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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 perfusion.A) Exemplary, representative time activity curve (TAC) of the arterial bloodpool and of both kidneys in a control rat on POD4. Areas under the curves during the first 30 seconds after 18F-fluoride injection were used to calculate the perfusion index (perfusion index left kidney: 310.7 (post IR), right kidney: 185.6 (CTR)). B) Comparison of the modified Hilson's Perfusion index between the two groups as assessed by 18F-PET. At the beginning of the reperfusion phase (POD0, post-ischemia) kidney perfusion was significantly impaired in both groups (CTR POD0: 192.8±25.7, n = 7 and HF POD0: 197.0±15.0, n = 8) compared with baseline values (CTR: 148.0±12.3, n = 9 vs. HF: 155.7±11.2, n = 9). However, kidney perfusion of the HF-treated rats has already recovered on POD4 while perfusion of the vehicle-treated group was still impaired (CTR POD4: 186.9±14.5, n = 6 vs. HF POD4: 152.4±11.6, n = 8) (§ p< 0.05 vs. baseline,* p<0.05 HF POD4 vs. CTR POD4). C) Mean systolic and diastolic blood-pressures of conscious rats before ischemia induction on POD0. No significant differences were detected between HF- and sham-treated animals (NxIRCTR 130.0±3.6 mmHg (systolic) 92.6±3.3 mmHg (diastolic), n = 8 vs. NxIRHF 118.8±4.8 (systolic) 82.7±4.2 (diastolic) n = 7, p<0.05). D) Relative phospho-MYPT1 expression in relation to GAPDH for HF- and sham treated animals as assessed by quantitative Western Blot analysis (NxIRCTR 0.46±0.06 vs. NxIRHF 0.31±0.03, n = 7, p<0.05).
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pone-0026419-g003: Effect of ischemia-reperfusion injury and ROCK-inhibition on renal perfusion.A) Exemplary, representative time activity curve (TAC) of the arterial bloodpool and of both kidneys in a control rat on POD4. Areas under the curves during the first 30 seconds after 18F-fluoride injection were used to calculate the perfusion index (perfusion index left kidney: 310.7 (post IR), right kidney: 185.6 (CTR)). B) Comparison of the modified Hilson's Perfusion index between the two groups as assessed by 18F-PET. At the beginning of the reperfusion phase (POD0, post-ischemia) kidney perfusion was significantly impaired in both groups (CTR POD0: 192.8±25.7, n = 7 and HF POD0: 197.0±15.0, n = 8) compared with baseline values (CTR: 148.0±12.3, n = 9 vs. HF: 155.7±11.2, n = 9). However, kidney perfusion of the HF-treated rats has already recovered on POD4 while perfusion of the vehicle-treated group was still impaired (CTR POD4: 186.9±14.5, n = 6 vs. HF POD4: 152.4±11.6, n = 8) (§ p< 0.05 vs. baseline,* p<0.05 HF POD4 vs. CTR POD4). C) Mean systolic and diastolic blood-pressures of conscious rats before ischemia induction on POD0. No significant differences were detected between HF- and sham-treated animals (NxIRCTR 130.0±3.6 mmHg (systolic) 92.6±3.3 mmHg (diastolic), n = 8 vs. NxIRHF 118.8±4.8 (systolic) 82.7±4.2 (diastolic) n = 7, p<0.05). D) Relative phospho-MYPT1 expression in relation to GAPDH for HF- and sham treated animals as assessed by quantitative Western Blot analysis (NxIRCTR 0.46±0.06 vs. NxIRHF 0.31±0.03, n = 7, p<0.05).

Mentions: ROCK-inhibition was verified by evaluating the phosphorylation status of the ROCK-substrate phospho-MYPT1 by quantitative Western Blot analysis. As depicted in Figure 3D, in NxIRHF ROCK-activity was significantly reduced in kidney samples taken immediately before the ischemia procedure (NxIRCTR 0.46±0.06 vs. NxIRHF 0.31±0.03, n = 7, p<0.05). Additionally, even though our results indicated a crucial role of ROCK-dependent leukocyte recruitment in renal IRI so far, the clinical field of application of ROCK-inhibitors (e.g., Fasudil) lies within cardiovascular diseases, where mainly vasodilatatory and therefore blood-pressure lowering effects are observed. Hence, one might assume that the post-ischemic functional improvement of kidneys was mediated by increased perfusion (via vasodilatation). To test this hypothesis, we assessed renal perfusion by 18F-PET and calculated a modified Hilson's Perfusion index (Hilson's PI, Figure 3, higher values indicate lower perfusion rates). At baseline renal perfusion did not differ between groups and kidneys (CTR: 148.0±12.3, n = 9 vs. HF: 155.7±11.2, n = 9). Ischemia led to a significant decrease of renal perfusion in the affected kidney only as assessed immediately after the ischemia inducing operation. Again, there was no difference between NxIRCTR POD0 (192.8±25.7, n = 7) and NxIRHF POD0 (197.0±15.0, n = 8). However, on POD4 renal perfusion in vehicle-treated animals was still impaired while the renal perfusion of HF-treated rats had completely recovered (NxIRCTR POD4: 186.9±14.5, n = 6 vs. NxIRHF POD4: 152.4±11.6, n = 8). In addition, blood-pressure lowering effects of HF were excluded by use of a non-invasive, volume pressure recording technique (Figure 3C) which is consistent with work published by Komers et al. [42].


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 perfusion.A) Exemplary, representative time activity curve (TAC) of the arterial bloodpool and of both kidneys in a control rat on POD4. Areas under the curves during the first 30 seconds after 18F-fluoride injection were used to calculate the perfusion index (perfusion index left kidney: 310.7 (post IR), right kidney: 185.6 (CTR)). B) Comparison of the modified Hilson's Perfusion index between the two groups as assessed by 18F-PET. At the beginning of the reperfusion phase (POD0, post-ischemia) kidney perfusion was significantly impaired in both groups (CTR POD0: 192.8±25.7, n = 7 and HF POD0: 197.0±15.0, n = 8) compared with baseline values (CTR: 148.0±12.3, n = 9 vs. HF: 155.7±11.2, n = 9). However, kidney perfusion of the HF-treated rats has already recovered on POD4 while perfusion of the vehicle-treated group was still impaired (CTR POD4: 186.9±14.5, n = 6 vs. HF POD4: 152.4±11.6, n = 8) (§ p< 0.05 vs. baseline,* p<0.05 HF POD4 vs. CTR POD4). C) Mean systolic and diastolic blood-pressures of conscious rats before ischemia induction on POD0. No significant differences were detected between HF- and sham-treated animals (NxIRCTR 130.0±3.6 mmHg (systolic) 92.6±3.3 mmHg (diastolic), n = 8 vs. NxIRHF 118.8±4.8 (systolic) 82.7±4.2 (diastolic) n = 7, p<0.05). D) Relative phospho-MYPT1 expression in relation to GAPDH for HF- and sham treated animals as assessed by quantitative Western Blot analysis (NxIRCTR 0.46±0.06 vs. NxIRHF 0.31±0.03, n = 7, p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198766&req=5

pone-0026419-g003: Effect of ischemia-reperfusion injury and ROCK-inhibition on renal perfusion.A) Exemplary, representative time activity curve (TAC) of the arterial bloodpool and of both kidneys in a control rat on POD4. Areas under the curves during the first 30 seconds after 18F-fluoride injection were used to calculate the perfusion index (perfusion index left kidney: 310.7 (post IR), right kidney: 185.6 (CTR)). B) Comparison of the modified Hilson's Perfusion index between the two groups as assessed by 18F-PET. At the beginning of the reperfusion phase (POD0, post-ischemia) kidney perfusion was significantly impaired in both groups (CTR POD0: 192.8±25.7, n = 7 and HF POD0: 197.0±15.0, n = 8) compared with baseline values (CTR: 148.0±12.3, n = 9 vs. HF: 155.7±11.2, n = 9). However, kidney perfusion of the HF-treated rats has already recovered on POD4 while perfusion of the vehicle-treated group was still impaired (CTR POD4: 186.9±14.5, n = 6 vs. HF POD4: 152.4±11.6, n = 8) (§ p< 0.05 vs. baseline,* p<0.05 HF POD4 vs. CTR POD4). C) Mean systolic and diastolic blood-pressures of conscious rats before ischemia induction on POD0. No significant differences were detected between HF- and sham-treated animals (NxIRCTR 130.0±3.6 mmHg (systolic) 92.6±3.3 mmHg (diastolic), n = 8 vs. NxIRHF 118.8±4.8 (systolic) 82.7±4.2 (diastolic) n = 7, p<0.05). D) Relative phospho-MYPT1 expression in relation to GAPDH for HF- and sham treated animals as assessed by quantitative Western Blot analysis (NxIRCTR 0.46±0.06 vs. NxIRHF 0.31±0.03, n = 7, p<0.05).
Mentions: ROCK-inhibition was verified by evaluating the phosphorylation status of the ROCK-substrate phospho-MYPT1 by quantitative Western Blot analysis. As depicted in Figure 3D, in NxIRHF ROCK-activity was significantly reduced in kidney samples taken immediately before the ischemia procedure (NxIRCTR 0.46±0.06 vs. NxIRHF 0.31±0.03, n = 7, p<0.05). Additionally, even though our results indicated a crucial role of ROCK-dependent leukocyte recruitment in renal IRI so far, the clinical field of application of ROCK-inhibitors (e.g., Fasudil) lies within cardiovascular diseases, where mainly vasodilatatory and therefore blood-pressure lowering effects are observed. Hence, one might assume that the post-ischemic functional improvement of kidneys was mediated by increased perfusion (via vasodilatation). To test this hypothesis, we assessed renal perfusion by 18F-PET and calculated a modified Hilson's Perfusion index (Hilson's PI, Figure 3, higher values indicate lower perfusion rates). At baseline renal perfusion did not differ between groups and kidneys (CTR: 148.0±12.3, n = 9 vs. HF: 155.7±11.2, n = 9). Ischemia led to a significant decrease of renal perfusion in the affected kidney only as assessed immediately after the ischemia inducing operation. Again, there was no difference between NxIRCTR POD0 (192.8±25.7, n = 7) and NxIRHF POD0 (197.0±15.0, n = 8). However, on POD4 renal perfusion in vehicle-treated animals was still impaired while the renal perfusion of HF-treated rats had completely recovered (NxIRCTR POD4: 186.9±14.5, n = 6 vs. NxIRHF POD4: 152.4±11.6, n = 8). In addition, blood-pressure lowering effects of HF were excluded by use of a non-invasive, volume pressure recording technique (Figure 3C) which is consistent with work published by Komers et al. [42].

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