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The sodium-hydrogen exchanger isoform 1 (NHE1) is a plasma membrane transporter with roles in transepithelial Na+/H+ transport, intracellular pH (pHi) and cell volume regulation. Within the kidney, NHE1 is previously documented to be expressed in all nephron segments. However, recent transcriptomic and proteomic analyses, and our staining results in mice, consistently find no expression of NHE1 in the proximal tubule or thick ascending limb, but in the distal nephron, including distal convoluted tubule, connecting tubule, and collecting duct (principal and intercalated cells, PC and IC, respectively). We hypothesized that basolateral NHE1 in IC (co-labelling with H+-ATPase B1 subunit, labeling all 3 types of IC: type A, B, and non-A-non-B) plays an important role in acid-base homeostasis. Due to lack of a suitable animal model, we generated kidney-specific NHE1 knockout (NHE1KS-KO) mice. NHE1KS-KO mice completely lack renal NHE1 protein expression compared to their control littermates (n=6 male mice/genotype). No differences were observed in body weight, food and water intake (determined in their home cages) or concentrations of blood Na+, K+, Ca2+, pH or HCO3-. Urine analysis showed no significant differences in electrolytes, minerals of pH. To mechanistically determine the role of NHE1 for pHi regulation in IC and PC we used confocal microscopy of acutely-split open cortical collecting ducts. This method allows for the analysis of a vast number of PC/IC simultaneously. To study the role of NHE1 we employed a NH4Cl pre-pulse (40 mM) and measured pHi recovery after switching back to a physiological solution. Intracellular pH recovery in PC (n=171) was significantly slower (~50%) in NHE1KS-KO mice compared to controls (n=85). Similar to PC, pHi recovery in type B IC of NHE1KS-KO mice (n=38) was significantly slower (~35%) compared to controls (n=40). No differences in type A IC were observed between NHE1KS-KO mice (n=35) and controls (n=29). Inhibition of ClC-K2 via NPPB (Cl-channel blocker) was used to confirm cell types: PC did not respond with a change in pHi but NPPB increased pHi in type A and decreased pHi in type B ICs. In summary, our results identify for the first time an important role of NHE1 for pHi regulation particularly in PCs and type B ICs. Funding to Dr. Rieg was provided by the NIDDK grant 1R01DK110621, VA Merit Review Award IBX004968A, AHA Transformational Research Award 19TPA34850116, and NIDDK Diabetic Complications Consortium grants DK076169 and DK115255. DK095029 and DK117865 (both to O. Pochynyuk). Dr. Thomas was supported by an AHA postdoctoral grant (828731), V. Tomilin by an AHA Career Development Award 19CDA34660148. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypertension is the major clinically relevant problem affecting millions of people in the US. The renal collecting duct is known to play a critical role in setting salt-sensitivity of blood pressure. Originally, the majority of clinical and basic effort has been devoted to defining the detrimental roles of elevated dietary Na+ in pathophysiology of hypertension. However, emerging evidence indicates that the dietary Cl- is as important for blood pressure control. The collecting duct has two independent types of cells: principal and intercalated having different functions and physiological roles. Principal cells regulate Na+ reabsorption, whereas intercalated cells are responsible for transport of Cl-. ClC-K2 is a Cl- permeable channel abundantly expressed on the basolateral membrane of several distal nephron segments from the thick ascending limb to the collecting duct. Loss-of-function mutations in ClC-Kb (human orthologue of ClC-K2) cause Bartter’s syndrome type 3, which is associated with urinary salt wasting and hypotension. While the function of ClC-Kb in the thick ascending limb and distal convoluted tubule are thought to be responsible for the salt-wasting phenotype in patients with Bartter’s syndrome, the role of the channel in the collecting duct intercalated cells is not known. To address this critical gap, we compared the blood pressure phenotype and systemic Cl- balance in newly created mice with deletion of ClC-K2 in the renal nephron (RN-KO) and selectively in intercalated cells (IC-KO) using cre-loxP strategy. Since the commonly used Na+ deficient diet only partially reduces chloride content and does not lead to appreciable reduction in blood pressure in rodents, we used the diet lacking both Na+ and Cl-. As anticipated, RN-KO ClC-K2 mice had lower blood pressure on both regular and NaCl deficient diets when compared to the respective genetic controls (WT). In addition, the mutants exhibited larger urinary volume and Cl- wasting in response to dietary Cl- restriction. Importantly, IC-KO ClC-K2 mice had comparable blood pressure on regular diet, but became more hypotonic when compared to WT in response to dietary NaCl deficiency. Moreover, patch clamp studies in freshly isolated collecting ducts from WT mice revealed that ClC-K2 activity is proportionally increased to the amount of Cl- reduction in a diet suggesting that ClC-K2 is instrumental in augmenting Cl- reabsorption during Cl- deficiency.In conclusion, our results demonstrate, for the first time, the significance of ClC-K2 function in the collecting duct intercalated cells in setting salt sensitivity of blood pressure. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-19CDA34660148 (to V. N. Tomilin). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanical stress arising from elevated fluid flow is an important regulator of the water-electrolyte transport in the collecting duct (CD). It is well established that mechano-sensitive TRPV4 Ca2+-permeable channel mediates intracellular Ca2+ ([Ca2+]i) responses to high flow in these cells. Piezo1, a Ca2+-permeable channel directly activated by the plasma membrane stretch, is a recent arrival at this scene. Piezo1 expression has been reported in the CD. However, its functional significance is yet to be determined. Here, we applied a combination of Ca2+ imaging and confocal microscopy in freshly isolated split-opened CDs to investigate a potential interplay between TRPV4 and Piezo1 in mediating [Ca2+]i signaling in the CD. Immunofluorescent analysis of split-opened CDs showed presence of Piezo1 in both AQP2-positive (principal cells, PC) and -negative (intercalated cells, IC) with higher intensity in the former. Importantly, Piezo1 was apparent on both apical and basolateral membrane of CD cells. Piezo1 agonists, Yoda-1 (20μM) and Jedi2 (500μM) acutely increase [Ca2+]i in both principal and intercalated cells. The responses were abolished when extracellular Ca2+ was buffered with EGTA suggesting a direct Ca2+ influx via Piezo1. Interestingly, we detected two disparate calcium responses to Piezo1 activation with the majority (70%) cells showing a fast transient response with a sustained plateau, whereas the remaining (30%) population had much slower gradual calcium increase to the same plateau level. Subsequent immunofluorescent analysis revealed that fast Ca2+ responses were attributed to PC, whereas responses with slow kinetics corresponded to IC. Consistently, PCs have ~3 fold higher Piezo-1 signal when compared to neighbor ICs. We next tested whether Piezo1 activity depends on TRPV4 status. Genetic deletion and pharmacological inhibition of TRPV4 with GSK2193874 did not affect Piezo1-dependent elevations in [Ca2+]i. However, high K+ diet, which is known to elevate the flow in the CD and augment both TRPV4 activity and expression, led to diminished Piezo1 responses to Yoda-1. In summary, our results are consistent with the view that Piezo1 and TRPV4 are functional in both principal and intercalated cells of the collecting duct and operate independently to modulate mechanosensitive [Ca2+]i signaling. We hypothesize that augmented TRPV4-dependent [Ca2+]i is compensated by the diminished activity of Piezo1 to account for elevated flow rate upon increased K+ intake. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-19CDA34660148 (to V. N. Tomilin). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanosensitive Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel plays a dominant role in maintaining intracellular Ca2+ ([Ca2+]i) homeostasis and flow-sensitive [Ca2+]i signaling in the distal segments of renal tubule and collecting duct. Polycystic kidney disease (PKD) manifests as progressive cyst growth with cystic cells exhibiting exacerbated cAMP-dependent fluid secretion along with deficient mechanosensitivity and reduced basal [Ca2+]i levels, and impaired TRPV4 activity. In this study, we tested how regulation of renal TRPV4 function by elevated dietary K+ intake modulates the rate of cystogenesis and mechanosensitive [Ca2+]i signaling in cystic cells of PCK453 rats (1 m.o.), a homologous model of human autosomal recessive PKD (ARPKD), where cyst development is restricted to the collecting duct. One month treatment with both high KCl (5% K+) and KB/C (5% K+ with bicarbonate to citrate in 4:1 ratio) diets significantly increased kidney TRPV4 levels in PCK453 rats when compared to the control group fed regular diet (0.9% K+). High KCl diet also caused an increased TRPV4-dependent Ca2+ influx, higher basal [Ca2+]i, and partial restoration of mechanosensitivity in freshly isolated monolayers of cystic cells. Unexpectedly, high KB/C diet induced an opposite effect by reducing TRPV4 activity and further worsening [Ca2+]i homeostasis. Importantly, high KCl diet decreased, whereas high KB/C diet further increased cAMP levels, as was assessed by the extent of cAMP-dependent translocation of AQP2 water channel to apical membrane in cystic cells. At the systemic level, high KCl diet fed PCK453 rats had significantly lower kidney-to-bodyweight ratio and reduced cystic area in renal sections. The beneficial effects of high KCl diet were negated by a concomitant administration of an orally active TRPV4 antagonist, GSK2193874, resulting in greater kidney weight and accelerated cystogenesis, when compared to these values in control rats. In a similar manner, high KB/C diet exacerbated renal manifestations of ARPKD, consistent with deficient TRPV4 activity in cystic cells. Moreover, KCl+GSK2193874 and high KB/C treated PCK453 rats have increased urinary levels of a renal injury marker, KIM-1.In conclusion, we demonstrate that TRPV4 channel activity negatively regulates cAMP levels in cystic cells upon variations in K+ intake thus attenuating (high activity) or accelerating (low activity) ARPKD progression in PCK453 rats. We propose that pharmacological or dietary means leading to TRPV4 stimulation might be useful to counteract PKD progression in the clinical setting. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-19CDA34660148 (to V. N. Tomilin). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Distal segments of the renal tubule have a major role in controlling whole body K+ homeostasis by performing flow-induced K+ secretion (FIKS) during K+ load and K+ reabsorption in response to systemic K+ deficiency. The transient receptor potential vanilloid type 4 (TRPV4) Ca2+-permeable channel serves as the sensor of tubular flow thus being well-suited to govern K+ transport in the distal renal tubule. In the current manuscript, we directly tested whether TRPV4 function in the renal tubule is significant in affecting K+ balance. For this, we employed balance metabolic cage studies and systemic measurements with different K+ feeding regimens: high (5% K+), regular (0.9% K+), and low (+) in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate genetic controls (TRPV4fl/fl). Successful deletion was verified by the absence of TRPV4 protein expression in renal homogenates and the lack of TRPV4-mediated Ca2+ influx in freshly-isolated split-opened collecting ducts. There were no differences in plasma electrolytes, baseline urinary volume, and K+ levels when both strains were fed regular K+ intake. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on both high and low K+ intake. This was associated with a marked decrease in 24 h urinary K+ levels in the knockout, whereas urinary volume and aldosterone levels were indistinguishable from the control TRPV4fl/fl littermates suggesting a direct decrease in the distal tubule K+ transport. Interestingly, we also detected significant differences in urinary pH levels indicative of the altered K+ reabsorption via H+-K+ ATPase in the collecting duct. Indeed, we detected a significantly faster pHi recovery after intracellular acidification with ammonium pulse in both acid secreting A- and base-secreting B-types of intercalated cells and to a lesser degree in principal cells in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice on low but not regular K+ intake, which is consistent with augmented H+-K+ ATPase activity and K+ reabsorption in the knockouts. In summary, our results demonstrate an indispensable pro-kaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-19CDA34660148 (to V. N. Tomilin). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the β and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. KEY POINTS: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel. We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability. Photoaffinity labelling experiments showed specific binding to the β and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes. Taurocholic acid regulation of ENaC was voltage-dependent, providing evidence for binding to pore-forming helices. Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems.

Tight regulation of K(+) balance is fundamental for normal physiology. Reduced dietary K(+) intake, which is common in Western diets, often leads to hypokalemia and associated cardiovascular- and kidney-related pathologies. The distal nephron, and, specifically, the collecting duct (CD), is the major site of controlled K(+) reabsorption via H(+)-K(+)-ATPase in the state of dietary K(+) deficiency. We (Mamenko MV, Boukelmoune N, Tomilin VN, Zaika OL, Jensen VB, O'Neil RG, Pochynyuk OM. Kidney Int 91: 1398–1409, 2017) have previously demonstrated that the transient receptor potential vanilloid type 4 (TRPV4) Ca(2+) channel, abundantly expressed in the CD, contributes to renal K(+) handling by promoting flow-induced K(+) secretion. Here, we investigated a potential role of TRPV4 in controlling H(+)-K(+)-ATPase-dependent K(+) reabsorption in the CD. Treatment with a K(+)-deficient diet (

Autosomal-dominant polycystic kidney disease (ADPKD) is a devastating disorder that is characterized by a progressive decline in renal function as a result of the development of fluid-filled cysts. Defective flow-mediated [Ca(2+)](i) responses and disrupted [Ca(2+)](i) homeostasis have been repeatedly associated with cyst progression in ADPKD. We have previously demonstrated that the transient receptor potential vanilloid type 4 (TRPV4) channel is imperative for flow-mediated [Ca(2+)](i) responses in murine distal renal tubule cells. To determine whether compromised TRPV4 function contributes to aberrant Ca(2+) regulation in ADPKD, we assessed TRPV4 function in primary cells that were cultured from ADPKD and normal human kidneys (NHKs). Single-channel TRPV4 activity and TRPV4-dependent Ca(2+) influxes were drastically reduced in ADPKD cells, which correlated with distorted [Ca(2+)](i) signaling. Whereas total TRPV4 protein levels were comparable in NHK and ADPKD cells, we detected a marked decrease in TRPV4 glycosylation in ADPKD cells. Tunicamycin-induced deglycosylation inhibited TRPV4 activity and compromised [Ca(2+)](i) signaling in NHK cells. Overall, we demonstrate that TRPV4 glycosylation and channel activity are diminished in human ADPKD cells compared with NHK cells, and that this contributes significantly to the distorted [Ca(2+)](i) dynamics. We propose that TRPV4 stimulation may be beneficial for restoring [Ca(2+)](i) homeostasis in cyst cells, thereby interfering with ADPKD progression.—Tomilin, V., Reif, G. A., Zaika, O., Wallace, D. P., Pochynyuk, O. Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca(2+)](i) homeostasis in human autosomal-dominant polycystic kidney disease cells.

ENaC-mediated sodium reabsorption in the collecting duct (CD) is a critical determinant of urinary sodium excretion. Existing evidence suggest direct stimulatory actions of Angiotensin II (Ang II) on ENaC in the CD, independently of the aldosterone- mineralocorticoid receptor (MR) signaling. Deletion of the major renal AT(1) receptor isoform, AT(1a)R, decreases blood pressure and reduces ENaC abundance despite elevated aldosterone levels. The mechanism of this insufficient compensation is not known. Here, we used patch clamp electrophysiology in freshly isolated split-opened CDs to investigate how AT(1a)R dysfunction compromises functional ENaC activity and its regulation by dietary salt intake. Ang II had no effect on ENaC activity in CDs from AT(1a)R −/− mice suggesting no complementary contribution of AT(2) receptors. We next found that AT(1a)R deficient mice had lower ENaC activity when fed with low (