Rost Sylvia, Daniel Christoph, Schulze-Lohoff Eckhard, Bäumert Hans G, Lambrecht Günter, Hugo Christian
Division of Nephrology, University of Erlangen-Nürnberg, Erlangen, Germany.
Kidney Int. 2002 Nov;62(5):1659-71. doi: 10.1046/j.1523-1755.2002.00621.x.
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.
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.
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.
These data strongly suggest an in vivo role for extracellular nucleotides in mediating early MC proliferation after MC injury.
尽管细胞外核苷酸已被证明可通过激活嘌呤能P2受体（P2Y受体）在培养的大鼠系膜细胞中发挥促有丝分裂作用，但迄今为止，这些发现的体内相关性尚不清楚。几乎所有细胞，尤其是血小板的致密颗粒都含有高水平的核苷酸，这些核苷酸在细胞损伤或血小板聚集时释放。在大鼠实验性系膜增生性肾小球肾炎（抗Thy1模型）中，系膜溶解和肾小球血小板聚集之后会出现明显的系膜细胞（MC）增殖反应，导致肾小球细胞增多。因此，我们使用P2受体拮抗剂PPADS研究了细胞外核苷酸及其相应受体在核苷酸刺激的培养系膜细胞和炎性肾小球疾病中的作用。
通过细胞计数和[3H]胸苷掺入试验测量PPADS对核苷酸或胎牛血清（FCS）刺激的培养MC增殖的影响。诱导抗Thy1模型后，大鼠接受不同剂量（15、30、60mg/kg体重）的P2受体拮抗剂PPADS注射。在抗Thy1疾病期间评估增殖的系膜细胞和非系膜细胞、系膜细胞活化、基质积累、炎性细胞流入、系膜溶解、微动脉瘤形成和肾功能参数。使用RT-PCR、实时PCR、Northern印迹分析、原位杂交和免疫组织化学评估P2Y-mRNA和蛋白表达。
在培养的系膜细胞中，PPADS以剂量依赖性方式抑制核苷酸刺激的增殖，但不抑制FCS刺激的增殖。在抗Thy1模型中，PPADS特异性且剂量依赖性地降低早期（第3天）而非晚期（第8天）的肾小球系膜细胞增殖以及系膜的表型活化和轻微的基质扩张。虽然在系膜溶解程度、炎性细胞流入、蛋白尿或血压方面未获得一致的效果，但PPADS治疗增加了抗Thy1大鼠的血清肌酐和尿素。在培养的MC和分离的肾小球中检测到P2Y受体表达（P2Y2和P2Y6），并在抗Thy1疾病期间显示出短暂的显著增加。
这些数据强烈表明细胞外核苷酸在介导MC损伤后早期MC增殖中具有体内作用。
