补体旁路途径过度活化在恶性高血压肾硬化中的作用

目的研究补体旁路途径(alternative complement pathway)过度活化在恶性高血压肾硬化中的作用。方法(1)选取本院经肾穿刺活检证实为恶性高血压肾硬化患者50例为病例组,零点行肾穿刺活检的供肾者25例为正常对照组,采用酶联免疫吸附法(ELISA)检测血浆及尿液中的补体旁路途径活化起始B因子、正向调节P因子、负向调节H因子及补体活化终末产物C3a、C5a水平。(2)免疫组化法检测补体活化终末产物C5b-9、C4d及凝集素途径活化产物甘露糖结合凝集素(MBL)在肾活检组织的沉积;免疫荧光双染检测C5b-9与CD34(内皮细胞标志物)在小动脉内皮及肾小球毛细血管内皮的沉积。结果(1)恶性高血压肾硬化患者血浆及尿液中补体B因子、P因子、C3a及C5a均高于正常对照组(均P<0.05),而H因子则低于正常对照组(P<0.05)。(2)恶性高血压肾硬化患者血浆中补体P因子与24 h尿蛋白量呈正相关(rs=0.465,P=0.001),而补体B因子、H因子、C3a、C5a与血肌酐及24 h尿蛋白量无明显相关性。恶性高血压肾硬化患者尿B因子/尿肌酐、尿P因子/尿肌酐、尿C3a/尿肌酐与血肌酐均呈正相关(rs=0.483,P<0.001;rs=0.352,P=0.012;rs=0.319,P=0.024),尿H因子/尿肌酐与血肌酐及24 h尿蛋白量均呈负相关(rs=-0.299,P=0.035;rs=-0.342,P=0.015),尿C5a/尿肌酐与血肌酐及24 h尿蛋白量均呈正相关(rs=0.525,P<0.001;rs=0.496,P<0.001)。(3)免疫组化显示,恶性高血压肾硬化患者C5b-9沉积于小动脉壁及肾小球毛细血管壁,而正常对照组肾组织中未见沉积。恶性高血压肾硬化患者肾脏C5b-9沉积强度评分与血肌酐及24 h尿蛋白量呈正相关(rs=0.791,P<0.001;rs=0.345,P=0.014)。双重免疫荧光标记法可见C5b-9、CD34沉积于小动脉内皮及肾小球毛细血管内皮。(4)恶性高血压肾硬化患者血浆中B因子与C3a(r=0.331,P=0.022)、P因子与C5b-9评分(rs=0.300,P=0.034)均呈正相关;尿液中补体旁路途径活化B因子与C3a、C5a及C5b-9均呈正相关(rs=0.311,P=0.028;rs=0.465,P=0.001;rs=0.428,P=0.002),P因子与C3a、C5a也均呈正相关(rs=0.307,P=0.030;rs=0.442,P=0.001)。恶性高血压肾硬化患者免疫组化可见C4d沉积于小动脉及肾小球,而未见凝集素途径活化产物MBL沉积。结论补体旁路途径过度活化可能参与恶性高血压肾硬化的发生。恶性高血压肾硬化严重程度与补体旁路途径的活化水平相关。

Role of alternative complement pathway overactivation in malignant nephrosclerosis

Objective To study the role of alternative complement pathway overactivation in malignant nephrosclerosis. Methods (1) Fifty patients with confirmed malignant nephrosclerosis by renal needle biopsy were enrolled. Meanwhile, twenty-five cases of time-zero renal needle biopsy were enrolled as control subjects. Enzyme linked immunosorbent assay (ELISA) was used to detect alternative complement pathway of the complement initiation factor B, positive regulation factor P, negative regulation factor H, and the complement end products C3a and C5a in the plasma and urine. (2) Immunohistochemistry was used to detect the deposition of the complement end product C5b-9, C4d and mannan binding lectin (MBL) of lectin pathway in the renal biopsies. Double immunofluorescence labeling method was used to assay the deposition of C5b-9 and CD34 (endothelial cell marker) in the arteriolar endothelium and glomerular capillary endothelium. Results (1) The plasma and urine levels of complement factor B, factor P, C3a and C5a in malignant nephrosclerosis patients were significantly higher than those in control subjects (all P＜0.05), while the plasma and urine levels of complement factor H in malignant nephrosclerosis patients were lower than those in control subjects (all P＜0.05). (2) The plasma level of factor P was positively correlated with 24 h urine protein (rs=0.465, P=0.001). Urinary factor B/urinary creatinine, urinary factor P/urinary creatinine and urinary C3a/ urinary creatinine were positively correlated with serum creatinine in malignant nephrosclerosis patients (rs=0.483, P＜0.001; rs=0.352, P=0.012; rs=0.319, P=0.024), while urinary factor H/urinary creatinine was negatively correlated with serum creatinine and 24 h urine protein (rs=-0.299, P=0.035; rs=-0.342, P=0.015). Urinary C5a/urinary creatinine was positively correlated with serum creatinine and 24 h urine protein (rs=0.525, P＜0.001; rs=0.496, P＜0.001). (3) Immunohistochemical results showed that there were C5b-9 deposited in the arterioles and glomerular capillary wall in malignant nephrosclerosis patients, and no deposition in control renal tissues. Meanwhile, the semi-quantitative scores showed that C5b-9 deposition intensity was positively correlated with serum creatinine and 24 h urine protein (rs=0.791, P＜0.001; rs=0.345, P=0.014). The double immunofluorescence labeling analysis showed that the C5b-9 and CD34 deposited in the arteriolar endothelium and glomerular capillary endothelium. (4) Plasma level of factor B in malignant nephrosclerosis patients was positively correlated with plasma C3a level (r=0.331, P=0.022). Plasma level of factor P was positively correlated with C5b-9 score (rs=0.300, P=0.034). Urinary B was positively correlated with urinary C3a, C5a and C5b-9 score (rs=0.311, P=0.028; rs=0.465, P=0.001; rs=0.428, P=0.002). Urinary factor P was also positively correlated with urinary C3a and C5a (rs=0.307, P=0.030; rs=0.442, P=0.001). Immunohistochemical result showed that there were C4d deposited in the arterioles and glomerular, and no deposition of MBL. Conclusion Complement activation via the alternative pathway may be involved in malignant nephrosclerosis and related to the severity of the disease.