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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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Effect of ischemia-reperfusion injury and ROCK-inhibition on matrix metalloproteinases expression.A) The mRNA-expression of Mmp2, Mmp7, Mmp8 and Mmp9 in whole kidney lysates as analyzed by real-time PCR. In vehicle-treated rats ischemia-reperfusion injury led to a significantly increased mRNA-expression of Mmp2 and Mmp7 on POD4, as well as decreased mRNA-expression of Mmp9 on POD1 and POD4 when compared to healthy and HF-treated animals. In turn, mRNA-expression of Mmp2 and Mmp7 of HF-treated animals did not change significantly compared to healthy animals. Differences in gene-expression were reflected by corresponding protein-expression as shown by immunohistochemistry of Mmp2 (B) and Mmp9 (C). Values are expressed as mean ± SEM, n = 4–5. a p<0.05 vs. CTR, b p<0.05 vs. NxIRCTR POD1, c p<0.05 vs. CTR and NxIRCTR POD1, d p<0.05 vs. NxIRCTR POD4, e p<0.05 vs. CTR and NxIRCTR POD4.
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pone-0026419-g005: Effect of ischemia-reperfusion injury and ROCK-inhibition on matrix metalloproteinases expression.A) The mRNA-expression of Mmp2, Mmp7, Mmp8 and Mmp9 in whole kidney lysates as analyzed by real-time PCR. In vehicle-treated rats ischemia-reperfusion injury led to a significantly increased mRNA-expression of Mmp2 and Mmp7 on POD4, as well as decreased mRNA-expression of Mmp9 on POD1 and POD4 when compared to healthy and HF-treated animals. In turn, mRNA-expression of Mmp2 and Mmp7 of HF-treated animals did not change significantly compared to healthy animals. Differences in gene-expression were reflected by corresponding protein-expression as shown by immunohistochemistry of Mmp2 (B) and Mmp9 (C). Values are expressed as mean ± SEM, n = 4–5. a p<0.05 vs. CTR, b p<0.05 vs. NxIRCTR POD1, c p<0.05 vs. CTR and NxIRCTR POD1, d p<0.05 vs. NxIRCTR POD4, e p<0.05 vs. CTR and NxIRCTR POD4.

Mentions: In the next step, we aimed to clarify if the infiltrates observed after IRI were harmful to the kidney. Therefore, we employed real-time polymerase chain reaction (PCR) and immunohistochemical analyses of matrix metalloproteinases (Mmp). The mRNA-expression results of Mmp2, Mmp7, Mmp8, and Mmp9 are presented in figure 5A. Whereas upon IRI-treatment, vehicle-treated animals demonstrated up-regulated levels of Mmp2 and Mmp7, Mmp9 was down-regulated when compared to HF-treated animals. Exemplary immunohistochemistry of Mmp2 and Mmp9 confirmed these findings also on the protein-expression level (Figure 5B and C).


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 matrix metalloproteinases expression.A) The mRNA-expression of Mmp2, Mmp7, Mmp8 and Mmp9 in whole kidney lysates as analyzed by real-time PCR. In vehicle-treated rats ischemia-reperfusion injury led to a significantly increased mRNA-expression of Mmp2 and Mmp7 on POD4, as well as decreased mRNA-expression of Mmp9 on POD1 and POD4 when compared to healthy and HF-treated animals. In turn, mRNA-expression of Mmp2 and Mmp7 of HF-treated animals did not change significantly compared to healthy animals. Differences in gene-expression were reflected by corresponding protein-expression as shown by immunohistochemistry of Mmp2 (B) and Mmp9 (C). Values are expressed as mean ± SEM, n = 4–5. a p<0.05 vs. CTR, b p<0.05 vs. NxIRCTR POD1, c p<0.05 vs. CTR and NxIRCTR POD1, d p<0.05 vs. NxIRCTR POD4, e p<0.05 vs. CTR and NxIRCTR POD4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198766&req=5

pone-0026419-g005: Effect of ischemia-reperfusion injury and ROCK-inhibition on matrix metalloproteinases expression.A) The mRNA-expression of Mmp2, Mmp7, Mmp8 and Mmp9 in whole kidney lysates as analyzed by real-time PCR. In vehicle-treated rats ischemia-reperfusion injury led to a significantly increased mRNA-expression of Mmp2 and Mmp7 on POD4, as well as decreased mRNA-expression of Mmp9 on POD1 and POD4 when compared to healthy and HF-treated animals. In turn, mRNA-expression of Mmp2 and Mmp7 of HF-treated animals did not change significantly compared to healthy animals. Differences in gene-expression were reflected by corresponding protein-expression as shown by immunohistochemistry of Mmp2 (B) and Mmp9 (C). Values are expressed as mean ± SEM, n = 4–5. a p<0.05 vs. CTR, b p<0.05 vs. NxIRCTR POD1, c p<0.05 vs. CTR and NxIRCTR POD1, d p<0.05 vs. NxIRCTR POD4, e p<0.05 vs. CTR and NxIRCTR POD4.
Mentions: In the next step, we aimed to clarify if the infiltrates observed after IRI were harmful to the kidney. Therefore, we employed real-time polymerase chain reaction (PCR) and immunohistochemical analyses of matrix metalloproteinases (Mmp). The mRNA-expression results of Mmp2, Mmp7, Mmp8, and Mmp9 are presented in figure 5A. Whereas upon IRI-treatment, vehicle-treated animals demonstrated up-regulated levels of Mmp2 and Mmp7, Mmp9 was down-regulated when compared to HF-treated animals. Exemplary immunohistochemistry of Mmp2 and Mmp9 confirmed these findings also on the protein-expression level (Figure 5B and C).

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