Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency.
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ABSTRACT:
SUBMITTER: Lin MH
PROVIDER: S-EPMC8564913 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.
| S-EPMC5198284 | biostudies-literature
Project description:ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ?10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells.
| S-EPMC5004338 | biostudies-literature
Project description:The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid-secreting A- and base secreting B-type of intercalated cells cannot be easily separated in functional studies. We used BCECF-sensitive intracellular pH (pHi ) measurements in split-opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell-specific ClC-K2-/- mice to demonstrate that ClC-K2 inhibition enables to distinguish signals from A- and B-intercalated cells. We show that ClC-K2 Cl- channel is expressed on the basolateral side of intercalated cells, where it governs Cl- -dependent H+ /HCO3- transport. ClC-K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC-K2-/- mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pHi in AE1-positive A-type and decreased pHi in pendrin-positive B-type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H+ secretion in A-type and decreased proton transport in B-type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance. Overall, we show that inhibition of ClC-K2 can be employed to discriminate between A- and B-type of intercalated cells in split-opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A- and B-type in response to systemic acid/base stimuli.
| S-EPMC9014849 | biostudies-literature
Project description:UnlabelledWe have utilized the persistent elevation of fractional excretion (FE) of urate, > 10%, to differentiate cerebral/renal salt wasting (RSW) from the syndrome of inappropriate antidiuretic hormone secretion (SIADH), in which a normalization of FEurate occurs after correction of hyponatremia. Previous studies suggest as well that an elevated FEurate with normonatremia, without pre-existing hyponatremia, is also consistent with RSW, including studies demonstrating induction of RSW in rats infused with plasma from normonatremic neurosurgical and Alzheimer's disease patients. The present studies were designed to test whether precipitates from the urine of normonatremic neurosurgical patients, with either normal or elevated FEurate, and patients with SIADH, display natriuretic activity. MethodsAmmonium sulfate precipitates from the urine of 6 RSW and 5 non-RSW Control patients were dialyzed (10 kDa cutoff) to remove the ammonium sulfate, lyophilized, and the reconstituted precipitate was tested for its effect on transcellular transport of (22)Na across LLC-PK1 cells grown to confluency in transwells.ResultsPrecipitates from 5 of the 6 patients with elevated FEurate and normonatremia significantly inhibited the in vitro transcellular transport of (22)Na above a concentration of 3 μg protein/ml, by 10-25%, versus to vehicle alone, and by 15-40% at concentrations of 5-20 μg/ml as compared to precipitates from 4 of the 5 non-RSW patients with either normal FEurate and normonatremia (2 patients) or with SIADH (2 patients).ConclusionThese studies provide further evidence that an elevated FEurate with normonatremia is highly consistent with RSW. Evidence in the urine of natriuretic activity suggests significant renal excretion of the natriuretic factor. The potentially large source of the natriuretic factor that this could afford, coupled with small analytical sample sizes required by the in-vitro bioassay used here, should facilitate future experimental analysis and allow the natriuretic factor to be investigated as a potential biomarker for RSW.
| S-EPMC3752684 | biostudies-literature
Project description:Endothelin (ET)-1 promotes natriuresis via the endothelin B receptor (ETB) within the renal medulla. In male rats, direct interstitial infusion of ET-1 into the renal medulla has no effect on renal sodium and water excretion but is associated with endothelin A receptor (ETA)-dependent reductions in medullary blood flow. Loss of ETB function leads to salt-sensitive hypertension. We hypothesized that HS intake would increase the natriuretic and diuretic response to renal medullary infusion of ET peptides.Male Sprague-Dawley (SD) rats were fed a normal (NS) or high (HS) salt diet for 7days. Rats were anesthetized and a catheter implanted in the renal medulla for interstitial infusion along with a ureteral catheter for urine collection. Medullary infusion of a low dose of ETB receptor agonist, sarafotoxin 6c (S6c; 0.15μg/kg/h), or ET-1 (0.45μg/kg/h) was used to determine changes in sodium excretion (UNaV).In HS fed rats, intramedullary infusion of a low dose of S6c induced a significant increase in UNaV, roughly 2-fold over baseline, compared to no response to this low dose in NS fed rats. In HS fed rats, intramedullary infusion of ET-1 induced a significantly greater increase in UNaV compared to NS fed rats, although this increase was not different from the HS time control studies.We conclude that high salt intake enhances the diuretic and natriuretic effects of ETB receptor activation in vivo consistent with a role for the ETB receptor in maintaining fluid-electrolyte homeostasis.
| S-EPMC5384466 | biostudies-other
Project description:The ability of animals to process dynamic sensory information facilitates foraging in an ever-changing environment. However, molecular and neural mechanisms underlying such ability remain elusive. The ClC anion channels/transporters play a pivotal role in cellular ion homeostasis across all phyla. Here, we find a ClC chloride channel is involved in salt concentration chemotaxis of Caenorhabditis elegans. Genetic screening identified two altered-function mutations of clh-1 that disrupt experience-dependent salt chemotaxis. Using genetically encoded fluorescent sensors, we demonstrate that CLH-1 contributes to regulation of intracellular anion and calcium dynamics of salt-sensing neuron, ASER. The mutant CLH-1 reduced responsiveness of ASER to salt stimuli in terms of both temporal resolution and intensity, which disrupted navigation strategies for approaching preferred salt concentrations. Furthermore, other ClC genes appeared to act redundantly in salt chemotaxis. These findings provide insights into the regulatory mechanism of neuronal responsivity by ClCs that contribute to modulation of navigation behavior.
| S-EPMC7834019 | biostudies-literature
Project description:The renal collecting duct plays a critical role in setting urinary volume and composition, with principal cells transporting Na+ and K+ and intercalated cells mediating Cl- reabsorption. Published evidence implies Angiotensin II (Ang II) is a potent regulator of the collecting duct apical transport systems in response to systemic volume depletion. However, virtually nothing is known about Ang II actions on the basolateral conductance of principal and intercalated cells. Here, we combined macroscopic and single channel patch clamp recordings from freshly isolated mouse collecting ducts with biochemical and fluorescence methods to demonstrate an acute stimulation of the basolateral Cl- conductance and specifically the ClC-K2 Cl- channel by nanomolar Ang II concentrations in intercalated cells. In contrast, Ang II did not exhibit measurable effects on the basolateral conductance and on Kir4.1/5.1 potassium channel activity in principal cells. Although both Ang II receptors AT1 and AT2 are expressed in collecting duct cells, we show that AT1 receptors were essential for stimulatory actions of Ang II on ClC-K2. Moreover, AT1R-/- mice had decreased renal ClC-K2 expression. We further demonstrated that activation of NADPH oxidases is the major signaling pathway downstream of Ang II-AT1R that leads to stimulation of ClC-K2. Treatment of freshly isolated collecting ducts with Ang II led to production of reactive oxygen species on the same timescale as single channel ClC-K2 activation. Overall, we propose that Ang II-dependent regulation of ClC-K2 in intercalated cells is instrumental for stimulation of Cl- reabsorption by the collecting duct, particularly during hypovolemic states.
| S-EPMC7949157 | biostudies-literature
Project description:Hypoxia inducible factor (HIF)-1alpha, a transcription factor, is abundantly expressed in the renal medulla and regulates many oxygen-sensitive genes such as nitric oxide synthase, cyclooxygenase-2, and heme oxygenase-1. Given the important roles of these genes in the control of arterial pressure, the present study was to test the hypothesis that HIF-1alpha-mediated gene activation serves as an antihypertensive pathway by regulating renal medullary function and sodium excretion. HIF-1alpha decoy oligodeoxynucleotides (ODNs) or scrambled ODNs were transfected into the renal medulla in uninephrectomized Sprague-Dawley rats. Two weeks after ODN transfection, the HIF-1alpha binding activities were significantly inhibited by 45%, and high salt-induced increases of nitric oxide synthase-2 and heme oxygenase-1 transcriptions were also inhibited by 70% and 61% in the renal medulla from decoy rats. The natriuretic responses and increases of renal medullary blood flow responding to the elevations of renal perfusion pressure were significantly blunted by 50% and 37% in decoy rats. Intravenously acute sodium loading increased medullary blood flow and urinary sodium excretion, which was remarkably attenuated in decoy rats. In decoy rats, high salt intake caused a greater positive sodium balance. Consequently, arterial pressure was remarkably increased (from 118+/-1.9 to 154+/-6.3 mm Hg) in decoy rats but not in control rats when the rats were challenged with a high salt diet. There was no blood pressure change in decoy rats that were maintained in normal salt diet. In conclusion, HIF-1alpha-mediated gene activation importantly participates in the regulation of renal medullary function and long-term arterial blood pressure.
| S-EPMC2649696 | biostudies-literature
Project description:UnlabelledAdult stem cell deficiency has been implicated in the pathogenic mechanism for various diseases. Renal medullary dysfunction is one of the major mechanisms for the development of hypertension in Dahl salt-sensitive (S) rats. The present study first detected a stem cell deficiency in the renal medulla in Dahl S rats and then tested the hypothesis that transplantation of mesenchymal stem cells (MSCs) into the renal medulla improves salt-sensitive hypertension in Dahl S rats. Immunohistochemistry and flowcytometry analyses showed a significantly reduced number of stem cell marker CD133+ cells in the renal medulla from Dahl S rats compared with controls, suggesting a stem cell deficiency. Rat MSCs or control cells were transplanted into the renal medulla in uninephrectomized Dahl S rats, which were then treated with a low- or high-salt diet for 20 days. High-salt-induced sodium retention and hypertension was significantly attenuated in MSC-treated rats compared with control cell-treated rats. Meanwhile, high-salt-induced increases of proinflammatory factors, monocyte chemoattractant protein-1, and interleukin-1β, in the renal medulla were blocked by MSC treatment. Furthermore, immunostaining showed that high-salt-induced immune cell infiltration into the renal medulla was substantially inhibited by MSC treatment. These results suggested that stem cell defect in the renal medulla may contribute to the hypertension in Dahl S rats and that correction of this stem cell defect by MSCs attenuated hypertension in Dahl S rats through anti-inflammation.Key messageStem cell defect in the renal medulla may contribute to salt-sensitive hypertension Stem cell therapy is a potential therapeutic strategy for salt-sensitive hypertension Normal stem cell inhibits the inflammatory response to high salt in the renal medulla.
| S-EPMC4334761 | biostudies-literature
Project description:COX (cyclooxygenase)-derived prostaglandins regulate renal hemodynamics and salt and water homeostasis. Inhibition of COX activity causes blood pressure elevation. In addition, chronic analgesic abuse can induce renal injury, including papillary necrosis. COX-2 is highly expressed in the kidney papilla in renal medullary interstitial cells (RMICs). However, its role in blood pressure and papillary integrity in vivo has not been definitively studied. In mice with selective, inducible RMIC COX-2 deletion, a high-salt diet led to an increase in blood pressure that peaked at 4 to 5 weeks and was associated with increased papillary expression of AQP2 (aquaporin 2) and ENac (epithelial sodium channel) and decreased expression of cystic fibrosis transmembrane conductance regulator. With continued high-salt feeding, the mice with RMIC COX-2 deletion had progressive decreases in blood pressure from its peak. After return to a normal-salt diet for 3 weeks, blood pressure remained low and was associated with a persistent urinary concentrating defect. Within 2 weeks of institution of a high-salt diet, increased apoptotic RMICs and collecting duct cells could be detected in papillae with RMIC deletion of COX-2, and by 9 weeks of high salt, there was a striking loss of the papillae. Therefore, RMIC COX-2 expression plays a crucial role in renal handling water and sodium homeostasis, preventing salt-sensitive hypertension and maintaining structural integrity of papilla.
| S-EPMC6207229 | biostudies-literature