Figure 1

Mechanical stress induces the expression of fibrogenic cytokines in tubular cells via MRTF- and TAZ-dependent mechanisms. LLC-PK1 cells were grown to confluence on Flexcell membranes and transfected with non-related (NR), MRTF-A + B- (50 nM each) or TAZ-specific (50 nM) siRNA for 48 h (a,c,e,g), or serum-deprived overnight and pretreated with vehicle (Veh), CCG-1423 (CCG, 5 μM) or verteporfin (VP, 1 μM) for 30 min (b,d,f,h), as shown. Cells were then left unchallenged (No stretch) or subjected to cyclic stretch (Stretch, 10% elongation at 1 Hz) for 3 h. The pharmacological inhibitors were present throughout the course of the experiment. Cells were lysed, total cellular mRNA was isolated and reverse-transcribed and qPCR was performed using primers targeting TGFβ1 (a,b), CTGF (c,d), PDGF-B (e,f) and IHH (g,h). The expression of cytokine mRNAs was normalized to GAPDH in the same samples, and data is presented as fold changes compared to the untreated and unchallenged samples (n = 3, in duplicate). To assess residual activation after inhibitory interventions, statistical analysis was also performed between no-stretch + inhibitor and stretch + inhibitor conditions. TGFβ: non-stretch siMRTF and siTAZ vs. stretch siMRTF and siTAZ, p < 0.05 and p < 0.0001; non-stretch CCG and VP vs. stretch CCG and VP, p < 0.01 and p < 0.05. CTGF: non-stretch siMRTF and siTAZ vs. stretch siMRTF and siTAZ, ns and p < 0.01; non-stretch CCG and VP vs. stretch CCG and VP, p < 0.0001 and p < 0.0001. PDGF: non-stretch siMRTF and siTAZ vs. stretch siMRTF and siTAZ, p < 0.0001 and p < 0.0001; non-stretch CCG and VP vs. stretch CCG and VP, ns and p < 0.001. IHH: non-stretch siMRTF and siTAZ vs. stretch siMRTF and siTAZ, p < 0.0001 and ns; non-stretch CCG and VP vs. stretch CCG and VP, ns and ns. The representative Western blots (inset in panel a) document the downregulation of MRTF and TAZ by the corresponding siRNAs.