The Effect Of Irbesartan On High Glucose Induced Mouse Podocyte TRPC6 Expression

Objective:Diabetic kidney disease is one of the major microvascular complications of diabetes mellitus,Micro-albuminuria is considered to the main clinical manifestations in early of disease?In recent years,abundant researches show us that podocyte injury is one of the main causes of proteinuria and become a hot research topic.Transient receptor potential cation channel 6 is one of the major components of the podocyte slit diaphragm proteins,which expression abnormalities can lead to the occurrence of urinary protein.In this experiment,we research the effect of expression irbesartan(angiotensinII receptor blockers)on high glucose induced the of TRPC6 in conditional immortalized mouse podocyte,and investigate the possible mechanism of irbesartan on protective podocyte in diabetic kidney disease.Method:To begin with,the mouse podocyte were divided into five groups: hypertonic control group(mannitol 30mmol/l+ glucose 5mmol/l),normal control group(5mmol/L glucose),experimental group(Glucose 15mmol/L,Glucose 25mmol/L and Glucose 35mmol/L),the morphological changes of the cells induced by different concentrations of glucose were observed under the inverted microscope,Cck-8 was used to detect Cell viability,Immuno-cytochemistry was used to detect changes which the distribution of TRPC6 expression in the podocytes induced by different concentrations of glucose,Western blotting was used to detect the expression of TRPC6 protein in the cells of different concentrations of glucose.Reverse transcription-polymerase chain reaction was used to detect the expression of podocyte TRPC6 mRNA in the cells of different concentrations of glucose.Next,high glucose(25mmol/l)respectively stimulate 0h ? 12 h ? 24 h and 48 h on podocyte.each time point were collected to extract the total cellular protein and total RNA by WB detection in high glucose induced podocyte at different time points caused by changes in the expression of TRPC6 protein and podocyte induced at different time points caused by changes in the expression of TRPC6 mRNA by RT-PCR assay for the detection of glucose.Finally,the cells were divided into five groups: normal control group(5mmol/l glucose),high glucose group,high glucose + irbesartan group(10-7 mol/l),high glucose + irbesartan group(10-6mol/l)and high glucose + irbesartan group(10-5mol/l),the morpholo-gical changes of the cells induced by the high glucose and irbesartan were observed under the inverted microscope,Cck-8 was used to detect Cell viability,the WB detection of irbesartan on high glucose induced podocyte 48 h after induced podocyte TRPC6 protein expression changes,the RT-PCR detection of irbesartan on high glucose induced podocyte 48 h after induced podocyte TRPC6 mRNA expression changes.Statisticl analysis is used by SPSS17.0 statistical software.Data were expressed as mean ± standard deviation()that the comparison between groups ANOVA,pairwise comparisons using LSD t test,test level ?= 0.05,P <0.05 was considered statistically significant.Results: 1.With the increase of glucose concentration,under the light microscope foot cell bodies became smaller and the foot processes were significantly decreased or disappeared,The inhibitory effect of CCK-8 on the activity of glucose was most obvious when 25mmol/l was detected;immunocyto-chemistry showed TRPC6 expressed mainly in the podocyte cell membrane,the foot process distribution is more obvious,WB and RT-PCR detected in TRPC6 protein and mRNA expression increased gradually,most significantly in 25mmol/l change.2.High glucose stimulation of podocytes,compared with the 0h group,with prolonging of stimulation time,the podocyte protein TRPC6 and mRNA expression was significantly increased(P<0.05),peaked at 48 h,was time dependent.3.Compared with the normal control group,high glucose induced TRPC6 expression increased significantly(P<0.05),compared with high glucose group,high glucose plus irbesartan group with increase of concentration of irbesartan,TRPC6 protein and mRNA expression was significantly down regulated(P<0.05)in a concentration dependent manner.Conclusion:High glucose can increase the expression of TRPC6 in podocytes.,irbesartan on high glucose induced podocyte injury has a protective effect,the mechanism may be realized by down regulating the expression of podocyte TRPC6.