| 研究実績の概要 |
In vitro study was conducted to determine the protective effects of (P)RR mAb. Conditionally immortalized mouse podocytes were cultured on collagen I coated plates in RPMI 1640 medium supplemented with 100 U/mL of recombinant mouse interferon-gamma (INF-γ) at 33 degree C, which is considered as growth-permissive conditions. To differentiate the podocytes, cells were placed at 37 degree C for 14 days without INF-γ. At 70-80% confluence, the differentiated cells were serum starved for overnight. Afterwards, these cells were cultured for 72 hours in medium containing 5 mM D-glucose plus 20 mM D-mannitol (osmotic control); 25 mM D-glucose (high glucose); and 25 mM D-glucose (high glucose) with 200 microgram/mL (P)RR neutralizing mAb (clone 48_8). Following intervention, cells were lysed and homogenized; extraction of RNA as well as preparation of cDNA were performed in accordance with our established protocol. Quantitative real time PCR was performed to check the mRNA expression of podocyte injury marker, nephrin. The gene expression data revealed that high glucose intervention reduced the mRNA expression of nephrin compared to the osmotic control group, revealing the podocyte injury which is in consistent with previous reports. In contrary, (P)RR mAb treatment caused the increase expression of nephrin mRNA, suggestive of the reduced podocyte injury. Therefore, these data are in line with our hypothesis that (P)RR mAb has the potential to protect the podocytes against injurious effects of high glucose in cell culture condition.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The research facility of the School of Medicine, International University of Health and Welfare, Narita Campus was opened in December, 2017. As a result of the delay in preparing the overall research atmosphere, the progression of research is somewhat late.
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| 今後の研究の推進方策 |
In-vitro studies:
(a) Functional assay of albumin permeability through podocyte monolayer will be assessed. (b) The intracellular signal molecules will be examined. (c) The mRNA/protein levels of the Wnt-β-catenin signaling pathway components will be measured.
In vivo studies:
(a) Male db/db mice will be used for in vivo study and will be treated with (P)RR neutralizing antibody systematically. (b) Urine will be collected every week interval to check the albuminuria. (c)PAS staining or IHC or mRNA expression for collagen IV, fibronectin, and desmin will be conducted. (d) Parameters related with inflammation will be measured in kidney tissue. (e) mRNA and/or protein levels of Wnt-β-catenin signaling pathway components will be examined.
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