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

1正常大鼠肾小球脂质结合蛋白的分析(1)本实验研究了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）、固醇载体蛋白2（SCP 2）、细胞视黄酸结合蛋白（CRABP）等脂质结合蛋白（LBP）。以大鼠全肾总RNA为模板，通过RT-PCR扩增这些LBP的特异性DNA片段。将每个特异性PCR产物连接到pGEM-T载体，亚克隆并测序。使用特异性DNA探针的北方印迹显示，所有这些LBP均在大鼠正常肾脏的肾小球和肾小管中表达（Kidney Int.1999）。(2)为了制备针对LBP的抗体，通过皮下注射PITP和SCP 2的合成肽免疫家兔。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.一个 关于我们 在大鼠和人肾脏的肾小球毛细血管中表达的110-kD FABP相关蛋白（110 p蛋白）的分析。(1)将与心脏型FABP免疫化学相关的110 p蛋白经双向电泳分离后，用CBB染色，从凝胶上切下，用电洗脱和HPLC进行部分纯化，纯化后的110 p蛋白用赖氨酰内肽酶酶切，酶切产物经反相HPLC纯化，用气相氨基酸序列测定仪测定其氨基酸序列。我们计划确定尽可能多的肽序列，并通过变性PCR获得I10 p蛋白的部分DNA序列。(2)心脏型FABP多克隆抗体免疫电镜显示110 p蛋白仅定位于肾小球脏上皮细胞足突的胞浆中.影响脂质代谢的基因多态性与晚期肾脏疾病的关系分析载脂蛋白E（Apo E）和胆固醇酯转运蛋白（CETP）可能分别通过影响LDL-C和HDL-C的代谢而与肾小球硬化的进展相关。纤溶酶原激活物-I（派- 1）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，我们研究这些基因的多态性是否与晚期肾脏疾病相关。(1)等位基因ε 4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）派-1 4G/5G多态性与NIDDM患者动脉粥样硬化并发症有关，但与糖尿病肾病进展无关（Kidney Int，1998）。(3)降低CETP活性的CETP D442 G突变是HDL-C水平低于中位数的透析患者血管疾病的显著风险因素，但不是糖尿病肾病或慢性肾小球肾炎的风险因素（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