两种不同病理类型肾病患者尿蛋白诱导肾小管上皮细胞上调表达趋化因子及其分子信号机制研究

目的 观察不同病理类型肾病患者尿蛋白对人近端肾小管上皮细胞（HK-2）分泌趋化因子的影响并探讨其分子信号机制。 方法 所有患者均经临床和肾穿刺活检确诊为原发性局灶节段性肾小球硬化（FSGS）或微小病变性肾病（MCD）。以超滤法浓缩提取患者尿中总蛋白，分别刺激体外培养的HK-2细胞。RT-PCR检测调节正常T细胞表达和分泌的细胞因子（RANTES）及巨噬细胞移动抑制因子（MIF）的mRNA水平。免疫荧光、ELISA及流式细胞仪检测蛋白水平的表达。Western 印迹法检测p38 MAPK和胞外信号调节激酶（ERK）水平。特异性抑制剂SB203580抑制p38磷酸化；PD98059则用于抑制ERK磷酸化。 结果 两种尿蛋白成分有差异，FSGS患者尿蛋白中含有更多的大分子量蛋白。两种尿蛋白均刺激HK-2细胞RANTES及MIF表达上调，而FSGS患者尿蛋白刺激作用更强。两种尿蛋白均显著激活HK-2细胞内ERK和p38 MAPK信号通路。特异性抑制剂分别抑制ERK或p38 MAPK的活化后，FSGS患者尿蛋白介导的RANTES和MIF的上调表达作用可被SB203580或PD98059抑制，而MCD患者尿蛋白的刺激作用却仅能被SB203580所阻断。 结论 FSGS肾病患者的尿蛋白比MCD患者的尿蛋白有更强的上调HK-2细胞RANTES及MIF表达的作用。两种尿蛋白介导趋化因子上调表达的分子信号机制存在差异。

Up-regulation of chemokines expression in renal proximal tubular epithelial cells induced by urinary protein from two pathological types of kidney disease and its molecular mechanism

Objective To investigate the molecular mechanism of chemokine released from renal proximal tubular epithelial cells (PTECs) induced by urine protein of minimal change disease (MCD) and focal segmental glomerular sclerosis (FSGS) patients. Methods Patients with MCD or FSGS were diagnosed by clinic and renal biopsy. Cultured PTECs were incubated with urine protein extracted from MCD or FSGS patients. Protein and gene levels of MIF and RANTES were detected by RT-PCR, flow cytometry and ELISA. MARK and extracellular regulated kinase (ERK) activation were measured by Western blotting. Pretreatment of PTECs with specific inhibitor SB203580 or PD98059 was performed to prevent the activation of MARK and ERK. Results Both of the two kinds of urine protein increased RANTES and MIF expression in time- and dose-dependent manner. Additionally, FSGS urine protein mediated a significantly greater secretion of chemokine in PTECs compared to MCD urine protein. Both of the two kinds of urine protein significantly activated p38 MARK and ERK pathway. Pretreatment with SB203580 or PD98059 prevented the effect of FSGS urine protein on RANTES and MIF production, while only SB203580 inhibited the effect of MCD urine protein. Conclusions Urinary protein of FSGS induces a potentially higher expression of chemokines in PTECs as compared to that of MCD through the distinct MAPKs signaling pathway. Component of proteinuria may play a role in determining the severity and progression of tubular inflammation and fibrosis.