title: Silencing and overexpression of the gamma-subunit of Na-K-ATPase directly affect survival of IMCD3 cells in response to hypertonic stress.
abstract: The gamma-subunit of Na-K-ATPase is robustly expressed in inner medullary collecting duct (IMCD)3 cells either acutely challenged or adapted to hypertonicity but not under isotonic conditions. Circumstantial evidence suggests that this protein may be important for the survival of renal cells in a hypertonic environment. However, no direct proof for such a contention has been forthcoming. The complete mRNA sequences of either gamma-subunit isoforms were spliced into an expression vector and transfected into IMCD3 cells. Multiple clones stably expressed gamma-subunit protein under isotonic conditions. Clones expressing the gamma(b) isoform showed enhanced survival at lethal acute hypertonicity compared with either gamma(a) isoform or empty vector (control) expressing clones. We also evaluated the loss of gamma-subunit expression on the survival of IMCD3 cells exposed to hypertonicity employing silencing RNA techniques. Multiple stable gamma-subunit-specific siRNA clones were obtained and exposed to sublethal hypertonicity. Under these conditions, both the level of gamma mRNA and protein was essentially undetectable. The impact of silencing gamma-subunit expression resulted in a 70% reduction at 48 h (P < 0.01) in cell survival compared with empty vector (control) clones. gamma siRNA clones showed a 45% decrease in myo-inositol uptake compared with controls after an 18-h exposure to sublethal hypertonicity. Taken together, these data demonstrate a direct and critical role of the gamma-subunit on IMCD3 cell survival and/or adaptation in response to ionic hypertonic stress.
authors.affiliation: Division of Renal Diseases and Hypertension, Univeristy of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262, USA.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Capasso
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Rivard
authors.forename: Tomas
authors.initials: T
authors.lastname: Berl
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D015870:Gene Expression; D020868:Gene Silencing; D006982:Hypertonic Solutions / Q000494:pharmacology; D007294:Inositol / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D009997:Osmotic Pressure; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D014882:Water-Electrolyte Balance / Q000502:physiology*
chemical_list: D006982:Hypertonic Solutions; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D007294:Inositol; C498816:Fxyd2 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase