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Abstract: TH-OR020

Anti-dsDNA Antibodies Bind to Ku70 in Proximal Renal Tubular Epithelial Cells and Increased Matrix Protein and Cytokine Secretion

Session Information

Category: CKD (Non-Dialysis)

  • 1903 CKD (Non-Dialysis): Mechanisms

Authors

  • Chan, Daniel Tak Mao, The University of Hong Kong, Hong Kong, China
  • Ho, Shirli S. K., The University of Hong Kong, Hong Kong, China
  • Tai, Chi pang, The University of Hong Kong, Hong Kong, China
  • Chan, Caleb C-Y, The University of Hong Kong, Hong Kong, China
  • Cheng, Wai-Ming, The University of Hong Kong, Hong Kong, China
  • Chau, Mel, The University of Hong Kong, Hong Kong, China
  • Yung, Susan, The University of Hong Kong, Hong Kong, China
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.

Funding

  • Government Support - Non-U.S.