Rosiglitazone increases PPARgamma in renal tubular epithelial cells and protects against damage by hydrogen peroxide.
OBJECTIVE
Thiazolidinediones (TZD) are ligands known to bind to and activate the nuclear peroxisome proliferator-activated receptor gamma (PPARgamma), 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 PPARgamma 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 microM rosiglitazone. The expression of PPARgamma 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 PPARgamma 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 PPARgamma 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 mM did not induce apoptosis as tested by DNA fragmentation assay, but significantly and dose-dependently reduced proliferation 15 h after injury as measured by [3H]thymidine incorporation. 25 microM 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 microM) increased PPARgamma mRNA and protein expression in bEPC in the absence of H2O2. Rosiglitazone failed to increase PPARgamma mRNA in cells with oxidative stress, but Western blots revealed an increase in cellular PPARgamma protein content in the presence of rosiglitazone and increasing concentrations of H2O2. This increase in PPARgamma protein content was almost totally abolished in the presence of 1 microg/ml cycloheximide, but was only marginally reduced by 0.1 microg/ml actinomycin D. EMSA showed a robust increase in nuclear PPARgamma protein binding in vitro to its consensus site after rosiglitazone whereas H2O2 treatment reduced PPARgamma activation. Rosiglitazone treatment of cells with oxidative stress preserved nuclear transactivation of PPARgamma.


CONCLUSIONS
Rosiglitazone increases the PPARgamma content in bEPC after H2O2-induced injury by a posttranscriptional mechanism. Activation of PPARgamma 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.
Sommer
Manfred
M
Department of Internal Medicine III, University of Jena, Jena, Germany.
Wolf
Gunter
G
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001399:Azure Stains; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007150:Immunohistochemistry; D007684:Kidney Tubules / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D047495:PPAR gamma / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000494:pharmacology*
D001399:Azure Stains; D007004:Hypoglycemic Agents; D047495:PPAR gamma; D012333:RNA, Messenger; D045162:Thiazolidinediones; D000077154:Rosiglitazone; D006861:Hydrogen Peroxide