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KCC3a expression is stimulated in alkalemia. This paper shows that bicarbonate itself is mediating this effect through a posttranscriptional mechanism. The paper also shows that this phenomenon is not mediated by aldosterone or angiotensin II.

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.

The renal response to acid-base disturbances involves phenotypic and remodeling changes in the collecting duct. This study examines whether the proximal tubule controls these responses. We examined mice with genetic deletion of proteins present only in the proximal tubule, either the A variant or both the A and B variants of isoform 1 of the electrogenic Na⁺-bicarbonate cotransporter (NBCe1). Both knockout (KO) mice have spontaneous metabolic acidosis. We then determined the collecting duct phenotypic responses to this acidosis and the remodeling responses to exogenous acid loading. Despite the spontaneous acidosis in NBCe1-A KO mice, Type-A intercalated cells in the inner stripe of the outer medullary collecting duct (OMCD

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.

Intercalated cells make up about a third of all cells within the connecting tubule and the collecting duct and are subclassified as type A, type B and non-A, non-B based on the subcellular distribution of the H + -ATPase, which dictates whether it secretes H + or HCO 3 − . Type B intercalated cells mediate Cl − absorption and HCO 3 − secretion, which occurs largely through the anion exchanger pendrin. Pendrin is stimulated by angiotensin II via the angiotensin type 1a receptor and by aldosterone through MR (mineralocorticoid receptor). Aldosterone stimulates pendrin expression and function, in part, through the alkalosis it generates. Pendrin-mediated HCO 3 − secretion increases in models of metabolic alkalosis, which attenuates the alkalosis. However, pendrin-positive intercalated cells also regulate blood pressure, at least partly, through pendrin-mediated Cl − absorption and through their indirect effect on the epithelial Na + channel, ENaC. This aldosterone-induced increase in pendrin secondarily stimulates ENaC, thereby contributing to the aldosterone pressor response. This review describes the contribution of pendrin-positive intercalated cells to Na + , K + , Cl − and acid-base balance.

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.

Background and objectives Although US physician-scientists have made enormous contributions to biomedical research, this workforce is thought to be getting smaller. However, among kidney researchers, changes have not been fully quantified. Design, setting, participants, & measurements We mined National Institutes of Health RePORTER to explore demographic changes of early-career and established physician and nonphysician principal investigators doing kidney-focused research. We searched for National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-funded K series and R01 awards focused on the kidney that were active between 1990 and 2020 and determined if their emphasis was basic or clinical science. We then used public databases available on the internet to determine if these funded investigators were physicians or nonphysicians, the year in which they received either their MD (physicians) or their terminal graduate degree (nonphysicians), their sex, and whether they received their terminal degree from a US or international institution. Results Kidney-focused R01-funded principal investigators are aging, particularly among physicians. Moreover, the relative representation of physicians among both early-career and established principal investigators is falling, particularly among those doing basic science research. In contrast, the number and relative representation of nonphysician-scientists are increasing. There is also greater representation of women and international graduates among physician and nonphysician R01-funded, kidney-focused NIDDK investigators. However, although there are greater numbers of women physician principal investigators doing both basic as well as clinical research, women physician principal investigators are increasingly more likely to do clinical rather than basic science research. Conclusions The physician-scientist workforce is increasingly made up of women and international medical graduates. However, the physician-scientist workforce is older and represents a smaller proportion of all principal investigators, particularly among those doing basic science research.

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.

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.

α-Ketoglutarate (α-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a Gq protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl−/[Formula: see text] exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl− absorption, we examined α-KG-stimulated Cl− absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl− absorption in CCDs from wild-type mice but had no effect on Cl− absorption in pendrin knockout mice. Because Gq protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl− absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl− absorption. Like α-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl− absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca2+ chelators (BAPTA, 10–20 μM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl− absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl− absorption. However, in SPAK null CCDs, Cl− absorption was not activated further by luminal α-KG application nor was Cl− absorption reduced with the PKC inhibitor GF109203 . Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl−/[Formula: see text] exchange through a mechanism dependent on PKC and Ca2+ that involves PKC-α and PKC-δ.

Ischemic acute kidney injury (AKI), a complication that frequently occurs in hospital settings, is often associated with hemodynamic compromise, sepsis, cardiac surgery, or exposure to nephrotoxins. Here, using a murine renal ischemia/reperfusion injury (IRI) model, we show that intercalated cells (ICs) rapidly adopted a proinflammatory phenotype after IRI. Wwe demonstrate that during the early phase of AKI either blockade of the proinflammatory P2Y14 receptor located on the apical membrane of ICs or ablation of the gene encoding the P2Y14 receptor in ICs (a) inhibited IRI-induced increase of chemokine expression in ICs, (b) reduced neutrophil and monocyte renal infiltration, (c) reduced the extent of kidney dysfunction, and (d) attenuated proximal tubule damage. These observations indicate that the P2Y14 receptor participates in the very first inflammatory steps associated with ischemic AKI. In addition, we show that the concentration of the P2Y14 receptor ligand UDP-glucose (UDP-Glc) was higher in urine samples from intensive care unit patients who developed AKI compared with patients without AKI. In particular, we observed a strong correlation between UDP-Glc concentration and the development of AKI in cardiac surgery patients. Our study identifies the UDP-Glc/P2Y14 receptor axis as a potential target for the prevention and/or attenuation of ischemic AKI.

Patient: Male, 82-year-old Final Diagnosis: End stage renal disease • thrombocytopenia • co-existing disea Symptoms: Fatigue • melena • weakness Medication: — Clinical Procedure: — Specialty: Nephrology Objective: Unusual or unexpected effect of treatment Background: Biocompatible hemodialysis membranes have greatly advanced the treatment of renal failure. Synthetic polysulfone dialysis membranes are considered to be very biocompatible because of their low propensity to activate complement. However, these membranes can reduce platelet count through platelet activation, although the mechanism of this activation is unknown. Case Report: We report the case of an 82-year-old man with a history of chronic kidney disease with recurrent gastrointestinal bleeding and worsening renal function who was initiated on renal replacement therapy with polysulfone dialysis membranes. On admission, the patient’s platelet count was normal at 233×103/μL. A significant fall in platelet count was observed following most dialysis treatments, reaching a nadir of 37×103/μL. With occasional dialysis treatments, his platelet count did not change. This dialysis-induced thrombocytopenia resolved following substitution with Cellentia-H cellulose triacetate single-use, hollow-fiber, high-flux hemodialyzer membrane. Conclusions: Polysulfone membranes are capable of activating platelets, which can result in severe thrombocytopenia. However, the magnitude of dialysis-induced thrombocytopenia varies from treatment to treatment. As such, it may not be evident when the pre- and postdialysis platelet counts are measured for a single treatment. Because the etiology of this platelet activation is unknown, substitution with cellulose triacetate membranes should be considered. These membranes have an unrelated chemical composition and a very low propensity to activate platelets.

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 CO 2 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 - /HCO 3 - 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 - /HCO 3 - 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 - /HCO 3 - exchange in ICs., Nedd4-2 gene ablation increases BP in part through its action in these cells.

Pendrin is a Na+-independent Cl−/HCO3−exchanger found in the apical regions of type B and non-A, non-B intercalated cells within the aldosterone-sensitive region of the nephron, i.e., the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD). Type B intercalated cells mediate Cl−absorption and HCO3−secretion primarily through pendrin-mediated Cl−/HCO3−exchange. This exchanger is upregulated with angiotensin II administration and in models of metabolic alkalosis, such as following administration of aldosterone or NaHCO3. In the absence of pendrin-mediated HCO3−secretion, an enhanced alkalosis is observed following aldosterone or NaHCO3administration. However, probably of more significance is the role of pendrin in the pressor response to aldosterone. Pendrin mediates Cl−absorption and modulates aldosterone-induced Na+absorption mediated by the epithelial Na channel (ENaC). Pendrin changes ENaC activity by changing both channel open probability ( Po) and surface density ( N), at least partly by altering luminal HCO3−and ATP concentration. Thus aldosterone and angiotensin II stimulate pendrin expression and function, which stimulates ENaC activity, thereby contributing to the pressor response of these hormones. However, pendrin may modulate blood pressure partly through its extrarenal effects. For example, pendrin is expressed in the adrenal medulla, where it modulates catecholamine release. The increase in catecholamine release observed with pendrin gene ablation likely contributes to the increment in vascular contractile force observed in the pendrin null mouse. This review summarizes the signaling mechanisms that regulate pendrin abundance and function as well as the contribution of pendrin to distal nephron function.

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.

New Findings What is the central question of this study? Pregnancy requires a robust plasma volume expansion driven by renal sodium retention. In the late-pregnant kidney, the aldosterone-responsive epithelial Na+ channel is increased, whereas the sodium–chloride cotransporter is decreased. Pendrin has been shown to support sodium reabsorption in the distal nephron and compensate for loss of the sodium–chloride cotransporter. We investigated the expression and abundance of pendrin in the pregnant kidney. What is the main finding and its importance? Pendrin protein, apical localization and thiazide sensitivity are increased in pregnancy. This implicates a possible role for pendrin in supporting the renal sodium chloride reabsorption and plasma volume expansion of pregnancy. Pregnancy is characterized by cumulative plasma volume expansion as a result of renal sodium retention, driven by activation of aldosterone. We previously reported that the abundance and activity of the aldosterone-responsive epithelial Na+ channel is increased, whereas the sodium–chloride cotransporter (NCC) is decreased in the kidney of the late-pregnant rat. The chloride–bicarbonate exchanger pendrin is also aldosterone responsive and has been shown to support activity of the aldosterone-responsive epithelial Na+ channel and compensate for the loss of NCC. Additionally, pendrin coupled to the sodium-dependent chloride–bicarbonate exchanger (NDCBE) mediates thiazide-sensitive sodium reabsorption in the cortical collecting duct. In this study, we investigated pendrin and NDCBE transcript expression, pendrin protein abundance, pendrin cellular localization and thiazide sensitivity in virgin, mid-pregnant and late-pregnant rats to test the hypothesis that increased pendrin activity might occur in pregnancy. By RT-PCR, NDCBE and pendrin mRNA expression was unchanged from virgins, whereas pendrin protein abundance determined by Western blotting was increased in both mid- and late-pregnant rats. The apical localization of pendrin was also increased in late-pregnant rats compared with virgins by immunohistochemistry. Pregnant rats displayed an increased natriuretic response to hydrochlorothiazide compared with virgins. Given that NCC expression is decreased in late pregnancy, an increased thiazide sensitivity may be due to inhibition of upregulated pendrin–NDCBE-coupled sodium reabsorption. Thus, increased pendrin in pregnant rats may compensate for the decreased NCC and aid in the renal sodium chloride reabsorption of pregnancy.

α-Ketoglutarate (α-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a G(q) protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl(−)/ [Formula: see text] exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl(−) absorption, we examined α-KG-stimulated Cl(−) absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl(−) absorption in CCDs from wild-type mice but had no effect on Cl(−) absorption in pendrin knockout mice. Because G(q) protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl(−) absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl(−) absorption. Like α-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl(−) absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca(2+) chelators (BAPTA, 10–20 μM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl(−) absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl(−) absorption. However, in SPAK null CCDs, Cl(−) absorption was not activated further by luminal α-KG application nor was Cl(−) absorption reduced with the PKC inhibitor GF109203. Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl(−)/ [Formula: see text] exchange through a mechanism dependent on PKC and Ca(2+) that involves PKC-α and PKC-δ.

Tacitly, Edemir (1) points to an important issue in the interpretation of data from single-cell RNA-sequencing experiments. Any given cell can exist in a variety of regulatory states that are affected both by extracellular signals and the prior history of the cell. Consequently, data from single-cell RNA sequencing, such as carried out in our paper (2), provide only a partial characterization of a given cell type. Workers engaged in large-scale attempts to identify transcriptomes of every cell type in the body (3) would do well to take note of this … [↵][1]1To whom correspondence should be addressed. Email: maurice_burg{at}nih.gov. [1]: #xref-corresp-1-1

Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.

Prior RNA sequencing (RNA-Seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume and extracellular fluid composition. To enrich these cell types, we used fluorescence-activated cell sorting (FACS) that employed well established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, viz. c-Kit. Single-cell RNA-Seq using the 1C- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs and PCs. The data were used to create a freely-accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type including cell-surface receptors, transcription factors, transporters and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing both aquapor¡n-2 and either anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g.Notch2chiefly in PCs vsJag1chiefly in ICs) suggesting signaling crosstalk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.SIGNIFICANCE STATEMENTA long-term goal in mammalian biology is to identify the genes expressed in every cell type of the body. In kidney, the expressed genes (“transcriptome”) of all epithelial cell types have already been identified with the exception of the cells that make up the renal collecting duct, responsible for regulation of blood pressure and body fluid composition. Here, a technique called "single-cell RNA-Seq" was used in mouse to identify transcriptomes for the major collecting-duct cell types: type A intercalated cells, type B intercalated cells and principal cells. The information was used to create a publicly-accessible online resource. The data allowed identification of genes that are selectively expressed in each cell type, informative for cell-level understanding of physiology and pathophysiology.

Background Nedd4–2 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 CO(2) 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(−)/HCO(3)(−) 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(−)/HCO(3)(−) 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(−)/HCO(3)(−) exchange in ICs., Nedd4–2 gene ablation increases BP in part through its action in these cells.

Pertussis disease, characterized by severe and prolonged coughing episodes, can progress to a critical stage with pulmonary inflammation and death in young infants. However, there are currently no effective treatments for pertussis. We previously studied the role of pertussis toxin (PT), an important Bordetella pertussis virulence factor, in lung transcriptional responses to B. pertussis infection in mouse models. One of the genes most highly upregulated in a PT-dependent manner encodes an epithelial transporter of bicarbonate, chloride, and thiocyanate, named pendrin, that contributes to asthma pathology. In this study, we found that pendrin expression is upregulated at both gene and protein levels in the lungs of B. pertussis -infected mice. Pendrin upregulation is associated with PT production by the bacteria and with interleukin-17A (IL-17A) production by the host. B. pertussis -infected pendrin knockout (KO) mice had higher lung bacterial loads than infected pendrin-expressing mice but had significantly reduced levels of lung inflammatory pathology. However, reduced pathology did not correlate with reduced inflammatory cytokine expression. Infected pendrin KO mice had higher levels of inflammatory cytokines and chemokines than infected pendrin-expressing mice, suggesting that these inflammatory mediators are less active in the airways in the absence of pendrin. In addition, treatment of B. pertussis -infected mice with the carbonic anhydrase inhibitor acetazolamide reduced lung inflammatory pathology without affecting pendrin synthesis or bacterial loads. Together these data suggest that PT contributes to pertussis pathology through the upregulation of pendrin, which promotes conditions favoring inflammatory pathology. Therefore, pendrin may represent a novel therapeutic target for treatment of pertussis disease.

Renal intercalated cells mediate the secretion or absorption of Cl−and OH−/H+equivalents in the connecting segment (CNT) and cortical collecting duct (CCD). In so doing, they regulate acid-base balance, vascular volume, and blood pressure. Cl−absorption is either electrogenic and amiloride-sensitive or electroneutral and thiazide-sensitive. However, which Cl−transporter(s) are targeted by these diuretics is debated. While epithelial Na+channel (ENaC) does not transport Cl−, it modulates Cl−transport probably by generating a lumen-negative voltage, which drives Cl−flux across tight junctions. In addition, recent evidence indicates that ENaC inhibition increases electrogenic Cl−secretion via a type A intercalated cells. During ENaC blockade, Cl−is taken up across the basolateral membrane through the Na+-K+−2Cl−cotransporter (NKCC1) and then secreted across the apical membrane through a conductive pathway (a Cl−channel or an electrogenic exchanger). The mechanism of this apical Cl−secretion is unresolved. In contrast, thiazide diuretics inhibit electroneutral Cl−absorption mediated by a Na+-dependent Cl−/HCO3−exchanger. The relative contribution of the thiazide and the amiloride-sensitive components of Cl−absorption varies between studies and probably depends on the treatment model employed. Cl−absorption increases markedly with angiotensin and aldosterone administration, largely by upregulating the Na+-independent Cl−/HCO3−exchanger pendrin. In the absence of pendrin [ Slc26a4(−/−)or pendrin null mice], aldosterone-stimulated Cl−absorption is significantly reduced, which attenuates the pressor response to this steroid hormone. Pendrin also modulates aldosterone-induced changes in ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function, at least in part, by altering luminal HCO3−. This review summarizes mechanisms of Cl−transport in CNT and CCD and how these transporters contribute to the regulation of extracellular volume and blood pressure.

Studies in rat aorta have shown that the Na-K-2Cl cotransporter NKCC1 is activated by vasoconstrictors and inhibited by nitrovasodilators, contributes to smooth muscle tone in vitro, and is upregulated in hypertension. To determine the role of NKCC1 in systemic vascular resistance and hypertension, blood pressure was measured in rats before and after inhibition of NKCC1 with bumetanide. Intravenous infusion of bumetanide sufficient to yield a free plasma concentration above the IC50for NKCC1 produced an immediate drop in blood pressure of 5.2% ( P < 0.001). The reduction was not prevented when the renal arteries were clamped, indicating that it was not due to a renal effect of bumetanide. Bumetanide did not alter blood pressure in NKCC1-null mice, demonstrating that it was acting specifically through NKCC1. In third-order mesenteric arteries, bumetanide-inhibitable efflux of86Rb was acutely stimulated 133% by phenylephrine, and bumetanide reduced the contractile response to phenylephrine, indicating that NKCC1 influences tone in resistance vessels. The hypotensive effect of bumetanide was proportionately greater in rats made hypertensive by a 7-day infusion of norepinephrine (12.7%, P < 0.001 vs. normotensive rats) but much less so when hypertension was produced by a fixed aortic coarctation (8.0%), again consistent with an effect of bumetanide on resistance vessels rather than other determinants of blood pressure. We conclude that NKCC1 influences blood pressure through effects on smooth muscle tone in resistance vessels and that this effect is augmented in hypertension.

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 ( VT). 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 VT. In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1aloxlox). 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 VTwas 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 VT. Finally, benzamil-sensitive Cl−flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

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 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.

Cystic fibrosis is caused by mutations in CFTR, the cystic fibrosis transmembrane conductance regulator gene. Disruption of CFTR-mediated anion conductance results in defective fluid and electrolyte movement in the epithelial cells of organs such as the pancreas, airways and sweat glands, but the function of CFTR in salivary glands is unclear. Salivary gland acinar cells produce an isotonic, plasma-like fluid, which is subsequently modified by the ducts to produce a hypotonic, NaCl-depleted final saliva. In the present study we investigated whether submandibular salivary glands (SMGs) in ΔF508 mice (CftrΔF/ΔF) display ion transport defects characteristic of cystic fibrosis in other tissues. Immunolocalization and whole-cell recordings demonstrated that Cftr and the epithelial Na+ (ENaC) channels are co-expressed in the apical membrane of submandibular duct cells, consistent with the significantly higher saliva [NaCl] observed in vivo in CftrΔF/ΔF mice. In contrast, Cftr and ENaC channels were not detected in acinar cells, nor was saliva production affected in CftrΔF/ΔF mice, implying that Cftr contributes little to the fluid secretion process in the mouse SMG. To identify the source of the NaCl absorption defect in CftrΔF/ΔF mice, saliva was collected from ex vivo perfused SMGs. CftrΔF/ΔF glands secreted saliva with significantly increased [NaCl]. Moreover, pharmacological inhibition of either Cftr or ENaC in the ex vivo SMGs mimicked the CftrΔF/ΔF phenotype. In summary, our results demonstrate that NaCl absorption requires and is likely to be mediated by functionally dependent Cftr and ENaC channels localized to the apical membranes of mouse salivary gland duct cells.

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 ∼4 to ∼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.

NKCC1 is a widely expressed isoform of the Na-2Cl-K cotransporter that mediates several direct and indirect vascular effects and regulates expression and release of renin. In this study, we used NKCC1-deficient (NKCC1−/−) and wild-type (WT) mice to assess day/night differences of blood pressure (BP), locomotor activity, and renin release and to study the effects of high (8%) or low (0.03%) dietary NaCl intake on BP, activity, and the renin/aldosterone system. On a standard diet, 24-h mean arterial blood pressure (MAP) and heart rate determined by radiotelemetry, and their day/night differences, were not different in NKCC1−/−and WT mice. Spontaneous and wheel-running activities in the active night phase were lower in NKCC1−/−than WT mice. In NKCC1−/−mice on a high-NaCl diet, MAP increased by 10 mmHg in the night without changes in heart rate. In contrast, there was no salt-dependent blood pressure change in WT mice. MAP reductions by hydralazine (1 mg/kg) or isoproterenol (10 μg/mouse) were significantly greater in NKCC1−/−than WT mice. Plasma renin (PRC; ng ANG I·ml−1·h−1) and aldosterone (aldo; pg/ml) concentrations were higher in NKCC1−/−than WT mice (PRC: 3,745 ± 377 vs. 1,245 ± 364; aldo: 763 ± 136 vs. 327 ± 98). Hyperreninism and hyperaldosteronism were found in NKCC1−/−mice during both day and night. High Na suppressed PRC and aldosterone in both NKCC1−/−and WT mice, whereas a low-Na diet increased PRC and aldosterone in WT but not NKCC1−/−mice. We conclude that 24-h MAP and MAP circadian rhythms do not differ between NKCC1−/−and WT mice on a standard diet, probably reflecting a balance between anti- and prohypertensive factors, but that blood pressure of NKCC1−/−mice is more sensitive to increases and decreases of Na intake.

The extracellular superoxide dismutase 3 (SOD3) is highly expressed in both blood vessels and lungs. In different models of pulmonary injury, SOD3 is reduced; however, it is unclear whether this contributes to lung injury. To study the role of acute SOD3 reduction in lung injury, the SOD3 gene was deleted in adult mice by using the Cre-Lox technology. Acute reduction of SOD3 led to a fivefold increase in lung superoxide, marked inflammatory cell infiltration, a threefold increase in the arterial-alveolar gradient, respiratory acidosis, histological changes similar to those observed in adult respiratory distress syndrome, and 85% mortality. Treatment with the SOD mimetic MnTBAP and intranasal administration of SOD-containing polyketal microparticles reduced mortality, prevented the histological alterations, and reduced lung superoxide levels. To understand how mice with the SOD3 embryonic deletion survived without lung injury, gene array analysis was performed. These data demonstrated the up-regulation of 37 genes and down-regulation of nine genes, including those involved in cell signaling, inflammation, and gene transcription in SOD3 −/− mice compared with either mice with acute SOD3 reduction or wild-type controls. These studies show that SOD3 is essential for survival in the presence of ambient oxygen and that acute loss of this enzyme can lead to severe lung damage. Strategies either to prevent SOD3 inactivation or to augment its levels might prove useful in the treatment of acute lung injury.

The H+/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

Purpose of review This review summarizes the contribution of the Cl−/HCO3− exchanger pendrin in the renal regulation of blood pressure. Recent findings Intercalated 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. Summary Aldosterone 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.

The low Ca2+concentration ([Ca2+]) of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported (Yamauchi et al., Biochem Biophys Res Commun 331: 1353–1357, 2005) that the epithelial Ca2+channels TRPV5 and TRPV6 (transient receptor potential types 5 and 6) are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D3, as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca2+], and transepithelial potential of the utricle were measured in Cl−/HCO3−exchanger pendrin ( SLC26A4) knockout mice in vivo. Slc26a4−/−mice exhibit reduced pH and utricular endolymphatic potential and increased [Ca2+]. Monolayers of primary cultures of rat semicircular canal duct cells were grown on permeable supports, and cellular uptake of45Ca2+was measured individually from the apical and basolateral sides. Net uptake of45Ca2+was greater after incubation with 1,25-dihydroxyvitamin D3. Net45Ca2+absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum, and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [Ca2+] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca2+absorption.

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−/HCO3−exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, JCl, in mouse CCD through a pendrin-dependent mechanism. JCland transepithelial voltage, VT, 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, VTand JClwere not different from zero either in the presence or absence of angiotensin II (10−8M) 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 VTand absorbed Cl−. With angiotensin II in the bath, Cl−absorption doubled although VTdid not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl−secretion and a VTof ∼0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished VTalthough JClfell only ∼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.

Mechanisms of K+secretion and absorption along the collecting duct are not understood fully. Because KCNQ1 participates in K+secretion within the inner ear and stomach, distribution of KCNQ1 in mouse kidney was studied using Northern and Western analyses, RT-PCR of isolated tubules, and immunohistochemistry. Northern blots demonstrated KCNQ1 transcripts in whole kidney. RT-PCR showed KCNQ1 mRNA in isolated distal convoluted tubule (DCT), connecting segment (CNT), collecting ducts (CD), and glomeruli. Immunoblots of kidney and stomach revealed a ∼75-kDa protein, the expected mobility for KCNQ1. KCNQ1 was detected by immunohistochemistry throughout the distal nephron and CD. Thick ascending limbs exhibited weak basolateral immunolabel. In DCT and CNT cells, immunolabel was intense and basolateral, although KCNQ1 label was stronger in late than in early DCT. Initial collecting tubule and cortical CD KCNQ1 immunolabel was predominantly diffuse, but many cells exhibited discrete apical label. Double-labeling experiments demonstrated that principal cells, type B intercalated cells, and a few type A intercalated cells exhibited distinct apical KCNQ1 immunolabel. In inner medullary CD, principal cells exhibited distinct basolateral KCNQ1 immunolabel, whereas intercalated cells showed diffuse cytoplasmic staining. Thus KCNQ1 protein is widely distributed in mouse distal nephron and CD, with significant axial and cellular heterogeneity in location and intensity. These findings suggest that KCNQ1 has cell-specific roles in renal ion transport and may participate in K+secretion and/or absorption along the thick ascending limb, DCT, connecting tubule, and CD.

What is the central question of this study? Pregnancy requires a robust plasma volume expansion driven by renal sodium retention. In the late-pregnant kidney, the aldosterone-responsive epithelial Na(+) channel is increased, whereas the sodium-chloride cotransporter is decreased. Pendrin has been shown to support sodium reabsorption in the distal nephron and compensate for loss of the sodium-chloride cotransporter. We investigated the expression and abundance of pendrin in the pregnant kidney. What is the main finding and its importance? Pendrin protein, apical localization and thiazide sensitivity are increased in pregnancy. This implicates a possible role for pendrin in supporting the renal sodium chloride reabsorption and plasma volume expansion of pregnancy. Pregnancy is characterized by cumulative plasma volume expansion as a result of renal sodium retention, driven by activation of aldosterone. We previously reported that the abundance and activity of the aldosterone-responsive epithelial Na(+) channel is increased, whereas the sodium-chloride cotransporter (NCC) is decreased in the kidney of the late-pregnant rat. The chloride-bicarbonate exchanger pendrin is also aldosterone responsive and has been shown to support activity of the aldosterone-responsive epithelial Na(+) channel and compensate for the loss of NCC. Additionally, pendrin coupled to the sodium-dependent chloride-bicarbonate exchanger (NDCBE) mediates thiazide-sensitive sodium reabsorption in the cortical collecting duct. In this study, we investigated pendrin and NDCBE transcript expression, pendrin protein abundance, pendrin cellular localization and thiazide sensitivity in virgin, mid-pregnant and late-pregnant rats to test the hypothesis that increased pendrin activity might occur in pregnancy. By RT-PCR, NDCBE and pendrin mRNA expression was unchanged from virgins, whereas pendrin protein abundance determined by Western blotting was increased in both mid- and late-pregnant rats. The apical localization of pendrin was also increased in late-pregnant rats compared with virgins by immunohistochemistry. Pregnant rats displayed an increased natriuretic response to hydrochlorothiazide compared with virgins. Given that NCC expression is decreased in late pregnancy, an increased thiazide sensitivity may be due to inhibition of upregulated pendrin-NDCBE-coupled sodium reabsorption. Thus, increased pendrin in pregnant rats may compensate for the decreased NCC and aid in the renal sodium chloride reabsorption of pregnancy.

Epithelial Na+channel (ENaC) blockade stimulates stilbene-sensitive conductive Cl−secretion in the mouse cortical collecting duct (CCD). This study's purpose was to determine the co-ion that accompanies benzamil- and DIDS-sensitive Cl−flux. Thus transepithelial voltage, VT, as well as total CO2(tCO2) and Cl−flux were measured in CCDs from aldosterone-treated mice consuming a NaCl-replete diet. We reasoned that if stilbene inhibitors (DIDS) reduce conductive anion secretion they should reduce the lumen-negative VT. However, during ENaC blockade (benzamil, 3 μM), DIDS (100 μM) application to the perfusate reduced net H+secretion, which increased the lumen-negative VT. Conversely, ENaC blockade alone stimulated H+secretion, which reduced the lumen-negative VT. Application of an ENaC inhibitor to the perfusate reduced the lumen-negative VT, increased intercalated cell intracellular pH, and reduced net tCO2secretion. However, benzamil did not change tCO2flux during apical H+-ATPase blockade (bafilomycin, 5 nM). The increment in H+secretion observed with benzamil application contributes to the fall in VTobserved with application of this diuretic. As such, ENaC blockade reduces the lumen-negative VTby inhibiting conductive Na+absorption and by stimulating H+secretion by type A intercalated cells. In conclusion, 1) in CCDs from aldosterone-treated mice, benzamil application stimulates HCl secretion mediated by the apical H+-ATPase and a yet to be identified conductive Cl−transport pathway; 2) benzamil-induced HCl secretion is reversed with the application of stilbene inhibitors or H+-ATPase inhibitors to the perfusate; and 3) benzamil reduces VTnot only by inhibiting conductive Na+absorption, but also by stimulating H+secretion.

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

NCC reabsorbs approximately 10% of filtered NaCl, an amount vital for maintaining extracellular electrolyte and fluid homeostasis. Thiazide inhibition of NCC is used clinically to reduce extracellu...

Pendrin, encoded by Slc26a4, is a Cl−/HCO3−exchanger expressed in the apical region of type B and non-A, non-B intercalated cells, which regulates renal NaCl excretion. Dietary Cl−restriction upregulates total pendrin protein expression. Whether the subcellular expression of pendrin and whether the apparent vascular volume contraction observed in Slc26a4 null mice are Cl−dependent, but Na+independent, is unknown. Thus the subcellular distribution of pendrin and its role in acid-base and fluid balance were explored using immunogold cytochemistry and balance studies of mice ingesting a NaCl-replete or a Na+-replete, Cl−-restricted diet, achieved through substitution of NaCl with NaHCO3. Boundary length and apical plasma membrane pendrin label density each increased by ∼60–70% in type B intercalated cells, but not in non-A, non-B cells, whereas cytoplasmic pendrin immunolabel increased ∼60% in non-A, non-B intercalated cells, but not in type B cells. Following either NaCl restriction or Cl−restriction alone, Slc26a4 null mice excreted more Cl−and had a higher arterial pH than pair-fed wild-type mice. In conclusion, 1) following dietary Cl−restriction, apical plasma membrane pendrin immunolabel increases in type B intercalated cells, but not in non-A, non-B intercalated cells; and 2) pendrin participates in the regulation of renal Cl−excretion and arterial pH during dietary Cl−restriction.

This review will summarize newly described novel functions of renal intercalated cells.Over the past 20 years, the importance of intercalated cells in the process of renal net acid excretion has been recognized. More recently, many of the molecular mechanisms responsible for this cellular function have been described. Functionally, type A and type B intercalated cells are largely mirror images in that type A intercalated cells are H secreting cells, whereas type B intercalated cells are OH secreting cells. Whether non-A, non-B intercalated cells represent H or OH secreting cells or whether they interconvert between these functions is unclear. Transporters such as pendrin (Slc26a4, Pds), AE1 (Slc4a1), the H-ATPase, and NBC3 (Slc4a7) contribute to the ability of intercalated cells to secrete H or OH equivalents. In addition to mediating secretion of H or OH equivalents, however, intercalated cells also regulate vascular volume, and hence blood pressure, likely by regulating renal Cl excretion.The molecular mechanisms of net H/OH secretion by intercalated cell subsets have been largely defined over the past decade. Moreover, targeted genetic disruption of these transporters has revealed novel roles, such as blood pressure regulation. Thus, some of these transporters might be the target of future antihypertensive drugs.