【作者】 唐荣；

【导师】 周巧玲；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 研究背景高血压病致肾损害的发病率逐年上升,已成为终末期肾脏疾病（End Stage Renal Disease,ESRD）的主要原因之一。以往对高血压肾损害的研究主要集中在肾小球,近年来认为肾小管间质损害程度比肾小球损害程度更能反映慢性肾功能衰竭的进展及预后,而肾小管上皮细胞凋亡在肾小管间质病变的发生发展中起重要作用。Klotho基因是新发现的与人类衰老相关的基因,主要表达于肾脏和大脑脉络膜中,尤以肾小管上皮细胞表达明显。Klotho基因缺陷小鼠可表现出一系列类似人类衰老的表型,慢性肾功能衰竭患者常出现以上许多类似表现。近年来已有学者在5/6肾切除大鼠、急性肾衰竭大鼠、ICR衍生性。肾小球肾炎小鼠、肾脏上皮细胞癌、慢性肾功能衰竭及其钙磷代谢紊乱等研究中对Klotho基因在相关肾脏疾病发病机制中的作用进行了探讨。但目前尚缺乏Klotho基因与高血压肾损害的相关研究。冬虫夏草（cordyceps sinensis,CS）是我国传统中药中的瑰宝,具有良好的肾保护作用,但其作用机制尚不清楚。本研究拟采用血管紧张素Ⅱ（AngiotensinⅡ,AngⅡ）干预的大鼠肾小管上皮细胞（NRK-52E）和自发性高血压大鼠（spontaneously hypertensive rat,SHR）作为研究对象,用冬虫夏草和血管紧张素Ⅰ型受体拮抗剂——氯沙坦（Losartan,Los）分别对其进行干预,观察Klotho基因及凋亡相关基因p53、p21等的表达和肾小管上皮细胞的凋亡情况,探讨Klotho在高血压肾损害肾小管上皮细胞凋亡中的作用及冬虫夏草肾脏保护作用的可能机制。第一章血管紧张素Ⅱ对NRK-52E中Klotho表达和凋亡的影响及冬虫夏草对其的干预作用目的研究AngⅡ干预NRK-52E后Klotho、p53和p21的表达和细胞凋亡情况以及冬虫夏草提取液对其的影响,探讨冬虫夏草对AngⅡ诱导的肾小管上皮细胞凋亡的影响及机制。方法1.NRK-52E按AngⅡ浓度梯度0（对照组）,10^-10,10^-9,10^-8,10^-7,10^-6mol/L分6组培养24小时,选择最佳的AngⅡ干预浓度后按时间梯度0（对照组）,6,12,18,24小时分5组培养,确定最佳干预时间。冬虫夏草提取液按浓度梯度0（对照组）,5,10,20,40,80mg/L单独或与AngⅡ共同培养NRK-52E 24,48,72小时,确定其最佳干预浓度及干预时间。采用MTT比色法检测细胞增殖;逆转录-多聚酶链反应（RT-PCR）检测Klotho mRNA表达;分光光度法检测caspase-3的活性;AnnexinV-PI双染法及流式细胞仪检测细胞凋亡。2.根据上述实验结果选择AngⅡ(10^-8mol/L)及冬虫夏草提取液（40mg/L）的最佳干预浓度培养NRK-52E 24小时,以氯沙坦(10^-5mol/L)作为阳性对照药物。设立对照组,AngⅡ组,AngⅡ+CS组,AngⅡ+Los组和AngⅡ+CS+Los组。分别采用RT-PCR及Western Blot检测Klotho、p53、p21 mRNA及蛋白表达;分光光度法检测caspase-3的活性;AnnexinV-PI双染法及流式细胞仪检测细胞凋亡。结果1.AngⅡ(10^-9mol/L～10^-6mol/L)可显著下调NRK-52E中KlothomRNA的表达（P＜0.01）;随着AngⅡ浓度增加(10^-10mol/L～10^-6mol/L)及干预时间延长（6h～24h）,NRK-52E细胞caspase-3活性逐渐增高,细胞凋亡率逐渐增加（P＜0.01）。2.冬虫夏草提取液（5mg/L～80mg/L）单独对NRK-52E中KlothomRNA表达无明显影响（P＞0.05）;冬虫夏草提取液（5mg/L～40mg/L）可拮抗AngⅡ诱导的Klotho基因低表达,浓度越高,其作用越明显（P＜0.05或P＜0.01）;与AngⅡ组比较,随着冬虫夏草提取液干预浓度增加（10mg/L～40mg/L）,NRK-52E细胞增殖逐渐增加,caspase-3活性及细胞凋亡率逐渐降低（P＜0.01）。3.AngⅡ组Klotho mRNA及蛋白表达明显下调,p53及p21表达上调,caspase-3活性增高,细胞凋亡率增加（P＜0.01）;经冬虫夏草或/和氯沙坦干预后,Klotho mRNA及蛋白表达上调,p53和p21表达下调,caspase-3活性及细胞凋亡率降低（P＜0.05或P＜0.01）。联合用药组较单用药组相比差异无统计学意义（P＞0.05）。小结1.AngⅡ可通过抑制NRK-52E中Klotho mRNA及蛋白的表达,上调p53及p21的表达,激活caspase-3,从而诱导肾小管上皮细胞凋亡,这可能是其在高血压肾损害肾小管细胞凋亡中的作用机制之一。2.冬虫夏草提取液单独对NRK-52E中Klotho mRNA表达无明显影响;冬虫夏草提取液可拮抗AngⅡ对NRK-52E细胞增殖的抑制和诱导的Klotho基因低表达及细胞凋亡,其抗凋亡作用可能与其增加肾小管上皮细胞Klotho的表达,抑制p53/p21凋亡通路和caspase-3的激活有关。第二章自发性高血压模型鼠肾脏Klotho的表达和肾小管上皮细胞凋亡情况及冬虫夏草对其的干预作用目的观察自发性高血压大鼠（SHR）肾脏Klotho的表达和肾小管上皮细胞凋亡情况以及冬虫夏草对其的影响,探讨Klotho基因在高血压肾损害肾小管上皮细胞凋亡中的作用,以及冬虫夏草肾保护作用的相关机制。方法20只22周龄雄性SHR随机分为4组（5只/组）:高血压模型组（SHR组）,冬虫夏草组（CS组,CS 5g/kg/d灌胃）,氯沙坦组（Los组,Los 50mg/kg/d灌胃）,冬虫夏草+氯沙坦组（CS+Los组,CS 5g/kg/d+Los 50mg/kg/d联合灌胃）;22周龄雄性Wistar-Kyoto大鼠（WKY组）6只作为对照组,共喂养8周。检测各组大鼠体重、尾动脉收缩压、24小时尿蛋白定量（Upro）、尿N-乙酰-β-D-氨基葡萄糖苷酶（NAG酶）、血尿素氮（BUN）、血肌酐（Scr）。采用HE及Masson染色方法观察。肾脏病理改变;原位缺口末端标记法（TUNEL法）检测肾小管上皮细胞凋亡;免疫组织化学法检测Klotho蛋白表达;RT-PCR检测Klotho、p53和p21 mRNA表达;Western Blot检测Klotho、p53、p21和活性caspase-3蛋白表达。结果1.实验第8周（30周龄）,SHR组大鼠的收缩压、Upro、尿NAG酶、BUN和Scr均明显高于WKY组,并出现肾脏小动脉管壁增厚及玻璃样变,肾小管上皮细胞空泡及颗粒变性,肾小管间质炎性细胞浸润,部分肾小球毛细血管缺血性皱缩、硬化等高血压肾损害的病理特征;经冬虫夏草或/和氯沙坦干预后,SHR的Upro、尿NAG酶、BUN和Scr均较未干预组降低,高血压引起的肾脏病理损害减轻（P＜0.05或P＜0.01）;冬虫夏草对血压变化无明显影响（P＞0.05）。2.Klotho蛋白主要表达于肾小管上皮细胞胞浆及胞核中,且以远曲小管上皮细胞为主。SHR组大鼠肾脏Klotho mRNA及蛋白表达较WKY组明显下调,同时p53和p21表达上调,活性caspase-3蛋白表达上调,肾小管上皮细胞凋亡率增加（13.316%±0.645%vs 0.810%±0.115%,P＜0.01）;经冬虫夏草或/和氯沙坦干预后,Klotho mRNA及蛋白表达上调,p53、p21和活性caspase-3表达下调,肾小管上皮细胞凋亡率降低（分别下降为7.559%±0.520%、7.928%±0.370%和7.373%±0.363%,P＜0.01）。而各干预组间以上各项观察指标的差异无统计学意义（P＞0.05）。小结1.SHR模型鼠血压升高,伴有尿蛋白排泄增多,尿NAG酶、血尿素氮和血肌酐升高,经冬虫夏草干预后其尿蛋白排泄减少,尿NAG酶、血尿素氮和血肌酐均降低;冬虫夏草对血压无明显影响。2.SHR模型鼠出现肾间质损伤的同时,肾脏Klotho mRNA及蛋白表达明显下调,p53和p21的表达上调,活性caspase-3表达上调,肾小管上皮细胞凋亡增多。3.冬虫夏草制剂在使SHR肾间质损伤减轻的同时,可上调肾脏Klotho mRNA及蛋白表达,抑制p53、p21的表达和caspase-3的活化,减少肾小管上皮细胞凋亡,其肾保护作用并非通过血压下降而实现。结论1.AngⅡ可使NRK-52E中Klotho基因的表达下调,同时使p53和p21的表达上调,活化caspase-3,从而诱导肾小管上皮细胞凋亡过程,这可能是其在高血压肾损害肾小管细胞凋亡中的作用机制之一。2.冬虫夏草提取液可拮抗AngⅡ诱导的肾小管上皮细胞凋亡,其抗凋亡作用可能与增加肾小管上皮细胞Klotho基因的表达,抑制p53/p21凋亡通路和caspase-3的激活有关。3.SHR模型鼠出现肾间质损伤的同时,肾脏Klotho基因的表达明显下调,p53和p21的表达上调,活性caspase-3表达上调,肾小管上皮细胞凋亡率增加。4.冬虫夏草制剂在使SHR肾间质损伤减轻的同时,可上调肾脏Klotho基因的表达,抑制p53、p21的表达和caspase-3的活化,降低肾小管上皮细胞凋亡率,且其肾保护作用并非通过血压的下降而实现。更多还原

【Abstract】 BackgroundThe rapidly increasing incidence of hypertensive renal damage has became one of the most important reasons of End Stage Renal Disease （ESRD）.In the past,research related to hypertensive renal damage has focused on the renal glomerulus.Recently,it has been considered that the progression and prognosis of chronic renal failure correlates better with tubulointerstitial damage than with glomerular damage.Renal tubular epithelial cell apoptosis plays an important role in this process.Klotho gene,an new anti-aging gene,is predominantly detected in the kidney and cerebullar choroid,significantly in renal tubular epithelial cells.Its deletion in mice results in phenotypes resembling human aging, features resembling clinical manifestation of patients with chronic renal failure.Recently,several studies have confirmed the association between Klotho gene and renal diseases including acute renal failure rat,5/6 nephrectomized rat,ICR-derived glomerulonephritis（ICGN） mice,renal cell carcinoma,chronic renal failure and abnormalities in calcium and phosphorus metabolism.So far,there is no research about the association between Klotho gene and hypertensive renal damage.Cordyceps sinensis（CS） is a type of rare traditional Chinese medicine with excellent reno-protective effects.However,less is known about the mechanisms of its reno-protective effects.In the present study, we used rat proximal renal tubular epithelial cells（NRK-52E） stimulated by AngiotensinⅡ（AngⅡ） and spontaneously hypertensive rat（SHR）, and drug intervention including cordyceps sinensis and the AT1 receptor antagonist Losartan（Los） to observe the expression of Klotho and apoptosis-related genes p53,p21,and tubular epithelial cells apoptosis. Then we investigated the role of Klotho in tubular epithelial cells apoptosis in hypertensive renal damage and mechanisms of reno-protective effects of cordyceps sinensis to provide evidence for treatment of hypertensive renal damage.Part 1 Effects of Cordyceps Sinensis Extract on Klotho expression and Apoptosis in NRK-52E Induced by AngiotensinⅡMethods1.NRK-52E cells were incubated with different concentrations of AngⅡ[0（control）,10^-10,10^-9,10^-8,10^-7,10^-6mol/L]for 24 hours.The optimal concentration of AngⅡwas selected to stimulate culture cells for 0（control）,6,12,18,24 hours to select the best cultivation time; NRK-52E were incubated with increasing concentrations of cordyceps sinensis extract[0（control）,5,10,20,40,80mg/L]with or without AngⅡfor 24,48,72 hours to select the optimal dose and time of cordyceps sinensis extract.Cell viability was determined by MTT assay; reverse transcription-polymerase chain reaction（RT-PCR） were applied to evaluate the Klotho mRNA expression;the caspase-3 activity was analyzed by caspase 3 activity assay kit;the cell apoptosis was assessed by Annexin V-FITC/PI double staining and flow cytometry.2.According to the above results,the optimal concentration of AngⅡ(10^-8mol/L) and cordyceps sinensis extract（40mg/L） were selected to incubated NRK-52E for the best cultivate time（24 hours）, Losartan(10^-5mol/L) as the positive control drug.Cells were divided into five groups,which were control、AngⅡ、AngⅡ+CS、AngⅡ+Los and AngⅡ+CS+Los.The mRNA and protein expression of Klotho、p53 and p21 were measured by RT-PCR and Western Blot;caspase-3 activity was evaluated by caspase-3 activity assay kit;cell apoptosis was determined by Annexin V-FITC/PI double staining and flow cytometry.Results1.AngⅡ(10^-9mol/L～10^-6mol/L) down-regulated Klotho mRNA expression obviously in NRK-52E compared with control（P<0.01）; caspase-3 activity and apoptotic rate in NRK-52E increased gradually as concentrations of AngⅡincreased(10^-10mol/L～10^-6mol/L) and time prolonged（6h～24h）（P<0.01）.2.There was no evidence that cordyceps sinensis extract（5mg/L～80mg/L） alone had any effect on Klotho mRNA expression in NRK-52E （P>0.05）;cordyceps sinensis extract（5mg/L～40mg/L） up-regulated Klotho mRNA expression reduced by AngⅡgroup,the effects increased gradually as the concentrations increased（P<0.05 or P<0.01）;as compared with AngⅡgroup,caspase-3 activity and apoptotic rate in NRK-52E increased gradually as concentrations of cordyceps sinensis increased（10mg/L～40mg/L）.3.AngⅡsignificantly down-regulated mRNA and protein expression of Klotho,enhanced p53 and p21 expression、caspase-3 activity and apoptotic rate of NRK-52E（P<0.01）.In the presence of either cordyceps sinensis or Losartan or both,down-regulation of Klotho expression was countered,the increment in p53 and p21 expression were inhibited,caspase-3 activity and apoptotic rate decreased（P<0.05 or P<0.01）.No cooperative effects were observed by these two drugs （P>0.05）.Conclusions1.AngⅡdown-regulates Klotho expression,up-regulates the expression of p53 and p21,increases caspase-3 activity,and induces apoptosis in NRK-52E,which suggests it maybe one of the mechanisms of AngⅡinvolved in renal tubular epithelial cells apoptosis in hypertensive renal damage.2.Different concentrations of cordyceps sinensis extract alone have no significant effect on Klotho mRNA expression in NRK-52E; cordyceps sinensis extract can increase cell proliferation and Klotho expression,and inhibit apoptosis induced by AngⅡin NRK-52E;The anti-apoptotic effects of cordyceps sinensis extract are related to increased expression of Klotho down-regulated by AngⅡ,inhibition of p53/p21 apoptotic pathway and caspase-3 activition. Part 2 Effects of Cordyceps Sinensis on Klotho Expression in Kidneys and Apoptosis in Renal Tubular Epithelial Cells of Spontaneously Hypertensive RatsObjective To observe renal Klotho expression and tubular epithelial cell apoptosis in spontaneously hypertensive rats and the effects of cordyceps sinensis on them,in order to study the role of Klotho in tubular epithelial cells apoptosis in hypertensive renal damage and mechanisms of reno-protective effects of cordyceps sinensis.Methods Twenty 22 week-old male SHR were randomly divided into four groups for five each:hypertensive group（SHR）,CS group（5g/kg/d）, Los group（50 mg/kg/d） and CS + Los group（treated with CS 5g/kg/d and Los 50mg/kg/d）;six 22 week-old male Wistar-Kyoto rats（WKY） as the control group.After 8 weeks,the body weigh,cuff-tail blood pressure, 24 hour urinary protein（Upro）,urinary N-acetyl-β-D-glucosaminidase （NAGase）,blood urea nitrogen（BUN） and serum creatinine（Scr） were measured.Renal pathological changes were viewed by HE and Masson staining;in situ detection of DNA fragmentation（TUNEL assay） was applied to evaluate renal tubular epithelial cell apoptosis;the mRNA expression of Klotho,p53 and p21 were detected by RT-PCR;the protein expression of Klotho,p53,p21 and cleaved-caspase-3 were tested by Western Blot.Results 1.At 30 weeks,the blood pressure,Upro,urine NAGase,BUN and Scr of untreated SHR were significantly higher than WKY rats,and typical pathological characteristics of hypertensive renal damage including thickness and hyalinization of glomerular arteriole walls, vacuolar and particulate degeneration of tubular cells,inflammatory cell infiltration in interstitial region,schemic atrophy and partial sclerosis of glomerular capillaries were founded in kidneys of SHR group（P<0.05）, cordyceps sinensis or/and Losartan reduced Upro,NAGase,BUN and Scr, and ameliorated renal pathological damage（P<0.05）.Cordyceps sinensis had no significant effect on blood pressure（P>0.05）.2.Positive stains of Klotho protein were mainly demonstrated in the cytoplasm and nucleus of renal distal convoluted tubule.Compared with WKY rats,Klotho mRNA and protein expression were significantly reduced in kidneys in SHR,concomitant with the increased p53、p21 expression,along with increased cleaved caspase-3 protein expression, and tubular cells apoptotic rate（13.316%±0.645%vs 0.810%±0.115%, P<0.01）.Aider treatment with cordyceps sinensis or/and Losartan, Klotho mRNA and protein expression were up-regulated,while the expression of p53,p21 and cleaved-caspase-3 were reduced,tubular cells apoptotic rate were decreased than SHR（decreased to 7.559%±0.520%, 7.928%±0.370%and 7.373%±0.363%separately,P<0.01）.No cooperative effects were observed by these two drugs（P>0.05）. Conclusions1.The blood pressure,Upro,urine NAGase,BUN and Scr are significantly increased in SHR,cordyceps sinensis or/and Losartan can decrease Upro,urine NAGase,BUN and Scr.cordyceps sinensis does not affect the blood pressure.2.The interstitial damage is appeared in kidneys of SHR, concomitantly the mRNA and protein expression of Klotho are down-regulated obviously,the expression of p53,p21 and cleaved-caspase -3 are up-regulated,tubular epithelial cells apoptotic rate is increased in kidneys.3.Cordyceps sinensis relieves renal interstitial damage of SHR, concomitantly increases renal Klotho expression,inhibits p53/p21 apoptotic pathway and decreases caspase-3 activation,then inhibits tubular epithelial cells apoptosis,its reno-protective effects are independent of blood pressure control.CONCLUSIONS1.AngⅡcan inhibit Klotho gene expression,increase p53,p21 expression and caspase-3 activity,and induce the apoptosis of renal tubular epithelial cells,which suggests it maybe one of the mechanisms of AngⅡinvolved in renal tubular epithelial cell apoptosis in hypertensive renal damage. 2.Cordyceps sinensis extract can reduce apoptosis induced by AngⅡ,its anti-apoptotic effects are related to increased expression of Klotho gene down-regulated by AngⅡ,inhibition of p53/p21 apoptotic pathway and caspase-3 activition.3.The interstitial damage is apparent in kidneys of SHR, concomitantly Klotho gene expression is down-regulated unambiguously, the expression of p53,p21 and cleaved-caspase-3 are up-regulated,while tubular cells apoptotic rate is increased in kidneys.4.Cordyceps sinensis relieves renal interstitial damage of SHR, concomitantly increases Klotho gene expression、inhibits p53/p21 apoptotic pathway and decreases caspase-3 activation,then inhibits tubular cell apoptosis,its reno-protective effects being independent of blood pressure control.更多还原