Inhibition Of NaCl Cotransporter Research Articles

DCT Transcriptional and Structural Remodeling: Impacts on Magnesium Handling

Loss of activity of the NaCl Cotransporter (NCC) in Gitelman syndrome and with chronic thiazide treatment results in hypomagnesemia but the underlying mechanism remains unresolved. The prevailing hypothesis is that it results indirectly from distal convoluted tubule (DCT) atrophy lowering magnesium (Mg)-reabsorbing capacity by this segment. We tested the hypothesis that NCC inhibition directly disrupts a cellular mechanism related to Mg handling independent of DCT atrophy. We used the NCC-Cre+−/−- INTACT+/- mouse line to allow enrichment of DCT nuclei via Cre-dependent GFP expression in DCT nuclei. Eight- to 10-week-old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days; littermate controls receiving only vehicle. After nuclei isolation, GFP+ events (DCT nuclei) were sorted by fluorescence assisted nuclei sorting. Purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat. Tubule morphometrics using optical tissue clearing was performed on kidneys from animals receiving the same treatment. MTZ-treated mice had lower plasma Mg compared to controls recapitulating thiazide-induced hypomagnesemia. Our snRNAseq dataset containing 45,953 cells from 3 MTZ-treated and 3 control mice showed clear separation into early DCT (DCT1) and late DCT (DCT2) based on canonical segment markers. Gene expression analysis showed the DCT1 contained a magnesium cassette consisting of known magnesiotropic genes Trpm6, Trpm7, Egf, Umod, Prox1, Fxyd2, Cnnm2, and Slc41a3 so we focused subsequent analyses on DCT1. NCC inhibition caused DCT1 degeneration as indicated by lower mean DCT1 score of DCT1 cells and a greater proportion of DCT1 cells with low differentiation state in the MTZ group. Similarly, MTZ-treated samples had lower DCT1/DCT2 cell proportion in our snRNAseq dataset and morphometrics revelead significantly shorter DCT1 tubules implying that the degeneration process eventually leads to DCT1 atrophy. Using the aggregate expression of the magnesium cassette genes, we computationally defined a Mg score as a surrogate marker for Mg reabsorption capacity for each individual cells. The average Mg score was lower in the MTZ-treated group compared to the controls. Our data supports the long-standing hypothesis that hypomagnesemia is a secondary consequence of the loss of effective surface area for Mg reabsorption. Additionally, we uncovered that even at the single cell level, the Mg handling capacity of the cell decreases as a result of NCC inhibition implying that a disruption in sodium reabsorption disrupts expression of the Mg reabsorption machinery. NIH DK51496, NIH DK054983, NIDDK DK132066, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP. This is the full abstract presented at the American Physiology Summit 2024 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.

Kir4.1 is involved in Bradykinin‐induced inhibition of NCC and natriuresis

Stimulation of type II bradykinin (BK) receptor (BK2R) has been shown to increase renal Na+ excretion. However, the mechanism by which BK stimulates natriuresis is not completely understood. Immunohistochemical study demonstrates that BK2R is highly expressed in both apical and basolateral membrane of Kir4.1 positive tubules including thick ascending limb, distal convoluted tubule (DCT) and cortical collecting duct. The aim of the present study is to explore the role of BK in regulating the basolateral K+ channel and apical Na‐Cl cotransporter (NCC) in the DCT. The single channel patch‐clamp experiments show that application of BK (1–10 μM) inhibited the basolateral 40 pS K+ channel (a Kir4.1/5.1 heterotetramer) and this effect was blocked by BK2R antagonist (HOE140) but not by BK1R antagonist. Whole‐cell recordings have also shown that BK decreased the basolateral K+ conductance of the DCT and depolarized the membrane. To examine the role of Kir4.1 in mediating BK‐induced natriuresis, we performed renal clearance experiment in WT and in kidney‐specific conditional Kir4.1 knockout (Ks‐Kir4.1 KO) mice. Acute application of BK increased urinary Na+ and K+ excretion. However, the BK‐induced natriuretic effect was completely abolished in Ks‐Kir4.1 KO mice. The results strongly suggest that Kir4.1 activity is required for BK‐induced natriuresis. Our previous experiments demonstrated that Kir4.1 plays an important role in determining NCC activity such that the deletion of Kir4.1 completely inhibited NCC in the DCT. To test the hypothesis that BK‐induced inhibition of Kir4.1 may cause the inhibition of NCC thereby increasing Na+ and K+ excretion, we examine the basolateral Kir4.1 activity, the expression of NCC and thiazide‐induced natriuresis in the mice with 3 day infusion of BK by an osmotic pump. The infusion of BK for 3 days significantly decreased the basolateral Kir4.1 conductance and the negativity of the DCT membrane. Western blot showed that infusion of BK decreased the expression of total NCC and phosphorylated NCC. Renal clearance experiments demonstrated that thiazide‐induced natriuresis was blunted in the mice receiving BK infusion, suggesting that BK infusion inhibited NCC function in the DCT. Consequently, mice received BK infusion for 3 days were hypokalemic, presumably due to increasing Na and volume delivery to the collecting duct. We conclude that BK2R plays a role in tonic inhibition of the basolateral Kir4.1 channels in the DCT and that BK‐induced inhibition of NCC is, at least in part, responsible for BK‐induced stimulation of Na excretion. We also conclude that BK‐induced inhibition of Kir4.1 in the DCT is an essential step for the inhibition of NCC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Epithelial Sodium Channel ENaC and the Apical Cl-/HCO3- Exchanger Pendrin in Compensatory Salt Reabsorption in the Setting of Na-Cl Cotransporter (NCC) Inactivation.

BackgroundThe absence of NCC does not cause significant salt wasting in NCC deficient mice under basal conditions. We hypothesized that ENaC and pendrin play important roles in compensatory salt absorption in the setting of NCC inactivation, and their inhibition and/or downregulation can cause significant salt wasting in NCC KO mice.MethodsWT and NCC KO mice were treated with a daily injection of either amiloride, an inhibitor of ENaC, or acetazolamide (ACTZ), a blocker of salt and bicarbonate reabsorption in the proximal tubule and an inhibitor of carbonic anhydrases in proximal tubule and intercalated cells, or a combination of acetazolamide plus amiloride for defined durations. Animals were subjected to daily balance studies. At the end of treatment, kidneys were harvested and examined. Blood samples were collected for electrolytes and acid base analysis.ResultsAmiloride injection significantly increased the urine output (UO) in NCC KO mice (from 1.3 ml/day before to 2.5 ml/day after amiloride, p<0.03, n = 4) but caused only a slight change in UO in WT mice (p>0.05). The increase in UO in NCC KO mice was associated with a significant increase in sodium excretion (from 0.25 mmol/24 hrs at baseline to 0.35 mmol/24 hrs after amiloride injection, p<0.05, n = 4). Daily treatment with ACTZ for 6 days resulted in >80% reduction of kidney pendrin expression in both WT and NCC KO mice. However, ACTZ treatment noticeably increased urine output and salt excretion only in NCC KO mice (with urine output increasing from a baseline of 1.1 ml/day to 2.3 ml/day and sodium excretion increasing from 0.22 mmole/day before to 0.31 mmole/day after ACTZ) in NCC KO mice; both parameters were significantly higher than in WT mice. Western blot analysis demonstrated significant enhancement in ENaC expression in medulla and cortex of NCC KO and WT mice in response to ACTZ injection for 6 days, and treatment with amiloride in ACTZ-pretreated mice caused a robust increase in salt excretion in both NCC KO and WT mice. Pendrin KO mice did not display a significant increase in urine output or salt excretion after treatment with amiloride or ACTZ.Conclusion1. ENaC plays an important role in salt reabsorption in NCC KO mice. 2. NCC contributes to compensatory salt reabsorption in the setting of carbonic anhydrase inhibition, which is associated with increased delivery of salt from the proximal tubule and the down regulation of pendrin. 3. ENaC is upregulated by ACTZ treatment and its inhibition by amiloride causes significant diuresis in NCC KO and WT mice. Despite being considered mild agents individually, we propose that the combination of acetazolamide and amiloride in the setting of NCC inhibition (i.e., hydrochlorothiazide) will be a powerful diuretic regimen.

Acute inhibition of NCC does not activate distal electrogenic Na+reabsorption or kaliuresis

Na+ reabsorption from the distal renal tubule involves electroneutral and electrogenic pathways, with the latter promoting K+ excretion. The relative activities of these two pathways are tightly controlled, participating in the minute-to-minute regulation of systemic K+ balance. The pathways are interdependent: the activity of the NaCl cotransporter (NCC) in the distal convoluted tubule influences the activity of the epithelial Na+ channel (ENaC) downstream. This effect might be mediated by changes in distal Na+ delivery per se or by molecular and structural adaptations in the connecting tubule and collecting ducts. We hypothesized that acute inhibition of NCC activity would cause an immediate increase in Na+ flux through ENaC, with a concomitant increase in renal K+ excretion. We tested this using renal clearance methodology in anesthetized mice, by the administration of hydrochlorothiazide (HCTZ) and/or benzamil (BZM) to exert specific blockade of NCC and ENaC, respectively. Bolus HCTZ elicited a natriuresis that was sustained for up to 110 min; urinary K+ excretion was not affected. Furthermore, the magnitude of the natriuresis was no greater during concomitant BZM administration. This suggests that ENaC-mediated Na+ reabsorption was not normally limited by Na+ delivery, accounting for the absence of thiazide-induced kaliuresis. After dietary Na+ restriction, HCTZ elicited a kaliuresis, but the natiuretic effect of HCTZ was not enhanced by BZM. Our findings support a model in which inhibition of NCC activity does not increase Na+ reabsorption through ENaC solely by increasing distal Na+ delivery but rather by inducing a molecular and structural adaptation in downstream nephron segments.

Familial Hyperkalemic Hypertension: A New Early-onset Pediatric Case

The Familial hyperkalemic hypertension (FHHt) syndrome (OMIM #145260), first described in 1964, and also known as Gordon’s syndrome or pseudohypoaldosteronism type II (PHA II), is a rare autosomal dominant disease characterized by hypertension, hyperkalemia, hyperchloremic metabolic acidosis, and normal renal and adrenal function, with suppressed plasma rennin activity. Thiazides correct the metabolic abnormalities and hypertension (1,2,3). FHHt syndrome may be due to mutations in the WNK1 and WNK4 genes, encoding the with-no-lysine kinases 1 and 4 respectively. WNK4 mutants fail to inhibit the thiazide-sensitive Na-Cl cotransporter (NCC), increasing reabsorption of chloride in the distal collecting tubule and causing volume expansion and hypertension, and fail to inhibit the ROMK1 channel, causing hyperkalemia. WNK1 mutants also cause an increase in chloride adsorption both through the activation of EnaC and reduced inhibition of NCC through its influence on the WNK4 activity (4,5,6). Three loci associated with FHHt have been identified (12p13.3 and 17p11-q21 and 1q31-q42), but other loci, not yet identified, are probably involved, demonstrating the genetic heterogeneity of FHH. The syndrome has been described primarily in adolescents and young adults, and in most cases, hypertension has been the presenting symptom; however, the investigations performed in the relatives of patients with the FHHt syndrome, showed that the metabolic disorders preceded hypertension, and no relationship between the severity of biochemical abnormalities and severity of hypertension was observed (7, 8). Short stature, hypercalciuria with urolithiasis and dental abnormalities have also been reported. The long-term prognosis remains uncertain because follow-up data are still limited.

Enhanced FGF23 Serum Concentrations and Phosphaturia in Gene Targeted Mice Expressing WNK-Resistant Spak

Background: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na⁺,K⁺,2Cl^- cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. Methods: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak^tg/tg) and in mice expressing wild type SPAK (spak^wt/wt). Results: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spak^tg/tg mice than in spak^wt/wt mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spak^tg/tg mice compared to spak^wt/wt mice. Treatment of spak^wt/wt mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. Conclusions: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca²⁺ and phosphate excretion.

Renal sympathetic nerve activity (RSNA) and adrenergic stimulation increase Na‐Cl cotransporter (NCC) phosphorylation and trafficking to the apical plasma membrane (APM)

RSNA plays a primary role in hypertension (HTN) pathogenesis: RSNA is increased in models of HTN, renal denervation alleviates HTN, and RSNA promotes Na reabsorption. Distal convoluted tubule (DCT) NCC reabsorbs only 5–10% of filtered NaCl yet NCC is a key blood pressure (BP), fluid and electrolyte control point: genetic or chemical inhibition of NCC provokes salt wasting and lowers BP while NCC activation can cause HTN. This study tested the hypothesis that acute RSNA and/or adrenergic stimulation increase the number of active NCC in the DCT APM. One of two stimulation protocols (and shams) was applied in Sprague Dawley rats: 1) RSNA was acutely increased by injecting 50 μl phenol into the cortex, 2) α1 agonist phenylephrine (PE, 10 mg/kg, 10–20 min) was infused into one renal artery. After the protocols, kidneys were removed and subjected to density gradient fractionation to resolve low density APM from high density intracellular membranes (ICM). 12 fractions were collected and assayed by immunoblot with anti-NCC and anti-NCCp antibodies. Both stimuli provoked redistribution of 20% of the total NCC from higher into lower density fractions enriched in APM. PE doubled the NCCp/total ratio in the APM fractions. These results demonstrate that activation of DCT NCC, mediated by trafficking and phosphorylation, may contribute to the anti-natriuretic, BP raising effects of RSNA. Supported by DK083785, ASN Scherbenske award

WNK Kinases and Renal Sodium Transport in Health and Disease

Hypertension affects 25% of the adult population in the developed world and is a major independent risk factor for stroke, myocardial infarction, and heart and kidney failure. Although many genetic and environmental contributors are involved, the kidney plays a dominant role, both in animal models,1 and in human essential hypertension.2 Most monogenic hypertensive syndromes result from increased Na+ transport along the aldosterone-sensitive distal nephron.3 The majority of these, however, are associated with hypokalemia, indicating that activation of the epithelial Na+ channel, ENaC, is a primary pathophysiologic process. In contrast, familial hyperkalemic hypertension (FHHt; also known as Gordon’s syndrome or type II pseudohypoaldosteronism) is characterized by hypertension with hyperkalemia, indicating that stimulated ENaC cannot be the primary event. FHHt was first described in 19644 and later shown to be inherited in an autosomal dominant manner.5,6 Patients with FHHt all exhibit hyperkalemia, which seems to be the most consistent feature of the disease. Hypertension, although commonly present and sometimes severe, often appears later in the natural history. Other characteristic features include mild metabolic acidosis, suppressed plasma renin activity, and aldosterone levels that are lower than would be expected, considering the hyperkalemia. Infusing the chloride salt of Na+ (NaCl) does not increase urinary potassium excretion in patients with FHHt, as it does in the normal individual, whereas infusing nonchloride salts of Na+ does increase K+ excretion in FHHt patients to normal levels.7,8 Patients are often remarkably sensitive to thiazide diuretics, which can correct both the hyperkalemia and hypertension, in many cases.9 In 2001, some cases of FHHt were shown to result from mutations in WNK1 and WNK4,10 identifying WNK kinases as previously undiscovered components of a novel electrolyte homeostasis pathway. Since that time, information about the physiological role of WNK …

Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport

With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt.

Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport

With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt.

WNK kinases regulate thiazide-sensitive Na-Cl cotransport.

Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder of hyperkalemia and hypertension. Mutations in two members of the WNK kinase family, WNK1 and WNK4, cause the disease. WNK1 mutations are believed to increase WNK1 expression; the effect of WNK4 mutations remains unknown. The clinical phenotype of PHAII is opposite to Gitelman syndrome, a disease caused by dysfunction of the thiazide-sensitive Na-Cl cotransporter. We tested the hypothesis that WNK kinases regulate the mammalian thiazide-sensitive Na-Cl cotransporter (NCC). Mouse WNK4 was cloned and expressed in Xenopus oocytes with or without NCC. Coexpression with WNK4 suppressed NCC activity by more than 85%. This effect did not result from defects in NCC synthesis or processing, but was associated with an 85% reduction in NCC abundance at the plasma membrane. Unlike WNK4, WNK1 did not affect NCC activity directly. WNK1, however, completely prevented WNK4 inhibition of NCC. Some WNK4 mutations that cause PHAII retained NCC-inhibiting activity, but the Q562E WNK4 demonstrated diminished activity, suggesting that some PHAII mutations lead to loss of NCC inhibition. Gain-of-function WNK1 mutations would be expected to inhibit WNK4 activity, thereby activating NCC, contributing to the PHAII phenotype. Together, these results identify WNK kinases as a previously unrecognized sodium regulatory pathway of the distal nephron. This pathway likely contributes to normal and pathological blood pressure homeostasis.