【作者】 刘佳；

【导师】 王笑云；

【作者基本信息】 南京医科大学 ， 肾脏病， 2010， 博士

【摘要】 随着慢性肾脏疾病（CKD）发病率的逐年上升,ESRD的发生率上升成必然趋势。导致CKD发生原因可以多种多样,但进展至ESRD的共同途径是肾脏纤维化。近几年来有关肾脏纤维化的发病机理及防治进展研究很多,虽然对已认识到的高血压,糖尿病,蛋白尿等危险因素制定了相应的治疗指南,但依然不能阻止的CKD上升趋势,说明许多研究还未能应用于临床,目前尚缺乏对CKD有效的防治措施。目的:1.探讨氟伐他汀对血管紧张素II诱导的肾小管上皮细胞增殖作用及其分泌纤维连接蛋白的影响。2.研究氟伐他汀对糖尿病肾病小鼠保护作用。3.探讨Rho激酶信号通路在糖尿病肾病间质纤维化中的作用及氟伐他汀保护机制4.探讨氟伐他汀对高糖环境下HK2 klotho蛋白表达及细胞增殖与凋亡的影响及作用机制方法:1①不同浓度(10^-9-10^-5 mol/L) AngII刺激体外培养的肾小管上皮细胞。②固定浓度AngII（10-6mol/L）刺激,不同浓度(10^-9-10^-5 mol/L)氟伐他汀干预。③在不同的时间点（24h、48h、72h）用苯唑氮蓝法（MTT）检测细胞增殖。④固定浓度AngII(10^-6mol/L)刺激肾小管上皮细胞6、12、24、48、72、96小时,不同浓度(10_-9-10^-5 mol/L)氟伐他汀干预48h,免疫印迹及细胞免疫荧光法检测纤维连接蛋白（FN）的表达。2建立糖尿病小鼠模型,观察不同剂量氟伐他汀对肾重/体重比值、肾功能、蛋白尿、IL-6、FN、平滑肌肌动蛋白（α-SMA）、足细胞相关蛋白WT-1作用及影响。研究方法有生物化学法检查血尿素氮、肌酐、血糖、胆固醇、甘油三酯等,ELISA法检测尿白蛋白/肌酐比、IL-6,Western blot检测FN、α-SMA、WT-1,肾脏病理（光镜、免疫组化）观察肾脏病理改变及FN、α-SMA在肾组织分布及量的改变。3①体外培养HK-2细胞,高糖刺激不同时间（6h、12h、24h）,western blot检测p-MYPT1（磷酸化的Rho激酶）、FN表达。②.选择高糖12h作用点,观察不同剂量氟伐他汀干预后HK-2细胞p-MYPT1、FN表达。③.高糖培养HK2细胞12h,分别观察Rho激酶激动剂LPA、甲羟戊酸及其下游异戊二烯类化合物GGPP、FPP对中剂量氟伐他汀上述作用的影响。4体外培养HK-2,观察高糖及不同浓度氟伐他汀、不同作用时间点对HK-2细胞增殖、凋亡及klotho、RhoA蛋白表达的影响,并观察分别合并MVA、FPP、GGPP对氟伐他汀作用klotho蛋白表达的影响。研究方法有苯唑氮蓝法（MTT）检测细胞增殖,Hoechst33258、检测细胞凋亡,western blot检测caspase-3、caspase-8、klotho、RhoA蛋白。结果1.①AngII促进肾小管上皮细胞增殖（P<0.05）,48小时作用最明显,且10^-5mol/L效果显著,随着浓度下降,促增殖作用下降。②氟伐他汀能够显著抑制AngⅡ的促增殖作用（P<0.05）,同样48小时抑制作用最明显,10^-5mol/L效果显著,随着浓度下降,抑制作用下降。③AngⅡ刺激肾小管上皮细胞12h后FN开始增加P<0.01),72h到达高峰。氟伐他汀干预组与AngⅡ刺激（未干预）组比较,FN表达明显下降（P<0.05）,浓度10^-5mol/L下降最明显,10^-6mol/L次之,10^-7-10^-9mol/L抑制作用没有明显的差别。2.①模型组血糖、尿白蛋白/肌酐、血尿素氮、肾重/体重比值、肾小球面积及系膜基质/肾小体面积比值,肾组织IL-6、FN、α-SMA检测均高于正常对照（P分别<0.05、0.01）,WT-1低于正常对照组（P<0.05、0.01）。各剂量氟伐他汀干预1月时,尿白蛋白/肌酐、血尿素氮、肾重/体重比值、肾小球面积及系膜基质/肾小体面积比值、IL-6水平与非干预组相比有所下降,但无显著性差异（P>0.05）;FN、α-SMA较非干预组下降有显著差异（P<0.01）,WT-1较对照组比上升有显著性（P<0.01）。中剂量连续干预2月和3月时,尿白蛋白/肌酐、血尿素氮、肾重/体重比值、肾小球面积及系膜基质/肾小体面积比值、IL-6、FN、α-SMA水平均明显低于非干预组（P分别﹤0.05、0.01）; WT-1明显高于非干预组（P<0.01）。②肾组织病理PAS显示糖尿病模型组小鼠肾小球面积和肾小体面积增大,随时间延长,系膜明显增生,细胞外基质显著增多。氟伐他汀干预后,与同期模型组小鼠相比,系膜增生及细胞外基质增多减轻。免疫组化病理显示模型组FN、α-SMA表达明显高于正常对照组,不同剂量氟伐他汀干预后FN、α-SMA表达较非干预组比均有明显下降（P<0.05、0.01）。③整个实验期间,血胆固醇及甘油三酯水平各组间均无显著差异（P>0.05）。3.高糖刺激HK2细胞后各时间点均可引起p-MYPT1、FN表达显著增加,与0h相比（P分别<0.05）,p-MYPT1表达12h最高、FN 24h达高峰,呈时间依赖（P分别<0.01）。不同剂量氟伐他汀(10^-7, 10^-6和10^-5 mol/L)干预后p-MYPT1和FN的表达均较未干预组降低,并呈浓度依赖性（P<0.05）。甲羟戊酸和GGPP可以逆转氟伐他汀的上述作用,使p-MYPT1和FN上升（P<0.05）,而FPP则不能（P>0.05）。LPA激动后可部分抵消氟伐他汀的上述作用,p-MYPT1、FN表达显著高于对照组（P<0.05）。4 HG培养HK-2至24⁴8h细胞增殖明显增加,与对照组比较P<0.05,而培养至72h时细胞增殖开始降低,并低于对照组P<0.05。高糖培养24～48h,细胞凋亡率及caspase3及caspase-8裂解片段增加（P分别<0.05）;高糖培养12h Klotho蛋白表达减少（P<0.05）、RhoA蛋白表达增加（P<0.05）,均呈时间依赖性。不同浓度氟伐他汀（0.1、1、10μmol/L）不同时间（12、24、48、72h）均明显抑制细胞增殖（P<0.05）,细胞增殖抑制率具有时间-浓度依赖性。各浓度氟伐他汀（0.1、1、10μmol/L）均增加Klotho蛋白表达（P<0.05）、减少RhoA蛋白表达（P<0.05）,呈浓度依赖性。减少细胞凋亡率及降低caspase-3、caspase-8的作用以浓度0.1、1、μmol/L明显（P<0.05）。氟伐他汀对HK2klotho蛋白的表达上调作用可被MVA或GGPP逆转（P<0.05）,而FPP则不能（P>0.05）。结论1氟伐他汀能抑制AngⅡ对肾小管上皮细胞的促增殖作用及促FN分泌作用,在一定范围内呈浓度依赖性。2氟伐他汀可以降低糖尿病小鼠蛋白尿和血尿素氮,抑制糖尿病小鼠肾脏IL-6 FN、α-SMA的表达,促进足细胞WT-1的表达,减少系膜细胞和细胞外基质的增多,改善糖尿病早期肾损害,减少肾脏纤维化,其肾保护作用独立于降血脂。3 Rho激酶可能是糖尿病肾病间质纤维化发生的起始信号之一,他汀类药物减少糖尿病肾病间质纤维化的作用机理与其抑制Rho激酶信号通路有关。4高糖可减少HK2 klotho蛋白表达,并同时促进细胞增殖及凋亡。他汀可部分纠正高糖对HK-2的上述作用,其机理可能与通过抑制Rho信号通路,上调HK-2 klotho蛋白有关。此结果为他汀类药物在糖尿病肾病的治疗中提供新靶点有积极作用。小结糖尿病肾病肾病发病机制复杂,炎性介质、细胞因子、各种信号通路等网络效应形成对糖尿病肾病治疗的挑战。他汀类药物独立于抗Ras、抗炎、抗氧化、抗纤维化、上调klotho的多效性作用,有望成为糖尿病肾病治疗的有力措施之一。更多还原

【Abstract】 Over the years, the number of end stage renal diseases （ESRD） has been going up steadily due to the increased incidences of chronic kidney disease （CKD）. Multiple causes can induce CKD but it is renal fibrosis that is commonly recognized as the single factor that leads to ESRD. Although there have been encouraging progress in studies regarding the mechanisms of renal fibrosis and numerous therapeutic approaches have been proposed in an attempt to control factors such as hypertension, diabetes, and proteinuria that may quicken the progression of renal disease, there seem to be no effective measures as yet in terms of preventing the development of CKD. This shows that most of the theoretical researches done so far cannot yet be applied to clinical use which really means that the prevention of CKD is still far from satisfying our expectations.Objective:1. To investigate the effects of Ffluvastatin on proliferation and fibronectin （FN） biosynthesis of human renal tubular epithelial cell induced by Angiotensin II.2. To observe Fluvastatin’s influence onto expression of IL-6 in kidney,renal interstitial fibrosis,potocyte, renal function, proteinuria and to discuss statins’protective effects on renal tissues damaged by diabetes mellitus （DM）.3. To explore the effect of Rho-kinase signal pathway in renal interstitial fibrosis of diabetic nephropathy（DN） and the renopective mechanism of fluvastatin.4. To investigate the effects and mechanism of fluvastatin on high glucose induced klotho expression, proliferation and opoptosis of human renal tubular epithelial cell（HK-2） Method:1①Cultured human renal tubular epithelial cells were treated with AngII at different concentrations (10^-9～10^-5 mol/L respectively).②Cells were treated with 10-6 mol/L AngII and different concentrations of fluvastatin(10^-9～10^-5 mol/L respectively).③The proliferation of renal tubular epithelial cells were determined by MTT colorimetry at different time（24h,.48h,72h）.④Cells were treated with AngII (10^-6 mol/L )for different time （6h, 12h, 24h, 48h, 72h, and 96h）, added different concentrations of fluvastatin(10^-9--10^-5mol/L respectively)for 48h. FN expression by renal tubular cell was detected by both western blot and immunofluorescence.2 Streptozocin（STZ）-induced diabetic model of CD-1 mice were studied. Diabitic mice were divided into three groups depend on different doses of Flufastatin treatment （5mg/kg/d,25mg/kg/d,125mg/kg/d）for 4 weeks and in dose 25mg/kg/d for 8 and 12weeks. All groups have the normal control and diabetic control. The mice were sacrificed at the end of the first, second and third month. Mouse body weight ,weight of one kidney, serum glucose（Glu）, cholesterol（ch）, triglyceride（TG）, urea nitrogen（BUN）, creatinine（Cr）, and ratio of urinary albumin /creatinine （Alb/cr）were measured before and were killed. IL-6, fibronecin （FN） ,α-Smooth muscle actin （α-SMA） and WT-1 in renal tissue were detected by ELISA or Western blotting and immunohistochemistry. PAS stain was used to observe pathomorphological changes in the renal tissues.3 Human renal proximal tubular epithelial cells（HK-2 cells） were cultured in vitro . Rho-kinase activity was expressed as phosphorylation of myosin-phosphatase target 1 （p-MYPT1）. The level of p-MYPT1 and fibronectin （FN） stimulated by high glucose was determined by Western blot at the time of 6h, 12h, 24h. Same marks were detected when high glucose cultured HK-2 cells treated with different concentrations (10^-7, 10^-6and 10^-5 mol/L) of fluvastatin for 12h,and when co-treated by10-5 mol/L of fluvastatin and lysophosphatidic acid （LPA）, MVA, GGPP, FPP respectively for 12h .4 HK-2 cells were incubated with normal glucose、high glucose、fluvastatin in different concentrations for different time. HK-2 cells proliferation and apoptosis were determined by MTT and Hoechst33258 respectively. The protein expression of caspase-3, klotho and RhoA were detected by western blotting. Furthermore, the effects of fluvastatin combined with or without mevalonate （MVA）, GGPP and FPP （essential molecules for isoprenylation of the small GTPase Rho） on klotho were observed.Result:1.①AngII can induce proliferation of human renal tubular epithelial cell（P<0.05）, it was the most significantly increased for 48h, especially at the concentration of 10-5 mol/L, this effect is reduced along with the lowering concentration.②Fluvastatin can attenuate the proliferation of human renal tubular epithelial cell induced by AngII （P<0.05）, that was significantly at the concentration of 10-5 mol/L for 48h.③Fibronectin synthesis by renal tubular epithelial cells cultured in AngII medium increased. The expression of FN started increasing after 12h, and achieved peak after 72h.④Fluvastatin inhibited FN expression by Ang II , the 10-5mol/L of fluvastatin had the most significant effect, and 10-6 mol/L had less effect than the 10-5mol/L, from 10-9 to 10-7mol/L, the fluvastatin had no effects on the expression of FN.2. （1） Blood sugar level, urinary albumin/creatinine ration, urea nitrogen in blood, kidney weight/body weight ratio, IL-6 , FN,α-SMA, renal corpuscle area, ratio of mesenterium matrix versus area of renal corpuscle in mice treated with STZ were higher than those in mice without STZ （P<0.05, 0.01 respectively ）, WT-1 in mice with STZ was lowered compared with those mice STZ free （P<0.05）. The marks mentioned above in all groups with Fluvastatin were decreased while WT-1 was increased compared to the group without Fluvastatin 1 or 2 months later （P<0.05, 0.01 respectively） . In addition, ratio of urinary albumin /creatinine as well as the IL-6 level tapered, presenting a time-dependent nature. （2） Renal pathology by PAS-stained show that STZ mice enlarged their area of glomerulus and area of renal corpuscle, and their mesenterium as well as extracellular matrix became proliferated obviously with time elapse. The mice treated with Fluvastatin alleviated their proliferation of mesenterium and extracellular matrix compared to those with the similar model but without Fluvastatin. Immunohistochemisty show that expression of FN,α-SMA in STZ mice were increased significantly compare with normal control （P<0.01） , and in STZ mice treated with Fluvastatin were decreased significantly compare to those mice without Fluvastatin （P<0.05,0.01）. （3） Serum cholesterol and triglyceride in all groups unchanged （P>0.05）.3. High glucose enhance the expression of p-MYPT1 and FN at the time of 6h, 12h, 24h compared with the time of 0h in culured HK-2cells（ P<0.05 respectively）. The increase of FN expression stimulated by high glucose was in time-dependent fashion and reached the peak at 24h（P<0.01）.The increased level of p-MYPT1 reached the peak at 12h（P<0.01）. Fluvastatin decreased high glucose-mediated level of p-MYPT1 and FN in dose-dependent manner （P<0.05）. The inhibitory effect of fluvastatin on up-regulation of p-MYPT1 and FN stimulated by high glucose was reversed by MVA, GGPP respectively （P<0.05） , partially neutralized by LPA（P<0.05） but was unchanged by FPP （P>0.05）.4. High glucos increased HK-2 cells proliferation at 24～48h（<0.05） while decreased proliferation at 72h （<0.05）. When after HK-2 incubated with high glucose 24～48h, the rate of apoptosis and cleaved caspase-3 increased, RhoA expression increased meanwhile klotho expression decreased at the time of 12h and in a time-dependent manner （<0.05 respectively）. Fluvastatin blunted the high glucose -induced response and ameliorated the decrease in klotho expression towards high levels in a dose-dependent （<0.05 respectively）. This regulatory effect on clotho was abolished by the addition of MVA and GGPP, but not FPP.Conclusion:1. Fluvastatin protects against AngII-induced proliferation and FN synthesis of human renal tubular epithelial cell in a dose-dependent fashion.2. Fluvastatin is able to reduce high glucose-induced proteinuria and improve renal function, alleviate proliferation of mesangial cells and extracellular matrix. The beneficial effect of Fluvastatin might be caused by inhibiting expression of IL-6, FN,α-SMA in renal tissue and preserving WT-1 expression in podocytes against high glucose-induced injury, so it can play the role of amelioration impairment of diabetic nephropathy early and prevent renal fibrosis. The renalprotective effects of Fluvastatin are independent on its lipid-lowering function.3. Rho-kinase may be one of the initiation signals of renal interstitial fibrosis of DN. The mechanism of decrease renal interstitial fibrosis in DN with Fluvastatin is associated with its effect of inhibiting Rho-kinase signaling pathway.4. High glucose can reduce HK-2 clotho expression and enhance cells proliferation and apoptosis。The mechanism of statins improving these effects by high glucose is related with statins inactivating the RhoA pathway, resulting in over-expression of klotho, which may contribute to the novel target of statins towards on treatment DN .In clousion, Challenges exist in the treatment of diabetic nephropathy due to its complicated pathologic mechanism and considerable cytokines, signal pathways and crosstalking. Statins is expected to play a potent role in the treatment of diabetic nephropathy because of its pleiotropic effects on Ras, inflammation, oxidative stress, renal fibrosis and klotho beyond lipid lowering. Key words:更多还原