肾小球基膜Ⅳ型胶原亚链定量分析在Alport综合征诊断中的意义

Quantitative analysis of type Ⅳ collagen alpha 3 and alpha 5 chain in the glomerular basement membrane in patients with Alport syndrome

目的:通过对肾组织Ⅳ型胶原亚链免疫组化染色未见异常的Alport综合征 (AS)患者进行Ⅳ型胶原亚链构成比的定量分析,探讨不典型AS的发病机制,提高AS的诊断水平。 方法:选取临床、病理及电镜符合AS、但肾组织Ⅳ型胶原亚链染色未见异常的患者 14例,运用免疫荧光双套色法,通过激光共聚焦荧光显微镜定量分析肾小球基膜(GBM)Ⅳ型胶原中α3链、α5链的含量。并选取 4例薄基膜肾病及 10例肾活检病理诊断为轻度系膜增生性病变(MsPGN)患者的组织切片作为对照。 结果:入选的 14例患者在电镜下皆以GBM病变为主。通过定量分析GBMⅣ型胶原亚链构成比,我们发现,AS患者GBMα3链和α5链在Ⅳ型胶原总体分布中所占的比例明显减少(P<0. 05, P<0. 01vsMsPGN)。根据肾组织Ⅳ型胶原亚链构成比的不同,可以将这 14例患者分为三组:①2例患者α5链 /Ⅳ型胶原比值降低,α3链 /Ⅳ型胶原比值正常。这组患者男女各 1例,无明确家族史;②9例患者α3 /Ⅳ及α5 /Ⅳ比值同时降低。这组患者女性发病明显多于男性,家系分析显示遗传方式为X性连锁显性遗传。③3例患者α3 /Ⅳ及α5 /Ⅳ比值皆正常。这组患者男性 2例,女性 1例。家系分析显示遗传方式亦为X性连锁显性遗传。我们还发现,蛋白尿的发生与α5链 /Ⅳ型胶原比值的降低程度密切相关。

Objective:Many genotypes of mutation and different phenotypes has been described in patients with Alport syndrome(AS) characterized by changes subchains of type Ⅳ collagen along the glomerular basement membrane(GBM). And in some of the patients, staining of GBM with antibodies to collagen subchains appeared positive/normal under immunohistochemistry studies. In this study, we studied such kind of atypical AS cases by quantitatively analyzed type Ⅳ collagen alpha 3 and alpha 5 chains in GBM, as to disclose the molecular pathogenesis of the disease and to provide a new method to diagnose these atypical AS. Methodology:This study involved 14 AS patients whose diagnosis were based on clinical feature,pathologic findings and electron microscopy study. All of them were positive for routine staining of subchains of type Ⅳ collagen. Immunofluorescence double staining and confocal microscopy were used to quantitatively determine the ratio of staining signal for α3(Ⅳ) or α5(Ⅳ) to Ⅳ along GBM. Four patients with thin basement membranous nephropathy (TBMN) and ten patients with mild mesangial proliferative glomerulonephritis (MsPGN) were used as the controls. Results:The GBM defect was the prominent finding under electron microscopy. Compared to MsPGN patients in the control group, the ratio of α3(Ⅳ)or α5(Ⅳ) to Ⅳ decreased significantly( P<0 05, P<0 01 vs MsPGN). Based on the results of quantitative analysis, patients were divided into three groups: Group 1, patients with reduced α5(Ⅳ)/Ⅳ but normal α3(Ⅳ) /Ⅳ. This group included 1 male and 1 female. Neither of them had positive family history of renal diseases. Group 2, patients with simultaneously reduced α5(Ⅳ)/Ⅳ and α3(Ⅳ) /Ⅳ. This group included 3 males and 6 females. Study of family history demonstrated most of the patients were X linked. Group 3, patients with normal α5(Ⅳ)/Ⅳ and α3(Ⅳ) /Ⅳ. This group included 1females and 2 males. Study of family history demonstrated most of the patients were X linked. Furthermore, we found that the reduced α5(Ⅳ)/Ⅳ correlated with proteinuria, which reflected the severity of glomerular involvement. Unlike AS, none of four patients with TBMN had decreases of α3(Ⅳ) /Ⅳ or α5(Ⅳ) /Ⅳ. Conclusion:Although the staining of subchains of type Ⅳ collagen along the GBM in some patients with AS looked like normal, the compositions of type Ⅳ collagen had altered and characterized by the decreased ratio of α3(Ⅳ)/Ⅳ or α5(Ⅳ)/Ⅳ. By applying this quantitative analysis, a new approach is available to the diagnosis of atypical AS cases. Data of this quantitative analysis is of clinical value for future classification of the genotypes and phenotypes of AS patients, and for the prognosis of these patients.