FUJII Hiroshi Niigata University, School of Medicine, Associate Professor, 医学部, 助教授 (90165340)
YOSHIDA Haruyoshi University of Fukui, Faculty of Medical Science, Professor, 医学部, 教授 (80135574)
|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2003 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 2002 : ¥1,800,000 (Direct Cost : ¥1,800,000)
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.
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-α
3.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