The Effect Of All-trans Retinoic Acid On The Glomerular P-cadherin Expression In Diabetic Nephropathy

Diabetic nephropathy ( DN ) is one of the most common chronic complication of Diabetes mellitus(DM).In the early DN,in addition to glomerular hypertrophy, endothelial cell dysfunction and extracellular matrix accumulation,the change of structure in visceral glomerular epithelial cells,that is,podocytes and their related factors also plays a very important role in the development of DN,and their injury are closely correlated with proteinuria generation and the progression of glomerulosclerosis in DN.Recent studies have indicated that p-cadherin,a transmembrane protein on the slit diaphragm of podocyte,plays an important role in the glomerular filtration barrier,its decreased expression causes proteinuria.It predicts that use effective drug to control the p-cadherin downregulation in early DN is effective to delay the DN progression.Objective:We apply biochemical,morphological and molecular biology techniques in this experiment to investigate the expression change of p-cadherin in early DN rat,clarify the effect of ATRA on above change,such as urinary albumin/urine creatinine (UAlb/UCr), kidney weight/body weight(KW/BW) and p-cadherin expression, observe the renal morphology change after treatment of ATRA, provide a theoretical basis for the prevention and treatment of DN.Methods:Experimental animal was divided into three groups. 30 adult male wistar rats, adaptability keeping one week,and checked blood glucose(BG) level.Randomly selected 10 rats as control group(Group N,n=10),The remaining 20 rats were given a intraperitoneal injection one time with STZ(55mg/kg, Dissolved in pH4.2,0.1mol/L citrate-sodium citrate buffer, prepared fresh in ice bath),group N was given a injection of equivalent citrate-sodium citrate buffer.After 48 hours, tested the BG of each rat from tail vein. BG of group N is normal,and the remaining 20 rat blood sugar were higher than 16.7mmol/L,DM model succeeded. The 20 rats were randomly divided into two groups: DN model group(Group DN,n=10) and DN+ATRA treatment group (Group ATRA,n=10).ATRA was given to Group DN and Group ATRA,20mg/kg/d,by intragastric administration, distilled water to Group N.BW and BG were measured at the start and after four weeks when the experiment finished.Urine was collected at the end of the experiment, and then the rats were sacrificed, measured kidney weight,collected blood and renal tissue, measured BG,BW,UAlb and UCr were assessed by ELISA,UAlb/UCr and KW/BW ratio were calculated.Colloidal gold immune electron microscopy was used to observe the location and quantity of p-cadherin expressed in podocyte.RT-PCR detected p-cadherin mRNA expression.Kidney histopathological changes by PAS staining were observed under light microscopy.P-cadherin expression in the kidney tissues were detected by immunohistochemistry.Results:The mental state of rats in Group N were good,they are strong, vivacious and their BW increased significantly;most of rats in Group DN emerged polydipsia, urorrhagia and became emaciated, depressed,sluggish,two of them died.Rats in Group ATRA were not so serious,one died.We considered that the cause of death was due to infection and high blood sugar of diabetic ketoacidosis.One rats died in group N,total mortality rate was 13.3%.1)The changes of BG,BW,KW,KW/BW,UAlb/UCr in each group:BG of Group DN was significantly increased than Group N(P<0.01);treated by ATRA,BG did not change significantly. BW of Group DN was decreased significantly compared with Group N(P<0.01);BW of Group ATRA did not change significantly compared with Group DN.KW of Group DN was evidently increased than Group N(P<0.01);KW of Group ATRA was decreased significantly compared with Group DN(P<0.01)and has no significant difference compared with Group N. KW/BW of Group DN was significantly increased than Group N(p<0.01);KW/BW of Group ATRA decreased significantly compared with Group DN(P<0.05).UAlb/UCr of Group DN was significantly increased than Group N(P < 0.01 ) and in Group ATRA,UAlb/UCr decreased significantly compared with Group DN(P<0.01).2)Result of immunoelectron microscope:Under electron microscope,we could see immuno-gold staining p-cadherin protein expressed on the slit diaphragm of podocyte,and compared with Group N,the expression of p-cadherin in Group DN was significantly decreased.3)Result of RT-PCR:Compared with Group N,the expression of p-cadherin mRNA in Group DN was significantly decreased(P<0.01);in Group ATRA,the expression of p-cadherin mRNA significantly increased than Group DN(P<0.01)and has no significant difference compared with Group N.4) Pathological changes:Under optical microscope,we detected in Group DN,the size of renal glomeruli increased, mesangial regions diffusely widened, extracellular matrix increased,part of the capillary basement membrane became thickened and capillaries cavity occluded.In Group ATRA,these changes mitigated. 5)Result of immunohistochemistry inspection:Under optical microscopy, in Group N,the expression of p-cadherin in the glomerular basement membrane shown to be a brown linear distribution, this expression in Group DN was significantly decreased. Treated by ATRA,the expression of p-cadherin increased and showed no significant decline compared with Group N.Conclusions:1)In a certain extent,ATRA could reduce the urinary albumin excretion in early DN rat,and also reduced its UAlb/UCr. 2)KW/BW in DN rats is higher than normal rats,it indicated that there were kidney hypertrophy in early DN,and ATRA could reduce the ratio of KW/BW to improve early kidney hypertrophy in DN.3)ATRA reversed the decrease of p-cadherin protein expression in early DN and protected podocyte and it's related molecules from being injured.4)ATRA could improve the pathological changes in DN,such as the decrease glomerular volume and extracellular matrix accumulation. These results proved that ATRA had a therapeutic effect on early DN.