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

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10839016
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 動物臨床医学
• Research Institution: Kitasato University
• Principal Investigator: HOSHI Fumio Kitasato University, School of Veterinary Medicine and Animal Science, Assistant Professor, 獣医畜産学部, 講師 (00219164)
• Co-Investigator(Kenkyū-buntansha): KAWAMURA seiichi Kitasato University, School of Veterinary Medicine and Animal Science, Professor, 獣医畜産学部, 教授 (60050530) ; HIGUCHI Seiichi. Kitasato University, School of Veterinary Medicine and Animal Science, Professor, 獣医畜産学部, 教授 (60095510)
• Project Period (FY): 1998 – 1999
• Keywords: membranous nephropathy / urinary pyotein / purification / amino acid sequence / feline urinary albumin / feline urinary IgG / cationized- BSA / hyperlipidemia

Research Abstract

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 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.