Alternative pathway complement activation induces proinflammatory activity in human proximal tubular epithelial cells

Background. Proximal tubular epithelial cells express a surface C3-convertase activity which induces C fixation and insertion of the C5b-9 membrane attack complex (MAC) into the cell plasma membrane. The physiopathological consequences of this phenomenon are unknown. Methods. The effect of C fixation on the production of inflammatory mediators by human proximal tubular epithelial cells in culture was explored. Results. Proximal tubular epithelial cells incubated with a sublytic amount of normal human serum as a source of C, but not with heat-inactivated human serum, showed a time-dependent calcium influx and a concomitant release of ¹⁴C-arachidonic acid (¹⁴C-AA). Eicosanoid synthesis following the arachidonic acid mobilization was studied as prostaglandin E2 release. Mg²⁺/EGTA, which did not prevent C activation by the C3-convertase, and p-bromodiphenacyl bromide, a phospholipase A2-inhibitor, inhibited mobilization of ¹⁴C-AA. These results suggest the activation of an extracellular Ca²⁺-dependent, phospholipase A2. Complement fixation was associated with the synthesis of proinflammaotry cytokines such as IL-6 and TNF-&agr;. Experiments with C6-deficient sera indicated that the release of ¹⁴C-AA and the production of cytokines were dependent on the insertion of the terminal components of complement in the plasma membrane. Indeed, the reconstitution of normal haemolytic activity of C6-deficient sera with purified C6 restored also the release of ¹⁴C-AA and the production of cytokines. Conclusion. In vitro complement activation on the proximal tubular cell surface triggers the generation of proinflammatory mediators, which may potentially contribute to the pathogenesis of tubulointerstitial injury.     

Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells.

BACKGROUND: Cellular and molecular mechanisms responsible for cisplatin-induced nephrotoxicity to renal tubular epithelial cells are not well understood. Although caspases play a critical role in the execution of the cell death pathway, their specific role in toxic injury to renal tubular epithelial cells has not been elucidated previously. METHODS: The role of caspases in cisplatin-induced injury was determined using caspase inhibitors and p35 transfected LLC-PK1 cells. The Akt/PKB phosphorylation pathway was studied for the regulation of caspase activation in these cells. RESULTS: The activation of initiator caspases-8, -9 and -2, and executioner caspase-3 began after eight hours of cisplatin treatment, thereafter markedly increased in a time (8 to 24 hours) and dose-dependent manner (0 to 200 micromol/L). Proinflammatory caspase-1 did not show cisplatin-induced activation. Inhibition of caspase-3 by over expressing cowpox virus p35 protein or alternatively by the peptide inhibitor DEVD-CHO provided marked protection against cell death and partial protection against DNA damage. We then examined the role of the Akt/PKB phosphorylation pathway in regulation of cisplatin-induced caspase activation. There was a marked induction of Akt/PKB phosphorylation in a time (0 to 8 hours) and dose-dependent (0 to 200 micromol/L) manner during the course of cisplatin injury. Cisplatin-induced Akt/PKB activation was associated with Bad phosphorylation, suggesting induction of a cell survival signal mediated by the Bcl-2 family member, Bad. Wortmannin or LY294002, two structurally dissimilar inhibitors of phosphatidylinositol 3'-kinase (PI-3 kinase), abolished both cisplatin-induced Akt phosphorylation and Bad phosphorylation, and promoted cisplatin-induced early and accelerated activation of caspase-3 and caspase-9, but not of caspase-8 and caspase-1, indicating that inhibition of the Akt/PKB phosphorylation pathway enhances the mitochondrial-dependent activation of caspases. The impact of enhanced activation of caspases by wortmannin or LY294002 was reflected on accelerated cisplatin-induced cell death. CONCLUSIONS: These studies demonstrate differential activation and role of caspases in cisplatin injury, and provide the first evidence of cisplatin-induced induction of the Akt/PKB phosphorylation pathway, inhibition of which enhances activation of caspase-3 and caspase-9.     

Cytokine cross-talk between tubular epithelial cells and interstitial immunocompetent cells

Immunostaining of renal biopsies of patients with interstitial rejection of allografts or other forms of interstitial inflammation has demonstrated the presence of activated T cells and monocytes/macrophages in the tubulointerstitial area. Cytokines that are produced by infiltrating cells are capable of activating tubular epithelial cells. In turn tubular epithelial cells can produce a wide variety of inflammatory mediators, including chemokines, which further regulate cellular influx. Interfering in this cross-talk between tubular epithelial cells and infiltrating cells might provide new options for therapeutic intervention.     

The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells.

BACKGROUND: Activation of urinary complement proteins in situ by proximal tubular epithelial cells (PTEC) may contribute to the mediation of tubulointerstitial injury in patients with significant proteinuria. However, the mechanism involved is unclear, and the role of changes in urinary pH and in the concentrations of urea or ammonia requires further clarification. METHODS: The protein fraction of urine samples from nine patients with proteinuria >1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.5-8.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition. RESULTS: Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components. CONCLUSIONS: These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a 'protected site' on their surface, rather than by the activation of C3 by convertase-like protease(s).     

肾小管上皮细胞ATP耗竭再恢复时热休克蛋白72与Paxillin 的相互作用

目的 研究肾小管上皮细胞ATP耗竭再恢复时热休克蛋白(HSP)72与桩蛋白(Paxillin)的相互作用和意义。方法 应用代谢抑制剂暂时性阻断肾小管上皮细胞ATP的生成，引起细胞内的ATP耗竭； 换用含10 mmol/L葡萄糖的DMEM培养液，使细胞内ATP再恢复；以热处理细胞或编码HSP72 RNA的腺病毒感染细胞，诱导细胞高表达HSP72。Western印迹检测HSP72水平；间接免疫荧光和Western印迹检测Paxillin在细胞内的分布变化。免疫共沉淀观察HSP72与Paxillin的相互作用。结果 肾小管上皮细胞ATP耗竭再恢复时，对照组细胞内的Paxillin由局部黏附结构区域向细胞浆内弥散分布；HSP72由细胞浆转移至细胞膜。细胞高表达HSP72后，HSP72在细胞浆和细胞膜中的蛋白含量均增加(P < 0.05)；Paxillin由细胞浆向细胞膜的转移明显减少(P < 0.05)；Paxillin在细胞内的正常分布改善；HSP72和Paxillin之间的相互作用显著增加(P < 0.05)。 结论 肾小管上皮细胞ATP耗竭再恢复时，HSP72可保持Paxillin在细胞内的正常分布特点和区域，其机制可能是HSP72增加与Paxillin的相互作用，发挥分子伴侣的功能。     

Role of ceramide synthase in oxidant injury to renal tubular epithelial cells.

Ceramide has been implicated to play an important role in the cell signaling pathway involved in apoptosis. Most studies that have used the apoptotic model of cellular injury have suggested that enhanced ceramide generation is the result of the breakdown of sphingomyelin by sphingomyelinases. However, the role of ceramide synthase in enhanced ceramide generation in response to oxidant stress has not been previously examined in any tissue. Hydrogen peroxide (H(2)O(2)) (1 mM) resulted in a rapid increase in ceramide generation (as measured by in vitro diacylglycerol kinase assay) in LLC-PK1 cells. The intracellular ceramide level was significantly increased at 5 min after exposure of cells to H(2)O(2) and thereafter continuously increased up to 60 min. H(2)O(2) also resulted in a rapid increase (within 5 min) in ceramide synthase activity (as measured by incorporation of [(14)C] from the labeled palmytoyl-CoA into dihydroceramide) in microsomes. In contrast, the exposure of cells to H(2)O(2) did not result in any significant change in sphingomyelin content or acid or neutral sphingomyelinase activity. An increase in ceramide production induced by H(2)O(2) preceded any evidence of DNA damage and cell death. The specific inhibitor of ceramide synthase, fumonisin B1 (50 microM), was able to suppress H(2)O(2)-induced ceramide generation and provided a marked protection against H(2)O(2)-induced DNA strand breaks, DNA fragmentation, and cell death. Taken together, these data provide the first evidence that H(2)O(2) is a regulator of ceramide synthase rather than sphingomyelinases and that ceramide synthase-dependent ceramide generation plays a key role in DNA damage and cell death in oxidant stress to renal tubular epithelial cells.     

Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.

Activation of alpha-smooth muscle actin-positive myofibroblast cells is a key event in the progression of chronic renal diseases that leads to end-stage renal failure. Although the origin of these myofibroblasts in the kidney remains uncertain, emerging evidence suggests that renal myofibroblasts may derive from tubular epithelial cells by a process of epithelial to mesenchymal transition. It was demonstrated that hepatocyte growth factor (HGF) exhibited a remarkable ability to block this phenotypic transition both in vitro and in vivo. HGF abrogated the alpha-smooth muscle actin expression and E-cadherin depression triggered by transforming growth factor-beta1 in tubular epithelial cells in a dose-dependent manner. HGF also blocked morphologic transformation of tubular epithelial cells and inhibited the expression and extracellular deposition of fibronectin. In a mouse model of renal fibrosis disease induced by unilateral ureteral obstruction, transforming growth factor-beta type I receptor expression was specifically increased in renal tubules, and myofibroblastically phenotypic transition of the tubules was evident in vivo. Remarkably, injections of exogenous HGF blocked myofibroblast activation and drastically prevented renal interstitial fibrosis in the obstructed kidneys. These results suggest that tubular epithelial to myofibroblast conversion may play an important role in the pathogenesis of renal fibrosis and that blocking this phenotypic transition could provide a novel therapeutic strategy for the treatment of fibrotic diseases.     

Role of autophagy in contrast media-induced renal tubular epithelial cells injury

Objective To observe the effect of contrast media on autophagy and apoptosis of renal tubular epithelial cells, evaluate the role of autophagy in contrast media-induced renal tubular epithelial cells injury. Methods NRK-52E cells were exposed to iopromide at different concentration for 1 hour or at 50 gI/L for variable incubation time. Rapamycin (1 μg/L) and 3-methyadenine (2 mmol/L) were further introduced to investigate the role of autophagy in the process. The formation of autophagy was observed by acridine orange staining and Green fluorescent protein tagged LC3 (GFP-LC3). The expression of autophagy protein LC3 and Beclin-1 was examined by Western blotting, and the apoptosis level was examined by flow cytometry and Hoechst 33342-staining. Results (1) Autophagy could be enhanced by contrast media in renal tubular epithelial cells. (2) The expression of LC3-Ⅱ/LC3-I in renal tubular epithelial cells rose at first and then dropped with the increase of iopromide stimulation time and concentration (P＜0.05). (3) Iopromide promoted renal tubular epithelial cell apoptosis in dose-and time-dependent manner (P＜0.05). (4) Co-culture with rapamycin further increased LC3-Ⅱ/LC3-I, Beclin-1 and GFP-LC3 expression, but obviously prevented iopromide-induced apoptosis of renal tubular epithelial cells (P＜0.05). On the contrary, Co-culture with 3-methyadenine reduced iopromide-induced LC3-II/LC3-I, Beclin-1 and GFP-LC3 overexpression, but aggravated the apoptosis induced by iopromide (P＜0.05). Conclusions Contrast media can induce renal tubular epithelial cells apoptosis as well as autophagy. Enhancing autophagy appropriately has a protective effect on iopromide-induced renal tubular epithelial cells apoptosis, which conforms that autophagy plays an important role in antagonizing iopromide-induced renal tubular epithelial cells injury.     

红细胞生成素抑制C3a介导的肾小管上皮细胞间充质转分化

目的 观察红细胞生成素(EPO)对补体成分C3片段C3a介导的肾小管上皮细胞间充质转分化(EMT)的影响.方法 将人近端肾小管上皮细胞(HK-2)分为6组:对照组、EPO组、TGF-β组、TGF-β+EPO组、C3a组、EPO+C3a组,分别用RT-PCR、Western印迹和细胞免疫荧光方法检测HK-2细胞α-SMA、E-cadherin、C3的mRNA和蛋白表达.结果 与对照组和EPO组比较,C3a或TGF-β干预HK2细胞后,αt-SMA mRNA和蛋白表达增强(均P＜0.05),E-cadherin mRNA和蛋白表达减少(均P＜ 0.05),补体C3 mRNA和蛋白表达增强(均P＜ 0.05);而同时给予EPO干预后,C3a或TGF-β的上述作用可被明显减弱(均P＜0.05).结论 EPO可抑制C3a介导的肾小管上皮细胞EMT.     

Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Up-regulation of tissue factor activity on human proximal tubular epithelial cells in response to Shiga toxin

BACKGROUND: The pathophysiology of hemolytic uremic syndrome (HUS) is incompletely established. Based on clinical studies demonstrating the presence of prothrombotic plasma markers in patients with HUS, we hypothesized that Shiga toxin might cause activation of the coagulation pathway by augmenting tissue factor, the major initiator of coagulation. METHODS: Human proximal tubular epithelial cells (PTECs) [human kidney-2 (HK-2 cells)] were exposed to Shiga toxin-1, and expression of tissue factor, cell detachment, protein synthesis, caspase-3 activity, and Shiga toxin-1 binding were examined. Results. HK-2 cells expressed constitutive surface tissue factor activity and increased their tissue factor expression upon exposure to Shiga toxin-1. Shiga toxin-1 bound to HK-2 cells and inhibited protein synthesis. The up-regulation of tissue factor was dose- and time-dependent and strongly correlated with cell detachment and increase in caspase-3 activity caused by Shiga toxin-1 exposure. A general caspase inhibitor simultaneously inhibited HK-2 cell detachment and tissue factor up-regulation while mutant Shiga toxin-1 neither caused cell detachment, protein synthesis inhibition, nor increase in tissue factor activity. Tissue factor activity elicited by Shiga toxin-1 was abrogated by a monoclonal antitissue factor antibody. Calphostin C, a protein kinase C (PKC) inhibitor, partially blocked tissue factor up-regulation, indicating possible involvement of PKC-dependent mechanism. CONCLUSION: These data, taken together, suggest a strong link between Shiga toxin-induced up-regulation of tissue factor activity, cytotoxicity, and apoptosis in HK-2 cells. The proximal tubule is a target of Shiga toxin in HUS, and it seems plausible that injured proximal tubular cells trigger the activation of the coagulation system, the formation of intrarenal platelet-fibrin thrombi, and the development of acute renal failure in HUS.     

Renal tubular epithelial cells injury induced by mannitol and its potential mechanism

Administration of mannitol with high dose could induce extensive isometric renal proximal tubular vacuolization and acute renal failure in clinic. We previously demonstrated that mannitol-induced human kidney tubular epithelial cell (HK-2) injury. The objective of our present work was to further study the cytotoxicity of mannitol in HK-2 cells and its potential mechanism. Cell viability was assessed by an MTT method. Cell morphological changes were observed. Furthermore, levels of malondialdehyde (MDA) and glutathione (GSH) were measured. Flow cytometry was performed to determine cell apoptosis by using Annexin V-FITC and PI. In addition, the F-actin of cells was labeled by FITC-Phalloidin for observation of cytoskeleton. The MTT assay displayed that the cell viability decreased significantly in a dose- and time-dependent manner. The morphological changes were observed, including cell membrane rapture and cell detachment. The GSH concentration in HK-2 cells decreased dramatically in mannitol treatment group, while MDA content increased significantly. The results of flow cytometry indicated that apoptotic percentages of HK-2 cells increased in 250?mmol/L mannitol treatment group. After treatment with 250?mmol/L mannitol for 48?h, HK-2 cells showed disorganization of cytoskeleton and even exhibited a totally destroyed cytoskeleton. Therefore, high dose of mannitol has a toxic effect on renal tubular epithelial cells, which might be attributed to oxidative stress, destroyed cellular cytoskeleton and subsequent cell apoptosis.     

中性粒细胞明胶酶相关脂质运载蛋白对大鼠肾脏缺血再灌注损伤肾小管上皮细胞的保护作用

目的 探讨中性粒细胞明胶酶相关脂质运载蛋白(NGAL)对大鼠缺血再灌注损伤肾脏肾小管上皮细胞凋亡的保护作用及机制.方法 建立大鼠肾脏缺血再灌注模型,雄性SD大鼠随机分为对照组、缺血再灌注模型组、NGAL组 ;HE染色观察3组大鼠肾组织病理变化 ;TUNEL法检测肾小管上皮细胞凋亡 ;实时定量PCR、Western印迹法检测凋亡蛋白fas、bcl-2的表达变化.结果 与缺血再灌注模型组比较,NGAL组肾小管上皮细胞凋亡数量显著减少[(8.6±3.4)/HP比(20.8±3.7)/HP,P＜0.05] ;NGAL组肾组织fas mRNA(2.34±0.51比6.84±2.34,P＜0.05)、fas蛋白(0.65±0.05比0.95±0.08,P＜0.05)表达显著下调,bcl-2蛋白(0.33±0.05比0.24±0.03,P＜0.05)表达显著上调,但bcl-2 mRNA表达无明显改变.结论 NGAL对大鼠缺血再灌注损伤肾小管上皮细胞有保护作用,其作用可能与减少细胞凋亡、改变凋亡蛋白的表达有关.     

转化生长因子β1诱导肾小管上皮细胞趋化因子的表达

近年来研究表明,肾间质损害的主要参与者炎性细胞在趋化因子招募下开始浸润至肾间质并进一步活化,因此趋化因子的表达与肾脏疾病,尤其是与肾小管间质病变的关系受到众多研究者的关注.