Figure 6

UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A. UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. ***p < 0.001. B. UCH-L1 tet-on podocytes were left untreated (-) or treated with doxycycline as in A (+) before PARP-1-reactive bands were detected by immunoblotting. Cell lysates from untreated and apoptotic L929Ts cells (Co, treated with 100 ng/ml TNF and 2 μg/ml CHX for 1 h) are shown as controls. Full-length and cleaved PARP-1 is marked by arrows. Detection of actin served as a loading control. C. Aliquots from the stimulations in B were also analyzed for caspase activity. As negative control, tet- podocytes were treated with doxycycline as in A; as positive control, lysates from doxycyline-treated tet- podocytes were incubated with cytochrome c and dATP to activate caspases. Subsequently, the activity of caspases-3 and -8 was determined by measuring the cleavage of fluorogenic substrates (zIETD-afc and zDEVD-afc) over 70 minutes. The Western blot below shows that UCH-L1 is indeed upregulated in doxycycline-treated but not untreated UCH-L1 tet-on podocytes and also not in doxycycline-treated negative control tet- podocytes. Treatment with doxycycline was performed as in A. UCH-L1 was detected with mAb UCH-L1, detection of actin served as a loading control. D. Cell death was induced in UCH-L1 tet-on podocytes by treatment with doxycycline as in A in the presence of 50 μM zVAD-fmk (+zVAD-fmk, middle and lower micrographs) or no inhibitor (upper micrographs). Micrographs show the morphology of dying cells within a monolayer of healthy cells (overlay, nuclei are stained blue), and in parallel the nuclear morphology of the same cells after staining with Hoechst dye (Hoechst). Original magnification: x 400.