Na-Cl Cotransporter Research Articles

Dysregulation of the WNK4-SPAK/OxSR1 pathway has a mild effect on NKCC2 activity

Background: The furosemide-sensitive Na + -K+ - 2Cl − cotransporter (NKCC2) reabsorbs 20% of filtered Na+ along the thick ascending limb (TAL), while and thiazide-sensitive NaCl cotransporter (NCC) reabsorbs 5-10% along the distal convoluted tubule (DCT). The WNK4-SPAK/OxSR1 pathway mediates NKCC2 phosphorylation at threonines 96 and 101 (pT96/pT101) and NCC phosphorylation at threonine 53 (pT53). This activates both cotransporters. A recent study reported that the commonly used pT96/pT101 pNKCC2 antibody cross-reacts with pT53 NCC in mice on the C57BL/6 background due to a 5 amino acid deletion, calling into question some previous findings regarding WNK4-SPAK/OxSR1 regulation of NKCC2. Although WNK4 is highly expressed along both TAL and DCT, dysregulation of WNK4-SPAK/OxSR1 pathway specifically causes Familial Hyperkalemic Hypertension (FHHt), which is exquisitely sensitive to thiazide diuretics, and thus a disease of NCC dysregulation. Using a new pNKCC2 antibody specific in C57BL/6 mice, we tested the hypothesis that the WNK4-SPAK/OxSR1 pathway more strongly activates NCC than it does NKCC2. Methods: A new pT96 NKCC2 antibody was validated in C57BL/6, 129Sv, and Slc12a3 −/− mice. The abundances of pT96 NKCC2 and pT53 NCC were evaluated in Wnk4 −/− , Oxsr1 −/− , Spak −/− , Oxsr1 −/− / Spak −/− mice, and several FHHt model, Cul3 +/−/Δ9 , Klhl3 −/− , and Klhl3 R528H/R528H mice. NKCC2 activity was confirmed by furosemide response test with thiazide pre-treatment in Klhl3 −/− mice. Both male and female were used and all genetically modified strains were on the C57BL/6 background. Results: The pT96 NKCC2 antibody detected pNKCC2 in C57BL/6 mice but not 129Sv mice, and did not cross-react with phosphorylated NCC. pT53 NCC was almost absent but pT96 NKCC2 was only slightly lower in Wnk4 −/− mice. pT53 NCC was almost absent with Spak deletion ( Spak −/− and Oxsr1 −/− / Spak −/− mice) but pT96 NKCC2 abundance did not differ from controls. Oxsr1 deletion ( Oxsr1 −/− and Oxsr1 −/− / Spak −/− mice) led to a modest lowering of pT96 NKCC2/total NKCC2 ratio but not pT96 NKCC2 abundance. Interestingly, immunofluorescence revealed that WNK4 expression was increased along not only DCT but also cortical TAL in Klhl3 −/− mice, but pT96 NKCC2 abundance was not changed. pT53 NCC abundance, but not pT96 NKCC2 abundance, was higher in two other FHHt mouse models with higher WNK4 ( Cul3 +/−/Δ9 and Klhl3 R528H/R528H mice). Consistent with no difference in pT96 NKCC2 abundance, urine Na + excretion after furosemide injection following thiazide treatment was similar between Klhl3 −/− and control mice. Conclusions: NCC is strongly phosphorylated by the WNK4-SPAK pathway, whereas NKCC2 is only mildly phosphorylated by the WNK4-OxSR1 pathway, suggesting another kinase can phosphorylate NKCC2. In FHHt models with Cul3 or Klhl3 mutations, NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide-sensitivity of FHHt. Y.M. received a postdoctoral award from the Uehara Foundation; J.A.M. is funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK098141; M.C.B. is funded by CONACyT, Mexico, Grant 101720. 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.

Resolving the thiazide-induced hypocalciuria conundrum using single cell transcriptomics

Thiazide diuretics lower urinary calcium excretion and are used to prevent calcium stone recurrence. Thiazides inhibit the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Hypocalciuria also occurs in Gitelman syndrome, caused by mutations in SLC12A3, encoding NCC. The mechanism by which loss of NCC function affects calcium handling remains very controversial. The two prevailing hypotheses invoked are 1) hypocalciuria results from volume-dependent increases in proximal tubule reabsorption; 2) thiazides activate calcium reabsorption along the DCT. Here we combined single nucleus RNA sequencing with physiologic analysis to unravel the causes of hypocalciuria.Eight to 10-week old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days. Littermates receiving only vehicle were used as controls. As DCT cells comprise a small percentage of the kidney mass, we used the NCC-Cre +/- - INTACT +/- mouse line to enrich for DCT via Cre-dependent GFP expression in DCT nuclei. After nuclei isolation, GFP+ events (DCT nuclei) were enriched by fluorescence assisted nuclei sorting. The purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat.The MTZ-treated mice showed lower urinary calcium excretion compared to controls recapitulating thiazide-induced hypocalciuria. Our snRNAseq integrated dataset contained 51,876 mapped genes across 45,953 samples from 3 MTZ-treated and 3 control mice. Roughly 99% of the nuclei were singlets expressing Slc12a3 showing successful enrichment for the DCT. Dimensional reduction analysis and projection confirmed two clusters, DCT1 and DCT2, based on canonical segment markers consistent with previously published transcriptomic atlases. Gene expression analysis showed the DCT2 contained a calcium cassette consisting of the known calciotropic genes Trpv5, Calb1, S100g, Slc8a1, Ryr2, Vdr, Casr, and Atp2b1. These genes were also expressed in the connecting tubule (CNT), as documented using the Calb1-Cre-INTACT system. From this set of genes, we defined a calcium score computed from the pooled average expression of all these genes in a single cell. Although all calcium cassette genes, and the average calcium score, were lower in the MTZ-treated group, the proportion of DCT2/DCT1 cells increased (0.27 ± 0.03 vs 0.35 ± 0.03; p = 0.027). These data suggest that lower calcium delivery to the DCT reduces calcium cassette expression per cell, but that distal fractional reabsorption is nonetheless increased owing to hypertrophy and elongation of the calcium transporting segments, DCT2 and CNT.We propose a modified mechanism to account for thiazide -induced hypocalciuria: 1) volume-dependent increase in proximal tubule reabsorption; 2) increased fractional (not absolute) DCT2 reabsorption owing to structural remodeling of DCT2 and CNT providing more surface area for calcium reabsorption. Funding: NIH DK51496, NIH DK054983, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP 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.

Dietary potassium restriction reprograms the distal tubule causing adverse metabolic consequences

Low potassium (K) intake has adverse health effects, including hypertension, osteoporosis, and nephrolithiasis. Dietary K restriction rapidly activates a K switch in the distal nephron to maintain homeostasis by reducing K excretion and activating the sodium chloride cotransporter (NCC). The distal convoluted tubule (DCT) is morphologically and functionally divided into DCT1 and DCT2, with each segment specialized for a different function. Here, we define the differences between DCT1 and DCT2 transcriptionally, examine their unique responses to potassium restriction, and detrimental physiological responses in mice.The K restriction effects were examined at 3 time points, overnight (acute), 4 days (short-term), and 4 weeks (chronic). Electrophysiology was used to assess effects of acute K deprivation on basolateral K channel (Kir4.1/5.1) activity. To determine effects of K restriction for 4 days, the DCT-targeted single-nucleus RNA-sequencing (snRNAseq) approach was applied. Female NCC-Cre-INTACT (PMID: 26087164) mice were provided either normal (NK) or K deficient (KD) diet for 4 days (short-term) and kidneys were snap-frozen for targeted snRNAseq (10X Chromium, NovaSeq) (3 mice per diet, 10,000 nuclei per mouse). Effects of chronic K-restriction were assessed by serum electrolytes, tissue clearing, and western blot.Acute K restriction stimulated basolateral K channel activity in DCT1, but not in DCT2, indicating that DCT1 and DCT2 respond differently. Although both DCT1 and DCT2 express NCC, snRNAseq data showed that DCT1 and DCT2 have distinct transcriptional signatures, with DCT1 expressing genes involved in magnesium reabsorption, and DCT2 expressing genes involved in calcium reabsorption and electrogenic sodium channel (ENaC). Four days of K restriction switched the DCT from a K-wasting state to K-sparing state, shown by reduction of gENaC ( Scnn1g) and Kallikrein ( Klk1) expression. Furthermore, K restriction decreased the expression of the calcium-handling genes Slc8a1, Calb and Vdr. When prolonged, K restriction led to increases in NCC, reduction in ENaC cleavage, and elongation of the DCT. It also led to calcium wasting (plasma iCa in NK, 1.38 ± 0.03, n=7; KD, 1.24 ± 0.01 mM, n=6) and reduction in calbindin D-28k protein.Our results suggest that DCT1 and DCT2 both express NCC, but have distinct transcriptomic signatures with unique roles in magnesium and calcium transport, respectively. Dietary K restriction reprograms DCT2 cells to be more like DCT1 cells, thereby maintaining K balance but contributing to calcium wasting as a complication. NIH DK51496, NIH DK054983, VA 1I01BX002228, LeDucq Fondation 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.

Dapagliflozin Attenuates Sodium Chloride Cotransporter (NCC) Protein Expression while Improving Urine Osmolality and Decreasing Blood Pressure in Hypertensive Diabetic db/db Mice

The prevalence of hypertension in diabetic patients varies depending on age, gender, ethnicity, body mass index (BMI), glycemic management history, and the presence of kidney disease. In addition, hypertension is a major contributor to the development of atherosclerotic cardiovascular disease (ASCV), heart failure, and microvascular consequences. In the kidney, NCC is expressed in cells of the distal convoluted tubules. About 10% of the filtered load of sodium is reabsorbed by renal NCC. Recently, dapagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, was introduced as an effective class of glucose-lowering agents for the treatment of type 2 diabetes. We hypothesize that dapagliflozin mitigates blood pressure by restoring normal renal functions. Twelve week-old male db/db mice with a blood glucose of more than 300 mg/dl were salt-loaded (4% NaCl) for 10 days to induce hypertension. The mice were subjected to metabolic cage studies for 24-hour urine collections to measure urinary electrolytes. Blood pressure was measured by the tail-cuff method to evaluate the effect of dapagliflozin (n=8), or vehicle (n=8) given by oral gavage (1 mg/kg of body weight per day for 2 weeks). Total NCC protein expression was reduced in kidney cortex membrane fractions, while there was increased urinary sodium levels, from dapagliflozin treated mice compared to vehicle treated mice. Urine osmolality was improved in the dapagliflozin treated group compared to the vehicle treated. Additionally, blood glucose, systolic blood pressure, and urinary potassium levels were reduced in the dapagliflozin treated group. Taken together, these results suggest blood pressure is normalized after dapagliflozin treatment in a mechanism involving reduced renal NCC and increased excretion of sodium. 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.

Dopamine D4 receptor inhibit sodium chloride cotransporter in renal distal convoluted tubules

Background: Dopamine D4 receptor (D4R) is expressed in the distal convoluted tubule (DCT) and mice lacking the D4R have kidney-related hypertension. Hypothesis: The D4R negatively regulates the sodium chloride cotransporter (NCC) in vitro and in vivo. Methods: NCC activities were assessed in vivo on urinary sodium excretion and systolic blood pressures (SBP) responded to NCC inhibitor hydrochlorothiazide (HCTZ, 30mg/kg/day, IP) in Drd4 null ( Drd4 -/- ) mice and wild-type ( Drd4 +/+ ) littermates (female 20 weeks old). Total and phosphorylation/ubiquitination of renal NCC were determined. The protein abundance of NCC was measured in the cultured immortalized mouse renal distal-convoluted-tubule cells (mDCT) treated by the D4R agonist PD168077 (PD), D4R antagonist L-745,870(L) or Drd4 siRNA respectively. D4R-related interaction, internalization, ubiquitination, degradation and sodium transport of NCC were also determined in vitro. T-test was used for 2 group comparisons while one-way ANOVA, Holm-Sidak test for multi-group comparisons (P<0.05 was considered significant). Results: HCTZ produced a stronger natriuretic effect in Drd4 -/- mice than in WT littermates in initial 6-hrs (16.3±2.2 vs 13.1±3.1μmol/mg of Cr, n=8). SBP were higher in Drd4 -/- mice than Drd4 +/+ littermates at baseline (117.8±5.2 vs 104.1±6.3 mmHg, n=8, tail-cuff) but normalized by HCTZ on days 4-7. The renal protein abundances of NCC by immunoblotting (230.2±51.12, % of control, n=4-5) and immunofluorescence were greater but ubiquitinated-NCC levels (65.8±12.8, %, n=4) were lower in KO than in WT mice and mRNA levels of NCC (88.5±23.8, %) and phosphorylation-NCC (87.7±10.6, %) were not altered. In the mDCT cells, inhibition of D4R by its siRNA (1.5nM, 48 hrs) increased the protein expression of NCC (151.9±33.6, %, n=4). PD (24 hrs) decreased NCC protein abundance concentration-dependently blocked by L. Total NCC (72.1±18.1,%) was decreased but ubiquitinated-NCC was increased remarkably (265.8±114.1, %) by PD. There was a co-immunoprecipitation of D4R with NCC that was increased by PD. Colocalization of them was seen with confocal microscopy and confirmed by Fret. Within 1 hr, PD internalized NCC by membrane biotinlaytion-method and increased the colocalization of NCC with lysosomal-associated membrane protein 1. PD-induced NCC decrease was reversed by lysosomal inhibitor chloroquine. 1 hr PD also decreased total NCC and NCC-dependent-intracellular sodium transport (74±1, % of Veh, n=7), which was blocked by L. PD decreased NCC under CHX-chase time-dependently (33.7±21.3, %, 8 hrs, n=5) suggesting a reduced NCC half-life. Conclusion: D4R inhibits NaCl transport in renal DCT by reducing NCC activity and protein abundance and shortening NCC half-life with promoting its internalization, ubiquitination and consequent lysosomal degradation. 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.

CD8+ T cells stimulate renal sodium transporters contributing to salt-sensitive hypertension

Impaired salt handling by the kidneys acts as one source of hypertension. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts), when adoptively transferred from salt-sensitive hypertensive mice to normotensive mice, stimulate Sodium Chloride Cotransporter (NCC). Through this stimulation, sodium retention occurs, and salt-sensitive hypertension develops. However, whether other sodium transporters within the nephron are affected by hypertensive CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize hypertensive CD8Ts will also have an impact on other sodium channels. Here, we demonstrate the impact of hypertensive CD8Ts on sodium-potassium-co-transporter (NKCC) and epithelial sodium channel (ENaCs) in the kidney and the potential of these regulations contributing to salt-sensitive hypertension.Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. With in vitro studies, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously.In the results of in vivo studies, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. In the results from in vitro studies, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological hormone levels or with additional corticosteroids.Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention. Although CD8Ts themselves can increase ENaC, this effect might be masked in physiologic conditions. NIH R01-HL146713 (Mu), AHA 15BGIA25730047 (Mu), and SPaT T32-GM10699 (Deck) 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.

Calcineurin inhibitors stimulate Kir4.1/Kir5.1 of the distal convoluted tubule to increase NaCl cotransporter.

We examine whether calcineurin or protein phosphatase 2B (PP2B) regulates the basolateral inwardly rectifying potassium channel Kir4.1/Kir5.1 in the distal convoluted tubule (DCT). Application of tacrolimus (FK506) or cyclosporine A (CsA) increased whole-cell Kir4.1/Kir5.1-mediated K+ currents and hyperpolarized the DCT membrane. Moreover, FK506-induced stimulation of Kir4.1/Kir5.1 was absent in kidney tubule-specific 12 kDa FK506-binding protein-knockout mice (Ks-FKBP-12-KO). In contrast, CsA stimulated Kir4.1/Kir5.1 of the DCT in Ks-FKBP-12-KO mice, suggesting that FK506-induced stimulation of Kir4.1/Kir5.1 was due to inhibiting PP2B. Single-channel patch-clamp experiments demonstrated that FK506 or CsA stimulated the basolateral Kir4.1/Kir5.1 activity of the DCT, defined by NPo (a product of channel number and open probability). However, this effect was absent in the DCT treated with Src family protein tyrosine kinase (SFK) inhibitor or hydroxyl peroxide. Fluorescence imaging demonstrated that CsA treatment increased membrane staining intensity of Kir4.1 in the DCT of Kcnj10fl/fl mice. Moreover, CsA treatment had no obvious effect on phosphorylated NaCl cotransporter (pNCC) expression in Ks-Kir4.1-KO mice. Immunoblotting showed acute FK506 treatment increased pNCC expression in Kcnj10fl/fl mice, but this effect was attenuated in Ks-Kir4.1-KO mice. In vivo measurement of thiazide-induced renal Na+ excretion demonstrated that FK506 enhanced thiazide-induced natriuresis. This effect was absent in Ks-FKBP-12-KO mice and blunted in Ks-Kir4.1-KO mice. We conclude that inhibition of PP2B stimulates Kir4.1/Kir5.1 of the DCT and NCC and that PP2B inhibition-induced stimulation of NCC is partially achieved by stimulation of the basolateral Kir4.1/Kir5.1.

Gitelman Syndrome – A Case Report

Hypokalaemia is an electrolyte disorder, commonly encountered in clinical practice. Sometimes, it may present with severe life-threatening complications. We present here a case of 22-year-old female, who was admitted to intensive care unit, for severe hypokalaemia associated with cardiac and neurological complications. She had recurrence of such episodes. On detailed clinical and biochemical evaluation, she was diagnosed to have Gitelman syndrome (GS). She was treated with intravenous potassium and magnesium supplementation under cardiac monitoring and other supportive management. She was discharged in stable condition, on oral supplements, and remained stable on follow-up visits. GS is an autosomal recessive and renal tubular disorder characterised by hypokalaemic metabolic alkalosis with hypomagnesaemia, hypocalciuria, secondary hyperreninemic aldosteronism and hypotension. The disease is caused by biallelic inactivating mutations within the SLC12A3 gene encoding the thiazide-sensitive sodium chloride cotransporter expressed within the apical membrane of cells, lining the distal convoluted tubule. Since GS is an autosomal recessive trait, the recurrence risk for people with an affected child is 25%. It is very important to completely evaluate a patient with hypokalaemia to find the underlying cause. There can be various etiological factors for hypokalaemia ranging from common to rare disorders. If underlying aetiology remains undiagnosed, there may be recurrence as well as increased morbidity and mortality. GS is one such disorder, which needs prompt diagnostic evaluation and treatment.

Cracking the natriuresis puzzle: Insight from structural biology

Sodium chloride cotransporter (NCC) plays a crucial role in regulating blood pressure through Na+ reabsorption. Recently, in Nature, Fan etal. determined the structure of human NCC and revealed the mechanism of action of thiazide diuretics, establishing the groundwork for future drug development.1.

High sodium intake does not worsen low potassium-induced kidney damage.

High sodium and low potassium intake have both been linked to poor cardiovascular health outcomes and increased mortality rates. A combination of the two is thought to be particularly detrimental. While mechanisms are multiple, the kidney is an important target of harmful effects and low potassium influences on both proximal and distal nephron segments are especially potent. We recently reported that a combined high sodium/low potassium diet causes kidney injury and that low potassium in isolation can have similar effects. However, how sodium intake alters this process is not well-understood. Here we tested the hypothesis that a high sodium intake amplifies effects of low dietary potassium on kidney injury. We observed adding high sodium to low potassium caused an expected increase in blood pressure, but did not worsen markers of kidney injury, inflammation, and fibrosis. It also did not increase abundance or phosphorylation of the sodium chloride cotransporter or its regulatory kinases, SPAK and OxSR1, known renal targets of low potassium. Findings support the claim that dietary potassium deficiency, and not high sodium, is a dominant factor affecting kidney injury in animal models of high sodium/low potassium intake. This suggests further investigation is required to identify optimal ranges of sodium and potassium intake in both healthy populations and in those with kidney disease.

Urinary extracellular vesicles carry valuable hints through mRNA for the understanding of endocrine hypertension.

Urinary extracellular vesicles (uEVs), released from cells of the urogenital tract organs, carry precious information about originating tissues. The study of molecules transported through uEVs such as proteins, lipids and nucleic acids provides a deeper understanding of the function of the kidney, an organ involved in the pathogenesis of hypertension and a target of hypertension-mediated organ damage. Molecules derived from uEVs are often proposed for the study of disease pathophysiology or as possible disease diagnostic and prognostic biomarkers. Analysis of mRNA loading within uEVs may be a unique and readily obtainable way to assess gene expression patterns of renal cells, otherwise achievable only by an invasive biopsy procedure. Interestingly, the only few studies investigating transcriptomics of hypertension-related genes through the analysis of mRNA from uEVs are inherent to mineralocorticoid hypertension. More specifically, it has been observed that perturbation in human endocrine signalling through mineralcorticoid receptors (MR) activation parallels changes of mRNA transcripts in urine supernatant. Furthermore, an increased copy number of uEVs-extracted mRNA transcripts of the 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene were detected among subjects affected by apparent mineralocorticoid excess (AME), a hypertension-inducing autosomal recessive disorder due to a defective enzyme function. Moreover, by studying uEVs mRNA, it was observed that the renal sodium chloride cotransporter (NCC) gene expression is modulated under different conditions related to hypertension. Following this perspective, we illustrate here the state of the art and the possible future of uEVs transcriptomics towards a deeper knowledge of hypertension pathophysiology and ultimately more tailored investigational, diagnostic-prognostic approaches.

Randomized Trial on the Effect of Oral Potassium Chloride Supplementation on the Thiazide-Sensitive Sodium Chloride Cotransporter in Healthy Adults

The putative "renal-K switch" mechanism links dietary potassium intake with sodium retention and involves activation of the sodium chloride (NaCl) cotransporter (NCC) in the distal convoluted tubule in response to low potassium intake, and suppression in response to high potassium intake. This study examined NCC abundance and phosphorylation (phosphorylated NCC [pNCC]) in urinary extracellular vesicles (uEVs) isolated from healthy adults on a high sodium diet to determine tubular responses to alteration in potassium chloride (KCl) intake. Healthy adults maintained on a high sodium (∼4.5 g [200 mmol]/d) low potassium (∼2.3 g [60 mmol]/d) diet underwent a 5-day run-in period followed by a crossover study, with 5-day supplementary KCl (active phase, Span-K 3 tablets (potassium 24 mmol) thrice daily) or 5-day placebo administrated in random order and separated by 2-day washout. Ambulatory blood pressure (BP) and biochemistries were assessed, and uEVs were analyzed by western blotting. Among the 18 participants who met analysis criteria, supplementary KCl administration (vs. placebo) was associated with markedly higher levels of plasma potassium and 24-hour urine excretion of potassium, chloride, and aldosterone. KCl supplementation was associated with lower uEV levels of NCC (median fold change (KCl/Placebo)= 0.74 [0.30-1.69], P< 0.01) and pNCC (fold change (KCl/Placebo)= 0.81 [0.19-1.75], P< 0.05). Plasma potassium inversely correlated with uEV NCC (R2= 0.11, P= 0.05). The lower NCC and pNCC in uEVs in response to oral KCl supplementation provide evidence to support the hypothesis of a functional "renal-K switch" in healthy human subjects.

Renal CD81 interacts with sodium potassium 2 chloride cotransporter and sodium chloride cotransporter in rats with lipopolysaccharide-induced preeclampsia.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

Cullin 3 and Blood Pressure Regulation: Insights From Familial Hyperkalemic Hypertension.

The study of rare monogenic forms of hypertension has led to the elucidation of important physiological pathways controlling blood pressure. Mutations in several genes cause familial hyperkalemic hypertension (also known as Gordon syndrome or pseudohypoaldosteronism type II). The most severe form of familial hyperkalemic hypertension is caused by mutations in CUL3, encoding CUL3 (Cullin 3)-a scaffold protein in an E3 ubiquitin ligase complex that tags substrates for proteasomal degradation. In the kidney, CUL3 mutations cause accumulation of the substrate WNK (with-no-lysine [K]) kinase and ultimately hyperactivation of the renal NaCl cotransporter-the target of the first-line antihypertensive thiazide diuretics. The precise mechanisms by which mutant CUL3 causes WNK kinase accumulation have been unclear, but several functional defects are likely to contribute. The hypertension seen in familial hyperkalemic hypertension also results from effects exerted by mutant CUL3 on several pathways in vascular smooth muscle and endothelium that modulate vascular tone. This review summarizes the mechanisms by which wild type and mutant CUL3 modulate blood pressure through effects on the kidney and vasculature, potential effects in the central nervous system and heart, and future directions for investigation.

WCN23-0205 Cyclosporin A-induced activation of the renal NaCl cotransporter depends on the presence of Kir4.1 in the distal convoluted tubule

WCN23-0205 Cyclosporin A-induced activation of the renal NaCl cotransporter depends on the presence of Kir4.1 in the distal convoluted tubule

Activation of Kir4.1/Kir5.1 contributes to the cyclosporin A-induced stimulation of the renal NaCl cotransporter and hyperkalemic hypertension.

Cyclosporin A (CsA) is a widely used immunosuppressive drug that causes hypertension and hyperkalemia. Moreover, CsA-induced stimulation of the thiazide-sensitive NaCl cotransporter (NCC) in the kidney has been shown to be responsible for the development of hyperkalemic hypertension. In this study, we tested whether CsA induces the activation of NCC by stimulating the basolateral Kir4.1/Kir5.1 channel in the distal convoluted tubule (DCT). Electrophysiology, immunoblotting, metabolic cages, and radio-telemetry methods were used to examine the effects of CsA on Kir4.1/Kir5.1 activity in the DCT, NCC function, and blood pressure in wild-type (WT) and kidney-specific Kir4.1 knockout (KS-Kir4.1 KO) mice. The single-channel patch clamp experiment demonstrated that CsA stimulated the basolateral 40 pS K+ channel in the DCT. Whole-cell recording showed that short-term CsA administration (2h) not only increased DCT K+ currents but also shifted the K+ current (IK ) reversal potential to the negative range (hyperpolarization). Furthermore, CsA administration increased phosphorylated NCC (pNCC) levels and inhibited renal Na+ and K+ excretions in WT mice but not in KS-Kir4.1 KO mice, suggesting that Kir4.1 is required to mediate CsA effects on NCC function. Finally, long-term CsA infusion (14 days) increased blood pressure, plasma K+ concentration, and total NCC or pNCC abundance in WT mice, but these effects were blunted in KS-Kir4.1 KO mice. We conclude that CsA stimulates basolateral K+ channel activity in the DCT and that Kir4.1 is essential for CsA-induced NCC activation and hyperkalemic hypertension.

Structure and thiazide inhibition mechanism of the human Na-Cl cotransporter.

The sodium-chloride cotransporter (NCC) is critical for kidney physiology1. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron2,3, and mutations in the NCC cause the salt-wasting disease Gitelman syndrome4. As a key player in salt handling, the NCC regulates blood pressure and is the target of thiazide diuretics, which have been widely prescribed as first-line medications to treat hypertension for more than 60 years5-7. Here we determined the structures of human NCC alone and in complex with a commonly used thiazide diuretic using cryo-electron microscopy. These structures, together with functional studies, reveal major conformational states of the NCC and an intriguing regulatory mechanism. They also illuminate how thiazide diuretics specifically interact with the NCC and inhibit its transport function. Our results provide critical insights for understanding the Na-Cl cotransport mechanism of the NCC, and they establish a framework for future drug design and for interpreting disease-related mutations.

The serine-threonine protein phosphatases that regulate the thiazide-sensitive NaCl cotransporter.

The activity of the Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC's activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K+ is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K+-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in in vitro models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.

CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.

The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.