Propionyl-L-carnitine prevents renal function deterioration due to ischemia/reperfusion

BACKGROUND: Ischemia-reperfusion injury after organ transplantation is a major cause of delayed graft function. Prevention of post-transplant ischemia acute renal failure is still elusive. METHODS: The present study was designed to examine whether propionyl-l-carnitine, an acyl derivative of carnitine involved in fatty acid oxidation pathway and adenosine 5'-triphosphate (ATP) generation of mitochondria, prevented renal function deterioration and structural injury induced by ischemia-reperfusion in an ex vivo rat model of isolated perfused kidney (IPK) preparation and in vivo in a model of syngeneic kidney transplantation. RESULTS: In the model of ischemia (20 or 40 min)/reperfusion (90 or 70 min) in IPK, untreated kidneys showed a marked reduction of glomerular filtration rate (GFR) and renal perfusate flow (RPF) as compared to baseline, when perfusion was established by restoring effective perfusion pressure to 100 mm Hg. Exposure of kidneys to propionyl-l-carnitine before establishing the ischemia insult to tissue, largely prevented renal function impairment. Pre-exposure of ischemic kidneys to propionyl-l-carnitine largely reduced the percent of lactate dehydrogenase (LDH), a cell injury marker, released into the perfusate after reperfusion as compared to untreated ischemic kidneys. Histologic findings showed very mild post-ischemic lesions in kidneys exposed to propionyl-l-carnitine as compared to untreated ischemic kidneys. Immunohistochemical detection of 4-hydroxynonenal protein adduct, a major product of lipid peroxidation, was very low in kidney infused with propionyl-l-carnitine and exposed to ischemia/reperfusion as compared to untreated ischemic kidneys. ATP levels were not affected by propionyl-l-carnitine treatment. Renal function of kidneys exposed for four hours to cold Belzer UW solution added with propionyl-l-carnitine and transplanted to binephrectomized recipients was largely preserved as compared to untreated ischemic grafts. Propionyl-l-carnitine almost completely prevented polymorphonuclear cell graft infiltration and reduced tubular injury at 16 hours post-transplant. CONCLUSIONS: These data indicate that propionyl-l-carnitine is of value in preventing decline of renal function that occurs during ischemia-reperfusion. The beneficial effect of propionyl-l-carnitine possibly relates to lowering lipid peroxidation and free radical generation that eventually results in the preservation of tubular cell structure. The efficacy of propionyl-l-carnitine to modulate ischemia-reperfusion injury in these models opens new perspectives for preventing post-transplant delayed graft function.     

The effect of a nitric oxide donor on endogenous endothelin-1 expression in renal ischemia/reperfusion injury

The balance between nitric oxide (NO) and endothelin-1 (ET-1) production is essential to the vascular function that controls organ perfusion. Elevated ET-1 levels in the peritubular capillary network following renal transplantation may be associated with renal allograft rejection. Administration of a nitric oxide donor during the preischemic period has been shown to protect kidney against ischemia-reperfusion injury, but the mechanism underlying this therapeutic benefit remains incompletely understood. We hypothesized that early administration of the NO donor sodium nitroprusside (SNP) may suppress ET-1, thereby improving renal function in an ischemia/reperfusion injury. Sprague-Dawley rats were subjected to 60 minutes of renal warm ischemia and contralateral nephrectomy. Renal biopsies were performed prior to ischemia and reperfusion, and at 1 hour and 48 hours after reperfusion. The animals were divided into four groups: sham group without warm ischemia; early SNP group (SNP given before ischemia); late SNP group (SNP given before reperfusion); and ischemic control. ET-1 expression was assessed by semiquantitative analysis with immunohistochemical stain using ET-1 monoclonal antibody and hematoxylin-eosin staining. Serum creatinine was measured at 48 hours after reperfusion. There were significant improvements in all parameters of the early compared with the late SNP group and the ischemic control, but there was no difference between the late SNP group and the ischemic control. These data suggest that early administration of SNP in renal ischemia-reperfusion improves renal function by suppressing ET-1 expression.     

Nitrite-derived nitric oxide protects the rat kidney against ischemia/reperfusion injury in vivo: role for xanthine oxidoreductase

In normal conditions, nitric oxide (NO) is oxidized to the anion nitrite, but in hypoxia, this nitrite may be reduced back to NO by the nitrite reductase action of deoxygenated hemoglobin, acidic disproportionation, or xanthine oxidoreductase (XOR). Herein, is investigated the effects of topical sodium nitrite administration in a rat model of renal ischemia/reperfusion (I/R) injury. Rats were subjected to 60 min of bilateral renal ischemia and 6 h of reperfusion in the absence or presence of sodium nitrite (30 nmol) administered topically 1 min before reperfusion. Serum creatinine, serum aspartate aminotransferase, creatinine clearance, fractional excretion of Na(+), and plasma nitrite/nitrate concentrations were measured. The nitrite-derived NO-generating capacity of renal tissue was determined under acidic and hypoxic conditions by ozone chemiluminescence in homogenates of kidneys that were subjected to sham, ischemia-only, and I/R conditions. Nitrite significantly attenuated renal dysfunction and injury, an effect that was abolished by previous treatment of rats with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (2.5 mumol intravenously 5 min before ischemia and 50 nmol topically 6 min before reperfusion). Renal tissue homogenates produced significant amounts of NO from nitrite, an effect that was attenuated significantly by the xanthine oxidoreductase inhibitor allopurinol. Taken together, these findings demonstrate that topically administered sodium nitrite protects the rat kidney against I/R injury and dysfunction in vivo via the generation, in part, of xanthine oxidoreductase-catalyzed NO production. These observations suggest that nitrite therapy might prove beneficial in protecting kidney function and integrity during periods of I/R such as those encountered in renal transplantation.     

The effect of verapamil on renal function after warm and cold ischemia in the isolated perfused rat kidney

The potential usefulness of verapamil, a calcium channel blocker to renal allograft preservation, was investigated using the isolated perfused rat kidney. Two models of ischemic injury were used. In the first model, rat kidneys were exposed to 40 min of 37 degrees C ischemia on the perfusion circuit. Addition of verapamil in doses of 2.5, 5, and 100 microM concentration to the perfusate significantly improved inulin clearance (Cin) and total sodium absorption (TNa) in the hour of reperfusion following ischemia. Regeneration of adenosine triphosphate (ATP) and total adenine nucleotide TAN levels during reperfusion following warm ischemia were also significantly higher in verapamil-treated kidneys. In the second model, rat kidneys were flushed in situ with Collins C2 solution and stored at 0 degrees C for 8 hr. After this period of cold ischemia, they were perfused on a perfusion circuit with perfusion media. Verapamil 2.5 microM was absent from both flush and perfusate (control), or added to just the flush, both the flush and perfusate, or just the perfusate. Addition of verapamil to the flush or the flush and perfusate significantly improved Cin, urine flow rate (V) and TNa during reperfusion, compared with control. Addition of verapamil to just the perfusate did not effect Cin, TNa, or V but did significantly increase RPF. These findings suggest the verapamil may protect against organ damage occurring during both warm and cold ischemia in the absence of any systemic effects and thus may be useful for renal allograft preservation.     

不同浓度左旋卡尼汀对离体兔缺血/再灌注心脏功能的保护作用

目的 观察不同浓度左旋卡尼汀(L-carnitine,L-CN)预处理对高钾停跳离体兔缺血/再灌注心脏功能的保护作用.方法 采用离体兔心Langendorff灌注实验模型,离体兔心24只随机等分成缺血/再灌注组、L-CN 2.5 mmol/L、L-CN 5.0 mmol/L、L-CN 10mmol/L 4组(n=6).缺血/再灌注损伤组:灌注K-H液25 min,(兔心)4℃标准St.Thomas停搏液(K~+16 mmol/L)至心脏停跳,45 min后恢复K-H液灌注20 min;不同浓度L-CN组:灌注K-H液10 min,再予不同浓度的L-CN续灌15 min,余步骤同缺血/再權注组.观察灌注过程中各组的血流动力学指标.心肌TTC染色测定缺血面积和梗死面积百分比.结果 再灌注后浓度5.0mmol/L和10.0 mmol/L的L-CN预处理的离体兔心心功能的恢复率均明显高于对照组(P＜0.05);其心肌存活面积百分比高于对照组(P＜0.05).结论 L-CN预处理对高钾停跳离体兔缺血/再灌注心脏具有较好的心功能保护作用.     

ATP-dependent K+ channels in renal ischemia reperfusion injury

ATP-dependent K+ channels (KATP) account for most of the recycling of K+ which enters the proximal tubules cell via Na, K-ATPase. In the mitochondrial membrane, opening of these channels preserves mitochondrial viability and matrix volume during ischemia. We examined KATP channel modulation in renal ischemia-reperfusion injury (IRI), using an isolated perfused rat kidney (IPRK) model, in control, IRI, IRI+200 microM diazoxide (a KATP opener), IRI + 10 microM glibenclamide (a KATP blocker) and IRI + 200 microM diazoxide + 10 microM glibenclamide groups. IRI was induced by 2 periods of warm ischemia, followed by 45 min of reperfusion. IRI significantly decreased glomerular filtration rate (GFR) and increased fractional excretion of sodium (FENa) (p < 0.01). Neither diazoxide nor glibenclamide had an effect on control kidney function other than an increase in renal vascular resistance produced by glibenclamide. Pretreatment with 200 microM diazoxide reduced the postischemic increase in FENa (p < 0.05). Adding 10 microM glibenclamide inhibited the diazoxide effect on postischemic FENa (p < 0.01). Histology showed that kidneys pretreated with glibenclamide demonstrated an increase in injury in the thick ascending limb of outer medulla (p < 0.05). Glibenclamide significantly decreased post ischemic renal vascular resistance (p < 0.05), but had no significant effect on other renal function parameters. Our results suggest that sodium reabsorption is improved by KATP activation and blockade of KATP channels during IRI has an injury enhancing effect on renal epithelial function and histology. This may be mediated through KATP modulation in cell and/or mitochondrial inner membrane.     

Attenuation of renal ischemia/reperfusion injury by a triple drug combination therapy

BACKGROUND: Renal ischemia is of great clinical interest because of its role in renal failure and renal graft rejection. The purpose of this study was to investigate the therapeutic effects of a combination therapy of: n-acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphormidon (P), an endothelin-1 converting enzyme inhibitor, on tissue protection against renal ischemia/reperfusion injury in the canine model. METHODS: In this study, 15-20 kg male dogs were subjected to 90 minutes of warm unilateral renal ischemia after removal of one kidney and then divided into control, ischemia alone and treatment groups. Blood samples were collected from these dogs for measurement of kidney function tests and the kidneys were harvested at different time intervals for morphological evaluation, immunostaining and Tunnel Assay. RESULTS: Kidney function tests (serum creatinine and blood urea nitrogen [BUN]) showed a significant difference between untreated and treated groups. ** P <0.01, * P< 0.0001 for treated versus untreated. The protective effect of the combination therapy is also supported by light microscopic studies, immunostaining of renal tissue sections for pro-inflammatory cytokines (TNF-alpha and IFN-gamma), iNOS, and apoptosis by TUNEL assay. CONCLUSIONS: Our results suggest that pre-administration of a combination of NAC, SNP, and P attenuates renal ischemia/reperfusion injury. This has potential application in preservation of donor kidney for transplantation by protecting cells against free radical damage.     

Renal protection by brief liver ischemia in rats

BACKGROUND: In this study, we evaluated the beneficial effect of brief ischemia and reperfusion, which was shown to have local effects on liver previously, on kidney as a remote organ in rats. METHODS: Male Wistar rats were divided into three groups: group I, sham; group II, renal ischemia for 45 min; and group III, 10 min of brief hepatic ischemia and 10 min of reperfusion after 45 min of renal ischemia. Biochemical determination, tumor necrosis factor (TNF)-alpha and tissue thiobarbituric acid-reactive substances (TBARS) levels, and histopathologic findings were evaluated at 45 min and 24 hr of reperfusion. RESULTS: Although blood urea nitrogen and creatinine levels were similar at 45 min in groups II and III, these levels were lower in group III at 24 hr. Creatine clearance values were higher and fraction excretion of sodium values were lower in group II than in group III at 24 hr. Lactate dehydrogenase levels of groups III and II were similarly elevated at 45 min, whereas group III values decreased more rapidly than those of group II at 24 hr. At 45 min of reperfusion, TNF-alpha and tissue TBARS levels were found lower in group III than in group II. Histopathologic parameters including congestion and tubular vacuolization, tubular cell detachment, and necrosis were significantly reduced in group III as compared with results of group II 45 min after ischemia. All histopathologic parameters were defined as statistically better in group II at 24 hr. CONCLUSIONS: The beneficial effect of brief ischemia of liver on renal ischemia as a remote organ was confirmed by biochemical, histopathologic, and ultrastructural findings.     

Influence of nitric oxide on microcirculation in pancreatic ischemia/reperfusion injury: an intravital microscopic study

Recently, protective effects of nitric oxide donors in pancreatic ischemia/reperfusion (IRI) injury have been described. Their role in post-ischemic microcirculation was previously not investigated. Ischemia reperfusion was induced in an isolated pancreatic tail segment in situ. Animals were randomized to four experimental groups (n=7 animals/group), the control group (CO) received saline as placebo. Treatment groups received either sodium nitroprusside (SN) 5 min before until 2 h after reperfusion, l-arginine (LA) 30 min before reperfusion until 2 h after reperfusion or sodium nitroprusside and l-arginine (SNLA) together. After induction of ischemia (2 h) post-ischemic microcirculation was observed for 2 h by intravital-fluorescence microscopy. Functional-capillary density (FCD), leukocyte adherence in post-capillary venules (LAV) and histological damage were analysed. After reperfusion FCD decreased in all groups (P<0.05). FCD was significantly restored in all groups with administration of nitric oxide donors after reperfusion (P<0.05) as compared to CO without significant difference between the individual nitric oxide donor groups. Leukocyte adherence was significantly increased 1 h and 2 h after reperfusion (P<0.001) as compared to baseline, which was lower in all nitric oxide donor groups. Histological damage in the pancreatic tail-segment was significantly reduced in nitric oxide donor groups (P<0.01). Administration of nitric oxide donors might be useful in ischemia-reperfusion injury of the pancreas by its protective effect on microcirculation and inflammatory reaction.     

DNA fragmentation reduced by antioxidants following ischaemia-reperfusion in the isolated perfused rat kidney

SUMMARY: The role of oxygen-derived free radicals (OFR) in modifying structure and function after ischaemia-reperfusion (IR) injury was studied in isolated perfused rat kidneys (IPRK). Control kidneys were studied after 20 min of ischaemia followed by 15 or 60 min of reperfusion. the xanthine oxidase inhibitor allopurinol and the hydroxyl radical scavenger dimethylthiourea (DMTU) were used to prevent OFR-related damage. Morphological injury was assessed in cortex, inner and outer medulla and compared with indices of global renal function (inulin clearance, fractional sodium excretion and renal vascular resistance). Apoptosis was assessed using both morphological criteria and in situ end-labelling (ISEL) to identify DNA fragmentation. Tubular damage, as evidenced by cellular blebbing, tubular cast formation, epithelial necrosis, and occasional apoptosis, was greatest in the straight proximal tubule and thick ascending limb (TAL) in the outer zone of the outer medulla. Pretreatment with allopurinol or DMTU did not significantly improve renal function. However, structural damage and luminal debris were diminished in allopurinol- and DMTU-treated kidneys. These changes may lead to functional improvement after more prolonged reperfusion. In situ end-labelling was more frequent in distal tubular epithelial cells after IR than either morphological evidence of apoptosis or necrosis. Decreased ISEL was observed after pretreatment with both allopurinol and DMTU. the data demonstrate that OFR produce DNA damage after IR, increasing ISEL. This probably represents reversible DNA damage rather than incipient apoptosis. Thus, antioxidants reduce or prevent DNA and cellular injury after IR and may reduce functional impairment after prolonged reperfusion.     

Intralobular heterogeneity of oxidative stress and cell death in ischemia-reperfused rat liver

The relationship between zonal oxidative stress and cell death after ischemia-reperfusion injury in rat liver was investigated. Oxidative stress was detected in situ by perfusion with nitroblue tetrazolium, which is converted to insoluble blue formazan by reducting agents. Cell death was detected in situ by perfusion with trypan blue. When isolated liver was perfused after 30 or 60 min of warm ischemia, oxidative stress was detected in periportal parenchymal cells after 30 min of reperfusion. It spread in the shape of a doughnut to midzonal cells after 60 min of reperfusion. On the other hand, cell death was observed in parenchymal cells that were within the doughnut-like area in which oxidative stress was detected. The extent of oxidative stress and cell death was higher after 60 min of ischemia than after 30 min of ischemia. In nonparenchymal cells, oxidative stress was observed in midzonal and pericentral regions after only 12 min of reperfusion, but minor cell death was observed only in periportal and midzonal regions after 30 min of reperfusion. Administration of allopurinol, an inhibitor of xanthine oxidase, suppressed oxidative stress and cell death in periportal parenchymal cells. These findings indicate that periportal and midzonal parenchymal cell death can be caused by zone-specific and xanthine-oxidase-mediated oxidative stress in parenchymal cells.     

Histological evaluation of proximal tubule cell injury in isolated perfused pig kidneys exposed to cold ischemia

BACKGROUND. Ischemic injury of the renal allograft before transplantation is a major cause of impaired graft function. Proton nuclear spectroscopy provides a useful technique for evaluating proximal tubular activity. In addition to this technique, we proposed a histological grading system for quantifying proximal tubule alterations. METHODS. The aim of this study was to evaluate the histological lesions of tubule epithelial cells in the model of isolated perfused pig kidneys following 48 to 72 h of cold storage in Euro-Collins solution. Normothermic isolated perfused pig kidneys were randomized in three experimental groups : Group 1, control group; cold flush with cold heparinized solution followed by immediate reperfusion (n = 6); Group 2, 48 h of cold storage in Euro-Collins followed by reperfusion (n = 6); Group 3, 72 h of cold storage in Euro-Collins followed by reperfusion (n = 6). Proton nuclear spectroscopy of urine and biochemical studies were performed for whole renal functional evaluation during reperfusion. Optical and electron microscopy analyses of the reperfused kidneys were performed by four investigators and the degree of cell injury was assessed using 8 different criteria in a 5-scale numerical score. RESULTS. Numerical scores corroborate the results from NMR spectroscopy and differed significantly between the three groups studied. The degree of proximal tubule cell damage was increased with prolonged cold ischemia as shown particularly in Group 3. CONCLUSION. The results from this study showed that analysis of cell injury based on an histological grading system in the model of isolated perfused kidney allows the quantification of the degree of proximal tubule injury. Thus, such morphological system analysis could be a useful method for quantifying tubule cell injuries observed under various physiopathological conditions.     

Decrease in core liver temperature with 10 degrees C by in situ hypothermic perfusion under total hepatic vascular exclusion reduces liver ischemia and reperfusion injury during partial hepatectomy in pigs

OBJECTIVE: We attempted to assess liver ischemia/reperfusion injury under a mild decrease in core liver temperature of 10 degrees C by in situ hypothermic perfusion during ischemia. METHODS: Liver ischemia was induced in pigs by total hepatic vascular exclusion with concomitant in situ perfusion with hypothermic (4 degrees C) Ringer-glucose (cold perfused group, core liver temperature maintained at 28 degrees C), with normothermic (38 degrees C) Ringer-glucose (warm perfused group) or without in situ perfusion (control group). RESULTS: In the cold perfused, warm perfused, and control groups, 24-hour survival was 5/5, 0/5, and 3/5, respectively. Hemodynamic parameters in the cold perfused group remained stable, whereas pigs in both other groups required circulatory support. Plasma AST and interleukin-6 levels were lower in the cold perfused group than in both other groups. Hepatocellular function was best preserved in the cold perfused group as indicated by complete recovery of bile production during reperfusion and no loss of indocyanine green clearance capacity. In both other groups, bile production and indocyanine green clearance capacity were reduced significantly. The hyaluronic acid uptake capacity of pigs in the cold perfused group or control group did not differ, indicating preserved sinusoidal endothelial cell function. Histopathologic injury scores during reperfusion were significantly lower in the cold perfused group when compared to both other groups. CONCLUSIONS: A mild decrease in core liver temperature of 10 degrees C by in situ hypothermic liver perfusion during ischemia protects the liver from ischemia/reperfusion injury. This protection appears to be related to cooling of the liver rather than to the washout of blood during perfusion.     

Effects of halothane,isoflurane and sevoflurane on ischemiareperfusion injury in the perfused liver of fasted rats

Background : Although intraoperative ischemia-reperfusion of the liver generally occurs under general anesthesia, little is known about the direct effect of anesthetic agents on hepatic injury due to this phenomenon. The effect of volatile anesthetics on ischemia-reperfusion injury was studied using isolated liver perfusion. Methods : The liver was isolated from 24-h-fasted male Sprague-Dawley rats and perfused through the portal vein with a modified Krebs-Ringer bicarbonate solution in a recirculating perfusion-aeration system. Ischemia was induced by reducing the baseline perfusion pressure from 1.2 to 0.2 kPa followed by reperfusion to baseline level. The ischemia-reperfusion injury was assessed by LDH release from the perfused liver. We studied the effect of halothane, isoflurane and sevoflurane on the ischemia-reperfusion injury during 20 min of control conditions, exposure of the liver to 60 min of ischemia and reperfusion for 90 min. Results : Ischemia was evident by reduced portal vein flow and oxygen consumption, and caused an increase in lactate production. Reperfusion caused a transient reduction in lactate production and a significant increase in LDH release. All anesthetics reduced hepatic oxygen consumption and increased the net lactate production during control conditions. Volatile anesthetics also significantly attenuated LDH release during reperfusion. The suppression of LDH release was observed even when isoflurane was administered during the reperfusion period, but not when it was administered only during ischemia. Conclusion : These results indicate that volatile anesthetics may protect the fasted liver from early, neutrophil-independent, ischemia-reperfusion injury by acting during the reperfusion phase.     

吗啡预处理-后处理对大鼠离体心脏缺血再灌注损伤的影响

目的 探讨吗啡预处理-后处理对大鼠离体心脏缺血再灌注损伤的影响.方法 雄性SD大鼠,体重180～200 g,应用Langendorff体外灌流装置,采用全心停灌45 min、再灌注60 min的方法制备大鼠离体心脏缺血再灌注模型.取模型制备成功的心脏40个,随机分为5组(n=8):缺血再灌注组(IR组)、吗啡预处理组(M1组)、吗啡后处理组(M2组)、吗啡预处理-后处理组(M1+M2组)、5-羟葵酸(5-HD)混合吗啡后处理组(5-HD+M2组).M1组全心停灌前30 min灌注含3.0 μmol/L吗啡的K-H液20 min,随后灌注K-H液10 min.M2组再灌注即刻灌注含3.0 μmol/L吗啡的K-H液10 min,随后灌注正常K-H液50 min.5-HD+M1组再灌注即刻灌注含3.0 μmol/L吗啡+10-4nunol/L 5-HD的K-H液10 min,随后灌注正常K-H液50 min.于再灌注60 min时,测定心肌肌酸激酶(CK-MB)活性,计算心肌梗死区与缺血危险区的比值(IS/AAR).结果 与IR组相比,其余各组IS/AAR减少,CK-MB活性降低(P<0.05);与M2组比较,5-HD+M2组CK-MB活性及IS/AAR升高(P<0.05);M1组、M2组和M1+M2组上述指标比较差异无统计学意义(P>0.05).结论 吗啡预处理.后处理虽然可减轻大鼠离体心脏缺血冉灌注损伤,但是与单独应用时效果相似,其原因可能是两者单独应用减轻心脏缺血再灌注损伤的机制均与开放线粒体ATP敏感性钾通道有关.     

Intravascular heparin protects muscle flaps from ischemia/reperfusion injury

Heparin has been found to decrease ischemia/reperfusion injury in skeletal muscle and other tissue/organ systems. The timing of heparin administration to the muscle vasculature has not been explored. We investigated the use of heparinized blood as a washout solution during ischemia to reduce ischemia/reperfusion injury. A rat cutaneous maximus muscle free flap was subjected to a 10-hr period of room temperature ischemia, then was heterotopically transplanted to the groin via microsurgical revascularization to the femoral vessels. In three experimental groups, flaps were subjected to brief ex vivo perfusion with autologous heparinized blood, at 2, 5, or 8 hr into the 10-hr ischemic interval. In the two other groups, the flaps were not perfused, and the animals were systemically heparinized either before ischemia or before transplantation, respectively. A control group underwent no flap perfusion or systemic heparinization. After transplantation, flaps were given a 48-hr period of in vivo reperfusion, then were harvested for evaluation. Flaps undergoing ex vivo perfusion or preischemic heparinization had no significant differences in weight gain (edema) compared with flaps receiving posttransplant heparinization or no heparinization (controls). The dehydrogenase staining of muscle biopsies was significantly faster (indicative of viable tissue) for perfused flaps and the flaps for which the animals received preischemic heparinization, when compared with flaps for which the animals received posttransplant heparinization or no heparinization. From these results, we conclude that heparin offers protection from ischemia/reperfusion injury when it can be introduced into the vascular network either prior to or during the ischemia period. These findings suggest the possibility of using heparinized washout solutions to enhance survival in amputated extremities. © 1995 Wiley-Liss, Inc.     

Protective effects of propofol against ischemia/reperfusion injury in rat kidneys

Aim: The purpose of this study was to investigate and compare the efficiency of propofol in the reduction of injury induced by free radicals in a rat model of renal ischemia/reperfusion (I/R). Method: Twenty-four Wistar rats were divided into four groups in our study. Rats in the sham group underwent laparotomy and were made to wait for 120 min without ischemia. Rats in the control group were given nothing with ischemia–reperfusion. Rats in the I/R groups were given propofol (25 mg/kg) and 10% intralipid (250 mg/kg) ip, respectively, 15 min before the ischemia for 60 min followed by reperfusion for 60 min. The kidney tissues of the rats were taken under anesthesia at the end of the reperfusion period. Evaluation of biochemical malondialdehyde (MDA), superoxide dismutase, and catalase activities and histopathological analysis were performed with these samples. Results: I/R significantly increased MDA levels (p < 0.05). Histopathological findings of the control group confirmed that there was renal impairment by tubular cell swelling, interstitial edema, medullary congestion, and tubular dilatation. MDA levels were lower in the propofol group compared to control group (p < 0.05). In the propofol group, the level of histopathological scores is significantly decreased than control and intralipid groups in ischemia–reperfusion. Conclusion: Our results demonstrate that I/R injury was significantly reduced in the presence of propofol. The protective effects of propofol may be due to their antioxidant properties. These results may indicate that propofol anesthesia protects against functional, biochemical, and morphological damage better than control in renal I/R injury.     

Mechanism of miR-21 in delayed ischemia preconditioning and its protection against subsequent ischemia reperfusion injury in kidney

Objective To investigate the molecular mechanism of protection of ischemia preconditioning on renal ischemia reperfusion injury. Methods Male C57/BL6N mice were randomly divided into two groups: in IR group, 35 min ischemia was induced by occlusion of both renal pedicles followed by 24 h perfusion (I/R). 15 min ischemia was induced 4 days before I/R in IPC group. Blood sample and kidney were collected in IR and IPC group after 24 h perfusion. Serum creatinine (Scr) and histological changes were used to evaluate the renal injury. PHD2 and HIF-1α were evaluated by Western blotting, miR-21 expression was confirmed by real-time PCR. In vitro, hypoxic model was established by 1% O2 in HK-2 cells. Knockdown of miR-21 in hypoxic model was perfermed by locked nucleic acid modified-anti-miR-21 transfection. The levels of miR-21, HIF-1α and PHD2 mRNA were confirmed by real-time PCR. The levels of HIF-1α and PHD2 proteins were tested by Western blotting. Results In vivo, Compared with IR group, the renal function and histological changes were improved in IPC group (P＜0.01). Compared with IR group, the expression of miR-21(P＜0.01) and HIF-1α (P＜0.05) were increased in IPC group, while PHD2 was reduced (P＜0.01). In vitro, hypoxia reduced miR-21. The inhibition of miR-21 could increased the expression of PHD2 (P＜0.05). Conclusions Ischemia preconditioning may exert protection against renal ischemia reperfusion injury by inhibiting PHD2.     

外源性硫化氢在大鼠肾脏缺血再灌注损伤中的保护作用

目的 观察不同剂量外源性硫化氢(H2S)供体硫氢化钠对大鼠肾脏缺血再灌注损伤( IRI)的保护作用.方法 健康雄性Wistar大鼠28只随机分为4组,即假手术组( Sham)、肾缺血再灌注(IR)组、硫氢化钠(NaHS)高剂量组、硫氢化钠低剂量组.大鼠右肾切除后,以NaHS作为硫化氢的供体,NaHS高、低剂量组分别经左肾动脉插管,按照1.5 μmol/min、300 nmol/min的剂量连续15 min给药,假手术组及IR组给予同体积生理盐水.停药5 min 后,NaHS组和IR组用无损伤微动脉夹夹闭左侧肾蒂45 min后解除阻断,建立大鼠急性IRI模型,假手术组不夹闭左肾动脉,其他操作同模型组.于肾脏恢复血流24h时留取血和肾组织标本,检测血清尿素氮(BUN)、血肌酐(Scr);半定量分析肾脏病理损伤;检测肾组织H2S生成率;采用实时定量PCR法检测胱硫醚-β-合成酶(CBS)、胱硫醚-γ-裂解酶(CSE )mRNA表达.结果 与假手术组相比,IR组H2S生成率显著降低(P＜0.01);CBS、CSE mRNA表达显著下降(P＜0.01 );Scr、BUN显著升高(P＜0.01);肾脏病理表现为急性肾小管坏死,且最严重.与IR组相比,NaHS预处理组H2S生成率升高(P＜0.05);CBS、CSE mRNA表达升高(P＜0.01 );Scr、BUN降低(P＜0.01);病理损伤明显减轻.NaHS两个剂量组之间差异无统计学意义.结论 外源性H2S对大鼠IRI具有保护作用.     

The effect of warm ischemic time on renal function and injury in the isolated hemoperfused kidney

BACKGROUND: The precise effect of warm ischemia on renal allograft function remains unclear and leads to variable warm ischemic time (WIT) limits advocated by transplant programs. This study aims to investigate the relationship between WIT, renal ischemia reperfusion injury, and graft function using a hemoperfused kidney model. METHODS: Porcine kidneys were perfused with normothermic blood on an isolated organ perfusion system. Kidneys were divided into four groups (n=6) and subjected to 7, 15, 25, and 40 min WIT. Physiological parameters were measured throughout the 6 hr perfusion period. Serum, tissue, and urine samples were analyzed for histological and biochemical markers of ischemia reperfusion injury. RESULTS: Creatinine clearance, urine output, renal hemodynamics, and oxygen consumption deteriorated proportionally with increasing WIT. A significant increase in plasma carbonyl levels during perfusion was seen after 25 and 40 min WIT only. Plasma 8-isoprostane levels were higher after 40 min WIT (2.5+/-1.6) vs. 7, 15, and 25 min WIT (0.65+/-0.43, 0.25+/-0.12, and 0.62+/-0.21, respectively; P<0.05). A negative correlation was shown between urine output and plasma carbonyls (r=-0.415, P<0.05) and between 8-isoprostane levels and creatinine clearance (r=-0.649, P<0.005). Caspase-3 activity was significantly higher after 7 min WIT compared with the other groups, correlating positively with creatinine clearance, urine output, and renal blood flow. CONCLUSION: The isolated organ perfusion system successfully delineated a clear WIT-dependent variation in renal function which correlated accurately with oxidative injury markers. This model may represent a clinically applicable tool for assessing graft viability.