Analysis of responses to chronic stress generated by proteinuria in renal proximal tubular cells.

蛋白尿与肾脏疾病的进展密切相关。我们发现蛋白尿增加了疾病模型肾脏中脑特异性蛋白胶质成熟因子-b（GMF）的表达。GMF的表达引起氧化损伤的脆弱性，导致在培养的肾近端小管（PT）细胞命名为mProx 24的凋亡。我们还发现，白蛋白在15分钟内产生氧化应激，导致STAT 1和STAT 5活化。因此，假设白蛋白可能因氧化应激而导致表达GMF的PT细胞（GMF-PT）凋亡。用不同浓度的白蛋白（1- 30 mg/ml）处理GMF-PT和野生型PT细胞72小时。MTS法检测细胞活力。结果表明，白蛋白可引起GMF-PT细胞中细胞死亡的显著和剂量相关性增加，但在野生型PT细胞中则无此现象。添加抗氧化剂N-乙酰-L-半胱氨酸、白藜芦醇、维生素C和E可抑制白蛋白引起的GMF-PT细胞死亡。白蛋白（30 mg/ml）处理24小时后，GMF-PT中Caspase-3活性增加。caspase-3抑制剂50 μM Z-VAD-favor和p38 MAPK抑制剂10 μM SB 203580可抑制该作用。这些结果表明，白蛋白，蛋白尿的主要成分，引起细胞凋亡的GMF表达PT细胞依赖于p38 MAPK，因为氧化应激的增加。已有研究表明，NF-κ B通过诱导抗凋亡基因的表达而在细胞凋亡中发挥重要作用。EMSA结果表明，GMF的表达降低了NF-κ B的结合活性。我们构建了转基因小鼠（GMF-TG），并研究了转基因小鼠体内表达GMF的影响，结果表明，GMF的表达在体内外均能诱导肾细胞凋亡。

It was well known that proteinuria involved in the progression of kidney diseases. We found that proteinuria increased the expression of brain specific protein, glia maturation factor-b (GMF) in the disease model kidney. The expression of GMF caused vulnerability to oxidative injury, leading to apoptosis in cultured renal proximal tubular (PT) cells named mProx24. We also found that albumin generated oxidative stress within 15min, resulting in activation of STAT1 and STAT5. Thus, it was hypothesized that albumin may cause apoptosis in GMF expressing PT cells (GMF-PT) because of oxidative stress. GMF-PT and wild type PT cells were treated with various concentration of albumin (1-30mg/ml) for 72hrs. Cell viability was examined by using MTS assay. It showed that albumin caused a significant and dose-related increase of cell death in GMF-PT cells, but not in wild-type PT cells. The addition of antioxidants N-Acetyl-L-Cysteine, resveratorol, vitamins C and E inhibited cell death of GMF-PT by albumin. Caspase-3 activity was increased in GMF-PT after 24hr treatment of albumin (30mg/ml). It was inhibited by the caspase-3 inhibitor 50 μM Z-VAD-fmk and the p38MAPK inhibitor 10 μM SB203580. These results demonstrated that albumin, a major component of proteinuria, caused apoptosis of GMF expressing PT cells dependent on p38MAPK because of the increased oxidative stress. It was reported that NF-kB played significant roles in apoptosis by inducing anti-apoptotic genes. EMSA demonstrated that expression of GMF decreased the binding activity of NF-kB. We constructed transgenic mice bearing GMF gene (GMF-TG) and have been investigating the effects of GMF expression in vivo.In conclusion, expression of GMF induced apoptosis of renal cells both in vivo and in vitro.