Klotho is highly expressed in the chief sites of regulated potassium secretion, and it is stimulated by potassium intake.

Klotho regulates many pathways in the aging process, but it remains unclear how it is physiologically regulated. Because Klotho is synthesized, cleaved, and released from the kidney; activates the chief urinary K+ secretion channel (ROMK) and stimulates urinary K+ secretion, we explored if Klotho protein is regulated by dietary K+ and the potassium-regulatory hormone, Aldosterone. Klotho protein along the nephron was evaluated in humans and in wild-type (WT) mice; and in mice lacking components of Aldosterone signaling, including the Aldosterone-Synthase KO (AS-KO) and the Mineralocorticoid-Receptor KO (MR-KO) mice. We found the specific cells of the distal nephron in humans and mice that are chief sites of regulated K+ secretion have the highest Klotho protein expression along the nephron. WT mice fed K+-rich diets increased Klotho expression in these cells. AS-KO mice exhibit normal Klotho under basal conditions but could not upregulate Klotho in response to high-K+ intake in the K+-secreting cells. Similarly, MR-KO mice exhibit decreased Klotho protein expression. Together, i) Klotho is highly expressed in the key sites of regulated K+ secretion in humans and mice, ii) In mice, K+-rich diets increase Klotho expression specifically in the potassium secretory cells of the distal nephron, iii) Aldosterone signaling is required for Klotho response to high K+ intake.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Dietary anions control potassium excretion: it is more than a poorly absorbable anion effect.

The urinary potassium (K+) excretion machinery is upregulated with increasing dietary K+, but the role of accompanying dietary anions remains inadequately characterized. Poorly absorbable anions, including [Formula: see text], are thought to increase K+ secretion through a transepithelial voltage effect. Here, we tested if they also influence the K+ secretion machinery. Wild-type mice, aldosterone synthase (AS) knockout (KO) mice, or pendrin KO mice were randomized to control, high-KCl, or high-KHCO3 diets. The K+ secretory capacity was assessed in balance experiments. Protein abundance, modification, and localization of K+-secretory transporters were evaluated by Western blot analysis and confocal microscopy. Feeding the high-KHCO3 diet increased urinary K+ excretion and the transtubular K+ gradient significantly more than the high-KCl diet, coincident with more pronounced upregulation of epithelial Na+ channels (ENaC) and renal outer medullary K+ (ROMK) channels and apical localization in the distal nephron. Experiments in AS KO mice revealed that the enhanced effects of [Formula: see text] were aldosterone independent. The high-KHCO3 diet also uniquely increased the large-conductance Ca2+-activated K+ (BK) channel ß4-subunit, stabilizing BKα on the apical membrane, the Cl-/[Formula: see text] exchanger, pendrin, and the apical KCl cotransporter (KCC3a), all of which are expressed specifically in pendrin-positive intercalated cells. Experiments in pendrin KO mice revealed that pendrin was required to increase K+ excretion with the high-KHCO3 diet. In summary, [Formula: see text] stimulates K+ excretion beyond a poorly absorbable anion effect, upregulating ENaC and ROMK in principal cells and BK, pendrin, and KCC3a in pendrin-positive intercalated cells. The adaptive mechanism prevents hyperkalemia and alkalosis with the consumption of alkaline ash-rich diets but may drive K+ wasting and hypokalemia in alkalosis.NEW & NOTEWORTHY Dietary anions profoundly impact K+ homeostasis. Here, we found that a K+-rich diet, containing [Formula: see text] as the counteranion, enhances the electrogenic K+ excretory machinery, epithelial Na+ channels, and renal outer medullary K+ channels, much more than a high-KCl diet. It also uniquely induces KCC3a and pendrin, in B-intercalated cells, providing an electroneutral KHCO3 secretion pathway. These findings reveal new K+ balance mechanisms that drive adaption to alkaline and K+-rich foods, which should guide new treatment strategies for K+ disorders.

Pendrin abundance, subcellular distribution, and function are unaffected by either αENaC gene ablation or by increasing ENaC channel activity.

The intercalated cell Cl-/HCO3- exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl- absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle's syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle's variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle's mutation increased total Cl- absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl- absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.

Pendrin regulation is prioritized by anion in high-potassium diets.

The Cl-/[Formula: see text] exchanger pendrin in the kidney maintains acid-base balance and intravascular volume. Pendrin is upregulated in models associated with high circulating aldosterone concentration, such as dietary NaCl restriction or an aldosterone infusion. However, it has not been established if pendrin is similarly regulated by aldosterone with a high-K+ diet because the effects of accompanying anions have not been considered. Here, we explored how pendrin is modulated by different dietary potassium salts. Wild-type (WT) and aldosterone synthase (AS) knockout (KO) mice were randomized to control, high-KHCO3, or high-KCl diets. Dietary KCl and KHCO3 loading increased aldosterone in WT mice to the same extent but had opposite effects on pendrin abundance. KHCO3 loading increased pendrin protein and transcript abundance. Conversely, high-KCl diet feeding caused pendrin to decrease within 8 h of switching from the high-KHCO3 diet, coincident with an increase in plasma Cl- and a decrease in [Formula: see text]. In contrast, switching the high-KCl diet to the high-KHCO3 diet caused pendrin to increase in WT mice. Experiments in AS KO mice revealed that aldosterone is necessary to optimally upregulate pendrin protein in response to the high-KHCO3 diet but not to increase pendrin mRNA. We conclude that pendrin is differentially regulated by different dietary potassium salts and that its regulation is prioritized by the dietary anion, providing a mechanism to prevent metabolic alkalosis with high-K+ base diets and safeguard against hyperchloremic acidosis with consumption of high-KCl diets.NEW & NOTEWORTHY Regulation of the Cl-/[Formula: see text] exchanger pendrin has been suggested to explain the aldosterone paradox. A high-K+ diet has been proposed to downregulate a pendrin-mediated K+-sparing NaCl reabsorption pathway to maximize urinary K+ excretion. Here, we challenged the hypothesis, revealing that the accompanying anion, not K+, drives pendrin expression. Pendrin is downregulated with a high-KCl diet, preventing acidosis, and upregulated with an alkaline-rich high-K+ diet, preventing metabolic alkalosis. Pendrin regulation is prioritized for acid-base balance.

Effects of Roxadustat on Erythropoietin Production in the Rat Body.

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

Pendrin-null mice develop severe hypokalemia following dietary Na+ and K+ restriction: role of ENaC.

Pendrin is an intercalated cell Cl-/[Formula: see text] exchanger thought to participate in K+-sparing NaCl absorption. However, its role in K+ homeostasis has not been clearly defined. We hypothesized that pendrin-null mice will develop hypokalemia with dietary K+ restriction. We further hypothesized that pendrin knockout (KO) mice mitigate urinary K+ loss by downregulating the epithelial Na+ channel (ENaC). Thus, we examined the role of ENaC in Na+ and K+ balance in pendrin KO and wild-type mice following dietary K+ restriction. To do so, we examined the relationship between Na+ and K+ balance and ENaC subunit abundance in K+-restricted pendrin-null and wild-type mice that were NaCl restricted or replete. Following a NaCl-replete, K+-restricted diet, K+ balance and serum K+ were similar in both groups. However, following a Na+, K+, and Cl--deficient diet, pendrin KO mice developed hypokalemia from increased K+ excretion. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. However, reducing ENaC activity also reduced blood pressure and increased apparent intravascular volume contraction, since KO mice had lower serum Na+, higher blood urea nitrogen and hemoglobin, greater weight loss, greater metabolic alkalosis, and greater NaCl excretion. We conclude that dietary Na+ and K+ restriction induces hypokalemia in pendrin KO mice. Pendrin-null mice limit renal K+ loss by downregulating ENaC. However, this ENaC downregulation occurs at the expense of intravascular volume.NEW & NOTEWORTHY Pendrin is an apical Cl-/[Formula: see text] exchanger that provides renal K+-sparing NaCl absorption. The pendrin-null kidney has an inability to fully conserve K+ and limits renal K+ loss by downregulating the epithelial Na+ channel (ENaC). However, with Na+ restriction, the need to reduce ENaC for K+ balance conflicts with the need to stimulate ENaC for intravascular volume. Therefore, NaCl restriction stimulates ENaC less in pendrin-null mice than in wild-type mice, which mitigates their kaliuresis and hypokalemia but exacerbates volume contraction.

Aldosterone Regulates Pendrin and Epithelial Sodium Channel Activity through Intercalated Cell Mineralocorticoid Receptor-Dependent and -Independent Mechanisms over a Wide Range in Serum Potassium.

BACKGROUND: Aldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium's role in this response and the receptor's effect on intercalated and principal cell function in the cortical collecting duct (CCD). METHODS: We measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell-specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice. RESULTS: Ablation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin's relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone's effect on pendrin total and apical abundance and subcellular distribution. CONCLUSIONS: With high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin's total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.

Blood collection in unstressed, conscious, and freely moving mice through implantation of catheters in the jugular vein: a new simplified protocol.

The mouse has become the most common mammalian animal model used in biomedical research. However, laboratory techniques used previously in rats and other larger animals to sample blood had to be adapted in mice due to their lower mouse plasma volume. Sampling is further confounded by the variability in plasma hormone and metabolite concentrations that can occur from the stress or the anesthesia that accompanies the collection. In this article, we describe in detail a protocol we developed for blood sampling in conscious, unrestrained mice. Our protocol implements the use of chronic indwelling catheters in the right external jugular vein, allowing the mice to recover fully in their home cages, untethered until the time of blood sampling. This protocol employs catheters that remain patent for days and does not require the purchase of expensive equipment. We validated this protocol by measuring the time course of plasma norepinephrine (NE) concentration during and after the relief of acute immobilization stress in wild type (WT) and pendrin knockout (KO) mice and compared these results with our previously published values. We found that following relief from immobilization stress, it takes longer for plasma NE concentration to return to basal levels in the pendrin KO than in the wild type mice. These results highlight the potential utility of this protocol and the potential role of pendrin in the neuroendocrine response to acute stress.

The Role of Intercalated Cell Nedd4-2 in BP Regulation, Ion Transport, and Transporter Expression.

BackgroundNedd4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between Nedd4-2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4-2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC Nedd4-2 knockout mice using Cre-lox technology and produced global pendrin/Nedd4-2 null mice by breeding global Nedd4-2 null (Nedd4-2-/- ) mice with global pendrin null (Slc26a4-/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl- and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC Nedd4-2 gene ablation markedly increased electroneutral Cl-/HCO3- exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl-/HCO3- exchanger (Slc4a8). However, IC Nedd4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4-2 knockout mice.Conclusions IC Nedd4-2 regulates Cl-/HCO3- exchange in ICs., Nedd4-2 gene ablation increases BP in part through its action in these cells.

Pendrin localizes to the adrenal medulla and modulates catecholamine release.

Pendrin (Slc26a4) is a Cl(-)/HCO3 (-) exchanger expressed in renal intercalated cells and mediates renal Cl(-) absorption. With pendrin gene ablation, blood pressure and vascular volume fall, which increases plasma renin concentration. However, serum aldosterone does not significantly increase in pendrin-null mice, suggesting that pendrin regulates adrenal zona glomerulosa aldosterone production. Therefore, we examined pendrin expression in the adrenal gland using PCR, immunoblots, and immunohistochemistry. Pendrin protein was detected in adrenal lysates from wild-type but not pendrin-null mice. However, immunohistochemistry and qPCR of microdissected adrenal zones showed that pendrin was expressed in the adrenal medulla, rather than in cortex. Within the adrenal medulla, pendrin localizes to both epinephrine- and norepinephrine-producing chromaffin cells. Therefore, we examined plasma catecholamine concentration and blood pressure in wild-type and pendrin-null mice under basal conditions and then after 5 and 20 min of immobilization stress. Under basal conditions, blood pressure was lower in the mutant than in the wild-type mice, although epinephrine and norepinephrine concentrations were similar. Catecholamine concentration and blood pressure increased markedly in both groups with stress. With 20 min of immobilization stress, epinephrine and norepinephrine concentrations increased more in pendrin-null than in wild-type mice, although stress produced a similar increase in blood pressure in both groups. We conclude that pendrin is expressed in the adrenal medulla, where it blunts stress-induced catecholamine release.

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 (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's 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 Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability.

Contractile force is enhanced in Aortas from pendrin null mice due to stimulation of angiotensin II-dependent signaling.

Pendrin is a Cl-/HCO3- exchanger expressed in the apical regions of renal intercalated cells. Following pendrin gene ablation, blood pressure falls, in part, from reduced renal NaCl absorption. We asked if pendrin is expressed in vascular tissue and if the lower blood pressure observed in pendrin null mice is accompanied by reduced vascular reactivity. Thus, the contractile responses to KCl and phenylephrine (PE) were examined in isometrically mounted thoracic aortas from wild-type and pendrin null mice. Although pendrin expression was not detected in the aorta, pendrin gene ablation changed contractile protein abundance and increased the maximal contractile response to PE when normalized to cross sectional area (CSA). However, the contractile sensitivity to this agent was unchanged. The increase in contractile force/cross sectional area observed in pendrin null mice was due to reduced cross sectional area of the aorta and not from increased contractile force per vessel. The pendrin-dependent increase in maximal contractile response was endothelium- and nitric oxide-independent and did not occur from changes in Ca2+ sensitivity or chronic changes in catecholamine production. However, application of 100 nM angiotensin II increased force/CSA more in aortas from pendrin null than from wild type mice. Moreover, angiotensin type 1 receptor inhibitor (candesartan) treatment in vivo eliminated the pendrin-dependent changes contractile protein abundance and changes in the contractile force/cross sectional area in response to PE. In conclusion, pendrin gene ablation increases aorta contractile force per cross sectional area in response to angiotensin II and PE due to stimulation of angiotensin type 1 receptor-dependent signaling. The angiotensin type 1 receptor-dependent increase in vascular reactivity may mitigate the fall in blood pressure observed with pendrin gene ablation.

Pendrin modulates ENaC function by changing luminal HCO3-.

The epithelial Na(+) channel, ENaC, and the Cl(-)/HCO(3)(-) exchanger, pendrin, mediate NaCl absorption within the cortical collecting duct and the connecting tubule. Although pendrin and ENaC localize to different cell types, ENaC subunit abundance and activity are lower in aldosterone-treated pendrin-null mice relative to wild-type mice. Because pendrin mediates HCO(3)(-) secretion, we asked if increasing distal delivery of HCO(3)(-) through a pendrin-independent mechanism "rescues" ENaC function in pendrin-null mice. We gave aldosterone and NaHCO(3) to increase pendrin-dependent HCO(3)(-) secretion within the connecting tubule and cortical collecting duct, or gave aldosterone and NaHCO(3) plus acetazolamide to increase luminal HCO(3)(-) concentration, [HCO(3)(-)], independent of pendrin. Following treatment with aldosterone and NaHCO(3), pendrin-null mice had lower urinary pH and [HCO(3)(-)] as well as lower renal ENaC abundance and function than wild-type mice. With the addition of acetazolamide, however, acid-base balance as well as ENaC subunit abundance and function was similar in pendrin-null and wild-type mice. We explored whether [HCO(3)(-)] directly alters ENaC abundance and function in cultured mouse principal cells (mpkCCD). Amiloride-sensitive current and ENaC abundance rose with increased [HCO(3)(-)] on the apical or the basolateral side, independent of the substituting anion. However, ENaC was more sensitive to changes in [HCO(3)(-)] on the basolateral side of the monolayer. Moreover, increasing [HCO(3)(-)] on the apical and basolateral side of Xenopus kidney cells increased both ENaC channel density and channel activity. We conclude that pendrin modulates ENaC abundance and function, at least in part by increasing luminal [HCO(3)(-)] and/or pH.

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.

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 increases chloride absorption in the cortical collecting duct in mice through a pendrin-dependent mechanism.

Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical Cl(-)/HCO(3)(-) exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, J(Cl), in mouse CCD through a pendrin-dependent mechanism. J(Cl) and transepithelial voltage, V(T), were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaCl-replete diet, V(T) and J(Cl) were not different from zero either in the presence or absence of angiotensin II (10(-8) M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative V(T) and absorbed Cl(-). With angiotensin II in the bath, Cl(-) absorption doubled although V(T) did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl(-) secretion and a V(T) of approximately 0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished V(T) although J(Cl) fell only approximately 50%. Thus substantial Cl(-) absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the H(+)-ATPase inhibitor bafilomycin abolished benzamil-insensitive Cl(-) absorption. In conclusion, angiotensin II increases transcellular Cl(-) absorption in the CCD through a pendrin- and H(+)-ATPase-dependent process.