Carbon Monoxide (CO) Protects Renal Tubular Epithelial Cells against Cold-Rewarm Apoptosis

Recent studies have suggested that carbon monoxide (CO) inhalation can reduce ischemia-reperfusion injury of kidneys. The purpose of the present study was to determine whether the direct application of CO using tricarbonylchloro (glycinato) ruthenium II (CORM3) would reduce cold-rewarm-associated apoptosis in renal tubular epithelial (RPTE) cells. RPTE cells were subjected to 48 hours of cold followed by 24 hours of rewarming with increasing concentrations (0–500 μM) of CORM3. CORM3 (100 μM) reduced apoptosis as determined by the TUNEL method from 21.6 ± 5.2 to 5.8 ± 1.1 % (untreated vs. treated, n?=?5; p < 0.001). We subsequently observed that the incubation of RPTE cells with CORM3 induced heme oxygenase (HO)-1 gene expression. As HO-1 itself can confer protection against cold rewarm injury, we investigated the role of HO-1 in the protective actions of CORM3 using siRNA oligonucleotides directed against HO-1. CORM3 treatment of RPTE cells caused a 4.9- fold increase in HO-1 gene expression as determined by real time PCR. Prior treatment of RPTE cells with siRNAs against HO-1 was able to completely abolish the CORM3 mediated induction of HO-1 mRNA and protein. The abolition of HO-induction with siRNAs did reduce CORM3-mediated protection against cold rewarm-induced apoptosis; however, CORM3 was able to significantly protect RPTE cells against cold-rewarm injury: apoptosis was 33.7 ± 0.9% vs. 15.4 ± 0.5% vs. 62.8 ± 1.5% vs. 23.5 ± 3.4 in control cold-rewarm vs. cold-rewarm + CORM3 (100 μM) vs. cold-rewarm + HO-1 siRNA vs. cold-rewarm + CORM3 (100 μM) + HO-1 siRNA (n?=?4). These results suggest that increased levels of CO alone can protect against cold-rewarm-induced apoptosis.     

肝细胞生长因子对人肾小管上皮细胞转分化抑制作用初步研究

目的：晚近研究提示肝细胞生长因子（hepatocyte growth factor,HGF)具有减轻实验性肾间质纤维化的作用，但其机制尚不十分清楚；本研究目的在于探讨HGF对人肾小管上皮细胞（HKC）转分化的作用。方法：将体外培养的HKC细胞分为：（1）无血清培养对照组；（2）阳性对照组（MCP－1＋AAⅠ）；（3）HGF组（HGF浓度为0.01,0.1,1.0,10.0ng/ml);(4)MCP-1 AAⅠ HGF组（HGF浓度0.1,1.0,10.0ng/ml)。应用间接免疫荧光法检测HKC细胞内α-平滑肌肌动蛋白（α-SMA）、波形蛋白、角蛋白表达的变化。应用流式细胞技术检测α-SMA（＋）的HKC细胞百分数。结果：不同浓度HGF分别作用于HKC细胞48h后，经间接免疫荧光法测定该组HKC细胞角蛋白、波形蛋白及α－SMA抗原表达,与无血清对照组均无显著差异；流式细胞术测得HGF组α-SMA表达阳性HKC细胞百分数，与无血清对照组（平均为3.1%)也无显著差异（P＞0.05)。MCP－1＋AAⅠ培养HKC48h后α－SMA（＋）的HKC细胞平均百分数为85.6%。MCP－1＋AAⅠ＋HGF（0.1,1.0,10.0ng/ml)培养HKC48h后，α－SMA（＋）的HKC细胞百分数分别为26.7%，2.0%,13.6%，比阳性对照组明显下降（P＜0.05)。结论：（1）HGF对正常HKC细胞分化无明显影响；（2）HGF对某些因素诱导下发生的增强的HKC细胞转化可能具有显著抑制作用。     

人近端肾小管上皮细胞黏附分子CD146表达对细胞凋亡的影响

目的：探讨人近端肾小管上皮细胞（HK-2）黏附分子CD146表达与细胞凋亡的关系。方法：体外培养的HK-2N胞在不同浓度葡萄糖和不同浓度甘露醇刺激下分别作用24h、48h、72h，经流式细胞仪（FCM）检测各组HK-2细胞CD146的表达和凋亡率的变化。结果：正常糖浓度下HK2微弱表达CD146，且延长作用时间对表达无影响；随糖浓度升高，CD146表达上调，细胞凋亡率也增高，延长刺激时间可促进CD146表达和细胞凋亡（P〈0．05）；当糖浓度达到40mmol／L，刺激时间达到48h，CD,46表达显著升高（P〈0．05），HK-2细胞凋亡率也显著上升（P〈0．05）；甘露醇对HK-2细胞CD146表达和细胞凋亡的促进作用较葡萄糖的作用显著降低（P〈0．01）。结论：HK-2组成性表达CD146，高糖诱导CD146表达，甘露醇诱导作用较高糖弱；高糖诱导HK-2凋亡，其凋亡作用与CD146表达增高联系密切，提示黏附分子CD146对细胞生存有重要影响。     

Antifibrotic Effects of Pirfenidone in Rat Proximal Tubular Epithelial Cells

Objective: Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. We previously investigated the renoprotective effects of the antifibrotic agent pirfenidone in a rat model of subtotal nephrectomy. Here, we further evaluated the antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells. Methods: NRK52E cells were incubated in a medium containing either transforming growth factor (TGF)-β1 (3 ng/mL) or platelet-derived growth factor (PDGF)-BB (5 Ang/mL) or both, with or without pirfenidone (0.1–1 mmol/L), for 24 h to assess mRNA expression, for 48 h to assess protein production, and for 1 h or various time (5–120 min) to assess phosphorylation of signal kinase. Results: TGF-β1, a key mediator in renal fibrosis, induced increases in the mRNA expression of various profibrotic factors and extracellular matrix, including plasminogen activator inhibitor type 1 (PAI-1), fibronectin, type 1 collagen, and connective tissue growth factor (CTGF)—increases which pirfenidone significantly inhibited. Specifically, pirfenidone potently inhibited TGF-β1-induced increases in the mRNA expression and protein secretion of PAI-1, an effect mediated, at least in part, via the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Further, PDGF-BB, which has been implicated in renal interstitial fibrosis, potently activated PAI-1 expression under TGF-β1 stimulation, and pirfenidone significantly inhibited TGF-β1- and PDGF-BB-induced increases in PAI-1 expression. Conclusions: Taken together, these results suggest that TGF-β1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules, such as PAI-1 and PDGF, in rat proximal tubular epithelial cells, and pirfenidone inhibits TGF-β1-induced fibrosis cascade and will therefore likely exert antifibrotic effects under pathological conditions.     

缬草油对高胆固醇血症大鼠肾小管上皮细胞巢蛋白表达的影响

目的：探讨缬草油对高胆固醇血症大鼠肾小管上皮细胞巢蛋白表达的影响。方法：大鼠随机分为正常组、高脂组、缬草油（25mg·kg-1.d-1）组和辛伐他汀（5mg·kg-1.d-1）组,用含4%胆固醇和1%胆酸钠的高脂饲料饲喂大鼠建立高脂模型。观察8、12、16周时各组大鼠血脂、尿蛋白、血肌酐和肾小管间质病理改变,免疫组化法检测肾小管上皮细胞巢蛋白和α-平滑肌肌动蛋白（α-SMA）表达。结果：随时间延长,高脂大鼠肾小管上皮细胞逐渐出现巢蛋白和α-SMA表达。巢蛋白表达与总胆固醇、低密度脂蛋白、24h尿蛋白、血肌酐正相关（r=0.963、0.830、0.944、0.706,P〈0.01）,与肾小管间质损伤指数正相关（r=0.974,P〈0.01）,与α-SMA呈正相关（r=0.804,P〈0.01）。缬草油能显著降低大鼠血总胆固醇、低密度脂蛋白、24h尿蛋白和血肌酐,下调肾小管上皮细胞巢蛋白和α-SMA表达（P〈0.01）。在巢蛋白表达减少同时,肾小管间质损伤和纤维化明显改善,其改善作用较辛伐他汀更明显（P〈0.05）。结论：缬草油可能通过降脂、下调肾小管上皮细胞巢蛋白表达减轻高胆固醇血症大鼠肾间质纤维化。     

Combined Deletion of Vhl and Kif3a Accelerates Renal Cyst Formation

A subset of familial and sporadic clear cell renal cell carcinomas (ccRCCs) is believed to develop from cystic precursor lesions. Loss of function of the von Hippel-Lindau tumor suppressor gene (VHL) predisposes renal epithelial cells to loss of the primary cilium in response to specific signals. Because the primary cilium suppresses renal cyst formation, loss of the cilium may be an initiating event in the formation of ccRCC. To test this hypothesis, we analyzed the consequences of inducible renal epithelium–specific deletion of Vhl together with ablation of the primary cilium via deletion of the kinesin family member 3A (Kif3a) gene. We developed a microcomputed tomography–based imaging approach to allow quantitative longitudinal monitoring of cystic burden, revealing that combined loss of Vhl and Kif3a shortened the latency of cyst initiation, increased the number of cysts per kidney, and increased the total cystic burden. In contrast with findings in other cystic models, cysts in Kif3a mutant mice did not display accumulation of hypoxia-inducible factor 1-α (HIF1α), and deletion of both Hif1a and Kif3a did not affect cyst development or progression. Vhl/Kif3a double mutation also increased the frequency of cysts that displayed multilayered epithelial growth, which correlated with an increased frequency of misoriented cystic epithelial cell divisions. These results argue against the involvement of HIF1α in promoting renal cyst growth and suggest that the formation of simple and atypical renal cysts that resemble ccRCC precursor lesions is greatly accelerated by the combined loss of Vhl and the primary cilium.     

1-Methylhydantoin Cytotoxicity on Renal Proximal Tubular Cells in Vitro

1-Methylhydantoin is produced by bacterial creatinine deaminase in the intestinal tract of uremic patients and retaken up into the body. The present study was designed to explore the toxic effect of 1-methylhydantoin on renal proximal tubular cells in vitro. HK-2 (Human renal proximal tubular cell line) was used as the subject. The cell viability was assessed by MTT assay. The cytotoxicity of 1-methylhydantoin to HK-2 was determined by NAG release test. Apoptosis of cultured HK-2 was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33258. Cells were exposed to 1-methylhydantoin (0.25mMol/L, 0.5mMol/L, or 1mMol/L), or creatinine (1mMol/L) for 24 h. 1-methylhydantoin induced a significant (p < 0.01) dose-dependent loss of cell viability. 1-methylhydantoin–treated HK-2 displayed characteristic microscopic features of apoptosis: reduced cell size, nuclear disintegration, and membrane bleb formation. FACS analysis demonstrated that 1-methylhydantoin induced apoptosis as well as cell changes consistent with necrosis. The proportion of cells with nuclear changes of apoptosis, identified by flow cytometry, increased significantly (p < 0.01) after 1-methylhydantoin (0.5mMol/L ) for 24 h. The results of the present study clearly demonstrate that both 1-methylhydantoin and creatinine are toxic for proximal tubular cells but that the damage resulting from the 1-methylhydantoin is more severe.     

肾小管上皮细胞内质网应激时NGAL表达改变的调控机制研究

目的:探讨人肾小管上皮细胞(HK-2细胞)发生内质网应激时,NGAL表达增加的上游调控机制。方法:将HK-2细胞分为对照组(正常HK-2细胞),TG(毒胡萝卜素,thapsigargin)组(5μmol/L TG处理8 h),单纯转染组(siRNAATF4试剂转染24 h),转染+TG组(siRNA-ATF4试剂转染24 h后,5μmol/L TG处理8 h),阴性对照组(siRNA-阴性对照物转染24 h),DMSO组(5μmol/L DMSO处理8 h)。采用Western blot检测各组细胞内质网源性转录因子(CHOP)、内质网分子伴侣葡萄糖调节蛋白78(GRP78)、中性粒细胞明胶酶相关性载脂蛋白(NGAL)、激活转录因子4(ATF4)的表达,采用Real-time PCR方法测得ATF4mRNA、NGALmRNA表达量。结果:与对照组相比,TG组细胞NGAL、ATF4、ATF4mRNA、NGALmRNA表达量显著提高(P <0. 05),而转染+TG组、单纯转染组、阴性对照组、DMSO组中ATF4及NGAL差异无统计学意义(P> 0. 05)。与TG组相比,转染+TG组ATF4、NGAL、ATF4mRNA及NGALmRNA表达量呈显著降低趋势(P <0. 05)。在TG组与转染+TG组细胞中,CHOP和GRP78呈过表达状态(P <0. 05),而转染+TG组细胞CHOP和GRP78提升趋势明显低于TG组细胞(P <0. 05)。结论:(1) TG可诱导人肾小管上皮HK-2细胞发生内质网应激反应。(2) HK-2细胞发生内质网应激反应时,抑制ATF4表达会引起NGAL降低,提示ATF4是NGAL表达的上游调控因子。(3) HK-2细胞发生内质网应激反应时,抑制ATF4不能阻止CHOP和GRP78发生过表达,但可降低其升高程度,提示ATF4及NGAL降低可能对内质网应激反应介导HK-2细胞损伤起到一定的缓解作用。     

Gene Expression of TGF-α, EGF and IL-6 in Cultured Renal Tubular Cells and Renal Cell Carcinoma

Background: Renal cell carcinoma (RCC) is thought to arise from the renal tubular cells (RTC). Assuming that proliferating RTC imply a premalignant change of RTC into RCC, messenger RNA expressions of growth factors in cultured RTC were compared to both cultured and frozen noncultured RCC.
Methods: The expression of transforming growth factor-alpha (TGF-α), epidermal growth factor (EGF), EGF receptor (EGFR) and Interleukin-6 (IL-6) were studied in surgically obtained RCC (n=17), cultured RCC (n=10), and autologous cultured RTC (n = 15). Quantitation of the PCR product was performed using a computer image analyzer which evaluated the intensity of each cytokine relative to β-actin.
Results: TGF-α, EGFR and IL-6 were detected in most of the cultured RTC, and both cultured and noncultured RCC were also expressed at high levels. In contrast to a high positivity of TGF-α, EGF was not strongly positive in all specimens.
Conclusions: Our results show that there is a predominant autocrine production of TGF-α in RCC and RTC, suggesting that TGF-α plays a distinct role in the proliferation of these cells. These studies also indicate that the mechanisms of proliferation and cytokine production of RCC and RTC are similar.     

Cisplatin-Induced Cytotoxicity in BSO-Exposed Renal Proximal Tubular Epithelial Cells: Sex,Age, and Species

Background. Cisplatin (CP)-induced kidney damage and effects of DL-buthionine-(S,R)-sulfoximine (BSO) on it are species- and age-different. It remains unclear whether CP-induced cytotoxicity in renal proximal tubular epithelial cells (RTEC), the main target cells of CP, is also species- and age-different; and whether CP-induced cytotoxicity varies with the difference in age and species, if any, is one of the questions. In the present study, the effects of BSO on CP-induced cytotoxicity in primary cultures of RTEC isolated from monkeys and different age and sex rats were studied. Methods. The RTEC were isolated from 3-week-old, 2-month-old, or 5-month-old rats, and 6-8 year-old monkeys. After subculturing, RTEC was inoculated into type I collagen-coated 96-well culture plates; after preincubation, 40 µM BSO was added, 16 hours later, varying concentrations of CP were added. At that time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were performed to test cell viability. Results. The concentrations of CP that inhibited 50% cell growth (IC₅₀) of RTEC from rats and monkeys were 1.11 and 3.03 mM at 8 hours, and 0.51 and 1.24 mM at 24 hours, respectively. The BSO made the IC₅₀s of RTEC from rats and monkeys lower, down to 0.07 and 0.48 mM at 8 hours, and 0.02 and 0.11 mM at 24 hours, respectively. The IC₅₀s of RTEC from different sex and age rats were almost same. Conclusion. These results suggested that CP-induced cytotoxicity was concentration- and time-dependent, with species-dependent differences, rat RTEC were more susceptible to CP than monkey RTEC, rat RTEC were more dependent on glutathione (GSH) during the stress state were than monkey cells; CP-induced cytotoxicity was without sex- and age-dependent differences in rat RTEC.     

不同病理类型肾综患者尿蛋白对肾小管上皮细胞增殖和凋亡相关蛋白Fas表达的影响

目的：观察不同病理类型肾病综合征（NS）患者尿蛋白对肾小管上皮细胞（RTECs）增殖和凋亡的影响,以进一步明确尿蛋白所致肾小管-间质损害的机制.方法：（1）从局灶-节段性肾小球硬化症（FSGS）、膜性肾病（MN）、微小病变肾病（MCN）三种不同病理类型的NS患者尿液中提取尿蛋白,经成份分析、灭菌等处理后以0.5 mg/ml、1.0 mg/ml、2 mg/ml、4 mg/ml、8 mg/ml浓度分别刺激体外培养的HK-2细胞,另设空白对照组.（2）MTT法检测不同病理类型NS患者尿蛋白刺激后细胞的增殖情况.（3）乳酸脱氢酶（LDH）释放实验检测不同病理类型NS患者尿蛋白的细胞毒作用.（4）Western Blotting法检测Fas蛋白表达.结果：各病理类型所提取的尿蛋白成分相同,主要为白蛋白、转铁蛋白、IgG等,但各病理类型组成比例不同;肾小管上皮细胞MTT值低浓度有明显增殖作用,高浓度时细胞过度增殖则导致凋亡;肾小管上皮细胞LDH释放率和Fas蛋白的表达水平随尿蛋白浓度的升高而升高;以上各项检测指标中FSGS患者尿蛋白对HK-2细胞的作用最强,MN次之,MCD最弱.结论：在体外条件下,尿蛋白对RTECs呈剂量依赖性的细胞毒作用,低剂量尿蛋白诱导RTECs异常增殖,较高剂量尿蛋白可诱导RTECs凋亡;除尿蛋白的量决定了损伤严重程度外,尿蛋白的性质也决定了损伤的严重程度.     

甘草酸对马兜铃酸致肾小管上皮细胞损害保护作用的初步研究

目的:探讨甘草酸(glycyrrhizin,GL)对马兜铃酸(aristolorchic acid,AA)肾损害的保护作用,观察GL是否可以减轻AA对培养的肾小管上皮细胞的损害.方法:5?S RPMI-1640培养肾小管上皮细胞(LLC-PK1),采用结晶紫染色法观察细胞增殖;荧光染色观察活细胞数目;酶动力学检测LDH活性,比色法检测NAG水平;透射电镜观察细胞超微结构.结果:(1)AA 40、80、160 μg/ml可明显抑制细胞增殖,结晶紫OD值显著减少,与无AA对照组比较(P<0.01).(2)GL在5、10、25 μg/ml时结晶紫OD值与无GL对照组比较(P>0.05);GL在50 μg/ml时P<0.05,100 μg/ml、200 μg/ml时P<0.01,提示大剂量GL可改善AA抑制细胞增殖的作用.(3)在AA 40 μg/ml作用下;随着时间延长,无GL组的细胞存活数目逐渐减少,而GL(100 μg/ml、200 μg/ml)作用组,活细胞数目明显增多,显示高浓度GL对细胞损伤有一定的保护作用.(4)GL在5、10、25 μg/ml时,LDH释放率、NAG酶水平与无GL对照组比较(P>0.05),GL在50 μg/ml时P<0.05,GL在100 μg/ml、200 μg/ml时LDH释放率、NAG酶明显减少,与无GL组比较(P<0.01).提示大剂量GL可减轻AA的细胞毒作用.(5)细胞超微结构观察:AA 40 μg/ml作用24 h后,细胞超微结构发生显著改变,以核变异最突出,出现核分叶、巨核、染色质浓染、核边集、核缺失、核膜卷曲增厚,线粒体肿胀等严重细胞损伤改变.GL 100 μg/ml、200 μg/ml组上述改变均较轻微.结论:GL可明显改善AA对肾小管上皮细胞增殖的抑制作用;改善细胞超微结构.     

三七总皂苷对TGF-β1诱导的HK-2细胞表型转分化的影响

目的:探讨三七总皂苷(PNS)对转化生长因子-β1(TGF-β1)诱导的人肾小管上皮细胞(HK-2)转分化的影响.方法:采用流式细胞仪(FCM)结合免疫荧光法(IF)检测PNS对TGF-β1诱导的HK-2细胞α-平滑肌肌动蛋白(α-SMA)阳性细胞百分率的影响;逆转录-聚合酶链反应(RT-PCR)检测PNS对TGF-β1诱导的HK-2细胞α-SMA信使核糖核酸(mRNA)表达.结果:流式细胞仪(FCM)结合免疫荧光法(IF)示:PNS 200～800 mg/L可使TGF-β1诱导的HK-2细胞增加的α-SMA阳性细胞百分率回降;RT-PCR示:PNS 200～800 mg/L剂量和时间依赖性地使TGF-β1诱导的HK-2细胞增加的α-SMA mRNA的基因表达回降.结论:PNS可通过抑制TGF-β1诱导的肾小管上皮细胞表型转分化,从而参与延缓肾间质纤维化的进程.     

Renal Clearance of Mineral Metabolism Biomarkers

CKD leads to disturbances in multiple interrelated hormones that regulate bone and mineral metabolism. The renal handling of mineral metabolism hormones in humans is incompletely understood. We determined the single-pass renal clearance of parathyroid hormone, fibroblast growth factor 23, vitamin D metabolites, and phosphate from paired blood samples collected from the abdominal aorta and renal vein in 17 participants undergoing simultaneous right and left heart catheterization and estimated associations of eGFR with the renal elimination of metabolites. The mean age ±SD of the study population was 71.4±10.0 years and 11 participants (65%) were male. We found a relatively large mean±SD single-pass renal extraction of parathyroid hormone (44.2%±10.3%) that exceeded the extraction of creatinine (22.1%±7.9%). The proportionate renal extraction of parathyroid hormone correlated with eGFR. The renal extraction of fibroblast growth factor 23 was, on average, lower than that of parathyroid hormone with greater variability across individuals (17.1%±19.5%). There were no differences in the mean concentrations of vitamin D metabolites across the renal vein and artery. In summary, we demonstrate substantial single-pass renal extraction of parathyroid hormone at a rate that exceeds glomerular filtration. Impaired renal clearance of parathyroid hormone may contribute to secondary hyperparathyroidism in CKD.     

Epithelial-to-Mesenchymal Transdifferentiation of Renal Tubular Epithelial Cell Mediated by Oxidative Stress and Intervention Effect of Probucol in Diabetic Nephropathy Rats

Objective: To elucidate the relationship of oxidative stress and specificity protein 1 (Sp1) in the process of epithelial-to-mesenchymal transdifferentiation (EMT) and also to investigate the molecular mechanism of protective effect of probucol on the pathogenesis of diabetic kidney disease (DKD). Methods: Thirty male Sprague–Dawley (SD) rats were randomly divided into control group, diabetic group, and diabetic group under probucol therapy (n = 10 per group). The biochemical indicators including 24-h urinary total protein (24-h UTP) excretion, blood glucose (BG), lipids [triglycerides (TGs), total cholesterol (TC)], serum creatinine (Scr), creatinine clearance rate (Ccr), kidney tissue malondialdehyde (MDA) level, and glutathione peroxidase (GSH-Px) activity were assessed in all groups. The renal pathological changes were evaluated by hematoxylin and eosin (HE) and Masson staining. The protein expression of Sp1, α-smooth muscle actin (α-SMA), and E-cadherin was also measured and analyzed by immunohistochemistry and Western blotting. Results: Compared with the control group, the BG, TC, Scr, 24-h UTP, and MDA level of renal tissue increased significantly and the Ccr reduced in the rats of diabetic group (all p < 0.01). The pathological scores and the expression of Sp1 and α-SMA in renal tissue were up-regulated (p < 0.01) and the expression of E-cadherin was down-regulated significantly in the diabetic animals (p < 0.01). In the diabetic animals treated with probucol, the renal injuries were alleviated (p < 0.01). Conclusions: Oxidative stress may play an important role in the EMT process of tubular epithelial cells. Probucol could ameliorate renal disease progression in this model of diabetic nephropathy, which might be due to an antioxidant action, down-regulation of Sp1 protein expression, and inhibition of renal tubular EMT.     

Increasing Resistance of Tubular Epithelial Cells to Apoptosis by shRNA Therapy Ameliorates Renal Ischemia-Reperfusion Injury

Renal tubular epithelial cells (TEC) die by apoptosis or necrosis in renal ischemia-reperfusion injury (IRI). Fas/Fas ligand-dependent fratricide is critical in TEC apoptosis, and Fas promotes renal IRI. Therefore, targeting Fas or caspase-8 may have therapeutic potential for renal injury in kidney transplant or failure. RNA silencing by short hairpin RNA (shRNA) is a novel strategy to down-regulate protein expression. Using this approach, silencing of Fas or caspase-8 by shRNA to prevent TEC apoptosis and IRI was evaluated. IRI was induced by renal artery clamping for 45 or 60 min at 32 degrees C in uninephrectomized C57BL/6 mice. Here, we showed that Fas or pro-caspase-8 expression was significantly knocked down in TEC by stable expression of shRNA, resulting in resistance to apoptosis induced by superoxide, IFN-gamma/TNF-alpha and anti-Fas antibody. Inferior vena cava delivery of pHEX-small interfering RNA targeting Fas or pro-caspase-8 resulted in protection of kidney from IRI, indicated by reduction of renal tubular injury (necrosis and apoptosis) and serum creatinine or blood urea nitrogen. Our data suggest that shRNA-based therapy targeting Fas and caspase-8 in renal cells can lead to protection of kidney from IRI. Attenuation of pro-apoptotic proteins using genetic manipulation strategies such as shRNA might represent a novel strategy to promote kidney allograft survival from rejection or failure.     

Impairment of Proteasome Function in Podocytes Leads to CKD

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PPARγ活化对TGF-β1诱导肾小管上皮细胞趋化因子表达的干预作用

目的：观察PPARγ活化对转化生长因子-β1（TGF-β1）诱导的肾小管上皮细胞炎症相关趋化因子表达的影响,探讨其在肾脏纤维化中的干预作用。方法：体外培养肾小管上皮细胞HK-2,LDH法检测15d-PGJ2和TGL对HK-2的细胞毒性,应用不同浓度的15d-PGJ2和TGL作用于TGF-β1诱导下的HK-2细胞,应用实时荧光定量PCR技术和ELISA方法检测趋化因子单核细胞趋化蛋白-1（MCP-1）、白细胞介素-8（IL-8）表达的变化。结果：5μmol/L15d-PGJ2和2.5μmol/LTGL不影响HK-2细胞MCP-1和IL-8mRNA基础表达和蛋白分泌。TGF-β1作用24h时,2.5μmol/L和5μmol/L15d-PGJ2及2.5μmol/LTGL均能有效干预TGF-β1诱导的MCP-1mRNA表达和蛋白的分泌（P〈0.05）。IL-8的表达与MCP-1相似,2.5μmol/L和5μmol/L15d-PGJ2能显著抑制TGF-β1诱导的IL-8表达,TGF-β1诱导24h时2.5μmol/LTGL能显著抑制IL-8mRNA表达（P〈0.05）。结论：PPARγ激动剂15d-PGJ2和TGL作用均能有效干预TGF-β1诱导的肾小管上皮细胞趋化因子MCP-1和IL-8的表达,可能具有有效干预肾间质炎症作用。     

来氟米特对人肾小管上皮细胞增殖和胶原合成的影响

目的探讨来氟米特对人肾小管上皮细胞增殖和胶原合成的作用。方法用不同浓度的来氟米特活性代谢产物A771726和重组人结缔组织生长因子（rhCTGF）分别以及共同作用于体外培养的人肾小管上皮细胞,用MTT法测定细胞增殖情况;ELISA法测定细胞合成Ⅰ、Ⅳ型胶原的含量。结果①经不同浓度的rhCTGF作用后,肾小管上皮细胞的增殖与对照组比较无显著性差异（P〉0．05）,而细胞合成Ⅰ、Ⅳ型胶原呈剂量依赖性增加（P〈0．01）;②来氟米特干预后,肾小管上皮细胞的增殖及胶原合成剂量依赖性减少（P〈0．01）;③来氟米特与rhcTGF共同作用的各组,细胞的增殖和胶原合成均较单用rhCTGF组减少（P〈0．05）。结论rhCTGF对肾小管上皮细胞的增殖无影响,但可使细胞合成Ⅰ、Ⅳ型胶原上调;来氟米特能够直接抑制肾小管上皮细胞增殖和Ⅰ、Ⅳ型胶原合成,并且对rhCTGF诱导的胶原合成增加有一定的阻断作用。     

IL‐17 Expression by Tubular Epithelial Cells in Renal Transplant Recipients with Acute Antibody‐Mediated Rejection

Acute rejection is still a common complication of kidney transplantation. IL‐17 is known to be associated with allograft rejection but the cellular source and the role of this cytokine remains unclear. We investigated IL‐17 graft expression in renal transplant recipients with acute antibody‐mediated rejection (ABMR), acute T‐cell‐mediated rejection (TCMR), interstitial fibrosis and tubular atrophy (IFTA) and acute tubular damage due to calcineurin‐inhibitor toxicity (CNI). In acute ABMR, tubular IL‐17 protein expression was significantly increased compared to TCMR, where most of the IL‐17⁺cells were CD4⁺graft infiltrating lymphocytes, IFTA and CNI control groups. The tubular expression of IL‐17 in acute ABMR colocalized with JAK2 phosphorylation and peritubular capillaries C4d deposition. In addition, IL‐17 tubular expression was directly and significantly correlated with the extension of C4d deposits. In cultured proximal tubular cells, C3a induced IL‐17 gene and protein expression along with an increased in JAK2 phosphorylation. The inhibition of JAK2 abolished C3a‐induced IL‐17 expression. The use of steroids and monoclonal antibodies reduced IL‐17 expression, JAK2 phosphorylation and C4d deposition in acute ABMR patients. Our data suggest that tubular cells represent a significant source of IL‐17 in ABMR and this event might be mediated by the complement system activation featuring this condition.