Acute Effects of Aldosterone on NCC Are Mediated by SPAK

Abstract

The sodium chloride cotransporter (NCC) is a major site of renal sodium reabsorption and plays a key role in blood pressure regulation. Using mDCT15 cells, we previously demonstrated that aldosterone (aldo) increases NCC activity and phosphorylation at 24 hrs. without affecting total or surface NCC abundance. Here we confirm those findings in vivo, examine the mechanisms underlying them and investigate additional time points. Adrenalectomized rats were given aldo or vehicle via osmotic minipumps. Kidney cortex from rats receiving aldo demonstrated no change in total NCC compared to control but showed a three‐fold change in phospho‐NCC (T53). Similarly, mDCT15 cells showed a three‐fold increase in phospho‐NCC at 12, 24, and 36 hours, corresponding to observed increases in NCC activity as measured by radiotracer uptake with no evidence of changes in total or surface NCC (n=6, p<0.05). mDCT15 cells were treated with shRNA specific for SPAK to generate a cell line with a 68±7% decrease in SPAK protein expression compared to controls. Stimulation of these cells with aldo had virtually no effect on NCC activity (6±3% increase after 24 hour aldo treatment in shRNA SPAK groups versus 52±5% increase after aldo treatment in control groups, n=4, p<0.01). This provides clear evidence that aldo acutely increases NCC activity without changing NCC abundance or distribution via a pathway involving SPAK and NCC phosphorylation.