The Role And Possible Mechanisms Of KLF4 In Proteinuria Occurred Under Type 2 Diabetic Nephropathy

Objective: Diabetic nephropathy(DN)is a severe complication of type 2diabetes.The development mechanism of proteinuria of DN has important clinical and social significane to prevent the DN,take corresponding measures to slow down the development of DN,and improve the quality of life and living standards of patients who has the type 2 diabetes.Podocytes will be damaged under type 2 diabetes could damage,main show is: Slit diaphragm(SD)structure disappears,foot process effacement,eventually lead to the occurrence of proteinuria.CD2 AP and nephrin are on the slit diaphragm of podocyte,?-actin4 is located in the foot process(FPs)of podocyte,integrin ?1 is a important adhesion molecule between podocyte and glomerular basement membrane.The loss of these molecules can cause podocyte damage and leading to the occurrence of proteinuria.Kruppel-like the factors(KLFs)belongs to the nuclear transcription factor superfamily,regulating cell of a variety of biological processes,including proliferation,differentiation,growth,development.In recent years,the study found that KLF4 has a important role in the development of the kidney and maintain the normal physiological function of kidney.In previous experiments,we found that KLF4 mRNA expression level was decreased significantly.Preliminary results suggest that KLF4 is likely associated with the occurrence of type 2 diabetic nephropathy.In the mice of kidney disease induced by adriamycin(ADM),the level of urine protein was increased significantly,at the same time,the levels of KLF4 and nephrin in podocytes were decreased significantly.Increased the expression of KLF4 in podocyte of mice under kidney disease can increase the expression of nephrin and reduce the level of urine protein.So,whether the decreased expression of KLF4 is associated with the occurrence of type 2 diabetic nephropathy proteinuria? What is the mechanism? in order to solve this problem,in this experiment,we used db/db mice of type 2 diabetes and podocyte in high lipid as experimental object to research the effect and mechanism of KLF4 in the occurrence of proteinuria of type 2 diabetic nephropathy,and provide the experimental data for understanding the molecular mechanism of the occurrence of proteinuria of type 2 diabetic nephropathy.Methods:1 Experimental AnimalsUsing the db/db mice as animal model of type 2 diabetes.Use the tail intravenous injection of KLF4 adenovirus to change the expression level of KLF4 in the kidney of db/db mice.2 Cell linesPodocytes of mice.Use palmitic acid(C16:0)to simulate a state of high lipid environment.Use KLF4 adenovirus to infect podocytes,change the expression level of KLF4 in podocytes.3 Samples collectionCollect urine and kidney tissue at 8 weeks,12 weeks and 16 weeks.Kidney tissue were used in Western blot,PCR,PAS staining and immunohistochemical experiment,etc.4 Cell collectionThe corresponding concentration of BSA,C16:0,C16:0+Ad-GFP,C16:0+Ad-GFP-KLF4 treat podocytes of logarithmic phase 48 hours,the podocytes were collected for Western blot and RT-PCR analysis experiment.5 Analysis of 24 h urine protein by Lowrry method.6 The mRNA levels of KLF4,nephrin and integrin ?1 were assayed by PCR.7 The protein levels of KLF4,nephrin,?-actin4 and integrin ?1 were assayed by Western blot.8 Observe glomerulus structure in mice by PAS stain.9 The protein levels of nephrin,CD2 AP,?-actin4,integrin ?1 and integrin ?3 were assayed by immunohistochemistry.10 Statistical analysisAll statistical analysis was performed using SPSS 17.0 software.All data were expressed as the means±SD.Statistical significance was set at P<0.05.Results:1 The expression of KLF4 was decreased in kidney glomerulus of db/db mice.1.1 The level of urine protein of type 2 diabetes mice.Compared with the db/m mice of same weeks(8 weeks: 5037.34 ±1037.73?g/24h;12 weeks: 2649.32 ± 87.07?g/24h;16 weeks: 34605.02 ±16160.58?g/24h),the urine protein level of db/db mice had no obvious change in 8 weeks(4777.18 ±1378.49?g/24h),but with the increase of weeks,the urine protein levels increased gradually in 12 weeks(18629.52 ±3885.96?g/24 h,P<0.01)and 16 weeks(323798.60±67766.10?g/24 h,P<0.01),and significantly higher than the control groups.1.2 The level of KLF4 in kidney glomerulus.PCR showed that compared with the db/m mice,the mRNA expression of KLF4 in renal cortex of db/db mice was decreased significantly.Western blot showed that compared with the db/m mice,the protein expression of KLF4 in renal cortex of db/db mice was decreased significantly.Immunohistochemical showed that compared with the db/m mice(275.50±30.26),the expression of KLF4(150.25±15.97,P<0.01)in glomerulus of db/db mice was decreased significantly.The above results suggested that the decline of the expression of KLF4 in glomerulus is likely associated with the occurrence of type 2 diabetic nephropathy proteinuria.2 After overexpression of KLF4 in glomerulus,the level of urine protein of db/db mice was decreased significantly.2.1 The level of KLF4 in glomerulus.The immunohistochemical showed that compared with the db/db+GFP mice(152.25±12.89),the expression of KLF4(317.5±33.25,P<0.01)was significantly increased in the glomerulus of db/db+GFP-KLF4 mice.2.2 After overexpression of KLF4 in glomerulus,the level of urine protein of db/db mice.Compared with the db/db+GFP mice(435255.6±10446.17?g/24h),the urine protein level of db/db+GFP+KLF4 mice(264330.80±33824.15?g/24 h,P<0.05)was decrease significantly.The above results indicated that the expression of KLF4 in glomeruli is one of the reasons for occurrence of type 2 diabetic nephropathy proteinuria.Increase the expression of KLF4 can improve proteinuria of type 2 diabetic nephropathy.3 KLF4 can improve proteinuria of type 2 diabetic nephropathy by raising the expression of nephrin,integrin ?1 and ?-actin4 in glomerulus of db/db mice.3.1 Glomerular structure of type 2 diabetes mice.The PAS staing showed the glomerulus structure of db/db mice had no obvious anomalies.3.2 The level of key proteins associated with Podocytes,such as nephrin,integrin ?1,integrin ?3,?-actin4 and CD2 AP.The immunohistochemical showed,compared with the db/m mice(nephrin: 0.208±0.016;integrin ?1: 0.180±0.008;?-actin4: 0.159±0.007),besides CD2 AP and integrin ?3,the expression of nephrin(0.178±0.006,P<0.01),integrin ?1(0.164±0.006,P<0.01)and ?-actin4(0.156±0.006,P<0.05)in glomerulus of db/db mice was decreased significantly.After overexpression of KLF4 in db/db mice,compared with db/db+Ad-GFP mice(nephrin: 0.148±0.003;integrin ?1: 0.109±0.009;?-actin4: 0.136±0.001),the expression of nephrin(0.178±0.006,P<0.01),integrin ?1(0.164±0.006,P<0.01)and ?-actin4(0.156±0.006,P<0.05)in glomerulus was increased significantly.Consistent with literature reports,integrin ?1,integrin ?3,?-actin4 and CD2 AP were also expressed in renal tubules,and compared with the db/m mice,the expresstion of ?-actin4 in renal tubules was low,the expression of integrin ?1,integrin ?3 and CD2 AP in renal tubules had no obvious change.after overexpression of KLF4 in db/db mice,the expresstion of integrin ?1,integrin ?3,?-actin4 and CD2 AP in renal tubules had no obvious change.nephrin expresses in glomerulus specially,but in this experiment,renal tubules also had a small amount of positive particles,may be because of less than optimal experiment conditions.These above results showed that the expression of nephrin,integrin ?1,integrin ?3 and ?-actin4 in glomerulus of type 2 diabetes mice was associated with the expression of KLF4.Lower expresstion of KLF4 was likely to affect the decreased expression of nephrin,integrin ?1 and ?-actin4 in glomerulus,and associated with the occurrence of type 2 diabetic nephropathy proteinuria.4 In podocytes of high lipid,KLF4 can adjust the expression of nephrin and integrin ?1.Under the different concentrations of C16:0,the expresstion of KLF4 in podocytes was decreased in concentration dependence.And after 48 hours of podocytes in the environment of 250 ?M of C16:0 concentration,along with KLF4 decline,the protein expression of nephrin and integrin ?1 was decreased significantly,the protein expresstion of ?-actin4 had no obvious change.After overexpression of KLF4 in podocytes of high lipid,the mRNA and protein expresstion of nephrin were increased significantly;the mRNA expression of integrin ?1 had no obvious change,the protein expresstion of integrin ?1 was increased significantly.These results showed,in podocytes of high lipid,KLF4 was likely regulate the expression of nephrin through transcription and increased the protein expression;KLF4 increased the protein expression of integrin ?1 through unknown way.The above results showed that the occurrence of type 2 diabetic nephropathy proteinuria was associated with the decreased expression of KLF4.The mechanism:(1)lower expresstion of KLF4 in podocytes affect the decreased expression of nephrin through transcription.(2)lower expresstion of KLF4 in podocytes affect the decreased expression of integrin ?1 through unknown way.In this experiment,the expression of KLF4 didn't affect the expression of?-actin4 in podocytes.Conclusions:The decreased expression of KLF4 in podocytes reduce the expression of nephrin,thus led to the occurrence of type 2 diabetic nephropathy proteinuria.Increased the expression of KLF4 in podocytes,can decrease the level of urine protein.