Dissecting the Effects of Aldosterone and Hypokalemia on the Epithelial Na+ Channel and the NaCl Cotransporter

Abstract

Primary hyperaldosteronism (PA) is characterized by aldosterone excess and hypertension. This may be linked to increased renal Na+ reabsorption via the epithelial Na+ channel (ENaC) and the NaCl cotransporter (NCC). The majority of PA patients have normal plasma K+ levels, but a subset of cases are associated with hypokalemia. High NCC levels observed in long-term studies with aldosterone-infused rodents have been attributed to direct effects of aldosterone. Aldosterone can also increase active phosphorylated NCC (pT58-NCC) acutely. However, direct effects of aldosterone on NCC have been contested by recent studies indicating that it is rather an indirect effect of hypokalemia. We therefore set out to determine isolated long-term aldosterone and K+ effects on ENaC and NCC using various in vivo and ex vivo approaches. In mice, aldosterone-induced hypokalemia was prevented by simultaneous amiloride infusion, coupled to increased cleavage of α- and γENaC but no effect on NCC. Regression analyses of in vivo data showed a positive correlation between aldosterone/K+ and αENaC but a negative correlation with NCC and pT58-NCC. Ex vivo, exposure of kidney tubules for 21 h to aldosterone increased cleavage of αENaC and γENaC, but no effects were observed on NCC or pT58-NCC. Exposure of tubules to low K+ media reduced αENaC but increased NCC and pT58-NCC. As hypokalemia can enhance cell proliferation markers in the distal convoluted tubule (DCT), we hypothesized that aldosterone infusion would increase proliferating cell nuclear antigen (PCNA) expression. Infusion of aldosterone in mice for 6 days greatly increased PCNA expression in the DCT. Collectively, in vivo and ex vivo data suggest that both aldosterone and K+ can increase ENaC directly. In contrast, the observed increase in abundance and phosphorylation of NCC in aldosterone-infused mice is likely an indirect effect of enhanced ENaC-mediated K+ secretion and subsequent hypokalemia. Thus, it is possible that NCC may only be increased in PA when the condition is associated with hypokalemia.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling

“…We observed an increase in γ -subunit protein in the aldosterone-alone treated group. Although against the commonly accepted role of aldosterone regulation in the kidney, our results support recently published studies showing changes in γ -ENaC expression with aldosterone, suggested to be caused by increased cell proliferation (Kristensen et al, 2022;Terker, Yarbrough, et al, 2016). Furthermore, we observed an increase in both βand γ -subunit message and protein in mice receiving HSD+Aldo.…”

“…For the exact P values, the following abbreviations are used to denote groups being compared: C, control; S, high salt diet; A, aldosterone; SA, high salt diet with aldosterone. [Colour figure can be viewed at wileyonlinelibrary.com] to measure the length of the ENaC + tubule within our cleared kidneys as a result of poor antibody penetration, the study by Kristensen et al (2022), coupled with our finding of increased DCT2 length in HSD+Aldo treated animals, suggests that proliferation of the late DCT help explain our results.…”

“…We examined transporter expression and tubular remodelling with chronic treatments and fixed doses of aldosterone and salt. Future studies should examine whether these changes are seen with acute treatment of HSD+Aldo because aldosterone alone has been shown to have differences in its effects on NCC phosphorylation at acute vs. chronic time points (Cheng et al, 2019;Kristensen et al, 2022). In addition, the amount of sodium in the diet and the time over which it is given may cause variable effects because a study with 4% NaCl diet given for 2 weeks found a reduction in total and phosphorylated NCC in mice (Torres-Pinzon et al, 2021), whereas our protocol revealed no differences in animals given HSD alone.…”

Regulation of distal tubule sodium transport: mechanisms and roles in homeostasis and pathophysiology

“…[ 81 ]. In the present context, it is notable that recent evidence strongly supports the conclusion that effects of aldosterone and SGK1 on NCC are indirect and due to ENaC-dependent lowering of plasma [K + ], which stimulates NCC [ 32 , 79 , 166 ].…”

Role of NCC in the pathophysiology of hypertension in primary aldosteronism

“…They observed that whereas αENaC abundance was significantly downregulated in MR -/- cells, no differences in NCC or pNCC levels were observed either in baseline or low Na + diet conditions [11]. Third, Kristensen et al [12 ▪ ] recently showed that co-administration of amiloride with aldosterone to prevent the K + wasting induced by ENaC activation did not result in increased NCC and pNCC. Moreover, in isolated tubules that were incubated ex vivo with aldosterone for 21 h, no effects on NCC of pNCC were observed.…”