Epithelial sodium channel regulation in the mouse kidney: Two novel approaches to a classic question.

摘要

Nearly one billion individuals worldwide have elevated blood pressure (BP) and 90% of these cases are due to unknown causes. Aldosterone is the primary hormone regulating Na+ reabsorption in the kidney and is therefore a major determinant of the long-term regulation of BP. The molecular mechanisms of aldosterone-induced Na+ transport are not entirely clear. The epithelial Na+ channel (ENaC) is the main molecular target of aldosterone and resides in the apical membrane of the principal cells of the renal cortical collecting duct (CCD), and other epithelial cells. ENaC activity is regulated by the abundance of channels in the membrane, and the open probability of those channels. This thesis focuses on proteins potentially involved in trafficking/recycling of ENaC to the membrane and its activation by proteolytic cleavage. We have determined that the transcript levels of melanophilin (MLPH), a vesicular trafficking protein, are rapidly and directly increased by aldosterone in CCD cells. This effect was near maximal at physiological aldosterone concentrations and was not prevented by the glucocorticoid receptor antagonist RU486, indicating that it is mediated by the mineralocorticoid receptor. To determine if this induction has functional consequences on transepithelial Na+ current (INa), we generated clonal CCD cell lines that express a tetracycline-inducible wild-type MLPH or one of two mutants that act as dominant negatives (DN) in melanocytes. Over-expression of the wild-type and point-mutated MLPH led to a relatively modest, but statistically significant increase in INa; conversely expression of a truncated MLPH led to a 60% decrease in INa. In addition, although MyosinVc, homologous to the MLPH-binding partner MyosinVa, has putative consensus sites for serum and glucocorticoid-induced kinase 1 (SGK1), MyosinVc is not phosphorylated by endogenous SGK1 in CCD cells. However, MyosinVc is probably involved in Na+ transport as over-expression of a DN MyoVc in M1 cells decreases INa by 25. We also conclude that while the serine protease epitheliasin cleaves ENaC subunits when coexpressed in HEK293 cells, it does not cleave endogenous ENaC in M1 cells. Furthermore, over-expression of epitheliasin in M1 cells does not affect INa suggesting it is not involved in processing/activation of ENaC in the renal CCD.