Vladimir Pech

The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl- in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl-/HCO3- exchanger (NDCBE/SLC4A8) and the Na+-independent Cl-/HCO3- exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

Angiotensin II Activates H+-ATPase in Type A Intercalated Cells

We reported previously that angiotensin II (AngII) increases net Cl(-) absorption in mouse cortical collecting duct (CCD) by transcellular transport across type B intercalated cells (IC) via an H(+)-ATPase-and pendrin-dependent mechanism. Because intracellular trafficking regulates both pendrin and H(+)-ATPase, we hypothesized that AngII induces the subcellular redistribution of one or both of these exchangers. To answer this question, CCD from furosemide-treated mice were perfused in vitro, and the subcellular distributions of pendrin and the H(+)-ATPase were quantified using immunogold cytochemistry and morphometric analysis. Addition of AngII in vitro did not change the distribution of pendrin or H(+)-ATPase within type B IC but within type A IC increased the ratio of apical plasma membrane to cytoplasmic H(+)-ATPase three-fold. Moreover, CCDs secreted bicarbonate under basal conditions but absorbed bicarbonate in response to AngII. In summary, angiotensin II stimulates H(+) secretion into the lumen, which drives Cl(-) absorption mediated by apical Cl(-)/HCO(3)(-) exchange as well as generates more favorable electrochemical gradient for ENaC-mediated Na(+) absorption.

Angiotensin II acts through the angiotensin 1a receptor to upregulate pendrin

Pendrin is an anion exchanger expressed in the apical regions of B and non-A, non-B intercalated cells. Since angiotensin II increases pendrin-mediated Cl(-) absorption in vitro, we asked whether angiotensin II increases pendrin expression in vivo and whether angiotensin-induced hypertension is pendrin dependent. While blood pressure was similar in pendrin null and wild-type mice under basal conditions, following 2 wk of angiotensin II administration blood pressure was 31 mmHg lower in pendrin null than in wild-type mice. Thus pendrin null mice have a blunted pressor response to angiotensin II. Further experiments explored the effect of angiotensin on pendrin expression. Angiotensin II administration shifted pendrin label from the subapical space to the apical plasma membrane, independent of aldosterone. To explore the role of the angiotensin receptors in this response, pendrin abundance and subcellular distribution were examined in wild-type, angiotensin type 1a (Agtr1a) and type 2 receptor (Agtr2) null mice given 7 days of a NaCl-restricted diet (< 0.02% NaCl). Some mice received an Agtr1 inhibitor (candesartan) or vehicle. Both Agtr1a gene ablation and Agtr1 inhibitors shifted pendrin label from the apical plasma membrane to the subapical space, independent of the Agtr2 or nitric oxide (NO). However, Agtr1 ablation reduced pendrin protein abundance through the Agtr2 and NO. Thus angiotensin II-induced hypertension is pendrin dependent. Angiotensin II acts through the Agtr1a to shift pendrin from the subapical space to the apical plasma membrane. This Agtr1 action may be blunted by the Agtr2, which acts through NO to reduce pendrin protein abundance.

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK-ERK pathway.

Role of pendrin in iodide balance: going with the flow

Pendrin is expressed in the apical regions of type B and non-A, non-B intercalated cells, where it mediates Cl(-) absorption and HCO3(-) secretion through apical Cl(-)/HCO3(-) exchange. Since pendrin is a robust I(-) transporter, we asked whether pendrin is upregulated with dietary I(-) restriction and whether it modulates I(-) balance. Thus I(-) balance was determined in pendrin null and in wild-type mice. Pendrin abundance was evaluated with immunoblots, immunohistochemistry, and immunogold cytochemistry with morphometric analysis. While pendrin abundance was unchanged when dietary I(-) intake was varied over the physiological range, I(-) balance differed in pendrin null and in wild-type mice. Serum I(-) was lower, while I(-) excretion was higher in pendrin null relative to wild-type mice, consistent with a role of pendrin in renal I(-) absorption. Increased H2O intake enhanced differences between wild-type and pendrin null mice in I(-) balance, suggesting that H2O intake modulates pendrin abundance. Raising water intake from approximately 4 to approximately 11 ml/day increased the ratio of B cell apical plasma membrane to cytoplasm pendrin label by 75%, although circulating renin, aldosterone, and serum osmolality were unchanged. Further studies asked whether H2O intake modulates pendrin through the action of AVP. We observed that H2O intake modulated pendrin abundance even when circulating vasopressin levels were clamped. We conclude that H2O intake modulates pendrin abundance, although not likely through a direct, type 2 vasopressin receptor-dependent mechanism. As water intake rises, pendrin becomes increasingly critical in the maintenance of Cl(-) and I(-) balance.

The interaction of pendrin an d the epithelial sodium channel in blood pressure regulation

Purpose of reviewThis review summarizes the contribution of the Cl−/HCO3− exchanger pendrin in the renal regulation of blood pressure. Recent findingsIntercalated cells are found in the distal convoluted tubule, the connecting tubule and the collecting duct. These cells regulate acid–base balance by secreting or absorbing OH−/H+ equivalents and regulate vascular volume and blood pressure by absorbing chloride ions. In type B and non-A, non-B intercalated cells chloride absorption and HCO3− secretion are accomplished through the apical sodium-independent Cl−/HCO3− exchanger pendrin. With increased circulating aldosterone, pendrin abundance and transport are upregulated. In the absence of functional pendrin (Slc26a4 (−/−) mice), aldosterone-stimulated chloride absorption is reduced, which attenuates the blood pressure response to this steroid hormone. Pendrin also regulates aldosterone-induced changes in epithelial sodium channel abundance and function through a kidney-specific mechanism that does not involve changes in concentration of a circulating hormone. In vitro, angiotensin II increases sodium chloride absorption in the collecting duct by increasing the driving force for pendrin-mediated chloride absorption and the epithelial sodium channel-mediated sodium absorption through greater electrogenic hydrogen secretion. SummaryAldosterone and angiotensin II modulate the renal regulation of blood pressure, in part, by regulating pendrin-mediated chloride absorption and the epithelial sodium channel-mediated sodium absorption. Pendrin also modulates stimulation of the epithelial sodium channel by aldosterone.

ENaC inhibition stimulates Cl− secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ∼66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

Nitric oxide reduces Cl⁻ absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism.

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na⁺ and Cl⁻ in the kidney, we asked whether NO regulates net Cl⁻ flux (JCl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl⁻ absorption. Cl⁻ absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced JCl and transepithelial voltage (VT) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N(G)-nitro-L-arginine methyl ester) increased JCl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in JCl, we examined the effect of NO on JCl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive JCl, NO modulates JCl independent of the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H⁺-ATPase and pendrin. In contrast, both total and NO-sensitive JCl and VT were abolished with application of an epithelial Na(+) channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl⁻ absorption in the CCD through a mechanism that is ENaC-dependent.

Pendrin gene ablation alters ENaC subcellular distribution and open probability

The present study explored whether the intercalated cell Cl(-)/HCO3(-) exchanger pendrin modulates epithelial Na(+) channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na(+) absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na(+) absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddles syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddles syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddles syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability.

ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. I. Stilbene-sensitive Cl− secretion

Inhibition of the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption in cortical collecting ducts (CCDs) from aldosterone-treated rats and mice. Since ENaC does not transport Cl(-), the purpose of the present study was to explore how ENaC modulates Cl(-) absorption in mouse CCDs perfused in vitro. Therefore, we measured transepithelial Cl(-) flux and transepithelial voltage in CCDs perfused in vitro taken from mice that consumed a NaCl-replete diet alone or the diet with aldosterone administered by minipump. We observed that application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid unmasks conductive Cl(-) secretion. During ENaC blockade, this Cl(-) secretion fell with the application of a nonselective Cl(-) channel blocker [DIDS (100 μM)] to the perfusate. While single channel recordings of intercalated cell apical membranes in split-open CCDs demonstrated a Cl(-) channel with properties that resemble the ClC family of Cl(-) channels, ClC-5 is not the primary pathway for benzamil-sensitive Cl(-) flux. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, and, second, benzamil application stimulates stilbene-sensitive conductive Cl(-) secretion, which occurs through a ClC-5-independent pathway.