**2. The concept of endogenous cardiotonic steroids (CTS) as natriuretic hormones**

CTS (also known as endogenous digitalis-like substances) are specific ligands and inhibitors of the Na/K-ATPase, which include plant-derived glycosides such as digoxin and ouabain and vertebrate-derived aglycones such as bufalin and marinobufagenin (MBG). Although the production and secretion of endogenous CTS are not completely understood, both ouabain and MBG have been identified as endogenous steroid hormones whose production and secretion can be regulated by multiple stimuli including angiotensin II and adrenocorticotropic hormone (ACTH) [30, 43–48]. Endogenous CTS are present in measurable amounts under

#### *The Na/K-ATPase Signaling Regulates Natriuresis in Renal Proximal Tubule DOI: http://dx.doi.org/10.5772/intechopen.92968*

normal physiological conditions and are markedly increased under a number of pathological conditions such as sodium imbalance, chronic renal failure, hyperaldosteronism, hypertension, congestive heart failure, acute plasma volume expansion, and preeclampsia [46, 49–59].

Even though digitalis-like drugs have been used to treat heart failure patients for over 200 years, studies have also revealed many extra-cardiac actions of these compounds, such as in response to salt loading in both animal models and human hypertensive patients [29, 57, 60–62]. In addition, low doses of CTS not only induced hypertension in rats but also caused a significant cardiovascular remodeling independent of their effect on blood pressure (BP) [63–66].

Bricker was the first to propose the existence of "the third factor" (named after the glomerular filtration rate as the first factor and the aldosterone as the second factor), and Dahl proposed the existence of a hormonal natriuretic factor that might cause a sustained increase in BP in salt-sensitive hypertensive rats [67, 68]. Subsequently, Bricker, de Wardener, and others proposed that this hormonal natriuretic factor inhibits the Na/K-ATPase, and Blaustein described how an increase in endogenous Na/K-ATPase inhibitors might cause a vascular contractility change and then a rise in BP [67, 69–72]. In 1980, de Wardener and MacGregor summarized the state of research at the time and proposed an insightful scheme explaining how the Na/K-ATPase inhibitor works as a natriuretic hormone [73]. In essence, it was contended that the Na/K-ATPase inhibitor (endogenous CTS) will rise in response to either a defect in renal Na<sup>+</sup> excretion or high salt intake. This increase, while returning Na+ balance toward normal by increasing renal Na<sup>+</sup> excretion, also causes hypertension through acting on the vascular Na/K-ATPase. With the advances in the field over the decades, much has been learned. The first unequivocal demonstration of ouabain-like substance in the human plasma was reported decades ago [46]. Blaustein and Hamlyn's laboratory has demonstrated how increases in endogenous CTS change vascular contractility and its effect on BP [74]. However, the pathophysiological significance of endogenous CTS (e.g., as a natriuretic hormone) has been a subject of debate since it was first proposed until Lingrel's laboratory reported their gene replacement in vivo studies, which unequivocally demonstrated that endogenous CTS play an important role in the regulation of renal Na+ excretion and BP through the Na/K-ATPase [75–77]. Specifically, Lingrel's group generated several lines of mice in which the mouse endogenous ouabain-insensitive α1 subunit is replaced by a mutant that alters the ouabain sensitivity of the Na/K-ATPase. For example, they generated a line of "humanized" α1 S/S mice where the endogenous ouabain-insensitive α1 is replaced by an ouabain-sensitive (human like) α1-mutant and used these mice to explore the role of endogenous CTS in the regulation of renal function and BP. Should endogenous CTS be important for these regulations, an increased CTS sensitivity in α1 S/S mice would make these mice more sensitive to conditions that raise circulating CTS. Indeed, when ACTH was administered to raise endogenous CTS, it caused much severe hypertension in α1 S/S mice than their control littermates. Moreover, expression of the ouabain-sensitive α1-mutant significantly increased renal Na+ excretion, confirming the natriuretic function of endogenous CTS as proposed by the pioneers of the field [67, 68, 70–73]. More evidences indicate that increases in endogenous CTS regulate both renal Na+ excretion and BP through the Na/K-ATPase [74–76, 78, 79].

### **3. The Na/K-ATPase signaling by specific ligands and ROS in RPTs**

Ouabain-stimulated protein-protein interaction and subsequent Na/K-ATPase signaling function were first demonstrated in rat neonatal myocytes, which were

further confirmed and developed in porcine LLC-PK1 cells (an immobilized RPT cell line) and other cell types. CTS-stimulated Na/K-ATPase signaling has been reviewed everywhere [22, 31, 32, 47, 80–83].

In LLC-PK1 cells, ouabain-stimulated Na/K-ATPase signaling increases ROS generation. Other than ouabain, exogenous H2O2 and glucose oxidase-induced H2O2 also activate Na/K-ATPase signaling pathways including phosphorylation of c-Src and ERK1/2, as well as protein carbonylation modification of Na/K-ATPase (direct carbonylation of two amino acid residues, Pro222 and Thr224, in the actuator domain of the α1 subunit) [84–87]. Pretreatment with antioxidant *N*-acetyl-l-cysteine (NAC) or disruption of the Na/K-ATPase/c-Src signaling complex attenuated ouabain- and glucose oxidase-stimulated Na/K-ATPase/c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase, and inhibition of active transepithelial 22Na+ transport. A basal level of ROS is critical in initiating ouabain-stimulated Na/K-ATPase/c-Src signaling, and carbonylation modification of the α1 subunit is involved in a feed-forward mechanism of the regulation of ouabain-mediated Na/K-ATPase signal function and subsequent Na+ transport. Furthermore, a stable overexpression of rat α1-mutant Pro224/Ala (Pro224 of rat α1 is the same as the Pro222 of pig α1) prevented ouabain-stimulated signal function of Na/K-ATPase, protein carbonylation, Na/K-ATPase endocytosis, and ouabain-induced inhibition of active transepithelial 22Na+ transport [79, 86, 87]. Taken together, in LLC-PK1 cells, there is a positive-feedback amplification loop of Na/K-ATPase signaling and ROS generation, in which carbonylation of the Pro222 of the α1 subunit is critical. In this working model, both Na/K-ATPase-specific ligands (such as ouabain) and ROS increases (induced by other stimuli like exogenous added glucose oxidase) could activate the Na/K-ATPase signaling, and the Na/K-ATPase/c-Src complex can function as a "receptor" of ROS signaling. This Na/K-ATPase signaling-ROS axis may explain the role of Na/K-ATPase signaling in the development of different pathophysiological conditions, including RPT sodium handling.

#### **4. Endocytosis of Na/K-ATPase**

Endocytosis is involved in many important cellular functions. Ouabaininduced endocytosis of the Na/K-ATPase was first observed by the laboratories of Cook and Lamb, which demonstrated that [3 H]-ouabain (bound to the Na/K-ATPase) was translocated from the plasmalemmal membrane surface to intracellular compartments (lysosomes) in HeLa cells, chick embryo heart cells, and Girardi heart cells [88–92].

#### **4.1 Dopamine and PTH**

One of the best-studied paradigms of hormonal natriuresis is the renal dopamine system [93–96]. Renal dopamine release increases in response to high salt intake or volume expansion. The activation of D1-like dopamine receptors stimulates PLC-γ and cAMP-PKA pathways and increases intracellular Ca2+. These pathways work in concert and produce the coordinated downregulation of NHE3 and the Na/K-ATPase and consequently natriuresis [93–95, 97, 98]. While Aperia's laboratory first revealed the pathways involved in dopamine-induced regulation of Na/K-ATPase activity [99–101] that is related to endocytosis of the Na/K-ATPase [102], Moe and others have mapped the pathways of NHE3 phosphorylation and trafficking [103–105]. In RPT, dopamine alters sodium handling by inducing Na/K-ATPase and NHE3 endocytosis. In RPT primary culture of Sprague-Dawley

*The Na/K-ATPase Signaling Regulates Natriuresis in Renal Proximal Tubule DOI: http://dx.doi.org/10.5772/intechopen.92968*

rats, dopamine-induced clathrin-dependent endocytosis of the rat Na/K-ATPase α1 subunit is triggered by activation of PI3K and subsequently phosphorylation of Ser-18 of rat α1 subunit [24, 106–109]. The activation of PI3K also stimulated phosphorylation of the Tyr537 of the α1 subunit that facilitates its binding with adaptor protein-2 (AP-2), providing the inclusion of the Na/K-ATPase into clathrin-coated pits (CCP) [24, 108]. However, Ser-18 is found only in rat α1 subunit and is not present in pig and dog α1 subunits [110]. Depending on the type of renal tubular epithelium, dopamine-induced endocytosis of the Na/K-ATPase may be mediated through PKC- or PKA-dependent mechanisms [108, 111–113]. Parathyroid hormone (PTH)-induced inhibition and endocytosis of the Na/K-ATPase were also demonstrated in opossum kidney (OK) cells, which is clathrin-mediated and requires ERK-dependent phosphorylation of Ser-11 of the α1 subunit [114].

#### **4.2 Ouabain-induced endocytosis of Na/K-ATPase through Na/K-ATPase signaling**

In LLC-PK1 cells, at the doses used, ouabain has no discernable effects on cell morphology, viability, transepithelial electrical resistance, tight junction integrity, and intracellular [Na<sup>+</sup> ] [115]. However, ouabain causes decreases in membrane-bound Na/K-ATPase without significantly affecting intracellular [Na<sup>+</sup> ] [116, 117]. As a specific ligand, nontoxic ouabain (~1/10th–1/20th of acute IC50) caused a dose- and time-dependent decrease in Na/K-ATPase ion-pumping activity (ouabain-sensitive 86Rb uptake), which is attributed to ouabain-stimulated clathrin-dependent endocytosis of the α1/β1-subunits, demonstrated by a decrease in cell surface biotinylated α1 subunit and a concomitant accumulation of α1/ β1-subunit and c-Src in early endosome (EE)/late endosome (LE) fractions. This leads to a net decrease in abundance of Na/K-ATPase in the plasma membrane and total ion-pumping activity of Na/K-ATPase and transcellular 22Na+ transport. This phenomenon was only observed when ouabain was applied to the basolateral, but not apical, aspect of Costar Transwell with membrane support for 12 hours, which indicates that this ouabain-induced endocytosis of the Na/K-ATPase is initiated by activating the receptor Na/K-ATPase/Src complex involving phosphorylation of c-Src and PI3K. The endocytosed [3 H]-ouabain/Na/K-ATPase/c-Src/EGFR complex can be detected in both EE and LE fractions.

To understand the molecular mechanism(s) involved in this process, studies were performed with LLC-PK1 as well as SYF and SYF + c-Src cells. SYF cells are triple Src kinase (c-Src, Yes, Fyn)-null mouse fibroblast cells, and SYF + c-Src are c-Src-rescued SYF cells. This pair of cells was used to determine the role of c-Src activation in ouabain-induced Na/K-ATPase signaling and endocytosis. While ouabain accumulates Na/K-ATPase α1 subunit content in clathrin-coated pits and EE/LE fractions, it also causes a translocation of the α1 subunit to nuclear fraction. Interestingly, the effects of ouabain are fully reversible in terms of ion-pumping activity, transepithelial 22Na+ flux, and cell surface Na/K-ATPase within 24 hours following the removal of ouabain with a fresh culture medium, suggesting a reversible process. Immunofluorescence showed that the Na/K-ATPase α1 subunit co-localized with clathrin both before and after ouabain treatment, and immunoprecipitation experiments indicated that ouabain stimulated interactions among the α1 subunit, AP-2, and clathrin heavy chain (CHC). Disruption and/or arresting of clathrin-coated pit formation (by potassium depletion with hypotonic shock [118] and chlorpromazine treatment [119]) significantly attenuated this ouabaininduced endocytosis, suggesting the involvement of a clathrin-coated pit. Inhibition of the ouabain-activated signaling with PP2 (a specific c-Src kinase inhibitor)

or wortmannin (a specific PI3K inhibitor) also significantly attenuated ouabaininduced endocytosis. Experiments performed in SYF cells and SYF + c-Src demonstrated that ouabain induces the endocytosis of the Na/K-ATPase in SYF + c-Src cells, but not in the SYF, indicating that ouabain-induced endocytosis of the Na/K-ATPase is c-Src-dependent.

Ouabain-stimulated Na/K-ATPase signaling also requires caveolin-1 (Cav-1) (a structural protein of caveolae, a subset of membrane lipid rafts) that functions as an anchoring protein for attracting the Na/K-ATPase α1 subunit into caveolae [120]. Accordingly, depletion of cholesterol (by methyl-β-cyclodextrin (Mβ-CD)) or caveolin-1 (by siRNA) blocked ouabain-induced endocytosis of the Na/K-ATPase, compartmentalization of signaling molecules in clathrin-coated pits, and early endosome. In addition, depletion of caveolin-1 also significantly reduced the protein-protein interactions among α1 subunit, AP-2, PI3K, and clathrin heavy chain, suggesting that caveolin-1 is involved in both ouabain-induced endocytosis of Na/K-ATPase and signal transduction [117].

These data demonstrate that ouabain stimulates a clathrin- and caveolin-1-dependent endocytosis of the Na/K-ATPase, a phenomenon requiring ouabaininduced Na/K-ATPase signaling function. Taken together, it is most likely that clathrin- and/or caveola-/lipid raft-mediated endocytosis of the Na/K-ATPase is a common phenomenon, but the mechanism and the relationship between the endocytosis of the Na/K-ATPase and signal transduction are still not fully understood. This is the first time to demonstrate that ligand-modulated endocytosis of the Na/K-ATPase is a mechanism by which RPT sodium transport is altered in a physiologically meaningful manner (**Figure 1**).

#### **Figure 1.**

*Illustration of activation of the Na/K-ATPase signaling-mediated endocytosis of the Na/K-ATPase. Both CTS and ROS can activate Na/K-ATPase signaling, which leads to translocation of cell surface Na/K-ATPase (α1- and β1-subunits), along with EGFR, c-Src, and ERK1/2, into clathrin-coated pits and early and late endosomes. This process is independent of change in intracellular Na<sup>+</sup> and Ca2+, but is dependent on activation of c-Src and PI3K, and the presence of caveolin-1. The activation of the Na/K-ATPase signaling also stimulates ROS generation which further activates the signaling. In LLC-PK1 cells, ouabain has no significant effect on recycling of endocytosed α1 subunit. AP-2, adaptor protein-2; Cav-1, caveolin-1; CCP, clathrin-coated pits; CHC, clathrin heavy chain; CTS, cardiotonic steroids; EE, early endosome; LE, late endosome; Na+ /X, Na+ dependent antitransporter; Na<sup>+</sup> /Y, Na+ -dependent cotransporter; NKA, Na/K-ATPase; TJ, tight junction.*
