Thorner P S, Baumal R, Valli V E, Mahuran D, Marrano P M, Jacobs R
Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada.
Virchows Arch A Pathol Anat Histopathol. 1992;421(6):467-75. doi: 10.1007/BF01606875.
Some patients with hereditary nephritis (HN) who have received a renal transplant have been shown to form antibody with specificity for the NC1 domain of collagen type IV, a major constituent of glomerular basement membranes (GBM). We attempted to duplicate this phenomenon in a family of dogs with X-linked HN, a model for human X-linked HN, by immunizing affected male dogs with normal dog NC1 domain. A collagenase digest was prepared from normal dog GBM, the NC1 domain was separated into dimer (approximately 50 kDa) and monomer (24 kDa and 26 kDa) components by SDS-PAGE, and injected into two affected male dogs. Antisera obtained from both dogs contained antibody which reacted with the NC1 domain of dog and human GBM by a plate-binding radioimmunoassay, bound to the dimer and 26 kDa monomer bands by Western blotting, and staining dog and human GBM by immunofluorescence (IF). The affected male dog antiserum reacted equally by radioimmunoassay with the NC1 domain isolated from GBM of unaffected, affected male, and carrier female dogs in the family with X-linked HN, and bound by Western blotting to dimers and the 26 kDa monomer band of the NC1 domain of GBM in each group of dogs. However, the affected male dog antiserum differentiated these dogs by IF; it produced global staining of GBM of unaffected dogs, failed to stain GBM of affected male dogs, and produced segmental staining of GBM of carrier female dogs. Absorption of the affected male dog antiserum with normal dog NC1 domain eliminated the staining of dog GBM by IF, whereas staining persisted after absorption with affected male dog NC1 domain. The abnormal staining patterns of GBM seen by IF in the affected male and carrier female dogs and the results of the absorption studies imply an abnormality of one or more determinants in the 26 kDa monomer band of the NC1 domain of their GBM. Amino acid sequencing of this band identified the alpha 1(IV) chain of collagen type IV, a finding that has implications for the pathogenesis of canine X-linked HN. Absent and segmental staining respectively were also seen by IF in GBM of a male and female patient with HN, using the affected male dog antiserum. Thus, the results obtained in affected male and carrier female dogs with X-linked HN may also be relevant to patients with this disease.
一些接受肾移植的遗传性肾炎（HN）患者已被证明会形成对IV型胶原NC1结构域具有特异性的抗体，IV型胶原是肾小球基底膜（GBM）的主要成分。我们试图在一个患有X连锁HN的犬类家族中重现这一现象，该家族是人类X连锁HN的模型，方法是用正常犬的NC1结构域免疫患病雄犬。从正常犬GBM制备胶原酶消化物，通过SDS-PAGE将NC1结构域分离为二聚体（约50 kDa）和单体（24 kDa和26 kDa）成分，并注射到两只患病雄犬体内。从两只犬获得的抗血清中均含有抗体，通过平板结合放射免疫测定法，该抗体可与犬和人GBM的NC1结构域发生反应，通过蛋白质印迹法与二聚体和26 kDa单体条带结合，并通过免疫荧光（IF）对犬和人GBM进行染色。患病雄犬抗血清通过放射免疫测定法与从患有X连锁HN的家族中未患病、患病雄犬和携带雌性犬的GBM中分离出的NC1结构域反应程度相同，并通过蛋白质印迹法与每组犬GBM的NC1结构域的二聚体和26 kDa单体条带结合。然而，患病雄犬抗血清通过IF区分了这些犬；它对未患病犬的GBM产生整体染色，对患病雄犬的GBM未染色，对携带雌性犬的GBM产生节段性染色。用正常犬NC1结构域吸收患病雄犬抗血清可消除IF对犬GBM的染色，而用患病雄犬NC1结构域吸收后染色仍持续存在。IF观察到的患病雄犬和携带雌性犬GBM的异常染色模式以及吸收研究结果表明，它们GBM的NC1结构域26 kDa单体条带中一个或多个决定簇存在异常。对该条带进行氨基酸测序鉴定出IV型胶原的α1(IV)链，这一发现对犬X连锁HN的发病机制具有重要意义。使用患病雄犬抗血清，在一名男性和一名女性HN患者的GBM中通过IF也分别观察到缺失和节段性染色。因此，在患有X连锁HN的患病雄犬和携带雌性犬中获得的结果可能也与该疾病患者相关。
