Figure 3.

Loss of RCAN1 increases CN activity and reduces viability in human podocytes. (A) Immunoblotting and (B) subsequent quantification revealed a significant reduction in RCAN1 expression in conditionally immortalized podocyte cell lines transduced with shRNA against RCAN1 (RCAN1 KD) when compared with lentiviral control podocytes (P<0.001, n=4, t test). (C) RCAN1 KD podocytes had increased levels of CN activity compared with controls (P=0.0003, n=12, t test). (D) RCAN1 KD podocytes exposed to serum starvation displayed an increased susceptibility to apoptosis, as measured by automated live-cell imaging of a fluorescent reporter of cleaved caspase-3 activity (P<0.01 for all time points after 30 hours, n>16 for each group, two-way ANOVA) and (E and F) immunoblotting (P=0.003, n=4, t test). This apoptosis could be rescued by CNI treatment (1 µM FK506), demonstrating the contributions of altered CN regulation to podocyte viability (P>0.35 for all time points).