Expression of nitric oxide and inducible nitric oxide synthase in acute renal allograft rejection in the rat

BACKGROUND: Recent studies have shown that nitric oxide (NO) synthases, particularly inducible nitric oxide synthase (i-NOS), are induced in acute rejection episodes following heart, liver, pancreas and kidney allotransplantation. Furthermore, tissue and cellular injury has been demonstrated to be mediated by peroxynitrite (ONOO-), a metabolite of NO as well as a potent oxidant. However, a detailed relationship between NO, i-NOS and graft injury in transplantation remains elusive. METHODS: The present study used the following models of renal transplantation in rats: allografts (n = 5, Brown-Norway to Lewis [LEW] rats), isografts (n = 5, LEW to LEW) and allografts treated with aminoguanidine (AG), an i-NOS inhibitor (n = 5). Blood urea nitrogen (BUN), serum creatinine (SCr) and urinary and serum nitrosocompounds (NOx) were measured on days 2, 4 and 7 post-transplant. Western blot analysis of i-NOS protein expression and measurement of i-NOS activity were carried out in grafts harvested on Day 7, along with immunohistochemical and histopathological examinations. RESULTS: In the allograft group, both BUN and SCr levels increased markedly on Day 7, in parallel with a sharp increase in NOx. A band stained by anti-i-NOS antibody was detected at approximately 130 kDa, along with high levels of i-NOS activity and diffusely distributed i-NOS-positive cells (macrophages). Histologically, an acute rejection episode was confirmed (Grade 3 according to Banff classifications). In the AG group, reduced renal function and graft injury were significantly less severe than in the allograft group. CONCLUSIONS: In rat renal allograft acute rejection, markedly increased levels of serum NOx were observed, along with enhanced tissue i-NOS activity, together resulting in graft injury. AG administration suppressed the increase of serum NOx levels, with concomitant mitigation of tissue injury and renal function impairment.     

Intrahepatic expression of inducible nitric oxide synthase in acute liver allograft rejection: Evidence of modulation by corticosteroids

Nitric oxide (NO) has been proposed to have an important role in the immune response. Plasma nitrate levels increase during acute rejection and decrease after treatment with corticosteroids, but little is known about its potential cellular source. We studied inducible NO synthase (iNOS) expression in liver biopsy specimens of 12 patients with acute rejection compared with biopsy specimens from the same patients after treatment with high doses of intravenous corticosteroids. We also compared iNOS expression during acute rejection with a control group (9 patients without histological rejection). iNOS expression was assessed by immunohistochemistry. Intrahepatic iNOS expression was only observed in the cytoplasm of hepatocytes, which were diffusely distributed throughout hepatic lobules. iNOS expression could not be shown in portal tracts, inflammatory cells, or endothelial and sinusoidal lining cells. In patients with acute rejection, iNOS expression was significantly stronger than in the control group (2 ± 0.7 v 0.6 ± 0.7; P < .05). After treatment with corticosteroids, iNOS expression decreased significantly (2 ± 0.7 v 1.3 ± 0.9; P < .05). In conclusion, the findings of the present study show that during acute liver rejection, hepatocytes are the main cellular source for NO production and treatment with corticosteroids induces significant downregulation of intrahepatic iNOS expression. (Liver Transpl 2001;7:16-21.)     

Osteopontin expression in acute renal allograft rejection

BACKGROUND: Osteopontin (OPN) is a potent chemoattractant for mononuclear cells that is up-regulated in various inflammatory states of the kidney. The role of OPN and its expression in human renal allograft rejection are unknown. METHODS: We examined by immunohistochemistry and in situ hybridization, renal biopsies from patients with acute rejection (N= 22), protocol biopsies without rejection (N= 9), and perioperative donor biopsies (N= 35) for intrarenal expression of OPN, and its correlation with clinical, laboratory, and histopathologic parameters. In the rejection biopsies, interstitial monocyte/macrophage infiltration, tubulointerstitial cell proliferation/regeneration and apoptosis were investigated. RESULTS: In the majority of rejection biopsies, OPN expression by proximal tubular epithelium was widespread, and tended to be enhanced in the tubules surrounded by numerous inflammatory cells. Conversely, in patients that did not experience episodes of rejection and in donor biopsies, OPN expression by proximal tubules was nil or weak. OPN mRNA was colocalized with its translated protein in the renal tubular epithelium. OPN expression positively correlated with the degree of interstitial inflammation (P < 0.05), CD68+ monocyte infiltration (P < 0.01), Ki-67+ regenerating tubular and interstitial cells (P < 0.05 and P < 0.005, respectively), but not with terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)-positive apoptotic tubular cells. CONCLUSION: These data suggest that inducible expression of OPN in the tubular epithelium may have a pathogenic role in acute renal allograft rejection by mediating interstitial monocyte infiltration and possibly tubular regeneration.     

Nitric oxide synthase in acute alteration of nitric oxide levels after subarachnoid hemorrhage

OBJECTIVE: Subarachnoid hemorrhage (SAH) is associated with acute decreases and subsequent recovery of cerebral nitric oxide (NO) levels, but the mechanisms of these alterations are not known. In this study, we measured NO synthase (NOS) protein and kinetics to determine its involvement in the alterations of cerebral NO levels after SAH. METHODS: The endovascular rat model of SAH was used. The number of NOS-1 (neuronal) and NOS-2 (inducible)-positive cells (0-96 h) was determined by counting immunoreactive cells in 8-microm cryostat sections. The tissue content of active NOS and its kinetic parameters were studied with an enzymatic l-citrulline assay. RESULTS: The number of NOS-1-positive cells increased between 1 and 3 hours after SAH, decreased to and below control values at 6 and 72 hours after SAH, and increased to control values 96 hours after SAH. The number of NOS-2-positive cells increased 1 hour after SAH, decreased to control values at 24 hours, and increased above control values 96 hours after SAH. The Michaelis-Menten kinetic parameters (V(max), K(m), slope) of NOS remained unchanged at 10 and 90 minutes after SAH. CONCLUSION: NOS-1 and -2 proteins undergo a triphasic alteration after SAH, whereas the amount of active NOS and its kinetic parameters remain unchanged during the first 90 minutes after SAH. Depletion of NOS is not involved in the acute alterations of cerebral NO levels after SAH.     

Nitric oxide production by bronchoalveolar cells during allograft rejection in the rat.

BACKGROUNDS: We reported the increased nitric oxide (NO) level in exhaled air of rat lung transplant recipients during acute rejection (AR). The aim of this study was to determine the site and level of NO production in the rejected graft. METHODS: Rat lung transplantation was performed in isografts and allografts. RESULTS: In isografts, no AR and no significant increase in NO production was identified. In allografts, grades I-II of AR was seen on postoperative day (POD) 3 and grade III on POD 5. NO produced by BAL cells increased on both POD 3 (11.8+/-2.0 parts per billion [ppb]) and POD 5 (115.3+/-66.9 ppb). There was a highly significant correlation between the level of NO and the severity of AR (p=0.862, P<0.005). BAL cells from allografts expressed iNOS mRNA. Among them, macrophages, lymphocytes and neutrophils were immunostained for iNOS. CONCLUSION: NO produced by BAL cells was detected in the early stages of rejection. Therefore, it may serve as a sensitive indicator of AR in lung transplantation.     

Nitric oxide synthase expression after human brain contusion

OBJECTIVE: Nitric oxide is a universal mediator of biological effects in the brain, and it has been implicated in the pathophysiological processes of traumatic brain injury. Experimental studies have indicated posttraumatic up-regulation of the three different isoforms of nitric oxide synthase (NOS) (i.e., inducible NOS [iNOS], endothelial NOS, and neuronal NOS) after brain trauma. This study was undertaken to investigate the cellular sources and tissue compartments of nitric oxide produced by human patients undergoing surgical treatment for contusional brain injuries. METHODS: Contused brain tissue specimens from eight consecutive patients who underwent surgical treatment for brain contusions 3 hours to 5 days after trauma were evaluated in immunohistochemical analyses. Double-staining assays were used to define which cells produced the different isoforms. RESULTS: Increases in iNOS-positive cells were detectable within 6 hours after trauma, with a peak at 8 to 23 hours. Expression of iNOS after trauma was detected in neurons, macrophages, neutrophils, astrocytes, and oligodendrocytes. The cellular sources of iNOS differed at different times after trauma. No detectable difference in the expression of the neuronal or endothelial isoforms was observed for trauma patients, compared with control subjects. CONCLUSION: iNOS expression was up-regulated in a time-dependent manner in human brain tissue obtained from patients undergoing surgical treatment for contusional trauma. Our human data largely parallel experimental findings in rats, indicating that such trauma models are relevant for experimental studies and treatment trials.     

一氧化氮合成酶在大鼠小肠移植急性排斥反应中的作用

目的 观察神经型(nNOS)和诱导型一氧化氮合成酶(iNOS)在大鼠小肠移植急性排斥反应(AR)中作用.方法 行大鼠原位小肠移植.实验分为2组.1组:同系移植组(Lewis→Lewis,12例);2组:同种移植组(DA→Lewis,12例).观察术后生存时间.再灌注30 min、术后1、3、5、7d检测血清一氧化氮(NO)浓度;开腹行麦芽糖吸收实验;切取移植肠管,苏木素-伊红(HE)染色后光镜检查.免疫组织化学法观察移植肠nNOS和iNOS的活性.逆转录-聚合酶链反应(RT-PCR)法检测移植肠nNOS mRNA和iNOS mRNA的表达.结果 A组生存时间＞30 d.B组生存时间为(6.83±0.75)d.再灌注后A组nNOS染色与mRNA表达明显减弱,此后nNOS染色和mRNA表达分别于术后3、7d恢复正常.再灌注后A组iNOS染色与mRNA表达增强,此后逐渐减弱.与A组比较,术后3～7 d,B组nNOS染色减弱,iNOS染色增强,血清NO水平明显升高(P＜0.05),血糖吸收值显著降低(P＜0.01);术后5、7d,B组nNOS mRNA表达显著下降(P＜0.001),iNOS mRNA表达明显增强(P＜0.01).结论 在AR过程中,nNOS可能调节了iNOS的表达;nNOS的活性和表达与移植肠管的结构和吸收功能密切相关;iNOS的激活是加重组织损伤的重要因素之一.     

一氧化氮供体及一氧化氮合酶抑制剂与动脉疾病

自血管内皮衍生舒张因子 (ｅｎｄｏｔｈｅｌｉｕｍ ｄｅｒｉｖｅｄｒｅｌａｘｉｎｇｆａｃｔｏｒ,ＥＤＲＦ)的化学本质被证实是ＮＯ以来[1] ,学者们对ＮＯ进行了大量的研究 ,证实ＮＯ在许多疾病的病理生理过程中具有重要功能。在血管系统研究方面 ,以往主要集中在ＮＯ的减少与冠心病、高血压相关的研究以及较新的冠脉ＰＴＡ术后再狭窄的基因治疗的研究等方面 ,而对ＮＯ在动脉扩张病及周围动脉硬化闭塞症的作用的研究较少。本文仅就ＮＯ供体和抑制剂在动脉瘤及周围动脉硬化闭塞症中的作用及应用的研究进展作一综述。1　ＮＯ生物学简介1.1　Ｎ…     

一氧化氮供体及一氧化氮合酶抑制剂与动脉疾病

自血管内皮衍生舒张因子(endothelium-derived relaxing factor,EDRF)的化学本质被证实是NO以来[1],学者们对NO进行了大量的研究,证实NO在许多疾病的病理生理过程中具有重要功能.在血管系统研究方面,以往主要集中在NO的减少与冠心病、高血压相关的研究以及较新的冠脉PTA术后再狭窄的基因治疗的研究等方面,而对NO在动脉扩张病及周围动脉硬化闭塞症的作用的研究较少.本文仅就NO供体和抑制剂在动脉瘤及周围动脉硬化闭塞症中的作用及应用的研究进展作一综述.     

肾移植术后早期排斥反应的处理

目的：探讨。肾移植术后早期排斥反应(AR)的处理方法。方法：对14例。肾移植术后3周内发生AR的患者,应用甲基泼尼松龙(MP)治疗7例,改硫唑嘌呤(Aza)为霉酚酸脂(MMF?)治疗5例,抗CD3治疗6例。结果：应用MP治疗的7例中5例AR逆转,2例无效;改Aza为MMF治疗的5例3例逆转,2例无效(其中包括MP无效的2例改Aza为MMF治疗后1例逆转1例无效)。抗CD3治疗6例4例逆转,2例无效(其中MP治疗无效改Aza为MMF治疗后仍无效的1例逆转,MP治疗无效的基础免疫为CsA加MMF?加Pred的1例无效)。结论：MP可使大部分术后早期AR逆转。应用Aza的患者改用MMP后可使早期AR逆转,MP不能完全逆转的AR改用MMF后也有良好的效果。及时应用抗CD3几乎可使术后早期AR全部逆转。     

Steroid-resistant acute allograft rejection in renal transplantation

Steroid-resistant rejection after pediatric renal transplantation forms a rare but severe complication with a guarded prognosis particularly if this occurs late after transplantation. There is a paucity of data on how to manage these challenging rejection episodes, particularly in the pediatric literature. Mohan Shenoy et al. published a case series of 15 patients who were treated with anti-thymocyte globulin for steroid-resistant acute allograft rejection over a 15-year period in a single center in this issue of Pediatric Nephrology. While the results for the early rejection group were encouraging, the results in the eight patients with late rejection episodes after transplantation were unfavorable and afflicted with a high incidence of side-effects. Important diagnostic tools such as C4d staining of the renal transplant biopsy and the measurement of donor-specific antibodies were underutilized. The editorial reviews the importance of the differentiation between humoral and cellular rejection and the challenges of treating late antibody-mediated acute rejection in these patients. A multi-center approach is required to establish a registry of these events and ideally prospective randomized interventions should be designed to provide some evidence base for the management of this challenging complication after pediatric renal transplantation.     

Increased expression of nitric oxide synthase in human lung transplants after nitric oxide inhalation

BACKGROUND: The effects of the ischemia-reperfusion process of organ transplantation on nitric oxide (NO) synthase (NOS) in humans are unknown. The effects of NO inhalation on endogenous NOS expression and activity are controversial. The authors hypothesized that NO inhalation may affect ischemia-reperfusion-induced alterations of the endogenous NOS system. METHODS: The authors performed lung biopsy on patients in a randomized phase II clinical trial of NO inhalation during lung transplantation. After lung implantation, 20 ppm of NO or placebo gas was administered 10 min after the start of reperfusion. Lung tissues were collected from 20 patients (NO, n=9; placebo, n=11) after cold and warm ischemia, 1 hr and 2 hr after reperfusion. The protein levels of NOS isoforms were analyzed by Western blotting and the total NOS activity was measured. RESULTS: The protein levels of inducible NOS did not change significantly in either of the groups. In contrast, during the 2-hr reperfusion period, constitutive NOS (neuronal NOS [nNOS] and endothelial NOS) tended to decrease in the placebo group, but gradually increased in the NO group. After 2 hr of reperfusion, the nNOS protein in the NO group was significantly higher than that in the placebo group (P <0.05). However, the total NOS activity remained at low levels in both groups. CONCLUSIONS: NO inhalation-induced increase of constitutive NOS proteins indicates the interaction between inhaled NO molecules and lung tissues. However, the activity of these newly synthesized NOS proteins remains suppressed during the ischemia-reperfusion period of lung transplantation.     

一氧化氮(NO)合酶和NO在延迟性异种移植排斥反应中的作用

目的利用小鼠至大鼠异位心脏移植模型,研究诱导性一氧化氮合酶(iNOS)和受体血清一氧化氮(NO)在延迟性异种移植排斥反应(DXR)中的作用.方法将大鼠随机分为4组A组(6只),空白对照;B组(5只),来氟米物(Lef)+环孢素A(CsA);C组(6只),氨基胍;D组(6只),氨基胍+Lef+CsA.利用免疫组织化学染色检测CD68和NOS2,原位杂交技术检测iNOS mRNA表达.于移植前3 d和移植心脏排斥时分别采集血清检测NO含量.结果所有被排斥心脏中均见巨噬细胞(MФ)浸润,Lef+CsA显著延长移植心脏存活(与A和C组相比,P＜0.05),单用氨基胍使移植心脏存活(3 83±1.47)d(与A组比较,P＜0.05),氨基胍联用Lef和CsA使移植心脏存活(8.67±1.76)d(与A、B和C组比较,P＜0.05).发生DXR时浸润的MФ均有NOS2蛋白和mRNA阳性表达,且不受氨基胍影响.发生DXR时大鼠血清NO水平较移植前显著升高(P＜0.01),氨基胍可显著降低排斥时NO水平.结论小鼠至大鼠心脏移植发生DXR时浸润的MФ表达iNOS增多,且血清NO升高.抑制iNOS活性,降低NO水平可显著延长移植物存活时间,提示iNOS和NO是DXR发生的可能机制之一.