Anti-dsDNA Antibodies Bind to Ku70 Leading to Increased MCP-1 and TGF-beta1 Expression in Proximal Tubular Epithelial Cells

Abstract

Introduction: Lupus nephritis is characterized by anti-dsDNA antibody production and their deposition in the kidney parenchyma resulting in immune-mediated tissue injury. Immune deposition along the tubular basement membrane is commonly observed but its pathogenic significance remains to be determined. We investigated the binding of anti-dsDNA antibodies to proximal tubular epithelial cells (PTEC) and its impact on inflammatory and fibrotic processes. Methods: Anti-dsDNA antibodies with high binding activity to PTEC were isolated from the sera of lupus nephritis patients using affinity chromatography. Cultured PTEC were incubated with serum free medium (SFM), control IgG, or anti-dsDNA antibodies in the presence or absence of DNase I, trypsin, exogenous dsDNA, histones and nucleosomes. PTEC plasma membrane proteins were isolated and immunoprecipitated with anti-dsDNA antibodies to identify cross-reactive antigens using LC-MS/MS. Results: DNase I, dsDNA, histones and nucleosomes had no effect on anti-dsDNA antibody binding to PTEC. Limited trypsin treatment of PTEC, which removed cell surface proteins without affecting cell attachment, resulted in a significant reduction in anti-dsDNA antibody binding (P<0.01). Anti-dsDNA antibodies bound to a 70 kDa protein identified as Ku70 by LC-MS/MS. Incubation of PTEC with anti-dsDNA antibodies increased Ku70 expression in a time-dependent manner, accompanied by increased MCP-1 and TGF-beta1 expression. Gene silencing with Ku70 specific RNAi suppressed Ku70 mRNA by approximately 85.0%, accompanied by 79.4% and 33.9% reduction in MCP-1 and TGF-beta1 gene expression respectively (P<0.05, for both). Kidney biopsies from lupus nephritis patients showed a marked increase in Ku70 expression, predominantly in proximal tubular epithelial cells. Conclusions: Our data showed that anti-dsDNA antibodies bound directly to PTEC through Ku70, and this binding was accompanied by downstream pro-inflammatory and pro-fibrotic processes. These findings could have important implications on tubulo-interstitial disease in lupus nephritis.