in NHE3-deficient mice with transgenic rescue of NHE3 in small intestines. Physiological Reports. 2015; **3**(11):e12605

[23] Li XC, Soleimani M, Zhu D, Rubera I, Tauc M, Zheng X, et al. Proximal tubule-specific deletion of the NHE3 (Na+ /H<sup>+</sup> exchanger 3) promotes the pressure-natriuresis response and lowers blood pressure in mice. Hypertension. 2018;**72**(6):1328-1336

[24] Tewksbury DA, Frome WL, Dumas ML. Characterization of human angiotensinogen. The Journal of Biological Chemistry. 1978;**253**(11): 3817-3820

[25] Tewksbury DA, Dart RA, Travis J. The amino terminal amino acid sequence of human angiotensinogen. Biochemical and Biophysical Research Communications. 1981;**99**(4): 1311-1315

[26] Bouhnik J, Clauser E, Strosberg D, Frenoy JP, Menard J, Corvol P. Rat angiotensinogen and des(angiotensin I) angiotensinogen: Purification, characterization, and partial sequencing. Biochemistry. 1981;**20**(24): 7010-7015

[27] Clouston WM, Evans BA, Haralambidis J, Richards RI. Molecular cloning of the mouse angiotensinogen gene. Genomics. 1988;**2**(3):240-248

[28] Kobori H, Nangaku M, Navar LG, Nishiyama A. The intrarenal renin-angiotensin system: From physiology to the pathobiology of hypertension and kidney disease. Pharmacological Reviews. 2007;**59**(3): 251-287

[29] Taugner R, Hackenthal E, Rix E, Nobiling R, Poulsen K. Immunocytochemistry of the reninangiotensin system: Renin, angiotensinogen, angiotensin I, angiotensin II, and converting enzyme in the kidneys of mice, rats, and tree shrews. Kidney International Supplements. 1982;**12**:S33-S43

[30] Darby IA, Sernia C. In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys. Cell and Tissue Research. 1995;**281**(2):197-206

[31] Kobori H, Harrison-Bernard LM, Navar LG. Expression of angiotensinogen mRNA and protein in angiotensin II-dependent hypertension. Journal of the American Society of Nephrology. 2001;**12**(3):431-439

[32] Kobori H, Harrison-Bernard LM, Navar LG. Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production. Kidney International. 2002;**61**(2): 579-585

[33] Kobori H, Harrison-Bernard LM, Navar LG. Enhancement of angiotensinogen expression in angiotensin II-dependent hypertension. Hypertension. 2001;**37**(5):1329-1335

[34] Matsusaka T, Niimura F, Shimizu A, Pastan I, Saito A, Kobori H, et al. Liver angiotensinogen is the primary source of renal angiotensin II. Journal of the American Society of Nephrology. 2012; **23**(7):1181-1189

[35] Tigerstedt R, Bergman PG. Niere und Kreislauf. Skandinavisches Archiv Für Physiologie. 1898;**8**:223-271

Gardiner-Calwell Communications

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

angiotensin-converting enzyme as revealed by gene targeting in mice. The Journal of Clinical Investigation. 1997;

[51] Bernstein KE. Views of the reninangiotensin system: Brilling, mimsy, and slithy tove. Hypertension. 2006;

[52] Bernstein KE, Ong FS, Blackwell WL, Shah KH, Giani JF, Gonzalez-Villalobos RA, et al. A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting enzyme. Pharmacological Reviews. 2012;**65**(1):

[53] Chai SY, Allen AM, Adam WR, Mendelsohn FA. Local actions of angiotensin II: Quantitative in vitro autoradiographic localization of angiotensin II receptor binding and angiotensin converting enzyme in target tissues. Journal of Cardiovascular Pharmacology. 1986;**8**(Suppl 10):

[54] Harrison-Bernard LM, Zhuo JL, Kobori H, Ohishi M, Navar LG. Intrarenal AT1 receptor and ACE binding in ANG II-induced

hypertensive rats. American Journal of Physiology. Renal Physiology. 2002;

[55] Bruneval P, Hinglais N, Alhenc-Gelas F, Tricottet V, Corvol P, Menard J, et al. Angiotensin I converting enzyme

in human intestine and kidney. Ultrastructural immunohistochemical localization. Histochemistry. 1986;**85**(1):

[56] Danilov SM, Faerman AI, Printseva OY, Martynov AV, Sakharov IY, Trakht IN. Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies. Histochemistry. 1987;**87**(5):

**99**:2375-2385

**47**(3):509-514

1-46

S35-S39

**282**(1):F19-F25

73-80

487-490

[43] Moe OW, Ujiie K, Star RA, Miller RT, Widell J, Alpern RJ, et al. Renin expression in renal proximal tubule. The Journal of Clinical Investigation. 1993;

[44] Chen M, Harris MP, Rose D, Smart A, He XR, Kretzler M, et al. Renin and renin mRNA in proximal tubules of the

[45] Tang SS, Jung F, Diamant D, Brown D, Bachinsky D, Hellman P, et al. Temperature-sensitive SV40

immortalized rat proximal tubule cell line has functional renin-angiotensin system. The American Journal of Physiology. 1995;**268**(3 Pt 2):F435-F446

[47] Iwao H, Nakamura N, Ikemoto F, Yamamoto K. Subcellular localization of exogenously administered renin in mouse kidney. Japanese Circulation Journal. 1983;**47**(10):1198-1202

[48] Soubrier F, Alhenc-Gelas F, Hubert C, Allegrini J, John M, Tregear G, et al. Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning.

Proceedings of the National Academy of

[49] Bernstein KE, Martin BM, Bernstein EA, Linton J, Striker L, Striker G. The isolation of angiotensin-converting enzyme cDNA. The Journal of Biological Chemistry. 1988;**263**(23):11021-11024

[50] Esther CR, Marino EM, Howard TE, Machaud A, Corvol P, Capecchi MR, et al. The critical role of tissue

Sciences of the United States of America. 1988;**85**(24):9386-9390

rat kidney. Journal of Clinical Investigation. 1994;**94**(1):237-243

[46] Taugner R, Hackenthal E, Inagami T, Nobiling R, Poulsen K. Vascular and tubular renin in the kidneys of mice. Histochemistry. 1982;

**75**(4):473-484

**99**

(Pacific) Ltd.; 1993

**91**(3):774-779

[36] Imai T, Miyazaki H, Hirose S, Hori H, Hayashi T, Kageyama R, et al. Cloning and sequence analysis of cDNA for human renin precursor. Proceedings of the National Academy of Sciences of the United States of America. 1983; **80**(24):7405-7409

[37] Taugner R, Hackenthal E, Nobiling R, Harlacher M, Reb G. The distribution of renin in the different segments of the renal arterial tree: Immunocytochemical investigation in the mouse kidney. Histochemistry. 1981;**73**(1):75-88

[38] Celio MR, Inagami T. Renin in the human kidney. Immunohistochemical localization. Histochemistry. 1981;**72**(1): 1-10

[39] Faraggiana T, Gresik E, Tanaka T, Inagami T, Lupo A. Immunohistochemical localization of renin in the human kidney. The Journal of Histochemistry and Cytochemistry. 1982;**30**(5):459-465

[40] Song K, Zhuo JL, Chai SY, Mendelsohn FA. A new method to localize active renin in tissues by autoradiography: Application to dog kidney. Kidney International. 1992; **42**(3):639-646

[41] Zhuo JL, Anderson WP, Song K, Mendelsohn FA. Autoradiographic localization of active renin in the juxtaglomerular apparatus of the dog kidney: Effects of sodium intake. Clinical and Experimental Pharmacology & Physiology. 1996; **23**(4):291-298

[42] Zhuo JL, Song K, Chai SY, Mendelsohn FA. Anatomical localization of components of the renin-angiotensin system in different organs and tissues. In: MacGregor GA, Sever PS, editors. Inhibition of the Renin-Angiotensin System: Recent Advances. Hong Kong:

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

Gardiner-Calwell Communications (Pacific) Ltd.; 1993

cloning of the mouse angiotensinogen gene. Genomics. 1988;**2**(3):240-248

*Selected Chapters from the Renin-Angiotensin System*

[35] Tigerstedt R, Bergman PG. Niere und Kreislauf. Skandinavisches Archiv Für Physiologie. 1898;**8**:223-271

[36] Imai T, Miyazaki H, Hirose S, Hori H, Hayashi T, Kageyama R, et al. Cloning and sequence analysis of cDNA for human renin precursor. Proceedings of the National Academy of Sciences of the United States of America. 1983;

[37] Taugner R, Hackenthal E, Nobiling R, Harlacher M, Reb G. The distribution of renin in the different segments of the renal arterial tree: Immunocytochemical investigation in the mouse kidney. Histochemistry. 1981;**73**(1):75-88

[38] Celio MR, Inagami T. Renin in the human kidney. Immunohistochemical localization. Histochemistry. 1981;**72**(1):

[39] Faraggiana T, Gresik E, Tanaka T, Inagami T, Lupo A. Immunohistochemical localization of renin in the human kidney. The Journal of Histochemistry and Cytochemistry. 1982;**30**(5):459-465

[40] Song K, Zhuo JL, Chai SY, Mendelsohn FA. A new method to localize active renin in tissues by autoradiography: Application to dog kidney. Kidney International. 1992;

[41] Zhuo JL, Anderson WP, Song K, Mendelsohn FA. Autoradiographic localization of active renin in the juxtaglomerular apparatus of the dog kidney: Effects of sodium intake. Clinical and Experimental

Pharmacology & Physiology. 1996;

Mendelsohn FA. Anatomical localization of components of the renin-angiotensin system in different organs and tissues. In: MacGregor GA, Sever PS, editors. Inhibition of the Renin-Angiotensin System: Recent Advances. Hong Kong:

[42] Zhuo JL, Song K, Chai SY,

**42**(3):639-646

**23**(4):291-298

**80**(24):7405-7409

1-10

[28] Kobori H, Nangaku M, Navar LG,

[29] Taugner R, Hackenthal E, Rix E,

Immunocytochemistry of the renin-

Nishiyama A. The intrarenal renin-angiotensin system: From physiology to the pathobiology of hypertension and kidney disease. Pharmacological Reviews. 2007;**59**(3):

Nobiling R, Poulsen K.

angiotensin system: Renin, angiotensinogen, angiotensin I, angiotensin II, and converting enzyme in the kidneys of mice, rats, and tree

shrews. Kidney International Supplements. 1982;**12**:S33-S43

[30] Darby IA, Sernia C. In situ

Navar LG. Expression of

579-585

**23**(7):1181-1189

**98**

hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys. Cell and Tissue Research. 1995;**281**(2):197-206

[31] Kobori H, Harrison-Bernard LM,

angiotensinogen mRNA and protein in angiotensin II-dependent hypertension. Journal of the American Society of Nephrology. 2001;**12**(3):431-439

[32] Kobori H, Harrison-Bernard LM, Navar LG. Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production. Kidney International. 2002;**61**(2):

[33] Kobori H, Harrison-Bernard LM,

angiotensin II-dependent hypertension. Hypertension. 2001;**37**(5):1329-1335

[34] Matsusaka T, Niimura F, Shimizu A, Pastan I, Saito A, Kobori H, et al. Liver angiotensinogen is the primary source of renal angiotensin II. Journal of the American Society of Nephrology. 2012;

Navar LG. Enhancement of angiotensinogen expression in

251-287

[43] Moe OW, Ujiie K, Star RA, Miller RT, Widell J, Alpern RJ, et al. Renin expression in renal proximal tubule. The Journal of Clinical Investigation. 1993; **91**(3):774-779

[44] Chen M, Harris MP, Rose D, Smart A, He XR, Kretzler M, et al. Renin and renin mRNA in proximal tubules of the rat kidney. Journal of Clinical Investigation. 1994;**94**(1):237-243

[45] Tang SS, Jung F, Diamant D, Brown D, Bachinsky D, Hellman P, et al. Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin-angiotensin system. The American Journal of Physiology. 1995;**268**(3 Pt 2):F435-F446

[46] Taugner R, Hackenthal E, Inagami T, Nobiling R, Poulsen K. Vascular and tubular renin in the kidneys of mice. Histochemistry. 1982; **75**(4):473-484

[47] Iwao H, Nakamura N, Ikemoto F, Yamamoto K. Subcellular localization of exogenously administered renin in mouse kidney. Japanese Circulation Journal. 1983;**47**(10):1198-1202

[48] Soubrier F, Alhenc-Gelas F, Hubert C, Allegrini J, John M, Tregear G, et al. Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proceedings of the National Academy of Sciences of the United States of America. 1988;**85**(24):9386-9390

[49] Bernstein KE, Martin BM, Bernstein EA, Linton J, Striker L, Striker G. The isolation of angiotensin-converting enzyme cDNA. The Journal of Biological Chemistry. 1988;**263**(23):11021-11024

[50] Esther CR, Marino EM, Howard TE, Machaud A, Corvol P, Capecchi MR, et al. The critical role of tissue

angiotensin-converting enzyme as revealed by gene targeting in mice. The Journal of Clinical Investigation. 1997; **99**:2375-2385

[51] Bernstein KE. Views of the reninangiotensin system: Brilling, mimsy, and slithy tove. Hypertension. 2006; **47**(3):509-514

[52] Bernstein KE, Ong FS, Blackwell WL, Shah KH, Giani JF, Gonzalez-Villalobos RA, et al. A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting enzyme. Pharmacological Reviews. 2012;**65**(1): 1-46

[53] Chai SY, Allen AM, Adam WR, Mendelsohn FA. Local actions of angiotensin II: Quantitative in vitro autoradiographic localization of angiotensin II receptor binding and angiotensin converting enzyme in target tissues. Journal of Cardiovascular Pharmacology. 1986;**8**(Suppl 10): S35-S39

[54] Harrison-Bernard LM, Zhuo JL, Kobori H, Ohishi M, Navar LG. Intrarenal AT1 receptor and ACE binding in ANG II-induced hypertensive rats. American Journal of Physiology. Renal Physiology. 2002; **282**(1):F19-F25

[55] Bruneval P, Hinglais N, Alhenc-Gelas F, Tricottet V, Corvol P, Menard J, et al. Angiotensin I converting enzyme in human intestine and kidney. Ultrastructural immunohistochemical localization. Histochemistry. 1986;**85**(1): 73-80

[56] Danilov SM, Faerman AI, Printseva OY, Martynov AV, Sakharov IY, Trakht IN. Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies. Histochemistry. 1987;**87**(5): 487-490

[57] Schulz WW, Hagler HK, Buja LM, Erdos EG. Ultrastructural localization of angiotensin I-converting enzyme (EC 3.4.15.1) and neutral metalloendopeptidase (EC 3.4.24.11) in the proximal tubule of the human kidney. Laboratory Investigation. 1988; **59**(6):789-797

[58] Navar LG, Kobori H, Prieto-Carrasquero M. Intrarenal angiotensin II and hypertension. Current Hypertension Reports. 2003;**5**(2): 135-143

[59] Navar LG, Carmines PK, Huang WC, Mitchell KD. The tubular effects of angiotensin II. Kidney International. Supplement. 1987;**20**:S81-S88

[60] Harris PJ, Navar LG. Tubular transport responses to angiotensin II. American Journal of Physiology. Renal Physiology. 1985;**248**:F621-F630

[61] Crowley SD, Gurley SB, Herrera MJ, Ruiz P, Griffiths R, Kumar AP, et al. Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(47): 17985-17990

[62] Crowley SD, Gurley SB, Oliverio MI, Pazmino AK, Griffiths R, Flannery PJ, et al. Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system. The Journal of Clinical Investigation. 2005;**115**(4):1092-1099

[63] Carey RM, Siragy HM. Newly recognized components of the reninangiotensin system: Potential roles in cardiovascular and renal regulation. Endocrine Reviews. 2003;**24**(3):261-271

[64] Navar LG, Kobori H, Prieto MC, Gonzalez-Villalobos RA. Intratubular renin-angiotensin system in hypertension. Hypertension. 2011;**57**(3): 355-362

[65] Nishiyama A, Seth DM, Navar LG. Renal interstitial fluid concentrations of angiotensins I and II in anesthetized rats. Hypertension. 2002;**39**(1):129-134

[73] Zou LX, Imig JD, von Thun AM, Hymel A, Ono H, Navar LG. Receptormediated intrarenal angiotensin II augmentation in angiotensin II-infused rats. Hypertension. 1996;**28**(4):669-677

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

Cloning and expression of a

2013;**4**:166

complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 1991;**351**(6323):230-233

[81] Zhuo JL, Ferrao FM, Zheng Y, Li XC. New frontiers in the intrarenal renin-angiotensin system: A critical review of classical and new paradigms. Frontiers in Endocrinology (Lausanne).

[82] Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ. Expression cloning of type 2 angiotensin II receptor reveals a unique

class of seven-transmembrane receptors. The Journal of Biological Chemistry. 1993;**268**(33):24539-24542

[84] Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H,

involved in phosphotyrosine

24543-24546

Hamakubo T, et al. Molecular cloning of a novel angiotensin II receptor isoform

phosphatase inhibition. The Journal of Biological Chemistry. 1993;**268**(33):

[85] Zhuo JL, Alcorn D, Harris PJ, Mendelsohn FA. Localization and properties of angiotensin II receptors in rat kidney. Kidney International. Supplement. 1993;**42**:S40-S46

[86] Zhuo JL, Song K, Harris PJ, Mendelsohn FA. In vitro

[87] Zhuo JL, Allen AM, Alcorn D, MacGregor D, Aldred GP, Mendelsohn FA. The distribution of angiotensin II receptors. In: Laragh JH, Brenner BM,

autoradiography reveals predominantly AT1 angiotensin II receptors in rat kidney. Renal Physiology and Biochemistry. 1992;**15**(5):231-239

[83] Nakajima M, Mukoyama M, Pratt RE, Horiuchi M, Dzau VJ. Cloning of cDNA and analysis of the gene for mouse angiotensin II type 2 receptor. Biochemical and Biophysical Research Communications. 1993;**197**(2):393-399

[74] Imig JD, Navar GL, Zou LX, O'Reilly KC, Allen PL, Kaysen JH, et al. Renal endosomes contain angiotensin peptides, converting enzyme, and AT1a receptors. The American Journal of Physiology. 1999;**277**(2 Pt 2):F303-F311

[75] Abadir PM, Foster DB, Crow M, Cooke CA, Rucker JJ, Jain A, et al. Identification and characterization of a functional mitochondrial angiotensin system. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**(36):

[76] Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, et al. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacological Reviews. 1993;**45**(2):

[77] de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. International union of pharmacology. XXIII. The

Pharmacological Reviews. 2000;**52**(3):

[78] Karnik SS, Unal H, Kemp JR, Tirupula KC, Eguchi S, Vanderheyden PM, et al. International union of basic and clinical pharmacology. XCIX. Angiotensin receptors: Interpreters of pathophysiological angiotensinergic stimuli. Pharmacological Reviews. 2015;

[79] Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor.

Nature. 1991;**16**(351):233-236

[80] Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, et al.

angiotensin II receptors.

14849-14854

205-251

415-472

**67**(4):754-819

**101**

[66] Siragy HM, Howell NL, Ragsdale NV, Carey RM. Renal interstitial fluid angiotensin. Modulation by anesthesia, epinephrine, sodium depletion, and renin inhibition. Hypertension. 1995; **25**(5):1021-1024

[67] Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E, Navar LG. Ang II accumulation in rat renal endosomes during Ang II-induced hypertension: Role of AT1 receptor. Hypertension. 2002;**39**(1):116-121

[68] Shaltout HA, Westwood BM, Averill DB, Ferrario CM, Figueroa JP, Diz DI, et al. Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: Evidence for ACE2 dependent processing of angiotensin II. American Journal of Physiology. Renal Physiology. 2007;**292**(1):F82-F91

[69] Prieto-Carrasquero MC, Kobori H, Ozawa Y, Gutierrez A, Seth D, Navar LG. AT1 receptor-mediated enhancement of collecting duct renin in angiotensin II-dependent hypertensive rats. American Journal of Physiology. Renal Physiology. 2005;**289**(3):F632- F637

[70] Zou LX, Hymel A, Imig JD, Navar LG. Renal accumulation of circulating angiotensin II in angiotensin II-infused rats. Hypertension. 1996;**27**(3 Pt 2): 658-662

[71] Chappell MC. Nonclassical reninangiotensin system and renal function. Comprehensive Physiology. 2012;**2**(4): 2733-2752

[72] Zhuo JL, Li XC. New insights and perspectives on intrarenal reninangiotensin system: Focus on intracrine/ intracellular angiotensin II. Peptides. 2011;**32**(7):1551-1565

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

[73] Zou LX, Imig JD, von Thun AM, Hymel A, Ono H, Navar LG. Receptormediated intrarenal angiotensin II augmentation in angiotensin II-infused rats. Hypertension. 1996;**28**(4):669-677

[57] Schulz WW, Hagler HK, Buja LM, Erdos EG. Ultrastructural localization of angiotensin I-converting enzyme (EC

*Selected Chapters from the Renin-Angiotensin System*

[65] Nishiyama A, Seth DM, Navar LG. Renal interstitial fluid concentrations of angiotensins I and II in anesthetized rats. Hypertension. 2002;**39**(1):129-134

[66] Siragy HM, Howell NL, Ragsdale NV, Carey RM. Renal interstitial fluid angiotensin. Modulation by anesthesia, epinephrine, sodium depletion, and renin inhibition. Hypertension. 1995;

[67] Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E, Navar LG. Ang II accumulation in rat renal endosomes during Ang II-induced hypertension: Role of AT1 receptor. Hypertension.

[68] Shaltout HA, Westwood BM, Averill DB, Ferrario CM, Figueroa JP, Diz DI, et al. Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: Evidence for ACE2 dependent processing of angiotensin II. American Journal of Physiology. Renal Physiology. 2007;**292**(1):F82-F91

[69] Prieto-Carrasquero MC, Kobori H, Ozawa Y, Gutierrez A, Seth D, Navar

enhancement of collecting duct renin in angiotensin II-dependent hypertensive rats. American Journal of Physiology. Renal Physiology. 2005;**289**(3):F632-

[70] Zou LX, Hymel A, Imig JD, Navar LG. Renal accumulation of circulating angiotensin II in angiotensin II-infused rats. Hypertension. 1996;**27**(3 Pt 2):

[71] Chappell MC. Nonclassical reninangiotensin system and renal function. Comprehensive Physiology. 2012;**2**(4):

[72] Zhuo JL, Li XC. New insights and perspectives on intrarenal renin-

angiotensin system: Focus on intracrine/ intracellular angiotensin II. Peptides.

LG. AT1 receptor-mediated

F637

658-662

2733-2752

2011;**32**(7):1551-1565

**25**(5):1021-1024

2002;**39**(1):116-121

metalloendopeptidase (EC 3.4.24.11) in the proximal tubule of the human kidney. Laboratory Investigation. 1988;

Carrasquero M. Intrarenal angiotensin II

[59] Navar LG, Carmines PK, Huang WC, Mitchell KD. The tubular effects of angiotensin II. Kidney International. Supplement. 1987;**20**:S81-S88

[60] Harris PJ, Navar LG. Tubular transport responses to angiotensin II. American Journal of Physiology. Renal Physiology. 1985;**248**:F621-F630

[61] Crowley SD, Gurley SB, Herrera MJ, Ruiz P, Griffiths R, Kumar AP, et al. Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(47):

[62] Crowley SD, Gurley SB, Oliverio MI, Pazmino AK, Griffiths R, Flannery PJ, et al. Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system. The Journal of Clinical Investigation. 2005;**115**(4):1092-1099

[63] Carey RM, Siragy HM. Newly recognized components of the reninangiotensin system: Potential roles in cardiovascular and renal regulation. Endocrine Reviews. 2003;**24**(3):261-271

[64] Navar LG, Kobori H, Prieto MC, Gonzalez-Villalobos RA. Intratubular

hypertension. Hypertension. 2011;**57**(3):

renin-angiotensin system in

355-362

**100**

[58] Navar LG, Kobori H, Prieto-

and hypertension. Current Hypertension Reports. 2003;**5**(2):

3.4.15.1) and neutral

**59**(6):789-797

135-143

17985-17990

[74] Imig JD, Navar GL, Zou LX, O'Reilly KC, Allen PL, Kaysen JH, et al. Renal endosomes contain angiotensin peptides, converting enzyme, and AT1a receptors. The American Journal of Physiology. 1999;**277**(2 Pt 2):F303-F311

[75] Abadir PM, Foster DB, Crow M, Cooke CA, Rucker JJ, Jain A, et al. Identification and characterization of a functional mitochondrial angiotensin system. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**(36): 14849-14854

[76] Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, et al. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacological Reviews. 1993;**45**(2): 205-251

[77] de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacological Reviews. 2000;**52**(3): 415-472

[78] Karnik SS, Unal H, Kemp JR, Tirupula KC, Eguchi S, Vanderheyden PM, et al. International union of basic and clinical pharmacology. XCIX. Angiotensin receptors: Interpreters of pathophysiological angiotensinergic stimuli. Pharmacological Reviews. 2015; **67**(4):754-819

[79] Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;**16**(351):233-236

[80] Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, et al. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 1991;**351**(6323):230-233

[81] Zhuo JL, Ferrao FM, Zheng Y, Li XC. New frontiers in the intrarenal renin-angiotensin system: A critical review of classical and new paradigms. Frontiers in Endocrinology (Lausanne). 2013;**4**:166

[82] Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ. Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. The Journal of Biological Chemistry. 1993;**268**(33):24539-24542

[83] Nakajima M, Mukoyama M, Pratt RE, Horiuchi M, Dzau VJ. Cloning of cDNA and analysis of the gene for mouse angiotensin II type 2 receptor. Biochemical and Biophysical Research Communications. 1993;**197**(2):393-399

[84] Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, et al. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. The Journal of Biological Chemistry. 1993;**268**(33): 24543-24546

[85] Zhuo JL, Alcorn D, Harris PJ, Mendelsohn FA. Localization and properties of angiotensin II receptors in rat kidney. Kidney International. Supplement. 1993;**42**:S40-S46

[86] Zhuo JL, Song K, Harris PJ, Mendelsohn FA. In vitro autoradiography reveals predominantly AT1 angiotensin II receptors in rat kidney. Renal Physiology and Biochemistry. 1992;**15**(5):231-239

[87] Zhuo JL, Allen AM, Alcorn D, MacGregor D, Aldred GP, Mendelsohn FA. The distribution of angiotensin II receptors. In: Laragh JH, Brenner BM,

editors. Hypertension: Pathology, Diagnosis & Management. 2nd ed. New York: Raven Press; 1995. pp. 1739-1762

[88] Gwathmey T, Shaltout HA, Pendergrass KD, Pirro NT, Figueroa JP, Rose JC, et al. Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production. American Journal of Physiology-Renal Physiology. 2009;**296**:F1484- F1493

[89] Gwathmey TM, Westwood BM, Pirro NT, Tang L, Rose JC, Diz DI, et al. Nuclear angiotensin-(1-7) receptor is functionally coupled to the formation of nitric oxide. American Journal of Physiology. Renal Physiology. 2010; **299**(5):F983-F990

[90] Wilson BA, Nautiyal M, Gwathmey TM, Rose JC, Chappell MC. Evidence for a mitochondrial angiotensin-(1-7) system in the kidney. American Journal of Physiology. Renal Physiology. 2016; **310**(7):F637-F645

[91] Zhuo JL, MacGregor D, Mendelsohn FA. Comparative distribution of angiotensin II receptor subtypes in mammalian adrenal glands. In: Vinson GP, Anderson DC, editors. Vascular, Adrenal and Hypertension. London: Journal of Endocrinology Pty Ltd.; 1995. pp. 53-68

[92] Ozono R, Wang ZQ, Moore AF, Inagami T, Siragy HM, Carey RM. Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney. Hypertension. 1997;**30**(5):1238-1246

[93] Kemp BA, Howell NL, Gildea JJ, Keller SR, Padia SH, Carey RM. AT(2) receptor activation induces natriuresis and lowers blood pressure. Circulation Research. 2014;**115**(3):388-399

[94] Touyz RM, Schiffrin EL. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular

smooth muscle cells. Pharmacological Reviews. 2000;**52**:639-672

Gomes DS, et al. Luminal ANG II is internalized as a complex with AT1R/AT2R heterodimers to target endoplasmic reticulum in LLC-PK1 cells. American Journal of Physiology. Renal Physiology. 2017;**313**(2):

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

through induction of transforming growth factor-beta expression in rat glomerular mesangial cells. The Journal of Clinical Investigation. 1994;**93**:2431-2437

[110] Wolf G, Ziyadeh FN, Stahl RA. Angiotensin II stimulates expression of transforming growth factor beta receptor type II in cultured mouse proximal tubular cells. Journal of Molecular Medicine. 1999;**77**(7):556-564

[111] Kurtz TW, Gardner DG.

380-386

852-859

**349**(3):365-370

2054-2057

Transcription-modulating drugs: A new frontier in the treatment of essential hypertension. Hypertension. 1998;**32**:

[112] Kurtz TW, Pravenec M. Moleculespecific effects of angiotensin IIreceptor blockers independent of the renin-angiotensin system. American Journal of Hypertension. 2008;**21**(8):

[113] Kurtz TW. Beyond the classic angiotensin-receptor-blocker profile. Nature Clinical Practice. Cardiovascular Medicine. 2008;**5**(Suppl 1):S19-S26

[114] Conchon S, Monnot C, Teutsch B, Corvol P, Clauser E. Internalization of the rat AT1a and AT1b receptors: Pharmacological and functional requirements. FEBS Letters. 1994;

[115] Schupp M, Janke J, Clasen R, Unger T, Kintscher U. Angiotensin type 1 receptor blockers induce peroxisome proliferator-activated receptor-gamma activity. Circulation. 2004;**109**(17):

[116] Kurtz TW, Klein U. Next

generation multifunctional angiotensin receptor blockers. Hypertension Research. 2009;**32**(10):826-834

[117] Mauer M, Zinman B, Gardiner R, Suissa S, Sinaiko A, Strand T, et al. Renal and retinal effects of enalapril and losartan in type 1 diabetes. The New

[104] Brasier AR, Jamaluddin M, Han Y, Patterson C, Runge MS. Angiotensin II induces gene transcription through celltype-dependent effects on the nuclear

factor-kappaB (NF-kappaB) transcription factor. Molecular and Cellular Biochemistry. 2000;**212**(1-2):

[105] Ruiz-Ortega M, Lorenzo O, Ruperez M, Konig S, Wittig B, Egido J. Angiotensin II activates nuclear

transcription factor κB through AT1 and AT2 in vascular smooth muscle cells: Molecular mechanisms. Circulation Research. 2000;**86**(12):1266-1272

[106] Li XC, Zhuo JL. Nuclear factor-κB as a hormonal intracellular signaling molecule: Focus on angiotensin IIinduced cardiovascular and renal injury. Current Opinion in Nephrology and Hypertension. 2008;**17**(1):37-43

[107] Takahashi M, Suzuki E, Takeda R, Oba S, Nishimatsu H, Kimura K, et al. Angiotensin II and tumor necrosis factor-alpha synergistically promote monocyte chemoattractant protein-1 expression: Roles of NF-κB, p38, and reactive oxygen species. American Journal of Physiology. Heart and Circulatory Physiology. 2008;**294**(6):

F440-F449

155-169

H2879-H2888

[108] Zhuo JL. Monocyte

2004;**22**(3):451-454

**103**

chemoattractant protein-1: A key mediator of angiotensin II-induced target organ damage in hypertensive heart disease? Journal of Hypertension.

[109] Kagami S, Border WA, Miller DE, Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis

[95] Mehta PK, Griendling KK. Angiotensin II cell signaling: Physiological and pathological effects in the cardiovascular system. American Journal of Physiology. Cell Physiology. 2007;**292**(1):C82-C97

[96] Baker KM, Booz GW, Dostal DE. Cardiac actions of angiotensin II: Role of an intracardiac renin-angiotensin system. Annual Review of Physiology. 1992;**54**:227-241

[97] Re RN. On the biological actions of intracellular angiotensin. Hypertension. 2000;**35**(6):1189-1190

[98] Re R. Intracellular renin-angiotensin system: The tip of the intracrine physiology iceberg. American Journal of Physiology-Heart and Circulatory Physiology. 2007;**293**(2):H905-H906

[99] Kumar R, Singh VP, Baker KM. The intracellular renin-angiotensin system: A new paradigm. Trends in Endocrinology and Metabolism. 2007; **18**(5):208-214

[100] Zhuo JL, Li XC. Novel roles of intracrine angiotensin II and signalling mechanisms in kidney cells. Journal of the Renin-Angiotensin-Aldosterone System. 2007;**8**(1):23-33

[101] De Mello WC, Danser AH. Angiotensin II and the heart: On the intracrine renin-angiotensin system. Hypertension. 2000;**35**(6):1183-1188

[102] Li XC, Zhuo JL. Intracellular ANG II directly induces in vitro transcription of TGF-β1, MCP-1, and NHE-3 mRNAs in isolated rat renal cortical nuclei via activation of nuclear AT1a receptors. American Journal of Physiology. Cell Physiology. 2008;**294**(4):C1034-C1045

[103] Ferrao FM, Cardoso LHD, Drummond HA, Li XC, Zhuo JL, *The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

Gomes DS, et al. Luminal ANG II is internalized as a complex with AT1R/AT2R heterodimers to target endoplasmic reticulum in LLC-PK1 cells. American Journal of Physiology. Renal Physiology. 2017;**313**(2): F440-F449

editors. Hypertension: Pathology, Diagnosis & Management. 2nd ed. New York: Raven Press; 1995. pp. 1739-1762

[88] Gwathmey T, Shaltout HA,

F1493

**299**(5):F983-F990

**310**(7):F637-F645

pp. 53-68

**102**

Pendergrass KD, Pirro NT, Figueroa JP, Rose JC, et al. Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production. American Journal of Physiology-Renal Physiology. 2009;**296**:F1484-

*Selected Chapters from the Renin-Angiotensin System*

smooth muscle cells. Pharmacological

Physiological and pathological effects in the cardiovascular system. American Journal of Physiology. Cell Physiology.

[96] Baker KM, Booz GW, Dostal DE. Cardiac actions of angiotensin II: Role of an intracardiac renin-angiotensin system. Annual Review of Physiology.

[97] Re RN. On the biological actions of intracellular angiotensin. Hypertension.

[98] Re R. Intracellular renin-angiotensin

physiology iceberg. American Journal of Physiology-Heart and Circulatory Physiology. 2007;**293**(2):H905-H906

[99] Kumar R, Singh VP, Baker KM. The intracellular renin-angiotensin system:

Endocrinology and Metabolism. 2007;

[100] Zhuo JL, Li XC. Novel roles of intracrine angiotensin II and signalling mechanisms in kidney cells. Journal of the Renin-Angiotensin-Aldosterone

system: The tip of the intracrine

A new paradigm. Trends in

System. 2007;**8**(1):23-33

[101] De Mello WC, Danser AH. Angiotensin II and the heart: On the intracrine renin-angiotensin system. Hypertension. 2000;**35**(6):1183-1188

[103] Ferrao FM, Cardoso LHD, Drummond HA, Li XC, Zhuo JL,

[102] Li XC, Zhuo JL. Intracellular ANG II directly induces in vitro transcription of TGF-β1, MCP-1, and NHE-3 mRNAs in isolated rat renal cortical nuclei via activation of nuclear AT1a receptors. American Journal of Physiology. Cell Physiology. 2008;**294**(4):C1034-C1045

**18**(5):208-214

Reviews. 2000;**52**:639-672

2007;**292**(1):C82-C97

1992;**54**:227-241

2000;**35**(6):1189-1190

[95] Mehta PK, Griendling KK. Angiotensin II cell signaling:

[89] Gwathmey TM, Westwood BM, Pirro NT, Tang L, Rose JC, Diz DI, et al. Nuclear angiotensin-(1-7) receptor is functionally coupled to the formation of nitric oxide. American Journal of Physiology. Renal Physiology. 2010;

[90] Wilson BA, Nautiyal M, Gwathmey TM, Rose JC, Chappell MC. Evidence for a mitochondrial angiotensin-(1-7) system in the kidney. American Journal of Physiology. Renal Physiology. 2016;

[91] Zhuo JL, MacGregor D, Mendelsohn

FA. Comparative distribution of angiotensin II receptor subtypes in mammalian adrenal glands. In: Vinson GP, Anderson DC, editors. Vascular, Adrenal and Hypertension. London: Journal of Endocrinology Pty Ltd.; 1995.

[92] Ozono R, Wang ZQ, Moore AF, Inagami T, Siragy HM, Carey RM. Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney. Hypertension. 1997;**30**(5):1238-1246

[93] Kemp BA, Howell NL, Gildea JJ, Keller SR, Padia SH, Carey RM. AT(2) receptor activation induces natriuresis and lowers blood pressure. Circulation

Research. 2014;**115**(3):388-399

[94] Touyz RM, Schiffrin EL. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular

[104] Brasier AR, Jamaluddin M, Han Y, Patterson C, Runge MS. Angiotensin II induces gene transcription through celltype-dependent effects on the nuclear factor-kappaB (NF-kappaB) transcription factor. Molecular and Cellular Biochemistry. 2000;**212**(1-2): 155-169

[105] Ruiz-Ortega M, Lorenzo O, Ruperez M, Konig S, Wittig B, Egido J. Angiotensin II activates nuclear transcription factor κB through AT1 and AT2 in vascular smooth muscle cells: Molecular mechanisms. Circulation Research. 2000;**86**(12):1266-1272

[106] Li XC, Zhuo JL. Nuclear factor-κB as a hormonal intracellular signaling molecule: Focus on angiotensin IIinduced cardiovascular and renal injury. Current Opinion in Nephrology and Hypertension. 2008;**17**(1):37-43

[107] Takahashi M, Suzuki E, Takeda R, Oba S, Nishimatsu H, Kimura K, et al. Angiotensin II and tumor necrosis factor-alpha synergistically promote monocyte chemoattractant protein-1 expression: Roles of NF-κB, p38, and reactive oxygen species. American Journal of Physiology. Heart and Circulatory Physiology. 2008;**294**(6): H2879-H2888

[108] Zhuo JL. Monocyte chemoattractant protein-1: A key mediator of angiotensin II-induced target organ damage in hypertensive heart disease? Journal of Hypertension. 2004;**22**(3):451-454

[109] Kagami S, Border WA, Miller DE, Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis

through induction of transforming growth factor-beta expression in rat glomerular mesangial cells. The Journal of Clinical Investigation. 1994;**93**:2431-2437

[110] Wolf G, Ziyadeh FN, Stahl RA. Angiotensin II stimulates expression of transforming growth factor beta receptor type II in cultured mouse proximal tubular cells. Journal of Molecular Medicine. 1999;**77**(7):556-564

[111] Kurtz TW, Gardner DG. Transcription-modulating drugs: A new frontier in the treatment of essential hypertension. Hypertension. 1998;**32**: 380-386

[112] Kurtz TW, Pravenec M. Moleculespecific effects of angiotensin IIreceptor blockers independent of the renin-angiotensin system. American Journal of Hypertension. 2008;**21**(8): 852-859

[113] Kurtz TW. Beyond the classic angiotensin-receptor-blocker profile. Nature Clinical Practice. Cardiovascular Medicine. 2008;**5**(Suppl 1):S19-S26

[114] Conchon S, Monnot C, Teutsch B, Corvol P, Clauser E. Internalization of the rat AT1a and AT1b receptors: Pharmacological and functional requirements. FEBS Letters. 1994; **349**(3):365-370

[115] Schupp M, Janke J, Clasen R, Unger T, Kintscher U. Angiotensin type 1 receptor blockers induce peroxisome proliferator-activated receptor-gamma activity. Circulation. 2004;**109**(17): 2054-2057

[116] Kurtz TW, Klein U. Next generation multifunctional angiotensin receptor blockers. Hypertension Research. 2009;**32**(10):826-834

[117] Mauer M, Zinman B, Gardiner R, Suissa S, Sinaiko A, Strand T, et al. Renal and retinal effects of enalapril and losartan in type 1 diabetes. The New

England Journal of Medicine. 2009; **361**(1):40-51

[118] van Kats JP, Schalekamp MA, Verdouw PD, Duncker DJ, Danser AH. Intrarenal angiotensin II: Interstitial and cellular levels and site of production. Kidney International. 2001;**60**(6): 2311-2317

[119] von Thun AM, Vari RC, El Dahr SS, Navar LG. Augmentation of intrarenal angiotensin II levels by chronic angiotensin II infusion. The American Journal of Physiology. 1994;**266**(1 Pt 2): F120-F128

[120] Zou LX, Imig JD, Hymel A, Navar LG. Renal uptake of circulating angiotensin II in Val<sup>5</sup> -angiotensin II infused rats is mediated by AT<sup>1</sup> receptor. American Journal of Hypertension. 1998;**11**(5):570-578

[121] Shao W, Seth DM, Navar LG. Augmentation of endogenous intrarenal angiotensin II levels in Val<sup>5</sup> -Ang II infused rats. American Journal of Physiology. Renal Physiology. 2009; **296**(5):F1067-F1071

[122] Li XC, Zhuo JL. Mechanisms of AT1a receptor-mediated uptake of angiotensin II by proximal tubule cells: A novel role of the multiligand endocytic receptor megalin. American Journal of Physiology. Renal Physiology. 2014;**307**(2):F222-F233

[123] Li XC, Gu V, Miguel-Qin E, Zhuo JL. Role of caveolin 1 in AT1a receptormediated uptake of angiotensin II in the proximal tubule of the kidney. American Journal of Physiology. Renal Physiology. 2014;**307**(8):F949- F961

[124] Li XC, Carretero OA, Navar LG, Zhuo JL. AT1 receptor-mediated accumulation of extracellular angiotensin II in proximal tubule cells: Role of cytoskeleton microtubules and tyrosine phosphatases. American

Journal of Physiology. Renal Physiology. 2006;**291**:F375-F383

American Journal of Physiology. Renal Physiology. 2001;**280**(4):F562-F573

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

Biological Chemistry. 1999;**274**(16):

[140] Thomas WG, Thekkumkara TJ, Baker KM. Molecular mechanisms of angiotensin II (AT1A) receptor

endocytosis. Clinical and Experimental

[141] Rajagopal K, Whalen EJ, Violin JD, Stiber JA, Rosenberg PB, Premont RT, et al. Beta-arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes. Proceedings of the National Academy of

[142] Rappoport JZ, Kemal S, Benmerah A, Simon SM. Dynamics of clathrin and adaptor proteins during endocytosis. American Journal of Physiology. Cell Physiology. 2006;**291**(5):C1072-C1081

[143] Zhang J, Ferguson SS, Barak LS, Menard L, Caron MG. Dynamin and beta-arrestin reveal distinct mechanisms

internalization. The Journal of Biological Chemistry. 1996;**271**(31):18302-18305

[144] Qian H, Pipolo L, Thomas WG. Association of beta-Arrestin 1 with the type 1A angiotensin II receptor involves

[145] Seachrist JL, Laporte SA, Dale LB, Babwah AV, Caron MG, Anborgh PH,

[146] Dale LB, Seachrist JL, Babwah AV, Ferguson SS. Regulation of angiotensin II type 1A receptor intracellular

retention, degradation, and recycling by

phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization. Molecular Endocrinology. 2001;**15**(10):1706-1719

et al. Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion. The Journal of Biological Chemistry. 2002;**277**(1):

679-685

for G protein-coupled receptor

Pharmacology & Physiology. Supplement. 1996;**3**:S74-S80

Sciences of the United States of America. 2006;**103**(44):16284-16289

10999-11006

[133] Zhai XY, Nielsen R, Birn H, Drumm K, Mildenberger S, Freudinger R, et al. Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney. Kidney International. 2000;**58**(4):1523-1533

[134] Kozyraki R, Fyfe J, Verroust PJ, Jacobsen C, Dautry-Varsat A, Gburek J, et al. Megalin-dependent cubilinmediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia. Proceedings of the National Academy of

Sciences of the United States of America. 2001;**98**(22):12491-12496

[135] Leheste JR, Rolinski B, Vorum H, Hilpert J, Nykjaer A, Jacobsen C, et al. Megalin knockout mice as an animal model of low molecular weight proteinuria. The American Journal of Pathology. 1999;**155**(4):1361-1370

[136] Gonzalez-Villalobos R, Klassen RB, Allen PL, Navar LG, Hammond TG. Megalin binds and internalizes angiotensin II. American Journal of Physiology. Renal Physiology. 2005;**288**:

[137] Anborgh PH, Seachrist JL, Dale LB, Ferguson SS. Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of β2 adrenergic and angiotensin II type 1A receptors. Molecular Endocrinology.

[138] Ferguson SS. Evolving concepts in G protein-coupled receptor endocytosis: The role in receptor desensitization and signaling. Pharmacological Reviews.

[139] Zhang J, Barak LS, Anborgh PH, Laporte SA, Caron MG, Ferguson SS. Cellular trafficking of G proteincoupled receptor/beta-arrestin endocytic complexes. The Journal of

F420-F427

2000;**14**(12):2040-2053

2001;**53**(1):1-24

**105**

[125] Li XC, Zhuo JL. Selective knockdown of AT1 receptors by RNA interference inhibits Val<sup>5</sup> -Ang II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells. American Journal of Physiology. Cell Physiology. 2007;**293**:C367-C378

[126] Zhuo JL, Carretero OA, Li XC. Effects of AT1 receptor-mediated endocytosis of extracellular Ang II on activation of nuclear factor-κB in proximal tubule cells. Annals of the New York Academy of Sciences. 2006;**1091**: 336-345

[127] Brown GP, Douglas JG. Angiotensin II binding sites on isolated rat renal brush border membranes. Endocrinology. 1982;**111**(6):1830-1836

[128] Douglas JG. Angiotensin receptor subtypes of the kidney cortex. The American Journal of Physiology. 1987; **253**(1 Pt 2):F1-F7

[129] Dulin NO, Ernsberger P, Suciu DJ, Douglas JG. Rabbit renal epithelial angiotensin II receptors. The American Journal of Physiology. 1994;**267**(5 Pt 2): F776-F782

[130] Becker BN, Cheng HF, Burns KD, Harris RC. Polarized rabbit type 1 angiotensin II receptors manifest differential rates of endocytosis and recycling. The American Journal of Physiology. 1995;**269**(4 Pt 1):C1048- C1056

[131] Thekkumkara TJ, Cookson R, Linas SL. Angiotensin (AT1a) receptormediated increases in transcellular sodium transport in proximal tubule cells. The American Journal of Physiology. 1998;**274**:F897-F905

[132] Christensen EI, Birn H. Megalin and cubilin: Synergistic endocytic receptors in renal proximal tubule.

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

American Journal of Physiology. Renal Physiology. 2001;**280**(4):F562-F573

England Journal of Medicine. 2009;

*Selected Chapters from the Renin-Angiotensin System*

[118] van Kats JP, Schalekamp MA, Verdouw PD, Duncker DJ, Danser AH. Intrarenal angiotensin II: Interstitial and cellular levels and site of production. Kidney International. 2001;**60**(6):

[119] von Thun AM, Vari RC, El Dahr SS, Navar LG. Augmentation of intrarenal angiotensin II levels by chronic angiotensin II infusion. The American Journal of Physiology. 1994;**266**(1 Pt 2):

[120] Zou LX, Imig JD, Hymel A, Navar


Journal of Physiology. Renal Physiology.

knockdown of AT1 receptors by RNA

endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells. American Journal of Physiology. Cell Physiology. 2007;**293**:C367-C378

[126] Zhuo JL, Carretero OA, Li XC. Effects of AT1 receptor-mediated endocytosis of extracellular Ang II on activation of nuclear factor-κB in proximal tubule cells. Annals of the New York Academy of Sciences. 2006;**1091**:

[127] Brown GP, Douglas JG.

**253**(1 Pt 2):F1-F7

F776-F782

C1056

Angiotensin II binding sites on isolated rat renal brush border membranes. Endocrinology. 1982;**111**(6):1830-1836

[128] Douglas JG. Angiotensin receptor subtypes of the kidney cortex. The American Journal of Physiology. 1987;

[129] Dulin NO, Ernsberger P, Suciu DJ, Douglas JG. Rabbit renal epithelial angiotensin II receptors. The American Journal of Physiology. 1994;**267**(5 Pt 2):

[130] Becker BN, Cheng HF, Burns KD, Harris RC. Polarized rabbit type 1 angiotensin II receptors manifest differential rates of endocytosis and recycling. The American Journal of Physiology. 1995;**269**(4 Pt 1):C1048-

[131] Thekkumkara TJ, Cookson R, Linas SL. Angiotensin (AT1a) receptormediated increases in transcellular sodium transport in proximal tubule cells. The American Journal of Physiology. 1998;**274**:F897-F905

[132] Christensen EI, Birn H. Megalin and cubilin: Synergistic endocytic receptors in renal proximal tubule.


2006;**291**:F375-F383

336-345

[125] Li XC, Zhuo JL. Selective

interference inhibits Val<sup>5</sup>


LG. Renal uptake of circulating

infused rats is mediated by AT<sup>1</sup> receptor. American Journal of Hypertension. 1998;**11**(5):570-578

[121] Shao W, Seth DM, Navar LG. Augmentation of endogenous intrarenal

infused rats. American Journal of Physiology. Renal Physiology. 2009;

[122] Li XC, Zhuo JL. Mechanisms of AT1a receptor-mediated uptake of angiotensin II by proximal tubule cells:

endocytic receptor megalin. American Journal of Physiology. Renal Physiology.

[123] Li XC, Gu V, Miguel-Qin E, Zhuo JL. Role of caveolin 1 in AT1a receptormediated uptake of angiotensin II in the

[124] Li XC, Carretero OA, Navar LG, Zhuo JL. AT1 receptor-mediated accumulation of extracellular

angiotensin II in proximal tubule cells: Role of cytoskeleton microtubules and tyrosine phosphatases. American

A novel role of the multiligand

proximal tubule of the kidney. American Journal of Physiology. Renal Physiology. 2014;**307**(8):F949-

2014;**307**(2):F222-F233

F961

**104**

angiotensin II levels in Val<sup>5</sup>

**296**(5):F1067-F1071

angiotensin II in Val<sup>5</sup>

**361**(1):40-51

2311-2317

F120-F128

[133] Zhai XY, Nielsen R, Birn H, Drumm K, Mildenberger S, Freudinger R, et al. Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney. Kidney International. 2000;**58**(4):1523-1533

[134] Kozyraki R, Fyfe J, Verroust PJ, Jacobsen C, Dautry-Varsat A, Gburek J, et al. Megalin-dependent cubilinmediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia. Proceedings of the National Academy of Sciences of the United States of America. 2001;**98**(22):12491-12496

[135] Leheste JR, Rolinski B, Vorum H, Hilpert J, Nykjaer A, Jacobsen C, et al. Megalin knockout mice as an animal model of low molecular weight proteinuria. The American Journal of Pathology. 1999;**155**(4):1361-1370

[136] Gonzalez-Villalobos R, Klassen RB, Allen PL, Navar LG, Hammond TG. Megalin binds and internalizes angiotensin II. American Journal of Physiology. Renal Physiology. 2005;**288**: F420-F427

[137] Anborgh PH, Seachrist JL, Dale LB, Ferguson SS. Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of β2 adrenergic and angiotensin II type 1A receptors. Molecular Endocrinology. 2000;**14**(12):2040-2053

[138] Ferguson SS. Evolving concepts in G protein-coupled receptor endocytosis: The role in receptor desensitization and signaling. Pharmacological Reviews. 2001;**53**(1):1-24

[139] Zhang J, Barak LS, Anborgh PH, Laporte SA, Caron MG, Ferguson SS. Cellular trafficking of G proteincoupled receptor/beta-arrestin endocytic complexes. The Journal of

Biological Chemistry. 1999;**274**(16): 10999-11006

[140] Thomas WG, Thekkumkara TJ, Baker KM. Molecular mechanisms of angiotensin II (AT1A) receptor endocytosis. Clinical and Experimental Pharmacology & Physiology. Supplement. 1996;**3**:S74-S80

[141] Rajagopal K, Whalen EJ, Violin JD, Stiber JA, Rosenberg PB, Premont RT, et al. Beta-arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(44):16284-16289

[142] Rappoport JZ, Kemal S, Benmerah A, Simon SM. Dynamics of clathrin and adaptor proteins during endocytosis. American Journal of Physiology. Cell Physiology. 2006;**291**(5):C1072-C1081

[143] Zhang J, Ferguson SS, Barak LS, Menard L, Caron MG. Dynamin and beta-arrestin reveal distinct mechanisms for G protein-coupled receptor internalization. The Journal of Biological Chemistry. 1996;**271**(31):18302-18305

[144] Qian H, Pipolo L, Thomas WG. Association of beta-Arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization. Molecular Endocrinology. 2001;**15**(10):1706-1719

[145] Seachrist JL, Laporte SA, Dale LB, Babwah AV, Caron MG, Anborgh PH, et al. Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion. The Journal of Biological Chemistry. 2002;**277**(1): 679-685

[146] Dale LB, Seachrist JL, Babwah AV, Ferguson SS. Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases. The Journal of Biological Chemistry. 2004; **279**(13):13110-13118

[147] Becker BN, Cheng HF, Harris RC. Apical ANG II-stimulated PLA2 activity and Na+ flux: A potential role for Ca2+ independent PLA. The American Journal of Physiology. 1997;**273**(4 Pt 2): F554-F562

[148] Elkjaer ML, Birn H, Agre P, Christensen EI, Nielsen S. Effects of microtubule disruption on endocytosis, membrane recycling and polarized distribution of Aquaporin-1 and gp330 in proximal tubule cells. European Journal of Cell Biology. 1995;**67**(1):57-72

[149] Schelling JR, Linas SL. Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis. The American Journal of Physiology. 1994;**266**(3 Pt 1):C669-C675

[150] Schelling JR, Hanson AS, Marzec R, Linas SL. Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells. The Journal of Clinical Investigation. 1992;**90**(6): 2472-2480

[151] Li XC, Hopfer U, Zhuo JL. AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through the microtubule-dependent endocytic pathway. American Journal of Physiology. Renal Physiology. 2009; **297**(5):F1342-F1352

[152] Robertson A, Khairallah P. Angiotensin II: Rapid localization in nuclei of smooth and cardiac muscle. Science. 1971;**172**:1138-1139

[153] Bianchi C, Gutkowska J, De Lean A, Ballak M, Anand-Srivastava MB, Genest J, et al. Fate of [125I]angiotensin II in adrenal zona glomerulosa cells. Endocrinology. 1986;**118**:2605-2607

[154] Cook JL, Mills SJ, Naquin RT, Alam J, Re RN. Cleavage of the angiotensin II type 1 receptor and nuclear accumulation of the cytoplasmic carboxy-terminal fragment. American Journal of Physiology. Cell Physiology. 2007;**292**(4):C1313-C1322

Sciences of the United States of America. 1990;**87**(20):7917-7920

F380

C1146

1496-1505

**57**(6):2457-2467

[161] Reilly AM, Harris PJ, Williams DA. Biphasic effect of angiotensin II on intracellular sodium concentration in rat proximal tubules. The American Journal of Physiology. 1995;**269**(3 Pt 2):F374-

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

[162] Jourdain M, Amiel C, Friedlander

activity by angiotensin II in opossum kidney cells. The American Journal of Physiology. 1992;**263**(6 Pt 1):C1141-

[163] Houillier P, Chambrey R, Achard JM, Froissart M, Poggioli J, Paillard M. Signaling pathways in the biphasic effect of angiotensin II on apical Na<sup>+</sup>

antiport activity in proximal tubule. Kidney International. 1996;**50**(5):

[164] Han HJ, Park SH, Koh HJ, Taub M.

[165] Tsuganezawa H, Preisig PA, Alpern RJ. Dominant negative c-Src inhibits angiotensin II induced activation of

[166] Du Z, Ferguson W, Wang T. Role of PKC and calcium in modulation of effects of angiotensin II on sodium transport in proximal tubule. American Journal of Physiology. Renal Physiology.

[167] Wang T, Chan YL. The role of phosphoinositide turnover in mediating the biphasic effect of angiotensin II on renal tubular transport. The Journal of Pharmacology and Experimental Therapeutics. 1991;**256**(1):309-317

[168] Karim Z, Defontaine N, Paillard M, Poggioli J. Protein kinase C isoforms in

Mechanism of regulation of Na<sup>+</sup> transport by angiotensin II in primary renal cells. Kidney International. 2000;

NHE3 in OKP cells. Kidney International. 1998;**54**(2):394-398

2003;**284**(4):F688-F692

**107**


*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*


C140

C1245

**13**(7):1750-1756

**84**(1):83-91

8098-8102

1743-1756

**79**(4):765-772

rat kidney proximal tubule: Acute effect of angiotensin II. The American Journal of Physiology. 1995;**269**(1 Pt 1):C134-

[169] Schelling JR, Singh H, Marzec R, Linas SL. Angiotensin II-dependent proximal tubule sodium transport is mediated by cAMP modulation of phospholipase C. The American Journal of Physiology. 1994;**267**(5 Pt 1):C1239-

[170] Lea JP, Jin SG, Roberts BR, Shuler

American Society of Nephrology. 2002;

[171] Liu FY, Cogan MG. Angiotensin II stimulates early proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate. The Journal of Clinical Investigation. 1989;

[172] Dulin NO, Alexander LD, Harwalkar S, Falck JR, Douglas JG. Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II. Proceedings of the National Academy of Sciences of the United States of America. 1998;**95**(14):

[173] Ruiz-Ortega M, Lorenzo O, Ruperez M, Blanco J, Egido J. Systemic

[174] Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects of intracellular angiotensin II in vascular smooth muscle cells. Circulation Research. 1996;

infusion of angiotensin II into normal rats activates nuclear factorkappaB and AP-1 in the kidney: Role of AT1 and AT2 receptors. The American Journal of Pathology. 2001;**158**(5):

Angiotensin II stimulates calcineurin activity in proximal tubule epithelia through AT-1 receptor-mediated tyrosine phosphorylation of the PLCgamma1 isoform. Journal of the

MS, Marrero MB, Tumlin JA.

G. Modulation of Na<sup>+</sup>

[155] Chen R, Mukhin YV, Garnovskaya MN, Thielen TE, Iijima Y, Huang C, et al. A functional angiotensin II receptor-GFP fusion protein: Evidence for agonist-dependent nuclear translocation. American Journal of Physiology. Renal Physiology. 2000; **279**(3):F440-F448

[156] Morinelli TA, Raymond JR, Baldys A, Yang Q, Lee MH, Luttrell L, et al. Identification of a putative nuclear localization sequence within the angiotensin II AT1a receptor associated with nuclear activation. American Journal of Physiology. Cell Physiology. 2007;**292**(4):C1398-C1408

[157] du Cheyron D, Chalumeau C, Defontaine N, Klein C, Kellermann O, Paillard M, et al. Angiotensin II stimulates NHE3 activity by exocytic insertion of the transporter: Role of PI 3 kinase. Kidney International. 2003; **64**(3):939-949

[158] Liu FY, Cogan MG. Angiotensin II stimulation of hydrogen ion secretion in the rat early proximal tubule. Modes of action, mechanism, and kinetics. Journal of Clinical Investigation 1988;**82**(2): 601-607

[159] Bloch RD, Zikos D, Fisher KA, Schleicher L, Oyama M, Cheng JC, et al. Activation of proximal tubular Na+ -H<sup>+</sup> exchange by angiotensin II. The American Journal of Physiology. 1992; **263**(1 Pt 2):F135-F143

[160] Geibel J, Giebisch G, Boron WF. Angiotensin II stimulates both Na<sup>+</sup> -H+ exchange and Na<sup>+</sup> /HCO3 cotransport in the rabbit proximal tubule. Proceedings of the National Academy of *The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

Sciences of the United States of America. 1990;**87**(20):7917-7920

Rab5, Rab7, and Rab11 GTPases. The Journal of Biological Chemistry. 2004;

*Selected Chapters from the Renin-Angiotensin System*

[154] Cook JL, Mills SJ, Naquin RT, Alam J, Re RN. Cleavage of the angiotensin II

[155] Chen R, Mukhin YV, Garnovskaya MN, Thielen TE, Iijima Y, Huang C, et al. A functional angiotensin II receptor-GFP fusion protein: Evidence

[156] Morinelli TA, Raymond JR, Baldys A, Yang Q, Lee MH, Luttrell L, et al. Identification of a putative nuclear localization sequence within the angiotensin II AT1a receptor associated with nuclear activation. American Journal of Physiology. Cell Physiology.

type 1 receptor and nuclear accumulation of the cytoplasmic carboxy-terminal fragment. American Journal of Physiology. Cell Physiology.

2007;**292**(4):C1313-C1322

for agonist-dependent nuclear translocation. American Journal of Physiology. Renal Physiology. 2000;

2007;**292**(4):C1398-C1408

**64**(3):939-949

601-607

[157] du Cheyron D, Chalumeau C, Defontaine N, Klein C, Kellermann O, Paillard M, et al. Angiotensin II stimulates NHE3 activity by exocytic insertion of the transporter: Role of PI 3 kinase. Kidney International. 2003;

[158] Liu FY, Cogan MG. Angiotensin II stimulation of hydrogen ion secretion in the rat early proximal tubule. Modes of action, mechanism, and kinetics. Journal of Clinical Investigation 1988;**82**(2):

[159] Bloch RD, Zikos D, Fisher KA, Schleicher L, Oyama M, Cheng JC, et al. Activation of proximal tubular Na+

[160] Geibel J, Giebisch G, Boron WF. Angiotensin II stimulates both Na<sup>+</sup>

/HCO3

Proceedings of the National Academy of

exchange by angiotensin II. The American Journal of Physiology. 1992;

in the rabbit proximal tubule.

**263**(1 Pt 2):F135-F143

exchange and Na<sup>+</sup>



cotransport

**279**(3):F440-F448

[147] Becker BN, Cheng HF, Harris RC. Apical ANG II-stimulated PLA2 activity and Na+ flux: A potential role for Ca2+ independent PLA. The American Journal of Physiology. 1997;**273**(4 Pt 2):

[148] Elkjaer ML, Birn H, Agre P, Christensen EI, Nielsen S. Effects of microtubule disruption on endocytosis, membrane recycling and polarized distribution of Aquaporin-1 and gp330 in proximal tubule cells. European Journal of Cell Biology. 1995;**67**(1):57-72

[149] Schelling JR, Linas SL. Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis. The American Journal of Physiology. 1994;**266**(3 Pt 1):C669-C675

[150] Schelling JR, Hanson AS, Marzec R, Linas SL. Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells. The Journal of Clinical Investigation. 1992;**90**(6):

[151] Li XC, Hopfer U, Zhuo JL. AT1 receptor-mediated uptake of

angiotensin II and NHE-3 expression in proximal tubule cells through the microtubule-dependent endocytic pathway. American Journal of Physiology. Renal Physiology. 2009;

**279**(13):13110-13118

F554-F562

2472-2480

**297**(5):F1342-F1352

**106**

[152] Robertson A, Khairallah P. Angiotensin II: Rapid localization in nuclei of smooth and cardiac muscle.

[153] Bianchi C, Gutkowska J, De Lean A, Ballak M, Anand-Srivastava MB, Genest J, et al. Fate of [125I]angiotensin II in adrenal zona glomerulosa cells. Endocrinology. 1986;**118**:2605-2607

Science. 1971;**172**:1138-1139

[161] Reilly AM, Harris PJ, Williams DA. Biphasic effect of angiotensin II on intracellular sodium concentration in rat proximal tubules. The American Journal of Physiology. 1995;**269**(3 Pt 2):F374- F380

[162] Jourdain M, Amiel C, Friedlander G. Modulation of Na<sup>+</sup> -H<sup>+</sup> exchange activity by angiotensin II in opossum kidney cells. The American Journal of Physiology. 1992;**263**(6 Pt 1):C1141- C1146

[163] Houillier P, Chambrey R, Achard JM, Froissart M, Poggioli J, Paillard M. Signaling pathways in the biphasic effect of angiotensin II on apical Na<sup>+</sup> -H+ antiport activity in proximal tubule. Kidney International. 1996;**50**(5): 1496-1505

[164] Han HJ, Park SH, Koh HJ, Taub M. Mechanism of regulation of Na<sup>+</sup> transport by angiotensin II in primary renal cells. Kidney International. 2000; **57**(6):2457-2467

[165] Tsuganezawa H, Preisig PA, Alpern RJ. Dominant negative c-Src inhibits angiotensin II induced activation of NHE3 in OKP cells. Kidney International. 1998;**54**(2):394-398

[166] Du Z, Ferguson W, Wang T. Role of PKC and calcium in modulation of effects of angiotensin II on sodium transport in proximal tubule. American Journal of Physiology. Renal Physiology. 2003;**284**(4):F688-F692

[167] Wang T, Chan YL. The role of phosphoinositide turnover in mediating the biphasic effect of angiotensin II on renal tubular transport. The Journal of Pharmacology and Experimental Therapeutics. 1991;**256**(1):309-317

[168] Karim Z, Defontaine N, Paillard M, Poggioli J. Protein kinase C isoforms in

rat kidney proximal tubule: Acute effect of angiotensin II. The American Journal of Physiology. 1995;**269**(1 Pt 1):C134- C140

[169] Schelling JR, Singh H, Marzec R, Linas SL. Angiotensin II-dependent proximal tubule sodium transport is mediated by cAMP modulation of phospholipase C. The American Journal of Physiology. 1994;**267**(5 Pt 1):C1239- C1245

[170] Lea JP, Jin SG, Roberts BR, Shuler MS, Marrero MB, Tumlin JA. Angiotensin II stimulates calcineurin activity in proximal tubule epithelia through AT-1 receptor-mediated tyrosine phosphorylation of the PLCgamma1 isoform. Journal of the American Society of Nephrology. 2002; **13**(7):1750-1756

[171] Liu FY, Cogan MG. Angiotensin II stimulates early proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate. The Journal of Clinical Investigation. 1989; **84**(1):83-91

[172] Dulin NO, Alexander LD, Harwalkar S, Falck JR, Douglas JG. Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II. Proceedings of the National Academy of Sciences of the United States of America. 1998;**95**(14): 8098-8102

[173] Ruiz-Ortega M, Lorenzo O, Ruperez M, Blanco J, Egido J. Systemic infusion of angiotensin II into normal rats activates nuclear factorkappaB and AP-1 in the kidney: Role of AT1 and AT2 receptors. The American Journal of Pathology. 2001;**158**(5): 1743-1756

[174] Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects of intracellular angiotensin II in vascular smooth muscle cells. Circulation Research. 1996; **79**(4):765-772

[175] Haller H, Lindschau C, Quass P, Luft FC. Intracellular actions of angiotensin II in vascular smooth muscle cells. Journal of the American Society of Nephrology. 1999;**10**(Suppl 11):S75-S83

[176] De Mello WC. Renin increments the inward calcium current in the failing heart. Journal of Hypertension. 2006; **24**(6):1181-1186

[177] Zhuo JL. Intracrine renin and angiotensin II: A novel role in cardiovascular and renal cellular regulation. Journal of Hypertension. 2006;**24**(6):1017-1020

[178] Baker KM, Kumar R. Intracellular angiotensin II induces cell proliferation independent of AT1 receptor. American Journal of Physiology. Cell Physiology. 2006;**291**(5):C995-C1001

[179] Baker KM, Chernin MI, Schreiber T, Sanghi S, Haiderzaidi S, Booz GW, et al. Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy. Regulatory Peptides. 2004;**120**(1-3):5-13

[180] van Kats JP, Methot D, Paradis P, Silversides DW, Reudelhuber TL. Use of a biological peptide pump to study chronic peptide hormone action in transgenic mice. Direct and indirect effects of angiotensin II on the heart. The Journal of Biological Chemistry. 2001;**276**(47):44012-44017

[181] Xu J, Carretero OA, Lin CX, Cavasin MA, Shesely EG, Yang JJ, et al. Role of cardiac overexpression of ANG II in the regulation of cardiac function and remodeling postmyocardial infarction. American Journal of Physiology. Heart and Circulatory Physiology. 2007;**293**(3):H1900-H1907

[182] Ding Y, Davisson RL, Hardy DO, Zhu LJ, Merrill DC, Catterall JF, et al. The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgenresponsive expression on the human angiotensinogen gene in transgenic mice. The Journal of Biological Chemistry. 1997;**272**(44): 28142-28148

[190] Moe OW. Acute regulation of proximal tubule apical membrane Na/H

*DOI: http://dx.doi.org/10.5772/intechopen.88054*

phosphorylation, protein trafficking, and regulatory factors. Journal of the American Society of Nephrology. 1999;

[191] Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, et al. Renal and intestinal absorptive defects in mice lacking the NHE3 Na<sup>+</sup>

H+ exchanger. Nature Genetics. 1998;

/H+ exchange. Adaptation to metabolic acidosis. The Journal of Biological Chemistry. 1996;**271**(51):

[193] Wang T, Yang CL, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, et al. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. The American Journal of

Physiology. 1999;**277**(2 Pt 2):F298-F302

[194] Ledoussal C, Lorenz JN, Nieman ML, Soleimani M, Schultheis PJ, Shull GE. Renal salt wasting in mice lacking

[195] Woo AL, Noonan WT, Schultheis PJ, Neumann JC, Manning PA, Lorenz JN, et al. Renal function in NHE3 deficient mice with transgenic rescue of small intestinal absorptive defect. American Journal of Physiology. Renal Physiology. 2003;**284**(6):F1190-F1198

[196] Noonan WT, Woo AL, Nieman ML, Prasad V, Schultheis PJ, Shull GE, et al. Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine. American Journal of Physiology. Regulatory, Integrative and

/H<sup>+</sup> exchanger but not in mice lacking NHE2. American Journal of Physiology. Renal Physiology. 2001;

[192] Wu MS, Biemesderfer D, Giebisch G, Aronson PS. Role of NHE3 in mediating renal brush border

/

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal…*

Comparative Physiology. 2005;**288**(3):

[197] Li XC, Zhu D, Chen X, Zheng XW, Zhao C, Zhang JF, et al. Proximal tubule-specific deletion of the NHE3

/H<sup>+</sup> exchanger 3) in the kidney

attenuates angiotensin II-induced hypertension in mice. Hypertension.

R685-R691

2019 (in press)

(Na+

exchanger NHE-3: Role of

**10**:2412-2425

**19**(3):282-285

32749-32752

NHE3 Na<sup>+</sup>

**109**

**281**(4):F718-F727

Na<sup>+</sup>

[183] Ding Y, Sigmund CD. Androgendependent regulation of human angiotensinogen expression in KAPhAGT transgenic mice. American Journal of Physiology. Renal Physiology. 2001;**280**(1):F54-F60

[184] Soler M, Tornavaca O, Sole E, Menoyo A, Hardy D, Catterall JF, et al. Hormone-specific regulation of the kidney androgen-regulated gene promoter in cultured mouse renal proximal-tubule cells. The Biochemical Journal. 2002;**366**(Pt 3):757-766

[185] Meseguer A, Catterall JF. Cellspecific expression of kidney androgenregulated protein messenger RNA is under multihormonal control. Molecular Endocrinology. 1990;**4**(8): 1240-1248

[186] Hediger MA, Rhoads DB. Molecular physiology of sodium-glucose cotransporters. Physiological Reviews. 1994;**74**(4):993-1026

[187] Weinman EJ, Shenolikar S. Regulation of the renal brush border membrane Na+ -H+ exchanger. Annual Review of Physiology. 1993;**55**:289-304

[188] Biemesderfer D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. The American Journal of Physiology. 1997; **273**(2 Pt 2):F289-F299

[189] Amemiya M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney International. 1995;**48**(4):1206-1215

*The Intratubular and Intracrine Renin-Angiotensin System in the Proximal… DOI: http://dx.doi.org/10.5772/intechopen.88054*

[190] Moe OW. Acute regulation of proximal tubule apical membrane Na/H exchanger NHE-3: Role of phosphorylation, protein trafficking, and regulatory factors. Journal of the American Society of Nephrology. 1999; **10**:2412-2425

[175] Haller H, Lindschau C, Quass P, Luft FC. Intracellular actions of angiotensin II in vascular smooth muscle cells. Journal of the American Society of Nephrology. 1999;**10**(Suppl

*Selected Chapters from the Renin-Angiotensin System*

cell-specific and highly androgenresponsive expression on the human

[183] Ding Y, Sigmund CD. Androgendependent regulation of human angiotensinogen expression in KAPhAGT transgenic mice. American Journal of Physiology. Renal Physiology.

[184] Soler M, Tornavaca O, Sole E, Menoyo A, Hardy D, Catterall JF, et al. Hormone-specific regulation of the kidney androgen-regulated gene promoter in cultured mouse renal proximal-tubule cells. The Biochemical Journal. 2002;**366**(Pt 3):757-766

[185] Meseguer A, Catterall JF. Cellspecific expression of kidney androgenregulated protein messenger RNA is under multihormonal control. Molecular Endocrinology. 1990;**4**(8):

[186] Hediger MA, Rhoads DB.

[187] Weinman EJ, Shenolikar S. Regulation of the renal brush border

1994;**74**(4):993-1026

**273**(2 Pt 2):F289-F299

membrane Na+

Molecular physiology of sodium-glucose cotransporters. Physiological Reviews.

Review of Physiology. 1993;**55**:289-304

[188] Biemesderfer D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. The American Journal of Physiology. 1997;

[189] Amemiya M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney International. 1995;**48**(4):1206-1215


angiotensinogen gene in transgenic mice. The Journal of Biological Chemistry. 1997;**272**(44):

2001;**280**(1):F54-F60

28142-28148

1240-1248

[176] De Mello WC. Renin increments the inward calcium current in the failing heart. Journal of Hypertension. 2006;

[177] Zhuo JL. Intracrine renin and angiotensin II: A novel role in cardiovascular and renal cellular regulation. Journal of Hypertension.

[178] Baker KM, Kumar R. Intracellular angiotensin II induces cell proliferation independent of AT1 receptor. American Journal of Physiology. Cell Physiology.

[179] Baker KM, Chernin MI, Schreiber T, Sanghi S, Haiderzaidi S, Booz GW, et al. Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy. Regulatory Peptides. 2004;**120**(1-3):5-13

[180] van Kats JP, Methot D, Paradis P, Silversides DW, Reudelhuber TL. Use of a biological peptide pump to study chronic peptide hormone action in transgenic mice. Direct and indirect effects of angiotensin II on the heart. The Journal of Biological Chemistry.

2001;**276**(47):44012-44017

[181] Xu J, Carretero OA, Lin CX, Cavasin MA, Shesely EG, Yang JJ, et al. Role of cardiac overexpression of ANG II in the regulation of cardiac function and remodeling postmyocardial infarction. American Journal of Physiology. Heart and Circulatory Physiology. 2007;**293**(3):H1900-H1907

[182] Ding Y, Davisson RL, Hardy DO, Zhu LJ, Merrill DC, Catterall JF, et al. The kidney androgen-regulated protein promoter confers renal proximal tubule

**108**

11):S75-S83

**24**(6):1181-1186

2006;**24**(6):1017-1020

2006;**291**(5):C995-C1001

[191] Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, et al. Renal and intestinal absorptive defects in mice lacking the NHE3 Na<sup>+</sup> / H+ exchanger. Nature Genetics. 1998; **19**(3):282-285

[192] Wu MS, Biemesderfer D, Giebisch G, Aronson PS. Role of NHE3 in mediating renal brush border Na<sup>+</sup> /H+ exchange. Adaptation to metabolic acidosis. The Journal of Biological Chemistry. 1996;**271**(51): 32749-32752

[193] Wang T, Yang CL, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, et al. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. The American Journal of Physiology. 1999;**277**(2 Pt 2):F298-F302

[194] Ledoussal C, Lorenz JN, Nieman ML, Soleimani M, Schultheis PJ, Shull GE. Renal salt wasting in mice lacking NHE3 Na<sup>+</sup> /H<sup>+</sup> exchanger but not in mice lacking NHE2. American Journal of Physiology. Renal Physiology. 2001; **281**(4):F718-F727

[195] Woo AL, Noonan WT, Schultheis PJ, Neumann JC, Manning PA, Lorenz JN, et al. Renal function in NHE3 deficient mice with transgenic rescue of small intestinal absorptive defect. American Journal of Physiology. Renal Physiology. 2003;**284**(6):F1190-F1198

[196] Noonan WT, Woo AL, Nieman ML, Prasad V, Schultheis PJ, Shull GE, et al. Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine. American Journal of Physiology. Regulatory, Integrative and

Comparative Physiology. 2005;**288**(3): R685-R691

[197] Li XC, Zhu D, Chen X, Zheng XW, Zhao C, Zhang JF, et al. Proximal tubule-specific deletion of the NHE3 (Na+ /H<sup>+</sup> exchanger 3) in the kidney attenuates angiotensin II-induced hypertension in mice. Hypertension. 2019 (in press)

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**1. Introduction**

**Chapter 6**

**Abstract**

Scientific Evidences Supporting

Renin-Angiotensin-Aldosterone

System during Estral Cycle and

In women and laboratory animals, local and circulating components of the renin-angiotensin-aldosterone system (RAAS) are related to specific reproductive functions that occur during the estrous cycle, such as folliculogenesis, ovulation, corpus luteum development, and steroidogenesis. Also, in pregnant females of these species, maternal cardiovascular and renal systems undergo intense modifications, with the aim of matching the increased energy requirements of the fetus and fetoplacental unit. Some of these changes can be the origin, and others the consequence of a new endocrine environment. The fetus and the placenta induce endocrine changes, with modifications in the protein, lipid, carbohydrate, and mineral metabolism, together with simultaneous cardiovascular changes derived from the uterine growth and its content. The participation of RAAS during this period is of vital importance to regulate these cardiovascular, hemodynamic, hematological, and metabolic adjustments imposed by pregnancy because they will have a direct influence on the correct development and viability of the fetus. In mares, our research team has been investigating the changes of RAAS in mares during the estral cycle and during pregnancy, and these results are presented in the current chapter, comparing with

the data previously reported for women and laboratory animals.

**Keywords:** estrous cycle, mare, pregnancy, renin, angiotensin, aldosterone

In nonpregnant females of various species, the components of the renin-angiotensin-aldosterone system (RAAS), i.e., prorenin, renin, angiotensin II (ANG-II), and aldosterone, are expressed in the tissues of reproductive organs, mainly the uterus and ovaries. These hormones have direct physiological relationships with specific reproductive functions, including folliculogenesis, oocyte maturation, ovulation, follicular atresia, corpus luteum development and luteolysis, steroidogenesis, angiogenesis, and expression of certain vasoactive substances [1–8].

A great body of literature has confirmed an increase in plasma activity of renin (PRA) and plasma concentrations of ANG-II and aldosterone in women during the

the Activation of the

Pregnancy in Mares

*Katy Satué and Ana Muñoz*

## **Chapter 6**
