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

1大鼠正常肾小球脂质结合蛋白分析。(1)观察正常大鼠肾小球中是否存在脂肪酸结合蛋白（FABP）、酰基辅酶a结合蛋白（ACBP）、磷脂酰肌醇转移蛋白（PITP）、甾醇载体蛋白2 （SCP2）、细胞维甲酸结合蛋白（CRABP）等脂质结合蛋白（lbp）。以大鼠全肾总RNA为模板，RT-PCR扩增出lbp的特异性DNA片段。将每个特异性PCR产物连接到pGEM-T载体上，进行亚克隆和测序。特异DNA探针的Northern blotting显示，这些lbp均在大鼠正常肾脏的肾小球和小管中表达（kidney Int. 1999）。(2)皮下注射合成的PITP和SCP2肽免疫家兔制备lbp抗体。利用这些抗体，我们计划研究lbp在大鼠正常和病变肾脏的组织内分布。110-kD fabp相关蛋白（110p蛋白）在大鼠和人肾肾小球毛细血管表达的进一步分析。(1)与心脏型FABP相关的110p蛋白经双向电泳免疫化学分离后，用CBB染色，从凝胶中切除，经电洗脱和HPLC部分纯化。纯化得到的110p蛋白用赖氨酸肽酶酶切，用反相高效液相色谱法进行酶切。用气相氨基酸测序仪测定纯化肽的氨基酸序列。我们计划确定尽可能多的肽的序列，并通过退化PCR获得i10p蛋白的部分DNA序列。(2)利用心脏型FABP多克隆抗体进行免疫电镜扫描，发现110p蛋白仅位于人肾小球内脏上皮细胞足突细胞质中。影响脂质代谢的遗传多态性与晚期肾脏疾病的关系分析。载脂蛋白E （Apo E）和胆固醇酯转移蛋白（CETP）可能分别通过影响LDL-C和HDL-C的代谢与肾小球硬化的进展有关。纤溶酶原激活物抑制剂- i （PAI- 1）与血清TG水平正相关，也可能影响肾小球硬化的进展。因此，我们研究了这些基因的多态性是否与晚期肾脏疾病有关。(1)等位基因epsilon4的存在是糖尿病肾病进展的重要保护因素，而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有关，但与糖尿病肾病的进展无关（Kidney Int, 1998）。(3) CETP D442G突变降低CETP活性是亚中位HDL-C水平透析患者血管疾病的重要危险因素，但不是糖尿病肾病或慢性肾小球肾炎的危险因素（J Am Soc Nephrol, 1999; Kidney Int. 1999）。少

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