BIOCHEMICAL AND HISTOCHEMICAL EXAMINATIONS ON LIPID-BINDING PROTEINS IN RAT AND HUMAN KIDNEY

大鼠和人肾中脂质结合蛋白的生化和组织化学检查

基本信息

批准号：
09671159
负责人：
KIMURA Hideki
金额：
$ 1.73万
依托单位：
FACULTY OF MEDICINE,FUKUI MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671159/
关键词：
glomeruli lipid-binding protein foot process apolipoprotein E PAI-1 CETP genetic polymorphism

项目摘要

1 Analysis of lipid-binding proteins in normal glomeruli of rat kidney.(1) We investigated whether or not lipid-binding proteins (LBPs) such as fatty acid binding protein (FABP), acyl CoA-binding protein (ACBP), phosphatidyl inositol transfer protein (PITP), sterol carrier protein 2 (SCP2), cellular retinoic acid-binding protein (CRABP) are present in normal glomeruli of rat kidney. Specific DNA fragments for these LBPs were amplified by RT-PCR using total RNA from rat whole kidney as a template. Each of the specific PCR products was ligated to pGEM-T vector, subcloned and sequenced. Northern blotting using the specific DNA probes showed that these all LBPs were expressed in glomeruli and tubules of rat normal kidney (Kidney Int. 1999).(2) In order to prepare antibodies for LBPs, rabbits were immunized by subcutaneous injection of synthetic peptides of PITP and SCP2. Using these antibodies, we plan to investigate intratissue distribution of LBPs in rat normal and diseased kidneys.2. An … More alysis of a 110-kD FABP-related protein (110p protein) expressed in glomerular capillary of rat and human kidney.(1) After the 110p protein immunochemically related to heart-type FABP was separated by two-dimensional electrophoresis, it was stained with CBB, excised from the gel, and partially purified by electroelution and HPLC.The 110p protein thus purified was digested with lysylendopeptidase, and the digest was submitted to reversed phase HPLC.Amino acid sequences of the purified peptides were determined with vapor-phased amino acid sequencer. We plan to determine the sequence of as many peptides as possible and to obtain a partial DNA sequence of the I 10p protein by degenerative PCR.(2) Immunoelectron micrography using polyclonal antibody for heart-type FABP showed that the 110p protein was exclusively located in the cytoplasm of footprocess of visceral epithelial cells of human glomeruli.3. Analysis of relationships between genetic polymorphisms affecting lipid metabolism and advanced renal disease.Apolipoprotein E (Apo E) and cholesteryl ester transfer protein (CETP) may be associated with progression of glomerulosclerosis by influencing metabolisms of LDL-C and HDL-C, respectively. Plasminogen activator inhibitor-I (PAI- 1) positively correlated with serum TG levels may also affect progression of glomerulosclerosis. Therefore, we examined whether or not polymorphisms of these genes are associated with advanced renal disease. (1) Presence of the allele epsilon4 was a significant protective factor for progression of diabetic nephropathy, but not chronic glomerulonephritis (Am J Kidney Dis, 1998). (2)PAI-1 4G/5G polymorphism was associated with atherosclerotic complications, but not progression of diabetic nephropathy in NIDDM patients (Kidney Int, 1998). (3) CETP D442G mutation reducing CETP activity was a significant risk factor for vascular disease in dialysis patients with sub-median HDL-C levels, but not for diabetic nephropathy or chronic glomerulonephritis(J Am Soc Nephrol, 1999 ; Kidney Int. 1999). Less

1 正常大鼠肾脏肾小球中脂质结合蛋白的分析。(1) 我们研究了脂肪酸结合蛋白(FABP)、酰基CoA结合蛋白(ACBP)、磷脂酰肌醇等脂质结合蛋白(LBP)的存在与否。大鼠肾正常肾小球中存在转运蛋白（PITP）、甾醇载体蛋白2（SCP2）、细胞视黄酸结合蛋白（CRABP）。使用大鼠整个肾脏的总 RNA 作为模板，通过 RT-PCR 扩增这些 LBP 的 DNA 片段，将每个特定 PCR 产物连接到 pGEM-T 载体，使用特定 DNA 探针进行亚克隆和测序。所有LBPs均在大鼠正常肾的肾小球和肾小管中表达(Kidney Int. 1999)。(2)为了制备LBPs抗体，免疫兔子通过皮下注射 PITP 和 SCP2 的合成肽，我们计划研究 LBP 在大鼠正常和患病肾脏中的组织内分布。2. 表达的 110-kD FABP 相关蛋白（110p 蛋白）的分析。 (1)通过二维分离与心型FABP免疫化学相关的110p蛋白后电泳后，用 CBB 染色，从凝胶上切下，并通过电洗脱和 HPLC 进行部分纯化。用赖氨酰内肽酶消化如此纯化的 110p 蛋白，并将消化物送至反相 HPLC。测定纯化肽的氨基酸序列我们计划用气相氨基酸测序仪确定尽可能多的肽序列，并通过退化获得 I 10p 蛋白的部分 DNA 序列。 (2)心型FABP多克隆抗体免疫电镜显示110p蛋白仅位于人肾小球内脏上皮细胞足突细胞质中。3．影响脂质代谢的基因多态性与晚期肾病的关系分析。载脂蛋白 E (Apo E) 和胆固醇酯转移蛋白 (CETP) 可能与肾小球硬化症的进展有关分别影响 LDL-C 和 HDL-C 的代谢，与血清 TG 水平呈正相关，也可能影响肾小球硬化的进展，因此，我们检查了这些基因的多态性是否与肾小球硬化症相关。 (1) 等位基因 epsilon4 的存在是糖尿病肾病进展的重要保护因素，但不是慢性肾小球肾炎。（Am J Kidney Dis，1998）（2）PAI-1 4G/5G 多态性与 NIDDM 患者的动脉粥样硬化并发症相关，但与糖尿病肾病的进展无关（Kidney Int，1998）（3）CETP D442G 突变降低 CETP 活性。是 HDL-C 水平低于中值的透析患者发生血管疾病的一个重要危险因素，但对于糖尿病肾病或慢性肾小球肾炎（J Am Soc Nephrol，1999；Kidney Int. 1999）。