|Budget Amount *help
¥3,900,000 (Direct Cost : ¥3,900,000)
Fiscal Year 2004 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 2003 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 2002 : ¥1,300,000 (Direct Cost : ¥1,300,000)
The initial step of the urine generation in the kidney is the glomerular filtration through the glomerular filtration barrier, which prevents protein leakage into the urine. In order study the pathophysiology of glomerular diseases, where the ultrastructure of the glomerular filtration barrier is damaged, it is necessary to establish a reliable model system to reproduce the structure and function of the barrier. The filtration barrier is composed of three components, i.e. the glomerular capillary endothelial cell, the glomerular basement membrane, and the podocyte, also known as the glomerular epitheliaI cell. This study has aimed to establish such a model using cell culture system.
With the conditionally immortalized mouse podocyte cell line which is kindly provided by our foreign collaborators, we have studied the signal transduction pathway regulating the podocyte morphogenesis (both process formation and cell-cell contact formation) with special reference on the RHO-family small GTP
ases (RhoA, Rac1, Cdc42) (Kobayashi N, 2002 ; Kobayashi et al., 2004 ; Gao et al., 2004 ; Gao et al., manuscript in preparation). Since in in vivo glomeruli, podocytes develop a cell-type specific intercellular junction, slit membrane, the formation of intercellular junctions between podocytes is thought to be the critical step in the development of the filtration barrier. We have shown that tight cell-cell contacts are induced between cultured podocytes by inhibition of RhoA with forskolin. Biochemical approach revealed the activation of other members of the RHO-family, Rac1 and Cdc42, during this cell-cell contact formation. It seems unlikely that a co-culture system (podocytes + endothelial cells) is absolutely necessary to establish the glomerular filtration barrier in vitro. We have also shown that pharmacological inhibition of RhoA and its downstream effector, ROCK, enhances process formation of the cultured podocytes. As planned at the beginning of this study, we are still trying to establish cell lines of renal glomerular endothelial cells. Less