Study Of Molecular Pathomechanism Of Renal Fibrosis In Chronic Allograft Nephropathy

Research backgroundKidney transplantation is the best therapy for end stage nephropathy.Due to the improvement of operation technique and tissue matching and organ preservation,and the application of novel immunosuppressive drugs,the complications during the perioperative period and early acute rejection decreased significantly,simultaneously the one year survival of grafts increased,while the half-life period of grafts was still rambling at 11 or 12 years.In the past two decades,we have seen considerable improvements in 1-year renal allograft survival rate over 80 percent for cadaveric donors and 90-95 percent for living related donors.These commendable outcomes could be largely attributed to the introduction of more potent and selective immunosuppressants,such as cyclosporine-A(CsA) and tacrolimus,as well as to improvements in understanding of the immunology of transplantation,tissue typing, organ preservation and the management of acute rejection.However,despite reducing the frequency and severity of acute rejection episodes,cyclosporin and tacrolimus are of no effect to the development of chronic allograft dysfunction.The half-life of renal allograft has remained unchanged for over a decade.Approximately 50 percent of transplanted kidneys develop progressive dysfunction within a few months post -transplantation,necessitating dialysis or retransplantation within a decade.Data from Leicester show that,despite a 1-year graft survival over 90 percent level,there is a gradual attrition of 4-5 percent of graft loss per year,leading to 5-and 10-year allograft survival rates of approximately 70 and 50 percent respectively.The most common cause of this decline in allograft survival rate is chronic rejection,perhaps more accurately described as chronic allograft nephropathy(CAN),now defined as interstital fibrosis and tubular atrophy not otherwise specified,is a near universal finding in transplant kidney biopsies by the end of the first decade posttransplantation. CAN remains the single most important cause of renal allograft failure after the first year following transplantation,accounting for some 50-80 percent of graft loss after this time.The most important characteristic of CAN renal allograft tissues is interstitial fibrosis of renal allograft.So it is very important to explore the mechanisms of renal allograft fibrosis and prevent the progressive fibrosis of renal allograft.Objective1.To study the histopathological characteristics of renal allograft tissue with chronic allograft nephropathy.2.To investigate the expression and significance of integrin-linked kinase(ILK), transforming growth factor-β1(TGF-β1),E-cadherin,α-smooth muscle action (α-SMA) and collagenⅣin the renal allografts tissue with CAN.3.To explore the effect and mechanism of ILK in the renal allografts tissue with CAN, further clarify the the molecular pathomechanisms of the renal allograft fibrosis with CAN for presenting the direction and theoretic evidences to treat the disease of CAN.Methods Thirty samples of renal tissues with proved by pathology were collected from renal allograft transplant recipients received allograft biopsy.These patients occur their renal hypofunction within 15 years posttransplantation,in which 22 of them were male,and 8 of them were female and their ages were during 21-59 years old, and 25 of them were treated with CsA+MMF+Pred,5 of them with FK506+ MMF+Pred.The average transplantation time of allograft was 4.5 years.The same blood type of donor-recipient,Lymphocytotoxicity cross-matching test(or CDC)＜10%,panel reactive antibody(PRA)＜10%and HLA of A,B,Dr,at least two sites match sites.The controlled group was made up of ten cases of normal renal tissues, were normal cadaveric renal donor preoperative biopsy specimens,no abnormal pathological examination,and their ages were during 25-50 years old.All the 40 samples were made paraffin blocks,then were stained by HE,PASM,Masson trichrome methods.And immunohistological staining for IgG,IgA,IgM,Clq and C4c was performed.The severity of the pathological disorders of renal allograft was classified as three grades according to Banff 2007 pathologically diagnostic classification standard(Interstitial fibrosis and tubular atrophy,no evidence of any specific etiology).GradeⅠ:Mild interstitial fibrosis and tubular atrophy(＜25%of cortical area).GradeⅡ:Moderate interstitial fibrosis and tubular atrophy(26-50%of cortical area).GradeⅢ:Severe interstitial fibrosis and tubular atrophy/loss(＞50% of cortical area)(may include non-specific vascular and glomerular sclerosis,but severity graded by tubulointerstitial features).In all the samples,immunohistological staining for ILK,TGF-β1,E-cadherin,α-SMA and collagenⅥwas performed with ELiVision^TM plus two-step method.The Leica Qwin(Type DMR+Q550) high acuity color pathologic analysis and report system was used to analyze images and assess the expression levels of above factors.For each sample,ten continuous fields of renal tubes and interstitium were observed(except glomerulus and large vessels).The ratio of the stained area to the whole tubulointerstitial area was calculated to represent the expression level of each factor.The values were expressed as mean±standard deviation(SD).SPSS 13.0 software was used in the statistical analysis of the data. The statistic of the expression levels of ILK,TGF-β1,E-cadherin,α-SMA and collagenⅥwere tested by homogeneity of variances.After the test of homogeneity of variances,One-way ANOVA was applied to analyze the data with homogeneitic variances and LSD used for two-two comparison.And Welch Test was applied to analyze the data with heterogeneity of variances and Dunnett' T3 used for two-two comparison.The Pearson or Spearman correlation coefficient was calculated for the analysis of the correlation between the factors(bilateral,α=0.05 as size of test). Statistical significance was considered at P＜0.05.Results1.Chronic allografi nephropathy in patients with renal histological changes in the main performanceRenal allograft interstitial diffuse fibrosis,associated with lymphocytes; plasmacytes,mononuclear cells infiltration;Varying degrees of renal tubular atrophy, with a more disordered,basement membrane thickening,medullary collecting duct some compensatory enlargement,protein cast common in tube;Mesangium matrix and mesangial cell proliferation,basement membrane thickening,glomerular capillaries segmental glomerular sclerosis or glomerular hyalinization completely, some cases of small arteries concentric fibrous proliferation,lumena stenosis or occlusion.2.The expression changes of ILK,TGF-β1,E-cadherin,α-SMA and collagenⅣin renal tubulointerstitiumIt is shown in our present experiment that,in normal renal tissue,ILK was slightly or negatively expressed in tubular epithelial cells;TGF-β1 slightly expressed mainly in the plasma of tubular epithelial cells;E-cadherin was largely expressed on renal tubular epithelial cell membrane and in the glomerulus,the expression of a small amount of cytoplasm;α-SMA was expressed only in the vascular smooth muscle cells,sometimes in the expression of tubulointerstitial and negatively in tubular epithelium;CollagenⅥwas mainly expressed in the glomerulus,tubular basement membrane,Bowman's capsule and mesangial area,partially in the interstitial area and negatively in tubular epithelium.In renal allograft tissue of CAN patients,ILK was expressed much more than normal tissues,and mainly located at glomerulus,tubular epithelial cell membrane, vascular smooth muscle cells and interstitium,distribution from the proximal tubule to the medullary collecting duct epithelial cells,Degeneration of renal tubular atrophy strongest expression.Its expression level shown an increasing tendency with the aggravation of the grade of IF/TA(r=0.802,P＜0.001).Its expression level was positively correlative with the serum creatinine(SCr) of the CAN patients(r=0.662, P＜0.001).The expression location of CollagenⅥin renal allograft tissue of CAN was like normal controls but the intensity was much stronger than normal controls.There was a tendency of increasing expression of CollagenⅣalong with the aggravation of the grade of IF/TA(r=0.887,P＜0.001)In normal controls there was large amount of expression of epithelial cell marker, E-cadherin,on renal tubular epithelial cell membrane and no expression of myofibroblast cell marker,α-SMA;While on the necrotic and atrophic renal tubular epithelial cell membrane of CAN patients,E-cadherine expression decreased significantly andα-SMA expression appeared,andα-SMA was universally distributed in the interstitium and seen in the cytoplasma of some destroyed and atrophic tubules. TGF-β1 was expressed evidently in renal allograft tissue of CAN patients, mainly expressed in tubular epithelial cytoplasma and membrane and partially in Bowman's capsule.Its expression level shown an increasing tendency along with the aggravation of the grade of IF/TA(r=0.939,P＜0.001),and was positively correlative with the expression level of myofibroblast maker,α-SMA(r=0.808,P＜0.001),while negatively correlative with E-cadherin expression(r=-0.779,P＜0.001).There was a positive correlation between the expression levels of TGF-β1 and CollagenⅥ(r=0.870,P＜0.001).3.Statistical analysis showed that The expression levels of ILK,TGF-β1,α-SMA, CollagenⅥin the renal tubule and interstitium of CAN patients were significantly increased,compared to normal controls(P＜0.01);but E-cadherine expression in the renal tubule epithelial cell membrane of CAN patients decreased significantly, compared to normal controls(P＜0.01),and was negatively correlative with the aggravation of the grade of IF/TA(r=-868,P＜0.001).The expression level of ILK in renal allograft of CAN patients was positively correlative respectively with the expression levels of TGF-β1,α-SMA and CollagenⅥ(r=0.815,r=0.700,r=0.727,P＜0.001),while negatively correlative with the expression level of E-cadherin(r=-0.618,P＜0.001).Conclusions1.The major histopathological disorders of CAN are characterized by intimal hyperplasia,glomerulosclerosis,tubular atrophy and interstitial fibrosis, associated with mononuclear cells infiltration.2.There was increased expression of ILK in the tubulointerstitium of renal allograft tissue of CAN and a tendency of increasing ILK expression along with the aggravation of the grade of IF/TA.Its expression level was positively correlative with the serum creatinine clearance rate of the CAN patients.It is suggested that increased ILK might be related to the mechanism of the pathological disorders and the deterioration of renal allograft function.3.The expression level of CollagenⅣin the tubulointerstitium of CAN patients was increased significantly and there was a tendency of increasing expression of CollagenⅣalong with the aggravation of the grade of IF/TA.It is suggested that abnormal CollagenⅣdeposit might be important manifestation of renal allograft fibrosis of CAN patients.4.In normal controls,there was large amount of expression of epithelial cell marker, E-eadherin,on renal tubular epithelial cell membrane and no expression of myofibroblast cell marker,α-SMA.While on the necrotic and atrophic renal tubular epithelial cell membrane of CAN patients,E-cadherine expression decreased significantly andα-SMA expression appeared,andα-SMA was universally distributed in the interstitium and seen in the cytoplasma of some destroyed and atrophic tubules.It is suggested that EMT might play a role in the development of renal allograft with CAN.5.There was increased expression of TGF-β1 in renal allograft and the expression level shown an increasing tendency along with the aggravation of the grade of IF/TA.Its expression level was positively correlative with the expression level of myofibroblast maker,α-SMA,while negatively correlative with E-cadherin expression.It is suggested that up-regulated expression of TGF-β1 might induce the EMT of tubular cells of renal allograft and play a role in the development of renal allograft fibrosis.6.The expression level of ILK in renal allograft of CAN patients was positively correlative respectively with the expression levels of TGF-β1,α-SMA and CollagenⅥ,while negatively correlative with the expression level of E-cadherin. It is suggested that ILK might take part in the process of TGF-β1 induced renal tubular EMT,finally resulting in renal allograft fibrosis,and play an important role in chronic renal allograft fibrosis progress.