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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 renal vascular permeability.A) Renal vascular permeability as assessed by Evans Blue (EB) tissue accrual after 30 min of reperfusion in renal sections. Kidney of untreated (NxIRCTR) rats exhibit thereby increased accumulation of EB compared with HF-treated rats (NxIRHF) as assessed by B) spectrophotometrical quantification at 620 nm (NxIRCTR: 16.7±1.3 µg/ml, n = 4, NxIRHF: 12.4±1,3 µg/ml, n = 5) (* p<0.05 vs. NxIRCTR). C) Evaluation of kidney wet weight/100 g body weight ratio. In comparison with healthy animals (CTR) kidney weight significantly increased in all groups. However, compared with uninephrectomized animals (Nx) the kidney wet weight/100 g body weight ratio only increased significantly in untreated ischemic animals (NxIRCTR 30 min & POD4), while there was no significant increase in the ROCK-inhibitor treated group (CTR: n = 18, Nx: n = 6, NxIRCTR 30 min: n = 7, NxIRHF 30 min: n = 8, NxIRCTR POD4: n = 3, NxIRHF POD4: n = 3; *  =  p<0,05 vs. CTR, # p<0,05 vs. Nx).
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pone-0026419-g004: Effect of ischemia-reperfusion injury and ROCK-inhibition on renal vascular permeability.A) Renal vascular permeability as assessed by Evans Blue (EB) tissue accrual after 30 min of reperfusion in renal sections. Kidney of untreated (NxIRCTR) rats exhibit thereby increased accumulation of EB compared with HF-treated rats (NxIRHF) as assessed by B) spectrophotometrical quantification at 620 nm (NxIRCTR: 16.7±1.3 µg/ml, n = 4, NxIRHF: 12.4±1,3 µg/ml, n = 5) (* p<0.05 vs. NxIRCTR). C) Evaluation of kidney wet weight/100 g body weight ratio. In comparison with healthy animals (CTR) kidney weight significantly increased in all groups. However, compared with uninephrectomized animals (Nx) the kidney wet weight/100 g body weight ratio only increased significantly in untreated ischemic animals (NxIRCTR 30 min & POD4), while there was no significant increase in the ROCK-inhibitor treated group (CTR: n = 18, Nx: n = 6, NxIRCTR 30 min: n = 7, NxIRHF 30 min: n = 8, NxIRCTR POD4: n = 3, NxIRHF POD4: n = 3; *  =  p<0,05 vs. CTR, # p<0,05 vs. Nx).

Mentions: Our results so far indicated a HF-related modulation of the inflammatory response affecting adhesive and migratory capabilities of immune cells. Additionally, HF-treatment modifies adhesion molecule expression and renal perfusion in the long run, which indicates HF-effects on endothelia. Thus, we aimed to test endothelial functionality by applying an Evans Blue (EB)-based vascular permeability assay during the initial 30 minutes of the reperfusion phase (Figure 4). Performing spectrophotometrical analyses of the renal content of EB after IRI, we assessed a distinct higher accumulation of EB in untreated (NxIRCTR: 16.7±1.3 µg/ml, n = 4) than in HF-treated rats (NxIRHF: 12.4±1.3 µg/ml, n = 5) (* p<0.05 vs. NxIRCTR).


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 renal vascular permeability.A) Renal vascular permeability as assessed by Evans Blue (EB) tissue accrual after 30 min of reperfusion in renal sections. Kidney of untreated (NxIRCTR) rats exhibit thereby increased accumulation of EB compared with HF-treated rats (NxIRHF) as assessed by B) spectrophotometrical quantification at 620 nm (NxIRCTR: 16.7±1.3 µg/ml, n = 4, NxIRHF: 12.4±1,3 µg/ml, n = 5) (* p<0.05 vs. NxIRCTR). C) Evaluation of kidney wet weight/100 g body weight ratio. In comparison with healthy animals (CTR) kidney weight significantly increased in all groups. However, compared with uninephrectomized animals (Nx) the kidney wet weight/100 g body weight ratio only increased significantly in untreated ischemic animals (NxIRCTR 30 min & POD4), while there was no significant increase in the ROCK-inhibitor treated group (CTR: n = 18, Nx: n = 6, NxIRCTR 30 min: n = 7, NxIRHF 30 min: n = 8, NxIRCTR POD4: n = 3, NxIRHF POD4: n = 3; *  =  p<0,05 vs. CTR, # p<0,05 vs. Nx).
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pone-0026419-g004: Effect of ischemia-reperfusion injury and ROCK-inhibition on renal vascular permeability.A) Renal vascular permeability as assessed by Evans Blue (EB) tissue accrual after 30 min of reperfusion in renal sections. Kidney of untreated (NxIRCTR) rats exhibit thereby increased accumulation of EB compared with HF-treated rats (NxIRHF) as assessed by B) spectrophotometrical quantification at 620 nm (NxIRCTR: 16.7±1.3 µg/ml, n = 4, NxIRHF: 12.4±1,3 µg/ml, n = 5) (* p<0.05 vs. NxIRCTR). C) Evaluation of kidney wet weight/100 g body weight ratio. In comparison with healthy animals (CTR) kidney weight significantly increased in all groups. However, compared with uninephrectomized animals (Nx) the kidney wet weight/100 g body weight ratio only increased significantly in untreated ischemic animals (NxIRCTR 30 min & POD4), while there was no significant increase in the ROCK-inhibitor treated group (CTR: n = 18, Nx: n = 6, NxIRCTR 30 min: n = 7, NxIRHF 30 min: n = 8, NxIRCTR POD4: n = 3, NxIRHF POD4: n = 3; *  =  p<0,05 vs. CTR, # p<0,05 vs. Nx).
Mentions: Our results so far indicated a HF-related modulation of the inflammatory response affecting adhesive and migratory capabilities of immune cells. Additionally, HF-treatment modifies adhesion molecule expression and renal perfusion in the long run, which indicates HF-effects on endothelia. Thus, we aimed to test endothelial functionality by applying an Evans Blue (EB)-based vascular permeability assay during the initial 30 minutes of the reperfusion phase (Figure 4). Performing spectrophotometrical analyses of the renal content of EB after IRI, we assessed a distinct higher accumulation of EB in untreated (NxIRCTR: 16.7±1.3 µg/ml, n = 4) than in HF-treated rats (NxIRHF: 12.4±1.3 µg/ml, n = 5) (* p<0.05 vs. NxIRCTR).

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