Wnt/β-catenin信号途径及ILK在糖尿病性肾小管间质纤维化发病中作用的研究
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摘要
目的:糖尿病肾病(diabetic nephropathy, DN)是糖尿病常见并发症,是引起终末期肾衰的主要原因之一,其主要病理变化为肾小球肥大,肾小球和肾小管基底膜增厚、细胞外基质聚集、肾小球硬化、肾小管萎缩及间质纤维化等。以往对DN的研究大多集中在肾小球病变,而对肾小管间质病变的发生发展及其机制研究较少。近来越来越多的研究表明糖尿病肾小管间质病变的严重程度与肾功能的进行性下降密切相关。高糖可诱导肾小管上皮细胞表型向间充质细胞转变,分泌并聚集细胞外基质而导致肾间质纤维化发生。糖尿病肾病小管间质病变是决定其肾功能下降水平及预后的重要因素。
Wnt/β-连环蛋白(β-catenin)信号途径参与细胞增殖、分化、存活、凋亡及细胞迁移等多种功能的调控,在胚胎发育、肿瘤发生和器官纤维化等重要生理及病理过程中发挥重要作用。已有研究表明Wnt/β-catenin信号途径调节高糖诱导的肾小球系膜细胞凋亡过程,在单侧输尿管梗阻(Unilateral Ureteral Obstruction,UUO)肾脏该途径表达上调,提示此途径可能参与了慢性肾脏病的发病。
整合素连接激酶(Integrin-linkedkinase,ILK)被认为是转化生长因子β1(transforming growth factorβ1,TGFβ1)下游最重要的致纤维化因子之一。在慢性肾脏病、糖尿病肾病及UUO模型梗阻侧肾脏均发现ILK过度表达加重细胞外基质的积聚及肾脏纤维化的程度。ILK过度表达可激活Wnt信号通路下游分子,也就是说通过抑制糖原合成酶激酶-3β(glycogen synthase kinase 3β,GSK-3β)活性促使β-catenin核聚集。ILK可能通过与Wnt信号途径“串话(cross talk)”而参与了肾小管间质纤维化的发病。
在糖尿病肾脏病变中Wnt/β-catenin信号转导途径的作用及该途径与ILK的关系尚有许多未知。本研究应用糖尿病大鼠模型和体外培养的人近端肾小管上皮细胞(HKC),系统观察了Wnt/β-catenin信号途径及ILK在早期糖尿病肾病以及高糖诱导HKC转分化中的表达;用ILK小干扰RNA(ILKsiRNA)沉默ILK基因表达,观察了Wnt/β-catenin途径下游因子在高糖诱导HKC转分化过程中的变化;并结合糖尿病肾病患者肾脏Wnt/β-catenin信号途径表达的变化,以进一步探讨Wnt途径在糖尿病肾病肾小管间质纤维化中的作用。以期为进一步阐明糖尿病肾小管间质纤维化发病机制及寻找新的治疗靶点提供依据。
方法:
1大鼠糖尿病模型的制备和ILK、Wnt4、GSK -3β、β-catenin蛋白及mRNA的检测
Wistar雄性大鼠均行右肾切除术,伤口愈合后随机分为对照组(CG)和糖尿病组(DM),DM组大鼠腹腔单次注射链脲佐菌素65mg/kg(STZ溶于0.1mol/L枸橼酸盐缓冲液中,pH值4.5),对照组只注射相同体积的枸橼酸盐缓冲液,48h后测定血糖和尿糖,血糖值≥16.7mmol/L,尿糖值+++~++++者确定为DM模型。成模后4、8、12周每组取6只大鼠,切取肾脏。取部分肾皮质组织置于4%多聚甲醛固定,用光镜及免疫组织化学染色法检测,免疫组化检测指标包括ILK、Wnt4、β-catenin、α-平滑肌肌动蛋白(α-SMA)表达;取部分肾皮质组织提取总蛋白及核蛋白,Western blot检测ILK、Wnt4、β-catenin、GSK-3β、磷酸化GSK-3β蛋白(P-GSK-3β)表达;取部分肾皮质组织提取总RNA,半定量RT-PCR检测Wnt4、β-catenin mRNA表达。
2细胞培养和ILK、Wnt4、GSK -3β、β-catenin蛋白及mRNA的检测
体外培养HKC,无血清DMEM培养基同步培养24h,细胞分成3组:正常糖组(NG,D-Glucose 5.5mmol/L ),甘露醇对照组(NG+M, D-Glucose 5.5 mmol/L+mannitol 24.5 mmol/L) ,高糖组(HG,D-Glucose 30 mmol/L ),培养12、24、48、72h后收集细胞提取细胞总蛋白、核蛋白及RNA。采用免疫细胞化学检测Wnt4、β-catenin及E-钙粘蛋白(E-cadherin),α-SMA的表达;Western blot检测ILK、Wnt4、GSK-3β、P-GSK-3β、β-catenin蛋白的表达;半定量RT-PCR检测细胞Wnt4、GSK-3β及β-catenin mRNA的水平。
3 HKC ILKsiRNA转染及ILK、GSK -3β、β-catenin表达的检测
针对HKC ILK编码序列分别设计3条体外转录的shRNA。应用lipofectamine 2000转染试剂将Pgenesil-1.1- ILKsiRNA真核表达载体转染入HKC,分成6组:①正常对照组(NG)②高糖组(HG)③高糖+阴性转染对照组:转染pGenesil-1.1-HK(HG+HK)④高糖+pGenesil-1.1-ILK-1(HG+ILK-1siRNA)组⑤高糖+ pGenesil-1.1-ILK-2 (HG+ILK-2siRNA)组⑥高糖+pGenesil-1.1-ILK-3 (HG+ILK-3siRNA)组。转染48h荧光倒置显微镜下观察绿色荧光蛋白(GFP)的表达。Western-blot及RT-PCR检测ILK蛋白及RNA表达水平,确定敲低效果最佳的ILK siRNA。免疫细胞化学法检测P-GSK -3β、β-catenin表达。Western blot检测GSK-3β、P-GSK -3β、β-catenin、E-cadherin和α-SMA表达。
4病例选择及肾组织Wnt4、P-GSK -3β、β-catenin的表达的检测
选择2006.10-2008.10在河北医大二院肾内科住院、经临床与肾活检诊断为糖尿病肾病患者33例(其中男性18例,女性15例,年龄28–70岁,48.7±10.3岁)。除外狼疮性肾炎、紫癜性肾炎、乙肝病毒相关性肾炎、甲状腺病相关性肾损害、类风湿性关节炎肾损害以及肿瘤相关性肾病等继发性肾脏病患者,并除外合并急性肾小管坏死和急、慢性间质性肾炎的患者。糖尿病肾病患者根据肾小管间质病变程度分为两组,轻中度组20例,重度组13例。10例肾肿瘤患者远离肿瘤的正常肾组织作为对照。详细收集患者临床资料,包括年龄、病程、血浆白蛋白、24小时尿蛋白定量、血肌酐等。免疫组化法检测Wnt4、P-GSK -3β、β-catenin蛋白的表达。
结果:
1糖尿病大鼠肾组织ILK、Wnt4、GSK-3β、β-catenin蛋白和mRNA的表达
①免疫组化显示ILK ,Wnt4在对照组部分肾小管上皮细胞弱表达,α-SMA仅见于血管平滑肌细胞。糖尿病组表达增强(P <0.05或P <0.01)。β-catenin在对照组表达于肾小管上皮细胞基底侧,糖尿病组胞浆表达增强,部分胞核表达。(P <0.05或P <0.01)。②Western blot结果显示ILK、Wnt4、P-GSK-3β及核β-catenin在糖尿病组表达较对照组均增强,并随时间延长更加明显(P<0.05);β-catenin总蛋白及GSK-3β在各组及各时间点无明显变化(P> 0. 05)。③半定量RT-PCR结果显示,Wnt4 mRNA在对照组有少量基础表达,糖尿病组表达增高(P<0. 05)。β-catenin mRNA在各组表达无明显差异(P> 0. 05)。
2高糖培养下HKC ILK、Wnt4、GSK -3β、β-catenin蛋白和mRNA的表达
①免疫细胞化学显示,Wnt4在正常糖及甘露醇对照组HKC胞浆有少量表达,高糖组表达明显增强;对照组β-catenin主要在HKC胞膜,胞浆有少量表达,高糖组β-catenin胞浆及胞核表达明显增强(P<0.01);E-cadherin在对照组肾小管上皮细胞表达明显,而高糖组表达减弱;α-SMA表达与E-cadherin相反(P<0.05)。②Western blot显示高糖组HKC Wnt4、p-GSK-3β、核β-catenin、ILK表达明显增强(P<0.05),Wnt4、p-GSK-3β、核β-catenin在高糖刺激24 h达高峰(P<0.05或P<0.01),ILK表达在高糖刺激48 h达高峰(P<0.05);总β-catenin、GSK-3β在各组之间及各时间点表达无明显差异(P>0.05)。③RT-PCR结果显示高糖组肾小管上皮细胞Wnt4 mRNA表达明显上调(P<0.05);GSK-3β及β-catenin mRNA在各组表达量基本一致,无统计学意义(P>0.05)。
3 ILKsiRNA瞬时转染HKC后ILK、β-catenin、GSK -3β的表达
①转染HKC细胞后绿色荧光蛋白表达:pGenesil-1.1-ILK-1siRNA、pGenesil-1.1-ILK-2siRNA、pGenesil-1.1-ILK-3 siRNA转染HKC细胞48小时后,荧光倒置显微镜下可见绿色荧光蛋白表达,证实质粒成功转染HKC细胞并能够表达绿色荧光蛋白②转染HKC细胞后ILK RNA及蛋白表达:与HG和阴性对照质粒组(HG+HK)相比,pGenesil-1.1-ILK-1 siRNA、pGenesil-1.1-ILK-2 siRNA、pGenesil-1.1-ILK-3 siRNA质粒转染组可见ILKmRNA水平下降,抑制率分别为36.47%、25.11%及43.15%;ILK蛋白表达下降,分别比HG及HG+HK减少41.59%,29.78%和56.12%,但较NG组表达仍高(p<0.05)。③免疫细胞化学显示:pGenesil-1.1-ILK-3转染HKC细胞48h后胞浆P-GSK-3β、胞浆及核β-catenin比HG及HG+HK组表达下降,但较NG组表达仍高(p<0.05)。④Western blot显示:pGenesil-1.1-ILK-3转染HKC细胞后P-GSK-3β、β-catenin核蛋白均较HG及HG+HK组表达下降(P<0.05);GSK-3β与总β-catenin在各组表达无明显差异( P>0.05);E-cadherin在HG+ILK-3siRNA的表达较HG及HG+HK组有所升高,α-SMA表达与E-cadherin相反(P<0.05或P<0.01)。
4糖尿病肾病患者肾组织病理变化及Wnt4、P-GSK -3β、β-catenin的表达
①肾组织的病理改变:轻中度组DN患者肾病理主要表现为肾小球的肥大,系膜细胞增生,系膜区增宽,肾小球及肾小管基底膜轻度增厚,局灶性肾小管上皮细胞颗粒变性、萎缩及间质纤维化。重度组DN患者肾组织出现典型的K-W结节,甚至球性硬化,弥漫性肾小管间质纤维化。②免疫组化结果:对照组Wnt4、p-GSK-3β仅微弱表达于部分肾小管上皮细胞,糖尿病肾病轻中度组表达增强,在重度组表达下降(P<0.05);β-catenin在对照组少量表达于肾小管上皮细胞基底膜侧,糖尿病肾病轻中度组肾小管上皮细胞及肾间质细胞胞浆表达明显增强,部分细胞胞核呈阳性表达。而在重度组表达减弱,尤其在重度纤维化病变处几乎不表达(P<0.05)。③Wnt4、p-GSK-3β、β-catenin表达均与24小时尿蛋白定量成正相关,而与估算的肾小球滤过率(eGFR)成负相关(P<0.05)。
结论:
1 Wnt/β-catenin信号途径在糖尿病肾病早期以及高糖诱导肾小管上皮细胞转分化过程中表达增高,该途径可能参与了糖尿病肾病肾小管间质纤维化的发生和进展。
2 ILK在糖尿病肾病早期肾小管间质病变及高糖诱导的肾小管上皮细胞转分化过程中表达增高;转染HKC ILKsiRNA能成功沉默ILK基因,使P-GSK-3β及核β-catenin表达下调,并使高糖诱导下调的E-cadherin水平升高,α-SMA水平降低而阻止肾小管细胞转分化,提示ILK可能通过调节Wnt/β-catenin途径下游因子活性参与肾小管细胞转分化过程。
3 Wnt/β-catenin信号途径在糖尿病肾病患者肾小管间质表达增高,但在重度肾小管间质纤维化病变表达下调,其变化可能参与了人类糖尿病肾病肾间质纤维化的发病与进展。
Objectives: Diabetic nephropathy(DN) is one of the most common complications of diabetes, which is histologically characterized by hypertrophy of glumeruli, increased thickness of basement membrane, overaccumulation of extracellular matrix, glomerulosclerosis, tubular atrophy, interstitial fibrosis and so on. Much more researches have focused on glomerular lesions, little emphasized the crucial role of tubulointerstitial injury in diabetic kidney in the past.It has recently been demonstrated that the development of tubulointerstitial fibrosis is more closely correlated with a progressive decline in renal function compared to glomerularsclerosis. Profibrotic switches in the phenotype of epithelial cells and epithelial–mesenchymal transition (EMT) can be induced by high glucose . Transdifferentiated epithelial cells become reprogrammed to secrete and excessively accumulate extracellular matrix which promote tubulointerstitial fibrosis in diabetic kidney. It is widely recognized that tubulointerstitial injury serves as an important mediator of chronic renal failure and predictor of outcome in patients with diabetic nephropathy.
Wnt/β-catenin signalling pathway plays an important role in mammalian developmental processes, tumorigenesis, and organic fibrosis by regulating a variety of cellular processes including cell proliferation, differentiation, survival and apoptosis.Wnt/β-catenin signalling pathway regulates apoptosis of mesangial cells induced by high glucose,and the pathway is activated after unilateral ureteral obstruction, suggesting that the Wnt/β-catenin signalling pathway is likely implicated in the pathogenesis of chronic kidney desease.
Integrin-linkedkinase (ILK) is one of the most important downstream effector of TGF-β1 in mediating renal fibrosis.Oversxpression of ILK aggravate extracellular matrix deposition and tubulointerstitial fibrosis in chronic kidney desease , diabetic nephropathy and mouse models of unilateral ureteral obstruction. Activation of the downstream components of the Wnt signaling pathway, namely inhibition of glycogen synthase kinase 3β(GSK -3β)activity and nuclear accumulation ofβ-catenin, has also been achieved through overexpression of ILK. Cross talk between ILK and Wnt signalling pathway likely contribute to the onset and progression of tubulointerstitial fibrosis.
Role for Wnt/β-catenin signaling pathway in mediating tubulointerstitial fibrogenesis in diabetic kidney and tubular epithelial–mesenchymal transition induced by high glucose remains to be unknown. Although ILK has been known to regulate the effector activity of Wnt signaling, direct interplay between ILK and the Wnt pathway in diabetic nephropathy has remained to be clarified.
In the present study, using STZ-induced diabetic model and HKC cells treated with high glucose ,we investigated the expression of Wnt/β-catenin signaling pathway and ILK and the relationship between them in early stage of DN and transdifferentiated tubular epithelial cells. Furthermore, we investigated the role of ILK in modulating the components expression of Wnt signaling in HKC cells by silencing ILK gene via specific ILK shRNA. At last, the expression of Wnt/β-catenin pathway was examined in renel tissues from patients with diabetic nephropathy to elucidate the potential role of this signaling pathway for tubulointerstitial fibrosis in diabetic kidney,in order to provide experimental and clinical evidences for explaining the mechanism of the development of tubulointerstitial disease in diabetic nephropathy and seeking new treatment target.
Methods:
1 Preparation of diabetic model and examination of ILK,Wnt4,GSK -3β,β-catenin protein and mRNA expression
Male Wistar rats were randomly divided into two groups after unilateral nephrectomy:control group(n=18), diabetic group(n=18). The rats of diabetic group were injected intraperitoneally with 65 mg/kg body weight STZ in 0.1mol/L sodium citrate solution (pH 4.5), and the rats in the control group were injected with 0.1mol/L sodium citrate solution. Diabetic model was considered to be successful when the blood glucose was≥16.7mmol/L and the urine glucose was +++~++++ after 48 hours of the injection. Six rats from each group were sacrificed respectively at weeks 4, 8 and 12 after STZ injection. Partial renal tissues were fixed in 4% formaldehyde for light microscopic and immunohistochemical staining. Partial renal cortices were used to extracte RNA and total and nuclear protein . The expressions of ILK,Wnt4,β-catenin andα-SMA in renal tissues were evaluated by immunohistochemistry . Western blot was used to examine the expression of ILK,Wnt4,GSK-3β,P-GSK -3β, total and nuclearβ-catenin . The mRNA levels of Wnt4 andβ-catenin were measured by reverse transcription and polymerase chain reaction (RT-PCR).
2 Cell culture and examination of ILK,Wnt4,GSK -3β,β-catenin protein and mRNA expression
Human kidney proximal tubular epithelial cell line (HKC) cells were incubated with serum-free DMEM for 24 hours to synchronize the cell growth,then the cells were divided into three groups:NG group (media containing 5.5mM glucose); mannitol control group (media containing 5.5mM glucose and 24.5mM mannitol); HG group (media containing 30mM glucose). The cells were collected to extracte total RNA and protein at 12, 24, 48 and 72 hours after incubation. The expression of Wnt4,β-catenin, E-cadherin andα-SMA were examined by immunocytochemistry. Western-blot was also used to detect the expression of ILK,Wnt4,GSK-3β,P-GSK-3β, total and nuclearβ-catenin proteins. The mRNA levels of Wnt4,GSK-3βandβ-catenin were examined by RT-PCR .
3 Transfection of ILKsiRNA into HKC cells and examination of ILK, GSK -3β,β-catenin protein and mRNA expression
Three small interfering RNA (siRNA) targeting human ILK gene sequences were synthesized to silence ILK gene expression.HKC cells were divided into six groups: NG group, HG group ,HK control group(a vector containing the non-specific siRNA was designed as negative control), pGenesil-1.1-ILK-1siRNA group, pGenesil-1.1-ILK-2siRNA group and pGenesil-1.1-ILK-3 siRNA group. Transient transfection of HKC cells was carried out using Lipofectamine 2000 according to the manufacturer’s instruction. Six hours after transfection, the medium of NG group was replaced by normal glucose and the medium of other groups was replaced by high glucose DMEM medium (30mM glucose) with 10% FBS .The cells were incubated for an additional 48h. Fluorescent microscopy was used to examine GFP expression . Then ILK protein and RNA expression was examined by Western-blot and RT-PCR. Immunocytochemistry was used to observe the expression of P-GSK-3βandβ-catenin. The expression of GSK-3β,P-GSK-3β, total and nuclearβ-catenin ,E-cadherin andα-SMA proteins was examined by Western-blot.
4 Patients and observation of Wnt4,P-GSK -3βandβ-catenin expression
Thirty three patients diagnosed as diabetic nephropathy by renal biopsy and clinical data from October 2006 to October 2008 at the Second Hospital of Hebei Medical University were included in this study.There were 18 male and 15 female patients whose ages were from 28 to 70(48.7±10.3)years old.The cases of lupus nephritis,Henoch-Schonlein nephritis, HBV associated glomerulonephritis, thyropathy associated glomerulonephritis,renal damage induced by rheumatoid arthritis and cancer associated nephropathy were excluded.Those cases complicated with acute renal tubular necrosis,acute or chronic interstitial nephritis were also excluded. The patients were divided into two groups according to the degrees of tubulointerstitial fibrosis: mild /moderate group (n=20)and advanced group(n=13). The renal tissues without evidence of renal disease (n=10) obtained from distant portions of kidneys surgically excised because of the presence of a localized neoplasm were used as control. Clinical data were collected including age, duration of diabetes, plasma albumin,urine protein excretion, serum creatinine and so on. Tubulointerstitial expression of Wnt4、P-GSK-3βandβ-catenin was assessed by immunhistochemical staining.
Results:
1 Protein and mRNA expression of ILK,Wnt4,GSK-3β,β-catenin in the renal tissues of diabetic rats
①Immunohistochemical positive staining of ILK and Wnt4 was observed in renal tubular epithelial cytoplasim whileα-SMA is restricted to the vascular smooth muscle cells in control group. They were all remarkably increased in diabetic group (P<0.05).In control group,β-catenin showed a basolateral localization within the proximal tubules,but was observed in cytoplasm and nuclei of tubular epithelial and renal interstitial cells in diabetic group (P<0.05).②From the results of Western blot analysis, the diabetic rats showed increased expression of ILK ,Wnt4,P-GSK-3βand nuclearβ-catenin in the kidney from week 4, and maintained at a higher level until week 12 (P<0.05). There is no significant difference of GSK-3βand totalβ-catenin protein expression between the groups (P>0.05).③The mRNA levels of Wnt4 in the kidney of diabetic rats increased compared with control group (P<0. 05). No significant difference ofβ-catenin mRNA expression was found between different groups(P> 0. 05).
2 The expression of ILK,Wnt4,GSK-3β,β-catenin in HKC cells incubated in HG
①Immunocytochemical staining showed that Wnt4 weakly expressed in HKC cytoplasm in NG group and mannitol control group while the expression enhanced after HG stimulation.β-catenin was mainly anchored at the membrane and faintly expressed in cytoplasm of HKC cells in control group . In HG group ectopic expression ofβ-catenin was found in cytoplasm and translocated into nuclei of HKC cells (P<0.05 or P<0.01). In HG group the expression of E-cadherin decreased compared with control groups. The expression ofα-SMA was opposite to that of E-cadherin(P<0.05).②Western blot analysis indicated that the expression of Wnt4,p-GSK-3β,nuclearβ-catenin and ILK were increased in HKC cells of HG group(P<0.05). Wnt4,p-GSK-3βand nuclearβ-catenin expression in HKC reached the peak at 24h and that of ILK peaked at 48h after HG stimulation(P<0.01). No significant difference of GSK-3βand totalβ-catenin protein expression was observed among the three groups (P>0.05).③The mRNA levels of Wnt4 was up-regulated in HG group compared with control group (P<0.05). There was no significant difference of GSK-3βandβ-catenin mRNA expression found among different groups(P>0.05).
3 The expression of ILK,GSK-3βandβ-catenin in HKC cells transfected with ILKsiRNA
①pGenesil-1.1-ILK-1siRNA, pGenesil-1.1-ILK-2siRNA and pGenesil-1.1-ILK-3 siRNA were transfected into HKC cells to silence ILK expression. At 48 hours after transfection, green fluorescent protein(GFP)was observed in about 60%-70% HKC cells, showing that the transfection is successful.②The ILK mRNA and protein were examined using the methods of RT-PCR and Western blot. Compared with HG and non-specific siRNA transfected control group(HK), the ILK mRNA was significantly inhibited by 36.47%、25.11% and 43.15% respectively in groups transfected with ILKsiRNA(P<0.05).Correspondingly,protein expression of ILK was inhibited by 41.59% , 29.78% and 56.12% respectively(P<0.05).③Immunocytochemical staining showed that P-GSK-3βas well as cytosolic and nuclearβ-catenin expression remarkbly reduced in HKC cells transfected with ILK siRNA compared with that of HG and non-specific siRNA transfected control group(P<0.05).④Western blot showed ILK siRNA suppressed phosphorylation of GSK-3βandβ-catenin translocation into nuclei, whereas the level of P-GSK-3βand nuclearβ-catenin remained increased in HG and non-specific siRNA transfected control group(P<0.05). No significant difference of GSK-3βand totalβ-catenin protein expression was observed among different groups (P>0.05). Downregulation of E-cadherin and upregulation ofα-SMA induced by HG were recovered to a certain degree in HG+ILK-3siRNA group(P<0.05).
4 Pathological changes and the expression of Wnt4,P-GSK-3,β-catenin in renal tissues of patients with diabetic nephropathy
①pathological changes including glomerular enlargement, mesangial matrix expansion, increase of glomerular and tubular basement membrane in thickeness, focal tubular epithelial vacuolar degeneration and atrophy as well as mild interstitial fibrosis were observed in the patients of mild/morderate group. Progressive glomerulosclerosis with the presence of Kimmelstiel–Wilson lesions, global sclerosis and diffused tubulointerstitial fibrosis could be found in the patients of advanced group.②Immunohistochemical staining displayed that Wnt4 and p-GSK-3βweakly expressed in renal tubular epithelial cytoplasim in control group. The expression remarkably increased in mild/morderate DN group,whereas decreased in advanced DN group(P<0.05). In control kidneys,β-catenin was found to be localized basolaterally within the proximal tubules. Increase of cytosolic and nuclearβ-catenin in both tubular epithelial and interstitial cells were observed in mild/morderate DN group. However, in advanced DN group cytosolic and nuclearβ-catenin is downregulated,and is barely detectable in severe tubulointerstitial lesions (P<0.05).③The expression of Wnt4, p-GSK-3β, nuclearβ-catenin had a positive correlation with urine protein excretion, and a negative correlation with estimated glomerular filtration rate(eGFR) (P<0.05) .
Conclusions:
1 Wnt/β-catenin signaling pathway activity is up-regulated in the early stage of DN and in the HKC cells incubated in high glucose medium,and the up-regulation is correlated with tubular epithelial–mesenchymal transition, suggesting that the pathway may be involved in the pathogenesis of tubulointerstitial fibrogenesis of DN.
2 The expression of ILK is also increased in the early stage of DN and in the HKC cells incubated in high glucose medium, and correlated with tubular epithelial–mesenchymal transition. ILKsiRNA transfection into HKC cells successfully silence ILK expression and reverse tubular epithelial–mesenchymal transition by downregulating the expression of P-GSK-3βand nuclearβ-catenin.Thus,the effect of ILK to modulate the activity of downstream componants in Wnt/β-catenin signaling pathway may be involved in the tubular epithelial–mesenchymal transition
3 The expression of Wnt/β-catenin signaling pathway is increased in the patients of mild/morderate DN group,but decreased in the patients of advanced DN group.These changes of the pathway may be contributable to the onset and progression of tubulointerstitial fibrosis of human diabetic nephropathy.
Wnt/β-连环蛋白(β-catenin)信号途径参与细胞增殖、分化、存活、凋亡及细胞迁移等多种功能的调控,在胚胎发育、肿瘤发生和器官纤维化等重要生理及病理过程中发挥重要作用。已有研究表明Wnt/β-catenin信号途径调节高糖诱导的肾小球系膜细胞凋亡过程,在单侧输尿管梗阻(Unilateral Ureteral Obstruction,UUO)肾脏该途径表达上调,提示此途径可能参与了慢性肾脏病的发病。
整合素连接激酶(Integrin-linkedkinase,ILK)被认为是转化生长因子β1(transforming growth factorβ1,TGFβ1)下游最重要的致纤维化因子之一。在慢性肾脏病、糖尿病肾病及UUO模型梗阻侧肾脏均发现ILK过度表达加重细胞外基质的积聚及肾脏纤维化的程度。ILK过度表达可激活Wnt信号通路下游分子,也就是说通过抑制糖原合成酶激酶-3β(glycogen synthase kinase 3β,GSK-3β)活性促使β-catenin核聚集。ILK可能通过与Wnt信号途径“串话(cross talk)”而参与了肾小管间质纤维化的发病。
在糖尿病肾脏病变中Wnt/β-catenin信号转导途径的作用及该途径与ILK的关系尚有许多未知。本研究应用糖尿病大鼠模型和体外培养的人近端肾小管上皮细胞(HKC),系统观察了Wnt/β-catenin信号途径及ILK在早期糖尿病肾病以及高糖诱导HKC转分化中的表达;用ILK小干扰RNA(ILKsiRNA)沉默ILK基因表达,观察了Wnt/β-catenin途径下游因子在高糖诱导HKC转分化过程中的变化;并结合糖尿病肾病患者肾脏Wnt/β-catenin信号途径表达的变化,以进一步探讨Wnt途径在糖尿病肾病肾小管间质纤维化中的作用。以期为进一步阐明糖尿病肾小管间质纤维化发病机制及寻找新的治疗靶点提供依据。
方法:
1大鼠糖尿病模型的制备和ILK、Wnt4、GSK -3β、β-catenin蛋白及mRNA的检测
Wistar雄性大鼠均行右肾切除术,伤口愈合后随机分为对照组(CG)和糖尿病组(DM),DM组大鼠腹腔单次注射链脲佐菌素65mg/kg(STZ溶于0.1mol/L枸橼酸盐缓冲液中,pH值4.5),对照组只注射相同体积的枸橼酸盐缓冲液,48h后测定血糖和尿糖,血糖值≥16.7mmol/L,尿糖值+++~++++者确定为DM模型。成模后4、8、12周每组取6只大鼠,切取肾脏。取部分肾皮质组织置于4%多聚甲醛固定,用光镜及免疫组织化学染色法检测,免疫组化检测指标包括ILK、Wnt4、β-catenin、α-平滑肌肌动蛋白(α-SMA)表达;取部分肾皮质组织提取总蛋白及核蛋白,Western blot检测ILK、Wnt4、β-catenin、GSK-3β、磷酸化GSK-3β蛋白(P-GSK-3β)表达;取部分肾皮质组织提取总RNA,半定量RT-PCR检测Wnt4、β-catenin mRNA表达。
2细胞培养和ILK、Wnt4、GSK -3β、β-catenin蛋白及mRNA的检测
体外培养HKC,无血清DMEM培养基同步培养24h,细胞分成3组:正常糖组(NG,D-Glucose 5.5mmol/L ),甘露醇对照组(NG+M, D-Glucose 5.5 mmol/L+mannitol 24.5 mmol/L) ,高糖组(HG,D-Glucose 30 mmol/L ),培养12、24、48、72h后收集细胞提取细胞总蛋白、核蛋白及RNA。采用免疫细胞化学检测Wnt4、β-catenin及E-钙粘蛋白(E-cadherin),α-SMA的表达;Western blot检测ILK、Wnt4、GSK-3β、P-GSK-3β、β-catenin蛋白的表达;半定量RT-PCR检测细胞Wnt4、GSK-3β及β-catenin mRNA的水平。
3 HKC ILKsiRNA转染及ILK、GSK -3β、β-catenin表达的检测
针对HKC ILK编码序列分别设计3条体外转录的shRNA。应用lipofectamine 2000转染试剂将Pgenesil-1.1- ILKsiRNA真核表达载体转染入HKC,分成6组:①正常对照组(NG)②高糖组(HG)③高糖+阴性转染对照组:转染pGenesil-1.1-HK(HG+HK)④高糖+pGenesil-1.1-ILK-1(HG+ILK-1siRNA)组⑤高糖+ pGenesil-1.1-ILK-2 (HG+ILK-2siRNA)组⑥高糖+pGenesil-1.1-ILK-3 (HG+ILK-3siRNA)组。转染48h荧光倒置显微镜下观察绿色荧光蛋白(GFP)的表达。Western-blot及RT-PCR检测ILK蛋白及RNA表达水平,确定敲低效果最佳的ILK siRNA。免疫细胞化学法检测P-GSK -3β、β-catenin表达。Western blot检测GSK-3β、P-GSK -3β、β-catenin、E-cadherin和α-SMA表达。
4病例选择及肾组织Wnt4、P-GSK -3β、β-catenin的表达的检测
选择2006.10-2008.10在河北医大二院肾内科住院、经临床与肾活检诊断为糖尿病肾病患者33例(其中男性18例,女性15例,年龄28–70岁,48.7±10.3岁)。除外狼疮性肾炎、紫癜性肾炎、乙肝病毒相关性肾炎、甲状腺病相关性肾损害、类风湿性关节炎肾损害以及肿瘤相关性肾病等继发性肾脏病患者,并除外合并急性肾小管坏死和急、慢性间质性肾炎的患者。糖尿病肾病患者根据肾小管间质病变程度分为两组,轻中度组20例,重度组13例。10例肾肿瘤患者远离肿瘤的正常肾组织作为对照。详细收集患者临床资料,包括年龄、病程、血浆白蛋白、24小时尿蛋白定量、血肌酐等。免疫组化法检测Wnt4、P-GSK -3β、β-catenin蛋白的表达。
结果:
1糖尿病大鼠肾组织ILK、Wnt4、GSK-3β、β-catenin蛋白和mRNA的表达
①免疫组化显示ILK ,Wnt4在对照组部分肾小管上皮细胞弱表达,α-SMA仅见于血管平滑肌细胞。糖尿病组表达增强(P <0.05或P <0.01)。β-catenin在对照组表达于肾小管上皮细胞基底侧,糖尿病组胞浆表达增强,部分胞核表达。(P <0.05或P <0.01)。②Western blot结果显示ILK、Wnt4、P-GSK-3β及核β-catenin在糖尿病组表达较对照组均增强,并随时间延长更加明显(P<0.05);β-catenin总蛋白及GSK-3β在各组及各时间点无明显变化(P> 0. 05)。③半定量RT-PCR结果显示,Wnt4 mRNA在对照组有少量基础表达,糖尿病组表达增高(P<0. 05)。β-catenin mRNA在各组表达无明显差异(P> 0. 05)。
2高糖培养下HKC ILK、Wnt4、GSK -3β、β-catenin蛋白和mRNA的表达
①免疫细胞化学显示,Wnt4在正常糖及甘露醇对照组HKC胞浆有少量表达,高糖组表达明显增强;对照组β-catenin主要在HKC胞膜,胞浆有少量表达,高糖组β-catenin胞浆及胞核表达明显增强(P<0.01);E-cadherin在对照组肾小管上皮细胞表达明显,而高糖组表达减弱;α-SMA表达与E-cadherin相反(P<0.05)。②Western blot显示高糖组HKC Wnt4、p-GSK-3β、核β-catenin、ILK表达明显增强(P<0.05),Wnt4、p-GSK-3β、核β-catenin在高糖刺激24 h达高峰(P<0.05或P<0.01),ILK表达在高糖刺激48 h达高峰(P<0.05);总β-catenin、GSK-3β在各组之间及各时间点表达无明显差异(P>0.05)。③RT-PCR结果显示高糖组肾小管上皮细胞Wnt4 mRNA表达明显上调(P<0.05);GSK-3β及β-catenin mRNA在各组表达量基本一致,无统计学意义(P>0.05)。
3 ILKsiRNA瞬时转染HKC后ILK、β-catenin、GSK -3β的表达
①转染HKC细胞后绿色荧光蛋白表达:pGenesil-1.1-ILK-1siRNA、pGenesil-1.1-ILK-2siRNA、pGenesil-1.1-ILK-3 siRNA转染HKC细胞48小时后,荧光倒置显微镜下可见绿色荧光蛋白表达,证实质粒成功转染HKC细胞并能够表达绿色荧光蛋白②转染HKC细胞后ILK RNA及蛋白表达:与HG和阴性对照质粒组(HG+HK)相比,pGenesil-1.1-ILK-1 siRNA、pGenesil-1.1-ILK-2 siRNA、pGenesil-1.1-ILK-3 siRNA质粒转染组可见ILKmRNA水平下降,抑制率分别为36.47%、25.11%及43.15%;ILK蛋白表达下降,分别比HG及HG+HK减少41.59%,29.78%和56.12%,但较NG组表达仍高(p<0.05)。③免疫细胞化学显示:pGenesil-1.1-ILK-3转染HKC细胞48h后胞浆P-GSK-3β、胞浆及核β-catenin比HG及HG+HK组表达下降,但较NG组表达仍高(p<0.05)。④Western blot显示:pGenesil-1.1-ILK-3转染HKC细胞后P-GSK-3β、β-catenin核蛋白均较HG及HG+HK组表达下降(P<0.05);GSK-3β与总β-catenin在各组表达无明显差异( P>0.05);E-cadherin在HG+ILK-3siRNA的表达较HG及HG+HK组有所升高,α-SMA表达与E-cadherin相反(P<0.05或P<0.01)。
4糖尿病肾病患者肾组织病理变化及Wnt4、P-GSK -3β、β-catenin的表达
①肾组织的病理改变:轻中度组DN患者肾病理主要表现为肾小球的肥大,系膜细胞增生,系膜区增宽,肾小球及肾小管基底膜轻度增厚,局灶性肾小管上皮细胞颗粒变性、萎缩及间质纤维化。重度组DN患者肾组织出现典型的K-W结节,甚至球性硬化,弥漫性肾小管间质纤维化。②免疫组化结果:对照组Wnt4、p-GSK-3β仅微弱表达于部分肾小管上皮细胞,糖尿病肾病轻中度组表达增强,在重度组表达下降(P<0.05);β-catenin在对照组少量表达于肾小管上皮细胞基底膜侧,糖尿病肾病轻中度组肾小管上皮细胞及肾间质细胞胞浆表达明显增强,部分细胞胞核呈阳性表达。而在重度组表达减弱,尤其在重度纤维化病变处几乎不表达(P<0.05)。③Wnt4、p-GSK-3β、β-catenin表达均与24小时尿蛋白定量成正相关,而与估算的肾小球滤过率(eGFR)成负相关(P<0.05)。
结论:
1 Wnt/β-catenin信号途径在糖尿病肾病早期以及高糖诱导肾小管上皮细胞转分化过程中表达增高,该途径可能参与了糖尿病肾病肾小管间质纤维化的发生和进展。
2 ILK在糖尿病肾病早期肾小管间质病变及高糖诱导的肾小管上皮细胞转分化过程中表达增高;转染HKC ILKsiRNA能成功沉默ILK基因,使P-GSK-3β及核β-catenin表达下调,并使高糖诱导下调的E-cadherin水平升高,α-SMA水平降低而阻止肾小管细胞转分化,提示ILK可能通过调节Wnt/β-catenin途径下游因子活性参与肾小管细胞转分化过程。
3 Wnt/β-catenin信号途径在糖尿病肾病患者肾小管间质表达增高,但在重度肾小管间质纤维化病变表达下调,其变化可能参与了人类糖尿病肾病肾间质纤维化的发病与进展。
Objectives: Diabetic nephropathy(DN) is one of the most common complications of diabetes, which is histologically characterized by hypertrophy of glumeruli, increased thickness of basement membrane, overaccumulation of extracellular matrix, glomerulosclerosis, tubular atrophy, interstitial fibrosis and so on. Much more researches have focused on glomerular lesions, little emphasized the crucial role of tubulointerstitial injury in diabetic kidney in the past.It has recently been demonstrated that the development of tubulointerstitial fibrosis is more closely correlated with a progressive decline in renal function compared to glomerularsclerosis. Profibrotic switches in the phenotype of epithelial cells and epithelial–mesenchymal transition (EMT) can be induced by high glucose . Transdifferentiated epithelial cells become reprogrammed to secrete and excessively accumulate extracellular matrix which promote tubulointerstitial fibrosis in diabetic kidney. It is widely recognized that tubulointerstitial injury serves as an important mediator of chronic renal failure and predictor of outcome in patients with diabetic nephropathy.
Wnt/β-catenin signalling pathway plays an important role in mammalian developmental processes, tumorigenesis, and organic fibrosis by regulating a variety of cellular processes including cell proliferation, differentiation, survival and apoptosis.Wnt/β-catenin signalling pathway regulates apoptosis of mesangial cells induced by high glucose,and the pathway is activated after unilateral ureteral obstruction, suggesting that the Wnt/β-catenin signalling pathway is likely implicated in the pathogenesis of chronic kidney desease.
Integrin-linkedkinase (ILK) is one of the most important downstream effector of TGF-β1 in mediating renal fibrosis.Oversxpression of ILK aggravate extracellular matrix deposition and tubulointerstitial fibrosis in chronic kidney desease , diabetic nephropathy and mouse models of unilateral ureteral obstruction. Activation of the downstream components of the Wnt signaling pathway, namely inhibition of glycogen synthase kinase 3β(GSK -3β)activity and nuclear accumulation ofβ-catenin, has also been achieved through overexpression of ILK. Cross talk between ILK and Wnt signalling pathway likely contribute to the onset and progression of tubulointerstitial fibrosis.
Role for Wnt/β-catenin signaling pathway in mediating tubulointerstitial fibrogenesis in diabetic kidney and tubular epithelial–mesenchymal transition induced by high glucose remains to be unknown. Although ILK has been known to regulate the effector activity of Wnt signaling, direct interplay between ILK and the Wnt pathway in diabetic nephropathy has remained to be clarified.
In the present study, using STZ-induced diabetic model and HKC cells treated with high glucose ,we investigated the expression of Wnt/β-catenin signaling pathway and ILK and the relationship between them in early stage of DN and transdifferentiated tubular epithelial cells. Furthermore, we investigated the role of ILK in modulating the components expression of Wnt signaling in HKC cells by silencing ILK gene via specific ILK shRNA. At last, the expression of Wnt/β-catenin pathway was examined in renel tissues from patients with diabetic nephropathy to elucidate the potential role of this signaling pathway for tubulointerstitial fibrosis in diabetic kidney,in order to provide experimental and clinical evidences for explaining the mechanism of the development of tubulointerstitial disease in diabetic nephropathy and seeking new treatment target.
Methods:
1 Preparation of diabetic model and examination of ILK,Wnt4,GSK -3β,β-catenin protein and mRNA expression
Male Wistar rats were randomly divided into two groups after unilateral nephrectomy:control group(n=18), diabetic group(n=18). The rats of diabetic group were injected intraperitoneally with 65 mg/kg body weight STZ in 0.1mol/L sodium citrate solution (pH 4.5), and the rats in the control group were injected with 0.1mol/L sodium citrate solution. Diabetic model was considered to be successful when the blood glucose was≥16.7mmol/L and the urine glucose was +++~++++ after 48 hours of the injection. Six rats from each group were sacrificed respectively at weeks 4, 8 and 12 after STZ injection. Partial renal tissues were fixed in 4% formaldehyde for light microscopic and immunohistochemical staining. Partial renal cortices were used to extracte RNA and total and nuclear protein . The expressions of ILK,Wnt4,β-catenin andα-SMA in renal tissues were evaluated by immunohistochemistry . Western blot was used to examine the expression of ILK,Wnt4,GSK-3β,P-GSK -3β, total and nuclearβ-catenin . The mRNA levels of Wnt4 andβ-catenin were measured by reverse transcription and polymerase chain reaction (RT-PCR).
2 Cell culture and examination of ILK,Wnt4,GSK -3β,β-catenin protein and mRNA expression
Human kidney proximal tubular epithelial cell line (HKC) cells were incubated with serum-free DMEM for 24 hours to synchronize the cell growth,then the cells were divided into three groups:NG group (media containing 5.5mM glucose); mannitol control group (media containing 5.5mM glucose and 24.5mM mannitol); HG group (media containing 30mM glucose). The cells were collected to extracte total RNA and protein at 12, 24, 48 and 72 hours after incubation. The expression of Wnt4,β-catenin, E-cadherin andα-SMA were examined by immunocytochemistry. Western-blot was also used to detect the expression of ILK,Wnt4,GSK-3β,P-GSK-3β, total and nuclearβ-catenin proteins. The mRNA levels of Wnt4,GSK-3βandβ-catenin were examined by RT-PCR .
3 Transfection of ILKsiRNA into HKC cells and examination of ILK, GSK -3β,β-catenin protein and mRNA expression
Three small interfering RNA (siRNA) targeting human ILK gene sequences were synthesized to silence ILK gene expression.HKC cells were divided into six groups: NG group, HG group ,HK control group(a vector containing the non-specific siRNA was designed as negative control), pGenesil-1.1-ILK-1siRNA group, pGenesil-1.1-ILK-2siRNA group and pGenesil-1.1-ILK-3 siRNA group. Transient transfection of HKC cells was carried out using Lipofectamine 2000 according to the manufacturer’s instruction. Six hours after transfection, the medium of NG group was replaced by normal glucose and the medium of other groups was replaced by high glucose DMEM medium (30mM glucose) with 10% FBS .The cells were incubated for an additional 48h. Fluorescent microscopy was used to examine GFP expression . Then ILK protein and RNA expression was examined by Western-blot and RT-PCR. Immunocytochemistry was used to observe the expression of P-GSK-3βandβ-catenin. The expression of GSK-3β,P-GSK-3β, total and nuclearβ-catenin ,E-cadherin andα-SMA proteins was examined by Western-blot.
4 Patients and observation of Wnt4,P-GSK -3βandβ-catenin expression
Thirty three patients diagnosed as diabetic nephropathy by renal biopsy and clinical data from October 2006 to October 2008 at the Second Hospital of Hebei Medical University were included in this study.There were 18 male and 15 female patients whose ages were from 28 to 70(48.7±10.3)years old.The cases of lupus nephritis,Henoch-Schonlein nephritis, HBV associated glomerulonephritis, thyropathy associated glomerulonephritis,renal damage induced by rheumatoid arthritis and cancer associated nephropathy were excluded.Those cases complicated with acute renal tubular necrosis,acute or chronic interstitial nephritis were also excluded. The patients were divided into two groups according to the degrees of tubulointerstitial fibrosis: mild /moderate group (n=20)and advanced group(n=13). The renal tissues without evidence of renal disease (n=10) obtained from distant portions of kidneys surgically excised because of the presence of a localized neoplasm were used as control. Clinical data were collected including age, duration of diabetes, plasma albumin,urine protein excretion, serum creatinine and so on. Tubulointerstitial expression of Wnt4、P-GSK-3βandβ-catenin was assessed by immunhistochemical staining.
Results:
1 Protein and mRNA expression of ILK,Wnt4,GSK-3β,β-catenin in the renal tissues of diabetic rats
①Immunohistochemical positive staining of ILK and Wnt4 was observed in renal tubular epithelial cytoplasim whileα-SMA is restricted to the vascular smooth muscle cells in control group. They were all remarkably increased in diabetic group (P<0.05).In control group,β-catenin showed a basolateral localization within the proximal tubules,but was observed in cytoplasm and nuclei of tubular epithelial and renal interstitial cells in diabetic group (P<0.05).②From the results of Western blot analysis, the diabetic rats showed increased expression of ILK ,Wnt4,P-GSK-3βand nuclearβ-catenin in the kidney from week 4, and maintained at a higher level until week 12 (P<0.05). There is no significant difference of GSK-3βand totalβ-catenin protein expression between the groups (P>0.05).③The mRNA levels of Wnt4 in the kidney of diabetic rats increased compared with control group (P<0. 05). No significant difference ofβ-catenin mRNA expression was found between different groups(P> 0. 05).
2 The expression of ILK,Wnt4,GSK-3β,β-catenin in HKC cells incubated in HG
①Immunocytochemical staining showed that Wnt4 weakly expressed in HKC cytoplasm in NG group and mannitol control group while the expression enhanced after HG stimulation.β-catenin was mainly anchored at the membrane and faintly expressed in cytoplasm of HKC cells in control group . In HG group ectopic expression ofβ-catenin was found in cytoplasm and translocated into nuclei of HKC cells (P<0.05 or P<0.01). In HG group the expression of E-cadherin decreased compared with control groups. The expression ofα-SMA was opposite to that of E-cadherin(P<0.05).②Western blot analysis indicated that the expression of Wnt4,p-GSK-3β,nuclearβ-catenin and ILK were increased in HKC cells of HG group(P<0.05). Wnt4,p-GSK-3βand nuclearβ-catenin expression in HKC reached the peak at 24h and that of ILK peaked at 48h after HG stimulation(P<0.01). No significant difference of GSK-3βand totalβ-catenin protein expression was observed among the three groups (P>0.05).③The mRNA levels of Wnt4 was up-regulated in HG group compared with control group (P<0.05). There was no significant difference of GSK-3βandβ-catenin mRNA expression found among different groups(P>0.05).
3 The expression of ILK,GSK-3βandβ-catenin in HKC cells transfected with ILKsiRNA
①pGenesil-1.1-ILK-1siRNA, pGenesil-1.1-ILK-2siRNA and pGenesil-1.1-ILK-3 siRNA were transfected into HKC cells to silence ILK expression. At 48 hours after transfection, green fluorescent protein(GFP)was observed in about 60%-70% HKC cells, showing that the transfection is successful.②The ILK mRNA and protein were examined using the methods of RT-PCR and Western blot. Compared with HG and non-specific siRNA transfected control group(HK), the ILK mRNA was significantly inhibited by 36.47%、25.11% and 43.15% respectively in groups transfected with ILKsiRNA(P<0.05).Correspondingly,protein expression of ILK was inhibited by 41.59% , 29.78% and 56.12% respectively(P<0.05).③Immunocytochemical staining showed that P-GSK-3βas well as cytosolic and nuclearβ-catenin expression remarkbly reduced in HKC cells transfected with ILK siRNA compared with that of HG and non-specific siRNA transfected control group(P<0.05).④Western blot showed ILK siRNA suppressed phosphorylation of GSK-3βandβ-catenin translocation into nuclei, whereas the level of P-GSK-3βand nuclearβ-catenin remained increased in HG and non-specific siRNA transfected control group(P<0.05). No significant difference of GSK-3βand totalβ-catenin protein expression was observed among different groups (P>0.05). Downregulation of E-cadherin and upregulation ofα-SMA induced by HG were recovered to a certain degree in HG+ILK-3siRNA group(P<0.05).
4 Pathological changes and the expression of Wnt4,P-GSK-3,β-catenin in renal tissues of patients with diabetic nephropathy
①pathological changes including glomerular enlargement, mesangial matrix expansion, increase of glomerular and tubular basement membrane in thickeness, focal tubular epithelial vacuolar degeneration and atrophy as well as mild interstitial fibrosis were observed in the patients of mild/morderate group. Progressive glomerulosclerosis with the presence of Kimmelstiel–Wilson lesions, global sclerosis and diffused tubulointerstitial fibrosis could be found in the patients of advanced group.②Immunohistochemical staining displayed that Wnt4 and p-GSK-3βweakly expressed in renal tubular epithelial cytoplasim in control group. The expression remarkably increased in mild/morderate DN group,whereas decreased in advanced DN group(P<0.05). In control kidneys,β-catenin was found to be localized basolaterally within the proximal tubules. Increase of cytosolic and nuclearβ-catenin in both tubular epithelial and interstitial cells were observed in mild/morderate DN group. However, in advanced DN group cytosolic and nuclearβ-catenin is downregulated,and is barely detectable in severe tubulointerstitial lesions (P<0.05).③The expression of Wnt4, p-GSK-3β, nuclearβ-catenin had a positive correlation with urine protein excretion, and a negative correlation with estimated glomerular filtration rate(eGFR) (P<0.05) .
Conclusions:
1 Wnt/β-catenin signaling pathway activity is up-regulated in the early stage of DN and in the HKC cells incubated in high glucose medium,and the up-regulation is correlated with tubular epithelial–mesenchymal transition, suggesting that the pathway may be involved in the pathogenesis of tubulointerstitial fibrogenesis of DN.
2 The expression of ILK is also increased in the early stage of DN and in the HKC cells incubated in high glucose medium, and correlated with tubular epithelial–mesenchymal transition. ILKsiRNA transfection into HKC cells successfully silence ILK expression and reverse tubular epithelial–mesenchymal transition by downregulating the expression of P-GSK-3βand nuclearβ-catenin.Thus,the effect of ILK to modulate the activity of downstream componants in Wnt/β-catenin signaling pathway may be involved in the tubular epithelial–mesenchymal transition
3 The expression of Wnt/β-catenin signaling pathway is increased in the patients of mild/morderate DN group,but decreased in the patients of advanced DN group.These changes of the pathway may be contributable to the onset and progression of tubulointerstitial fibrosis of human diabetic nephropathy.
引文
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