Phosphorylation of the thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular potassium concentration. High extracellular [K+] is believed to activate specific phosphatases to dephosphorylate NCC, thereby reducing its activity. This process is defective in the human disease familial hyperkalemic hypertension, in which extracellular [K+] fails to dephosphorylate NCC, suggesting an interplay between the NCC-activating and inactivating switches. Here, we explored the role of SPAK and intracellular chloride concentration in the rapid effects of extracellular K+ on NCC phosphorylation. SPAK was found to be rapidly dephosphorylated in vitro in HEK cells and ex vivo in kidney slices by high [K+]. Acute high K+ challenge resulted in DCT1-specific SPAK dephosphorylation in vivo and dissolution of WNK bodies. In line with the postulate of interplay between activating and inactivating switches, we found that the ON switch, represented by WNK4-SPAK, must be turned off for rapid NCC dephosphorylation by high [K+]. Longer term WNK-SPAK mediated stimulation, however, altered the sensitivity of the system, as it attenuated rapid NCC dephosphorylation due to acute K+ loading. Although blocking PP1 increased NCC phosphorylation at baseline, neither PP1 nor PP3, singly or in combination, were essential for NCC dephosphorylation. Overall our data suggest that NCC phosphorylation is regulated by a dynamic equilibrium between activating kinases and inactivating phosphatases; with kinase inactivation playing a key role in the rapid NCC dephosphorylation by high extracellular K+.

Aims Salt-sensitive (SS) hypertension is accompanied by impaired vasodilation in the systemic and renal circulation. However, the causal relationship between vascular dysfunction and salt-induced hypertension remains controversial. We sought to determine whether primary vascular dysfunction, characterized by a failure to vasodilate during salt loading, plays a causal role in the pathogenesis of SS hypertension. Methods and results Mice selectively expressing a peroxisome proliferator-activated receptor γ dominant-negative mutation in vascular smooth muscle (S-P467L) exhibited progressive SS hypertension during a 4 week high salt diet (HSD). This was associated with severely impaired vasodilation in systemic and renal vessels. Salt-induced impairment of vasodilation occurred as early as 3 days after HSD, which preceded the onset of SS hypertension. Notably, the overt salt-induced hypertension in S-P467L mice was not driven by higher cardiac output, implying elevations in peripheral vascular resistance. In keeping with this, HSD-fed S-P467L mice exhibited decreased smooth muscle responsiveness to nitric oxide (NO) in systemic vessels. HSD-fed S-P467L mice also exhibited elevated albuminuria and a blunted increase in urinary NO metabolites which was associated with blunted renal blood flow and increased sodium retention mediated by a lack of HSD-induced suppression of NKCC2. Blocking NKCC2 function prevented the salt-induced increase in blood pressure in S-P467L mice. Conclusion We conclude that failure to vasodilate in response to salt loading causes SS hypertension by restricting renal perfusion and reducing renal NO through a mechanism involving NKCC2 in a mouse model of vascular peroxisome proliferator-activated receptor γ impairment.

Cl--sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT). Cl- enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl- channel ClC-Kb or a K-Cl cotransporter (KCC). While KCC is electroneutral, Cl- exit via ClC-Kb is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl- movement across the basolateral membrane. Although a role for intracellular Cl- in the regulation of WNK and NCC has been established, intracellular Cl- concentrations ([Cl-]i) have not been directly measured in mammalian DCT. Therefore, to measure [Cl-]i in DCT cells, we have generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl- fluorescent imaging in the isolated DCT. Basal measurements indicate that the mean [Cl-]i is approximately 7 mM. Stimulation of chloride exit with low chloride hypotonic solutions decreased [Cl-]i, whereas inhibition of KCC by DIOA or inhibition of ClC-Kb by NPPB increased [Cl-]i, suggesting roles for both KCC and ClC-Kb modulating [Cl-]i . Blocking the basolateral K+ channels, Kir4.1/5.1, with barium significantly increased the [Cl-]i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl-]i whereas raising extracellular K+ transiently increased [Cl-]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl-]i. We conclude that the alteration in ClC-Kb, KCC and Kir4.1/5.1 activity influences the [Cl-]i in the DCT.

Background: Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) cause the disease Familial Hyperkalemic Hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine [K] Kinase 4 (WNK4), inappropriately activating Sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK), which then phosphorylates and hyperactivates the Na+-Cl- cotransporter (NCC). The precise mechanism by which CUL3-Δ9 causes FHHt has been unclear. We tested the hypothesis that reduced abundances of CUL3 and of Kelch-like 3 (KLHL3), the CUL3 substrate adaptor for WNK4, are mechanistically important. Since JAB1, an enzyme that inhibits CUL3 activity by removing the ubiquitin-like protein NEDD8, cannot interact with CUL3-Δ9, we also determined whether Jab1 disruption mimicked the effects of CUL3-Δ9 expression. Methods: We used an inducible renal tubule-specific system to generate several mouse models expressing CUL3-Δ9, mice heterozygous for both CUL3 and KLHL3 (Cul3+/−/Klhl3+/−), and mice with short-term Jab1 disruption (to avoid renal injury associated with long-term disruption). Results: Renal KLHL3 was higher in Cul3−/− mice, but lower in Cul3−/−/Δ9 mice and in the Cul3+/−/Δ9 FHHt model, suggesting KLHL3 is a target for both WT and mutant CUL3. Cul3+/−/Klhl3+/− mice displayed increased WNK4-SPAK activation and phospho-NCC abundance, and an FHHt-like phenotype with increased plasma [K+] and salt-sensitive blood pressure. Short-term Jab1 disruption in mice lowered abundances of CUL3 and KLHL3, and increased abundances of WNK4 and phospho-NCC. Conclusions:Jab1-/- mice and Cul3+/−/Klhl3+/− mice recapitulated the effects of CUL3-Δ9 expression on WNK4-SPAK-NCC. Our data suggest that degradation of both KLHL3 and CUL3 plays a central mechanistic role in CUL3-Δ9-mediated FHHt.

Background: MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone. Methods: We studied AS (aldosterone synthase)-deficient (AS −/− ) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR −/− ) mice. KS-MR −/− mice were used to compare MR expression and ENaC localization and cleavage with AS −/− mice. Results: MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR −/− mice but were fully preserved along DCT2 and were partially reduced along CCD in AS −/− mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS −/− mice and were not increased along CCD after low salt. AS −/− mice exhibited transient Na + wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS −/− mice led to hyperkalemia and decreased body weight with higher Na + excretion, mimicking the phenotype of MR −/− mice. Conclusions: Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS −/− mice.