ET-1 Induced Downregulation of MRP2 via miRNA 133a - A Marker for Tubular Nephrotoxicity?

Abstract

Background: Multiple drug resistance (MDR), known from treating malignant tumors with chemotherapy, increases the efflux of reabsorbed reagents in tumor cells. This mechanism has been reported in the renal proximal tubule and may prevent therapeutic tubular protection in proteinuria. Since endothelin-1 (ET-1), a major component in the urine of proteinuric patients, stimulates proximal tubules, its influence on MDR was analyzed with emphasis on the multidrug resistance-associated protein 2 (MRP2), a prominent transporter in the human proximal tubule and microRNA (miRNA) 133a. Methods: ET-1 stimulated, cultured human renal proximal tubule cells (RPTECs), were analyzed via Western blot for the expression of MRP2 and via qRT-PCR for miRNA 133a. For direct interaction between the miRNA 133a and the 3′UTR of MRP2, an immunoprecipitation was performed using FITC-labelled miRNA 133a as capture, followed by MRP2 PCR analysis and Sanger sequencing. Murine Adriamycin nephropathic model and human proteinuric samples showed high levels of miRNA 133a but low levels of MRP2. The increasing miRNA 133a levels were detectable in urine samples of humans and animals. Results: ET-1 activates the miRNA 133a, which can bind to the 3′UTR of MRP2 and is therefore responsible for the detectable decrease of MRP2. Conclusion: This is the first report to analyze the correlation between ET-1-induced miRNA 133a overexpression in proteinuria resulting in MRP2 downregulation, which is a contributing factor for renal cytotoxicity. The detection of the miRNA 133a in urine samples can be possibly used as a monitor for cytotoxicity.