The influence of charge and size on antigen binding to the rat glomerular basement membrane (GBM) was investigated. Chemically cationized ovalbumin, human serum albumin (HSA), human immunoglobulin G (Hu IgG), horse spleen ferritin and human immunoglobulin M (Hu IgM) were injected into rats intravenously. By immunofluorescence significant glomerular binding occurred when the pI exceeded a threshold value of 8.5 to 9.5. At a given pI antigen binding increased with molecular size. Cationized Hu IgM bound only weakly to the glomerular capillary wall, presumably excluded due to size. Subepithelial immune deposits were formed only when antibody was injected subsequently. Detailed electron microscopic studies on in situ formation of immune complexes were performed using cationized horse spleen ferritin. Early on subendothelial deposits were very marked, giving way to subepithelial deposits with time. Under the conditions employed, it appears that deposits can be formed directly at the subepithelial locus but that complexes are also formed subendothelially, dissociating into free molecules or small complexes and then migrating through the lamina densa and reforming.

Podocytes are crucial for the permeability of the glomerular filtration barrier. In glomerular disease, however, reactive oxygen species (ROS) may be involved in podocyte injury and subsequent proteinuria. Here, we describe ROS-dependent gene induction in differentiated podocytes stimulated with H(2)O(2) or xanthine/xanthine-oxidase. Superoxide anions and H(2)O(2) increased mRNA and protein expression of GAS5 (growth arrest-specific protein 5) and CHOP (C/EBP homology protein). Cultured podocytes overexpressing CHOP showed increased generation of superoxide anions compared to controls. In addition, the expression of alpha(3)/beta(1) integrins, crucial for cell-matrix interaction of podocytes, was down-regulated, leading to increased cell-matrix adhesion and cell displacement. The altered cell-matrix adhesion was antagonized by the ROS scavenger 1,3-dimethyl-2-thiourea, and the increase in cell displacement could be mimicked by stimulating untransfected podocytes with puromycin, an inductor of ROS. We next performed immunohistochemical staining of human kidney tissue (normal, membranous nephropathy, focal segmental glomerulosclerosis, and minimal change nephropathy) as well as sections from rats with puromycin nephrosis, a model of minimal change nephropathy. CHOP was weakly expressed in podocytes of control kidneys but up-regulated in most proteinuric human kidneys and in rat puromycin nephrosis. Our data suggest that CHOP-via increased ROS generation-regulates cell-matrix adhesion of podocytes in glomerular disease.

These results suggest an important role for quantitative and qualitative mtDNA alterations through the reduction of mtDNA-encoded respiratory chain function and induction of superoxide in doxorubicin-induced renal lesions.

Microarray analysis of rejected kidneys may help to define different entities of transplant nephropathy, reflecting the multifactorial cause of chronic rejection.