The Study for Clinical Pathophysiolongy of Feline Membranous Nephropathy and Diagnostic Significance of Urinary Micro-Protein Analyses.

每周3次静脉注射阳离子牛血清白蛋白建立膜性肾病模型，连续3个月。5例中4例表现为肾小球基底膜增生、棘波形成、免疫球蛋白沉积。血生化检查中，血浆总蛋白、白蛋白降低，F-CHO、Cho-E、PL升高，NEFA、TG无明显变化。血清脂蛋白分析显示乳糜粒和极低密度脂蛋白无变化，而低密度脂蛋白和高密度脂蛋白升高。提示本病为IIa型高脂血症。即使Cre、UN和内源性Cre清除量没有变化，24小时尿蛋白浓度、尿蛋白Cre指数和尿蛋白排泄量也增加。提示尿蛋白分析对膜性肾病的诊断有一定价值。尿蛋白的SDSPAGE结果显示，相对分子质量为160.5 kDa、113.2 kDa a…的蛋白条带相对浓度增加分子量分别为131.4 kDa、77.8 kDa、42.5 kDa、39.8 kDa、31.5 kDa和24.6 kDa。从9条蛋白条带中筛选出160.5 kDa的蛋白(160UP)和113.2 kDa的蛋白(113UP)。并通过氨基酸序列分析对这些蛋白质进行了鉴定。采用丙烯酰胺凝胶电洗脱法和高效液相色谱法相结合的方法对~(113)UP进行纯化。其氨基酸序列与猫血清白蛋白(FSA)一致，但其相对分子质量与FSA不一致。该蛋白与二硫苏糖醇共孵育后的SDSPAGE结果显示，其分子量分别为113.2 kDa、69.7 kDa、68.3 kDa、65 kDa。检测到1 kDa和60.3 kDa。这5条蛋白带的氨基酸序列与FSA的氨基酸序列完全一致。这些结果表明，113UP可能是二硫键形成的FSA代谢产物的二聚体。经丙烯酰胺凝胶电洗脱法可进一步纯化160UP。160UP与DTT共孵育后的SDS-PAGE显示，160UP由相对分子质量分别为50.5 kDa、47.8 kDa和25.0 kDa的片段组成。这些片段的氨基酸序列与其他物种的免疫球蛋白同源，免疫印迹表明160UP是免疫性的猫科动物的免疫球蛋白。本研究证实猫尿白蛋白和猫尿免疫球蛋白可作为膜性肾病的诊断工具。较少

The membranous nephropathy model producted by cationized-BSA administration in vein three times a week for three months. Four of them from 5 showed hyperplasia of glomerular basement membrane, formation of spike, deposition of IgG. In blood biochemistry examination, the plasma TP and Alb decreased, and plasma F-Cho, Cho-E and PL increased, but NEFA and TG showed no change. The serum lipoprotein analyses revealed that chylomicron and VLDL showed no change but LDL and HDL increased. This results demonstrated that this disease state was Iia type of hyperlipidemia. Even if the Cre, UN and Endogenous Cre clearance did not change, urinary protein concentration, Cre index of urinary protein and excretion of urinary protein for 24hrs increased. This results demonstrated that urinary protein analyses is useful for diagnosis of the membranous nephropathy. In a result of SDS-PAGE of urinary protein, the relative concentration of protein bands increased on the molecular weight 160.5kDa, 113.2kDa a … More nd 67.7kDa, and new the protein bands were able to detect on the molecular weight 131.4kDa, 77.8kDa, 42.5kDa, 39.8kDa, 31.5kDa and 24.6kDa. The 160.5 kDa protein (160UP) and the 113.2 kDa protein (113UP) among these 9 protein bands were selected and purified. Furthermore, these proteins were identified by amino acid sequence analyses. 113UP was able to purify by the combination method of electric elution form acryramide gels and HPLC. Its amino acid sequence agreed with feline serum albumin (FSA), but its molecular weight did not agree with FSA. In the result of SDS-PAGE which the protein incubated with dithiothreitol (DTT), 5 protein bands of molecular weight 113.2 kDa, 69.7 kDa, 68.3 kDa, 65. 1 kDa and 60.3kDa were detected. The amino acid sequence of these 5 protein bands were homologous with that of FSA. These results demonstrated that 113UP would be a dimer of FSA metabolite formed by disulfide linkage. Further 160UP was able to purify by the electric elution form acryramide gels. The SDS-PAGE of 160UP incubated with DTT revealed that 160UP consist of the some fragment of molecular weight 50.5 kDa, 47.8 kDa and 25.0kDa. The amino acid sequences of these fragments were homologous with IgG of other species, Furthermore, Western blotting used Anti-feline IgG revealed that 160UP is immunological feline IgG. This research was proved that feline urinary albumin and feline urinary IgG would be a tool for diagnosis of membranous nephropathy. Less