Ischemic preconditioning and kidney transplantation: in vivo nitric oxide monitoring in a rat ischemia-reperfusion experimental model

Direct nitric oxide measurement in live tissue would help us to understand its role in ischemia-reperfusion injury and its relationship to ischemic preconditioning (IP). We constructed four experimental groups of ischemia-reperfusion in the rat kidney: G1 were controls; G2, 1 hour of renal ischemia; G3 and G4: one or two 15/10 minute cycles, respectively, of IP prior to 1 hour of ischemia. Real-time in vivo nitric oxide measurements were compared with functional parameters of kidney damage at 24 hours. The peaks of nitric oxide production in the IP periods increased less in the rising curve of nitric oxide production during the 1 hour ischemia time. No improvement in the IP groups was observed based on serum creatinine levels at 24 hours.     

Endogenous nitric oxide and exogenous nitric oxide supplementation in hepatic ischemia-reperfusion injury in the rat

BACKGROUND: Although nitric oxide (NO) is thought to be beneficial in hepatic ischemia-reperfusion (I/R), the mechanisms for this effect are not well established. METHODS: To investigate the effects of endogenous NO and exogenous NO supplementation on hepatic I/R injury and their pathogenic mechanisms, serum ALT and hyaluronic acid (endothelial cell damage), and hepatic malondialdehyde and H2O2 (oxidative stress), myeloperoxidase activity (leukocyte accumulation), and endothelin (vasoconstrictor peptide opposite to NO) were determined at different reperfusion periods in untreated rats and rats receiving L-NAME, L-NAME+L-arginine, and spermine NONOate (exogenous NO donor). RESULTS: After reperfusion every parameter increased in untreated animals. Endogenous NO synthesis inhibition by L-NAME increased hepatocyte and endothelial damage as compared to untreated rats, which was reverted and even improved by the addition of L-arginine. Spermine NONOate also improved this damage. However, different mechanisms account for the beneficial effect of endogenous and exogenous NO. Oxidative stress decreased by both L-NAME and L-NAME+L-arginine, but remained unmodified by spermine NONOate. Myeloperoxidase increased by L-NAME and this effect was reverted by the addition of L-arginine, whereas no change was observed with spermine NONOate. Endothelin levels were not modified by L-NAME and L-NAME+L-arginine, but decreased with spermine NONOate. CONCLUSIONS: These results suggest that, although both endogenous and exogenous NO exert a protective role in experimental hepatic I/R injury, the mechanisms of the beneficial effect of the two sources of NO are different.     

Multiparametric monitoring of ischemia-reperfusion in rat kidney: effect of ischemic preconditioning

BACKGROUND: Microelectrode technology is a promising tool for monitoring kidney ischemia and the changes induced by its therapeutic management. Ischemic preconditioning, that is, brief ischemic periods before sustained ischemia, has been shown to protect several organs, including the kidney, from ischemia-reperfusion injury. We tested whether the effect of preconditioning could be appraised by real-time measurement of parameters representative of tissue hypoxia. METHODS: In a sample of pentobarbital-anesthetized and mechanically ventilated rats, we studied the effect of renal ischemic preconditioning (10-min ischemia and 10-min reflow interval) on subsequent ischemia-reperfusion (45 min and 60 min). Renal tissue electrical impedance, extracellular pH, and potassium concentration [K+] were measured continuously by implanted microelectrodes. RESULTS: Ischemia induced an early, rapid rise in extracellular potassium and impedance module, followed by a phase of slower increase, whereas pH decreased rapidly, reaching a plateau. Preconditioning treatment did not cause significant changes in interstitial pH and [K+] but increased ischemic tissue impedance. During reperfusion, the three variables recovered progressively; however, after a decline, electrical impedance showed a clear postischemic increase. This rise was suppressed by preconditioning. CONCLUSIONS: Real-time measurement of any of the three parameters showed capability for early detection of ischemia. In contrast with findings in myocardial tissue, preconditioning in the kidney did not increase potassium cell loss during ischemia or improve ischemic acidosis or tissue impedance. Electrical impedance increased for a second time during reperfusion, indicating the presence of a postischemic cellular edema; concealing this episode was the most noticeable effect of the preconditioning treatment.     

一氧化氮在大鼠小肠移植缺血再灌注损伤和急性排斥反应中的作用

目的 探讨一氧化氮(NO)在大鼠小肠移植缺血再灌注损伤(IRI)和急性排斥反应(AR)中作用.方法 建立同种大鼠原位小肠移植模型,采用随机数字表法将受鼠分为4组.移植对照组、左旋精氨酸(L-Arg)组、左旋硝基精氨酸甲酯(L-NAME)Ⅰ组(Ⅰ组)和L-NAMEⅡ组(Ⅱ组)受鼠于手术当天开始分别每天给予生理盐水、L-Arg 150 mg·kg-1 ·d-1、L-NAME 4和8 mg·kg-1·d-1.术后观察各组受鼠的存活时间,行HE染色观察移植小肠的组织病理学改变,采用免疫组织化学法观察移植小肠一氧化氮合酶(NOS)的活性,以及检测血糖吸收功能和血清NO浓度.结果 移植对照组、L-Arg组、Ⅰ组及Ⅱ组受鼠的存活时间分别为(11.7±1.2)d、(10.2±1.0)d、(12.3±1.5)d和(17.3±1.9)d,Ⅱ组受鼠的存活时间明显延长(P＜0.01).与移植对照组相比,L-Arg组和Ⅰ组IRI的Park评分下降,IRI减轻;Ⅱ组Park评分显著升高(P＜0.01),IRI加重,但AR明显减轻.与移植对照组相比,IRI期间,Ⅰ组iNOS染色减弱,Ⅱ组iNOS和nNOS染色均减弱;AR期间,Ⅱ组iNOS染色明显减弱.各组血清NO浓度于再灌注后30min逐渐升高.与移植对照组相比,Ⅱ组血 NO浓度的升高延缓.与移植对照组相比,L-Arg组血糖吸收值于再灌注30 min至术后3d明显增高(P＜0.01);Ⅰ组和Ⅱ组血糖吸收值术后处于较低水平.结论 NO在大鼠小肠移植IRI中起到了细胞毒和细胞保护的双重作用;在AR中加重了组织损伤.术后早期补充L-Arg可促进移植肠管对糖类的吸收.     

应用电子顺磁共振技术研究大鼠肾移植缺血再灌注过程中的NO水平及变化

目的应用电子顺磁共振(EPR)技术动态监测大鼠移植肾缺血再灌注过程中一氧化氮(NO)的产生及其作用. 方法雄性LEW大鼠75只,8～10周龄,体重200～230 g.30只作为供体,供肾0 ℃保存24 h.其余45只分3组,每组15只.第1组为对照组,麻醉后开腹,暴露30 min后关腹;第2组为单纯肾移植组,行同基因肾移植,移植肾再灌注同时切除双肾;第3组为肾移植加N-硝基-L-精氨酸甲脂(L-NAME)组,移植肾再灌注同时切除双肾,供体和受体术前2 h分别给予L-NAME 30 mg/kg.应用EPR动态测定移植肾恢复血流前及之后各时间点NO水平.测定恢复血流后24 h和120 h血肌酐、肾小球滤过率及肾组织羰基含量. 结果单纯肾移植组再灌注后15 min NO开始显著增加并持续上升,120 min达较高水平后迅速下降,到210 min恢复再灌注前水平;肾移植加L-NAME组呈类似变化趋势,但NO水平明显低于前组.L-NAME组的24 h和120 h血肌酐水平均显著高于单纯移植组(P<0.05);24 h(P<0.05)和120 h(P<0.01)肾小球滤过率均显著低于移植组.L-NAME组的24h组织羰基含量显著低于单纯移植组(P<0.05),120 h高于单纯移植组(P<0.05). 结论冷缺血移植肾再灌注过程中,NO早期增加并迅速下降,对移植肾以保护为主.应用L-NAME抑制NO后不利于移植肾功能恢复.     

一氧化氮缺乏性高血压大鼠肾脏损害的病理机制

目的研究一氧化氮(NO)缺乏性高血压大鼠肾脏损害的特点及可能的病理生理机制.方法较大剂量的一氧化氮合酶(NOS)抑制剂L-NAME造成NO缺乏性高血压大鼠模型.测定尿蛋白及尿β2微球蛋白(β2-M)排泄.8周后电子和普通光学显微镜观察肾脏病理变化.测定血液和肾组织中NO及血管紧张素Ⅱ(Ang-Ⅱ)水平.Western印迹和免疫组化分析转化生长因子β1(TGF-β1)在肾脏的表达差异.结果大鼠4周后形成稳定的NO缺乏性高血压模型,8周后收缩压明显升高(P＜O.05).与对照组相比,给予L-NAME第3～7天尿蛋白和β2-M排泄形成一早期高峰(P＜0.05),后迅速下降;4周后尿蛋白和β2-M排泄持续升高.8周时肾小球滤过膜严重受损,肾小球内细胞数明显增多,细胞外基质(ECM)明显沉积,肾小球硬化和间质纤维化指数评分增高(P＜0.01),形成肾小球硬化和间质纤维化.血液和肾组织NO较对照组明显下降,血中AngⅡ水平无明显变化,但肾脏局部AngⅡ和TGF-β1水平明显升高(P＜0.05),尤以肾髓质为甚,TGF-β1水平为对照组的近3倍.结论长期大剂量给予NOS抑制剂可造成NO缺乏性高血压肾损害模型,其机制可能与"AngⅡ-TGF-β1-ECM"轴有关,是一种较好的研究高血压性肾损害模型.     

Downregulation of neuronal nitric oxide synthase in the rat remnant kidney

Chronic renal failure is associated with disturbances in nitric oxide (NO) production. This study was conducted to determine the effect of 5/6 nephrectomy (5/6 Nx) on expression of intrarenal neuronal nitric oxide synthase (nNOS) in the rat. In normal rat kidney, nNOS protein was detected in the macula densa and in the cytoplasm and nuclei of cells of the inner medullary collecting duct by both immunofluorescence and electron microscopy. Western blot analysis revealed that 2 wk after 5/6 Nx, there were significant decreases in nNOS protein expression in renal cortex (sham: 95.42+/-15.60 versus 5/6 Nx: 47.55+/-12.78 arbitrary units, P<0.05, n = 4) and inner medulla (sham: 147.70+/-26.96 versus 5/6 Nx: 36.95+/-17.24 arbitrary units, P<0.005, n = 8). Losartan treatment was used to determine the role of angiotensin II (AngII) AT1 receptors in the inhibition of nNOS expression in 5/6 Nx. Losartan had no effect on the decreased expression of nNOS in the inner medulla, but partially increased nNOS protein expression in the cortex of 5/6 Nx rats. In contrast, in sham rats losartan significantly inhibited nNOS protein expression in the cortex (0.66+/-0.04-fold of sham values, P<0.05, n = 6) and inner medulla (0.74+/-0.12-fold of sham values, P<0.05, n = 6). nNOS mRNA was significantly decreased in cortex and inner medulla from 5/6 Nx rats, and the effects of losartan on nNOS mRNA paralleled those observed on nNOS protein expression. These data indicate that 5/6 Nx downregulates intrarenal nNOS mRNA and protein expression. In normal rats, AngII AT1 receptors exert a tonic stimulatory effect on expression of intrarenal nNOS. These findings suggest that the reduction in intrarenal nNOS expression in 5/6 Nx may play a role in contributing to hypertension and altered tubular transport responses in chronic renal failure.     

The effect of nitric oxide in testicular ischemia-reperfusion injury

This experiment was carried out to investigate the effect of endogenous nitric oxide (NO) on the ischemia-reperfusion injury of testis.Testicular ischemia was achieved by twisting the right testis and spermatic cord 1080 counter-clockwise for 30 minutes and reperfusion was allowed for 30 minutes after detorsion of 33 rats. Animals were treated with normal saline in controls just before detorsion,NG-nitro-L-arginine methyl ester (L-NAME), and L-arginine (L-arg) in others.The tissue damage was evaluated with light microscopy, malondialdehyde (MDA) level in tissue, and the blood flow measurement using ¹³³xenon (Xe) clearence technique.MDA indicator of reperfusion injury increased 25% after detorsion when only normal saline was given, L-NAME further increased MDA, L-arginin decreased MDA to control level.Conclusion: L-arginin infusion during the detorsion reduced the reperfusion injury of testis and improved the testicular blood flow after the detorsion.     

The role of nitric oxide in testicular ischemia-reperfusion injury

PURPOSE: This study was designed to determine the role of nitric oxide (NO) in the ischemia-reperfusion (I/R) injury process in testes. METHODS: Fifty prepubertal male rats were divided into 5 groups each containing 10 rats. After 4-hour torsion and 4-hour detorsion, bilateral orchiectomies were performed for measurement of tissue malondialdehyde (MDA) level and histopathologic examination. The results were compared statistically. The groups were labeled as group 1, basal values of biochemical parameters in testes; group 2 (control group), torsion plus detorsion; group 3, torsion plus N-monomethyl-L-arginine (L-NMMA) plus detorsion; group 4, torsion plus L-arginine plus detorsion; group 5, sham operation. RESULTS: The highest MDA values were determined in the L-arginin group in ipsilateral testes. Group 3 and group 4 were statistically different from control group. Histological examination showed that specimens from group 4 had a significantly (P < .05) greater histological injury than group 3, and contralateral testes showed normal testicular architecture in all groups. CONCLUSIONS: These results suggest that NO plays an important role in damaging the testis with I/R. Although inhibition of NO synthesis with L-NMMA significantly improves I/R injury in testes, enhancing NO production by providing excess of L-arginine increases such damage. In the early periods of detorsion, there is no damage to contralateral testes after unilateral testicular torsion.     

依布西林对大鼠无心跳供体肺的保护作用

目的 观察依布西林在大鼠无心跳供体(NHBD)肺保护中的作用.方法 将60只SD大白鼠随机分为A组:有心跳供体(HBD)组;B组:NHBD组;C组:NHBD+依布硒林(Ebselen)组.B组、C组供体处死后维持辅助呼吸,放置室温中30 min,再灌注低钾右旋糖苷(LPD)液.受体鼠行"原位左肺移植术".C组受体在肺移植前1 h给予Ebselen.结果 C组移植后肺顺应性为0.1740±0.0100,结扎右肺门后15、30 min动脉血氧分压分别为(93.97±5.94)、(92.30±6.57)mm-Hg,肺组织丙二醛(MDA)含量为(0.63±0.23)nmol/mg蛋白,肺组织能量代谢物总量为(821.51±29.70)mol/g,与B组比较差异均有统计学意义(P＜0.05).结论 给予受体一定浓度的Ebselen可改善NHBD肺保护作用.

Abstract：

Objective To evaluate the protective effect of ebselen on the rat lungs from non-heartbeating donors (NHBD). Methods Sixty Sprague-Dawley rats were randomly divided into 3 groups:group A, heart-beating donor; group B, NHBD with 30 min of warm ischemia time (WIT); group C, NHBD with 30 min of WIT and administration of ebselen. The donor lungs in groups B and C maintained ventilation at room temperature for 30 min after asystolia and then were flushed with LPD solution. The recipient rats underwent left lung transplantation. The recipients in group C were administered with ebselen 1 h before transplantation. Results All the recipients survived during the observation period. The pulmonary compliance of group C was 0. 1740 ±0. 0100. The PaO2 at 15 min and 30 min after the ligation of the right pulmonary hila was (93.97 ±5.94), (92. 30 ±6. 57) mmHg, respectively. Malondialdehyde (MDA) of the pulmonary tissue was (0. 63 ±0. 23) nmol/mg pro and the energy metabolism was (821.51 ±29.70)mol/g. The difference between group B and group C was significant (P ＜ 0. 05 ). Conclusion The administration of ebselen is a safe and effective treatment in the preservation of the rat lungs from NHBD.     

Changes in nitric oxide production in the rat kidney due to CaOx nephrolithiasis

AIMS: The aim of the study was to test the hypothesis that the renal nitric oxide (NO) system is involved in the animal model of nephrolithiasis by evaluating the relationship between nitric oxide synthase (NOS) and oxidative stress. METHODS: Deposition of renal calculi was induced by adding 0.75% ethylene glycol (EG) to the drinking water of male Wistar rats. After 42 days of treatment, urinary biochemistry and urinary levels of oxalate, NO metabolites (nitrate and nitrite), cGMP, and lipid peroxides, and markers for renal damage and oxidative stress in the kidney were examined. In the second part of the experiment, two diuretic stimuli (intrarenal infusion of l-arginine or saline loading) were applied to test the renal NO system response. Finally, levels of three isoforms of NOS in renal tissues were evaluated by immunostaining. RESULTS: In the EG-treated rats, increased urinary excretion of enzymes and lipid peroxides and increased nitrotyrosine levels and oxidative injury markers in the kidneys indicated that peroxynitrite formation occurred during oxidative stress, while the 24-hr urinary excretion of NO metabolites and cGMP remained unchanged. In contrast to control rats, urinary excretion and NO metabolites and cGMP excretion were unresponsive to intrarenal l-arginine infusion; in response to saline loading, an increase in these factors was seen, but the increase was only 50% of that seen in the identically treated control group. A significant decrease in eNOS expression and increase in iNOS expression were observed in the renal medulla of the EG-treated group, whereas expression of nNOS was not affected. CONCLUSIONS: Although basal renal NO production remained unchanged, excessive peroxynitrite formation in the kidney was noted in this model. A decreased response of the NOS system was noted when diuretic stimuli were applied. How the imbalance between eNOS and iNOS expression influences CaOx stone formation requires detailed evaluation.     

Differential nitric oxide synthase expression during hepatic ischemia-reperfusion

BACKGROUND: In recent years the important role of nitric oxide in hepatic ischemia-reperfusion injury has been increasingly recognised. The prevailing consensus is that reperfusion injury may be partly the result of decreased production of nitric oxide from endothelial nitric oxide synthase and excessive production of nitric oxide from the inducible isoform. We therefore undertook this study to characterize the expression of different nitric oxide synthase isoforms during hepatic reperfusion. METHODS: Male Wistar rats (n = 6) were subjected to 45 minutes of partial hepatic ischemia (left lateral and median lobes) followed by 6 hours of reperfusion. Control animals (n = 6) were subjected to sham laparotomy. The expression of endothelial and inducible nitric oxide synthase was examined using immunohistochemistry and Western blotting. Liver sections were also stained with nitrotyrosine antibody, a specific marker of protein damage induced by peroxynitrite (a highly reactive free radical formed from nitric oxide). RESULTS: Liver sections from all the control animals showed normal expression of the endothelial isoform and no expression of inducible nitric oxide synthase. Livers from all the animals subjected to hepatic ischemia showed decreased expression of endothelial nitric oxide synthase, and all but one animal from this group showed expression of the inducible isoform both in inflammatory cells and in hepatocytes. Western blotting confirmed these findings. Staining with the antinitrotyrosine antibody was also confined to five liver sections from animals subjected to hepatic ischemia. CONCLUSIONS: During the reperfusion period after hepatic ischemia, endothelial nitric oxide synthase is downregulated while inducible nitric oxide synthase is expressed in both hepatocytes and inflammatory cells. The presence of nitrotyrosine in livers subjected to hepatic ischemia-reperfusion suggests that the expression of inducible nitric oxide synthase plays an important role in mediating reperfusion injury in this model.     

一氧化氮对大鼠肝缺血-再灌注损伤中肝细胞凋亡的影响

目的探讨一氧化氮对肝缺血-再灌注损伤及肝细胞凋亡的影响。方法在一氧化氮合酶（NOS）增强剂和抑制剂条件下,观察大鼠肝缺血-再灌注后1、3、6、24h肝的血清门冬氨酸氨基转移酶（AST）和丙氨酸氨基转移酶（ALT）变化,同时进行肝细胞胞浆诱导型一氧化氮合酶（iNOS）表达及肝细胞凋亡的免疫组织化学分析。结果在再灌注6h点AST、ALT、肝细胞凋亡水平上,L-精氨酸（L-arg）组值分别为（341.88±111.24）IU/L、（311.75±139.41）IU/L和2.80±1.79,显著低于L-硝基精氨酸甲酯（L-NAME）组（2080.88±241.98）IU/L、（1153.00±110.92）IU/L和5.50±0.71（P值均＜0.05）。而L-arg组的肝细胞内iNOS表达灰度值152.07±3.46显著高于L-NAME组灰度值180.45±4.46（P＜0.05）。结论一氧化氮可减少肝缺血-再灌注损伤后肝酶的释放,抑制肝细胞的凋亡,改善肝缺血-再灌注损伤。     

Oxidative stress and nitric oxide in kidney function

PURPOSE OF REVIEW: Nitric oxide is a potent, endogenous vasodilator that regulates systemic blood pressure and renal function, among other functions. The bioactivity of nitric oxide is reduced by superoxide, a major reactive oxygen species. Overproduction of superoxide and other related reactive oxygen species resulting in oxidative stress reduces the biological effects of nitric oxide. Though both of these highly reactive species have distinct roles in other pathways, their interaction is emerging as a major regulatory factor in normal and pathological renal function. The purpose of this review is to highlight the recent studies on oxidative stress and nitric oxide in the kidney, focusing on their interaction in normal and pathological conditions. RECENT FINDINGS: Studies have focused on pro-oxidant pathways and nitric oxide defense systems in normal and pathological conditions. The oxidant potential of uncoupled nitric oxide synthases is gaining interest as a pro-oxidant system. Both animal and clinical studies have attempted to identify strategies to intervene at various stages of the oxidant-nitric oxide pathways to improve function during renal failure. SUMMARY: Several new approaches and provocative findings have emerged over the last year. A regulatory role for nitric oxide in the control of the renal microcirculation and as a participant in tubule function is further described. New information of the cause and possible prevention of acute and chronic renal failure has also been produced in the last year. These advances demonstrate the value of research in the normal and pathological roles of oxidative stress and nitric oxide in the kidney.     

Distribution of postsynaptic density proteins in rat kidney: relationship to neuronal nitric oxide synthase

BACKGROUND: Neuronal nitric oxide synthase (nNOS) is expressed in skeletal muscle beneath the sarcolemma associated with dystrophin complex. In brain, nNOS is anchored to synaptic membranes by specific postsynaptic density proteins (PSD)-95 and PSD-93. We have investigated the cellular and subcellular localization of these PSD proteins in the kidney and their relationship to nNOS and the cell membrane. METHODS: Kidneys from male Sprague-Dawley rats were processed for light and electron microscopic immunohistochemistry with polyclonal antibodies against PSD and nNOS proteins. RESULTS: Western blot analysis of rat kidney revealed a specific band for PSD-93 at the molecular weight of 103 kDa. Immunostaining for PSD-93 was located in the thick ascending limb of the loop of Henle, macula densa cells, distal convoluted tubules, cortical collecting ducts, outer and inner medullary collecting duct, glomerular epithelium, and Bowman's capsule. A pre-embedding electron microscopic immunoperoxidase procedure localized PSD-93 to the basolateral membrane of these tubular cells. Using different sized immunogold particles, a portion of nNOS in the macula densa colocalized with PSD-93 adjacent to cytoplasmic vesicles and the basolateral membrane. In contrast, PSD-95 protein was detected only weakly in the cortex by Western blot. Immunostaining for PSD-95 was located only faintly in the apical membrane of the thick ascending limb, macula densa, distal convoluted tubule and cortical collecting duct cells. CONCLUSION: PSD-93 is the predominant PSD expressed in the rat kidney. It is located primarily in the basolateral membranes of distal nephron and colocalizes with a pool of nNOS in cytoplasmic vesicles and basolateral membranes of macula densa cells.