| 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.Methods1Determination of Notch pathway in renel tissues from patients with diabetic nephropathyThirty 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 miceMale 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 glucoseTo 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.Results1pathological manifestation and Notch pathway expression of patientswith diabetic nephropathy①There 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 injury①Diabetic 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 injury①Immunocytochemical 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.Conclusions1The 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. |
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