| BackgroundDiabetes mellitus is a metabolic disease characterized by high blood sugar,which is caused by the deficiency of insulin secretion or insulin action.Continuous high blood sugar and long-term metabolic disorder can lead to a variety of complications,especially in eyes,kidneys,cardiovascular and nervous system damage,end to dysfunction and failure.Diabetic nephropathy(DN)is one of the most common microvascular complications of diabetes,and its clinical manifestations includes hypertension,edema,severe proteinuria,low serum albumin and progressive renal dysfunction.Renal pathology showes nodular and/or diffuse mesangial cell proliferation,basement membrane(GBM)thicken,mesangial extracellular matrix accumulation,podocytes fusion,glomerular sclerosis and renal tubular interstitial fibrosis.Diabetic nephropathy is first reason leading to end-stage renal disease(ESRD)in worldwide,and increasing of national health care expenditures.The incidence rate in China and in chronic dialysis nephropathy in proportion is also increasing,and the chronic dialysis nephropathyseriously affects the quality of life of patients.Therefore,it is of great significance to explore the pathogenesis of diabetic nephropathy and to seek more effective prevention and treatment measures for the majority of patients with diabetic nephropathy.The pathogenesis of DN is very complex,it involves the role of glucose,lipid metabolism disorders,inflammatory mediators release,hemodynamic abnormalities,cytokines,oxidative stress and apoptosis.Recent studies indicated that structure and function of glomerular filtration barrier changes,especially podocyte injury,played a pivotal role in progression of diabetic nephropathy.The podocyte injury was considered to be one key factor to cause DN proteinuria and glomerular sclerosis.Therefore,study of injury of the podocytes brought many new opportunities for prevention and treatment of DN.The podocytes,also known as the visceral glomerular epithelial cells,attached to outside of the glomerular basement membrane.Podocytes,glomerular basement membrane and endothelial cells constitute glomerular filtration barrier together.The main function of podocytes is to play an important role in the filtration of protein and the renewal of GBM components.Protein leakage increase,thereby increase the incidence of renal failure.Proteinuria and podocytes injury aggravate when the podocyte damage because the podocytes are highly differentiated cells and the proliferation ability of podocytes is poor.In the development of DN,podocyte changes include podocyte number decreasing,apoptosis increasing and enhancing mobility.At last the podocyte detachment is excreted in the urine from GBM,resulting in GBM exposed,formation of focal adhesion and glomerular sclerosis.The mechanisms of Podocyte damage in DN is complex,which is not induced by single factor,but the joint action of multiple factors.The mechanisms mainly include the nephrin,hyperglycemia integrin,mechanical stress,inflammatory reaction,the activation of renin angiotensin aldosterone system,the accumulation of AGEs,insulin like growth factor GH-(insulin-like growth factor,IGF)-1 axis,vascular endothelial growth factor,heparan sulfate proteoglycan,wT1,podocalyxin,perox-isome proliferatoractivated receptor,adiponectin,miRNA,TGF-β1,oxidative stress and cell apoptosis,etc.Studies showed that the IQ domain of GTP protein 1 was a protein binding domain containing a protein binding domain.Recent studies have found that IQGAP1 plays an important role in cell adhesion,migration,cell morphology,and regulation of cell apoptosis.Whether IQGAP1 participates in apoptosis of podocytes or not by high glucose has not been reported.Based on the above theoretical basis,we designed and carried out this the present study.This study was divided into two parts.Part 1,in vitro,human podocytes cells(HPC)were cultured,and we observed the expression and distribution of IQGAP1 and the apoptosis of the HPC by high glucose stimulation.Part 2,further study was to observed the effect of IQGAP1 and mitogen activated protein kinase(MAPK)signaling pathway and renin angiotensin aldosterone system(RAAS)with a total in podocyte apoptosis,then to investigate the molecular mechanism of high glucose induced podocyte apoptosis,so as to provide theoretical support for better treatment of diabetic nephropathy.Part One:Effects of high glucose stimulation on the expression and distribution of IQGAP1 and apoptosis in HPC.Objectives:1.To observe the expression of IQGAP1 in HPC induced by high glucose stimulation in vitro.2.To observe the distribution of IQGAP1 in HPC induced by high glucose stimulation in vitro.3.To observe the apoptosis of HPC induced by high glucose stimulation in vitro.Methods:HPC were incubated in RPMI 1640 medium containing 10%Fetal Bovine Serum(FBS)and y-interferon(10 U/ml)and then cultured in incubators under 33℃ and with a content of 5%CO 2 for proliferation.Then cells passage culture was continued with RPMI 1640 medium only containing 10%FBS under the conditions of 37℃ and 5%CO 2 containing.After incubated for 14 days,the differentiated HPC was collected for subsequent experiments.HPC was randomly divided into 3 groups:group A:normal control group(Control NC),group B:mannitol control group(Manitol + Control Glucose,MG)and group C:high glucose group(Glucose High,HG).Group A were given D-glucose5.5mmol/L.In order to control the cell experiment results by high osmotic pressure,group B were given D-glucose5.5mmol/L +24.5mol/L and mannitol as control group.Group C were given D-glucose 30mmol/L HG.The cells were cultured for 0,12,24,48 and 72 hours respectively,then were collected.The expression and distribution of IQGAP1 were observed in different environment in podocytes by immunofluorescence.The IQGAP1mRNA was detected by real-time PCR in different groups.Apoptosis of each group was detected by flow cytometry.Results:1.From Fig.1-1 IQGAP1 expressed in the cytoplasm and the nucleus in podocytes,but mainly in the nucleus,2.Fig.1-2,Fig.1-3A,3B,3C display,in 0 hours,There was no significant difference between normal control group,mannitol control group and high glucose group.In 48 hours,the immunofluorescence showed:compared with normal glucose groupthe,the expression of IQGAPI did not vary in mannitol group and the difference was not statistically significant.But the expression level of IQGAPI was significantly reduced by high glucose.Compared with the normal control group,the expression level of IQGAPI was significantly decreased and the difference was statistically significant.3.Fig.1-4A、B、C、D showed the apoptosis of the HPC.The apoptosis was detected by flow cytometry.The early apoptotic cells in the lower right quadrant and the late apoptotic cells in the upper right quadrant.The two aspets cells were considered total apoptotic cells.In 0 hours,the apoptosistic rate of normal sugar group was 1.73%+ 0.13%,the apoptostic rate of mannitol group was 2.1%+0.15%,and the rate of high glucose group was 2.1%+ 0.12%.There was no significant difference in apoptosis rate between the groups.In 24 hours,the apoptosis rate of normal glucose group was 26.39%+ 4.57%,the apoptosis rate of mannitol group was 30.44%+ 5.15%,the apoptosisticrate of high glucose group was 56.15%+ 6%.In 48 hours,podocyte apoptosistic rate of normal glucose group was 37.76%+ 5.6%,the apoptosisticrate of mannitol group was 36.77%+ 4.27%,the apoptosistic rate of high glucose group was 68.9%+ 6.64%.There was no significant change in theapoptosistic rate of mannitol group compared with normal glucose group,the apoptosistic rate was significantly increased in the high glucose group compared with normal glucose group,There is significant statistically difference(P<0.05).Conclusions:1.IQGAP1 expressed in the cytoplasm and nucleus of podocytes,maily in nucleus by normal glucose group,mannitol group and high glucose group.2、The expression of IQGAP1 was down regulated significantly by High glucose and regulation decreased with time.3.High glucose could significantly increase the apoptosis of the HPC,and the apoptosis rate increased with time.Part two:The role of IQGAP1 in high glucose-induced apoptosis of podocytes and its possible mechanismsObjectives:1.By flow cytometry,the apoptosis of HPC and the relationship between IQGAP1 and apoptosis were observed by high glucose stimulation in vitro.2.To observe the expression of IQGAP1,p-ERK 1/2 and ERK1/2 by RT-PCR、Western blotting.Further study of between IQGAP1 and p-ERK1/2 and ERK1/2 in mitogen activated protein kinase(MAPK)signaling pathway.3.To observe the expression of IQGAP1,p-ERK 1/2,ERK 1/2 protein in high glucose group culture podocytes adds benazepril(10 mol/L),and to explore the relationship between IQGAP1 and renin angiotensin aldosterone system as well as the role of cell apoptosis in the HPC.4.To observe the expression of IQGAP1,p-ERK1/2,ERK1/2 protein and apoptosis of HPC when added the specific inhibitor of U0126(10 mol/L)joined ERK 1/2 in high glucose group in podocyte cultured.Methods:The HPC were randomly divided into 5 groups:group A:Normal sugar control group(5.5 glucose mmol/L,NC),group B:mannitol control group(24.5 mmol/L)Mannitol mmol/L glucose +5.5,MG),group C:high glucose group(30 glucose mmol/L,HG),group D:Benner Pury group(30 glucose +10 mol/L benazepril mmol/L),group E:U0126 Inhibitor group(30 glucose +10 mol/L U0126 mmol/L).Cells in each group were collected after 24 hours and 48 hours.The expression and distribution of IQGAP1 in HPC were observed by immunofluorescence assay.The expression of IQGAP1mRNA in HPC was observed by real-time PCR.The expression of IQGAP1,ERK1/2 and P-ERK1/2 were observed by Western blotting.Apoptosis of each group were detected by flow cytometry.Results:1.HG inhibited the expression of ERK1,increased the expression of IQGAP1/2,and promoted the apoptosis of HPC.2.Benazepril could reduce the inhibition of IQGAP1 by HG,raise the expression of IQGAP1 and reduce the expression of P-ERK1/2.Benazepril could relieve the apoptosis of HPC induced by high glucose.3.U0126 had no effect on the expression of IQGAP1.The apoptosis rate of E group was 3.19%+ 1.68%,and the rate of apoptosis was significantly decreased than that in group C(68.9%±6.64%).This phenomenon suggested that U0126 could significantly decrease the apoptosis rate of HPC by HG.Conclusions:1、High glucose promoted the apoptosis of HPC by down-regulating the expression of IQGAP1 and up-regulatingthe expression of p-ERK protein.2、Benazepril could increase the expression of IQGAP1 and decrease the apoptosis of HPC induced by high glucose.3、U0126 inhibitors could reduce the apoptosis induced by high glucose,but there was no effect in the expression of IQGAP1.4、The IQGAP1 played an important role on podocyte apoptosis interacting with ERK1/2 MAPK signaling pathway and the renin angiotensin aldosterone system(RAAS).It developed an important role in the progression of diabetic nephropathy. |
