Binding of anti-dsDNA antibodies to major vault protein of proximal renal tubular epithelial cells resulted in increased fibronectin expression and MCP-1 secretion

Abstract

Background: Anti-dsDNA antibodies deposit in the kidney parenchyma in lupus nephritis. In addition to complement activation by immune complexes and activation of downstream pro-inflammatory mechanisms, whether these antibodies could directly induce organ damage remains controversial. We investigated the binding of human antidsDNA antibodies to proximal renal tubular epithelial cells (PTEC) and the downstream impact on cell functions. Methods: Human polyclonal anti-dsDNA antibodies were isolated from the sera of lupus nephritis patients using affinity chromatography and those with high binding affinity to PTEC were selected for further studies. PTEC membrane, cytosolic and nuclear proteins were isolated and immunoprecipitated with anti-dsDNA antibodies to identify cross-reactive antigens using liquid chromatography-mass spectrometry (LC-MS). Results: Anti-dsDNA antibodies bound to a 100 kDa protein, identified as major vault protein (MVP), which was present in the cytosolic and nuclear fractions, but not in the plasma membrane fraction. Incubation of PTEC with anti-dsDNA antibodies increased MVP expression, as determined by Western blot analysis, in a time-dependent manner (P<0.001, for 24h), and accompanied by increased fibronectin expression (P<0.01) and MCP-1 secretion (P<0.05) compared to cells incubated with IgG from healthy controls. Immunohistochemical studies showed predominantly perinuclear localization of MVP and weak intracellular expression of fibronectin under basal conditions. MVP overexpression in PTEC, by transfection with MVP plasmid, resulted in clustering of MVP in the cytoplasm, which was accompanied by fibronectin accumulation in the extracellular matrix and increased MCP-1 secretion (P<0.01). MVP gene silencing using RNAi resulted in 40% reduction in fibronectin expression and 53% reduction in MCP-1 secretion. Kidney biopsies from lupus nephritis patients showed markedly increased MVP expression, predominantly on proximal tubular epithelial cells. Conclusions: Our data showed that anti-dsDNA antibody binding to MVP in PTEC was associated with downstream inflammatory and fibrogenic processes.