|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥2,200,000 (Direct Cost : ¥2,200,000)
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.
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