P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangial proliferative glomerulonephritis.
BACKGROUND: Although extracellular nucleotides have been shown to confer mitogenic effects in cultured rat mesangial cells through activation of purinergic P2 receptors (P2Y receptors), thus far the in vivo relevance of these findings is unclear. Virtually all cells and in particular the dense granules of platelets contain high levels of nucleotides that are released upon cell injury or platelet aggregation. In experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), mesangiolysis and glomerular platelet aggregation are followed by a pronounced mesangial cell (MC) proliferative response leading to glomerular hypercellularity. Therefore, we examined the role of extracellular nucleotides and their corresponding receptors in nucleotide-stimulated cultured mesangial cells and in inflammatory glomerular disease using the P2 receptor antagonist PPADS. METHODS: The effects of PPADS on nucleotide- or fetal calf serum (FCS)-stimulated proliferation of cultured MC were measured by cell counting and [3H]thymidine incorporation assay. After induction of the anti-Thy1 model, rats received injections of the P2-receptor antagonist PPADS at different doses (15, 30, 60 mg/kg BW). Proliferating mesangial and non-mesangial cells, mesangial cell activation, matrix accumulation, influx of inflammatory cells, mesangiolysis, microaneurysm formation, and renal functional parameters were assessed during anti-Thy1 disease. P2Y-mRNA and protein expression was assessed using RT-PCR and real time PCR, Northern blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: In cultured mesangial cells, PPADS inhibited nucleotide, but not FCS-stimulated proliferation in a dose-dependent manner. In the anti-Thy1 model, PPADS specifically and dose-dependently reduced early (day 3), but not late (day 8), glomerular mesangial cell proliferation as well as phenotypic activation of the mesangium and slightly matrix expansion. While no consistent effect was obtained in regard to the degree of mesangiolysis, influx of inflammatory cells, proteinuria or blood pressure, PPADS treatment increased serum creatinine and urea in anti-Thy1 rats. P2Y receptor expression (P2Y2 and P2Y6) was detected in cultured MC and isolated glomeruli, and demonstrated a transient marked increase during anti-Thy1 disease. CONCLUSION: These data strongly suggest an in vivo role for extracellular nucleotides in mediating early MC proliferation after MC injury.