Alterations of PPARα and its coactivator PGC-1 in cisplatin-induced acute renal failure

Abstract

In this study we examined whether a recently characterized coactivator of Peroxisome proliferator activated receptor alpha (PPARalpha), Peroxisome proliferator activated receptor-gamma-coactivator-1 (PGC-1) plays a role in the regulation of fatty acid oxidation during cisplatin-induced nephrotoxicity. Studies in mouse kidneys used quantitative reverse transcription-polymerase chain reaction (RT-PCR) to measure peroxisomal acyl coenzyme A (acyl-CoA) and PGC-1 mRNA levels and in situ hybridization to localize PGC-1 mRNA. Studies in LLCPK1 cells used quantitative RT-PCR and biochemical assays to measure mRNA levels and enzyme activities of peroxisomal acyl-CoA, mitochondrial carnitine palmitoyl transferase (CPT) and PGC-1. Eletrophoretic mobility shift assays (EMSA) and Western blot analysis of nuclear extracts, and transient transfection of PGC-1 were used to examine the effect of cisplatin on PPARalpha-regulated fatty acid oxidation. Cisplatin decreased mRNA levels of peroxisomal acyl-CoA enzyme in mouse kidney and also reduced the mRNA levels and enzyme activities of acyl-CoA and mitochondrial CPT-1 in LLCPK1 cells. DNA-protein binding studies demonstrated that exposure to cisplatin reduces PPARalpha/retinoid X receptor (RXRalpha) binding activity. Immunoblotting studies demonstrated that cisplatin had no effect on nuclear levels of PPARalpha or RXRalpha protein. In situ hybridization studies in mouse kidney demonstrated the localization of PGC-1 mRNA to proximal tubules and thick ascending limb of Henley (TALH) cells. Cisplatin diminished the expression of PGC-1 mRNA levels in mouse kidney and also in LLCPK1 cells. Transient expression of PGC-1 shows the nuclear localization of PGC-1 protein and increased PPARalpha transcriptional activity in LLCPK1 cells. These results demonstrate that cisplatin deactivates PPARalpha by reducing its DNA binding activity and the availability of its tissue specific coactivator PGC-1.