Increased nitrotyrosine staining in kidneys from patients with diabetic nephropathy

Background. Proximal tubular cells produce nitric oxide (NO.). We have shown that under hyperglycemic conditions, cultured proximal tubular cells express cytochrome P450 2E(1), which is capable of producing superoxide (O-2(.)) NO. and O-2(.) react to form peroxynitrite (ONOO.), a powerful oxidant. ONOO. nitrosylates tyrosine moieties on proteins causing tissue damage. Our hypothesis is that ONOO. plays a role in early diabetic tubular damage and perhaps disease progression. Methods. Renal biopsies from patients with diabetic nephropathy (DM), acute allograft rejection (AAR), acute allograft tubular necrosis (ATN), and glomerulonephritis (GN) were obtained. Normal kidney specimens were taken from nephrectomy samples (N = 10 for each group). The tissues were examined for the presence of nitrotyrosine using an immunoperoxidase technique with a polyclonal antibody. Samples were then arbitrarily scored, and the results analyzed (analysis of variance and Student's t-test for unpaired data). The number of apoptotic cells in a sample of tubules in each biopsy was also assessed. Results. The DM biopsies showed increased staining for nitrotyrosine in proximal tubules (P = 0.0001) and in the thin limb of the loop of Henle (P = 0.0006) compared with all other groups. There was increased staining in the ascending and distal tubules in GN as compared to DM and ATN (P = 0.01). Nitrotyrosine was also found in all distal tubules and collecting ducts, including normals. There was no difference in the number of apoptotic tubular cells in diabetics compared with controls. Conclusion. To our knowledge, these data provide the first evidence for the presence of nitrotyrosine in both normal and diseased kidneys. The significance of the findings in normals is unclear, but could be due to activation of constitutive NOS. However, the study clearly demonstrates increased production of ONOO. in proximal tubules of patients with DM, and suggests that oxidant injury of the proximal tubules plays an important part in the pathogenesis of DM.