Background and objectives Systemic lupus erythematosus (SLE) causes inflammation and damage of various organs, often of the kidney (Lupus nephritis, LN). The occurrence of antibodies against double stranded DNA (adsDNAabs) is a hallmark of the disease, and serum levels of adsDNAabs correlate directly with the risk for LN during SLE. Podocytes are the filtrating cells of the renal glomerulus, and podocyte damage is a key factor in the development of LN-associated proteinuria. However, the molecular pathogenesis of LN as well as the question of how dsDNAabs act on podocytes during LN are not completely understood.
Methods Human adsDNAabs originally isolated from SLE patients were produced recombinantly, and podocytes (AB8 cell line) were incubated with these adsDNAabs as well as control antibodies. The interaction of the antibodies with the AB8 cells, their internalisation and subcellular localisation were analysed by immunofluorescence, Western blot, and electron microscopy. To decompose potential antibody-antigen complexes, adsDNAabs were pre-incubated with different amounts of DNaseI. To study potential internalisation mechanisms, endocytosis was inhibited by MDC, Nystatin, and Chlorpromazine. Primary human podocytes from urine of LN patients were harvested, fixed and stained for immunofluorescence.
Results Podocytes internalised adsDNAabs in a time and dose dependent manner, whereas no uptake of non-selfreactive and anti-SSA/Ro antibodies was observed. These adsDNAabs were seen in intracellular vesicle-like structures, and internalisation depended on the formation of complexes of adsDNA antibodies with DNA. Inhibition of clathrin dependent endocytosis reduces the number of IgG positive vesicles within the cell by 75%, while inhibition of caveolin dependent endocytosis had no effects. Podocytes of LN patients were found in urine and exhibited human IgG positive aggregates, indicating a disease relevant role of antibody internalisation by podocytes.
Conclusion Podocytes are postmitotic cells, therefore damage and loss of podocytes during LN is an irreversible process, leading to proteinuria. We could show that these cells internalise adsDNAabs selectively in complex with their target. A similar pattern of IgG positive structures could be found in podocytes isolated from the urine of LN patients. The internalisation takes place in a clathrin dependent manner and could be part of the pathogenesis of LN.