The activity of the thiazide-sensitive sodium-chloride cotransporter NCC within the kidney distal convoluted tubule (DCT) is crucial for modulating blood pressure (BP). Emerging evidence also suggests that alterations in NCC activity play a pivotal role in the effects of dietary potassium (K⁺) on BP. Greater dietary K⁺ intake lowers NCC abundance and activity, which is often associated with reduced BP. Our previous studies have demonstrated that during high K⁺ intake there is greater ubiquitin-dependent NCC degradation, a process that involves an interaction between heat shock protein 70 (Hsp70) and NCC, and is potentially orchestrated by the ubiquitin E3 ligase CHIP (carboxy-terminus of Hsc70-interacting protein) (1). To validate this mechanism, we investigated the role of CHIP in modulating NCC activity in vivo and its implications for BP control.

Methods: CHIP knockout (KO) and wildtype (WT) control mice were fed diets with variable K⁺ content; low (0% K⁺), normal (1%), or high (5%) for 5 days. BP measurements on the various diets or after treatment with the NCC inhibitor hydrochlorothiazide (HCTZ) (37.5 mg/kg BW) or the epithelial sodium channel (ENaC) inhibitor amiloride (5 mg/kg BW) were obtained using telemetry (24 h readings) or tail cuff plethysmography (early evening). To examine NCC half-life, ex vivo kidney tubule suspensions from KO and control mice were incubated in control media (4.0 mM K⁺) containing cycloheximide and actinomycin for various time points and NCC expression was evaluated by immunoblotting.

Results: On a normal K⁺ diet, CHIP KO mice had elevated NCC protein levels (n = 5/ group; p<0.0001), but NCC mRNA levels were not significantly different between the genotypes. CHIP KO mice had higher BP compared to WT controls (n = 8/group; p<0.001) on a normal diet. Inhibition of NCC activity using hydrochlorothiazide normalized BP in the CHIP KO mice to similar levels as WT mice (n = 4-5/group; p<0.001), emphasizing NCC's predominant role in the observed higher BP phenotype. Inhibition of ENaC activity with amiloride had a similar effect to lower BP in WT and CHIP KO mice (n = 4-5/group; p<0.05). The ability of a high K⁺ diet to reduce NCC abundance was attenuated in CHIP KO mice (n = 4-5/group; p<0.0001). In ex vivo tubule suspensions from CHIP KO mice, NCC half-life was prolonged compared to WT controls (n = 3/group with 3 technical replicates; p <0.05), suggesting a role for CHIP in NCC degradation.

Conclusions: Our findings highlight the critical involvement of CHIP-mediated NCC ubiquitylation for regulating NCC abundance and BP. Preliminary studies also suggest a role for CHIP in mediating the effects of higher dietary potassium intake on NCC. Understanding the molecular mechanisms governing NCC regulation may offer valuable insights into hypertension pathophysiology and facilitate the development of targeted therapeutic interventions.