Translational regulation of Kidney Injury Molecule-1 MRNA: presence of an internal ribosomal entry site

Abstract

Kidney Injury Molecule-1 (KIM-1) is a type I transmembrane glycoprotein that is highly upregulated on regenerating dedi erentiated renal proximal tubular epithelial cells as a consequence of toxic or ischemic injury. KIM-1 is a recognized biomarker of nephrotoxicity by United States Food and Drug Administration (FDA) and Europe Medicines Agency; therefore, understanding its transcriptional and translational regulation under chemotoxic stress is of the utmost importance. As chemotoxic stress inducers we used three xenobiotics, Ochratoxin A (OTA), Gentamicin (GM) and Cisplatin (CP) which are known to cause damage to kidney cells. The e ects of these three xenobiotics on viability, proliferation, necrosis and apoptosis are examined using the immortilized human proximal tubule epithelial HK-2 cell line. The transcriptional start region of Kim-1 gene is determined by Rapid Ampli cation of cDNA Ends (RACE). The changes in KIM-1 mRNA and protein levels are measured by Quantitative Real Time PCR and Western Blotting, respectively. Although there was a signi cant decrease in global protein synthesis upon xenobiotic treatment, KIM-1 protein levels increased. Furthermore, no signi cant change in KIM-1 mRNA levels can be detected which led us to suspect the existence of a possible post-transcriptional regulatory mechanism by which KIM-1 mRNA is translated via an Internal Ribosome Entry Site (IRES). This hypothesis is tested in vitro using bicistronic reporter vector pRF containing the 5'UTR of Kim-1 gene. Our results con rm our hypothesis of a possible IRES localized around 300 bp upstream of the translation initiation codon.