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Hydroxyfasudil-mediated inhibition of ROCK1 and ROCK2 improves kidney function in rat renal acute ischemia-reperfusion injury.

Kentrup D, Reuter S, Schnöckel U, Grabner A, Edemir B, Pavenstädt H, Schober O, Schäfers M, Schlatter E, Büssemaker E - PLoS ONE (2011)

Bottom Line: Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity.Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals.Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, (18)F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine D, Experimental Nephrology, University of Münster, Münster, Germany.

ABSTRACT
Renal ischemia-reperfusion (IR) injury (IRI) is a common and important trigger of acute renal injury (AKI). It is inevitably linked to transplantation. Involving both, the innate and the adaptive immune response, IRI causes subsequent sterile inflammation. Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity. Considering the important role of cytoskeletal reorganization, mainly regulated by RhoGTPases, in the development of IRI we hypothesized that a preventive, selective inhibition of the Rho effector Rho-associated coiled coil containing protein kinase (ROCK) by hydroxyfasudil may improve renal IRI outcome. Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals. In addition, renal perfusion (as assessed by (18)F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, (18)F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis. We conclude from our study that ROCK-inhibition by hydroxyfasudil significantly improves kidney function in a rat model of acute renal IRI and is therefore a potential new therapeutic option in humans.

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Related in: MedlinePlus

Effect of ischemia-reperfusion injury and ROCK-inhibition on mRNA-expression.Fold change in mRNA-expression compared to uninephrectomized animals (whole kidney lysates) as analyzed by real-time PCR (CD4, CD8a, CD20/Ms4a1, CD25/Il2ra, CD56/Ncam1, CD66a/Ceacam1, Fcnb, Foxp3, CD80, CD86, Kim1, Icam1, Vcam1) on POD1 (A) and POD4 (B). Following IRI, we observed a distinct up-regulation of marker mRNA, B-cells (CD20), neutrophil granulocytes (CD66a), activated B-cells/monocytes (CD80), antigen presenting cells (CD86) and the monocyte/macrophage activity marker ficolin B (Fcnb). IRI also led to a distinct up-regulation of mRNA-levels of vascular adhesion molecules (Icam1, Vcam1) and, especially, of Kim1. ROCK-inhibition significantly attenuated the up-regulation of CD66a, CD80, CD86, FcnB, Icam1, Vcam1 and Kim1. CD86 and Vcam1 were not significantly different in NxIRCTR and NxIRHF. Values are expressed as mean ± SEM, n = 4–6. a p<0.05 vs. Nx, b p<0.05 vs. NxIRCTR POD1 or NxIRCTR POD4, respectively.
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pone-0026419-g006: Effect of ischemia-reperfusion injury and ROCK-inhibition on mRNA-expression.Fold change in mRNA-expression compared to uninephrectomized animals (whole kidney lysates) as analyzed by real-time PCR (CD4, CD8a, CD20/Ms4a1, CD25/Il2ra, CD56/Ncam1, CD66a/Ceacam1, Fcnb, Foxp3, CD80, CD86, Kim1, Icam1, Vcam1) on POD1 (A) and POD4 (B). Following IRI, we observed a distinct up-regulation of marker mRNA, B-cells (CD20), neutrophil granulocytes (CD66a), activated B-cells/monocytes (CD80), antigen presenting cells (CD86) and the monocyte/macrophage activity marker ficolin B (Fcnb). IRI also led to a distinct up-regulation of mRNA-levels of vascular adhesion molecules (Icam1, Vcam1) and, especially, of Kim1. ROCK-inhibition significantly attenuated the up-regulation of CD66a, CD80, CD86, FcnB, Icam1, Vcam1 and Kim1. CD86 and Vcam1 were not significantly different in NxIRCTR and NxIRHF. Values are expressed as mean ± SEM, n = 4–6. a p<0.05 vs. Nx, b p<0.05 vs. NxIRCTR POD1 or NxIRCTR POD4, respectively.

Mentions: Because ROCK-inhibition considerably improved renal recovery after IRI and reduced inflammation, we aimed to analyze the expression of possibly involved genes. Although earlier analyses have linked ROCK-inhibition to reduced infiltration of macrophages into the (post-ischemic) kidney [11], [12], [43] a precise analysis of the immune cell types, i.e. neutrophiles, monocytes, as well as B- and T-cells, involved is lacking. Recently, it has been shown that ROCK-inhibition generally negatively influences the ability of leukocytes to adhere and to (trans-) migrate [25], [26], [28], [30], [32]. Moreover, ROCK-inhibition significantly influences the integrity and permeability of endo- and epithelia. In a first step, we analyzed marker genes of specific leukocyte subpopulations on POD1 and POD4 (Figure 6) to characterize the post-ischemic infiltrate and the effect of HF on different immune cell types. On POD1 following IRI, there was no significant difference between the HF treated and untreated IRI rats, besides the mRNA of the up-regulated B-cell marker CD20 and the neutrophil granulocytes marker Ceacam1, which was downregulated in the HF group. On POD4, HF treatment attenuated the up-regulation of Ceacam1, CD80 (activated B-cells/monocytes), CD86 (antigen presenting cells and the monocyte/macrophage activity marker ficolin B. As a second gene set, we measured mRNA-expression of pro-inflammatory cytokines such as interferon gamma (Infg), tumor necrosis factor (Tnf), tumor necrosis factor (ligand) superfamily member 15 (Tnfsf15), adhesion molecules (Icam1, Vcam1), and kidney injury molecule 1 (Kim1). On POD1, we assessed a distinct up-regulation of Kim1 only, whereas on POD4 Icam1, Vcam1, and Kim1 were up-regulated. HF, treatment attenuated the up-regulation of Kim1 (POD1 and 4), Icam1 and Vcam1 (POD4). However, in the latter case (Vcam1) expression was only attenuated in NxIRHF when compared to uninephrectomized animals but failed to be significantly downregulated when compared to NxIRCTR. Ifng, Tnf and Tnfsf15 were not significantly different expressed on POD1 and 4; Figure 6 and Figure S1 (for Figure S1 see supplemental data)).


Hydroxyfasudil-mediated inhibition of ROCK1 and ROCK2 improves kidney function in rat renal acute ischemia-reperfusion injury.

Kentrup D, Reuter S, Schnöckel U, Grabner A, Edemir B, Pavenstädt H, Schober O, Schäfers M, Schlatter E, Büssemaker E - PLoS ONE (2011)

Effect of ischemia-reperfusion injury and ROCK-inhibition on mRNA-expression.Fold change in mRNA-expression compared to uninephrectomized animals (whole kidney lysates) as analyzed by real-time PCR (CD4, CD8a, CD20/Ms4a1, CD25/Il2ra, CD56/Ncam1, CD66a/Ceacam1, Fcnb, Foxp3, CD80, CD86, Kim1, Icam1, Vcam1) on POD1 (A) and POD4 (B). Following IRI, we observed a distinct up-regulation of marker mRNA, B-cells (CD20), neutrophil granulocytes (CD66a), activated B-cells/monocytes (CD80), antigen presenting cells (CD86) and the monocyte/macrophage activity marker ficolin B (Fcnb). IRI also led to a distinct up-regulation of mRNA-levels of vascular adhesion molecules (Icam1, Vcam1) and, especially, of Kim1. ROCK-inhibition significantly attenuated the up-regulation of CD66a, CD80, CD86, FcnB, Icam1, Vcam1 and Kim1. CD86 and Vcam1 were not significantly different in NxIRCTR and NxIRHF. Values are expressed as mean ± SEM, n = 4–6. a p<0.05 vs. Nx, b p<0.05 vs. NxIRCTR POD1 or NxIRCTR POD4, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198766&req=5

pone-0026419-g006: Effect of ischemia-reperfusion injury and ROCK-inhibition on mRNA-expression.Fold change in mRNA-expression compared to uninephrectomized animals (whole kidney lysates) as analyzed by real-time PCR (CD4, CD8a, CD20/Ms4a1, CD25/Il2ra, CD56/Ncam1, CD66a/Ceacam1, Fcnb, Foxp3, CD80, CD86, Kim1, Icam1, Vcam1) on POD1 (A) and POD4 (B). Following IRI, we observed a distinct up-regulation of marker mRNA, B-cells (CD20), neutrophil granulocytes (CD66a), activated B-cells/monocytes (CD80), antigen presenting cells (CD86) and the monocyte/macrophage activity marker ficolin B (Fcnb). IRI also led to a distinct up-regulation of mRNA-levels of vascular adhesion molecules (Icam1, Vcam1) and, especially, of Kim1. ROCK-inhibition significantly attenuated the up-regulation of CD66a, CD80, CD86, FcnB, Icam1, Vcam1 and Kim1. CD86 and Vcam1 were not significantly different in NxIRCTR and NxIRHF. Values are expressed as mean ± SEM, n = 4–6. a p<0.05 vs. Nx, b p<0.05 vs. NxIRCTR POD1 or NxIRCTR POD4, respectively.
Mentions: Because ROCK-inhibition considerably improved renal recovery after IRI and reduced inflammation, we aimed to analyze the expression of possibly involved genes. Although earlier analyses have linked ROCK-inhibition to reduced infiltration of macrophages into the (post-ischemic) kidney [11], [12], [43] a precise analysis of the immune cell types, i.e. neutrophiles, monocytes, as well as B- and T-cells, involved is lacking. Recently, it has been shown that ROCK-inhibition generally negatively influences the ability of leukocytes to adhere and to (trans-) migrate [25], [26], [28], [30], [32]. Moreover, ROCK-inhibition significantly influences the integrity and permeability of endo- and epithelia. In a first step, we analyzed marker genes of specific leukocyte subpopulations on POD1 and POD4 (Figure 6) to characterize the post-ischemic infiltrate and the effect of HF on different immune cell types. On POD1 following IRI, there was no significant difference between the HF treated and untreated IRI rats, besides the mRNA of the up-regulated B-cell marker CD20 and the neutrophil granulocytes marker Ceacam1, which was downregulated in the HF group. On POD4, HF treatment attenuated the up-regulation of Ceacam1, CD80 (activated B-cells/monocytes), CD86 (antigen presenting cells and the monocyte/macrophage activity marker ficolin B. As a second gene set, we measured mRNA-expression of pro-inflammatory cytokines such as interferon gamma (Infg), tumor necrosis factor (Tnf), tumor necrosis factor (ligand) superfamily member 15 (Tnfsf15), adhesion molecules (Icam1, Vcam1), and kidney injury molecule 1 (Kim1). On POD1, we assessed a distinct up-regulation of Kim1 only, whereas on POD4 Icam1, Vcam1, and Kim1 were up-regulated. HF, treatment attenuated the up-regulation of Kim1 (POD1 and 4), Icam1 and Vcam1 (POD4). However, in the latter case (Vcam1) expression was only attenuated in NxIRHF when compared to uninephrectomized animals but failed to be significantly downregulated when compared to NxIRCTR. Ifng, Tnf and Tnfsf15 were not significantly different expressed on POD1 and 4; Figure 6 and Figure S1 (for Figure S1 see supplemental data)).

Bottom Line: Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity.Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals.Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, (18)F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine D, Experimental Nephrology, University of Münster, Münster, Germany.

ABSTRACT
Renal ischemia-reperfusion (IR) injury (IRI) is a common and important trigger of acute renal injury (AKI). It is inevitably linked to transplantation. Involving both, the innate and the adaptive immune response, IRI causes subsequent sterile inflammation. Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity. Considering the important role of cytoskeletal reorganization, mainly regulated by RhoGTPases, in the development of IRI we hypothesized that a preventive, selective inhibition of the Rho effector Rho-associated coiled coil containing protein kinase (ROCK) by hydroxyfasudil may improve renal IRI outcome. Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals. In addition, renal perfusion (as assessed by (18)F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, (18)F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis. We conclude from our study that ROCK-inhibition by hydroxyfasudil significantly improves kidney function in a rat model of acute renal IRI and is therefore a potential new therapeutic option in humans.

Show MeSH
Related in: MedlinePlus