SUN-042 PREGNANE X RECEPTOR (PXR) IS A NOVEL THERAPEUTIC TARGET FOR THE TREATMENT OF CISPLATIN-INDUCED NEPHROTOXICITY IN MICE

Abstract

As a widely used chemotherapeutic agent for treating malignancies, the clinical application of cisplatin (CP) is limited by the toxicity to several vital human organs, especially nephrotoxicity. The pregnane X receptor (PXR) is a ligand-dependent transcriptional factor belonging to the nuclear receptor superfamily. PXR is highly expressed in the liver, intestine and kidney, where it plays an important role in xenobiotic and endobiotic metabolism. PXR is constitutively expressed in normal kidney with relatively high levels in the proximal tubular cells, which are initially affected in CP-induced nephrotoxicity. In this study, we aimed to investigate the role of PXR in CP-induced nephrotoxicity. In vivo, wild-type male C57BL/6 mice were orally pretreated with pregnenolone 16α-carbonitrile (PCN, 40mg/kg), a well-known mouse PXR agonist, for 3 days. After a single nephrotoxic dose of CP (20mg/kg) was administered at day 4, the mice were treated daily with PCN for another 3 days. Levels of blood urea nitrogen (BUN), serum creatinine (sCr), urinary N-acetyl-D-glucosaminidase (NAG) and urinary albumin were measured by commercial kits, high performance liquid chromatography (HPLC), fluorescence probe examination or enzyme linked immunosorbent assay (ELISA). Renal histopathologic features were evaluated by hematoxylin and eosin (H&E) staining on formalin-fixed kidney sections. Caspase 3 activity was measured in kidney homogenates, and TUNEL staining was performed on formalin-fixed sections. Expression levels of important genes involved in apoptosis, oxidative stress, and inflammation were investigated in kidney homogenates by immunoblot and real-time PCR. Whole transcriptome sequencing was applied to reveal the potential mechanisms of PXR protective effects to CP-induced nephrotoxicity. In vitro, PXR expression and activity were modulated by PCN, overexpression, or knockdown by shRNAs in mouse renal tubular epithelial (MCT) cells. A significant increase in BUN, sCr, urinary NAG and albumin levels induced by CP, was markedly prevented by PCN treatment. Histologically, PXR activation ameliorated CP-induced renal morphologic damage, especially in the proximal tubules, characterized by a large number of apoptotic cells. TUNEL-positive cells and renal activity of caspase 3 were both decreased in PCN treated mice. PXR activation abolished CP-induced p53 transcription and phosphorylation, and significantly reduced p53 protein levels. PXR activation also dramatically prevented oxidative and inflammatory stress in the kidneys of mice treated with CP. Whole transcriptome sequencing revealed that protective effects of PXR were associated with multiple crucial signaling pathways, including fatty acid metabolism and degradation, metabolism of xenobiotics by cytochrome P450, protein processing in endoplasmic reticulum, biosynthesis of unsaturated fatty acids, carbon metabolism, steroid hormone biosynthesis, PI3K-Akt signaling pathway, PPAR signaling pathway. Notably, we discovered that PXR protects against CP-induced nephrotoxicity by translational regulating multidrug and toxin extrusion 1 (MATE1), an important transporter mediating cellular excretion of CP, in the kidney. Our data provide evidence that PXR activation preserves renal function after CP treatment, and suggest the possibility of PXR as a novel therapeutic target for CP-induced nephrotoxicity.