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Silencing and overexpression of the -subunit of Na-K-ATPase directly affect survival of IMCD3 cells in response to hypertonic stress

Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, School of Medicine, Denver, Colorado

Submitted 3 March 2006 ; accepted in final form 23 June 2006

The -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 -subunit isoforms were spliced into an expression vector and transfected into IMCD3 cells. Multiple clones stably expressed -subunit protein under isotonic conditions. Clones expressing the _b isoform showed enhanced survival at lethal acute hypertonicity compared with either _a isoform or empty vector (control) expressing clones. We also evaluated the loss of -subunit expression on the survival of IMCD3 cells exposed to hypertonicity employing silencing RNA techniques. Multiple stable -subunit-specific siRNA clones were obtained and exposed to sublethal hypertonicity. Under these conditions, both the level of mRNA and protein was essentially undetectable. The impact of silencing -subunit expression resulted in a 70% reduction at 48 h (P < 0.01) in cell survival compared with empty vector (control) clones. 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 -subunit on IMCD3 cell survival and/or adaptation in response to ionic hypertonic stress.

hypertonicity; osmoregulation; small-interference RNA