Change And Significance Of Different Endothelin Receptors In Renal Tissue Of Patients With Diabetic Nephropathy During Different Period Of The Disease

Objective: Diabetic Nephropathy (Diabetic Nephropathy, DN) is oneof the whole body microvascular complications of diabetes, Both type1and type2diabetes are associated with severe complications including d-iabetic nephropathy.With the rapidly rising number of patients with diab-etes worldwide,diabetic nephropathy is becoming a major cause of endstage renal disease in most countries.because diabetic nephropathy develo-ps in30%to40%of patients with a duration of10to20years,no matter how old.Multigenetic predisposition contributes in the developmentofdiabetic nephropathy, but the exact progression is only partly understood.once developed to the end stage, it is more difficult to been treeat thanother forms of kidney disease.Including the glomerular hemodynamicalt-erations,biochemical metabolic disorders, Vascular endothelial lesion,hemorheological alterations, increased oxidative stress, cytokines,and genetics-usceptibility factor.The content of plasma endothelin is higher whenVascular endothelial was injured.Endothelin (Endothelin, ET)is a potent vasoconstrictor peptide prod-uced by vascular endothelium,ET‐1produces its actions by acting on e-ndothelin ETA and ETB receptors.The kidney is known to synthesize ET-1and to possess both ETA and ETB receptors[3-5],both of which maybe involved in physiologic and pathophysiologic actions of ET-1in thekidney.the ratio of the numbers of ETAR and ETBR was30:70.The a-ffinity of ETA receptors for ET-1and ET-2is100-fold higher than forET-3,whereas ETB receptors bind ET isopeptides with a similar affinityETAR receptor mediates vasoconstriction and cell proliferation,whereas ETBR receptors are important for the clearance of ET-1,on endothelial c- ells mediate vasodilation through the production of nitric oxide and pro-stacyclin originally identified as"endothelial derived relaxing factor",promoting the excretion of urinary sodium.It was shown that diabetes induced elevated level of renal ET‐1may induce glomerular hyperperfusionand damage tubulointerstitium in rats.Studies on the localization of ET-1andETAR in the rat kidney wi-th diabetic nephropathy is distributed in renal tubular epithelial cells,end-othelial cells and glomerular cell,and the number of ET-1and ETARwere higher than it is in the normal control group.Endothelin receptor an-tagonists may be of benefit in improving renal hemodynamics and redu-cing proteinuria,may inhibitor inflammatory and oxidative stress,ET is li-kely also involved in the progression of renal disease.Endothelin recept-or antagonists can improve blood flow dynamics of renal,reduce the co-ntent of urinary protein,reduce the inflammatory response,play a protecti-vee effect on the kidneys, but Whether selective ETARor non-selectiveETAR/ETBR receptor antagonists are preferable is still a matter of debate.So Study the localization of endothelin receptor in kid-ney tissues ofpatients with Diabetic Nephropathy to guide treatment is especially important.It was shown that the level of endothelin in plasmaurine renal ET‐1were elevated in rats and in patients with diabetic ne-phropathy,but the study about the relationship between the distribution of endothelin recept-or in kidney of patients with diabetes Nephropathy and the severityof the disease is still lesser.The purpose of this study is to explore the relationship between th-e changes of the endothelin receptors and the development of diabetic nephropathy,through observing the distribution and expression level of theendothelin receptors ETAR, ETBR in the kidney of patients with type2diabetes nephropathy, finally to Provide the experimental evidence for theselective of Endothelin receptor antagonists.Methods: This study was approved by the Department of Nephrolo-gy,the second hospital of Hebei Medical University.We choose28cases of diabetic nephropathy(13males and15females).The mean age of them was51.14±9.65years.They were definited by renal biopsy from Oct-ober2010to February2012.Patients with hypertension,LupusNephritis,HBV-GN associated glomerulonephritis,renal damage induced by polyarthriti-s destruens,neoplasms associated nephropathand those on glucocorticoid,Heart-failure,infection,obesity,pregnancy,liver disease,Drug-induced renal da-magewere excluded from this study. Pathologic classification of diabeticnephropathy patients were divided into3groups by Pathologic classific-ationof diabetic nephropathy[1](ⅠMild or nonspecific LM changes and EMproven GBM thickening,Biopsy does not meet any of the criteria, ment-ionedbelow for class II,III,or IV, GBM>395nm in female and>430nmin male individuals9years of age and older;IIa Mild mesangial expansion Biopsy does not meet criteria for class III or IV,Mild mesangial ex-pansion in>25%of the observed mesangium;IIb Severe mesangial expa-nsion Biopsy does not meet criteria for classIII or IV, Severe mesangialexpansion in>25%of the observed mesangium; III Nodular sclerosis(Kimmelstiel–Wilson lesion),Biopsy does not meet criteria for classIV,Atleast oneconvincing Kimmelstiel–Wilson lesion IV Advanced diabetic glomerulos-clerosis Global glomerular sclerosis in>50%of glomeruli Lesionsfrom classes I through III):diabetic nephropathyⅠgrade (S1,n=8),diabetic nephropathyⅡgrade (S2, n=10) diabetic nephropathy Ⅲ grade (S3, n=10);5specimens as control group(2males and3females,the mean agewas51.2±11.0years)were collected from the normal renal tissue apart fr-om kidney tumor. All tissues were diagnosed as normal which evaluatedby lighe microscopy and immunofluorescence.HE staining: under the light microscope (100×) Observed and calc-ulate the rate of glomerulosclerosis=hardening of the ball/the total thenumber of glomerular; Immunohistochemistry was was used for normalportions of kidneys and kidneys with diabetic nephropathy for ETAR ETBR expression in kidney tissues The semiquantitative analysis of renaltiss ue immunohistochemistry were analyzed. Collecting detailed clinical andpathological datas.Then the expression of ETAR ETBR was analyzed w-ith clinical and pathological datas.Double-labeling immunofluorescence and confocal laser scanning microscopy for the distribution of ETAR andETBR receptors,The markers included Lotus tetragonolobus agglutinin(LTL),which labels proximal tubules;Tamm-Horsfall protein (THP),which labels thick ascending limbs ofloops of Henle and distal tubules;and Dolichos biflorus agglutinin (DBA),which labels collecting ducts[2].The resultswere semiquantitatively analyzed with an image-processingsystem. Allthedata were analysed by SPSS13.0statistics software,P value<0.05was considered to have statistical significance.Result:1.Chemical examine:as the disease progresses,Upro in S3gr-oup was obviously higher than in S1groups(P<0.01) There was no sig-nificant difference between S2group and S1group;2.Renal pathology: light microscopyshowed thatⅠMild or nonspecif-ic LM changes and EMproven GBM thickening;II Mild to Severe mesa-ngial expansion>25%of the observed mesangium;III Nodular sclerosis(Kimmelstiel-Wilson lesion), At least one convincing Kimmelstiel–Wilso-n lesion.The rate of glomerulosclerosis in diabetic nephropathy Ⅲ gradegroup was obviously higher than in diabetic nephropathyⅠgrade and Ⅱgrade groups(P<0.05).There was no significant difference between S2gr-oupand S1group;3.Immunohistochemistry:1.It has been shown that the positive staini-ng for ETAR and ETBR was observed in the glomerular proximal tubu-lar in normal controls group and diabetic nephropathy groups,the expre-ssionlevel of ETAR and ETBR are significant higher than in C group(P<0.01,P<0.05)In the renal tubular,the level of ETAR ETBR were less v-isible in C group,but in In diabetic nephropathy groups;2.InC group, thelevel of ETBR is higher than ETAR(p<0.01)ETAR/ETBR=0.37;in S3gr-oup, the level of ETAR was higher than ETBR(p<0.01)ETAR/ETBR=2.1,In S3group,the expression level of ETAR was the highest than in Cgro- up(p<0.01),S1ETAR/ETBR=0.83group(p<0.01),S2group ETAR/ETBR=1.23(p<0.01),while in S2group,it was more higher than in C group(p<0.01)and S1group(p<0.01);The expression level of ETBR in S2group we-re significantly higher,in S3group,it was the lowest,but the difference b-etween each group showed no significant difference(p>0.05).In glomerul-ar,the different expression level of ETAR and ETBR between each gro-up were no significant difference;4.A significant positive correlation was found between the expressi-on of ETAR and the excretion of Urine protein(r=0.730,P<0.01),the rateof glomerulosclerosisin (r=0.783,P<0.01) diabetic nephropathy. there wasno correlation between the the expression of ETBR and the rate of glo-meruloscleros,and the excretion of Urine protein.5.Double-labeling immunofluorescence and confocal laser scanningmicroscopy ETAR and ETBR receptor are both labeled in proximal tub-ules,and the expression level of ETAR is higher than ETBR,ETAR arelabeled fewer in distal tubules and collecting ducts; ETBR are not been observed in distal tubules and collecting ducts;Conclusion:1.ETAR and ETBR are both distributed in glomerulus,proximal tubular in human kidney tissues; In normal renal tissue,the content of ETAR is fewer than it of ETBR; yet the expression of ETAR and ETBR were much more compared in the patients with diabetes nephropathy;2. With the diabetic nephropathy progressing,the content of ETAR increased, shows a positive correlation with the excretion of Urine protein andthe rate of glomerulosclerosis, in grade three,the expression level of ETAR is highest,ETBR is the lowest; the ETBR may increased firstlyand then decreased.