Study on the Role of Bone Marrow Stem Cells in the Recovery of Acute Renal Failure and Nephropathy Using Fluorescence Gene Transfected Mice

2006 Fiscal Year Final Research Report Summary

• Project/Area Number: 17590176
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General anatomy (including Histology/Embryology)
• Research Institution: Tokai University
• Principal Investigator: TUN Aung Tokai University, Medical Research Institute, Postdoctoral Research Associate, 総合医学研究所, 研究員 (50398749)
• Co-Investigator(Kenkyū-buntansha): SAITO Akira Tokai University, School of Medicine, Prof, 医学部, 教授 (60307296)
• Project Period (FY): 2005 – 2006
• Keywords: Gene-transfected mouse / Acute renal failure / Bone marrow stem cell

Research Abstract

Kidneys of the KHNEICC transgenic mice were analyzed for renal tubule-specific expression of HcRed1 and podocyte-specific expression of EGFP. Although ECFP was expressed under control of CAG promoter in glomeruli and renal tubules, HcRed1 expression under control of Ksp-cadherin promoter appeared to be confined to renal tubules. Moreover, EGFP expression under control of nephrin promoter was confined to podocytes. However, HcRed1 expression was not confirmed under detailed genetical analyses. Sequence analysis of KHNEICC plasmid showed that a new translational start codon accidentally appeared between the Ksp-cadherin promoter region and the HcRed1, and there was also a big insert between the insulator and the CAG promoter. Bone marrow cells of KHNEICC transgenic mouse were transplanted into podocyte-specific injury transgenic mouse induced by immunotoxin injection. After 2, 4, 6, and 8 weeks of post-transplantation, kidneys of the recipient mice were examined. After 4weeks, there was some histological improvement of renal injury. However, no fluorescent cell was observed in glomeruli of the recipient kidneys. Many fluorescent cells that seem to be the result of auto-fluorescence were observed in renal tubules. The wavelength of EGFP is close to that of ECFP, and accordingly, it is difficult to distinguish these 2 proteins by fluorescence microscopy. Therefore, 2 fluorescent genes with widely-separated wavelengths, i.e. EGFP with CAG promoter and DsRed2 with Ksp-cadherin promoter, were reconstructed, and transfected into mouse bone marrow mesenchymal stem cells. These cells were transplanted into cisplatin-induced acute renal failure mice. After 2, 4, 6, and 8 weeks of post-transplantation, kidneys of the recipient mice were examined histologically, immunohistochemically, and by fluorescence microscopy. At 2 weeks, EGFP and Ksp-cadherin or DsRed2 double-stained cells were observed in renal tubules and their number increased after 4 weeks onwards. However, possibility of auto-fluorescence and cross-reaction of antibodies could not be excluded and detailed molecular biological analyses are ongoing.