**5.1 NHE3 regulation**

In RPT, NHE3 resides in the apical membrane of S1 and S2 segments, mediating transcellular reabsorption of Na+ and HCO3 <sup>−</sup> and fluid reabsorption [36, 37]. Moreover, vesicular NHE3 activity regulates endosomal pH and consequently affects receptor-mediated endocytosis as well as endocytic vesicle fusion [41, 42]. Consistent with its cellular function, upregulation of NHE3 activity and expression is associated with the development of hypertension [121–124]. Conversely, the reduction of NHE3 surface expression or NHE3 activity occurs during pressure natriuresis in rats [125–128]. As expected, NHE3-deficient mice are hypotensive [129–131] because of reduced Na+ reabsorption and increased Na+ excretion. Interestingly, NHE3-deficient mice also develop acidosis since the blunted H+ secretion through NHE3, which links to greatly reduced RPT HCO3 <sup>−</sup> reabsorption (please see Introduction for the linkage of NHE3 H<sup>+</sup> secretion and HCO3 <sup>−</sup> reabsorption), could not be compensated by H+ -ATPase and AE1 (anion exchanger-1, SLC4A1) Cl<sup>−</sup>/HCO3 <sup>−</sup> exchanger, compared with wild-type mice [131, 132]. These observations put renal Na+ reabsorption through NHE3 in a central position in the development and control of salt loading- and volume expansion-mediated hypertension. Structurally, NHE3 has a predicted N-terminal hydrophobic ion-translocating domain and a variable C-terminal hydrophilic domain which contains regulatory sequences [133].

The NHE3 activity is regulated at various levels through different mechanisms, mainly via phosphorylation, trafficking, and transcriptional regulation [34, 35, 103]. The surface expression of NHE3 is mainly regulated by changes in endocytosis/exocytosis and is the primary regulatory mechanism of NHE3 activity. NHE3 has been found to traffic between the plasma membrane and EE/LE fractions via a clathrinand PI3K-dependent pathway [41, 134–141]. The NHE3 activity can be stimulated by exocytosis [141–143] or inhibited by endocytosis [105, 125, 144]. The activation of c-Src, PKA, and PKC and increase in intracellular Ca2+ are involved in the regulation of NHE3 trafficking.

NHE3 has been shown to be redistributed under a hypertensive state, accompanying reversible downregulation of the Na/K-ATPase activity in the renal cortex [125, 127, 145]. This raised the possibility that the basolateral-localized Na/K-ATPase and apically localized NHE3 work in concert to regulate renal sodium handling in response to the Na/K-ATPase signaling. The coordinated regulation of NHE3 and the Na/K-ATPase is critical in maintaining intracellular Na<sup>+</sup> homeostasis and extracellular fluid volume. It is believed that the apical Na+ entry through NHE3 is the rate-limiting step because the functional reserve of the Na/K-ATPase in the nephron is more than sufficient even under some pathological conditions.

#### **5.2 Chronic NHE3 regulation by Na/K-ATPase signaling**

In LLC-PK1 cells, chronic, low-concentration ouabain (50 and 100 nM, 24 hours) treatment in the basolateral aspect, but not in apical aspect, did not change intracellular [Na+ ] but decreased apical NHE3-mediated Na<sup>+</sup> absorption, NHE3 promoter activity, and NHE3 protein and mRNA abundance. Pretreatment with specific inhibitors against c-Src and PI3K attenuates ouabain-induced downregulation of NHE3 activity and NHE3 mRNA [146]. In caveolin-1 knockdown LLC-PK1 cells, ouabain failed to reduce NHE3 mRNA and NHE3 promoter activity, in which ouabain-induced Na/K-ATPase signaling reduced Sp1 and TR DNA

binding activity and consequently decreased NHE3 expression and activity [146]. These effects are abolished by inhibition of either c-Src or PI3K. Promoter mapping identified that ouabain-response elements reside in a region between −450 and −1194 nt and that ouabain reduces the binding of transcriptional factor *Sp1* to its cognate *cis*-element.

#### **5.3 Acute NHE3 regulation by Na/K-ATPase signaling**

Acute application of ouabain (1 hour) in the basolateral, but not apical, aspect significantly reduced NHE3 activity (22Na+ uptake) and active transepithelial 22Na+ transport. This is accompanied by a reduced NHE3 content on cell surface and an increased NHE3 content in EE/LE fractions, as seen in the case of the Na/K-ATPase α1 subunit. These changes are independent of change in the integrity of tight junctions and the intracellular Na<sup>+</sup> concentration [115]. Ouabain-induced NHE3 trafficking was abolished by either PI3K or c-Src inhibition. Disruption of caveolae/ lipid rafts by cholesterol depletion prevented ouabain-induced accumulation of NHE3 and Na/K-ATPase α1 in early endosomes, and cholesterol repletion restored the ouabain-induced endosomal accumulation of NHE3 and Na/K-ATPase α1. Moreover, pretreatment of cells with the intracellular Ca2+ chelator BAPTA-AM attenuated ouabain-induced NHE3 trafficking, suggesting Ca2+ might link the Na/K-ATPase signaling to NHE3 regulation which is in agreement with observations that intracellular Ca2+ can regulate NHE3 activity and trafficking [147, 148]. These changes indicate that ouabain acutely stimulates NHE3 trafficking, like Na/K-ATPase, by activating the basolateral Na/K-ATPase signaling complex [115]. In RPT cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1), results further indicate that ouabain-induced inhibition of transcellular 22Na+ transport

#### **Figure 2.**

*Illustration of activation of the Na/K-ATPase signaling-mediated endocytosis of NHE3. Activation of the Na/K-ATPase signaling leads to intracellular Na+ -independent NHE3 endocytosis. However, like Na/K-ATPase signaling-mediated Na/K-ATPase endocytosis, the NHE3 endocytosis is dependent on intracellular Ca2+, activation of c-Src and PI3K, and caveolin-1. In LLC-PK1 cells, ouabain inhibits the endocytic recycling of endocytosed NHE3. Since the Na/K-ATPase and NHE3 reside on basolateral and apical membrane in monolayer, respectively, it is still unclear how the basolateral Na/K-ATPase signaling is transmitted to NHE3 regulation. There are several possible pathways as illustrated, as proposed in the text (please see Figure 1 for abbreviations).*

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

as well as trafficking of the α1 subunit and NHE3 is not a species-specific phenomenon. Furthermore, in LLC-PK1 cells, ouabain inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit [149].

Taken together, by activating the basolateral receptor Na/K-ATPase/c-Src complex, ouabain can simultaneously and coordinately regulate trafficking of basolateral Na/K-ATPase and apical NHE3, leading to inhibition of transepithelial Na<sup>+</sup> transport. This mechanism may be important to RPT Na<sup>+</sup> handling during conditions associated with increases in circulating endogenous CTS. However, it remains to be established whether ouabain-induced regulation of NHE3 trafficking comes from the endocytosed Na/K-ATPase/c-Src complex or directly from the plasma membrane, since ouabain still binds to endocytosed Na/K-ATPase (**Figure 2**).

#### **6. Ouabain-induced regulation of Na/K-ATPase α1 subunit and NHE3 is independent of intracellular [Na+ ]**

High concentrations of ouabain are known to increase intracellular [Na<sup>+</sup> ], depolarize the proximal tubule, and affect the tight junction of epithelial cells. In LLC-PK1 cells, ouabain (up to 100 nM) has no acute effect on intracellular [Na+ ], transepithelial electrical resistance, and tight junction integrity, suggesting that in the concentration, ouabain is not likely to increase passive Na<sup>+</sup> transport by depolarizing LLC-PK1 monolayers [115]. To further define whether the effects of ouabain on the Na/K-ATPase and NHE3 are independent of intracellular [Na<sup>+</sup> ], the change in intracellular transporters after the equilibrium of intracellular [Na<sup>+</sup> ] with extracellular [Na+ ] was achieved by using conventional "Na<sup>+</sup> -clamping" methods [150]. LLC-PK1 cells (both control and ouabain-treated) are pretreated either with 20 μM monensin or with 10 μM monensin plus 5 μM gramicidin for 30 min. Both "clamping" methods raise basal levels of α1 and NHE3 in EE/LE fractions (monensin is known to accumulate proteins in intracellular compartments). However, ouabain is still able to further accumulate more α1 and NHE3 in EE/LE. These observations indicate that ouabain-induced trafficking of α1 and NHE3 can be independent of intracellular [Na+ ] change [115]*.*
