肾小管上皮细胞线粒体氧化损伤在肾间质纤维化中的病理学机制研究

目的：研究肾小管上皮细胞线粒体氧化损伤在肾间质纤维化中的病理学机制。方法：80只雄性大鼠随机平分为假手术组和UUO模型组,每组40只,行大鼠左侧输尿管结扎术,假手术组游离左侧输尿管但不予结扎,其余手术与UUO模型组相同。分别于术后d3、d7、d10、d14、d21每组处死8只,取大鼠左侧肾脏。①检测并比较假手术组和UUO模型组大鼠线粒体相关基因的表达水平;②检测两组大鼠肾脏组织COX和SOD含量变化;③检测两组大鼠肾脏组织RIF指数变化;④检测并比较肾体比、肾功能和肾组织Hyp含量动态变化。结果：与假手术组大鼠相比,①UUO模型组大鼠的线粒体相关基因的表达水平显著升高（P＜0.05）,包括线粒体mtDNA、再生基因（PGC1a、NRF1和Tfam）、断裂基因（Mfn1、Mfn2和Opa1）和融合基因（Drp1和Fis1）;②UUO模型组大鼠在造模后10d、14d和21d,肾脏组织中的COX水平显著升高（P＜0.05）,SOD水平显著降低（P＜0.05）,造模时间越长,UUO模型组大鼠COX水平越高（P＜0.05）,SOD水平越低;③UUO模型组大鼠造模后10d、14d和21d,肾脏组织RIF指数均显著升高（P＜0.05）;④UUO模型组大鼠肾体比明显升高且一直维持在较高水平（P＜0.05）,造模时间越长,模型大鼠血清Scr和BUN水平及肾组织Hyp含量水平逐渐升高,与假手术组相比均具有显著差异（P＜0.05）。结论：与假手术组大鼠相比,UUO模型组大鼠肾组织中的线粒体更活跃,线粒体基因、再生基因、断裂基因和融合基因含量升高,COX含量升高,SOD含量降低,RIF指数升高,肾体比、血清Scr和BUN和肾组织Hyp水平显著升高。     

Berberine ameliorates renal interstitial inflammation and fibrosis in mice with unilateral ureteral obstruction

Berberine acts via multiple pathways to alleviate fibrosis in various tissues and shows renoprotective effects. However, its role and underlying mechanisms in renal fibrosis remain unclear. Herein, we aimed to investigate the protective effects and molecular mechanisms of berberine against unilateral ureteric obstruction-induced renal fibrosis. The results indicated that berberine treatment (50 mg/kg/day) markedly alleviated histopathological alterations, collagen deposition and inflammatory cell infiltration in kidney tissue and restored mouse renal function. Mechanistically, berberine intervention inhibited NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and the levels of the inflammatory cytokine IL-1β in the kidneys of unilateral ureteric obstruction mice. In addition, berberine relieved unilateral ureteric obstruction-induced renal injury by activating adenosine monophosphate-activated protein kinase (AMPK) signalling and promoting fatty acid β-oxidation. In vitro models showed that berberine treatment prevented the TGF-β1-induced profibrotic phenotype of hexokinase 2 (HK-2) cells, characterized by loss of an epithelial phenotype (alpha smooth muscle actin [α-SMA]) and acquisition of mesenchymal marker expression (E-cadherin), by restoring abnormal fatty acid β-oxidation and upregulating the expression of the fatty acid β-oxidation related-key enzymes or regulators (phosphorylated-AMPK, peroxisome proliferator activated receptor alpha [PPARα] and carnitine palmitoyltransferase 1A [CPT1A]). Collectively, berberine alleviated renal fibrosis by inhibiting NLRP3 inflammasome activation and protected tubular epithelial cells by reversing defective fatty acid β-oxidation. Our findings might be exploited clinically to provide a potential novel therapeutic strategy for renal fibrosis.     

红花黄色素对实验性肾间质纤维化大鼠肾小管上皮细胞凋亡的影响

目的观察红花黄色素注射液对肾间质纤维化大鼠肾小管上皮细胞凋亡的的影响,探讨其肾脏保护机制。方法45只雄性SD大鼠,随机分为假手术组（A组）、单侧输尿管梗阻（unilateral ureteral obstruction;UUO）组（B组）、UUO联合红花黄色素治疗组（C组）,各15只。C组红花黄色素5mg/（kg·d）,造模术前1d开始腹腔注射给药,各组术后10d处死大鼠,处死后取术侧肾组织行HE、Masson染色,TUNEL法检测肾小管上皮细胞凋亡。结果B组、C组可见明显肾间质纤维化的病理改变,可检测到凋亡细胞。C组与B组相比,肾间质纤维化病变程度较轻,凋亡细胞明显减少（P〈0.01）。结论注射用红花黄色素对大鼠肾间质纤维化有保护作用,这种作用可能通过抑制肾小管细胞过度凋亡实现。     

Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAI-treated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53-independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.     

雷帕霉素对肾小管上皮细胞增殖和凋亡的影响

目的 探讨雷帕霉素对肾小管上皮细胞增殖和凋亡的影响。方法 体外培养肾小管上皮细胞(HK-2细胞),选择100, 200, 400, 800 ng/ml雷帕霉素作用于HK-2细胞24、48、72h。利用 MTT实验分析雷帕霉素对HK-2细胞增殖的影响,并计算各浓度及不同时间的增殖抑制率优化雷帕霉素作用的浓度和时间;选择最佳的作用浓度和作用时间处理HK-2细胞,通过流式细胞分析技术,分析雷帕霉素对HK-2细胞的凋亡的影响。结果 MTT实验显示不同浓度的雷帕霉素,对增殖有抑制作用,且表现浓度依赖性和时间依赖性;RAPA可以促进HK-2细胞的凋亡。结论 通过HK-2细胞模型研究,发现雷帕霉素可抑制肾小管上皮细胞增殖、促进细胞凋亡。     

The cell cycle inhibitor P21 promotes the development of pulmonary fibrosis by suppressing lung alveolar regeneration

The cell cycle inhibitor P21 has been implicated in cell senescence and plays an important role in the injury–repair process following lung injury. Pulmonary fibrosis (PF) is a fibrotic lung disorder characterized by cell senescence in lung alveolar epithelial cells. In this study, we report that P21 expression was increased in alveolar epithelial type 2 cells (AEC2s) in a time-dependent manner following multiple bleomycin-induced PF. Repeated injury of AEC2s resulted in telomere shortening and triggered P21-dependent cell senescence. AEC2s with elevated expression of P21 lost their self-renewal and differentiation abilities. In particular, elevated P21 not only induced cell cycle arrest in AEC2s but also bound to P300 and β-catenin and inhibited AEC2 differentiation by disturbing the P300–β-catenin interaction. Meanwhile, senescent AEC2s triggered myofibroblast activation by releasing profibrotic cytokines. Knockdown of P21 restored AEC2-mediated lung alveolar regeneration in mice with chronic PF. The results of our study reveal a mechanism of P21-mediated lung regeneration failure during PF development, which suggests a potential strategy for the treatment of fibrotic lung diseases.     

Cyclophilin D Promotes Acute,but Not Chronic,Kidney Injury in a Mouse Model of Aristolochic Acid Toxicity

The plant-derived toxin, aristolochic acid (AA), is the cause of Chinese Herb Nephropathy and Balkan Nephropathy. Ingestion of high dose AA induces acute kidney injury, while chronic low dose ingestion leads to progressive kidney disease. Ingested AA is taken up by tubular epithelial cells of the kidney, leading to DNA damage and cell death. Cyclophilin D (CypD) participates in mitochondrial-dependent cell death, but whether this mechanism operates in acute or chronic AA-induced kidney injury is unknown. We addressed this question by exposing CypD-/- and wild type (WT) mice to acute high dose, or chronic low dose, AA. Administration of 5 mg/kg AA to WT mice induced acute kidney injury 3 days later, characterised by loss of kidney function, tubular cell damage and death, and neutrophil infiltration. All of these parameters were significantly reduced in CypD-/- mice. Chronic low dose (2 mg/kg AA) administration in WT mice resulted in chronic kidney disease with impaired renal function and renal fibrosis by day 28. However, CypD-/- mice were not protected from AA-induced chronic kidney disease. In conclusion, CypD facilitates AA-induced acute kidney damage, but CypD does not contribute to the transition of acute kidney injury to chronic kidney disease during ongoing AA exposure.     

普罗布考对甲基胍诱导纤维化相关因子在HK-2细胞表达的影响

目的研究降脂药普罗布考对小分子尿毒素甲基胍诱导纤维化相关因子在人肾小管上皮细胞表达的影响。方法 HK-2细胞分3组培养,A组（正常对照,不加干预因素）;B组培养基中加入0.5mmol.L-1甲基胍;C组培养基中加入0.5mmol.L-1甲基胍和20μmol.L-1普罗布考。各组细胞培养48h后用免疫细胞化学、RT-PCR检测TGF-β1、CTGF和FN,并用流式细胞仪对以上因子作定量检测,以观察其在HK-2细胞中的表达。结果在甲基胍刺激后,细胞免疫化学染色,HK-2细胞质中着色,显示TGF-β1、CTGF和FN均有表达。正常对照组HK-2细胞质中无着色。测定结果显示甲基胍组TGF-β1、CTGF和FN显著高于正常对照组（P〈0.01）,加入普罗布考后,表达显著下降（P〈0.01）。结论甲基胍能促进纤维化相关因子在肾小管上皮细胞中表达;普罗布考能抑制甲基胍诱导纤维化相关因子在肾小管上皮细胞的表达。     

酸性成纤维细胞生长因子对体外培养肾小管上皮细胞存活与增殖的影响

目的探讨酸性成纤维细胞因子(aFGF)对体外培养的大鼠肾小管上皮细胞生长的影响。方法通过酶消化法获取肾小管上皮细胞。将aFGF(低、中、高浓度)加入培养液中作为实验组,不加aFGF的作为对照组。分别在12、24、48和72h进行细胞计数及形态学观察。结果从大鼠肾成功地获取了肾小管上皮细胞。干预72h内,实验组和对照组的肾小管上皮细胞都有不同程度地增殖,12h内各组无明显区别,24、48、72h实验组与对照组相比差异有统计学意义(P<0.05)。结论aFGF有明显的促进肾小管上皮细胞存活与增殖的作用,存在剂量-效应和时间-效应关系。     

缬沙坦对人肾近端小管上皮细胞TGF-β/Smad信号途径的影响

目的观察缬沙坦对TGF-β刺激人肾近端小管上皮细胞表达Smad的影响。方法以人肾小管上皮细胞(HKC)为对象,分为正常对照组;TGF-β1刺激组;缬沙坦组。Western blot检测p-Smad2/3、smad2/3、Smad7以及细胞上清ColⅠ蛋白含量;RT-PCR检测Smad2 mRNA和Smad7 mRNA水平。MTT法检测刺激组及缬沙坦组在不同时间、不同药物浓度对细胞增殖状态的影响。结果①Western blot显示,与TGF-β1刺激组相比,缬沙坦使Smad2/3、p-Smad2/3蛋白的表达下降,ColⅠ分泌显示相同趋势,而Smad7蛋白的表达没有变化。②半定量RT-PCR显示,与TGF-β1刺激组相比,缬沙坦组Smad2 mRNA降低,Smad7 mRNA无变化。③MTT法检测显示不同浓度缬沙坦对TGF-β1刺激所引起的细胞增殖受抑制现象均有改善,并且随药物浓度的增加该作用增强。结论提示缬沙坦的肾脏保护作用可能部分是通过抑制TGF-β/Smads信号途径实现的。     

全反式维甲酸对高糖诱导肾小管上皮细胞增殖及凋亡的影响

摘要： 目的 探讨全反式维甲酸 （ATRA） 对高糖诱导人 HK-2 细胞增殖及凋亡的影响, 以期延缓糖尿病肾病（DN）。方法 体外培养 HK-2 细胞, 随机分为 6 组： 空白组 （未加任何刺激物）、 高糖组 （加 D-葡萄糖 30 mmol/L）、 高渗组 （加入甘露醇 24.5 mmol/L）、 低浓度 ATRA 组 （加入 ATRA 1×10-7 mol/L+D-葡萄糖 30 mmol/L）、 中浓度 ATRA 组（加入 ATRA 1×10-6 mol/L+D-葡萄糖 30 mmol/L）、 高浓度 ATRA 组 （加入 ATRA 1×10-5 mol/L+D-葡萄糖 30 mmol/L）。各组细胞培养 48 h。MTT 法检测各组细胞增殖情况, 流式细胞术检测各组细胞凋亡情况。结果 空白组与高渗组细胞光密度 （OD） 值和凋亡率差异无统计学意义。高糖组较空白组、 高渗组细胞增殖减少, 凋亡率增加; 低浓度、 中浓度、 高浓度 ATRA 组细胞增殖均较高糖组增加, 且低、 中及高浓度 ATRA 组依次增加, 凋亡率较高糖组减少, 且低、 中及高浓度 ATRA 组依次减少 （均 P＜0.05）。结论 ATRA 可促进高糖诱导的 HK-2 细胞增殖, 抑制其凋亡, 并与 ATRA 浓度可能具有一定的依赖性。     

SB203580对缺氧/复氧损伤诱导的大鼠肾小管上皮细胞凋亡的保护作用及其机制

目的研究SB203580(吡啶咪唑类抗炎药)对大鼠肾小管上皮细胞凋亡的保护作用及机制。方法SD大鼠肾小管上皮细胞原代培养,取第2代细胞实验;共分为3组:对照组,正常培养细胞;模型组,细胞缺氧1 h后,复氧6、12、24、48 h培养;SB203580预处理组,在缺氧损伤前1 h,用不同剂量SB203580(0.2、2、20μmol·L~(-1))预处理细胞,尔后进行缺氧/复氧损伤处理,各细胞用Hoechst 33258、Tunnel染色、流式细胞仪及Western blot方法,观察缺氧/复氧后细胞凋亡及p38MAPK的磷酸化水平。结果与对照组相比,缺氧/复氧后,实验组细胞的凋亡率显著增加,且细胞内p38MAPK磷酸化水平明显升高;而应用SB203580预处理细胞,可显著降低实验组细胞的凋亡率及细胞内p38MAPK的磷酸化水平。结论SB203580通过抑制p38MAPK的活性,对缺氧/复氧诱导的肾小管上皮细胞凋亡有保护作用。     

Possible role of mitochondrial injury in Caulis Aristolochia manshuriensis-induced chronic aristolochic acid nephropathy

Context: The proximal tubular epithelial cells (PTECs) are the primary target of aristolochic acids and especially vulnerable to mitochondrial injury from insults of toxic xenobiotics. Objectives: This study aimed to investigate the possible role of mitochondrial injury in Caulis Aristolochia manshuriensis (CAM)-induced aristolochic acid nephropathy (AAN). Materials and methods: Male Sprague-Dawley rats were gavaged with CAM extract every other week for 1, 4, 8 and 12 weeks, respectively. Results: The rats in the model group showed chronic AAN as evidenced by worsening kidney function evaluated by blood urea nitrogen, creatinine and proteinuria levels, and severe tubulointerstitial injury marked by massive tubular atrophy and interstitial fibrosis in kidney tissues. Moreover, overt apoptosis and impaired regeneration of PTECs were observed in AAN rats. Furthermore, the study revealed that mitochondria in PTECs were fragmented into small, punctuate suborganelles in AAN rats. Two mitochondrial respiratory chain proteins, mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit ? (COX-?) and nuclear DNA-encoded nicotinamide adenine dinucleotide dehydrogenase (ubiquinone)-1β subcomplex 8 (NDUFβ8), were both down-regulated after one week of CAM treatment. However, with AAN progression, NDUFβ8 level restored, while COX-? level maintained low. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), master regulator of mitochondrial biogenesis, was significantly down-regulated at week 4 and week 8, but significantly up-regulated at week 12. In addition, mtDNA copy number reduced markedly along with AAN progression. Discussion and conclusion: A rat model of chronic AAN was successfully reproduced by gavage with CAM extract. Dynamic changes of mitochondrial injury induced by CAM might contribute to the AAN progression.     

Pivotal role of pericytes in kidney fibrosis

1. Kidney pericytes were recently identified as collagen Iα1-producing cells in healthy kidney, but the developmental, physiological and pathological roles of kidney pericytes remain poorly understood. Pericytes are stromal-derived cells that envelop and have intimate connections with adjacent capillary endothelial cells (EC). Recent studies in the eye and brain have revealed that pericytes are crucial for angiogenesis, vascular stability and vessel integrity. 2. In response to kidney injury, pericytes promptly migrate away from the capillary wall into the interstitial space. Here, pericytes are activated and differentiate into scar-forming myofibroblasts. In the absence of pericytes, peritubular capillaries are destabilized, leading to vascular regression. Consequently, capillary loss and fibrosis following kidney injury are intimately linked and hinge centrally around pericyte detachment from EC. 3. Kinetic mathematical modelling has demonstrated that pericytes are the major source of myofibroblasts in the fibrotic kidney. Comprehensive genetic fate mapping studies of nephron epithelia or kidney stroma has demonstrated that epithelial cells do not migrate outside of the epithelial compartment to become myofibroblasts; rather, interstitial pericytes are progenitors of scar-forming myofibroblasts. Bidirectional signalling between pericytes and EC is necessary for pericyte detachment from peritubular capillaries. 4. In the present review, we summarize the pathologically vital roles of kidney pericytes in fibrosis, including our new findings. The study of kidney pericytes and endothelial-pericyte cross-talk will identify novel therapeutic targets for currently incurable chronic kidney diseases.     

丹酚酸B对高糖干预下的人肾小管上皮细胞自噬相关蛋白的影响

目的 探究丹酚酸B对高糖干预下的人肾小管上皮细胞自噬相关蛋白的影响.方法 根据人近端肾小管上皮细胞HK-2细胞培养方式的不同,分为对照组、葡萄糖组和实验组,对照组加入5.5 mmol/L的葡萄糖;葡萄糖组加入葡萄糖的浓度分为10、20和30 mmol/L;实验组在培养基中分别加入丹酚酸B和葡萄糖(30 mmol/L),丹酚酸B的浓度分为0.1、1、10和100 μmol/L,培养24 h.使用蛋白提取试剂盒分别提取3组培养细胞中的总蛋白,采用Bradford法测定不同培养组细胞自噬相关蛋白的含量,进行分析比较.结果 随着葡萄糖刺激浓度的增高,p62蛋白表达增加.24 h后,高糖可诱导HK-2细胞自噬相关蛋白p62表达增加,LC3表达减少,且呈剂量依赖性.与对照组相比,随着丹酚酸B干预剂量的增高,24 h后的细胞LC3的表达增加,不同剂量丹酚酸B干预可减少高糖诱导下的p62蛋白的表达增加,同时LC3的表达得到明显恢复,且呈剂量依赖性(P<0.05).Logistic回归分析的结果显示,LC3蛋白含量、p62蛋白含量是影响细胞自噬的独立影响因素.结论 在高糖的干预下,不同剂量丹酚酸B干预可减少高糖诱导下的p62蛋白的表达增加,同时LC3的表达得到明显恢复,且呈剂量依赖性.     

对比剂诱导巨噬细胞来源外泌体对肾小管上皮细胞的作用及甘草酸苷的干预效应#br#

目的　探讨对比剂诱导巨噬细胞来源外泌体对肾小管上皮细胞的作用及甘草酸苷的干预效应。方法　分别用PBS、碘海醇、碘海醇+甘草酸苷刺激THP-1细胞,获取3种外泌体（NC-Exo、CIN-Exo、CIN-GL-Exo）。透射电镜观察外泌体的形态;蛋白免疫印迹（Western blot）检测CD63、TSG101、calnexin的表达;荧光标记法检测外泌体的摄取。将HK-2细胞随机分为4组：Con组、NC-Exo组、CIN-Exo组、CIN-GL-Exo组。Western blot检测3种外泌体中高迁移率族蛋白1（HMGB1）表达量,以及4组细胞中凋亡相关蛋白及HMGB1/Toll样受体4（TLR4）/核因子κB（NF-κB）信号通路相关蛋白的表达;Annexin Ⅴ-FITC/PI双染法检测4组细胞的凋亡率;实时荧光定量PCR检测4组细胞炎性因子［白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α］mRNA的表达。结果　THP-1细胞来源的外泌体为圆形或类圆形小囊泡,表达标志物蛋白CD63、TSG101,而不表达内质网蛋白calnexin。标记DiR荧光素的外泌体可被HK-2细胞摄取。CIN-Exo中HMGB1表达量高于NC-Exo和CIN-GL-Exo（P＜0.05）。CIN-Exo组的细胞凋亡率以及Bax、Cleaved caspase-3表达量高于Con组、NC-Exo组、CIN-GL-Exo组,而Bcl-2表达量低于其他3组（P＜0.05）。CIN-Exo组细胞中IL-1β、IL-6、TNF-α mRNA水平,以及HMGB1、TLR4、NF-κB表达量高于其他3组（P＜0.05）。结论　对比剂可通过巨噬细胞来源外泌体诱导肾小管上皮细胞凋亡和炎症反应,可能与外泌体HMGB1水平上调有关,甘草酸苷可抑制该途径而发挥保护效应。     

阿米卡星对肾小管上皮细胞的损伤及热休克蛋白70表达的影响

目的:观察阿米卡星对肾小管上皮细胞的损伤及热休克蛋白70表达的影响。方法:常规培养人肾小管上皮细胞系HK-2细胞,给予阿米卡星(100μg·mL-1)损伤或MnCl2(2μg·mL-1)保护,于24,48,72,96 h时分光光度法测定细胞增殖,乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、丙二醛(MDA)、N-乙酰-β氨基葡萄糖苷酶(NAG)含量,逆转录多聚酶链式反应(RT-PCR)法检测HSP70 mRNA表达,Western-blot检测HSP70蛋白表达。结果:阿米卡星损伤HK-2细胞后,细胞增殖显著减少,LDH释放量、NAG含量、MDA含量增加显著,SOD活力明显下降,HSP70 mRNA及其蛋白的表达显著增加(与对照组比,P<0.01);给予MnCl2干预后,HK-2细胞的细胞增殖明显有所恢复,LDH释放量、NAG含量、MDA含量有所下降,SOD活力有所增加,HSP70 mRNA及其蛋白的表达有所下降(与损伤组比,P<0.01)。结论:阿米卡星的肾脏毒性与HSP70的表达存在密切的联系。