Submitted on March 3, 2006
Accepted on June 23, 2006
Silencing and overexpression of the 
subunit of Na/K-ATPase directly affects survival of IMCD3 cells in response to hypertonic stress
Juan Capasso1, Christopher John Rivard1, and Tomas Berl1*
1 School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, United States
* To whom correspondence should be addressed. E-mail: tomas.berl{at}uchsc.edu.
The 
subunit of Na/K-ATPase is robustly expressed in IMCD3 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 as compared to 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 sub-lethal 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 as compared with empty vector (control) clones. siRNA clones showed a 45% decrease in myo-inositol uptake when compared to controls after an 18 h exposure to sub-lethal 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.
