Molecular Cloning of a 70‐kiloDalton (kDa) Nuclear Protein That Binds to Insulin Responsive Element (IRE) of Rat Angiotensinogen (ANG) Gene Promoter and Modulates ANG Gene Expression in Kidney Proximal Tubular Cells

Abstract

We reported previously an IRE in rat ANG gene promoter that binds to two nuclear proteins with apparent molecular weights of 48- and 70-kDa from rat immortalized renal proximal tubular cells (IRPTCs). We recently identified the 48-kDa nuclear protein as the 46 kDa heterogenous nuclear ribonucleoprotein F (hnRNP F) that binds to rANG-IRE and inhibits ANG gene expression in IRPTCs. The present studies aimed to clone the 70-kDa nuclear protein and to study its action on ANG gene expression. Nuclear proteins isolated from IRPTCs were subjected to 2-dimensional gel electrophoresis. The 70-kDa nuclear protein was detected by Southwestern blotting and subsequently identified by mass spectrometry, which revealed that it was identical to 65-kDa hnRNP K. Transient transfection of hnRNP K cDNA inhibits ANG mRNA expression and ANG gene promoter activity in IRPTCs. Most interestingly, hnRNP K pulls down with hnRNP F. Co-transfection of hnRNP K with hnRNP F positively regulates ANG gene expression in IRPTCs. In conclusion, our studies demonstrate that 65-kDa hnRNP K is a novel nuclear protein that interacts with hnRNP F and binds to IRE of the rat ANG gene promoter and subsequently modulates ANG gene expression in IRPTCs. These data indicate that 65-kDa hnRNP K plays an important role in modulating intrarenal ANG gene expression and renin-angiotensin system activation and subsequently kidney injury. These observations open a novel research route to study renal ANG gene regulation.