Renal progenitors: Roles in kidney disease and regeneration

Kidney disease is a devastating condition that affects millions of people worldwide, and its prevalence predicted to significantly increase. The kidney is complex organ encompassing many diverse cell type organized in a elaborate tissue architecture, makin regeneration a challenging feat. In recent years, there ha been a surge in the field of stem cell research to develo regenerative therapies for various organ systems. Here we review some recent progressions in characterizing th role of renal progenitors in development, regeneration and kidney disease in mammals. We also discuss how the zebrafish provides a unique experimental anima model that can provide a greater molecular and genet understanding of renal progenitors, which may contribut to the development of potential regenerative therapies fo human renal afflictions.

Role of adult resident renal progenitor cells in tubular repair after acute kidney injury

Acute kidney injury is a serious global health problem and determinant of morbidity and mortality. Recent advancements in the fi eld of stem cell research raise hopes for stem cell-based regenerative approaches to treat acute kidney diseases. In this review, the authors summarized the latest research advances of the adult resident renal progenitor cells（ARPCs） on kidney repair, the role of ARPCs on tubular regeneration after acute kidney injury, the current understanding of the mechanisms rela ted to ARPC activation and modulation, as well as the challenges that remain to be faced.

CD34^(+)内皮祖细胞和内皮抑素在肾细胞癌组织中的表达

目的探讨CD34^(+)内皮祖细胞(EPCs)、内皮抑素(ES)在肾细胞癌(RCC)组织中的表达及其在RCC进展中的意义。方法应用免疫组化S-P法检测96例RCC组织和20例正常肾组织中CD34^(+)EPCs、ES蛋白的表达,并对其与各临床病理特征的关系进行分析。结果RCC组织中CD34^(+)EPCs、ES阳性率均高于正常肾组织的(χ^(2)=47.126,P<0.001;χ^(2)=41.184,P<0.001)。RCC组织中CD34^(+)EPCs、ES表达的与肿瘤大小、临床分期、病理分级有关(χ^(2)=13.942,P<0.001;χ^(2)=21.009,P<0.001;χ^(2)=11.107,P=0.004)。RCC组织中CD34^(+)EPCs与ES表达呈正相关(r=0.226,P=0.027)。结论CD34^(+)EPCs和ES在RCC发生、发展的过程中有着重要的作用,两者联合检测对RCC患者诊断、治疗和预后评估有指导意义。

Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis

AIM: To investigate whether fetal kidney stem cells(f KSC) ameliorate cisplatin induced acute renal failure(ARF) in rats and promote renal angiogenesis.METHODS: The f KSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3rd passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of f KSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of f KSC in ARF, f KSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nickend labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor(VEGF), hypoxia-inducible factor(HIF)-1α and endothelial nitric oxide synthase(eN OS) by western blot.RESULTS: The fK SC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well asrenal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin(CK)18 and CK19 positive epithelial cells. Administration of fK SC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3(2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7(0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fK SC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis(P < 0.05) of renal cells. The kidneys of fK SC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields(HPF), P < 0.05], and on day 7(21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fK SC treated rats had an upregulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eN OS on day 3(P < 0.05).CONCLUSION: Our study shows that fK SC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.

Renal stem cell reprogramming: Prospects in regenerative medicine

Stem cell therapy is a promising future enterprise for renal replacement in patients with acute and chronic kidney disease, conditions which affect millions worldwide and currently require patients to undergo lifelong medical treatments through dialysis and/or organ transplant. Reprogramming differentiated renal cells harvested from the patient back into a pluripotent state would decrease the risk of tissue rejection and provide a virtually unlimited supply of cells for regenerative medicine treatments, making it an exciting area of current research in nephrology. Among the major hurdles that need to be overcome before stem cell therapy for the kidney can be applied in a clinical setting are ensuring the fidelity and relative safety of the reprogrammed cells, as well as achieving feasible efficiency in the reprogramming processes that are utilized. Further, improved knowledge about the genetic control of renal lineage development is vital to identifying predictable and efficient reprogramming approaches, such as the expression of key modulators or the regulation of geneactivity through small molecule mimetics. Here, we discuss several recent advances in induced pluripotent stem cell technologies. We also explore strategies that have been successful in renal progenitor generation, and explore what these methods might mean for the development of cell-based regenerative therapies for kidney disease.

保元汤对慢性肾衰患者红系祖细胞的作用及临床疗效观察

用保元汤治疗虚证慢性肾功能衰竭(CRF)患者12例,观察该方对红系集落形成的作用,同时作临床疗效分析。结果显示该方能降低血肌酐,显效4例,有效6例。CRF血清对正常骨髓红系集落形成有抑制作用,治疗后这种抑制作用减弱,患者Hb增高。且Hb与红系集落形成呈正相关,与血肌酐呈负相关。提示保元汤改善CRF贫血与提高红系造血祖细胞增殖、分化能力及降低血肌酐有关。

细胞生长因子联合诱导ES细胞向肾脏前体细胞分化的实验研究

目的:探讨细胞生长因子联合诱导胚胎干细胞(embryonic stem cell,ES细胞)向肾脏前体细胞定向分化的实验技术,为应用胚胎干细胞进行肾脏再生提供实验基础。方法:小鼠ES细胞悬浮培养2d制备成拟胚体,RA、activin-A、Bmp7三种生长因子诱导培养5d设为A组,不加生长因子培养设为对照(B组),生长因子培养5天后继续以肾脏上皮细胞生长培养液培养7d设为C组,免疫荧光检测Pax2、Bry、WT1蛋白表达情况,流式细胞技术检测Pax2、Bry、CD24阳性细胞比例,realtimePCR检测Pax2、Bry、Osr1、Lim1、WT1、Six2、Sall1、AQP1、CD24、PDGFR基因表达情况。结果:免疫荧光染色结果显示A组细胞Pax2、Bry、WT1的表达明显高于B组;流式细胞技术检测显示A组Pax2、Bry、CD24阳性细胞比例较B组增加,C组表达Pax2、Bry的阳性细胞比例较A组明显升高;realtimePCR检测显示A组Pax2、Bry、Osr1、Lim1、WT1表达较B组明显增加(P<0.05或P<0.01),C组表达CD24基因较A组明显升高(P<0.05)。结论:胚胎干细胞在体外生长因子联合诱导条件下能够分化为早期肾脏前体细胞,并表达部分成熟肾脏细胞的标记物。

Stem cells for spine surgery

In the past few years, stem cells have become the focus of research by regenerative medicine professionals and tissue engineers. Embryonic stem cells, although capable of differentiating into cell lineages of all three germ layers, are limited in their utilization due to ethical issues. In contrast, the autologous harvest and subsequent transplantation of adult stem cells from bone marrow, adipose tissue or blood have been experimentally utilized in the treatment of a wide variety of diseases ranging from myocardial infarction to Alzheimer's disease. The physiologic consequences of stem cell transplantation and its impact on functional recovery have been studied in countless animal models and select clinical trials. Unfortunately, the bench to bedside translation of this research has been slow. Nonetheless, stem cell therapy has received the attention of spinal surgeons due to its potential benefits in the treatment of neural damage, muscle trauma, disk degeneration and its potential contribution to bone fusion.

C3aR在调节人壁层肾祖细胞源性的足细胞再生中的作用和机制研究

目的:探讨C3a受体(C3a receptor,C3aR)在人壁层肾祖细胞(CD24^(+)CD133^(+)PEC)向足细胞分化中的作用及其机制。方法:分离和培养原代人CD24^(+)CD133^(+)PEC,诱导分化为足细胞并检测C3aR在分化过程中的表达。构建C3a分泌性表达慢病毒,感染CD24^(+)CD133^(+)PEC,获得C3a过表达细胞株(C3a过表达组)。检测C3a过表达组、空载体组和未感染组细胞的活力、分化情况及E-cadherin、p-ERK1/2的表达。结果:成功分离出CD24^(+)CD133^(+)PEC并诱导其分化为表达Wilms肿瘤蛋白1(Wilms tumor protein 1,WT1)的足细胞,C3aR在分化过程中表达显著降低。C3a过表达组的细胞活力较空载体组和未感染组显著增强(P<0.01);诱导分化第2天,C3a过表达组细胞WT1的表达较空载体组和未感染组显著降低(P<0.01),E-cadherin及p-ERK1/2的表达较空载体组和未感染组显著升高(P<0.01)。结论:C3aR参与了人CD24^(+)CD133^(+)PEC向足细胞分化的过程,C3a过表达促进CD24^(+)CD133^(+)PEC的增殖并抑制其向足细胞分化,可能是通过ERK1/2信号通路发挥作用的。

血必净注射液在内皮祖细胞修复脂多糖诱导的肾内皮细胞损伤中的作用及机制

目的探究血必净注射液在内皮祖细胞修复脂多糖诱导的肾内皮细胞损伤中的作用及机制。方法提取并鉴定大鼠内皮祖细胞和肾脏微血管内皮细胞(RMEC),收集内皮祖细胞的培养上清作为条件培养基(CM)。取肾脏微血管内皮细胞,分为6组,空白组正常培养,模型组给予脂多糖(LPS)培养,CM+LPS组、CM+LPS+血必净12.5 mg/mL组、CM+LPS+血必净25 mg/mL组、CM+LPS+血必净50 mg/mL组给予相应干预。MTT法检测各组细胞增殖情况,Transwell小室法检测细胞迁移能力,小管形成实验检测体外血管新生能力,流式细胞术检测细胞凋亡情况,qRT-PCR法检测血管内皮生长因子(VEGF)和碱性成纤维生长因子(bFGF)表达情况,Western blot法检测NF-κB表达情况。结果模型组细胞增殖检测OD值及VEGF、bFGF相对表达量均明显低于空白组,细胞迁移数、小管形成数均明显少于空白组,细胞凋亡率及NF-κB相对表达量明显高于空白组,差异均有统计学意义(P均<0.05);CM+LPS组及血必净各组细胞增殖检测OD值及VEGF、bFGF相对表达量均明显高于模型组,细胞迁移数、小管形成数均明显多于模型组,细胞凋亡率及NF-κB相对表达量明显低于模型组,且血必净各组上述指标改善情况均明显优于CM+LPS组(P均<0.05)。结论血必净注射液可通过抑制NF-κB信号通路而促进内皮祖细胞修复脂多糖诱导的肾内皮细胞损伤。

红细胞生成素对慢性肾衰竭大鼠外周血内皮祖细胞SDF-1及其受体表达的影响

目的:观察红细胞生成素(EPO)对慢性肾衰竭(CRF)大鼠外周血内皮祖细胞(EPCs)归巢因子表达的影响。方法:采用分阶段5/6肾切除制备大鼠CRF模型。实验动物随机分为3组:假手术组、CRF组和EPO治疗组。从第3周开始,治疗组大鼠每次皮下注射重组人EPO 50 U/kg,每周3次,共6周。8周时取其外周血分离与培养EPCs,并检测EPCs的功能。采用实时荧光定量PCR和Western blotting检测外周血EPCs基质细胞衍生因子1(SDF-1)及其受体(CXCR4)、EPO及其受体(EPOR)mRNA和蛋白的表达。结果:与CRF组比较,EPO治疗可上调外周血EPCs中SDF-1和CXCR4 mRNA及蛋白的表达(均P<0.05);此外,EPO还可上调CRF大鼠外周血EPCs的EPO及EPOR mRNA和蛋白的表达(均P<0.05)。结论:EPO对慢性肾衰竭大鼠外周血EPCs的动员可能与其增加外周血EPCs的SDF-1及其受体表达有关。

成体肾脏干/祖细胞参与急性肾脏损伤的修复

人类肾脏对于缺血和毒性损伤均非常敏感,肾小管坏死和炎症反应的激发均将导致急性肾脏损伤。尽管肾小管在遭遇损伤后有一定的再生能力,但在多数情况下肾脏的修复能力无法满足再生的需要。成体肾脏干/祖细胞可能通过分化并最终融合入肾组织的方式参与肾脏修复,或者由旁分泌因子诱导、促进肾脏的修复和再生。上皮细胞可暂时获得祖细胞的表型而进一步增殖,最终取代损伤的肾小管细胞。

免疫磁珠法分离纯化人肾癌CD133^+细胞实验研究

目的:本研究通过分析CD133+细胞在肾细胞癌患者肿瘤组织的表达情况,寻找肾癌中CD133+细胞分离纯化方法,探寻CD133+细胞与肾癌发生中的作用。方法:①应用胎盼蓝拒染试验检测细胞活性。②应用免疫磁珠法分离纯化肿瘤组织中CD133+细胞。③应用流式细胞仪检测肾透明细胞癌标本中CD133+细胞表达情况。结果:30例肾透明细胞癌中CD133+比为(13.08±1.39)%,与病理分级、临床分期无关。免疫磁珠法分离纯化CD133+纯度为(75±2.4)%,细胞活性不受影响。结论:免疫磁珠技术纯化肾癌CD133+细胞成熟有效,CD133+细胞可能是肾癌干细胞。

阿托伐他汀对慢性肾衰竭大鼠外周血内皮祖细胞的影响

目的探讨阿托伐他汀对慢性肾衰竭大鼠外周血内皮祖细胞(endothelial progenitor cells,EPCs)数量和功能的影响。方法采用分阶段5/6肾切除制备大鼠慢性肾衰竭模型。实验动物随机分为4组:假手术组、慢性肾衰竭组(模型组)、8mg.kg-1.d-1阿托伐他汀干预组(小剂量组)、16mg.kg-1.d-1阿托伐他汀干预组(大剂量组)。大鼠阿托伐他汀灌胃8周后,取其外周血分离与培养EPCs,并检测EPCs数量及其增殖、黏附能力。结果与假手术组比较,慢性肾衰竭大鼠外周血EPCs数量及其增殖、黏附能力均下降(P<0.05)。应用阿托伐他汀能明显增加慢性肾衰竭大鼠外周血EPCs数量,改善外周血EPCs增殖、黏附能力(P<0.05),并且呈剂量依赖性。结论阿托伐他汀可改善慢性肾衰竭大鼠外周血EPCs的数量和功能。

红细胞生成素对慢性肾衰竭大鼠外周血内皮祖细胞Ang-1表达的影响

目的:观察红细胞生成素(EPO)对慢性肾衰竭大鼠(CRF)外周血内皮祖细胞(EPC)血管生成素-1(Ang-1)表达的影响。方法:采用分阶段5/6肾切除制备大鼠CRF模型。实验动物随机分为3组:假手术组、CRF模型组、EPO治疗组。从第3周开始,治疗组大鼠每次皮下注射重组人EPO 50 U/kg,每周3次,共6周。8周时取其外周血分离与培养EPC,并检测EPC的功能。采用实时荧光定量PCR和western印迹方法检测外周血EPC的Ang-1 mRNA和蛋白的表达。结果:与模型组比较,EPO治疗能提高CRF大鼠外周血EPC数量及其增殖、黏附与形成血管腔样结构的能力(均P<0.05);并可上调外周血EPC的Ang-1 mRNA及蛋白的表达(均P<0.05)。结论:EPO可动员慢性肾衰竭大鼠外周血EPC,并能增加外周血EPC血管生成素1表达。

血液透析患者内皮祖细胞的变化及其与心血管危险因素的关系

目的检测血液透析患者内皮祖细胞数量(EPCs)和功能的改变,并探讨其与心血管危险因素的关系。方法采用密度梯度离心法分离培养慢性肾功能衰竭长期血液透析患者和健康组外周血单个核细胞,将其接种在人纤维连接蛋白包被培养板,7d后取贴壁细胞进行Di-LDL和FITC-UEA-I双染色,并通过流式细胞仪检测其表面标志CD34、CD133、KDR,以鉴定EPCs。采用改良的Boy-den小室、黏附功能检测评价其迁移和黏附能力,并与性别、高血压、糖尿病、吸烟及高脂血症等心血管危险因素和Framingham危险评分进行统计学分析。结果长期血液透析患者EPCs的数量和迁移、黏附功能都低于正常健康者,差异有统计学意义(P<0.05);有糖尿病的患者EPCs的数量和黏附、迁移功能明显降低(P<0.05),有高血压的患者EPCs的数量和迁移能力明显降低(P<0.05),而性别、血脂和吸烟对EPCS有一定的影响但差异无统计学意义;Framingham危险评分与患者EPCs的数量和迁移、黏附功能分别呈明显负相关(r=-0.623,-0.658,-0.625,P<0.05)。结论长期血液透析患者的EPCs数量和功能降低,存在血管新生和内皮修复的缺陷,增加了患冠心病的风险。

长期血液透析患者内皮祖细胞变化与同型半胱氨酸的关系

目的检测长期血液透析患者内皮祖细胞（EPCs）数量和功能的改变，并探讨其与同型半胱氨酸的关系。方法采用密度梯度离心法分离培养长期血液透析患者和健康者的外周血单个核细胞，将其接种在人纤维连接蛋白包被培养板，7d后取贴壁细胞进行Dil—LDL和FITC—UEA-I双染色，并通过流式细胞仪检测其表面标志CD34、CD133、KDR，以鉴定EPCs。采用改良的Boyden小室、粘附功能检测评价其迁移和粘附能力，并与患者血清半胱氨酸含量进行相关性分析。结果患者EPCs的数量和迁移、粘附功能都低于健康者，差异有统计学意义（P〈0．05）；患者血清半胱氨酸含量[（57．10±24．76）umol／L]显著高于健康者[（6．75±3．58）umol／L]（P〈0．05），并且含量与患者EPCs的数量和迁移、粘附功能分别呈明显负相关（P〈0．05）。结论长期血液透析患者的EPCs数量减少，功能降低，可能与患者的高同型半胱氨酸血症有关。

长期血液透析慢性肾功能衰竭患者内皮祖细胞的变化及其与Framingham危险评分的关系

目的检测血液透析患者内皮祖细胞数量(EPCs)和功能的改变,并探讨其与Framingham危险评分的关系。方法采用密度梯度离心法分离培养慢性肾功能衰竭长期血液透析患者和对照组外周血单个核细胞,将其接种在人纤维连接蛋白包被培养板,7 d后取贴壁细胞进行Di-LDL和FITC-UEA-I双染色.并通过流式细胞仪检测其表面标志CD34、CD133、KDR,以鉴定EPCs。采用改良的Boyden小室、黏附功能检测评价其迁移和黏附能力,并与10 a Framingham危险评分进行统计学分析。结果长期血液透析患者EPCs的数量和迁移、黏附功能均低于对照组,差别有统计学意义(P<0.05);10 a Framingham危险评分与患者EPCs的数量和迁移、黏附功能分别呈负相关(r=-0.623,-0.658,-0.625,P<0.05)。结论长期血液透析患者的EPCs数量和功能降低,存在血管新生和内皮修复的缺陷,增加了患冠心病的风险。

血管内皮祖细胞移植对大鼠重症急性胰腺炎肾损伤的保护作用

目的:探讨内皮祖细胞(endothelial progenitor cells,EPCs)对大鼠重症急性胰腺炎(severe acute pancreatitis,SAP)合并肾损伤的保护作用。方法:通过共沉淀法制备超顺磁性氧化铁(superparamagnetic iron oxide,SPIO);密度梯度离心法分离培养SD大鼠骨髓来源EPCs;使用SPIO标记EPCs;将72只SD大鼠随机分为对照组、SAP组和SPIOEPCs组,采用胰腺被膜下均匀注射5%牛磺胆酸钠制作SAP模型。对照组和SAP组经尾静脉注射生理盐水,SPIOEPCs组注射等量的SPIO-EPCs;分别于造模2,6,12 h检测各组大鼠血清中淀粉酶、尿素氮、肌酐和肿瘤坏死因子-α(TNF-α)的水平;苏木精染色观察肾组织各时相病理变化及进行病理评分,普鲁士蓝染色观察肾组织各时相铁颗粒含量。结果:SAP组2,6,12 h的血清淀粉酶、尿素氮、肌酐和TNF-α的水平较对照组明显升高(P<0.01);而SPIO-EPCs组均显著低于SAP组(P<0.01)。与对照组比较,SAP组2,6,12 h肾病理改变明显加重,肾病理评分明显提高(P<0.05);与SAP组比较,SPIO-EPCs组肾病理改变明显减轻,肾病理评分明显降低(P<0.05)。普鲁士蓝染色见SPIOEPCs组在2,6和12 h肾组织血管内皮铁颗粒含量逐渐增多。结论:移植EPCs可减少炎症介质释放,减轻SAP肾的血管内皮损伤,对大鼠SAP肾损伤具有保护作用。