【作者】 彭琳琳；

【导师】 宁建平；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 研究背景与目的：糖尿病肾病(diabetic nephropathy, DN)已成为目前终末期肾病(End-stage renal disease, ESRD)的主要基础病之一,其主要病理改变为肾间质细胞外基质聚积最终导致肾间质纤维化。既往研究表明,肾小管上皮细胞-肌成纤维细胞转分化(tubular epithelial myofibroblast transdifferentiation, TEMT)在其过程中发挥了重要作用。整合素连接激酶(integrin-linked kinase, ILK)是一种与整合素细胞内区域相结合的丝氨酸／苏氨酸蛋白激酶,在调节细胞粘附、迁移、基质积聚等过程中起重要作用,与肾脏疾病的发生发展密切相关。研究表明ILK是TGF-β1/Smad信号传导途径的重要下游效应分子,也是该信号传导途径诱导TEMT的关键步骤,参与ECM聚积过程。基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)可降解包括IV型胶原在内的多种ECM成分。金属蛋白酶组织抑制齐(?)-1(tissue inhibitor of metalloproteinase-1, TIMP-1)作为MMP--9的抑制物可与其特异性结合,保持动态平衡。发生TEMT时,MMP-9表达增多,引起MMP-9/TIMP-1失衡。高表达的MMP-9可特异性过度降解小管基底膜的Ⅳ型胶原,破坏基底膜。转分化的肾小管上皮细胞随后穿过断裂的基底膜,到达肾间质成为MyoF,促进间质纤维化的进一步发展。MMP-9/TIMP-1失衡可能是TEMT过程中的促进因素。大黄酸(rhein)作为大黄的主要有效成分,已被证实对肾脏有保护作用。本课题组通过前期研究发现,大黄酸可以抑制糖尿病肾病TEMT的发生,但其作用是否与调节ILK表达及影响MMP-9/TIMP-1比值,抑制TEMT及纤维化的进展有关?尚不清楚。本研究拟从糖尿病肾病患者、糖尿病肾病大鼠、与体外高糖环境下人肾小管上皮细胞培养三个研究层面入手,观察在DN小管间质病变中,肾小管上皮细胞转分化与ILK、MMP-9/TIMP-1表达的改变,同时采用ILK小分子干扰RNA技术(siRNA)特异性抑制ILK的表达,进而探讨ILK对MMP-9/TIMP-1的调控机制及其在TEMT中的作用；同时用大黄酸干预处理,观察大黄酸对DN的治疗作用及其对上述过程的影响,旨在从TEMT的角度探讨DN发病的具体机制及中药大黄防治DN的可能新靶点。方法：1.收集糖尿病肾病患者肾穿刺活检标本,使用HE染色、PASM染色及Masson染色方法,了解其肾脏损伤程度；免疫组织化学方法检测肾组织中E-cadherin、α-SMA的表达水平,并与正常肾组织比较,以观察人糖尿病肾病发病过程中是否存在TEMT。2.饲养8周龄健康雄性Wistar大鼠,随机分为正常组(12只),糖尿病组(12只)、大黄酸组(12只)及缬沙坦组(12只)。分别于第8、16周末,每组各处死6只大鼠,肾脏原位灌洗后,取肾组织,固定于蜡块中并切片。HE染色检测肾小管间质损伤程度,Masson染色方法观察肾间质胶原相对面积,免疫组织化学方法检测E-cadherin、α-SMA、TGF-β1、ILK、MMP-9、TIMP-1及AP-1的表达水平。3.培养人近端肾小管上皮细胞(HK-2),并分为：正常对照组、高糖组、高糖+抑制ILK表达组、高糖+无序siRNA组、高糖+大黄酸低浓度干预组、高糖+大黄酸中浓度干预组、高糖+大黄酸高浓度干预组、高渗对照组。各组细胞培养48小时后,分别提取细胞总RNA、总蛋白,使用Real-time RT PCR法、Western blot法及细胞免疫组织化学染色法评定各组细胞E-cadherin、α-SMA、TGF-β1、 ILK、MMP-9、TIMP-1的相对表达量。结果：1.与正常肾组织相比,糖尿病肾病患者肾组织中可观察到肾小管间质损伤及胶原沉积,其肾小管上皮细胞胞膜及胞浆中E-Cadherin表达下降,α-SMA表达上升,评分值有显著差异(P<0.05)。2.与正常组相比,糖尿病肾病大鼠肾间质胶原相对面积增加,肾小管上皮细胞E-cadherin表达减少、α-SMA表达增加(P<0.05)。与糖尿病组相比,大黄酸组及缬沙坦组大鼠肾间质胶原相对面积减少,E-cadherin表达增加、α-SMA表达减少(P<0.05),大黄酸组与缬沙坦组之间无明显差异(P>0.05)。与正常组相比,糖尿病肾病大鼠肾小管上皮细胞MMP-9、 TIMP-1表达随病情发展出现变化,MMP-9/TIMP-1比值紊乱。与糖尿病组相比,大黄酸组及缬沙坦组大鼠肾小管上皮细胞MMP-9/TIMP-1比值更接近于正常组(P<0.05),大黄酸组与缬沙坦组之间无明显差异(P>0.05)。与正常组相比,糖尿病肾病大鼠肾小管上皮细胞TGF-β1、ILK、 AP-1的表达随病情发生均呈进行性升高(P<0.05)。与糖尿病组相比,大黄酸组及缬沙坦组TGF-β1、ILK、AP-1的表达均降低(P<0.05),大黄酸组与缬沙坦组之间无明显差异(P>0.05)。糖尿病三组大鼠肾小管上皮细胞中AP-1表达与MMP-9/TIMP-1比值呈负相关。3.与正常组相比,高糖环境下培养的HK-2细胞、TGF-β1刺激的HK-2细胞均出现E-cadherin mRNA与蛋白表达减少,α-SMA、ILK mRNA与蛋白表达增加(P<0.05)。使用ILK-siRNA特异性抑制ILK表达后,HK-2细胞出现E-cadherin mRNA与蛋白表达上调,α-SMA、ILK mRNA与蛋白表达减少(P<0.05)。在高糖培养的HK-2细胞中加入大黄酸后,HK-2细胞出现与加入ILK-siRNA类似的改变,二者无明显差异(P>0.05)。与正常组相比,高糖环境下培养的HK-2细胞出现MMP-9/TIMP-1比值紊乱。使用ILK-siRNA特异性抑制ILK表达后,HK-2细胞的MMP-9/TIMP-1比值回归到接近正常(P<0.05),高糖+大黄酸组亦出现类似改变,二者无明显差异(P>0.05)。结论：1.在人糖尿病肾病病变发展过程中,可能存在肾小管上皮细胞转分化现象。2.高糖环境下HK-2细胞与糖尿病肾病大鼠肾小管上皮细胞E-cadherin表达减少,α-SMA表达增加,表明在糖尿病肾病(DN)肾小管间质病变进程中,存在肾小管上皮细胞转分化(TEMT)。3.在DN病理状态下,肾小管上皮细胞的ILK表达增高；ILK参与、介导了DN的TEMT过程,特异性抑制ILK可阻抑TEMT过程,调节紊乱的MMP-9/TIMP-1比值,且对TEMT有部分逆转作用。4.大黄酸可下调DN大鼠肾小管上皮细胞及高糖环境下HK-2细胞ILK的表达,改善MMP-9/TIMP-1比值的失衡,并阻抑TEMT的进展；大黄酸对DN中ILK高表达的抑制及其对MMP-9/TIMP-1比值的调节可能是大黄酸肾脏保护作用的机制之一。更多还原

【Abstract】 Background and Objectives:With the rising incidence of diabetes, diabetic nephropathy (DN), the main pathological change of which is renal interstitial fibrosis, is one of the main primary diseases to result in the end-stage renal disease (ESRD). Tubular epithelial myofibroblast transdifferentiation (TEMT), along with extracellular matrix accumulating, play important roles in renal interstitial fibrosis.Integrin-linked kinase (ILK) is a serine/threonine protein kinase, combinated with the intracellular region of integrin, regulates cell adhesion, migration, extracellular matrix accumulation, and is closely related with the pathogenesis of kidney diseases. Studies have shown that ILK, which participates in ECM accumulation, is an important downstream effector molecule of TGF-β1/Smad signaling pathway, and the committed step of TGF-β1/Smad induced TEMT.Matrix metalloproteinases-9(MMP-9) can specifically degrade the main ingredient-collagen IV in tubular epithelial basement membrane. Tissue inhibitor of metalloproteinase-1(TIMP-1) can specifically combine with MMP-9and keep their activity in a state of homeostasis. Recently scholars have been focused on the ratio of MMP-9/TIMP-1and considered it as a potential index for evaluating MMP-9effect in the process of renal fibrosis. In the condition of TEMT, the normal MMP-9/TIMP-1ratio is disturbed. Excessive MMP-9degradates type IV collagen in tubular basement membrane, trough which the transdifferentiated tubular epithelial cells pass and reach the renal interstitium, promotes further development of interstitial fibrosis.Rhein is a precursor component isolated from rhubarb anthraquinone derivatives, and a main renoprotective ingredient of rhubarb. Cumulative evidence suggests that some Chinese herbal medicines, including rhubarb, have a beneficial role in slowing the progression of CKD. Our previous studies suggested that rhein inhibited the pathogenesis of TEMT, however, whether this effect was related with regulation of ILK and MMP-9/TIMP-1ratio, is still unclear.This research studied from three aspects:chronic kidney disease patients, diabetic nephropathy rats and high glucose environment cultured human proximal tubular epithelial cells; meanwhile inhibited ILK expression with small interfering RNA (siRNA), and intervened the above process with rhein. The aims of this research were:observe TEMT in DN tubulointerstitial lesions, and the changes of ILK expression and MMP-9/TIMP-1ratio; explore the regulating effects of ILK on MMP-9/TIMP-1ratio in TEMT; investigate the therapeutic effect of rhein on DN and its impact on the above-mentioned process, elucidate the specific pathogenesis and the possible new target of DN prevention.Methods:1. Renal biopsy specimens of diabetic nephropathy patients (n=6) were collected. HE, PASM, Masson staining were used to observe renal pathological changes. Immunohistochemistry was used to detect E-cadherin, a-SMA expression in renal biopsy specimens and compared with the control group (n=6); the aim was to observe whether there is TEMT process in diabetic nephropathy patients. 2. Healthy male Wistar rats of8weeks old were randomly divided into normal group (n=12), diabetic nephropathy group (n=12), rhein intervention group (n=12) and valsartan intervention group (n=12). Six rats of each group were killed at the end of the8th and16th week. The renal interstitial injuries and the relative area of renal interstitial collagen were measured by HE and Masson staining. The protein expression of E-cadherin, a-SMA, TGF-β1, ILK, MMP-9, TIMP-1and AP-1in renal tubular epithelial cells were measured by immunohistochemistry.Human proximal tubular epithelial cells (HK-2) were cultured and divided into the following groups:normal group; high glucose group; ILK-siRNA group; negative siRNA group; high glucose+low concentration rhein group; high glucose+medium concentration rhein group; high glucose+high concentration rhein group; and hypertonic group. After cultured for48hours, total cell RNA and total cell protein were extracted. The mRNA and protein expression of E-cadherin, a-SMA, TGF-β1, ILK, MMP-9, TDVIP-1was detected by Real-time RT PCR, Western blot and cytoimmunohistochemistry.Results:1. HE, Masson and PASM staining showed that there were tubular and interstitial lesions in the kidneys of DN patients. Comparing to the patients of the control group, the E-cadherin expression in the cell membrane and cytoplasm decreased, and a-SMA expression increased in the renal tubular cells of diabetic nephropathy patients, with significant difference (P<0.05). 2. Comparing to the normal group, the relative area of renal interstitial collagen of diabetic nephropathy rats increased. The expression of E-cadherin in renal tubular epithelial cells decreased and the expression of a-SMA significantly increased at the level of P<0.05. Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed increases in the relative area of renal collagen. The expression of E-cadherin increased, while the expression of α-SMA significantly decreased. There is no significant difference between rhein intervention group and valsartan intervention group at the P>0.05level.Comparing to the normal group, the expressions of MMP-9and TIMP-1in renal tubular epithelial cells of diabetic nephopathy rats changed while the ratio of MMP-9/TIMP-1was disturbed. Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed significant decrease in MMP-9/TIMP-1ratio. There is no significant difference between rhein intervention group and valsartan intervention group at the P>0.05level.Comparing to the normal group, the expressions of TGF-β1, ILK, AP-1in renal tubular epithelial cells of diabetic nephopathy rats increased significantly with the disease progression (P<0.05). Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed significant decrease in expression of TGF-β1, ILK, AP-1(P<0.05). There is no significant differnce between rhein intervention group and valsartan intervention group at the P>0.05level.3. The mRNA and protein expression of cultured HK-2cells were evaluated by Real-time RT PCR, Western blot and cytoimmunohistpchemistry respectively. The results indicated: Comparing to the normal group, the expression of E-cadherin decreased; the a-SMA and ILK expression significantly increased in the high glucose group (P<0.05). Inhibition of ILK by ILK-siRNA resulted in the increased expression of E-cadherin, decreased expression of a-SMA and ILK at the P<0.05level. In the rhein intervention groups, the HK-2cells showed a similar change with the ILK-siRNA group and there was no significant difference between these groups (P>0.05).Comparing to the normal group, the MMP-9/TIMP-1ratio was disturbed in the high glucose group (P<0.05). Inhibition of ILK by ILK-siRNA resulted in a normal return of MMP-9/TIMP-1ratio. Similar change was observed in the rhein intervention groups and there was no significant difference between them (P>0.05).Conclusions:1. TEMT may exist in the process of human diabetic nephropathy.2. E-cadherin expression decreased and a-SMA expression increased in high glucose environment cultured HK-2cells and the tubular epithelial cells of DN rats, indicating that TEMT exists in the process of tubulointerstitial lesions.3. In the pathological state of DN, ILK expression of renal tubular cells increased; ILK participated and mediated the process of TEMT; ILK inhibition could counteract with TEMT, and regulate the disturbed ratio of MMP-9/TIMP-1, thus inhibit TEMT. 4. Rhein could down-regulate ILK expression in HK-2cells under high glucose environment and in renal tubular epithelial cells of DN rats, improve the status of MMP-9/TIMP-1ratio imbalance, and attenuate the progress of TEMT. The inhibition effect of rhein towards the ILK overexpression in DN and its regulating role to MMP-9/TIMP-1ratio may be one of its renoprotective mechanisms.更多还原