| Project/Area Number |
14571010
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Kidney internal medicine
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| Research Institution | Akita University |
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Principal Investigator |
WAKUI Hideki Akita University, School of Medicine, Lecturer, 医学部, 講師 (70240463)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Ryoji Kagawa University, Faculty of Medicine, Professor, 医学部, 教授 (00020917)
ITOH Hideaki Akita University, Faculty of Engineering and Resource Science, Professor, 工学資源学部, 教授 (80168369)
KOMATSUDA Atsushi Akita University, School of Medicine, Lecturer, 医学部, 講師 (70272044)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | glomerulus / filtration barrier / actinin-4 / nephrotic syndrome / puromycin aminonucleoside / focal segmental glomerulosclerosis / humanin / two-hybrid system / ネフローゼ惹起物質 |
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| Research Abstract |
1.Renal alpha-actinin-4 : Purification and puromycin aminonucleoside-(PAN)-binding property (Reference 1)
We established an efficient purification method for actinin-4 from the mammalian kidney. The purified actinin-4 possessed the known function of the protein, and tightly bound to PAN that can induce nephrosis in animals. Based on the results of analysis of digested fragments of actinin-4, the PAN-binding site was mapped within the central rod spectrin-like repeats of the protein. It is possible that PAN has adverse effects on the function of actinin-4, by binding to the spectrin-like repeats, in the PAN nephrosis animal model.
2.Analysis of mutations in alpha-actinin-4 (ACTN4) and podocin genes of patients with chronic renal failure due to sporadic focal segmental glomerulosclerosis (FGS) (Reference 2)
We isolated mRNA from blood samples of five patients with chronic renal failure due to sporadic FGS, and then prepared cDNA. Using specific primers for ACTN-4 and podocin, polymareae chain reaction (PCR) was performed. We analyzed mutations in ACTN4 and podocin genes, by direct sequencing PCR products. Mutations resulting in amino acid substitutions were not found in both genes. This suggests that sporadic FSG is a heterogenous disease.
3.Interaction of the spectrin-like repeats of alpha-actinin-4 with humanin peptide (Reference 3)
We performed a bacterial two-hybrid screening of human kidney cDNA library using as a bait human actinin-4 spectrin-like repeats, and identified humanin peptide as an interacting molecule. We further verified this interaction by in vitro affinity assays and immunofluorescent studies of cultured cells. PAN had no effect on the interactions. In situ hybridization and Immunohistochemical studies showed the expression of humanin in podocytes. These results suggest that humanin is a novel binding partner of actini-4 spectrin-like repeats. Humanin and PAN are unlikely to compete for the same binding surface in actinin-4.
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