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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 histological changes.Shown are representative PAS-stainings (cortex and medulla) and 18F-FDG-autoradiographies of post-ischemic kidneys on POD4 (NxIRCTR, NxIRHF), as well as PAS-stainings of kidneys from healthy (CTR) and uninephrectomized animals (Nx). Following IRI kidneys presented with signs of ATN, i.e. tubular dilation, swelling and necrosis in addition to intraluminal brush border debris and protein casts. Leukocyte infiltration was moderate and emphasized in the outer medulla. Kidneys of HF-treated animals (NxIRHF) showed reduced signs of ATN including attenuated interstitial infiltration when compared with vehicle-treated animals (NxIRCTR). In congruence, we found distinct 18F-FDG-uptake (quantified as counts per second (cps)/mm2) in the outer medulla region of NxIRCTR (arrows in autoradiography) with ATN whereas this was absent in the NxIRHF group. This was statistically verified by calculating the ratio (E/OM), between the 18F-FDG-uptake in the entire organ slice (E) compared to that in the outer medulla (OM). While there is no statistical difference between uninephrectomized (Nx: 1.2±0.02) and ROCK-inhibitor treated (NxIRHF: 1.12±0.04) animals, the E/OM ratio of untreated ischemic animals (NxIRCTR: 0.88±0.06) is significantly different from both aforementioned. Values are expressed as mean ± SEM, n = 3. * p<0.05 vs. Nx & NxIRHF). Non-marked, intensively stained areas are artifacts due to renal 18F-FDG excretion.
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pone-0026419-g002: Effect of ischemia-reperfusion injury and ROCK-inhibition on histological changes.Shown are representative PAS-stainings (cortex and medulla) and 18F-FDG-autoradiographies of post-ischemic kidneys on POD4 (NxIRCTR, NxIRHF), as well as PAS-stainings of kidneys from healthy (CTR) and uninephrectomized animals (Nx). Following IRI kidneys presented with signs of ATN, i.e. tubular dilation, swelling and necrosis in addition to intraluminal brush border debris and protein casts. Leukocyte infiltration was moderate and emphasized in the outer medulla. Kidneys of HF-treated animals (NxIRHF) showed reduced signs of ATN including attenuated interstitial infiltration when compared with vehicle-treated animals (NxIRCTR). In congruence, we found distinct 18F-FDG-uptake (quantified as counts per second (cps)/mm2) in the outer medulla region of NxIRCTR (arrows in autoradiography) with ATN whereas this was absent in the NxIRHF group. This was statistically verified by calculating the ratio (E/OM), between the 18F-FDG-uptake in the entire organ slice (E) compared to that in the outer medulla (OM). While there is no statistical difference between uninephrectomized (Nx: 1.2±0.02) and ROCK-inhibitor treated (NxIRHF: 1.12±0.04) animals, the E/OM ratio of untreated ischemic animals (NxIRCTR: 0.88±0.06) is significantly different from both aforementioned. Values are expressed as mean ± SEM, n = 3. * p<0.05 vs. Nx & NxIRHF). Non-marked, intensively stained areas are artifacts due to renal 18F-FDG excretion.

Mentions: To estimate renal damage and infiltration (also for validation of 18F-FDG data) we evaluated renal histology (Figure 2) in addition to metabolic parameters. Subsequent to IRI (POD4), kidneys presented with signs of ATN, i.e. tubular dilation, swelling, and necrosis, in addition to intraluminal brush border debris and protein casts. Leukocyte infiltration was moderate and emphasized in the outer medulla. Corresponding to improved renal function, kidneys of NxIRHF showed reduced signs of ATN including attenuated interstitial infiltration when compared with NxIRCTR. Examination of inflamed tissue by autoradiography has shown that 18F-FDG-accumulation is correlated to the degree of inflammatory infiltration in different pathophysiological scenarios [38], [41]. Thus, we amended histological evaluation by autoradiographic assessment of mid-coronary kidney slices in order to visualize inflamed tissue areas. Exemplary measures are given in figure 2. In congruence to histological data, we found a distinct 18F-FDG-uptake in the outer medulla region of NxIRCTR with ATN whereas this was absent in NxIRHF. Both, histologic and autoradiographic evaluation of kidneys showed distinct amelioration of renal damage (less structural damage, less inflammation) in the HF-treated group.


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 histological changes.Shown are representative PAS-stainings (cortex and medulla) and 18F-FDG-autoradiographies of post-ischemic kidneys on POD4 (NxIRCTR, NxIRHF), as well as PAS-stainings of kidneys from healthy (CTR) and uninephrectomized animals (Nx). Following IRI kidneys presented with signs of ATN, i.e. tubular dilation, swelling and necrosis in addition to intraluminal brush border debris and protein casts. Leukocyte infiltration was moderate and emphasized in the outer medulla. Kidneys of HF-treated animals (NxIRHF) showed reduced signs of ATN including attenuated interstitial infiltration when compared with vehicle-treated animals (NxIRCTR). In congruence, we found distinct 18F-FDG-uptake (quantified as counts per second (cps)/mm2) in the outer medulla region of NxIRCTR (arrows in autoradiography) with ATN whereas this was absent in the NxIRHF group. This was statistically verified by calculating the ratio (E/OM), between the 18F-FDG-uptake in the entire organ slice (E) compared to that in the outer medulla (OM). While there is no statistical difference between uninephrectomized (Nx: 1.2±0.02) and ROCK-inhibitor treated (NxIRHF: 1.12±0.04) animals, the E/OM ratio of untreated ischemic animals (NxIRCTR: 0.88±0.06) is significantly different from both aforementioned. Values are expressed as mean ± SEM, n = 3. * p<0.05 vs. Nx & NxIRHF). Non-marked, intensively stained areas are artifacts due to renal 18F-FDG excretion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198766&req=5

pone-0026419-g002: Effect of ischemia-reperfusion injury and ROCK-inhibition on histological changes.Shown are representative PAS-stainings (cortex and medulla) and 18F-FDG-autoradiographies of post-ischemic kidneys on POD4 (NxIRCTR, NxIRHF), as well as PAS-stainings of kidneys from healthy (CTR) and uninephrectomized animals (Nx). Following IRI kidneys presented with signs of ATN, i.e. tubular dilation, swelling and necrosis in addition to intraluminal brush border debris and protein casts. Leukocyte infiltration was moderate and emphasized in the outer medulla. Kidneys of HF-treated animals (NxIRHF) showed reduced signs of ATN including attenuated interstitial infiltration when compared with vehicle-treated animals (NxIRCTR). In congruence, we found distinct 18F-FDG-uptake (quantified as counts per second (cps)/mm2) in the outer medulla region of NxIRCTR (arrows in autoradiography) with ATN whereas this was absent in the NxIRHF group. This was statistically verified by calculating the ratio (E/OM), between the 18F-FDG-uptake in the entire organ slice (E) compared to that in the outer medulla (OM). While there is no statistical difference between uninephrectomized (Nx: 1.2±0.02) and ROCK-inhibitor treated (NxIRHF: 1.12±0.04) animals, the E/OM ratio of untreated ischemic animals (NxIRCTR: 0.88±0.06) is significantly different from both aforementioned. Values are expressed as mean ± SEM, n = 3. * p<0.05 vs. Nx & NxIRHF). Non-marked, intensively stained areas are artifacts due to renal 18F-FDG excretion.
Mentions: To estimate renal damage and infiltration (also for validation of 18F-FDG data) we evaluated renal histology (Figure 2) in addition to metabolic parameters. Subsequent to IRI (POD4), kidneys presented with signs of ATN, i.e. tubular dilation, swelling, and necrosis, in addition to intraluminal brush border debris and protein casts. Leukocyte infiltration was moderate and emphasized in the outer medulla. Corresponding to improved renal function, kidneys of NxIRHF showed reduced signs of ATN including attenuated interstitial infiltration when compared with NxIRCTR. Examination of inflamed tissue by autoradiography has shown that 18F-FDG-accumulation is correlated to the degree of inflammatory infiltration in different pathophysiological scenarios [38], [41]. Thus, we amended histological evaluation by autoradiographic assessment of mid-coronary kidney slices in order to visualize inflamed tissue areas. Exemplary measures are given in figure 2. In congruence to histological data, we found a distinct 18F-FDG-uptake in the outer medulla region of NxIRCTR with ATN whereas this was absent in NxIRHF. Both, histologic and autoradiographic evaluation of kidneys showed distinct amelioration of renal damage (less structural damage, less inflammation) in the HF-treated group.

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