Purification and localization of a 25-kD porcine renal puromycin aminonucleoside-binding protein

T. Kodama, H. Wakui, A. Komatsuda, H. Imai, A. B. Miura, Y. Tashima
1997 Nephrology, Dialysis and Transplantation  
Introduction Background. Reactive oxygen species ( ROS) are considered to have a role in the progression of puromycin Reactive oxygen species (ROS ), including free radicals aminonucleoside (PAN ) nephrosis. However, the exact such as superoxide anion and hydroxyl radical, are mechanism by which PAN induces ROS in this model mediators of ischaemic, toxic, and immune-mediated is little known. In the present study, we attempted to tissue injury. Numerous studies have shown that ROS purify a
more » ... ROS purify a candidate for the target protein from PAN are implicated in the progression of various kidney nephrotoxicity. diseases in experimental animal models [1-4 ]. Methods. Using PAN-aÃnity column chromato-A single intravenous injection of puromycin aminography, a series of PAN-binding proteins was isolated nucleoside (PAN ) induces massive proteinuria in from porcine renal extracts. We produced a specific animals, and early histological changes are similar to antibody against a 25-kD protein eluted from the those of human minimal change nephrotic syndrome PAN-aÃnity matrix, and then developed a method to [5] . Thus, PAN nephrosis has been widely used as an purify this protein. A partial amino acid sequence of animal model for human nephrotic syndrome. It has the 25-kD PAN-binding protein was determined, and been demonstrated that ROS are also involved in the its tissue distribution was examined by immunoblot pathogenesis of PAN nephrosis [1, 2, 4] . Indeed, the and immunohistochemical studies. administration of antioxidants, superoxide dismustase Results. The purified 25-kD PAN-binding protein was and allopurinol, can reduce proteinuria and prevent identified as a renal homolog of a new member of glomerular injury in PAN nephrosis [6 ] . However, the NAD(P)H:quinone oxidoreductases (NQOs, EC exact mechanism by which PAN induces ROS in this ) that suppress the semiquinone and superoxide model is little known. anion formation in cells, designated NQO 2 . We have attempted to isolate renal target proteins Immunoblot analysis revealed a higher expression of from PAN nephrotoxicity, and recently purified a the 25-kD PAN-binding protein in the kidney, brain, 17-kD PAN-binding protein from porcine kidney [7 ] . and liver among porcine major organs. Immuno-This protein was identified as the reported 17-kD histochemical studies showed an intrarenal distriprotein kinase C inhibitor, but its function remained bution of this protein in epithelial cells of the glomeruli unclear. In the present study, we have purified another and tubules, mesangial cells, and vascular smooth porcine renal PAN-binding protein with a molecular muscle cells. mass of 25 kD. This PAN-binding protein was identi-Conclusions. We have purified the renal homolog of fied as a renal homolog of a new member of NQO 2 as a PAN-binding protein, and shown its unique NAD(P)H:quinone oxidoreductases ( NQOs) [8], also tissue expression. PAN may bind to the NQO 2 homoknown as DT-diaphorases (EC Although log and inhibit its function in the renal target cells. this NQO member has not been purified or character-This is presumed to result in an increase of ROS in ized as a protein so far, a role against oxygen toxicity the kidney with PAN nephrosis. is suggested [8, 9] . We also examined the tissue distribution and intrarenal localization of the protein, by Key words: animal model; NAD(P)H:quinone oxidoreimmunoblot analysis and immunohistochemical study ductase; nephrotic syndrome; puromycin aminonucleousing a specific antibody. Amongst porcine organs, a side; reactive oxygen species higher expression of the 25-kD PAN-binding protein was found in the kidney, mainly expressed in epithelial cells of the glomeruli and mesangial cells. We discuss Correspondence and oÂprint requests to: Dr H. Wakui, Third the possibility that this new NQO member might be a
doi:10.1093/ndt/12.7.1453 pmid:9249785 fatcat:q7urebzhoja4hgybrddaegaxte