Inhibition of complement factor C5 protects against renal ischemia-reperfusion injury: inhibition of late apoptosis and inflammation

BACKGROUND: Complement has been implicated in the pathophysiology of renal ischemia-reperfusion (I/R) injury. However, the mechanism underlying complement-mediated renal I/R injury is thus far unknown. To investigate the involvement of complement in I/R injury, we studied the activation and deposition of complement in a murine model of renal I/R injury. Furthermore, we examined the effect of inhibition of complement-factor C5 on renal I/R injury. METHODS: Mice were subjected to 45 min of unilateral ischemia and subsequent contralateral nephrectomy and reperfusion for 2, 12, or 24 hr. Mice were control treated or treated with BB5.1, a monoclonal antibody that prevents cleavage of complement factor C5, thereby preventing C5a generation and formation of the membrane attack complex (MAC). RESULTS: Renal I/R induced extensive deposition of C3 early after reperfusion, whereas C6 and C9 deposition (MAC formation) occurred relatively late. I/R-induced complement deposition was mainly localized to tubular epithelium. Treatment with BB5.1 totally prevented MAC formation but also reduced C3 deposition. Inhibition of C5 strongly inhibited late inflammation, as measured by neutrophil influx and induction of the murine CXC chemokines macrophage inflammatory protein-2, KC, and lipopolysaccharide-induced CXC chemokine. Anti-C5 treatment furthermore abrogated late I/R-induced apoptosis, whereas early apoptosis was not affected. Moreover, BB5.1 treatment significantly protected against I/R-induced renal dysfunction. CONCLUSIONS: Renal I/R is followed by activation of the complement system and intrarenal deposition of C3 and MAC. Complement activation plays a crucial role in the regulation of inflammation and late apoptosis. Complement inhibition, by preventing C5 activation, abrogates late apoptosis and inflammation, being strongly protective against renal function loss.     

The many effects of complement C3- and C5-binding proteins in renal injury

The complement system is an important component of the innate immune system and a modulator of adaptive immunity. The entire complement system is focused on C3 and C5. Thus, there are proteins that activate C3 and C5, those that regulate this activation, and those that transduce the effects of C3 and C5 activation products; each can affect the kidney in renal injury. The normal kidney has the inherent capacity to protect itself from complement activation through cellular expression of decay-accelerating factor, membrane cofactor protein (in human beings), and Crry (in rodents). In addition, plasma factor H protects vascular spaces in the kidney. Although the main function of these proteins is to limit complement activation, there is now considerable evidence that they can transduce signals on engagement in immune cells. The G-protein-coupled 7-span transmembrane receptors for C3a and C5a, and the integral membrane complement receptors (CR) for C3b, iC3b, and C3dg, are expressed outside the kidney, particularly in cells of hematopoietic and immune lineage. These are important in renal injury through their infiltration of the kidney and/or by affecting kidney-directed immune responses. There is mounting evidence that intrinsic glomerular and tubular cell C3aR and C5aR expression and activation also can affect renal injury. CR1 on podocytes and the beta2 integrins CR3 and CR4 in kidney dendritic cells have functions that remain poorly defined. Cells of the kidney also have the capacity to produce and activate their own complement proteins. Thus, intrinsic renal cells express decay-accelerating factor, membrane cofactor protein, Crry, C3aR, C5aR, CR1, CR3, and CR4. These can be engaged by C3 and C5 activation products derived from systemic and local pools in renal injury. Given their capacity to provide signals that influence kidney cellular behavior, their activation can have substantial effects in renal injury. Defining these in a cell- and disease-specific fashion is an exciting challenge for future research.     

Distinct roles for C3a and C5a in complement-induced tubulointerstitial injury

To prevent injury to host tissues, complement activation is regulated by a number of plasma and membrane-associated proteins, most of which limit C3 and C5 activation. An influx of circulating C3 from a syngeneic host into donor kidneys deficient in Crry (a membrane protein that reduces C3 convertase activity) causes spontaneous complement activation, primarily in the tubulointerstitum, leading to renal failure. To determine the roles of the C3a and C5a anaphylatoxins in tubulointerstitial inflammation and fibrosis, kidneys from Crry-/-C3-/- mice were transplanted into hosts lacking the C3a and/or C5a receptor. While unrestricted complement activation in the tubules was not affected by receptor status in the transplant recipient, C3a receptor deficiency in the recipients led to significantly reduced renal leukocyte infiltration and the extent of tubulointerstitial inflammation and fibrosis, all of which led to preserved renal function. The absence of C5a receptors in recipients was not only inconsequential, but the protective effect of C3a receptor deficiency was also eliminated, suggesting distinct roles of C3a and C5a receptor signaling in this model. There was significant infiltration of the tubulointerstitum with 7/4+F4/80+CD11b+ myelomonocytic cells and Thy1.2+ T cells along injured tubules, and interstitial collagen I and III deposition, all of which were C3a receptor dependent. Thus, blockade of C3a receptor signaling is a possible treatment to reduce renal inflammation and preserve renal function associated with complement activation.     

Aging aggravates long-term renal ischemia-reperfusion injury in a rat model

Aim

Ischemia-reperfusion injury (IRI) has been considered as the major cause of acute kidney injury and can result in poor long-term graft function. Functional recovery after IRI is impaired in the elderly. In the present study, we aimed to compare kidney morphology, function, oxidative stress, inflammation, and development of renal fibrosis in young and aged rats after renal IRI.

Materials and methods

Rat models of warm renal IRI were established by clamping left pedicles for 45 min after right nephrectomy, then the clamp was removed, and kidneys were reperfused for up to 12 wk. Biochemical and histologic renal damage were assessed at 12 wk after reperfusion. The immunohistochemical staining of monocyte macrophage antigen-1 (ED-1) and transforming growth factor beta 1 (TGF-β1) and messenger RNA level of TGF-β1 in the kidney were analyzed.

Results

Renal IRI caused significant increases of malondialdehyde and 8-hydroxydeoxyguanosine levels and a decrease of superoxide dismutase activity in young and aged IRI rats; however, these changes were more obvious in the aged rats. IRI resulted in severe inflammation and tubulointerstitial fibrosis with decreased creatinine (Cr) clearance and increased histologic damage in aged rats compared with young rats. Moreover, we measured the ratio of Cr clearance between young and aged IRI rats. It demonstrated that aged IRI rats did have poor Cr clearance compared with the young IRI rats. ED-1 and TGF-β1 expression levels in the kidney were significantly higher in aged rats than in young rats after IRI.

Conclusion

Aged rats are more susceptible to IRI-induced renal failure, which may associate with the increased oxidative stress, increased histologic damage, and increased inflammation and tubulointerstitial fibrosis. Targeting oxidative stress and inflammatory response should improve the kidney recovery after IRI.     

PPARγ与肾脏缺血再灌注损伤的研究进展

过氧化物酶体增殖物激活受体(peroxisome proliferator aetivated receptor gamma,PPARγ)核转录因子超家族成员之一[1],主要参与糖脂代谢、脂肪细胞分化、机体免疫及增强胰岛素敏感性.与糖尿病、肥胖、高血压、动脉粥样硬化、炎症性疾病、休克、肿瘤、不育等疾病密切相关.     

Effects of trapidil on renal ischemia-reperfusion injury

There is increasing evidence to suggest that reactive oxygen and nitrogen species play a role in the pathogenesis of renal ischemia-reperfusion (I/R) injury. This study was designed to determine the possible protective effects of trapidil treatment against oxidative and nitrosative tissue injury of kidney induced by I/R.A renal I/R injury was induced by a left renal pedicle occlusion by ischemia for 45 minutes, followed by 1 hour of reperfusion with contralateral nephrectomy in I/R and I/R + trapidil groups. Trapidil (8 mg/kg intravenously) was administrated immediately before reperfusion phase. At the end of the reperfusion period, rats were killed. Then, renal tissue samples were taken for biochemical analysis and histopathological evaluation, and blood samples were obtained to determinate serum urea, aspartate aminotransferase (AST), and tumor necrosis factor-α (TNF-α) levels. Ischemia-reperfusion injury caused significant increases in myeloperoxidase activity and malondialdehyde and 3-nitrotyrozine levels in renal tissue and elevated serum urea, AST, and TNF-α levels. In addition, severe deterioration of renal morphology was seen in the I/R group. Trapidil treatment significantly reduced in biochemical parameters, as well as serum urea, AST, and TNF-α levels. Furthermore, renal tissue injury was markedly attenuated with trapidil treatment. These data suggest that reactive oxygen species and reactive nitrogen species play a causal role in I/R-induced renal tissue, and trapidil has a renoprotective effect against oxidative and nitrosative kidney damage.     

Carnitine as a preventive agent in experimental renal ischemia-reperfusion injury

Reactive oxygen species generated during the reperfusion of ischemic kidney, as well as any other tissue, cause lipid peroxidation damaging the cell membrane. The aim of this study was to investigate the effect of carnitine in reperfusion injury of the kidney. Male albino rabbits were subjected to unilateral renal 1-h warm ischemia followed by 15 min of reperfusion. Group I (n=9): control group received 3 cc of isotonic saline solution and group II (n=9): carnitine group received 100 mg/kg of carnitine. Blood samples were collected at the 15th min of reperfusion from the left renal vein selectively. Preischemic and post-reperfusion serum and renal tissue MDA levels were measured by thiobarbituric acid reactive substances (TBARS) spectrophotometric analysis. The preischemic serum and tissue MDA values (sham values) for groups I and II were statistically comparable (P > 0.01). Serum and tissue MDA levels were markedly elevated after 15 min of reperfusion in group I (P < 0.01), while the values remained in the baseline levels following reperfusion in group II (P > 0.01). In group I, the major histological differences observed in the reperfused kidneys were marked edema and congestion whereas glomerular and tubular cellular integrity were well preserved in group II. Pre-treatment with carnitine in solid organ transplantations, preschock states, surgical procedures that require temporary vascular clamping etc. may be helpful to minimize the reperfusion injury in the involved tissue, reducing morbidity and mortality. Received: 22 May 2000 / Accepted: 1 February 2001     

左卡尼汀减轻大鼠肾缺血再灌注损伤及其机制的研究

目的 研究左卡尼汀对大鼠肾缺血再灌注损伤(IRI)的影响及其机制.方法 将Wistar大鼠分为3组:L组大鼠制成IRI模型,于夹闭肾动静脉前5 min及松开动脉夹后30 min,分2次经尾静脉注射左卡尼汀,各500mg/kg;I组大鼠制成IRI模型,仅注射生理盐水;C组仅分离双侧肾动、静脉,注射生理盐水.分别于再灌注后3、6和24 h处死各组大鼠.处死前经下腔静脉取血,检测血清肌酐(Cr)、尿素氮( BUN)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量.获取肾组织样本,进行病理学观察;应用逆转录聚合酶链反应检测肾组织核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、γ-谷氨酰半胱氨酸合成酶(γ-GCS)的mRNA水平;蛋白质印迹法检测细胞核中Nrf2含量;免疫组织化学法检测肾组织中Nrf2的表达及定位.结果 再灌注后3h时,L组与I组血清Cr和BUN均高于C组(P＜0.01);再灌注后6h时,L组血清Cr和BUN高于C组(P＜0.01),而低于I组(P＜0.01);再灌注后24 h时,L组血清Cr和BUN仍低于I组(P＜0.05).再灌注后6和24 h时,L组与I组SOD水平低于C组(P＜0.05),MDA水平高于C组(P＜0.05).再灌注后各时间点,L组SOD水平均高于I组(P＜0.05),MDA水平均低于I组(P＜0.05).再灌注后24 h时,L组肾组织病理改变较I组轻.再灌注后6h时,I组Nrf2、HO-1、γ-GCS的mRNA相对含量均高于C组(P＜0.05),而L组各基因mRNA的相对含量高于I组(P＜0.05).L组细胞核内Nrf2的相对含量高于I组(P＜0.05).结论 左卡尼汀可减轻大鼠肾脏IRI,其机制可能与激活Nrf2-ARE通路,进而增强下游抗氧化基因的表达有关.     

雷帕霉素对肾脏缺血再灌注损伤的影响

移植肾在经历缺血再灌注损伤后可以产生不同程度的损伤,而雷帕霉素作为临床上常用的免疫抑制剂,又可对再灌注的移植肾产生多方面的影响.本文就雷帕霉素对肾脏缺血再灌注损伤的影响作一综述.     

NLRP3炎症小体与相关炎症信号通路在肾缺血-再灌注损伤中的作用

肾缺血-再灌注损伤（IRI）常见于肾移植术中,是引起急性肾衰竭的重要病理生理过程,严重影响受者预后。炎症反应在IRI的发病机制及病理过程中占据着重要地位。活化的NOD样受体蛋白3（NLRP3）炎症小体可通过介导多种促炎因子的成熟与释放,调节机体炎症反应及相关细胞功能。本文对肾IRI中的NLRP3炎症小体及其相关炎症信号通路的作用机制进行了总结,旨在为临床肾IRI的防治提供新思路。     

Tissue factor antisense oligonucleotides prevent renal ischemia-reperfusion injury

BACKGROUND: Tissue factor (TF) expression is induced on macrophages and endothelial cells during the immune response. We designed an antisense (AS) phosphorothioate oligodeoxynucleotide (ODN) to specifically inhibit the expression of rat TF to study the effects of the AS ODN on renal ischemia-reperfusion injury in the rat. METHOD: AS-1 ODN for TF was delivered intravenously to inhibit the expression of TF in endothelial cells. After 8 hr, the right kidney was harvested and the left renal artery and vein were clamped. The kidney was reperfused after 90 min of ischemia, and rats were killed at 0, 1.5, 5, 12, and 24 hr after reperfusion. TF expression was analyzed by immunohistochemical staining using monoclonal antibody. RESULTS: In the untreated ischemic group, 0 of 20 rats survived beyond day 3. However, treatment with AS-1/TF led to 12 of 20 rats surviving beyond day 4. TF was detected on distal tubular epithelial cells, endothelial cells, and blood vessels but not on necrotic and proximal tubular epithelial cells. The necrotic area extended and encompassed nearly all of the ischemic kidney within 12 hr after reperfusion. The necrotic area and the grade of TF staining were more significantly reduced in the AS-1/TF-treated group than in the control group. Furthermore, fluorescein isothiocyanate-labeled AS-1/TF was significantly intense in tubular epithelial cells 8 hr after intravenous administration. CONCLUSIONS: The results indicate that AS-1/TF inhibited the ischemia-reperfusion injury of the kidney. Microcirculatory incompetence resulting from microthrombus may cause the formation and development of necrosis.     

Decreased renal ischemia-reperfusion injury by IL-16 inactivation

T-cell-mediated renal injury is a major cause of kidney transplant rejection and renal failure; hence, understanding T-cell migration within the kidney is important for preventing renal injury. Interleukin (IL)-16 is a T-cell chemoattractant produced by leukocytes. Here we measured IL-16 expression in the kidney and its role in renal ischemia-reperfusion injury induced by different conditions in several strains of mice. IL-16 was strongly expressed in distal and proximal straight tubules of the kidney. The IL-16 precursor protein was cleaved to a chemotactic form in cultured tubular epithelial cells. Inactivation of IL-16 by antibody therapy or IL-16 deficiency prevented ischemia-reperfusion injury as shown by reduced levels of serum creatinine or blood urea nitrogen compared to control mice. Further studies indicated that fewer CD4-cells infiltrated the post-ischemic kidneys of IL-16-deficient mice and that the protective effect of IL-16 antibody treatment was lymphocyte-dependent. Our results suggest that IL-16 is a critical factor in the development of inflammation-mediated renal injury and may be a therapeutic target for prevention of ischemia-reperfusion injury of the kidney.     

Study of cyclooxygenase-2 in renal ischemia-reperfusion injury

The pathogenesis of ischemia-reperfusion (I/R) injury is known to involve cytokines and particularly surface adhesion molecules, the expression of which initiates the attachment of inflammatory cells. Cyclooxygenase (COX)-1 and COX-2 catalyze the initial key enzymatic steps in the metabolism of arachidonic acid. COX-1 is constitutively expressed in most tissues, whereas COX-2 is induced in response to proinflamamatory cytokines and stress. In this study we examined the expression of COX-1 and COX-2 in the rat after 90 minutes of warm-I/R injury. Rats were sacrificed at 0, 1.5, 3, 5, 12, and 24 hours after reperfusion. COX-2 expressions were analyzed by immunohistochemical staining, which was graded on a scale of 0 to 4. All results are presented as the mean values +/- SD. Data analyses used analysis of variance. COX-2 expression was most intense on endothelial cells at 3 and 5 hours after reperfusion. From 12 to 24 hours after reperfusion COX-2 expression on endothelial cells gradually became weaker. COX-2 expression scores were significantly higher at 1.5, 3, 5, 12, and 24 hours after reperfusion than at 0 hours. However, there were no differences in COX-1 expression after reperfusion. Several hours after the maximum of COX-2 expression the maximum renal I/R injury was observed. These results suggest a relationship between COX-2 expression and renal I/R injury.     

Hyperbaric oxygenation attenuates renal ischemia-reperfusion injury in rats

BACKGROUND: Oxygen-derived free radicals play an important role in ischemia-reperfusion injury (IR). Hyperbaric oxygenation (HO) decreases free radical production. The aim of this study was to determine the effect of HO treatment on renal ischemia-reperfusion injury in rats. METHODS: Rats were divided into four groups. All groups underwent right nephrectomy. Group I served as the control group; group II had left renal ischemia-reperfusion; group III was pretreated with HO; and group IV, ischemia-reperfusion and HO pretreatment. Tissue malondialdehyde (MDA) and glutathione (GSH) levels were measured, and histopathologic damage scored. RESULTS: HO pretreatment significantly decreased tissue MDA levels and histopathologic scores among rats with IR. There was an increased GSH in HO-pretreated rats with IR; however, the difference was not significant. CONCLUSION: HO prior to ischemia displayed a beneficial effect on renal IR by reducing oxygen radical peroxidation of lipid membranes.     

Folate receptor-targeted antioxidant therapy ameliorates renal ischemia-reperfusion injury

Antioxidant therapy can protect against ischemic injury, but the inability to selectively target the kidney would require extremely high doses to achieve effective local concentrations of drug. Here, we developed a directed therapeutic that specifically targets an antioxidant to renal proximal tubule cells via the folate receptor. Because a local increase in superoxide contributes to renal ischemic injury, we created the folate-antioxidant conjugate 4-hydroxy-Tempo (tempol)-folate to target folate receptors, which are highly expressed in the proximal tubule. Dihydroethidium high-performance liquid chromatography demonstrated that conjugated tempol retained its efficacy to scavenge superoxide in proximal tubule cells. In a mouse model of renal ischemia-reperfusion injury, tempol-folate reduced renal superoxide levels more effectively than tempol alone. Furthermore, electron spin resonance revealed the successful targeting of the tempol-folate conjugate to the kidney and other tissues expressing folate receptors. Administration of tempol-folate protected the renal function of mice after ischemia-reperfusion injury and inhibited infiltration of macrophages. In conclusion, kidney-specific targeting of an antioxidant has therapeutic potential to prevent renal ischemic injury. Conjugation of other pharmaceuticals to folate may also facilitate the development of treatments for other kidney diseases.