Left Renal Artery Clamping Research Articles

#2339 All-trans retinoic acid enhances the efficacy of mesenchymal stem cells in acute kidney injury

Abstract Background and Aims Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic strategy for acute kidney injury (AKI), but still suffer from the drawbacks of low survival rate, low homing rate, and uncertain differentiation. In search of better therapeutic strategies, we explored all-trans retinoic acid (ATRA) pretreatment of MSCs to observe whether it could improve the therapeutic efficacy on AKI. Method We established a renal ischemia/reperfusion (I/R) injury model induced by clamping of left renal artery for 30 minutes and contralateral nephrectomy. C57BL/6 mice were randomly divided into 4 groups: sham group, IRI group, MSCs group, and ATRA-MSCs group. MSCs or ATRA-pretreated MSCs were injected via tail vein. GFR was measured by transcutaneous monitoring the clearance of FITC-sinistrin. These mice were sacrificed 3 days after surgery, and the serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured. The renal pathological changes were examined by Periodic Acid-Schiff staining (PAS). The in vitro hypoxia/reoxygenation (H/R) model was established using human proximal tubular epithelial cells (HK-2 cells). Co-culturing HK-2 cells with MSCs or ATRA-pretreated MSCs. Western blot, quantitative real time-PCR (qRT-PCR), and immunohistochemistry were used to detect mRNA or protein expression. We performed RNA sequencing (RNA-seq) analysis in ATRA-pretreated MSCs. The content of hyaluronic acid (HA) in the supernatant of ATRA-pretreated MSCs culture was detected by ELISA. Hyaluronan synthase mRNA expression level in ATRA-pretreated MSCs assessed by qRT-PCR analysis. The HAS2 promoter-binding transcription factors were forecasted by the JASPAR Transcription Factor Binding Site database. Western blot was used to detect whether P13K/AKT pathway was activated by ATRA-pretreated MSCs. Whether this nephroprotective effect could be reversed were explored by silencing HAS2 or CD44 antibodies. Results The AKI mice showed a significant decrease in GFR and even complete loss of renal function. Administration of MSCs significantly increased GFR, and MSCs pretreated with ATRA further increased GFR. Mice injected with ATRA-pretreated MSCs had significantly lower levels of SCr, BUN and ATN scores compared with untreated MSCs. Western blot, quantitative real-time PCR (qRT-PCR), and immunohistochemistry showed that ATRA pretreated MSCs were more effective in reducing AKI inflammation and apoptosis and promoting proliferative repair than untreated MSCs. GO enrichment analysis demonstrated that the hyaluronan biosynthetic process may be a significantly expressed. HAS2 expression and HA content of MSCS were significantly increased after the addition of ATRA. Analysis for potential transcription factor binding sites revealed that the HAS2 promoter region has multiple potential binding sites for the retinoic acid receptor. Based on the predictions from the JASPAR database, the RXR-RAR heterodimer had the highest prediction score. We then performed ChIP analysis to experimentally verify the binding of RXR-RAR heterodimer transcription factor to HAS2 promoters. We further found that ATRA-pretreated MSCs activated the PI3K/AKT signaling pathway in AKI. As expected, we found that ATRA-pretreated MSCs anti-inflammatory, anti-apoptosis, and pro-proliferation effects were substantially reversed by silencing HAS2 or blocking CD44. Conclusion ATRA promotes HA secretion from MSCs and activates the PI3K/AKT pathway, which in turn enhances the therapeutic efficacy of MSCs on AKI.

Heat Shock Protein 70 Is Involved in the Efficiency of Preconditioning with Cyclosporine A in Renal Ischemia Reperfusion Injury by Modulating Mitochondrial Functions.

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.

Value of T2 Mapping in the Dynamic Evaluation of Renal Ischemia-Reperfusion Injury

To explore the value of T2 mapping in the dynamic quantitative evaluation of renal ischemia- reperfusion injury (IRI). Forty-eight healthy New Zealand rabbits were randomly divided into IRI group (n = 40) and control group (n = 8). Rabbits in the IRI group underwent left renal artery clamping for 60 minutes. Rabbits underwent MRI examinations (T2WI and T2 mapping) before and 1, 12, 24, and 48 hours after IRI. The inter-observer and intra-observer reproducibility of the T2 values were assessed using the intraclass correlation coefficient (ICC) with 95% confidence interval (CI). Correlations between the T2 value of the renal outer medulla and injury scores were assessed by Spearman correlation analysis. The repeated measures analysis of variance was used to compare the differences in T2 values of the IRI and control group across the different time points. Both of the intra-observer (ICC = 0.97, 95% CI 0.95-0.99) and inter-observer reproducibility (ICC = 0.92, 95% CI 0.86-0.96) were excellent for T2 values. The T2 value of the renal outer medulla was moderately positive correlated with tubular epithelial edema (ρ = 0.686, p < 0.001). In IRI group, T2 values of the renal outer medulla were increase at 1 h after IRI (p = 0.001) and were decrease from 1 h to 12 h (p = 0.002). At 1 h after IRI, the T2 values of the renal outer medulla for the IRI group were higher than those for the control group (p < 0.001). T2 mapping can reflect the dynamic changes of renal parenchyma in an animal model of IRI and be used to assess the early renal IRI.

Effect of unilateral renal ischemia on the contralateral kidney assessed by Caspase 3 expression.

BackgroundStudies have demonstrated with histological analysis and Doppler flow measurement analysis that unilateral renal ischemia, which is performed in some surgeries, interfered with the contralateral kidney, identifying the phenomenon of kidney-kidney crosstalk.ObjectivesTo identify the effects on the ischemic and contralateral kidney of renal ischemia induced by two types of clamping technique by analyzing the volume of kidney cells positive for Caspase 3.MethodsSixteen pigs were divided into 2 groups, as follows: A (n = 8) – clamping of left renal artery only and AV (n = 8) – clamping of left renal artery and vein. Immunohistochemical analyses (anti Caspase 3) were conducted with biopsy specimens collected from the ischemic and contralateral kidney at 0, 30, 60, and 90 minutes of ischemia and morphometric analysis was performed, taking the mean to represent the volume of the Caspase 3 positive area (%).ResultsMorphometric analysis of specimens collected at 30, 60, and 90 minutes of ischemia showed that the mean area marked for Caspase 3 was statistically larger in the contralateral kidney than the ischemic kidney in both groups: clamped renal artery (A) and clamped renal artery and vein (AV). Comparing the ischemic and contralateral kidney, there was no statistically significant difference in the area marked for Caspase 3 between the two types of clamping.ConclusionsIn the experimental model of unilateral renal ischemia, the non-ischemic kidney exhibited cell damage, demonstrated by Caspase 3 expression. The type of hilum clamping does not appear to influence the area marked for Caspase 3.

Evaluation of renal ischemia-reperfusion injury by magnetic resonance imaging texture analysis: An experimental study.

To explore the value of MRI texture analysis in evaluating the presence and severity of early renal ischemia-reperfusion injury (IRI). Healthy New Zealand rabbits were used (IRI group, N=54; control group, N=8). Rabbits in the IRI group underwent left renal artery clamping for 60minutes. Magnetic resonance imaging was performed before and at 1, 12, 24, and 48hours after IRI. The relationship between MRI texture features and histopathology parameters was assessed using Pearson's correlation coefficients. The diagnostic performance of texture features in kidney differentiation at different time points was assessed by receiver operating characteristic curve analysis. T2 WI_S(3,-3)Inverse_Difference_Moment had the strongest correlation with brush border destruction, tubular epithelial edema, necrosis, and cast (r=0.56, -0.58, 0.62, and 0.69, respectively; all P<.001). BOLD_S(4,-4)Correlation had the strongest correlation with interstitial inflammatory cell infiltration (r=0.63, P<.001). SWI_S(4,4)Difference_Entropy had the strongest correlation with microvessel density (r=0.61, P<.001). The areas under the curve for T2 WI_S(3,-3)Inverse_Difference_Moment, SWI_S(4,4)Difference_Entropy, and BOLD_S(4,-4)Correlation in kidney differentiation before IRI and that at 1 and 12hours after reperfusion were 0.76, 0.72, and 0.70, respectively; the values before IRI and at 24 and 48hours after reperfusion were 0.84, 0.81, and 0.69, respectively. The area under the curve for T2 WI_S(3,-3)Inverse_Difference_Moment in kidney differentiation at 1 and 12hours after reperfusion and that at 24 and 48hours after reperfusion was 0.66. Magnetic resonance imaging texture analysis can be used for evaluating the presence and severity of early renal IRI.

Remote Ischemic Preconditioning Ameliorates Renal Fibrosis After Ischemia-Reperfusion Injury via Transforming Growth Factor beta1 (TGF-β1) Signalling Pathway in Rats.

BackgroundThe present study was conducted to explore the influence of remote ischemic preconditioning (RIPC) on the adjustment of renal fibrosis after ischemia-reperfusion injury (IRI).Material/MethodsMale Sprague-Dawley rats were randomly assigned to 3 groups following right-side nephrectomy: the Sham group (without renal artery clamping), the IRI group (45 min left renal artery clamping), and the RIPC group (rats were treated daily with 3 cycles of 5 min of limb ischemia and 5 min of reperfusion on 3 consecutive days before left renal artery occlusion). After 3 months of reperfusion, the renal function and the extent of tubular injury and renal fibrosis were assessed. The expressions of transforming growth factor beta1 (TGF-β1), p-Smad2, Smad2, p-Smad3, and Smad3 were also evaluated.ResultsThere was no significant difference in renal function and tubular damage among the 3 groups after 45 min of kidney ischemia followed by 3 months of reperfusion. However, an obvious increase of extracellular matrix components and α-SMA could be observed in the kidney tissues of the IRI group, and the changes were significantly ameliorated in rats treated with enhanced RIPC. Compared with the IRI group, the expression of TGF-β1 and the level of p-Smad2 and p-Smad3 were decreased after the intervention of enhanced RIPC.ConclusionsEnhanced RIPC ameliorated renal fibrosis after IRI in rats, which appears to be associated with inhibition of the TGF-β1/p-Smad2/3 signalling pathway.

A Pilot Study of Postoperative Animal Welfare as a Guidance Tool in the Development of a Kidney Autotransplantation Model With Extended Warm Ischemia.

Background.This pilot study aimed to maintain acceptable animal welfare in the development of a porcine autotransplantation model with severe and incremental renal ischemic injury, a model for usage in future intervention studies. Secondary aims were to develop and test methods to collect blood and urine without the need to restrain or use sedative and avoid transportation to optimize welfare of the pig.Methods.Kidneys from 7 female pigs were subjected to incremental durations of warm ischemia (WI) 30, 45, or 75 minutes by left renal artery and vein clamping. After static cold storage, contralateral nephrectomy was performed, and the injured graft was autotransplanted and animals observed for 14 days. Animal welfare was assessed and recorded using a structured scoring sheet before and 4 days after the kidney autotransplantation. Furthermore, blood samples were drawn daily the first week and every second day the following week using a semi-central venous catheter. An ostomy bag around the genitals was tested for urine collection. Measured glomerular filtration rate was calculated using renal clearance of chromium-51-labeled ethylenediamine tetraacetic acid on day 14.Results.None of the 7 animals died during the follow-up. The animal welfare was moderately affected when applying 75 minutes of WI (n = 2), and for that reason WI was not further increased. Pigs with lower WI had no observed welfare issues. With 75 minutes of WI peak, plasma creatinine was 1486 and 1317 µmol/L, reached on day 4. Lowest glomerular filtration rate levels were observed in the pigs with 75 minutes of WI.Conclusions.WI up to 75 minutes caused the intended severely impaired renal function without significantly compromising animal welfare. Blood and urine was collected postoperatively without sedation of the pigs or use of a metabolic cage.

Duplex scan and histologic assessment of acute renal injury in a kidney-kidney crosstalk swine experimental model

ObjectiveThe objective of this study was to identify the effect of two left renal vasculature occlusion strategies on the duplex ultrasound-assessed rheology and histology of the contralateral kidney. MethodsPigs were randomly assigned to one of two groups: left renal artery-only clamping (A group, n = 8) or left renal artery and vein clamping (AV group, n = 9). Bilateral renal parenchymal biopsy specimens were taken every 10 minutes for 90 minutes. Duplex ultrasound resistive index (RI) and pulsatility index (PI) were measured. Mixed models with normal distribution and first-order autoregressive correlation structure and generalized estimating equation models were used. Results are presented as adjusted means with standard errors, estimated proportions with standard errors, and line plots with 95% confidence intervals. ResultsRI and PI increased in the nonischemic kidney. In A group animals, RI values increased significantly (P < .01) after 30 minutes of ischemia and PI increased significantly (P < .04) from 30 to 60 minutes of ischemia. The number of histologic abnormalities was higher in A group than in AV group biopsy specimens. The percentage of lesions increased significantly after 10 minutes in A group nonischemic kidneys (P < .02) and between 50 and 80 minutes in AV group nonischemic kidneys (P < .01). ConclusionsNonischemic kidneys were acutely affected by contralateral ischemia. Their function was more adversely affected by unilateral renal artery occlusion with preserved renal vein patency (A group).

Magnetic resonance imaging evaluation of renal ischaemia-reperfusion injury in a rabbit model.

What is the central question of this study? Renal ischaemia-reperfusion injury occurs in various clinical settings. The clinical diagnosis of ischaemia-reperfusion injury is routinely based on biochemical and haematological tests, which cannot evaluate the function of a single kidney. New magnetic resonance imaging techniques to identify the pathophysiological changes in the renal outer medulla were evaluated. What is the main finding and its importance? This study demonstrated that susceptibility-weighted imaging is a feasible non-invasive tool for imaging and evaluating physipathological changes in the renal outer medulla after ischaemia-reperfusion injury. The aim was to evaluate the feasibility of susceptibility-weighted imaging (SWI) as a tool to identify the changes in the renal outer medulla (OM) in a rabbit model of renal ischaemia-reperfusion injury (IRI). New Zealand rabbits were used (control group n=10; IRI group n=40). The rabbits in the IRI group were subjected to left renal artery clamping for 60min. T2-weighted (T2WI) and SWI examinations were performed at 1, 12, 24 or 48h after reperfusion (each n=10). After the examinations, the kidneys were submitted to histological evaluation. The contrast-to-noise ratio (CNR) for the left renal OM was measured using T2WI and SWI. The T2WI and SWI scores of the integrity of the renal OM were evaluated. There were significant differences between T2WI CNRs and SWI CNRs in the control group and the IRI 1, 12 and 48h time points (all P<0.05). No significant difference was found between T2WI and SWI CNRs at IRI 24h (P>0.05). The mean SWI scores of renal OM in the IRI 1 and 12h subgroups were both significantly lower than that in the control group (all P<0.05). The only significant difference in the mean T2WI scores of renal OM was observed between the control and IRI 1h groups (P<0.05). Susceptibility-weighted imaging has a significant advantage in evaluation of healthy renal OM over conventional magnetic resonance imaging, and it is a feasible non-invasive tool for imaging and evaluating changes in the renal OM after IRI.

Glomerular Morphology and Renal Function after Warm Ischemia by Main Artery or Selective Clamping in a Porcine Model

Objective: The study aimed to evaluate the ischemic and non-ischemic areas after selective arterial occlusion by using stereological analysis of glomeruli, and to compare them with main arterial clamping and sham-operated animals. Materials and Methods: Twenty-four male pigs were used in the study. The animals were divided into 3 groups with 8 animals in each as follows: group sham, submitted to laparoscopic dissection of the renal pedicle but not submitted to ischemia; group arterial (A), submitted to left renal artery clamping; and group selective (S), submitted to left renal artery caudal branch occlusion. Groups A and S underwent 30 min of warm ischemia. Left and right kidneys were collected after 21 days and renal fragments were processed for stereological evaluation. Glomerular volume density (Vv[glom]), mean glomerular volume (MGV), and glomerular density were measured. Serum creatinine and urea were assessed preoperatively, 10 days after surgery, and before euthanasia. Results: There was no significant difference among groups with regard to renal function. Renal weight and volume were similar among groups. Also, no difference was observed between the groups with regard to Vv[glom], MGV, and glomerular density, both when compared to its right control or when left kidneys were compared. Conclusions: Selective arterial clamping technique was neither superior nor inferior to main artery clamping.

Involvement of the endothelin and nitric oxide systems in the pathogenesis of renal ischemic damage in an experimental diabetic model

AimsIschemic acute kidney injury (iAKI) in experimental diabetes mellitus (DM) is associated with a rapid kidney dysfunction more than in non-diabetic rats. We hypothesize that this vulnerability is due to excessive endothelin-1 (ET-1) expression along with dysregulation of nitric oxide synthase (NOS) isoforms. The aim of the present study was to assess the impact of ischemia on renal function in diabetic rats as compared with non-diabetic rats, and to investigate the involvement of ET-1 and NO systems in the susceptibility of diabetic kidney to ischemic damage. Main methodsDM was induced by Streptozotocin. iAKI was induced by clamping of left renal artery for 30min. Right intact kidney served as control. 48h following ischemia, clearance protocols were applied to assess glomerular filtration rate (GFR), urinary flow (V) and sodium excretion (UNaV) in both kidneys. The renal effects of ABT-627, ETA antagonist; A192621.1, ETB antagonist; L-NAME, NOS non-selective inhibitor; 1400W, inducible NOS (iNOS) inhibitor; and NPLA, neuronal NOS (nNOS) inhibitor, were assessed following ischemic renal injury in diabetic rats. Key findingsInduction of iAKI in diabetic and non-diabetic rats caused significant reductions in GFR, V, and UNaV, which were greater in diabetic than non-diabetic rats. While, treatment with ABT-627 decreased V and UNaV, and increased GFR, A192621.1 decreased all these parameters. L-NAME, 1400W, and NPLA improved GFR in the ischemic diabetic kidney. SignificanceExcessive vasoconstrictive effects of ET-1 via ETA and upregulation of iNOS, are partly responsible for the impaired recovery of renal function following ischemia in diabetic rats.

Effects of the anti-ulcer agents ecabet sodium, cimetidine and sucralfate on acetylsalicylic acid-induced gastric mucosal damage deteriorated by renal failure in rats.

Renal failure including post-renal transplantation increases the susceptibility of the upper gastrointestinal mucosa to injury. The aim of this study was to confirm the influence of renal failure on gastric mucosal barrier and the protective effect of various anti-ulcer agents in rats. Renal failure (RF) was induced by 45-min left renal artery clamping and right-uninephrectomy. Four days after surgery, gastric mucosal lesions were induced by intragastric administration of acetylsalicylic acid (ASA, CAS 50-78-2) (100 mg/kg). Anti-ulcer agents were given orally 30 min before ASA administration. RF induced moderate gastric mucosal damages, but significantly worsened the ASA-induced gastric lesions. Ecabet sodium (CAS 86408-72-2) and cimetidine (CAS 51481-61-9) significantly inhibited ASA-induced gastric lesions in RF rats, whereas sucralfate (CAS 54182-58-0) tended to inhibit it. ASA and all of these anti-ulcer agents had no effect on the serum creatinine and blood urea nitrogen levels increased by RF. The gastric mucosa of RF rats is more susceptible to damage induced by ASA. Ecabet and cimetidine potently inhibited gastric lesions in RF rats suggesting its utility for the gastric mucosal damage in patients with RF including post-renal transplant.

Dexmedetomidine and S(+)-ketamine in ischemia and reperfusion injury in the rat kidney

To investigate blood creatinine and renal histology in rats anesthetized with S(+)-ketamine (keta) or dexmedetomidine (dex) and submitted to kidney ischemia/reperfusion injury (IRI). Under intraperitoneal (ip) S(+)-ketamine, 20 male Wistar rats were divided into two groups (n=10): maintenance with iv S(+)-ketamine or dex (keta and dex groups), and submitted to right (R) nephrectomy and left (L) renal artery clamping for 45 min. Blood creatinine was measured before ischemia (T1) and 48h after reperfusion (T2), when L nephrectomy was performed. Histological analysis was performed in all kidneys. Blood creatinine was significantly higher at T2 in both groups, but dex group results were lower than those of keta group. Histological changes: between groups, R kidneys did not differ; there were significant high scores for vascular dilation: keta L kidneys; for vascular congestion, tubular dilation, and necrosis: L kidneys from both groups; for tubular degeneration: keta R kidneys. S(+)-ketamine plus IRI were aggressive to rat kidneys, according to histological changes, and dexmedetomidine may have not totally protected the kidneys from these injuries, despite the better results of blood creatinine.

Effect of sildenafil in renal ischemia/reperfusion injury in rats

To evaluate the effect of sildenafil, administered prior to renal ischemia/reperfusion (I/R), by scintigraphy and histopathological evaluation in rats. Twenty-four rats were divided randomly into two groups. They received 0.1 ml of 99mTechnetium-etilenodicisteine intravenous, and a baseline (initial) renal scintigraphy was performed. The rats underwent 60 minutes of ischemia by left renal artery clamping. The right kidney was not manipulated. The sildenafil group (n=12) received orally 1 mg/kg of sildenafil suspension 60 minutes before ischemia. Treatment with saline 0.9% in the control group (n=12). Half of the rats was assessed after 24 hours and half after seven days I/R, with new renal scintigraphy to study differential function. After euthanasia, kidneys were removed and subjected to histopathological examination. For statistical evaluation, Student t and Mann-Whitney tests were used. In the control group rats, the left kidneys had significant functional deficit, seven days after I/R, whose scintigraphic pattern was consistent with acute tubular necrosis, compared with the initial scintigraphy (p<0.05). Sildenafil treatment resulted in better differential function of the left kidneys 24h after reperfusion, compared with controls. Histopathologically, the left kidney of control rats (24 hours after I/R) showed a higher degree of cellular necrosis when compared with the sildenafil treated rats (p<0.05). Sildenafil had a protective effect in rat kidneys subjected to normothermic I/R, demonstrated by scintigraphy and histomorphometry.

Effects of sirolimus alone or in combination with cyclosporine A on renal ischemia/reperfusion injury

Calcineurin inhibitors exacerbate ischemic injury in transplanted kidneys, but it is not known if sirolimus protects or exacerbates the transplanted kidney from ischemic injury. We determined the effects of sirolimus alone or in combination with cyclosporin A (CsA) on oxygenated and hypoxic/reoxygenated rat proximal tubules in the following in vitro groups containing 6-9 rats per group: sirolimus (10, 50, 100, 250, 500, and 1000 nanog/mL); CsA (100 microg/mL); sirolimus (50 and 250 nanog/mL) + CsA (100 microg/mL); control; vehicle (20% ethanol). For in vivo studies, 3-week-old Wistar rats (150-250 g) were submitted to left nephrectomy and 30-min renal artery clamping. Renal function and histological evaluation were performed 24 h and 7 days after ischemia (I) in five groups: sham, I, I + SRL (3 mg x kg(-1) x day(-1), po), I + CsA (3 mg x kg(-1) x day(-1), sc), I + SRL + CsA. Sirolimus did not injure oxygenated or hypoxic/reoxygenated proximal tubules and did not potentiate the tubular toxic effects of CsA. Neither drug affected the glomerular filtration rate (GFR) at 24 h. GFR was reduced in CsA-treated rats on day 7 (0.5 +/- 0.1 mL/min) but not in rats receiving sirolimus + CsA (0.8 +/- 0.1 mL/min) despite the reduction in renal blood flow (3.9 +/- 0.5 mL/min). Acute tubular necrosis regeneration was similar for all groups. Sirolimus alone was not toxic and did not enhance hypoxia/reoxygenation injury or CsA toxicity to proximal tubules. Despite its hemodynamic effects, sirolimus protected post-ischemic kidneys against CsA toxicity.

Protective Effects of Toll-like Receptor 4 Inhibitor Eritoran on Renal Ischemia-Reperfusion Injury

Background Ischemia-reperfusion injury (I/R) has a negative effect on renal allograft survival. Using a rat model of kidney IR injury, we demonstrated inhibition of Toll-like receptor (TLR) 4 with erotoran may shed new light on I/R therapy. Methods All 44 Fisher rats were anesthetized with ethrane. Animals were randomly divided into the S group (sham, n = 11) that received only right kidney nephrectomy or the I/R group of right kidney nephrectomy and ichemia for 40 minutes by clamping of left renal artery ( n = 11). In addition, the E group (Eritoran, n = 11) and the V group (vehicle, n = 11) received eritoran (5 mg · kg −1) and vehicle pretreatment, respectively. Analysis of renal histology, function, cytokine/chemokine production, as well as animal mortality was performed in parallel groups by ribonuclease protection assay (RPA). Results At 24 hours, the creatinine value 1.49 ± 0.2 mg/dL in the eritoran group was significantly lower than untreated controls (2.17 ± 0.4 mg/dL). Histological findings showed tubular loss and morphological stutus as well as animal survival post-I/R injury compared to vehicle-treated rats; the difference between the S versus E groups was significant. Eritoran administration significantly attenuated monocyte infiltration into the kidney. RPA assays showed the following fold increase over sham normalized to that of GAPDH mRNA expression of tumor necrosis factor-α (4.67 ± 1.52 vs 1.37 ± 0.05), interleukin (IL)-1β (5.11 ± 1.17 vs 1.92 ± 0.27), IL-6 (4.20 ± 0.29 vs 1.21 ± 0.37) and monocyte chemoattractant protein-1 (8.77 ± 1.24 vs 2.57 ± 1.59). GAPDH was markedly reduced by eritoran treatment (eritoran vs vehicle group). Conclusion These data demonstrated that inhibition of TLR4 with eritoran reduced I/R-related inflammatory responses and improved the course of kidney I/R injury.

Remifentanil, Isoflurane, and Preconditioning Attenuate Renal Ischemia/Reperfusion Injury in Rats

Background The purpose of this investigation was to examine the effect of isoflurane, remifentanil, and preconditioning in renal ischemia/reperfusion injury (IRI). Methods All 52 male Wistar rats were anesthetized with isoflurane, intubated and mechanically ventilated. The animals were randomly divided into: S group (sham; n = 11) that underwent only right nephrectomy; as well as the I group of right nephrectomy and ischemia for 45 minutes by clamping of left renal artery. (n = 11); the IP (n = 9), the R (n = 10), and the RP (n = 11) groups. In addition, the R and RP animals received remifentanil (2 μg · kg −1 · min −1) during the entire experiment. The IP and RP group underwent ischemic preconditioning (IPC = three cycles of 5 minutes). Serum creatinine values were determined before and after IRI, as well as 24 hours later. In addition to an Histological study, cells from the left kidney were evaluated for apoptosis by flow cytometry (FCM). Results The Creatinine value of 0.8 ± 0.2 mg/dl in the S group was significantly lower at 24 hours than the I 3.9 ± 1.5 mg/dl; IP 2.6 ± 1.7 mg/dl; R 3.3 ± 2.8 mg/dl; or RP 1.8 ± 0.5 mg/dl groups. The RP group value was significantly lower than those of the I, IP, and R groups (p < 0.05). The S group showed less proximal tubular cell damage than the I, IP, R, and RP groups (p < 0.05). The percentages of apoptotic cells (FITC +/PI −) were: S group = 11.6 ± 6.5; I = 16.7 ± 7.3; IP = 37.0 ± 28.4; R = 11.7 ± 6.6, and RP = 8.8 ± 1.5. The difference between the IP vs RP group was significant. Similar percentages of necrotic cells (FITC +/PI +) and intact cells (FITC −/PI −) were observed among the groups. Conclusions Ischemic preconditioning showed no protective effect in the isoflurane group (IP) but when isoflurane was administered associated with remifentanil (RP), there was a beneficial effect on the kidney, as demonstrated by flow cytometry and serum creatinine values.

Renal Protection by Ischemic Preconditioning Is Associated with p50/p50 Homodimers

Background: Although recent studies indicate that renal ischemia preconditioning (IPC) protects the kidney from ischemia-reperfusion (I/R) injury, the precise protection mechanism is still unknown. It has been widely recognized that the activity of nuclear factor-ĸB (NF-ĸB) is altered upon I/R injury. However, few studies have focused on the compositional change of NF-ĸB complexes. In the current study, we observed different NF-ĸB dimers in I/R and IPC, and postulated that p50 homodimers might represent the predominant NF-ĸB activation in renal IPC. Methods: 24 male SD rats were treated with a sham operation, I/R or IPC. While the right kidney was removed in all animals, I/R was induced by left renal artery clamping for 45 min, and IPC was induced by left renal artery clamping for 2 min, reperfused for 5 min, and repeated for 3 cycles before 45 min occlusion. After 24 h of reperfusion, we assessed NF-ĸB activities, composition of NF-ĸB, and expression of IL-18. Results: Compared to the I/R group, NF-ĸB activity decreased significantly in the IPC group. Supershift assays were then performed to examine the NF-ĸB subunits in the binding complexes. In both the I/R and IPC groups, the composition of NF-ĸB contained p65 and p50. The densities of these two supershift bands were almost equivalent in the I/R group, suggesting the NF-ĸB composition was p50/p65 dimers. However, the densities of p50 bands were more than twice that of p65 in the IPC group and the levels of Bcl-3 increased in parallel in these rats, suggesting that p50/p50 homodimers might dominate the NF-ĸB activities in renal IPC. Finally, the expression of IL-18, a marker of acute kidney injury, was significantly increased in the area of tubulointerstitium in the I/R group and attenuated in the IPC group. Conclusion: In conclusion, our investigation suggests that: (1) the general activity of NF-ĸB is attenuated in IPC kidneys, and (2) the remaining activation of NF-ĸB is dominated with p50/p50 homodimer. Both of these effects might subsequently downregulate IL-18 expression, as well as provide renal protective effects of IPC. Though we focused on the phenomenological observations in this study, the detailed mechanism is still to be determined.

Protective Effect of Parenteral Vitamin E on Ischemia-reperfusion Injury of Rabbit Kidney

Objectives To evaluate the effect of parenteral vitamin E on renal ischemia-reperfusion injury. Methods Twenty German rabbits weighing 1.5-1.9 kg were selected and divided randomly into study and control groups. Each group contained 5 male and 5 female rabbits. Intravenous vitamin E was administered to the study group 5 minutes before left renal artery clamping and the same volume of normal saline was administered to the control group. Ischemia was maintained for 60 minutes. After 48 hours, nephrectomy was performed and kidneys were sent to the pathology laboratory. Histopathologic sections were evaluated by the pathologist and graded by the extent of tissue injury to normal, mild, and moderate to severe injury. Results Histopathologic evaluation of the sections revealed that in the control group 50% of the sections had signs of moderate to severe injury and 50% were categorized as mild injury, whereas 50% of the study group sections developed no sign of ischemia-reperfusion injury and 50% developed mild injury ( P = .03). No significant correlation was found between sex and the extent of cell injury ( P = .99). Conclusions Parenteral injection of vitamin E significantly protects cells against ischemia-reperfusion injury in German rabbits.

Protective effects of hydroxyethyl starch (130/0.4) against ischemia reperfusion injury of kidney: experiment with rats

To explore the protective effects of hydroxyethyl starch (HES)(130/0.4) against ischemia reperfusion injury of kidney and its impact on the expression of intercellular adhesion molecule-1 (ICAM-1). Forty SD rats underwent resection of the right kidney and clamping of the left renal artery for 60 min to establish renal ischemia reperfusion model, and then were randomly divided into 4 equal groups: Group S, undergoing infusion of normal saline 20 mg/kg 20 min before the reopening of the renal artery, Group W1, undergoing infusion of HES 5 mg/kg 20 min before the reopening of the renal artery, Group W2, undergoing infusion of HES 10 mg/kg, and Group W3, undergoing infusion of normal saline 20 mg/kg. Ten rats underwent sham operation and were used as controls (Group C). 24 h after reperfusion arterial blood samples were collected to detect 6 the levels of serum creatinine (Cr) and blood urea nitrogen (BUN), and then the rats were killed with their kidneys taken out to undergo pathological examination and calculation of Paller's score. Immunohistochemistry was used to detect the expression of intercellular adhesion molecule-1 (ICAM-1). The levels of BUN and Cr of Group S were 152 +/- 22 and 3.17 +/- 1.00 mmol/L respectively, both significantly higher than those of Group C, and the levels of BUN and Cr of Groups W2 and W3 were 99 +/- 23 and 1.82 +/- 0.86 mmol/L and 92 +/- 28 and 1.57 +/- 0.70 mmol/L respectively, all significantly lower than those of Group S (P < 0. 05 or P < 0.01). The Paller's scores of W2 and W3 were 33.6 +/- 16.6 and 29.2 +/- 12.3 respectively,both significantly lower that of Group S (43.2 +/- 15. 8, both P < 0.05). The absorption levels of ICAM-1 of Groups W2 and W3 were 182 +/- 22 and 161 +/- 25 respectively, both significantly lower than that of Group S (212 +/- 32, P < 0.01). Infusion of 10-20 ml/kg HES (130/0.4) significantly alleviates the ischemia-reperfusion injury, probably by reducing the ICAM-1 expression.