**Section 2**

**Novel Therapeutic Molecules in Diabetic Nephropathy** 

82 Diabetic Nephropathy

[124] Feldman DL, Jin L, Xuan H, Contrepas A, Zhou Y, Webb RL, Mueller DN, Feldt S,

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[126] Feldman DL: New insights into the renoprotective actions of the renin inhibitor aliskiren in experimental renal disease. Hypertens Res 2010; 33: 279–287. [127] Pimenta E, Oparil S: Role of aliskiren in cardiorenal protection and use in

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[130] Huang Y, Wongamorntham S, Kasting J, Mc-Quillan D, Owens RT, Yu L, Noble NA,

[131] Yusuf S, Teo KK, Pogue J, Dyal L, Copland I, Hamilton ON, Schumacher H, Ingelheim

[132] Marc S. Weinberg, MD, Nicholas Kaperonis, MD, and George L. Bakris, MD. How High

[134] Anderson S, Rossing P, Juhl TR, Deinum J, Parving HH. Optimal dose of losartan for

[135] Andersen S, Frans A, Nieuwenhoven V, Tarnow L, Rossing P, Rossing K, Wieten L,

[136] Imig JD. ACE inhibition and bradykinin-mediated renal vascular responses.

Diabetic Nephropathy? Current Hypertension Reports 2003; 5:418–425. [133] Dahlof B, Devereux R, de Faire U. The Losartan Intervention For Endpoint reduction

diabetic TG(mRen-2)27 rats. Hypertension 2008; 52: 130–136.

effects by the (pro)renin receptor? Hypertens Res 2010; 33:4–10.

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Goldschmeding R, Parving HH. Reduction of urinary connective tissue growth factor by Losartan in type 1 patients with diabetic nephropathy. Kidney Int. 2005;

**5** 

Uh-Hyun Kim

*Republic of Korea* 

**Kidney ADP-Ribosyl Cyclase Inhibitors as a** 

ADP-ribosyl cyclases (ADPR-cyclases)/CD38 have emerged as effecter molecules for generating novel Ca2+ signaling messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (1, 2) (see Figure 1). Mounting evidence has indicated that G protein-coupled receptors, including the angiotensin II (Ang II) receptor, mediate activation of ADPR-cyclase to generate Ca2+ signaling messengers (3-5). We have studied Ang II receptor-mediated activation of ADPR-cyclase, resulting in Ca2+ dysfunction

Fig. 1. CD38/ADPR-cyclase-catalyzed reactions for the production of two Ca2+ mobilizing

second messengers, NAADP and cADPR.

**1. Introduction** 

**Therapeutic Tool for Diabetic Nephropathy** 

*Department of Biochemistry and the Institute of Cardiovascular Research,* 

*Chonbuk National University Medical School, Jeonju* 

## **Kidney ADP-Ribosyl Cyclase Inhibitors as a Therapeutic Tool for Diabetic Nephropathy**

#### Uh-Hyun Kim

*Department of Biochemistry and the Institute of Cardiovascular Research, Chonbuk National University Medical School, Jeonju Republic of Korea* 

#### **1. Introduction**

ADP-ribosyl cyclases (ADPR-cyclases)/CD38 have emerged as effecter molecules for generating novel Ca2+ signaling messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (1, 2) (see Figure 1). Mounting evidence has indicated that G protein-coupled receptors, including the angiotensin II (Ang II) receptor, mediate activation of ADPR-cyclase to generate Ca2+ signaling messengers (3-5). We have studied Ang II receptor-mediated activation of ADPR-cyclase, resulting in Ca2+ dysfunction

Fig. 1. CD38/ADPR-cyclase-catalyzed reactions for the production of two Ca2+ mobilizing second messengers, NAADP and cADPR.

Kidney ADP-Ribosyl Cyclase Inhibitors as a Therapeutic Tool for Diabetic Nephropathy 87

social behavior (28). CD38 acts mainly as an NAD glycohydrolase therewith regulating intracellular NAD levels (29, 30). CD38 was initially identified as a cell surface marker on thymocytes and T lymphocytes, showing discrete expression during lymphocyte differentiation (31). Further studies revealed that CD38 expression is ubiquitous in the immune system as well as in various organs, including prostate epithelial cells, pancreatic islet cells, and brain cells (32-35). From a study on new intracellular messengers in the sea mollusk *Aplysia*, a surprising finding of the striking similarity between human CD38 and the ADPRcyclase enzyme purified from *Aplysia* was made (36). ADPR-cyclase generates two important Ca2+-mobilizing second messengers, cADPR and NAADP, from NAD+ and NADP+, respectively (37-39). The second messenger, cADPR, increases intracellular Ca2+ concentration ([Ca2+]i) through the release of Ca2+ from intracellular endoplasmic reticulum (ER) stores via ryanodine receptors and/or Ca2+ influx through plasma membrane Ca2+ channels (5, 39,40, 41). The other second messenger, NAADP, increases intracellular Ca2+ concentration ([Ca2+]i) through the release of Ca2+ from a discrete intracellular store, called acidic organelles, via Twopore channels (TPCs) (42). Production of NAADP by ADPR-cyclases including CD38 is

stimulated by various G protein-coupled receptors (GPCRs), including, AT1R (43, 44).

(4) are different due to different ADPR-cyclases (see below).

**4. The role of ADPR-cyclase/CD38 in GPCR-mediated Ca2+ signaling** 

Evidence from our and other laboratories has indicated that various G protein-coupled receptors (GPCRs) mediate the activation of ADP-ribosyl cyclase (ADPR-cyclase) (3-6). ADPR-cyclase-involved GPCRs include the -adrenergic receptor, muscarinic receptor, interleukin 8 receptor (IL8R) and AT1R. The mechanism by which GPCR activates ADPRcyclase was discovered from the functional loop involving IL-8 and CD38 in lymphokineactivated killer (LAK) cells (5). Stimulation of IL8R results in protein kinase G-dependent phosphorylation of nonmuscle myosin heavy chain IIA (MHCIIA) and the association of

Mounting evidence has indicated that ADPR-cyclase(s) other than CD38 may exist in the kidney, brain, and the heart (40, 45), including various cells (30, 45-47). The first clues to the existence of novel ADPR-cyclase(s) emerged from experiments of the comparison of tissue cADPR levels in CD38 wild type and knockout mice (40). Levels of cADPR in spleen, bone marrow and lungs of CD38 knockout mice were significantly decreased, compared to those of CD38 wild type mice, whereas levels of cADPR in brain, heart and kidneys of CD38 knockout mice were comparable to those of CD38 wild type mice (40). These results suggest that ADPR-cyclase(s) other than CD38 may exist in the kidney, brain, and the heart. We recently demonstrated that Ang II-stimulated Ca2+ signals were not significantly different between CD38 wild type and CD38 knockout cardiomyocytes (48). However a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR) completely inhibited the Ang II-induced sustained Ca2+ increase. These findings indicate that cADPR is generated by a novel unidentified ADPR-cyclase other than CD38. In addition, a bisphenyl compound 4,4' dihydroxyazobenzene (4-DAB) has been shown to inhibit kidney ADPR-cyclase, but not CD38, with a high potency (47). The kidney ADPR-cyclase inhibitor inhibits kidney ADPRcyclase activity with a 10,000-fold more potency than it does with heart ADPR-cyclase activity. However, an analog of 4-DAB, 2,2'-dihydroxyazobenzene (2-DAB), inhibits kidney and heart ADPR-cyclase activity with similar effects (see below). These results suggest that ADPR-cyclases in the kidney and the heart are different. Therefore, the signaling pathways of Ang II-induced ADPR-cyclase activation in rat cardiomyocytes (48) and mesangial cells

which plays an important role in the pathogenesis of renal failure using an in vitro and an in vivo model (4, 6). In this review article, I would like to give an overview on the current worldwide status of diabetic nephropathy (DN) as a leading cause of end-stage renal disease (ESDR), the causative role of renin-angiotensin-aldosterone system (RAAS) for DN, the role of ADPR-cyclase in pathogenesis of DN and a potential therapeutic tool for DN by the intervention of Ang II receptor-mediated Ca2+ signaling with a kidney-specific ADPR-cyclase inhibitor.

#### **2. Diabetic nephropathy and the renin-angiotensin-aldosterone system**

Chronic kidney disease (CKD) is a major worldwide public-health problem affecting about 10% of the population (7). CKD has an increased annual incidence rate of about 5–8% (8). A leading cause of CKD is diabetic nephropathy (DN) throughout much of the world. This disease is characterized by the thickening of the glomerular basement membrane and mesangial matrix expansion (9). The early stage of DN is associated with glomerular hyperfiltration and glomerular hypertrophy, but not the collapse of the glomerular capillaries. DN results from an interaction between metabolic and hemodynamic factors. Glucose-dependent pathways are activated within the diabetic kidney, such as increasing oxidative stress, polyol formation, and advanced glycation end product accumulation (10).

In addition to elevated blood glucose, hypertension and inappropriate activation of the RAAS have been identified as contributing to the development and progression of diabetic renal disease (11). Clinical studies have demonstrated an important role for blood glucose control in reducing the development and progression of DN (12, 13) and they also have shown the importance of blood pressure reduction (14, 15) and the blockade of the RAAS (16–18) in slowing the progression of renal dysfunction in diabetes.

The pharmacological inhibition of the RAAS with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor antagonists (ARBs) are the first-line treatments for CKD patients. Despite several advantages of these agents, a number of side-effects do occur (19- 21). Moreover, the incidence of end-stage renal disease as a result of diabetes continues to rise in the world.

RAAS is a major regulatory system of cardiovascular and renal function. The final step of the RAAS cascade is the activation of Ang II receptors by Ang II. In the kidney, Ang II plays critical roles in the regulation of the glomerular filtration rate (GFR) and renal blood flow, and salt water retention (22-24). Effects of Ang II are mediated by at least two structurally and pharmacologically distinct Ang II type 1 and 2 receptors (AT1R and AT2R, respectively) (23, 24). The physiological and pathophysiological effects of Ang II are mainly exerted by AT1R activation (24-26) via complex interacting signaling pathways involving the primary stimulation of phospholipase C (PLC) and Ca2+ mobilization and the secondary activation of protein tyrosine kinase (PTK), extracellular signal-regulated kinases-1 and -2, and phosphatidylinositol 3-kinase (PI3K)-dependent kinase Akt (23-26). We extended these signaling pathways mainly focusing on the molecular basis of Ca2+ signaling by ADPR-cyclase activation in Ang II signaling in murine mesangial cells (MMCs) and other cells (see below).

#### **3. ADP-ribosyl cyclase (ADPR-cyclase)/CD38**

CD38, a type II transmembrane glycoprotein, represents a mammalian ADPR-cyclase and is involved in T cell activation (27) and oxytocin secretion, which is closely associated with

which plays an important role in the pathogenesis of renal failure using an in vitro and an in vivo model (4, 6). In this review article, I would like to give an overview on the current worldwide status of diabetic nephropathy (DN) as a leading cause of end-stage renal disease (ESDR), the causative role of renin-angiotensin-aldosterone system (RAAS) for DN, the role of ADPR-cyclase in pathogenesis of DN and a potential therapeutic tool for DN by the intervention of Ang II receptor-mediated Ca2+ signaling with a kidney-specific ADPR-cyclase inhibitor.

**2. Diabetic nephropathy and the renin-angiotensin-aldosterone system** 

(16–18) in slowing the progression of renal dysfunction in diabetes.

**3. ADP-ribosyl cyclase (ADPR-cyclase)/CD38** 

rise in the world.

Chronic kidney disease (CKD) is a major worldwide public-health problem affecting about 10% of the population (7). CKD has an increased annual incidence rate of about 5–8% (8). A leading cause of CKD is diabetic nephropathy (DN) throughout much of the world. This disease is characterized by the thickening of the glomerular basement membrane and mesangial matrix expansion (9). The early stage of DN is associated with glomerular hyperfiltration and glomerular hypertrophy, but not the collapse of the glomerular capillaries. DN results from an interaction between metabolic and hemodynamic factors. Glucose-dependent pathways are activated within the diabetic kidney, such as increasing oxidative stress, polyol formation, and advanced glycation end product accumulation (10). In addition to elevated blood glucose, hypertension and inappropriate activation of the RAAS have been identified as contributing to the development and progression of diabetic renal disease (11). Clinical studies have demonstrated an important role for blood glucose control in reducing the development and progression of DN (12, 13) and they also have shown the importance of blood pressure reduction (14, 15) and the blockade of the RAAS

The pharmacological inhibition of the RAAS with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor antagonists (ARBs) are the first-line treatments for CKD patients. Despite several advantages of these agents, a number of side-effects do occur (19- 21). Moreover, the incidence of end-stage renal disease as a result of diabetes continues to

RAAS is a major regulatory system of cardiovascular and renal function. The final step of the RAAS cascade is the activation of Ang II receptors by Ang II. In the kidney, Ang II plays critical roles in the regulation of the glomerular filtration rate (GFR) and renal blood flow, and salt water retention (22-24). Effects of Ang II are mediated by at least two structurally and pharmacologically distinct Ang II type 1 and 2 receptors (AT1R and AT2R, respectively) (23, 24). The physiological and pathophysiological effects of Ang II are mainly exerted by AT1R activation (24-26) via complex interacting signaling pathways involving the primary stimulation of phospholipase C (PLC) and Ca2+ mobilization and the secondary activation of protein tyrosine kinase (PTK), extracellular signal-regulated kinases-1 and -2, and phosphatidylinositol 3-kinase (PI3K)-dependent kinase Akt (23-26). We extended these signaling pathways mainly focusing on the molecular basis of Ca2+ signaling by ADPR-cyclase activation in Ang II signaling in murine mesangial cells (MMCs) and other cells (see below).

CD38, a type II transmembrane glycoprotein, represents a mammalian ADPR-cyclase and is involved in T cell activation (27) and oxytocin secretion, which is closely associated with social behavior (28). CD38 acts mainly as an NAD glycohydrolase therewith regulating intracellular NAD levels (29, 30). CD38 was initially identified as a cell surface marker on thymocytes and T lymphocytes, showing discrete expression during lymphocyte differentiation (31). Further studies revealed that CD38 expression is ubiquitous in the immune system as well as in various organs, including prostate epithelial cells, pancreatic islet cells, and brain cells (32-35). From a study on new intracellular messengers in the sea mollusk *Aplysia*, a surprising finding of the striking similarity between human CD38 and the ADPRcyclase enzyme purified from *Aplysia* was made (36). ADPR-cyclase generates two important Ca2+-mobilizing second messengers, cADPR and NAADP, from NAD+ and NADP+, respectively (37-39). The second messenger, cADPR, increases intracellular Ca2+ concentration ([Ca2+]i) through the release of Ca2+ from intracellular endoplasmic reticulum (ER) stores via ryanodine receptors and/or Ca2+ influx through plasma membrane Ca2+ channels (5, 39,40, 41). The other second messenger, NAADP, increases intracellular Ca2+ concentration ([Ca2+]i) through the release of Ca2+ from a discrete intracellular store, called acidic organelles, via Twopore channels (TPCs) (42). Production of NAADP by ADPR-cyclases including CD38 is stimulated by various G protein-coupled receptors (GPCRs), including, AT1R (43, 44).

Mounting evidence has indicated that ADPR-cyclase(s) other than CD38 may exist in the kidney, brain, and the heart (40, 45), including various cells (30, 45-47). The first clues to the existence of novel ADPR-cyclase(s) emerged from experiments of the comparison of tissue cADPR levels in CD38 wild type and knockout mice (40). Levels of cADPR in spleen, bone marrow and lungs of CD38 knockout mice were significantly decreased, compared to those of CD38 wild type mice, whereas levels of cADPR in brain, heart and kidneys of CD38 knockout mice were comparable to those of CD38 wild type mice (40). These results suggest that ADPR-cyclase(s) other than CD38 may exist in the kidney, brain, and the heart. We recently demonstrated that Ang II-stimulated Ca2+ signals were not significantly different between CD38 wild type and CD38 knockout cardiomyocytes (48). However a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR) completely inhibited the Ang II-induced sustained Ca2+ increase. These findings indicate that cADPR is generated by a novel unidentified ADPR-cyclase other than CD38. In addition, a bisphenyl compound 4,4' dihydroxyazobenzene (4-DAB) has been shown to inhibit kidney ADPR-cyclase, but not CD38, with a high potency (47). The kidney ADPR-cyclase inhibitor inhibits kidney ADPRcyclase activity with a 10,000-fold more potency than it does with heart ADPR-cyclase activity. However, an analog of 4-DAB, 2,2'-dihydroxyazobenzene (2-DAB), inhibits kidney and heart ADPR-cyclase activity with similar effects (see below). These results suggest that ADPR-cyclases in the kidney and the heart are different. Therefore, the signaling pathways of Ang II-induced ADPR-cyclase activation in rat cardiomyocytes (48) and mesangial cells (4) are different due to different ADPR-cyclases (see below).

#### **4. The role of ADPR-cyclase/CD38 in GPCR-mediated Ca2+ signaling**

Evidence from our and other laboratories has indicated that various G protein-coupled receptors (GPCRs) mediate the activation of ADP-ribosyl cyclase (ADPR-cyclase) (3-6). ADPR-cyclase-involved GPCRs include the -adrenergic receptor, muscarinic receptor, interleukin 8 receptor (IL8R) and AT1R. The mechanism by which GPCR activates ADPRcyclase was discovered from the functional loop involving IL-8 and CD38 in lymphokineactivated killer (LAK) cells (5). Stimulation of IL8R results in protein kinase G-dependent phosphorylation of nonmuscle myosin heavy chain IIA (MHCIIA) and the association of

Kidney ADP-Ribosyl Cyclase Inhibitors as a Therapeutic Tool for Diabetic Nephropathy 89

[3H]thymidine and [3H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in Ang II signaling for cell proliferation and protein synthesis. The Ang II-induced ADPR-cyclase activation has also been observed in rat cardiomyocytes (48) and MMCs (4), and hepatic stellate cells (50), although the signaling pathways in those

Fig. 3. Variation on the theme of angiotensin II-induced Ca2+ signaling. AT1R, angiotensin II

**5. The discovery of a small-molecule inhibitor for kidney ADPR-cyclase and** 

In order to get small-molecule inhibitors of kidney ADPR-cyclase, which make it possible to elucidate the involvement of ADPR-cyclase/cADPR in Ang II signaling in the kidney (4, 6), we screened a chemical library of approximately 10,000 compounds using a partially purified ADPR-cyclase from rat kidneys (47). This screen resulted in the selection of 4-DAB as a small molecule inhibitor (Figure 4). The compound was able to inhibit the generation of cGDPR and -ADPR from NGD+ and -NAD+, respectively, by the kidney ADPR-cyclase in a concentration-dependent manner. These data suggest that the compound may bind to the active site of the enzyme. Half maximal inhibition (IC50) of the enzyme activity was approximately 100 M. CD38 and ADPR-cyclases partially purified from rat brain, heart,

Although a number of GPCRs have been shown to utilize ADPR-cyclase in the regulation of [Ca2+]i, we chose the extracellular calcium ion ([Ca2+]o)-sensing receptor (CaSR) to test 4-DAB as a possible candidate inhibitor of ADPR-cyclase in MMCs. Stimulation of CaSR with [Ca2+]o resulted in a significant increase of [cADPR]i and a generation of long-lasting increase of [Ca2+]i, involving an initial peak rise followed by a sustained increase that was gradually

type 1 receptor; MMC, mouse mesangial cell; HSC, hepatic stellate cell.

**its application to diabetic nephropathy** 

and spleen tissues were insensitive to 4-DAB at 200 M.

cells are different from each other (see below, Figure 3).

phosphorylated MHCIIA with CD38 through Lck, which are essential for CD38 internalization for cADPR formation (49). Ensuing cADPR-mediated Ca2+ release from ER stores induces NAADP production by Rap1 activation via cAMP/Epac/PKA, resulting in the release of Ca2+ from lysosome-related acidic organelles (44). Although the result of IL8 mediated CD38 activation mechanism in LAK cells shows us one representative model, whether a similar mechanism by which other GPCRs use to activate ADPR-cyclase in other cells as that in IL8R-LAK cells remains to be clarified.

Initially we assumed that ADPR-cyclase plays a role in Ang II receptor-mediated Ca2+ signaling in the kidney. Therefore, we chose mouse mesangial cells (MMCs) as a model system to study Ang II signaling because MMCs are believed to be the center for the pathogenesis of CKD (4). Treatment of MMCs with Ang II induced an increase in intracellular Ca2+ concentrations through a transient Ca2+ release via an inositol 1,4,5 trisphosphate receptor (IP3R) and a sustained Ca2+ influx via L-type Ca2+ channels. The sustained Ca2+ signal, but not the transient Ca2+ signal, was blocked by 8-Br-cADPR, and an ADPR cyclase inhibitor, 4-DAB. In support of the results, 4-DAB inhibited Ang II-induced cADPR production. Application of pharmacological inhibitors revealed that the activation of ADPR-cyclase by Ang II involved AT1R, PI3K, PTK, and PLC-1 (Figure 2).

Fig. 2. Schematic model of ADPR-cyclase activation in Ang II signaling pathway (adopted from [4]). Stimulation of AT1R by Ang II leads to sequential activation of PI3K, PTK, and PLC1, in turn causing a Ca2+ release by IP3R from ER, resulting in activation of ADPRcyclase. Activation of ADPR-cyclase induces Ca2+ influx via L-type calcium channels, Akt phosphorylation, NFAT nuclear translocation, cell proliferation, and protein synthesis. 4- DAB abrogates the sustained Ca2+ signal, thereby blocking downstream events.

Moreover, 4-DAB as well as 8-Br-cADPR abrogated Ang II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell (NFAT), and the uptake of

phosphorylated MHCIIA with CD38 through Lck, which are essential for CD38 internalization for cADPR formation (49). Ensuing cADPR-mediated Ca2+ release from ER stores induces NAADP production by Rap1 activation via cAMP/Epac/PKA, resulting in the release of Ca2+ from lysosome-related acidic organelles (44). Although the result of IL8 mediated CD38 activation mechanism in LAK cells shows us one representative model, whether a similar mechanism by which other GPCRs use to activate ADPR-cyclase in other

Initially we assumed that ADPR-cyclase plays a role in Ang II receptor-mediated Ca2+ signaling in the kidney. Therefore, we chose mouse mesangial cells (MMCs) as a model system to study Ang II signaling because MMCs are believed to be the center for the pathogenesis of CKD (4). Treatment of MMCs with Ang II induced an increase in intracellular Ca2+ concentrations through a transient Ca2+ release via an inositol 1,4,5 trisphosphate receptor (IP3R) and a sustained Ca2+ influx via L-type Ca2+ channels. The sustained Ca2+ signal, but not the transient Ca2+ signal, was blocked by 8-Br-cADPR, and an ADPR cyclase inhibitor, 4-DAB. In support of the results, 4-DAB inhibited Ang II-induced cADPR production. Application of pharmacological inhibitors revealed that the activation of

Fig. 2. Schematic model of ADPR-cyclase activation in Ang II signaling pathway (adopted from [4]). Stimulation of AT1R by Ang II leads to sequential activation of PI3K, PTK, and PLC1, in turn causing a Ca2+ release by IP3R from ER, resulting in activation of ADPRcyclase. Activation of ADPR-cyclase induces Ca2+ influx via L-type calcium channels, Akt phosphorylation, NFAT nuclear translocation, cell proliferation, and protein synthesis. 4-

Moreover, 4-DAB as well as 8-Br-cADPR abrogated Ang II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell (NFAT), and the uptake of

DAB abrogates the sustained Ca2+ signal, thereby blocking downstream events.

ADPR-cyclase by Ang II involved AT1R, PI3K, PTK, and PLC-1 (Figure 2).

cells as that in IL8R-LAK cells remains to be clarified.

[3H]thymidine and [3H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in Ang II signaling for cell proliferation and protein synthesis. The Ang II-induced ADPR-cyclase activation has also been observed in rat cardiomyocytes (48) and MMCs (4), and hepatic stellate cells (50), although the signaling pathways in those cells are different from each other (see below, Figure 3).

Fig. 3. Variation on the theme of angiotensin II-induced Ca2+ signaling. AT1R, angiotensin II type 1 receptor; MMC, mouse mesangial cell; HSC, hepatic stellate cell.

#### **5. The discovery of a small-molecule inhibitor for kidney ADPR-cyclase and its application to diabetic nephropathy**

In order to get small-molecule inhibitors of kidney ADPR-cyclase, which make it possible to elucidate the involvement of ADPR-cyclase/cADPR in Ang II signaling in the kidney (4, 6), we screened a chemical library of approximately 10,000 compounds using a partially purified ADPR-cyclase from rat kidneys (47). This screen resulted in the selection of 4-DAB as a small molecule inhibitor (Figure 4). The compound was able to inhibit the generation of cGDPR and -ADPR from NGD+ and -NAD+, respectively, by the kidney ADPR-cyclase in a concentration-dependent manner. These data suggest that the compound may bind to the active site of the enzyme. Half maximal inhibition (IC50) of the enzyme activity was approximately 100 M. CD38 and ADPR-cyclases partially purified from rat brain, heart, and spleen tissues were insensitive to 4-DAB at 200 M.

Although a number of GPCRs have been shown to utilize ADPR-cyclase in the regulation of [Ca2+]i, we chose the extracellular calcium ion ([Ca2+]o)-sensing receptor (CaSR) to test 4-DAB as a possible candidate inhibitor of ADPR-cyclase in MMCs. Stimulation of CaSR with [Ca2+]o resulted in a significant increase of [cADPR]i and a generation of long-lasting increase of [Ca2+]i, involving an initial peak rise followed by a sustained increase that was gradually

Kidney ADP-Ribosyl Cyclase Inhibitors as a Therapeutic Tool for Diabetic Nephropathy 91

We utilized the specific inhibitor for kidney ADPR-cyclase to corroborate the evidence that there are ADPR-cyclases different from CD38. We utilized a human T cell-derived cell line, Jurkat T cell, which exclusively expresses CD38 that is regulated by CD3/TCR (51). Treatment of Jurkat T cells with OKT3, which is a ligand for CD3/TCR, showed a typical biphasic increase of [Ca2+]i, involving an initial peak rise followed by a sustained increase. Pre-treatment with 8-Br-cADPR inhibited only the sustained Ca2+ rise. In contrast, 4-DAB

Fig. 6. Light microscopic appearance of glomeruli. (adopted from [6]). A: Representative photomicrographs of the kidney sections stained with periodic acid-Schiff (PAS). Scale bars; 50 m. B: Quantification of glomerular size from A. Glomerular cross-sectional areas were determined by using a computer-assisted color image analyzer. MAG; mean area of glomeruli. C: Quantification of extracellular mesangial matrix expansion is expressed as PAS-positive mesangial material per total glomerular tuft cross-sectional area (mesangial area/total glomerular tuft area X 100). Values are means ± SE from 25 individual glomeruli in kidney sections from 6 mice in each group. \*P < 0.05 vs. control; #P < 0.05 vs. STZ.

did not show any effects on OKT3-mediated Ca2+ rise even at 10 M.

decreased. The sustained Ca2+ signal, but not the initial peak, was blocked by pre-treatment with 8-Br-cADPR. On the basis of these results that show the stimulation of CaSR activates ADPR-cyclase in MMC, we next evaluated 4-DAB as a possible candidate inhibitor of ADPRcyclase. This compound was able to inhibit [Ca2+]o-mediated later sustained elevation of [Ca2+]i but not the initial rise of [Ca2+]i in a dose-dependent manner. Further, [Ca2+]o -induced production of cADPR was also blocked by pre-treatment of 4-DAB in a concentrationdependent manner. IC50 was approximately 2.5 nM. In addition, since it has been reported that CaSR-mediated Ca2+ signals is involved in MMC proliferation, we examined whether 4-DAB inhibits the [Ca2+]o-induced MMC proliferation and demonstrated that the [Ca2+]o-induced increment of proliferation was also inhibited by 4-DAB in a similar range of concentrations observed in the inhibition of the sustained Ca2+ signal.

Fig. 4. Structure of 4,4'-dihydroazobenzene (4-DAB), left, and 2,2'-dihydroazobenzene (2-DAB), right.

Fig. 5. Effect of 4,4'-dihydroazobenzene (DHAB) on streptozotocin (STZ)-treated mice. (adopted from [6]). A: Plasma glucose level (PG), B: Ratio of kidney weight per body weight (KW/BW), C: Creatinine clearance (CCr) level, and D: Urinary albuminuria (UA) of 6 wk diabetic and control mice after DHAB treatment. Data are means ± SE. \*P < 0.05 vs. control, #P < 0.05 vs. STZ group.

decreased. The sustained Ca2+ signal, but not the initial peak, was blocked by pre-treatment with 8-Br-cADPR. On the basis of these results that show the stimulation of CaSR activates ADPR-cyclase in MMC, we next evaluated 4-DAB as a possible candidate inhibitor of ADPRcyclase. This compound was able to inhibit [Ca2+]o-mediated later sustained elevation of [Ca2+]i but not the initial rise of [Ca2+]i in a dose-dependent manner. Further, [Ca2+]o -induced production of cADPR was also blocked by pre-treatment of 4-DAB in a concentrationdependent manner. IC50 was approximately 2.5 nM. In addition, since it has been reported that CaSR-mediated Ca2+ signals is involved in MMC proliferation, we examined whether 4-DAB inhibits the [Ca2+]o-induced MMC proliferation and demonstrated that the [Ca2+]o-induced increment of proliferation was also inhibited by 4-DAB in a similar range of concentrations

Fig. 4. Structure of 4,4'-dihydroazobenzene (4-DAB), left, and 2,2'-dihydroazobenzene

Fig. 5. Effect of 4,4'-dihydroazobenzene (DHAB) on streptozotocin (STZ)-treated mice. (adopted from [6]). A: Plasma glucose level (PG), B: Ratio of kidney weight per body weight (KW/BW), C: Creatinine clearance (CCr) level, and D: Urinary albuminuria (UA) of 6 wk diabetic and control mice after DHAB treatment. Data are means ± SE. \*P < 0.05 vs. control,

observed in the inhibition of the sustained Ca2+ signal.

(2-DAB), right.

#P < 0.05 vs. STZ group.

We utilized the specific inhibitor for kidney ADPR-cyclase to corroborate the evidence that there are ADPR-cyclases different from CD38. We utilized a human T cell-derived cell line, Jurkat T cell, which exclusively expresses CD38 that is regulated by CD3/TCR (51). Treatment of Jurkat T cells with OKT3, which is a ligand for CD3/TCR, showed a typical biphasic increase of [Ca2+]i, involving an initial peak rise followed by a sustained increase. Pre-treatment with 8-Br-cADPR inhibited only the sustained Ca2+ rise. In contrast, 4-DAB did not show any effects on OKT3-mediated Ca2+ rise even at 10 M.

Fig. 6. Light microscopic appearance of glomeruli. (adopted from [6]). A: Representative photomicrographs of the kidney sections stained with periodic acid-Schiff (PAS). Scale bars; 50 m. B: Quantification of glomerular size from A. Glomerular cross-sectional areas were determined by using a computer-assisted color image analyzer. MAG; mean area of glomeruli. C: Quantification of extracellular mesangial matrix expansion is expressed as PAS-positive mesangial material per total glomerular tuft cross-sectional area (mesangial area/total glomerular tuft area X 100). Values are means ± SE from 25 individual glomeruli in kidney sections from 6 mice in each group. \*P < 0.05 vs. control; #P < 0.05 vs. STZ.

Kidney ADP-Ribosyl Cyclase Inhibitors as a Therapeutic Tool for Diabetic Nephropathy 93

[1] Malavasi, F., Deaglio, S., Funaro, A., Ferrero, E., Horenstein, A. L., Ortolan, E., Vaisitti,

[3] Higashida H, Zhang JS, Hashi M, Shintaku M, Higashida C, and Takeda Y. (2000)

[4] Kim SY, Gul R, Rah SY, Kim SH, Park SK, Im MJ, Kwon HJ, and Kim UH. (2008)

[6] Kim SY, Park KH, Gul R, Jang KY, and Kim UH. (2009) Role of kidney ADP-ribosyl cyclase in diabetic nephropathy. Am J Physiol Renal Physiol 296: F291–F297

[8] El-Nahas M (2005) The global challenge of chronic kidney disease. Kidney Int 68:2918–

[9] Vestra MD, Saller A, Mauer M, and Fioretto P. (2001) Role of mesangial expansion in the

[10] Cooper ME. Interaction of metabolic and haemodynamic factors in mediating experimental diabetic nephropathy. (2001) Diabetologia 44: 1957–1972 [11] Coresh J, Astor BC, Greene T, Eknoyan G, and Levey AS (2003) Prevalence of chronic

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Based on our earlier observation that 4-DAB was a potent inhibitor of kidney ADPR-cyclase and could protect Ang II-mediated mesangial cell growth (4, 47), we further investigated the effects of 4-DAB on a mouse model of DN (6). Male mice were randomly assigned to normal control and diabetic groups of comparable age. The diabetic group received 45 g/kg of 4- DAB for 6 wk via daily intraperitoneal injections. Alterations of mesangial cell proliferation and extracellular matrix (ECM) production are believed to play predominant roles in the pathogenesis of progressive glomerulosclerosis which leads to ESRD (52, 53). In the process of tissue development and wound healing, TGF-1 plays a crucial role in controlling ECM deposition and remodeling: TGF-1 stimulates the synthesis of major components of ECM proteins, such as collagen and fibronectin (54-56). In diabetic kidneys, the overexpression of TGF-1 is believed to be the major mediator responsible for early pathological changes of DN, including glomerular basement membrane thickening and mesangial matrix expansion (52, 55).

4-DAB treatment significantly ameliorated albuminuria and downregulated the expression of fibrogenic factor TGF-1, subsequently reducing mesangial matrix protein production in diabetic mice kidney, without, however, changing serum glucose levels (Figures 5 and 6, Ref. 6). ADPR-cyclase was significantly activated, and cADPR levels were also increased in diabetic kidneys, which were prevented by 4-DAB treatment. On the other hand, plasma and kidney Ang II levels were elevated in both the diabetic and 4-DAB -treated diabetic mice group. This result suggests that 4-DAB affects only ADPR-cyclase activation, but not plasma and kidney Ang II levels in the diabetic experimental model. Furthermore, 4-DAB inhibited the phosphorylation of Akt and the NFAT3 nuclear translocation in the kidneys of the diabetic group. These findings indicate a crucial role of ADPR-cyclase signaling in the renal pathogenesis of diabetes and provide a therapeutic tool for the treatment of renal diseases.

#### **6. Perspectives**

A potent small-molecule inhibitor 4-DAB, that inhibits specifically the kidney ADPRcyclase, has been discovered. The discovery of the specific inhibitor for the enzyme enables us to provide further evidence that there are ADPR-cyclases different from CD38. Benefits of the kidney ADPR-cyclase specific inhibitor are several folds: the use of 4-DAB may facilitate in the understanding of kidney functions involving the regulation of Ca2+ homeostasis; the inhibitor may help to understand the pathogenesis of the kidney; this compound can be the basis for the development of tissue specific inhibitors of ADPR-cyclases; and finally, the compound may be applied for therapeutic purposes for the prevention and management of human CKD. Furthermore, a similar strategy can be applied for the development of tissue specific inhibitors of ADPR-cyclases with the intent to intervene in other diseases, such as hypertension. For instance, the identification of an inhibitor for ADPR-cyclase of arterial smooth muscle cells can be a potential anti-hypertensive drug.

#### **7. Acknowledgments**

This work was supported by the Korea Science and Engineering Foundation (National Research Laboratory Grant R0A-2007-000-20121-0). The author thanks Dr. Gabor Raffai and John Kang for critically reading the manuscript.

#### **8. References**

92 Diabetic Nephropathy

Based on our earlier observation that 4-DAB was a potent inhibitor of kidney ADPR-cyclase and could protect Ang II-mediated mesangial cell growth (4, 47), we further investigated the effects of 4-DAB on a mouse model of DN (6). Male mice were randomly assigned to normal control and diabetic groups of comparable age. The diabetic group received 45 g/kg of 4- DAB for 6 wk via daily intraperitoneal injections. Alterations of mesangial cell proliferation and extracellular matrix (ECM) production are believed to play predominant roles in the pathogenesis of progressive glomerulosclerosis which leads to ESRD (52, 53). In the process of tissue development and wound healing, TGF-1 plays a crucial role in controlling ECM deposition and remodeling: TGF-1 stimulates the synthesis of major components of ECM proteins, such as collagen and fibronectin (54-56). In diabetic kidneys, the overexpression of TGF-1 is believed to be the major mediator responsible for early pathological changes of DN, including glomerular basement membrane

4-DAB treatment significantly ameliorated albuminuria and downregulated the expression of fibrogenic factor TGF-1, subsequently reducing mesangial matrix protein production in diabetic mice kidney, without, however, changing serum glucose levels (Figures 5 and 6, Ref. 6). ADPR-cyclase was significantly activated, and cADPR levels were also increased in diabetic kidneys, which were prevented by 4-DAB treatment. On the other hand, plasma and kidney Ang II levels were elevated in both the diabetic and 4-DAB -treated diabetic mice group. This result suggests that 4-DAB affects only ADPR-cyclase activation, but not plasma and kidney Ang II levels in the diabetic experimental model. Furthermore, 4-DAB inhibited the phosphorylation of Akt and the NFAT3 nuclear translocation in the kidneys of the diabetic group. These findings indicate a crucial role of ADPR-cyclase signaling in the renal pathogenesis of diabetes and provide a therapeutic

A potent small-molecule inhibitor 4-DAB, that inhibits specifically the kidney ADPRcyclase, has been discovered. The discovery of the specific inhibitor for the enzyme enables us to provide further evidence that there are ADPR-cyclases different from CD38. Benefits of the kidney ADPR-cyclase specific inhibitor are several folds: the use of 4-DAB may facilitate in the understanding of kidney functions involving the regulation of Ca2+ homeostasis; the inhibitor may help to understand the pathogenesis of the kidney; this compound can be the basis for the development of tissue specific inhibitors of ADPR-cyclases; and finally, the compound may be applied for therapeutic purposes for the prevention and management of human CKD. Furthermore, a similar strategy can be applied for the development of tissue specific inhibitors of ADPR-cyclases with the intent to intervene in other diseases, such as hypertension. For instance, the identification of an inhibitor for ADPR-cyclase of arterial

This work was supported by the Korea Science and Engineering Foundation (National Research Laboratory Grant R0A-2007-000-20121-0). The author thanks Dr. Gabor Raffai and

thickening and mesangial matrix expansion (52, 55).

tool for the treatment of renal diseases.

smooth muscle cells can be a potential anti-hypertensive drug.

John Kang for critically reading the manuscript.

**6. Perspectives** 

**7. Acknowledgments** 


Kidney ADP-Ribosyl Cyclase Inhibitors as a Therapeutic Tool for Diabetic Nephropathy 95

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**6** 

*1Japan 2Egypt* 

**Significance of Advanced Glycation** 

**AGE (RAGE) in Diabetic Nephropathy** 

*Kanazawa University Graduate School of Medical Science, Kanazawa* 

*1Department of Biochemistry and Molecular Vascular Biology* 

*2On leave from Kafr EL-Sheikh University* 

**End-Products (AGE) and the Receptor for** 

Tarek Kamal1,2, Yasuhiko Yamamoto1,\* and Hiroshi Yamamoto1

Diabetic nephropathy is a life-threatening complication of *diabetes mellitus* and the leading cause of end-stage renal disease (ESRD) in developed countries. Diabetes is responsible for over 40% of all new cases with ESRD in the United States and Japan, eventually undergoing renal dialysis or transplantation. Diabetic nephropathy is characterized by glomerular hyperfiltration and thickening of glomerular basement membranes, followed by expansion of extracellular matrix in mesangial area. There are many factors and pathways that are involved in the pathogenesis of diabetic nephropathy. In this chapter, we will focus on advanced glycation end-products (AGE) and the receptor for AGE (RAGE) in the

Diabetic nephropathy occurs in 20-40% of patients with diabetes and accounts for disabilities and the high mortality rate in patients with diabetes (1). In proportion to the rapid increase of diabetic population, diabetic nephropathy is now the major cause of ESRD in developed countries. There are many factors influencing the development of diabetic nephropathy, this including genetic, hemodynamic, environmental, and metabolic factors. The epidemiological studies have revealed that hyperglycemia *per se* is the most important factor in the onset and progression of diabetic vascular complications (2). Potential mechanisms underlying diabetic nephropathy include activations of polyol and hexosamine pathways, oxidative and nitrosative stress, ER stress, protein kinase C activation, poly(ADPribose) polymerase activation, and inflammation (3). Extensive intracellular and extracellular formation of AGE can also become a pathogenic factor in sustained hyperglycemia-induced kidney injuries. Both receptor-dependent and -independent

**2. Possible molecular mechanisms for the development of diabetic** 

mechanisms are involved in AGE-induced cellular dysfunction and tissue damage.

**1. Introduction** 

**nephropathy** 

 \*

Corresponding Author

development and prevention of diabetic nephropathy.

