Anti-dsDNA antibodies bind to Ku70 in proximal renal tubular epithelial cells and increased matrix protein and cytokine secretion

Abstract

Background: Anti-dsDNA antibodies can lead to immune-mediated kidney injury through complement activation or trigger downstream pro-inflammatory or fibrotic responses after cellular binding. Immune deposition along tubular basement membrane is commonly observed. We investigated the binding of anti-dsDNA antibodies to proximal renal tubular epithelial cells (PTEC). Methods: Human polyclonal anti-dsDNA antibodies were isolated from the sera of lupus nephritis patients using affinity chromatography and samples demonstrating high binding affinity to PTEC were studied. Cultured PTEC were incubated with serum-free medium, control IgG, or anti-dsDNA antibodies for up to 48h. PTEC plasma membrane proteins were isolated and immuno-precipitated with anti-dsDNA antibodies to identify cross-reactive antigens using LC-MS/MS. The role of Ku70 in inflammatory and fibrotic processes was investigated by gene silencing with RNAi. Results: The 70 kDa on PTEC cell membrane that bound anti-dsDNA antibodies was identified as Ku70 by LC-MS/MS. Exogenous DNA, histones, or nucleosomes did not affect its binding by anti-dsDNA antibodies. Anti-dsDNA antibodies binding to Ku70 was accompanied by increased fibronectin and laminin expression, and increased MCP-1 and IL-6 secretion (P<0.05, for all). Ku70 gene silencing with ninety percent efficacy resulted in reduced PTEC binding by anti-dsDNA antibodies as shown by immunohistochemistry. Ku70 knockdown significantly decreased the expression of fibronectin, laminin, and Bax, and and also the secretion of MCP-1 and IL-6, induced by TGF-β1, MCP-1, IL-1β, 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 Conclusion: Our data shows that Ku70 mediates the binding of anti-dsDNA antibodies to PTEC, which is accompanied by downstream pro-inflammatory and pro-fibrotic cellular responses.