In proliferative glomerulonephritis, both macrophages and mesangial cells generate reactive oxygen species (ROS), contributing to the development of glomerular injury. We have attempted to determine which cell produces ROS during anti-Thy1 nephritis (ATN) in rats. The generation of ROS was studied using luminol amplified chemiluminescence (GCL) on isolated glomeruli. Immunohistochemical studies used avidin-biotin complex (ABC) to label macrophages and mesangial cells. Immediately after ATN induction, mesangiolysis and infiltration with ED-1 positive cells (referred to as macrophage) was noted with a peak at day 1. After day 4, mesangial proliferation appeared with a decrease of the ED-1 positive cells and a prominent increase of PCNA positive cells (regarded as mesangial cells). In the early phase of ATN, GCL, reflecting ROS generation, increased along with the appearance of ED-1 positive cells. GCL subsequently decreased as mesangial cells increased. This suggested that macrophage were the principal participants in ROS generation in the early phase of ATN although mesangial cells cannot be completely disregarded in the generation of ROS and development of glomerular injury.

Depletion of serum complement decreases subepithelial EDDs as well as the number of sites with decreased anionic charge underlying the EDDs. Thus, the size of subepithelial EDDs plays a pivotal role in the onset of albuminuria.

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Background. Compositional changes in the extracellular matrix (ECM) and the role of transforming growth factor 1 (TGF-1 ) in the process of mesangial expansion have been widely investigated in experimental animals, but much less attention has been focused on such changes in human renal biopsy specimens, particularly in regard to quantitative studies. Methods. In 28 biopsy specimens with varying degrees of mesangial expansion, we performed conventional ECM staining and immunohistochemical staining for ECM components, including cellular fibronectin containing extra domain A (EDA(ϩ)cFn), tenascin, and type IV collagen, as well as investigating mRNA and protein expression of TGF-1 . Results for ECM components stained were evaluated quantitatively. Results. By multiple regression analyses, mesangial expansion, as quantitated by conventional silver, but not by periodic acid Schiff (PAS) staining, correlated well with EDA(ϩ)cFn accumulation. In specimens with increased mesangial expansion estimated by silver staining, the ratio of the EDA(ϩ)cFn-positive area to the silver-positive area was greater than the ratio of the type IV collagen-positive area to the silver-positive area; the type IV collagenpositive area, however, was always larger than the EDA(ϩ)cFn-positve area. In glomeruli with moderate mesangial expansion, expression of TGF-1 mRNA and protein was enhanced. Where mesangial expansion was severe, TGF-1 protein and mRNA expression was decreased in the mesangial area but increased in neighboring epithelium and endothelium. Conclusion.Our results indicate that silver staining, but not PAS, reflects the accumulation of mesangial ECM proteins and that while the proportion of EDA(ϩ)cFn is altered in the course of ECM expansion, type IV collagen remains the major ECM component. Expression of TGF-1 was suppressed at sites of massive mesangial ECM accumulation.

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