The Role And Regulatory Mechanism Of GLUT1 Expression In Mesangial Cells Of Diabetic Kidney Disease

Background and AimsDiabetic kidney disease（DKD）is a serious microvascular complication of diabetes mellitus（DM）as well as the most common cause of chronic kidney disease and end-stage kidney disease,but its exact pathogenesis remains unclear.Mesangial cells are the main cellular components of the glomerulus,and their lesions play an important role in the progression of DM glomerulosclerosis.Dysfunction of glucose uptake by cells is an important pathophysiological mechanism of DKD renal injury.Glucose cannot directly pass through the bilayer cell membrane and needs to be transported by the glucose transporter（GLUT）on the cell membrane.GLUT1 is the main glucose transport channel in mesangial cells,and the change of its expression level is the main limiting factor of glucose metabolism in mesangial cells.However,the change of GLUT1 expression in DKD mesangial cells and its role in the occurrence and development of DKD have not been completely clarified.The increased expression of 12-lipoxygenase（12-LO）is closely related to the progression of DKD.12-LO is expressed highly in DKD glomeruli or mesangial cells stimulated by high glucose（HG）.12-LO and its metabolite,12（S）-hydroxyeicosatetraenoic acid[12（S）-HETE],regulate the expression of several key proteins closely related to the development of DKD through a series of complex mechanisms.It was found that the expression of 12-LO in vascular endothelial cells and vascular smooth muscle cells was gradually increased with the HG stimulation time,while the expression of GLUT1 was gradually decreased.However,the decreased expression of GLUT1 was reversed after treatment with 12-LO inhibitor,suggesting that 12-LO may have a regulatory effect on GLUT1 in DM micro-environment.This study we evaluated GLUT1 expression in mesangial cells in different types of DKD and during different stages of DKD by using renal biopsy tissue samples from DKD patients,rat models at early and late stages of type 1 DKD（T1DKD）and type 1 DKD（T2DKD）,and rat mesangial cells stimulated by HG at different times,to confirm the regulatory effect of12-LO on GLUT1 and clarify the mechanism of 12-LO regulation of GLUT1 expression in DKD mesangial cells,which provides important theoretical basis for development of GLUT1 as a therapeutic target for delayed DKD.Methods1.To evaluate the change of GLUT1 expression in DKD glomeruli:（1）Renal biopsy wax samples from 40 patients were selected,including normal controls（NC）group,DKD glomerular pathology Grade II group and DKD glomerular pathology grade III group.GLUT1 expression in renal tissues was detected by immunohistochemistry（IHC）staining and double immunofluorescence staining.（2）A T1DKD rat model was established by high-dose streptozotocin（STZ）.The total model time was 12 weeks.It was defined as the early model of T1DKD when the rats showed kidney damage at 4 weeks after model establishment,and the late model of T1DKD when the rats showed severe kidney damage at12 weeks after model establishment.Rats at different periods were divided into control group（CON group）and DKD group.Samples of rats at the early and late stages of DKD were respectively collected at 4 weeks and 12 weeks after model was successfully established,and glomeruli were extracted by serial screening method.GLUT1 expression was evaluated by RT-q PCR,western blot,IHC staining and double immunofluorescence staining.（3）The T2DKD rat model was established by high-fat diet combined with low-dose STZ.The total model time was 8 weeks.It was defined as the early model of T1DKD when the rats showed kidney damage at 4 weeks after model establishment,and the late model of T1DKD when the rats showed severe kidney damage a at 8 weeks.Rats were divided into CON group and DKD group at different periods.The glomeruli of rats in the early and late stages of DKD were respectively collected at 4 weeks and 8 weeks after model was successfully established,and the GLUT1 expression was evaluated by RT-q PCR,western blot,IHC staining and double immunofluorescence staining.（4）Mesangial cells were cultured in vitro,stimulated with HG of 30 mmol/L concentration,and divided into CON group and HG group.GLUT1 expression was detected by RT-q PCR,western blot,and immunofluorescence staining after HG stimulation for 12 h and 48 h.2.Elucidation of the involvement of 12-LO in regulating GLUT1 expression in DKD mesangial cells.（1）Early-stage and late-stage T1DKD rat model were established and divided into CON group,DKD group and CDC（cinnamyl-3,4-dihydroxy-α-cynanocinnamate,12-LO inhibitor）treatment group（DKD+CDC group）.DKD+CDC group was given subcutaneous injection of CDC 8 mg/kg 3 times a week,administered to rats in the early stage of DKD for 4 weeks and rats in the late stage of DKD for 12 weeks respectively,and the glomeruli were extracted by serial screening method.RT-q PCR,western blot,IHC staining and double immunofluorescence staining were used to evaluate the GLUT1 expression in the glomeruli of the three groups.（2）After the establishment of early-stage and late-stage T2DKD model,the rats were divided into CON group,DKD group and DKD+CDC group.The DKD+CDC group was given subcutaneous injection of CDC 8mg/kg 3 times a week,administered to rats in the early stage of DKD for 4 weeks,and rats in the late stage of DKD for 8 weeks,respectively and the glomeruli were extracted by serial screening method.RT-q PCR,western blot,IHC staining and double immunofluorescence staining were used to evaluate the GLUT1 expression in the three groups of rats.（3）Cultured mesangial cells were divided into three groups:CON group,HG group,HG+CDC group.GLUT1 expression was detected by RT-q PCR,western blot and immunofluorescence staining after HG stimulation for 12 h and 48 h.The intracellular 12（S）-HETE concentration was detected by ELISA.（4）Cultured mesangial cells were directly stimulated with12（S）-HETE,a metabolite of 12-LO,and divided into CON group and 12（S）-HETE group(10^-7mol/L).GLUT1 expression was detected by RT-q PCR and western blot after 12 h and48 h stimulation.3.Exploring the mechanism of 12-LO-12（S）-HETE regulation on GLUT1 expression in DKD mesangial cells:（1）Western blot and IHC staining were used to evaluate the differences in MAPK pathway protein phosphorylation levels in the CON,DKD and DKD+CDC groups at 4 and 12 weeks of model establishment for type 1 DM,at 4 and 8weeks for type 2 DM.（2）Western blot was used to detect the differences in MAPK pathway protein phosphorylation levels in CON group,HG group and HG+CDC group when HG stimulated glomerular mesangial cells for 12 h and 48 h.Mesangial cells were cultured in vitro were given MAPK inhibitor along with HG stimulation and divided into three groups:CON group,HG group and HG+p38MAPK inhibitor group.GLUT1 expression was determined by western blot after HG stimulation for 48 h.4.Evaluating the effect of decreased GLUT1 expression in mesangial cells on renal injury in the late stage of DKD:（1）The membrane GLUT1 level of glomeruli in CON group,DKD group and DKD+CDC group was detected by western blot at 12 weeks of model establishment for type 1 DM.Observe the differences of glomerular hypertrophy,renal interstitial fibrosis,and renal injury indexes of proteinuria between CON group and DKD group in the late stage of T1DKD,and the degree of renal injury remission after application of CDC.（2）The membrane GLUT1 level of glomeruli in CON group,DKD group and DKD+CDC group was detected by western blot at 8 weeks of model establishment for type2 DM.Observe the differences of glomerular hypertrophy,renal interstitial fibrosis,and renal injury indexes of proteinuria between CON group and DKD group in the late stage of T2DKD,and the degree of renal injury remission after application of CDC..Results1.The results of IHC staining and double immunofluorescence showed that compared with the NC group,the level of GLUT1 protein in the glomeruli of DKD patients with grade II glomerular pathology was higher,and the level of GLUT1 protein in the glomeruli of DKD patients with grade III glomerulus pathology was lower.At 4 weeks of model establishment for type 1 DM and type 2 DM,GLUT1 expression in mesangial cells in DKD group was higher than that in CON group;at 12 weeks of model establishment for type 1DM and 8 weeks for type 2 DM,GLUT1 expression in mesangial cells of rats in DKD group was lower than that in CON group;12-LO expression consistently increased at 4 and 12weeks of model establishment for type 1 DM,at 4 and 8 weeks for type 2 DM.After HG stimulation,GLUT1 expression in mesangial cells was increased at 12 h,decreased at 48 h,and 12-LO expression was consistently increased.2.At 4 weeks of model establishment for both type 1 DM and type 2 DM.,the expression level of GLUT1 in glomeruli of rats in DKD+CDC group was lower than that in DKD group.GLUT1 expression level in glomeruli of DKD+CDC group was higher than that of DKD group at 12 weeks of model establishment for type 1 DM and 8 weeks for type2 DM.After 12 h of HG stimulation,the elevated GLUT1 expression level in the HG group turned down in the HG+CDC group,and after 48 h of HG stimulation,the decreased GLUT1level in the HG group was rebounded in the HG+CDC group.After 12 h stimulation with12（S）-HETE,GLUT1 level in mesangial cells was significantly higher than that of CON group.After 12（S）-HETE stimulation for 48 h,GLUT1 level was significantly decreased compared with CON group.3.Compared with CON group,the phosphorylation level of p38MAPK in mesangial cells of rats in DKD group was increased significantly at 12 weeks of model establishment for type 1 DM and 8 weeks for type 2 DM.Compared with DKD group,the phosphorylation level of p38MAPK in DKD+CDC group was decreased.When stimulated by HG for 48 h,the phosphorylation level of p38MAPK was significantly increased,and the increased phosphorylation level of p38MAPK was significantly decreased after application of CDC.After treatment with p38MAPK inhibitor,the level of down-regulated GLUT1 in mesangial cells was increased significantly after 48 h of HG stimulation.4.At 12 weeks of model establishment for type 1 DM and 8 weeks for type 2 DM,compared with the CON group,the level of membrane GLUT1 in glomeruli of rats in DKD group was significantly decreased,which was accompanied by obvious enlarged glomerular volume,mesangial extracellular matrix accumulation and increased proteinuria;Compared with the DKD group,membrane GLUT1 level in glomeruli was increased significantly,the enlarged glomerular volume decreased significantly,mesangial matrix proliferation decreased significantly,the proportion of renal tubule interstitial fibrosis decreased significantly,and 24h UTP level decreased significantly in DKD+CDC group.Conclusions1.The level of GLUT1 expression in DKD mesangial cells is increased at the early stage of DKD and decreased at the late stage of DKD.2.12-LO-12（S）-HETE is involved in regulating the phasic expression of GLUT1 in mesangial cells of DKD,which up-regulates the expression of GLUT1 in the early stage and down-regulates the expression of GLUT1 through p38MAPK signaling pathway in the late stage.3.The low expression of GLUT1 in mesangial cells is involved in the pathophysiology of late-stage DKD renal injury and may play an important role in promoting the progression of DKD.