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The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na+ balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial [Na.sup.+] transport via the epithelial [Na.sup.+] channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently [Na.sup.+] reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced [Na.sup.+] transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with [Na.sup.+] reabsorption and hypertension. 17-allylamino-17-demethoxygeldanamycin; COOH terminus of Hsp70-interacting protein; sodium reabsorption; nephron; proteasome doi: 10.1152/ajprenal.00285.2010

Adjustment of [Na.sup.+] balance in extracellular fluids is achieved by regulated [Na.sup.+] transport involving the amiloride-sensitive epithelial [Na.sup.+] channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and [Na.sup.+] channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Uspl0), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Uspl0 in ENaC-transfected HEK293 cells causes a more than fivefold increase in amiloride-sensitive [Na.sup.+] currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of [alpha]- and [gamma]-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Uspl0 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Uspl0 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Uspl0. In [mCCD.sub.c11] cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC. sorting nexin 3; epithelial sodium channel