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The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice.

Reviriego-Mendoza MM, Santy LC - Physiol Rep (2015)

Bottom Line: Immunohistochemistry showed that HGF treatment promoted recovery of tubule structure, and had enhanced levels of active, GTP-bound Arf6 and GTP-Rac1.Additionally, SecinH3 counteracted the renal reparative effects of HGF.Our results support the conclusion that cytohesin function is required for HGF-stimulated renal IRI repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania.

No MeSH data available.


Related in: MedlinePlus

HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μm.
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fig03: HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μm.

Mentions: Cytohesins act to promote epithelial migration by stimulating the activation of Arf6 and the subsequent activation of Rac1 by Dock180 (Santy et al. 2001, 2005; Santy and Casanova 2002). We have shown that inhibiting cytohesin function or Dock180 function impairs HGF-stimulated migration and HGF-stimulated Rac1 activation in MDCK cells (Attar and Santy 2013). We thus tested whether this signaling cascade is activated in the kidney upon HGF treatment of injured mice. Kidney samples from mice subjected to IRI were collected 96 h after surgery and processed for cryosectioning. Tissues were then immunostained to detect either GTP-bound Rac1 or GTP-bound Arf6, and either actin or the proximal tubule marker aquaporin 1 (Aqp1). Kidney sections from injured mice treated with HGF showed high levels of GTP-Arf6 and GTP-Rac1 in the basal surfaces of proximal tubule cells when compared to untreated IRI mice, demonstrating that HGF does induce activation of both proteins (Fig.2, compare panels c and g with panels a and e). When treating IRI mice with HGF in combination with SecinH3, however, the levels of both GTP-Arf6 and GTP-Rac1 diminish dramatically in the tubules (Fig.2, panels d and h), and are comparable to those of untreated or SecinH3-treated samples (Fig2, compare panels a and e, with panels b and f). Perhaps, SecinH3 treatment alone might reduce the levels of these active proteins even further when compared to levels observed in untreated IRI mice kidney sections (Fig.2, compare panels b and f, with panels a and e). Similar results were observed in tissue samples stained against GTP-Arf6 and GTP-Rac1 and the proximal tubular marker Aqp1 (Fig.3). The beginning of migration and dedifferentiation of surviving tubular cells to reepithelialize the tubules occurs right after inflammation, approximately 48 h after injury (Bonventre 2003). We thus wanted to investigate the levels of active Arf6 and active Rac1 in kidney tissue from injured mice harvested 48 h after surgery. Kidney sections from injured mice treated with HGF experienced high levels of active Arf6 and Rac1 that localized at the basal surfaces of tubular cells (Fig.4, compare panels a and e with panels c and g). To the contrary, kidney samples from injured mice treated with SecinH3 showed reduced levels of these active proteins and were similar to those found in the kidneys from untreated mice (Fig.4, compare panels b and f with panels c and d). Interestingly, kidney tissues from injured mice treated with both HGF and SecinH3 showed a partial reduction in the levels of GTP-bound Arf6 and Rac1 when compared to those of injured kidney tissues from mice treated with HGF only (Fig.4, compare panels d and h with panels c and g). This observation suggests that HGF treatment elicits a rapid response in the pathway that activates Arf6 and subsequently Rac1, and that a single treatment with SecinH3 does not fully inhibit HGF action. Alternatively, it is plausible that early in damaged kidney recovery HGF activates Arf6 and Rac1 through additional cytohesin-independent pathways. Together, these data demonstrate that HGF promotes recovery in part by stimulating the activation of Arf6 by cytohesins. This event leads to activation of Rac1 and migration of renal tubular epithelial cells to repopulate the damaged areas.


The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice.

Reviriego-Mendoza MM, Santy LC - Physiol Rep (2015)

HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μm.
Mentions: Cytohesins act to promote epithelial migration by stimulating the activation of Arf6 and the subsequent activation of Rac1 by Dock180 (Santy et al. 2001, 2005; Santy and Casanova 2002). We have shown that inhibiting cytohesin function or Dock180 function impairs HGF-stimulated migration and HGF-stimulated Rac1 activation in MDCK cells (Attar and Santy 2013). We thus tested whether this signaling cascade is activated in the kidney upon HGF treatment of injured mice. Kidney samples from mice subjected to IRI were collected 96 h after surgery and processed for cryosectioning. Tissues were then immunostained to detect either GTP-bound Rac1 or GTP-bound Arf6, and either actin or the proximal tubule marker aquaporin 1 (Aqp1). Kidney sections from injured mice treated with HGF showed high levels of GTP-Arf6 and GTP-Rac1 in the basal surfaces of proximal tubule cells when compared to untreated IRI mice, demonstrating that HGF does induce activation of both proteins (Fig.2, compare panels c and g with panels a and e). When treating IRI mice with HGF in combination with SecinH3, however, the levels of both GTP-Arf6 and GTP-Rac1 diminish dramatically in the tubules (Fig.2, panels d and h), and are comparable to those of untreated or SecinH3-treated samples (Fig2, compare panels a and e, with panels b and f). Perhaps, SecinH3 treatment alone might reduce the levels of these active proteins even further when compared to levels observed in untreated IRI mice kidney sections (Fig.2, compare panels b and f, with panels a and e). Similar results were observed in tissue samples stained against GTP-Arf6 and GTP-Rac1 and the proximal tubular marker Aqp1 (Fig.3). The beginning of migration and dedifferentiation of surviving tubular cells to reepithelialize the tubules occurs right after inflammation, approximately 48 h after injury (Bonventre 2003). We thus wanted to investigate the levels of active Arf6 and active Rac1 in kidney tissue from injured mice harvested 48 h after surgery. Kidney sections from injured mice treated with HGF experienced high levels of active Arf6 and Rac1 that localized at the basal surfaces of tubular cells (Fig.4, compare panels a and e with panels c and g). To the contrary, kidney samples from injured mice treated with SecinH3 showed reduced levels of these active proteins and were similar to those found in the kidneys from untreated mice (Fig.4, compare panels b and f with panels c and d). Interestingly, kidney tissues from injured mice treated with both HGF and SecinH3 showed a partial reduction in the levels of GTP-bound Arf6 and Rac1 when compared to those of injured kidney tissues from mice treated with HGF only (Fig.4, compare panels d and h with panels c and g). This observation suggests that HGF treatment elicits a rapid response in the pathway that activates Arf6 and subsequently Rac1, and that a single treatment with SecinH3 does not fully inhibit HGF action. Alternatively, it is plausible that early in damaged kidney recovery HGF activates Arf6 and Rac1 through additional cytohesin-independent pathways. Together, these data demonstrate that HGF promotes recovery in part by stimulating the activation of Arf6 by cytohesins. This event leads to activation of Rac1 and migration of renal tubular epithelial cells to repopulate the damaged areas.

Bottom Line: Immunohistochemistry showed that HGF treatment promoted recovery of tubule structure, and had enhanced levels of active, GTP-bound Arf6 and GTP-Rac1.Additionally, SecinH3 counteracted the renal reparative effects of HGF.Our results support the conclusion that cytohesin function is required for HGF-stimulated renal IRI repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania.

No MeSH data available.


Related in: MedlinePlus