Rosiglitazone increases PPAR gamma in renal tubular epithelial cells and protects against damage by hydrogen peroxide

Background/Aims: Thiazolidinediones (TZD) are ligands known to bind to and activate the nuclear peroxisome proliferator-activated receptor gamma (PPAR gamma), and are currently used as insulin sensitizers in type 2 diabetes. Recently, several studies have shown that TZD may have a role in renal protection in various experimental models. However, the precise mechanisms by which TZD may possibly affect tubular cell survival after injury remain unclear. We studied the influence of the TZD rosiglitazone on PPAR gamma expression and cell function with cellular damage induced by increasing hydrogen peroxide (H2O2) concentrations in bovine renal tubular epithelial cells (bEPC) to determine whether rosiglitazone is cytoprotective under these conditions. Methods: bEPC were cultured in the presence of H2O2 after pretreatment with or without 25 mu M rosiglitazone. The expression of PPAR gamma mRNA and protein were determined using RT-PCR or Western blots, respectively, after 6 and 24 h. Some cells also received actinomycin D or cycloheximide and PPAR gamma protein expression was tested. Proliferation rates of cultures were compared after 15 h and after a recovery phase of 6 days. Apoptosis was assessed by DNA fragmentation. Nuclear PPAR gamma activity was evaluated by electrophoretic mobility shift assay (EMSA), and the cellular location was detected using immunofluorescence. Results: Incubation of bEPC with H2O2 concentrations up to 0.75 m M did not induce apoptosis as tested by DNA fragmentation assay, but significantly and dose-dependently reduced proliferation 15 h after injury as measured by [H-3] thymidine incorporation. 25 mu M rosiglitazone alone also reduced proliferation and failed to attenuate the H2O2-mediated inhibition of proliferation. However, rosiglitazone facilitates recovery of tubular cells 6 days after H2O2 induced injury. Rosiglitazone (25 mu M) increased PPAR gamma mRNA and protein expression in bEPC in the absence of H2O2. Rosiglitazone failed to increase PPAR gamma mRNA in cells with oxidative stress, but Western blots revealed an increase in cellular PPAR gamma protein content in the presence of rosiglitazone and increasing concentrations of H2O2. This increase in PPAR gamma protein content was almost totally abolished in the presence of 1 mu g/ml cycloheximide, but was only marginally reduced by 0.1 mu g/ml actinomycin D. EMSA showed a robust increase in nuclear PPAR gamma protein binding in vitro to its consensus site after rosiglitazone whereas H2O2 treatment reduced PPAR gamma activation. Rosiglitazone treatment of cells with oxidative stress preserved nuclear transactivation of PPAR gamma. Conclusions: Rosiglitazone increases the PPAR gamma content in bEPC after H2O2-induced injury by a posttranscriptional mechanism. Activation of PPAR gamma facilitates the long-term recovery of tubular cells 6 days after oxidative injury, but had no effect on the attenuated proliferation shortly after injury. TZD cannot prevent oxidative injury to tubular cells, but may be important mediators to enhance cellular recovery after oxidative stress. Copyright (c) 2007 S. Karger AG, Basel.