Abstract:Fibroblast growth factor 2 (FGF2) augments podocyte injury, which induces glomerulosclerosis, although the mechanisms remain obscure. In this study, we investigated the effects of FGF2 on cultured podocytes with interdigitating cell processes in rats. After 48 h incubation with FGF2 dynamic changes in the shape of primary processes and cell bodies of podocytes resulted in the loss of interdigitation, which was clearly shown by time-lapse photography. FGF2 reduced the gene expressions of constituents of the sli… Show more
“…Therefore, we examined the role of FilGAP in primary rat podocyte cells using a primary culture system reported previously. 38 In this culture, cells spread and exhibited abundant stress fibers in the cell bodies when the cells were cultured at a low density (Figure 6A,C), as seen in other cell lines and primary F I G U R E 4 FilGAP regulates the process formation through the inactivation of Rac1 in C7 podocyte cells. (A) DIC images of FSKtreated C7 cells.…”
Section: Fa and Process Formations In Primary Podocyte Cellsmentioning
confidence: 53%
“…Second, primary podocyte cells isolated from rat glomeruli were cultured in the presence of retinoic acid at a confluent density to promote process formation, as previously reported. 38 Depletion of FilGAP inhibited in vivo-like process formation in the primary cultured cells as well as C7 cells (Figure 6). Thus, the inactivation of Rac1 by FilGAP is responsible for the process formation of podocytes.…”
Section: Discussionmentioning
confidence: 93%
“…71 In addition, retinoic acid is required to promote process formation in the primary culture used in this study. 38 Retinoic acid is known to have renal-protective effects in animal models of kidney disease and activates the cAMP pathway through its receptor. 72 These findings suggest that cAMP signaling is involved in the process formation of podocytes.…”
Section: Discussionmentioning
confidence: 99%
“…The primary culture of rat podocytes was derived from cells growing from isolated glomeruli as previously described. 38 The glomerular-derived cells were seeded and cultured on laminin-521 (Corning)-coated glass slides printed with a highly water-repellent mark with wells 5 mm in diameter (Matsunami Glass, Osaka, Japan) in DMEM/F12 supplemented with 5% FBS, 0.5% ITS, and 50 U/mL penicillin/streptomycin at 37°C. After 6 h, when most cells were attached to the glass slides, culture media were changed to DMEM/F12 supplemented with 0.5% FBS, 0.5% ITS, 0.4 mg/mL dextran sulfate, and 0.6 μM all-trans retinoic acid (ATRA).…”
The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin‐driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase‐activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin‐based protein complex closely associated with stress fibers, that mediates cell‐extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process‐like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP‐depleted podocyte cells was rescued by inhibition of Rac1 or P21‐activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell‐ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.
“…Therefore, we examined the role of FilGAP in primary rat podocyte cells using a primary culture system reported previously. 38 In this culture, cells spread and exhibited abundant stress fibers in the cell bodies when the cells were cultured at a low density (Figure 6A,C), as seen in other cell lines and primary F I G U R E 4 FilGAP regulates the process formation through the inactivation of Rac1 in C7 podocyte cells. (A) DIC images of FSKtreated C7 cells.…”
Section: Fa and Process Formations In Primary Podocyte Cellsmentioning
confidence: 53%
“…Second, primary podocyte cells isolated from rat glomeruli were cultured in the presence of retinoic acid at a confluent density to promote process formation, as previously reported. 38 Depletion of FilGAP inhibited in vivo-like process formation in the primary cultured cells as well as C7 cells (Figure 6). Thus, the inactivation of Rac1 by FilGAP is responsible for the process formation of podocytes.…”
Section: Discussionmentioning
confidence: 93%
“…71 In addition, retinoic acid is required to promote process formation in the primary culture used in this study. 38 Retinoic acid is known to have renal-protective effects in animal models of kidney disease and activates the cAMP pathway through its receptor. 72 These findings suggest that cAMP signaling is involved in the process formation of podocytes.…”
Section: Discussionmentioning
confidence: 99%
“…The primary culture of rat podocytes was derived from cells growing from isolated glomeruli as previously described. 38 The glomerular-derived cells were seeded and cultured on laminin-521 (Corning)-coated glass slides printed with a highly water-repellent mark with wells 5 mm in diameter (Matsunami Glass, Osaka, Japan) in DMEM/F12 supplemented with 5% FBS, 0.5% ITS, and 50 U/mL penicillin/streptomycin at 37°C. After 6 h, when most cells were attached to the glass slides, culture media were changed to DMEM/F12 supplemented with 0.5% FBS, 0.5% ITS, 0.4 mg/mL dextran sulfate, and 0.6 μM all-trans retinoic acid (ATRA).…”
The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin‐driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase‐activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin‐based protein complex closely associated with stress fibers, that mediates cell‐extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process‐like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP‐depleted podocyte cells was rescued by inhibition of Rac1 or P21‐activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell‐ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.
“… 96–98 The nephrin and podocin linear staining between the foot processes has been confirmed in the primary rat podocytes ( Figure 7D ), suggesting that the serum-free and laminin 521 scaffolds are essential for the structure of the podocyte foot processes. 96 , 98 The application of these approaches for culturing the podocytes in GoCs and demonstrating the localization of the dendritic intermediate diameter filaments and slit diaphragm proteins between the foot processes and cell cycle migration quiescence would be useful for improving the GoCs. Figure 7 Interdigitating the foot process formation with cultured podocytes ( A and B ) Induction of the process formation with heat-sensitive mouse podocytes (HSMP), vimentin (red), F-actin (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue); scale bar 100 µm.…”
Glomerulopathy, characterized by a dysfunctional glomerular capillary wall, results in proteinuria, leading to end-stage renal failure and poor clinical outcomes, including renal death and increased overall mortality. Conventional glomerulopathy research, including drug discovery, has mostly relied on animal experiments because in-vitro glomerulus models, capable of evaluating functional selective permeability, was unavailable in conventional in-vitro cell culture systems. However, animal experiments have limitations, including time- and cost-consuming, multi-organ effects, unstable reproducibility, inter-species reliability, and the social situation in the EU and US, where animal experiments have been discouraged. Glomerulus-on-a-chip, a new in-vitro organ model, has recently been developed in the field of organ-on-a-chip research based on microfluidic device technology. In the glomerulus-on-a-chip, the podocytes and endothelial cells are co-cultured in a microfluidic device with physical stimuli that mimic the physiological environment to enhance cell function to construct a functional filtration barrier, which can be assessed by permeability assays using fluorescently labeled molecules including inulin and albumin. A combination of this glomerulus-on-a chip technology with the culture technology to induce podocytes and endothelial cells from the human pluripotent stem cells could provide an alternative organ model and solve the issue of animal experiments. Additionally, previous experiments have verified the difference in the leakage of albumin using differentiated podocytes derived from patients with Alport syndrome, such that it could be applied to intractable hereditary glomerulopathy models. In this review, we provide an overview of the features of the existing glomerulus-on-a-chip systems, focusing on how they can address selective permeability verification tests, and the challenges they involved. We finally discuss the future approaches that should be developed for solving those challenges and allow further improvement of glomerulus-on-a-chip technologies.
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