Role of calcium in polycystic kidney disease: From signaling to pathology

Figure 2 Downregulation of PKD1 and PKD2 genes increases fetal bovine serum-induced calcium oscillations in HEK293 cells. A: The stable transfection of HEK293 cells with plasmids containing specific anti-PKD1 and anti-PKD2 sequences causes a partial (+/-) or complete (-/-) downregulation of PC1 and PC2 expression compared with HEK293 cells stably transfected with scramble sequences (control). PKD1 and PKD2 gene silencing was evaluated by Western blotting using anti-PC1 and anti-PC2 antibodies. Calcium oscillations were increased in both partially (+/-) and fully (-/-) cells silenced for the PKD1 gene, as well as in fully (-/-) PKD2-silenced cells, as compared with scramble-treated cells (control). The number of oscillations/15 min were: 12 ± 1.5 in PKD1^(+/-) cells, 12.2 ± 1.42 in PKD1^(-/-) cells and 11.13 ± 1.79 in PKD2^(-/-) cells, vs 6.39 ± 1.09 in control cells (^bP < 0.01; ^aP < 0.05); B: The expression of full-length exogenous PC2 fused with GFP in PKD2^(-/-) cells restores normal calcium oscillations (11.13 ± 1.79 oscillations/15 min in PKD2^(-/-) cells vs 7.72 ± 1.07 in PKD2^(-/-) cells transiently transfected with PKD2-GFP cDNA; ^aP < 0.05). Western blotting, oscillation recording and cell imaging were performed as previously reported[16]. Data, obtained from three different experiments analyzing at least 45 cells for every HEK293 clone, are represented as mean ± standard deviation. Analysis of data was performed using Student’s t test, and differences were considered significant at a value of P < 0.05. PKD: Polycystic kidney disease; HEK293: Human embryonic kidney cells; GFP: Green fluorescent protein; PC: Polycystin.