Anti-dsDNA antibodies bind to Ku70 in proximal renal tubular epithelial cells and increase matrix protein synthesis and MCP-1 secretion

Abstract

Introduction: We previously reported that anti-dsDNA antibodies from patients with lupus nephritis could bind to renal tubular epithelial cells (PTEC) and induce downstream changes that promote inflammation and fibrosis. We proceeded to identify the cell membrane antigen that mediated anti-dsDNA binding. Methods: Human polyclonal anti-dsDNA antibodies were isolated from the sera of patients with lupus nephritis using affinity chromatography. Samples that showed significant binding to PTEC were selected for subsequent experiments. PTEC plasma membrane proteins were isolated and immuno-precipitated with anti-dsDNA antibodies to identify cross-reactive antigens using LC-MS/MS. After obtaining the identity of the cell membrane protein, gene silencing with RNAi was used to investigate its roles relevant to inflammation and fibrosis. Results: Human anti-dsDNA antibodies bound to a 70 kDa PTEC plasma membrane protein that was identified as Ku70 by LC-MS/MS. The binding was not affected by exogenous DNA, histones, or nucleosomes. Anti-dsDNA binding to PTEC was accompanied by increased fibronectin and laminin expression, and increased MCP-1 secretion (P<0.05, for all). Gene silencing of Ku70 resulted in a marked reduction in antidsDNA antibody binding as demonstrated with immunohistochemistry. Ku70 knockdown decreased fibronectin, laminin and Bax expression, and MCP-1 secretion that were induced by TGF-beta1, MCP-1, IL-1beta, and IL-6 (P<0.05, for all). Kidney specimens from lupus nephritis patients and NZB/W F1 mice showed markedly increased Ku70 expression in the proximal tubules. Conclusions: Our data shows that Ku70 mediates anti-dsDNA antibody binding to PTEC, and Ku70 may have a role in mediating inflammatory and fibrosis in tubulointerstitial disease.