Regulation of NKCC2 and renal NaCl transport by protein-protein interactions

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Abstract In the kidney, the thick ascending limb (TAL) of the loop of Henle is critical for NaCl homeostasis and blood pressure regulation. In humans and animal models of salt-sensitive hypertension, NaCl absorption is abnormally increased in the TAL, where NaCl absorption depends on the renal transporter NKCC2, an apical Na+/K+/2Cl- co-transporter. We showed that the presence of NKCC2 at the TAL apical membrane controls NaCl absorption in this epithelium. The molecular mechanisms that control apical membrane NKCC2 levels involve endocytosis, recycling and exocytic insertion. Inhibition of endocytic retrieval causes NKCC2 accumulation at the membrane and increased NaCl absorption. Any gene or protein affecting NKCC2 endocytosis could potentially influence NKCC2 activity and renal salt transport but only few proteins are known to bind NKCC2. Using a targeted proteomics screen, we identified ALMS1 (Alström Syndrome 1) and ACTN4 (alpha-actinin 4) as interacting partners of NKCC2. We also found that ALMS1 and ACTN4 interact with each other, raising the possibility that these proteins form a complex. Single nucleotide polymorphisms in ALMS1 and ACTN4 are associated with hypertension and decreased kidney function. We found that ALMS1 knockout rats have higher surface NKCC2 and high blood pressure. We found that ACTN4, a protein involved in podocyte biology, is also expressed throughout the nephron, including the TAL. The roles of ALMS1 and ACTN4 in renal NaCl handling by the TAL and their role in blood pressure regulation are unknown. We hypothesize that ALMS1 controls surface NKCC2 levels and NKCC2-mediated NaCl absorption by binding the carboxyl-terminus of NKCC2 and ACTN4 to mediate NKCC2 endocytosis from the apical membrane. A decrease in ALMS1 or ACTN4 expression in the TAL increases surface NKCC2, NKCC2-mediated NaCl reabsorption, tubulo-glomerular feedback (TGF) sensitivity and leads to salt-sensitive hypertension. Our long-term goal is to increase our understanding of the role of ALMS1 and ACTN4 in kidney NaCl transport.