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Investigation on anti-inflammatory effects of lipid-binding proteins in progression of renal injury.

脂质结合蛋白在肾损伤进展中的抗炎作用的研究。

基本信息

批准号：
14571021
负责人：
KIMURA Hideki
金额：
$ 2.18万
依托单位：
Faculty of Medical Sciences, University of Fukui
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571021/
关键词：
PPAR lipid-binding protein bile acid-binding protein PPAR knockout mice PAI-1 renal tubular cells reporter assay reporter_assy

项目摘要

1.Investigation on the expression of Lipid-binding proteins, PPARs and FXRs in cultured human proximal renal tubular epithelial cells (HPTECs).(1)Expression of L type (L-) fatty acid-binding protein (FABP), heart-type FABP, and PPAR-α,β,γ was examined in cultured HPTECs. Western blot analysis showed that L-FABP and PPAR-α,β,γ but not H-FABP or bile acid binding protein were identified in the cell lyates.(2)Total RNA was extracted from HPTECs incubated in DMEM for 48 hours under normoxic conditions. Then, cDNA was generated and submitted to cDNA array analysis. The gene expression array analysis revealed that mRNA expression of PPAR-α,β,γ, FXR and RXR was expressed in HPTECs under normoxic conditions.2.Investigation on effects of hypoxia and inflammatory cytokines (TNF-α) on gene expressions of HPTECs.(1)In HPTECs, 24-hour treatments with hypoxia (1% O2) and TNF-α(10 ng/ml) increased expression of PAI-1 mRNA by 3.5-fold and secretion rate of PAI-1 protein by about 2-fold, respectively. … More A PPAR-α agonist, fenofibrate, induced an about 30% decrease in the secretion rate under basal conditions and a 15-20% decrease in that during treatments with hypoxia or TNF-α, indicating an anti-inflammatory effect of PPAR-α activation in HPTECs.(2)Expression of transcriptional factors with lipid affinity was analyzed in HPTECs using a cDNA array analysis. Under normoxic conditions, mRNA expression of FXR, RXR, and PPAR-α,β,γ was identified with FXR and RXR mRNA expressed at the largest amount, while hypoxia reduced expression of all these genes. A real time PCR assay showed that hypoxia produced an about 60% decrease in PPAR-α3.Comparison of renal fibrosis status induced by UUO between PPAR-α deficit mice and control mice.PPAR-α deficit mice were purchased from Jackson Lab and mated. Fasting treatment for only 3 days resulted in fatty liver and lipid accumulation of proximal renal tubular cells, suggesting the severe impairment of free fatty acid oxidation and ATP production. UUO operation was performed in PPAR-α deficit mice and control mice (S 129) aged 12-16 weeks. Scores of renal interstitial fibrosis and infiltration of macrophage appeared to be higher in the deficit mice than the control mice. Detailed experiments are now ongoing. Less

1.研究脂质结合蛋白、PPARs和FXRs在培养的人近端肾小管上皮细胞（HPTECs）中的表达。（1）检测体外培养的HPTECs中L型（L-）脂肪酸结合蛋白（FABP）、心脏型FABP和过氧化物酶体增殖物激活受体-α、β、γ的表达。Western blot分析显示，细胞裂解液中检测到L-FABP和PPAR-α、β、γ，但未检测到H-FABP和胆汁酸结合蛋白。（2）从在常氧条件下在DMEM中孵育48小时的HPTEC中提取总RNA。然后，产生cDNA并进行cDNA阵列分析。基因表达谱分析显示，常氧条件下，HPTECs表达PPAR-α、β、γ、FXR和RXR的mRNA。2.研究缺氧和炎性细胞因子（TNF-α）对HPTECs基因表达的影响。(1)In缺氧（1%O2）和TNF-α（10 ng/ml）处理24 h可使HPTECs派-1 mRNA表达增加3.5倍，派-1蛋白分泌增加约2倍。 ...更多信息在基础条件下，PPAR-α激动剂非诺贝特诱导分泌率降低约30%，在缺氧或TNF-α处理期间降低15-20%，表明PPAR-α活化在HPTEC中具有抗炎作用。（2）利用cDNA阵列分析HPTEC中具有脂质亲和力的转录因子的表达。在常氧条件下，FXR、RXR和PPAR-α、β、γ的mRNA表达被鉴定为FXR和RXR mRNA表达量最大，而缺氧降低所有这些基因的表达。真实的荧光定量PCR结果显示，缺氧可使小鼠肾组织中PPAR-α 3的表达降低约60%。仅禁食治疗3天就导致脂肪肝和近端肾小管细胞的脂质积聚，表明游离脂肪酸氧化和ATP产生严重受损。对12-16周龄的PPAR-α缺陷小鼠和对照小鼠（S129）进行UUO手术。肾间质纤维化评分和巨噬细胞浸润在缺陷小鼠中似乎高于对照小鼠。目前正在进行详细的实验。少