Notch信号通路在糖尿病肾病足细胞损伤中的研究
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摘要
目的：糖尿病肾病（diabetic nephropathy, DN）是糖尿病的常见并发症，是引起终末期慢性肾功能衰竭的主要原因之一。DN的发病机制非常复杂，糖脂代谢紊乱、血流动力学改变、氧化应激反应及多种细胞因子等是其主要起始因素。在这些起始因素的下游，又有多条信号传导通路在其中发挥着重要作用。近年来发现Notch信号通路在DN的发生、发展过程中起着重要作用。
已有研究表明，Notch通路决定胚胎发育、细胞增殖和分化，在胚胎后肾形成过程中对足细胞和肾小管的分化中起着重要的作用。近年来在肾脏疾病领域的研究证实，Notch信号通路与糖尿病肾病、局灶节段性肾小球硬化、肾病综合症等多种肾小球疾病有关，参与肾小球硬化的发生。在哺乳动物体内Notch信号通路有四种受体和五种配体，其中受体为Notch1-Notch4，配体分别为Jagged1、Jagged2、Delta样配体（Dll）1、Dll3和Dll4。配体与Notch受体的结合导致受体构象发生改变，随后在γ-分泌酶（γ-secretase）的介导下发生蛋白水解作用，释放出Notch胞内域（Notch intracellular domain, NICD），NICD转移至细胞核内，活化Hes1和Hey1等分化拮抗基因的转录，阻碍分化效应基因的表达，影响细胞的分化、增殖和凋亡。
既往研究认为，DN的主要病理特征是肾小球基底膜（glomerularbasement membrane，GBM）增厚、细胞外基质（extracellular matrix, ECM）堆积、肾小球硬化、肾小管萎缩及肾间质纤维化。近年研究发现，足细胞损伤在DN发生、发展过程中起重要作用。足细胞是终末分化的肾小球上皮细胞，它附着在GBM外侧，是肾小球滤过膜的重要组成部分。由于足细胞是高度分化细胞，增殖能力较差，当足细胞受到损伤时，Nephrin和Podocin等足细胞相关蛋白在足细胞裂孔膜（slit diaphragm，SD）丢失，使肾小球滤过电荷屏障减弱，促进蛋白尿的发生。足盂蛋白（podocalyxin，PCX）是足突顶膜区主要的带负电荷跨膜蛋白，当足细胞损伤后PCX表达减少，破坏了足突间的排斥作用使之发生粘附、融合，导致足细胞易于脱落。现已知主要的凋亡通路有Bcl-2、p53、NF-κB等，在DN发生时，足细胞内的这些凋亡通路可被激活，诱导足细胞凋亡。同时受损的足细胞对ECM生成的调节作用发生紊乱，转化生长因子β1（transforming growthfactor-β1, TGF-β1）等促进基质生成的因素增加，造成Ⅳ型胶原等基膜样基质增加，导致肾小球硬化，加速DN的进展。
许多学者通过不同方法证实了Notch信号通路在足细胞分化中的作用，Cheng等使用γ-分泌酶抑制剂（γ-secretase inhibitor，GSI）抑制小鼠后肾形成过程中Notch通路的活化，发现近端小管细胞和肾小球中足细胞的形成明显减少，而伴随非上皮细胞的增多。如果在足细胞发育过程中出现异常Notch通路的活化，可抑制足细胞的终末分化，引起小鼠肾小球硬化。我们通过收集临床糖尿病肾病标本、建立糖尿病小鼠模型及足细胞高糖（high glucose, HG）培养，检测Notch信号通路家族成员（Jagged1、Notch1、NICD1、Hes1及Hey1）的表达情况，并通过应用血管紧张素II1受体阻断剂（angiotensin II type1receptor antagonism, AT1Ra）、基因干扰技术及Notch信号通路抑制剂（GSI）抑制Notch通路，来观察是否Notch信号通路参与DN时的足细胞损伤，促进足细胞凋亡和细胞外基质沉积，为糖尿病肾病的治疗提供一条新的思路。
方法
1糖尿病肾病患者肾组织中Notch信号通路检测
收集2010年10月~2012年10月在河北医科大学第三医院经病史、临床检查及肾穿刺活检病理诊断为糖尿病肾病患者34例（diabeticnephropathy group），既往无其它肾脏病史，以10例肾肿瘤患者远离肿瘤的瘤旁组织做为对照组（control group）。留取血和尿标本检测血糖（Glu）、糖化血红蛋白（HbA1）、24小时尿蛋白定量（UPE）、肌酐（Scr）、估算肾小球滤过率（eGFR）；采用免疫组织化学检测Jagged1、Notch1、NICD1、Hes1及Hey1蛋白表达。
2Notch信号通路在糖尿病小鼠肾组织中的作用
雄性CD-1小鼠随机分为3组：对照组（control group）、糖尿病组（diabetic group）、糖尿病+缬沙坦组（diabetic+Valsartan group）。糖尿病模型小鼠腹腔单次注射链脲佐菌素（streptozotocin, STZ，150mg/kg），对照组只注射相同体积的枸橼酸盐缓冲液，72小时后测定血糖和尿糖，血糖值≥16.7mmol/L,尿糖值+++~++++者确定为糖尿病模型成功。糖尿病+缬沙坦组小鼠在糖尿病模型建立后，以缬沙坦（40mg/kg）灌胃，对照组和糖尿病组以等体积蒸馏水灌胃。分别于成模后1、2、4、8周每组取6只小鼠，收集血液及24小时尿液，用于生化指标检测，ELISA方法检测尿液PCX的含量；切取肾脏4%中性甲醛固定用于组织化学、TUNEL及免疫组织化学染色；取部分肾皮质组织置于4%戊二醛用于电镜观察；部分肾皮质组织用于提取蛋白及RNA，Western blot检测Jagged1、Notch1、NICD1、Hes1、Hey1、Bax、Bcl-2、cleaved Caspase-3、p-p53及p53蛋白表达，Real-time PCR检测Jagged1、Notch1、Hes1及Hey1mRNA表达；另取部分肾皮质组织于70%酒精固定，用于流式细胞术检测细胞凋亡率。
3高糖对小鼠足细胞Notch信号通路的作用
小鼠足细胞在5%CO2，33°C CO2培养箱中，以含有10%胎牛血清及γ-干扰素（10U/ml）的RPMI1640培养液培养细胞使其增殖，传代后更换为不含γ-干扰素的RPMI1640培养液，置入5%CO2，37°C CO2培养箱中培养10~14天，促进细胞分化成熟。将实验细胞分成7组：正常糖对照组（5.5mmol/L glucose, NG）、正常糖+高渗对照组（5.5mmol/Lglucose+24.5mmol/L mannitol, NM）、高糖组（30mmol/L glucose，HG）、高糖+阴性质粒组（30mmol/L glucose+sh-Scramble，HG+C）、高糖+sh-Jagged1质粒组（30mmol/L glucose+sh-Jagged1，HG+J1）、高糖+sh-Notch1质粒组（30mmol/L glucose+sh-Notch1，HG+N1）、高糖+GSI组（30mmol/L glucose+1μmol/L GSI，HG+GSI）。分别经细胞同步化、分组干预刺激0、12、24、48、72小时后收集细胞，免疫细胞化学检测Jagged1、Notch1、NICD1、Hes1和Hey1蛋白表达；Western blot检测Jagged1、Notch1、NICD1、Hes1、Hey1、Bax、Bcl-2、cleaved Caspase-3、p-p53及p53蛋白表达；Real-time PCR检测Jagged1、Notch1、Hes1及Hey1mRNA表达；TUNEL和AnnexinⅤ/PI染色检测足细胞凋亡率。
结果
1糖尿病肾病患者临床病理表现及Notch信号通路检测
①光镜下观察，对照组肾小球、肾小管及肾间质未见明显异常；糖尿病肾病组肾小球体积增大，基底膜弥漫或不规则增厚，细胞外基质增多，K-W结节形成，部分肾小管上皮细胞出现空泡变性，间质纤维化。②糖尿病肾病组空腹血糖、糖化血红蛋白、尿蛋白量、肌酐较对照组均明显增多，而肾小球滤过率糖尿病肾病组患者比对照组下降。③免疫组化显示Jagged1、Notch1、NICD1、Hes1及Hey1在肾小球及肾小管均有表达，糖尿病肾病组较对照组表达增多。
2糖尿病小鼠肾组织Notch信号通路的表达及缬沙坦对Notch信号通路和足细胞损伤的影响
①光镜及电镜下观察，糖尿病组小鼠轻微肾小球体积增大，系膜基质增多，基底膜不规则增厚，足细胞数量减少，足突融合，部分肾小管上皮细胞出现空泡变性。②与对照组相比，糖尿病组小鼠尿蛋白（Upro）从2周开始明显升高，随病程延长呈逐渐升高的趋势；糖尿病小鼠血糖（Glu）、尿素氮（BUN）、肌酐（Scr）比对照组小鼠增加，随病程延长呈逐渐升高的趋势；与对照组相比，糖尿病组小鼠尿蛋白和尿PCX含量明显升高，给予缬沙坦治疗后尿蛋白和PCX含量降低。③免疫组化结果显示Jagged1、Notch1、NICD1、Hes1和Hey1在对照组肾小球及肾小管有少量表达，在糖尿病组表达明显增加；Western blot检测显示，与对照组比较，糖尿病组小鼠肾组织中Jagged1、NICD1、Hes1及Hey1从1周时开始升高，表达最高点在4周，8周时略有下降；Notch1蛋白在糖尿病组比对照组表达增加，在糖尿病组各周次间表达无明显差异；糖尿病组Jagged1、Notch1、Hes1和Hey1mRNA水平与对照组相比表达增加，最高点在2周，随后下降。④Western blot和Real-time PCR显示，糖尿病组小鼠肾小球组织Jagged1、Notch1、NICD1、Hes1和Hey1蛋白及Jagged1、Notch1、Hes1和Hey1mRNA水平与对照组相比增加，给予缬沙坦治疗后降低。⑤TUNEL染色显示在糖尿病组小鼠肾组织中观察到凋亡细胞；Bax、p-p53及cleaved Caspase-3蛋白在糖尿病组小鼠肾小球组织中表达比对照组增加，给予缬沙坦治疗后降低；Bcl-2蛋白表达水平糖尿病组较对照组降低，给予缬沙坦后表达升高；p53蛋白在各组间表达无明显差异；流式细胞术显示糖尿病组小鼠肾小球内细胞凋亡增加，糖尿病+缬沙坦组降低，但仍高于对照组。
3足细胞体外高糖培养对Notch信号通路表达及足细胞损伤的影响
①免疫细胞化学显示，Jagged1、Notch1、NICD1、Hes1和Hey1在正常糖对照组足细胞中有少量表达，给予高糖刺激后表达量明显增加；Western blot检测显示，Jagged1和NICD1蛋白在高糖刺激足细胞12小时升高，48小时表达最高，72小时略有下降；Notch1蛋白在高糖刺激后其它时间点的表达量是高糖刺激0小时表达量的2倍，高糖刺激12~72小时足细胞中Notch1蛋白水平表达无明显差异；Hes1蛋白在高糖刺激24小时升高，48小时达到顶点，72小时有所降低；Hey1表达在高糖刺激12和24小时升高，然后随着刺激时间延长表达下降；高糖刺激呈时间依赖性引起Jagged1、Notch1和Hes1mRNA升高，最高峰在48小时；Hey1mRNA最高点在高糖刺激24小时，随后随着刺激时间延长而下降。②Western blot和Real-time PCR检测发现，高糖刺激引起Jagged1、Notch1、NICD1、Hes1、Hey1蛋白及Jagged1、Notch1、Hes1、Hey1mRNA表达增加，sh-Jagged1和sh-Notch1转染足细胞后表达下降，GSI可抑制足细胞NICD1、Hes1、Hey1蛋白及Hes1、Hey1mRNA在高糖刺激后的过表达现象，但Jagged1、Notch1蛋白及mRNA在高糖+GSI组和高糖组之间无明显差异。③Western blot检测发现高糖呈时间依赖性刺激Bax蛋白表达，12小时开始增加，72小时达到最高点，而Bcl-2蛋白在高糖刺激后表达逐渐下降，最低点在72小时；cleaved Caspase-3在高糖刺激12小时开始增加，24小时为最高点，之后逐渐减少；p-p53蛋白在高糖刺激下表达逐渐增加，最高点在72小时；p53蛋白在高糖刺激各时间点之间表达无明显差异。④足细胞高糖培养并应用GSI或sh-Jagged1、sh-Notch1质粒转染后发现Bax、p-p53及cleaved Caspase-3蛋白较高糖组表达下降，但较正常糖对照组表达仍高，而Bcl-2蛋白较高糖组表达增强，但较正常糖对照组表达仍低，p53蛋白在各组间表达无明显差异。⑤TUNEL荧光染色及AnnexinⅤ/PI染色显示高糖组较正常糖对照组细胞凋亡率升高，转染组及高糖+GSI组细胞凋亡率比高糖组降低。
结论
1在糖尿病肾病患者肾组织、糖尿病小鼠肾组织、高糖培养足细胞中发现Notch信号通路成员有过表达现象，提示Notch通路在糖尿病肾病足细胞中激活并可能参与足细胞损伤过程。
2缬沙坦抑制糖尿病小鼠肾组织中Notch信号通路的活化，同时抑制蛋白尿和足细胞脱落，抑制凋亡相关蛋白表达，减少细胞凋亡，抑制肾小球内细胞外基质沉积。提示缬沙坦可通过抑制Notch信号通路来产生对糖尿病肾病的治疗效果。
3在高糖培养的足细胞中，应用Notch信号通路干扰质粒及抑制剂抑制Notch信号通路的活化后，可抑制凋亡相关蛋白表达，减少足细胞凋亡。提示Notch信号通路参与高糖诱导的足细胞损伤。
Objectives: Diabetic nephropathy (DN) is one of the most commoncomplications of diabetes and has become the most frequent cause ofend-stage renal disease. The pathogenesis of diabetic nephropathy is verycomplicated, which original factors encompass glucose and lipid metabolicdisorder, abnormal hemodynamics, oxidative stress, more cytokines. Manykinds of signal pathways pay an important role in DN. Recent research foundthat Notch pathway had a close relation with DN.
The family of Notch pathway play important roles in embryonicdevelopment, cellular proliferation and differentiation. Detailed analyses ofthe expression pattern of Notch and related genes in differentiation ofproximal tubule and podocyte during nephrogenesis have been performed.Recent studies in renal disease found that Notch pathway was relevant withglomerular disease, including DN, focal segmental glomerulosclerosis andhydropigenous nephritis, to participate in glomerular sclerosis. In mammalthere are four receptors, Notch1-Notch4, and five ligands, Jagged1, Jagged2,Delta-like (Dll)1, Dll3, and Dll4. The binding of ligand and Notch receptorinduces a conformational change in the Notch receptor. This allows theγ-secretase–mediated protease to release the Notch intracellular domain(NICD). NICD travels into the nucleus where it activates the transcription ofdownstream genes such as Hes1and Hey1genes and affects cellulardifferentiation, proliferation and apoptosis.
The major characteristics of DN includes glomerular basement membrane(GBM) thickening, accumulation of extracellular matrix (ECM), glomerularsclerosis, tubular atrophy and interstitial fibrosis. It is believed thatpodocytes injury can lead to the development of DN. Since podocytes formeda critical part of the glomerular filtration barrier are terminally differentiated cells, which are unable to divide. Loss of slit diaphragm-associated proteinssuch as nephrin and podocin that weaken the electrostatic barrier ofglomerular slit diaphragm (SD) and result in proteinuria. Podocalyxin (PCX),as a protein rich in negative charge, is expressed in an epiphragm of footprocess. Down-regulation of podocalyxin expression in podocytes injurydestroys the rejection of foot process and results in podocytes detachmentfrom the glomerular basement membrane. In the development of DN,podocytes apoptosis is induced via activation of apoptotic pathways, such asBcl-2, p53, NF-κB pathway and so on. Impaired podocytes also have theimbalance between ECM synthesis and degradation, increase transforminggrowth factor-β1(TGF-β1) level to generate ground substance, lead toincreasing expression of Type Ⅳ collagen, cause glomerulosclerosis inprogression of DN.
Detailed analyses of the expression pattern of Notch and related genesduring nephrogenesis have been performed. Cheng et al used γ-secretaseinhibitor (GSI) to depress the activation of notch pathway in mousemetanephros, then found fewer renal epithelial structures, low quantity ofproximal tubule and glomerular podocyte, accompanied by an increase inintervening nonepithelial cells. Ectopic activation of Notch pathway indeveloping podocytes on the other hand caused glomerulosclerosis indeveloping murine kidneys and opposed terminal differentiation of podocytes.In the present study, we investigated the expression of Notch pathway(Jagged1, Notch1, NICD1, Hes1, Hey1) on renel tissues from patients withdiabetic nephropathy, diabetic mice and podocytes treated with high glucose(HG). Furthermore, we inhibited Notch pathway by angiotensin II type1receptor antagonism (AT1Ra), chemical inhibitor or specific short hairpinRNA vector to investigate whether Notch pathway participated in podocytesinjury and induced cell apoptosis and accumulation of extracellular matricalcomponents. These provide a new treatment target for diabetic nephropathy.
Methods
1Determination of Notch pathway in renel tissues from patients with diabetic nephropathy
Thirty four patients diagnosed as diabetic nephropathy by renal biopsyand clinical data from October2010to October2012at the third Hospital ofHebei Medical University were included in this study. Other nephropathy wasexcluded. The renal tissues (n=10) obtained from distant portions of kidneyssurgically excised because of the presence of a localized neoplasm were usedas control. Blood and urinary samples was collected to detect Glu, HbA1, UPE,Scr and eGFR. Protein expression of Jagged1, Notch1, NICD1, Hes1andHey1was assessed by immunhistochemical staining.
2Role of Notch pathway in diabetic mice
Male CD-1mice were randomly divided into three groups: control group,diabetic group and diabetic+Valsartan group. The mice of diabetic modelreceived a single intraperitoneal injection of streptozotocin (STZ) at a dose of150mg/kg body weight. The mice of control group only received an injectionof the same volume of sodium citrate. The model of diabetes was consideredto be successful when the blood glucose was≥16.7mmol/L and the glucose inurine was+++~++++after72hours of the injection. After the diabetic modelwas affirmed to be successful, the mice of diabetic+Valsartan group wereadministered daily with Valsartan (40mg/kg) by gavage. The mice of controlgroup and diabetic group were only administered daily with the same volumeof diatilled water by gavage. Six mice from every group were respectivelysacrificed at weeks1,2,4and8after STZ injection. Blood and24h urinesamples were collected for biochemical indicator and enzyme linkedimmunosorbent assay (ELISA). Partial renal tissures were fixed in4%neutralformalin for histochemical, TUNEL and immunohistochemical staining.Partial renal cortices were fixed in4%glutaraldehyde for electron microscopicobservation. Protein and RNA were extracted from partial renal cortices forWestern blot and Real-time PCR. The expression of Jagged1, Notch1, NICD1,Hes1, Hey1, Bax, Bcl-2, cleaved Caspase-3, p-p53and p53protein wasrespectively evaluated by Western blot. The mRNA levels of Jagged1, Notch1,Hes1and Hey1were evaluated by Real-time PCR. Partial renal cortices were fixed in70%alcohol for flow cytometry.
3Role of Notch pathway in mouse podocytes induced by high glucose
To induce proliferation, mouse podocytes were cultured at33°C in ahumidified atmosphere of5%CO2in RPMI1640containing10%fetal bovineserum and10U/ml-IFN under growth permissive conditions, and then cellswere cultured at37°C in RPMI1640without-IFN under growth restrictiveconditions for10~14days to induce quiescence and the differentiatedphenotype. Podocytes were randomly divided into seven groups: normalglucose group (5.5mmol/L glucose, NG), normal glucose+mannitol group(5.5mmol/L glucose+24.5mmol/L mannitol, NM), high glucose group (30mmol/L glucose, HG), high glucose+negative control vector (30mmol/Lglucose+sh-Scramble, HG+C), high glucose+sh-Jagged1vector (30mmol/Lglucose+sh-Jagged1, HG+J1), high glucose+sh-Notch1vector (30mmol/Lglucose+sh-Notch1, HG+N1) and high glucose+GSI (30mmol/L glucose+1μmol/L GSI, HG+GSI). The seven groups were cultured for0,12,24,48and72hours respectively, and then podocytes were harvested. The proteinexpression of Jagged1, Notch1, NICD1, Hes1and Hey1was detected byimmunocytochemistry. The expression of Jagged1, Notch1, NICD1, Hes1,Hey1, Bax, Bcl-2, cleaved Caspase-3, p-p53and p53protein was respectivelyevaluated by Western blot. The mRNA levels of Jagged1, Notch1, Hes1andHey1were evaluated by Real-time PCR. The TGF-β1, Type Ⅳ collagen andLaminin proteins were examined by ELISA. Podocytes apoptosis was detectedby TUNEL and AnnexinⅤ/PI staining.
Results
1pathological manifestation and Notch pathway expression of patientswith diabetic nephropathy
①There were no abnormal changes in glomerulus, renal tubule andinterstitium of control group by light microscopy. Pathological changesincluding glomerular enlargement, increase of glomerular basementmembrane in thickeness, increase of ECM, the presence ofKimmelstiel–Wilson lesions, focal tubular epithelial vacuolar degeneration as well as interstitial fibrosis were observed in the patients of diabeticnephropathy.②Compared with control group, the levels of Glu, HbA1, UPEand Scr in diabetic nephropathy group were increased significantly. The eGFRlevel of diabetic nephropathy group was decreased than control group.③Immunohistochemical staining displayed that Jagged1, Notch1, NICD1, Hes1and Hey1weakly expressed in renal glomerular and tubular epithelium incontrol group, whereas remarkably increased in diabetic nephropathy group.
2The expression of Notch pathway in renal tissues of diabetic mice andthe effect of Valsartan on Notch pathway and podocytes injury
①Diabetic mice showed slightly glomeruli hypertrophy, increasingmesangium matrix, thickened glomerular basement membrane, reduction inthe number of podocytes, fusion of foot process, partial tubular epithalialvacuolar degeneration by light and electron microscope.②Compared withcontrol group, the Glu, BUN and Scr levels upregulated in a time-dependentmanner in diabetic group.24h urine protein was significantly increased fromweek2to week8in diabetic group than control group.24h urine protein andthe concentration of podocalyxin in urine of diabetic mice were higher thanthat in control group. Compared with diabetic group,24h urine protein andthe concentration of podocalyxin in urine of diabetic+Valsartan group weresignificantly decreased.③By immunohistochemical staining, the proteinexpression of Jagged1, Notch1, NICD1, Hes1and Hey1was increased inglomeruli and tubule cells of diabetic group than that in control group.Compared with control group, Western blot indicated that the proteinexpression of Jagged1, NICD1, Hes1and Hey1began to increase at week1,reached the peak at week4and slightly decreased at week8in diabetic group.Notch1protein expression of diabetic mice increased than control mice. Nodifferences of Notch1was found in diabetic group among different time points.The mRNA levels of Jagged1, Notch1, Hes1and Hey1began to increase atweek1, reached the peak at week2and slightly decreased in diabetic group.④By Western blot analysis, glomerular tissues of diabetic mice showedincreased expression of Jagged1, Notch1, NICD1, Hes1and Hey1, but Valsartan decreased their protein expression in diabetic mice. The expressionof Jagged1, Notch1, Hes1and Hey1mRNA was the same with the expressionof protein.⑤Apoptotic cells by TUNEL were observed in renal tissues ofthe diabetic kidney. Glomerular tissues of dabetic mice had a significantup-regulation in Bax, p-p53and cleaved Caspase-3expression anddown-regulation in Bcl-2expression compared with control group. However,the alternations of Bax, p-p53, cleaved Caspase-3and Bcl-2protein levels indiabetic group were reversed by addition of Valsartan. No change of p53protein expression was found in different groups. Apoptotic rate of glomerulartissues in diabetic group increased compared to control group by flowcytometry. Apoptotic rate was significantly lower in the diabetic+Valsartangroup than that in diabetic group.
3The effects of high glucose in podocytes on the expression of Notchpathway and podocytes injury
①Immunocytochemical staining showed that the expression of Jagged1,Notch1, NICD1, Hes1and Hey1increased in podocytes induced by highglucose. By Western blot, the protein expression of Jagged1and NICD1beganto increase at12h after the stimulation of HG, reached the peak at48h andslightly decreased at72h. Notch1protein expression at12-72h within HGstimulation was2-fold greater than that of the0h of HG stimulation. Nochange of Notch1protein expression was found in HG-induced podocytes at12~72h. The Hes1protein significantly increased in podocytes stimulated byHG for24h, continuously increased up to48h and slightly decreased at72h.The increased Hey1protein expression was confirmed after12and24h ofstimulation with HG separately, and gradually decreased with prolongedstimulation. The effects of HG on mRNA levels of Jagged1, Notch1and Hes1were revealed at12h and peaked at48h. HG also induced Hey1mRNAexpression and peaked at24h.②Compared with the cells of the normalglucose group, the protein levels of Jagged1, Notch1, NICD1, Hes1and Hey1significantly increased in high glucose group by Western blot. Thetransfection with sh-Jagged1or sh-Notch1vector respectively decreased high glucose-induced the protein overexpression of Jagged1, Notch1, NICD1, Hes1and Hey1in podocytes. Real-time PCR showed the similar changes ofJagged1, Notch1, Hes1and Hey1mRNA after transfection. The protein levelsof NICD1, Hes1and Hey1were markedly higher in podocytes stimulated withhigh glucose than the cells treated with normal glucose and were dramaticallyreduced in response to GSI. Real-time PCR showed the similar changes ofHes1and Hey1mRNA after treatment with GSI. Western blot and Real-timePCR analysis revealed that GSI did not inhibite Jagged1and Notch1overexpression induced by high glucose.③HG notably increased proteinlevel of Bax in time-dependent manner and peaked at72h. HG stimulationdecreased Bcl-2protein level in time-dependent manner and the cellsstimulated by HG for72h showed the minimum expression. The expressionof the HG-induced cleaved Caspase-3significantly increased at12h, peakedat24h, and gradually decreased with prolonged HG stimulation. HGstimulation increased p-p53protein level in time-dependent manner and themaximum expressions were at72h after stimulation of HG. However, therewas no difference of p53expression among all time spots in HG-inducedpodocytes.④High glucose stimulation in podocytes decreased Bcl-2proteinlevel than in normal glucose medium. The protein expression of the highglucose-induced Bax, p-p53and cleaved Caspase-3was significantlyincreased than normal glucose stimulation in podocytes. Compared with thecells treated with high glucose, Bax, p-p53and cleaved Caspase-3proteinlevels significantly decreased in cells transfected with sh-Jagged1orsh-Notch1vector and pretreated with GSI, while Bcl-2level increased. Nochange of p53protein expression was found in the cultured podocytes ofdifferent groups.⑤The results of TUNEL and AnnexinⅤ/PIstainingshowed that the apoptotic podocytes in high glucose stimulation weremarkedly higher than that in normal group, which were inhibited bysh-Jagged1or sh-Notch1vector and GSI.
Conclusions
1The overexpression of Notch pathway member is found in renel tissues from patients with diabetic nephropathy, diabetic mice and mouse podocytesinduced by high glucose, which suggests that activation of notch pathway maymediate podocyte injury of diabetic nephropathy.
2Valsartan inhibits activation of Notch apthway in renel tissues ofdiabetic mice. Valsartan also suppresses proteinuria and podocyte detachment,inhibits expression of apoptotic associated protein, decreases cell apoptosis,and inhibits accumulation of extracellular matrical components in glomeruli.These findings suggest that Valsartan provides a treatment for diabeticnephropathy via inhibiting Notch pathway.
3Podocytes transfected with specific shRNA vector and treated with GSIrespectively inhibits activationg of Notch pathway in high glucose-inducedpodocytes, inhibits expression of apoptotic associated protein, decreasespodocytes apoptosis. These findings suggest that activation of notch pathwaymediates podocyte injury in high glucose-induced podocytes.
已有研究表明，Notch通路决定胚胎发育、细胞增殖和分化，在胚胎后肾形成过程中对足细胞和肾小管的分化中起着重要的作用。近年来在肾脏疾病领域的研究证实，Notch信号通路与糖尿病肾病、局灶节段性肾小球硬化、肾病综合症等多种肾小球疾病有关，参与肾小球硬化的发生。在哺乳动物体内Notch信号通路有四种受体和五种配体，其中受体为Notch1-Notch4，配体分别为Jagged1、Jagged2、Delta样配体（Dll）1、Dll3和Dll4。配体与Notch受体的结合导致受体构象发生改变，随后在γ-分泌酶（γ-secretase）的介导下发生蛋白水解作用，释放出Notch胞内域（Notch intracellular domain, NICD），NICD转移至细胞核内，活化Hes1和Hey1等分化拮抗基因的转录，阻碍分化效应基因的表达，影响细胞的分化、增殖和凋亡。
既往研究认为，DN的主要病理特征是肾小球基底膜（glomerularbasement membrane，GBM）增厚、细胞外基质（extracellular matrix, ECM）堆积、肾小球硬化、肾小管萎缩及肾间质纤维化。近年研究发现，足细胞损伤在DN发生、发展过程中起重要作用。足细胞是终末分化的肾小球上皮细胞，它附着在GBM外侧，是肾小球滤过膜的重要组成部分。由于足细胞是高度分化细胞，增殖能力较差，当足细胞受到损伤时，Nephrin和Podocin等足细胞相关蛋白在足细胞裂孔膜（slit diaphragm，SD）丢失，使肾小球滤过电荷屏障减弱，促进蛋白尿的发生。足盂蛋白（podocalyxin，PCX）是足突顶膜区主要的带负电荷跨膜蛋白，当足细胞损伤后PCX表达减少，破坏了足突间的排斥作用使之发生粘附、融合，导致足细胞易于脱落。现已知主要的凋亡通路有Bcl-2、p53、NF-κB等，在DN发生时，足细胞内的这些凋亡通路可被激活，诱导足细胞凋亡。同时受损的足细胞对ECM生成的调节作用发生紊乱，转化生长因子β1（transforming growthfactor-β1, TGF-β1）等促进基质生成的因素增加，造成Ⅳ型胶原等基膜样基质增加，导致肾小球硬化，加速DN的进展。
许多学者通过不同方法证实了Notch信号通路在足细胞分化中的作用，Cheng等使用γ-分泌酶抑制剂（γ-secretase inhibitor，GSI）抑制小鼠后肾形成过程中Notch通路的活化，发现近端小管细胞和肾小球中足细胞的形成明显减少，而伴随非上皮细胞的增多。如果在足细胞发育过程中出现异常Notch通路的活化，可抑制足细胞的终末分化，引起小鼠肾小球硬化。我们通过收集临床糖尿病肾病标本、建立糖尿病小鼠模型及足细胞高糖（high glucose, HG）培养，检测Notch信号通路家族成员（Jagged1、Notch1、NICD1、Hes1及Hey1）的表达情况，并通过应用血管紧张素II1受体阻断剂（angiotensin II type1receptor antagonism, AT1Ra）、基因干扰技术及Notch信号通路抑制剂（GSI）抑制Notch通路，来观察是否Notch信号通路参与DN时的足细胞损伤，促进足细胞凋亡和细胞外基质沉积，为糖尿病肾病的治疗提供一条新的思路。
方法
1糖尿病肾病患者肾组织中Notch信号通路检测
收集2010年10月~2012年10月在河北医科大学第三医院经病史、临床检查及肾穿刺活检病理诊断为糖尿病肾病患者34例（diabeticnephropathy group），既往无其它肾脏病史，以10例肾肿瘤患者远离肿瘤的瘤旁组织做为对照组（control group）。留取血和尿标本检测血糖（Glu）、糖化血红蛋白（HbA1）、24小时尿蛋白定量（UPE）、肌酐（Scr）、估算肾小球滤过率（eGFR）；采用免疫组织化学检测Jagged1、Notch1、NICD1、Hes1及Hey1蛋白表达。
2Notch信号通路在糖尿病小鼠肾组织中的作用
雄性CD-1小鼠随机分为3组：对照组（control group）、糖尿病组（diabetic group）、糖尿病+缬沙坦组（diabetic+Valsartan group）。糖尿病模型小鼠腹腔单次注射链脲佐菌素（streptozotocin, STZ，150mg/kg），对照组只注射相同体积的枸橼酸盐缓冲液，72小时后测定血糖和尿糖，血糖值≥16.7mmol/L,尿糖值+++~++++者确定为糖尿病模型成功。糖尿病+缬沙坦组小鼠在糖尿病模型建立后，以缬沙坦（40mg/kg）灌胃，对照组和糖尿病组以等体积蒸馏水灌胃。分别于成模后1、2、4、8周每组取6只小鼠，收集血液及24小时尿液，用于生化指标检测，ELISA方法检测尿液PCX的含量；切取肾脏4%中性甲醛固定用于组织化学、TUNEL及免疫组织化学染色；取部分肾皮质组织置于4%戊二醛用于电镜观察；部分肾皮质组织用于提取蛋白及RNA，Western blot检测Jagged1、Notch1、NICD1、Hes1、Hey1、Bax、Bcl-2、cleaved Caspase-3、p-p53及p53蛋白表达，Real-time PCR检测Jagged1、Notch1、Hes1及Hey1mRNA表达；另取部分肾皮质组织于70%酒精固定，用于流式细胞术检测细胞凋亡率。
3高糖对小鼠足细胞Notch信号通路的作用
小鼠足细胞在5%CO2，33°C CO2培养箱中，以含有10%胎牛血清及γ-干扰素（10U/ml）的RPMI1640培养液培养细胞使其增殖，传代后更换为不含γ-干扰素的RPMI1640培养液，置入5%CO2，37°C CO2培养箱中培养10~14天，促进细胞分化成熟。将实验细胞分成7组：正常糖对照组（5.5mmol/L glucose, NG）、正常糖+高渗对照组（5.5mmol/Lglucose+24.5mmol/L mannitol, NM）、高糖组（30mmol/L glucose，HG）、高糖+阴性质粒组（30mmol/L glucose+sh-Scramble，HG+C）、高糖+sh-Jagged1质粒组（30mmol/L glucose+sh-Jagged1，HG+J1）、高糖+sh-Notch1质粒组（30mmol/L glucose+sh-Notch1，HG+N1）、高糖+GSI组（30mmol/L glucose+1μmol/L GSI，HG+GSI）。分别经细胞同步化、分组干预刺激0、12、24、48、72小时后收集细胞，免疫细胞化学检测Jagged1、Notch1、NICD1、Hes1和Hey1蛋白表达；Western blot检测Jagged1、Notch1、NICD1、Hes1、Hey1、Bax、Bcl-2、cleaved Caspase-3、p-p53及p53蛋白表达；Real-time PCR检测Jagged1、Notch1、Hes1及Hey1mRNA表达；TUNEL和AnnexinⅤ/PI染色检测足细胞凋亡率。
结果
1糖尿病肾病患者临床病理表现及Notch信号通路检测
①光镜下观察，对照组肾小球、肾小管及肾间质未见明显异常；糖尿病肾病组肾小球体积增大，基底膜弥漫或不规则增厚，细胞外基质增多，K-W结节形成，部分肾小管上皮细胞出现空泡变性，间质纤维化。②糖尿病肾病组空腹血糖、糖化血红蛋白、尿蛋白量、肌酐较对照组均明显增多，而肾小球滤过率糖尿病肾病组患者比对照组下降。③免疫组化显示Jagged1、Notch1、NICD1、Hes1及Hey1在肾小球及肾小管均有表达，糖尿病肾病组较对照组表达增多。
2糖尿病小鼠肾组织Notch信号通路的表达及缬沙坦对Notch信号通路和足细胞损伤的影响
①光镜及电镜下观察，糖尿病组小鼠轻微肾小球体积增大，系膜基质增多，基底膜不规则增厚，足细胞数量减少，足突融合，部分肾小管上皮细胞出现空泡变性。②与对照组相比，糖尿病组小鼠尿蛋白（Upro）从2周开始明显升高，随病程延长呈逐渐升高的趋势；糖尿病小鼠血糖（Glu）、尿素氮（BUN）、肌酐（Scr）比对照组小鼠增加，随病程延长呈逐渐升高的趋势；与对照组相比，糖尿病组小鼠尿蛋白和尿PCX含量明显升高，给予缬沙坦治疗后尿蛋白和PCX含量降低。③免疫组化结果显示Jagged1、Notch1、NICD1、Hes1和Hey1在对照组肾小球及肾小管有少量表达，在糖尿病组表达明显增加；Western blot检测显示，与对照组比较，糖尿病组小鼠肾组织中Jagged1、NICD1、Hes1及Hey1从1周时开始升高，表达最高点在4周，8周时略有下降；Notch1蛋白在糖尿病组比对照组表达增加，在糖尿病组各周次间表达无明显差异；糖尿病组Jagged1、Notch1、Hes1和Hey1mRNA水平与对照组相比表达增加，最高点在2周，随后下降。④Western blot和Real-time PCR显示，糖尿病组小鼠肾小球组织Jagged1、Notch1、NICD1、Hes1和Hey1蛋白及Jagged1、Notch1、Hes1和Hey1mRNA水平与对照组相比增加，给予缬沙坦治疗后降低。⑤TUNEL染色显示在糖尿病组小鼠肾组织中观察到凋亡细胞；Bax、p-p53及cleaved Caspase-3蛋白在糖尿病组小鼠肾小球组织中表达比对照组增加，给予缬沙坦治疗后降低；Bcl-2蛋白表达水平糖尿病组较对照组降低，给予缬沙坦后表达升高；p53蛋白在各组间表达无明显差异；流式细胞术显示糖尿病组小鼠肾小球内细胞凋亡增加，糖尿病+缬沙坦组降低，但仍高于对照组。
3足细胞体外高糖培养对Notch信号通路表达及足细胞损伤的影响
①免疫细胞化学显示，Jagged1、Notch1、NICD1、Hes1和Hey1在正常糖对照组足细胞中有少量表达，给予高糖刺激后表达量明显增加；Western blot检测显示，Jagged1和NICD1蛋白在高糖刺激足细胞12小时升高，48小时表达最高，72小时略有下降；Notch1蛋白在高糖刺激后其它时间点的表达量是高糖刺激0小时表达量的2倍，高糖刺激12~72小时足细胞中Notch1蛋白水平表达无明显差异；Hes1蛋白在高糖刺激24小时升高，48小时达到顶点，72小时有所降低；Hey1表达在高糖刺激12和24小时升高，然后随着刺激时间延长表达下降；高糖刺激呈时间依赖性引起Jagged1、Notch1和Hes1mRNA升高，最高峰在48小时；Hey1mRNA最高点在高糖刺激24小时，随后随着刺激时间延长而下降。②Western blot和Real-time PCR检测发现，高糖刺激引起Jagged1、Notch1、NICD1、Hes1、Hey1蛋白及Jagged1、Notch1、Hes1、Hey1mRNA表达增加，sh-Jagged1和sh-Notch1转染足细胞后表达下降，GSI可抑制足细胞NICD1、Hes1、Hey1蛋白及Hes1、Hey1mRNA在高糖刺激后的过表达现象，但Jagged1、Notch1蛋白及mRNA在高糖+GSI组和高糖组之间无明显差异。③Western blot检测发现高糖呈时间依赖性刺激Bax蛋白表达，12小时开始增加，72小时达到最高点，而Bcl-2蛋白在高糖刺激后表达逐渐下降，最低点在72小时；cleaved Caspase-3在高糖刺激12小时开始增加，24小时为最高点，之后逐渐减少；p-p53蛋白在高糖刺激下表达逐渐增加，最高点在72小时；p53蛋白在高糖刺激各时间点之间表达无明显差异。④足细胞高糖培养并应用GSI或sh-Jagged1、sh-Notch1质粒转染后发现Bax、p-p53及cleaved Caspase-3蛋白较高糖组表达下降，但较正常糖对照组表达仍高，而Bcl-2蛋白较高糖组表达增强，但较正常糖对照组表达仍低，p53蛋白在各组间表达无明显差异。⑤TUNEL荧光染色及AnnexinⅤ/PI染色显示高糖组较正常糖对照组细胞凋亡率升高，转染组及高糖+GSI组细胞凋亡率比高糖组降低。
结论
1在糖尿病肾病患者肾组织、糖尿病小鼠肾组织、高糖培养足细胞中发现Notch信号通路成员有过表达现象，提示Notch通路在糖尿病肾病足细胞中激活并可能参与足细胞损伤过程。
2缬沙坦抑制糖尿病小鼠肾组织中Notch信号通路的活化，同时抑制蛋白尿和足细胞脱落，抑制凋亡相关蛋白表达，减少细胞凋亡，抑制肾小球内细胞外基质沉积。提示缬沙坦可通过抑制Notch信号通路来产生对糖尿病肾病的治疗效果。
3在高糖培养的足细胞中，应用Notch信号通路干扰质粒及抑制剂抑制Notch信号通路的活化后，可抑制凋亡相关蛋白表达，减少足细胞凋亡。提示Notch信号通路参与高糖诱导的足细胞损伤。
Objectives: Diabetic nephropathy (DN) is one of the most commoncomplications of diabetes and has become the most frequent cause ofend-stage renal disease. The pathogenesis of diabetic nephropathy is verycomplicated, which original factors encompass glucose and lipid metabolicdisorder, abnormal hemodynamics, oxidative stress, more cytokines. Manykinds of signal pathways pay an important role in DN. Recent research foundthat Notch pathway had a close relation with DN.
The family of Notch pathway play important roles in embryonicdevelopment, cellular proliferation and differentiation. Detailed analyses ofthe expression pattern of Notch and related genes in differentiation ofproximal tubule and podocyte during nephrogenesis have been performed.Recent studies in renal disease found that Notch pathway was relevant withglomerular disease, including DN, focal segmental glomerulosclerosis andhydropigenous nephritis, to participate in glomerular sclerosis. In mammalthere are four receptors, Notch1-Notch4, and five ligands, Jagged1, Jagged2,Delta-like (Dll)1, Dll3, and Dll4. The binding of ligand and Notch receptorinduces a conformational change in the Notch receptor. This allows theγ-secretase–mediated protease to release the Notch intracellular domain(NICD). NICD travels into the nucleus where it activates the transcription ofdownstream genes such as Hes1and Hey1genes and affects cellulardifferentiation, proliferation and apoptosis.
The major characteristics of DN includes glomerular basement membrane(GBM) thickening, accumulation of extracellular matrix (ECM), glomerularsclerosis, tubular atrophy and interstitial fibrosis. It is believed thatpodocytes injury can lead to the development of DN. Since podocytes formeda critical part of the glomerular filtration barrier are terminally differentiated cells, which are unable to divide. Loss of slit diaphragm-associated proteinssuch as nephrin and podocin that weaken the electrostatic barrier ofglomerular slit diaphragm (SD) and result in proteinuria. Podocalyxin (PCX),as a protein rich in negative charge, is expressed in an epiphragm of footprocess. Down-regulation of podocalyxin expression in podocytes injurydestroys the rejection of foot process and results in podocytes detachmentfrom the glomerular basement membrane. In the development of DN,podocytes apoptosis is induced via activation of apoptotic pathways, such asBcl-2, p53, NF-κB pathway and so on. Impaired podocytes also have theimbalance between ECM synthesis and degradation, increase transforminggrowth factor-β1(TGF-β1) level to generate ground substance, lead toincreasing expression of Type Ⅳ collagen, cause glomerulosclerosis inprogression of DN.
Detailed analyses of the expression pattern of Notch and related genesduring nephrogenesis have been performed. Cheng et al used γ-secretaseinhibitor (GSI) to depress the activation of notch pathway in mousemetanephros, then found fewer renal epithelial structures, low quantity ofproximal tubule and glomerular podocyte, accompanied by an increase inintervening nonepithelial cells. Ectopic activation of Notch pathway indeveloping podocytes on the other hand caused glomerulosclerosis indeveloping murine kidneys and opposed terminal differentiation of podocytes.In the present study, we investigated the expression of Notch pathway(Jagged1, Notch1, NICD1, Hes1, Hey1) on renel tissues from patients withdiabetic nephropathy, diabetic mice and podocytes treated with high glucose(HG). Furthermore, we inhibited Notch pathway by angiotensin II type1receptor antagonism (AT1Ra), chemical inhibitor or specific short hairpinRNA vector to investigate whether Notch pathway participated in podocytesinjury and induced cell apoptosis and accumulation of extracellular matricalcomponents. These provide a new treatment target for diabetic nephropathy.
Methods
1Determination of Notch pathway in renel tissues from patients with diabetic nephropathy
Thirty four patients diagnosed as diabetic nephropathy by renal biopsyand clinical data from October2010to October2012at the third Hospital ofHebei Medical University were included in this study. Other nephropathy wasexcluded. The renal tissues (n=10) obtained from distant portions of kidneyssurgically excised because of the presence of a localized neoplasm were usedas control. Blood and urinary samples was collected to detect Glu, HbA1, UPE,Scr and eGFR. Protein expression of Jagged1, Notch1, NICD1, Hes1andHey1was assessed by immunhistochemical staining.
2Role of Notch pathway in diabetic mice
Male CD-1mice were randomly divided into three groups: control group,diabetic group and diabetic+Valsartan group. The mice of diabetic modelreceived a single intraperitoneal injection of streptozotocin (STZ) at a dose of150mg/kg body weight. The mice of control group only received an injectionof the same volume of sodium citrate. The model of diabetes was consideredto be successful when the blood glucose was≥16.7mmol/L and the glucose inurine was+++~++++after72hours of the injection. After the diabetic modelwas affirmed to be successful, the mice of diabetic+Valsartan group wereadministered daily with Valsartan (40mg/kg) by gavage. The mice of controlgroup and diabetic group were only administered daily with the same volumeof diatilled water by gavage. Six mice from every group were respectivelysacrificed at weeks1,2,4and8after STZ injection. Blood and24h urinesamples were collected for biochemical indicator and enzyme linkedimmunosorbent assay (ELISA). Partial renal tissures were fixed in4%neutralformalin for histochemical, TUNEL and immunohistochemical staining.Partial renal cortices were fixed in4%glutaraldehyde for electron microscopicobservation. Protein and RNA were extracted from partial renal cortices forWestern blot and Real-time PCR. The expression of Jagged1, Notch1, NICD1,Hes1, Hey1, Bax, Bcl-2, cleaved Caspase-3, p-p53and p53protein wasrespectively evaluated by Western blot. The mRNA levels of Jagged1, Notch1,Hes1and Hey1were evaluated by Real-time PCR. Partial renal cortices were fixed in70%alcohol for flow cytometry.
3Role of Notch pathway in mouse podocytes induced by high glucose
To induce proliferation, mouse podocytes were cultured at33°C in ahumidified atmosphere of5%CO2in RPMI1640containing10%fetal bovineserum and10U/ml-IFN under growth permissive conditions, and then cellswere cultured at37°C in RPMI1640without-IFN under growth restrictiveconditions for10~14days to induce quiescence and the differentiatedphenotype. Podocytes were randomly divided into seven groups: normalglucose group (5.5mmol/L glucose, NG), normal glucose+mannitol group(5.5mmol/L glucose+24.5mmol/L mannitol, NM), high glucose group (30mmol/L glucose, HG), high glucose+negative control vector (30mmol/Lglucose+sh-Scramble, HG+C), high glucose+sh-Jagged1vector (30mmol/Lglucose+sh-Jagged1, HG+J1), high glucose+sh-Notch1vector (30mmol/Lglucose+sh-Notch1, HG+N1) and high glucose+GSI (30mmol/L glucose+1μmol/L GSI, HG+GSI). The seven groups were cultured for0,12,24,48and72hours respectively, and then podocytes were harvested. The proteinexpression of Jagged1, Notch1, NICD1, Hes1and Hey1was detected byimmunocytochemistry. The expression of Jagged1, Notch1, NICD1, Hes1,Hey1, Bax, Bcl-2, cleaved Caspase-3, p-p53and p53protein was respectivelyevaluated by Western blot. The mRNA levels of Jagged1, Notch1, Hes1andHey1were evaluated by Real-time PCR. The TGF-β1, Type Ⅳ collagen andLaminin proteins were examined by ELISA. Podocytes apoptosis was detectedby TUNEL and AnnexinⅤ/PI staining.
Results
1pathological manifestation and Notch pathway expression of patientswith diabetic nephropathy
①There were no abnormal changes in glomerulus, renal tubule andinterstitium of control group by light microscopy. Pathological changesincluding glomerular enlargement, increase of glomerular basementmembrane in thickeness, increase of ECM, the presence ofKimmelstiel–Wilson lesions, focal tubular epithelial vacuolar degeneration as well as interstitial fibrosis were observed in the patients of diabeticnephropathy.②Compared with control group, the levels of Glu, HbA1, UPEand Scr in diabetic nephropathy group were increased significantly. The eGFRlevel of diabetic nephropathy group was decreased than control group.③Immunohistochemical staining displayed that Jagged1, Notch1, NICD1, Hes1and Hey1weakly expressed in renal glomerular and tubular epithelium incontrol group, whereas remarkably increased in diabetic nephropathy group.
2The expression of Notch pathway in renal tissues of diabetic mice andthe effect of Valsartan on Notch pathway and podocytes injury
①Diabetic mice showed slightly glomeruli hypertrophy, increasingmesangium matrix, thickened glomerular basement membrane, reduction inthe number of podocytes, fusion of foot process, partial tubular epithalialvacuolar degeneration by light and electron microscope.②Compared withcontrol group, the Glu, BUN and Scr levels upregulated in a time-dependentmanner in diabetic group.24h urine protein was significantly increased fromweek2to week8in diabetic group than control group.24h urine protein andthe concentration of podocalyxin in urine of diabetic mice were higher thanthat in control group. Compared with diabetic group,24h urine protein andthe concentration of podocalyxin in urine of diabetic+Valsartan group weresignificantly decreased.③By immunohistochemical staining, the proteinexpression of Jagged1, Notch1, NICD1, Hes1and Hey1was increased inglomeruli and tubule cells of diabetic group than that in control group.Compared with control group, Western blot indicated that the proteinexpression of Jagged1, NICD1, Hes1and Hey1began to increase at week1,reached the peak at week4and slightly decreased at week8in diabetic group.Notch1protein expression of diabetic mice increased than control mice. Nodifferences of Notch1was found in diabetic group among different time points.The mRNA levels of Jagged1, Notch1, Hes1and Hey1began to increase atweek1, reached the peak at week2and slightly decreased in diabetic group.④By Western blot analysis, glomerular tissues of diabetic mice showedincreased expression of Jagged1, Notch1, NICD1, Hes1and Hey1, but Valsartan decreased their protein expression in diabetic mice. The expressionof Jagged1, Notch1, Hes1and Hey1mRNA was the same with the expressionof protein.⑤Apoptotic cells by TUNEL were observed in renal tissues ofthe diabetic kidney. Glomerular tissues of dabetic mice had a significantup-regulation in Bax, p-p53and cleaved Caspase-3expression anddown-regulation in Bcl-2expression compared with control group. However,the alternations of Bax, p-p53, cleaved Caspase-3and Bcl-2protein levels indiabetic group were reversed by addition of Valsartan. No change of p53protein expression was found in different groups. Apoptotic rate of glomerulartissues in diabetic group increased compared to control group by flowcytometry. Apoptotic rate was significantly lower in the diabetic+Valsartangroup than that in diabetic group.
3The effects of high glucose in podocytes on the expression of Notchpathway and podocytes injury
①Immunocytochemical staining showed that the expression of Jagged1,Notch1, NICD1, Hes1and Hey1increased in podocytes induced by highglucose. By Western blot, the protein expression of Jagged1and NICD1beganto increase at12h after the stimulation of HG, reached the peak at48h andslightly decreased at72h. Notch1protein expression at12-72h within HGstimulation was2-fold greater than that of the0h of HG stimulation. Nochange of Notch1protein expression was found in HG-induced podocytes at12~72h. The Hes1protein significantly increased in podocytes stimulated byHG for24h, continuously increased up to48h and slightly decreased at72h.The increased Hey1protein expression was confirmed after12and24h ofstimulation with HG separately, and gradually decreased with prolongedstimulation. The effects of HG on mRNA levels of Jagged1, Notch1and Hes1were revealed at12h and peaked at48h. HG also induced Hey1mRNAexpression and peaked at24h.②Compared with the cells of the normalglucose group, the protein levels of Jagged1, Notch1, NICD1, Hes1and Hey1significantly increased in high glucose group by Western blot. Thetransfection with sh-Jagged1or sh-Notch1vector respectively decreased high glucose-induced the protein overexpression of Jagged1, Notch1, NICD1, Hes1and Hey1in podocytes. Real-time PCR showed the similar changes ofJagged1, Notch1, Hes1and Hey1mRNA after transfection. The protein levelsof NICD1, Hes1and Hey1were markedly higher in podocytes stimulated withhigh glucose than the cells treated with normal glucose and were dramaticallyreduced in response to GSI. Real-time PCR showed the similar changes ofHes1and Hey1mRNA after treatment with GSI. Western blot and Real-timePCR analysis revealed that GSI did not inhibite Jagged1and Notch1overexpression induced by high glucose.③HG notably increased proteinlevel of Bax in time-dependent manner and peaked at72h. HG stimulationdecreased Bcl-2protein level in time-dependent manner and the cellsstimulated by HG for72h showed the minimum expression. The expressionof the HG-induced cleaved Caspase-3significantly increased at12h, peakedat24h, and gradually decreased with prolonged HG stimulation. HGstimulation increased p-p53protein level in time-dependent manner and themaximum expressions were at72h after stimulation of HG. However, therewas no difference of p53expression among all time spots in HG-inducedpodocytes.④High glucose stimulation in podocytes decreased Bcl-2proteinlevel than in normal glucose medium. The protein expression of the highglucose-induced Bax, p-p53and cleaved Caspase-3was significantlyincreased than normal glucose stimulation in podocytes. Compared with thecells treated with high glucose, Bax, p-p53and cleaved Caspase-3proteinlevels significantly decreased in cells transfected with sh-Jagged1orsh-Notch1vector and pretreated with GSI, while Bcl-2level increased. Nochange of p53protein expression was found in the cultured podocytes ofdifferent groups.⑤The results of TUNEL and AnnexinⅤ/PIstainingshowed that the apoptotic podocytes in high glucose stimulation weremarkedly higher than that in normal group, which were inhibited bysh-Jagged1or sh-Notch1vector and GSI.
Conclusions
1The overexpression of Notch pathway member is found in renel tissues from patients with diabetic nephropathy, diabetic mice and mouse podocytesinduced by high glucose, which suggests that activation of notch pathway maymediate podocyte injury of diabetic nephropathy.
2Valsartan inhibits activation of Notch apthway in renel tissues ofdiabetic mice. Valsartan also suppresses proteinuria and podocyte detachment,inhibits expression of apoptotic associated protein, decreases cell apoptosis,and inhibits accumulation of extracellular matrical components in glomeruli.These findings suggest that Valsartan provides a treatment for diabeticnephropathy via inhibiting Notch pathway.
3Podocytes transfected with specific shRNA vector and treated with GSIrespectively inhibits activationg of Notch pathway in high glucose-inducedpodocytes, inhibits expression of apoptotic associated protein, decreasespodocytes apoptosis. These findings suggest that activation of notch pathwaymediates podocyte injury in high glucose-induced podocytes.
引文
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11Guarani V, Deflorian G, Franco CA, et al. Acetylation-dependentregulation of endothelial Notch signalling by the SIRT1deacetylase.Nature,2011,473(7346):234~238
12Wong NK, Fuller M, Sung S, et al. Heterogeneity of breast cancer stemcells as evidenced with Notch-dependent and Notch-independentpopulations. Cancer Med,2012,1(2):105~113
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19Cheng HT, Kopan R. The role of Notch signaling in specification ofpodocyte and proximal tubules within the developing mouse kidney.Kidney Int,2005,68(5):1951~1952
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22Bigas A, Robert-Moreno A, Espinosa L. The Notch pathway in thedeveloping hematopoietic system. Int J Dev Biol,2010,54(6-7):1175~1188
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25Carbery ID, Ji D, Harrington A, et al. Targeted genome modification inmice using zinc-finger nucleases. Genetics,2010,186(2):451~459
26Qu X, Afelik S, Jensen JN, et al. Notch-mediated post-translational controlof Ngn3protein stability regulates pancreatic patterning and cell fatecommitment. Dev Biol,2013,376(1):1~12
27Shih HP, Kopp JL, Sandhu M, et al. A Notch-dependent molecularcircuitry initiates pancreatic endocrine and ductal cell differentiation.Development,2012,139(14):2488~2499
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30Kolev V, Mandinova A, Guinea-Viniegra J, et al. EGFR signalling as anegative regulator of Notch1gene transcription and function inproliferating keratinocytes and cancer. Nat Cell Biol,2008,10(8):902~911
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