Expression And Significance Of Serum Chemokine CXCL9 And CXCL10 For Acute Rejection Following Kidney Transplantation In Rats

BACKGROUNDKidney transplantation is regarded as the best treatment for patients with end-stage renal failure.It represents an important breakthrough in the field of life sciences in the 20th century,and it is also one of the major therapies for end-stage renal diseases.Development and clinical use of immunosuppressants andamelioration of surgical techniques and medical care have improved the survival of the transplant patients and the transplanted kidney in the past ten years.However,the high incidence and repeated recurrence of acute rejection(AR)post kidney transplantation results in reduced graft survival.At present,there is still a lack of the specific and early indicator for the diagnosis of AR following kidney transplantation,making it difficult to prevent AR in the early stage and the graft damage post kidney transplantation.Therefore,it is necessary to find out an early diagnostic indicator for AR,which will help the surgeon react promptly to protect the graft function post kidney transplantation.Transplant renal biopsy is the gold standard for the diagnosis ofAR after kidney transplantation.But this method cannot promptly reflect the emergence of AR,since the graft kidney has sufferedvarying degrees of damage and its function has also been affected when the histopathological diagnosis is made..For timely diagnosing AR,clinicians suggested the renal allograft biopsy should be acted as a regular evaluation in kidney transplant recipients.However,the renal allograft biopsy is an invasive method which may cause biopsy-associated complications.Furthermore it is very difficult for patients who have been discharged to regularly receive the biopsy.Therefore,looking for a newly noninvasive method with high sensitivity and specificity in the diagnosis of AR has become the focus in the international transplantation research.Anglicheau et al.described the development of AR as a continuum,with initial events identified by the subtle change of specific molecules and histological changes and clinical manifestations being relatively latter events.Thus,monitoring the change of specific molecules in transplant recipientswould play a pivotal role in the early diagnosis of AR post kidney transplantation.Chemokine is a kind of polypeptide with molecular weight of 8～12 KDa,which contains four conservative cysteine residues and induces chemotaxis of responsive cells.On the basis of the configuration of a conserved amino proximal cysteine-containing motif,they are classified into four subtypes:C,CC,CX3C and CXC(X can be any amino acid).The main function of chemokine is to recruit immune cells to the responsive site,where immune regulation as well as the pathological reaction is induced.CXCL9 and CXCL10 induced by IFN-γ belong to ELR-chemokine superfamily and serve as an important factor in angiogenesis inhibition both in vivo and in vitro.CXCL9 emerges in the early process of inflammation,which makes it a valuable candidate for the prediction of transplant complicationsCXCL10 also plays an important role in inflammation and is the first ligands detected follwing kidney transplant.Both CXCL9 and CXCL10 are signal molecules in the downstream of IFN-γ pathway.It is suggested that the recruitment of CXC chemokine receptor 3(CXCR3 +)T cells into the graft by CXCL9 and CXCL10 plays a key role in the development of AR.As more T cells produce more CXCL9 and CXCL10 and vice versa more T cells are recruited into the graft.the effect is amplified.Gene expression of CXCR3which is the receptor of CXCL9 and CXCL10r,has been foundto be closely related to TH1 T cell activation and proved to be of a high value for the prediction of early graft rejection.In addition,researchers haves reported that CXCL9 and CXCL10 may be associated with the early AR.As chemokine play a key role in driving immune cell differentiation,migration and proliferation when acute rejection occurs,its level in the blood circulation is closely related to immune status of transplant recipients.Serum CXCL9,CXCL10 may not only have high diagnostic value for early acute rejection in renal transplantation,but also be an ideal targets for intervention.However,there is still no report on that.Some scholars put forward that more research is needed to establish whether post-transplant serum CXCR3-binding chemokines(CXCL9 and CXCL10)are predictive for graft outcome,especially AR.Analysis of the objective,verifiability and unified standard is very important in the study of biomarkers.Currently,due to large scale population sample research with these characteristics can not to be implemented,a certain number of animal experimental model on the study of serum CXCL9 and CXCL10 levels in acute rejection after renal transplant is necessary.In the early stage of the work,we have successfully established rat,rabbit renal transplantation model and rat liver,kidney,pancreas organs cluster transplantation model,confirmed that the CD44 is a predictor of early acute rejection and dynamic monitoring of CD3,CD69,CD25 and CD71 expressions could help in the early diagnosis of acute rejection.OBJECTIVEThis study focused on the expression and significant of serum CXCL9 and CXCL10 levels in acute rejection after kidney transplantation in rats.To provide optional biomarkers for early diagnosis of acute rejection after kidney transplantation,rat renal acute rejection model established by improving operative technique,the pathological grading and diagnosis was performed on the allograft,serum CXCL9 and CXCL10 levels were tested using enzyme-linked immunosorbent assay(ELISA)method for acute rejection after kidney transplantation in rats.METHODS1 Established the animal model of acute renal allograft rejection1.1 Animal groupsAdult male Brown Norway(BN)and Lewis inbred rats,200-250g,were randomly divided into two groups:①allogeneic transplantation for acute rejection group(ARx,n=15),BN rats as donors and Lewis rats as receptors;②homogenic transplantation for control group(con,n=15),Lewis rats as donors and receptors.Animals were feeding in air-conditioned animal room which maintained at 22℃ on a 12 h light-12 h dark schedule with food and water available and libitum.All experiments were done after the animals could fit with their surroundings for one week.1.2 Renal transplantationRat renal artery anastomosis by the end-side continuous suture;renal vein with end-to-end anastomosis,on one side of the venous wall took continuous suture,while the other side with interrupted suture;using donor ureter with bladder flap and receptors’ bladder for urinary tract reconstruction;all the allograft orthotopic renal transplantation surgery were perform on the left side of the donors and receptors.Postoperative observation of rat general status,including mental status,diet,activity,weight change,stool properties,and so on.For successful operation rats,mental state were gradually improving on the first day of postoperative,free to drink,good activity,sensitive response to external stimuli,should prevent the occurrence of postoperative complications and survival more than 3 days suggested a surgical success.2 Expression of serum chemokine CXCL9 and CXCL10 after kidney transplantation in rats2.1 sample collectionEach group in 3,5,and 7 days after operation executed five rats respectively,achieved the graft,each collected 6-10 ml of abdominal aortic blood,4000 RPM after centriffuge for 10 min,took 0.3 ml to test the serum creatinine(Cr),the remaining serum cryopreserved in-80 ℃waiting for inspection.2.2 histological examinationAllograft tissues in 10%of formaldehyde solution fixed,serial section(3 microns)before HE staining,the transplant renal pathological diagnosis reference banff97 standard and the severity of acute rejection are evaluated using semi-quantitative score(0 =normal,1 = borderline change,2 = IA,3 = IB,4 = IIA,5=IIB,6-III).2.3 serum CXCL9.CXCL10 detectionUsing ELISA method to detect the expression of serum CXCL9,CXCL10,according to the instruction manual,and read the OD value in 450 nm,under the condition of using "Curve Exert 1.4" software to calculate sample concentration,all samples are in duplicate.RESULTSI Establishing rat renal acute rejection modelWe performed 45 rat allograft renal transplantation surgeries,the success rate was 86.7%.A total of 6 rats dead,1 for anesthesia accident,1 for low blood volume,2 for thrombosis,2 for hypothermia.Artery anastomosis time(15±1.5)min,venous anastomosis time(15± 1)min,the total time(30±2)min.2 Pathological changesSemi-quantitative score showed that there was a significant difference observed in the severity of acute rejection between ARx and con group in 3,5,and 7 days after operation(P<0.01).In con group,comparison of semi-quantitative score of 3,5,and 7 days each other,there were no significant statistical difference(P>0.05).In ARx group,comparison of semi-quantitative score of 3 days with 5,7 days were statistically significant differences(P<0.05),while there was no statistically significant difference between 5 days and 7 days(P>0.05).Based on the semi-quantitative score averages,in ARx group,3,5,and 7 days showed light,medium and severe rejection,respectively;the control group showed no obvious rejection.3 Changes of serum creatinine after renal transplantationIn ARx group,3,5,and 7 days serum Cr expression continue to rise,the expression of between different times were statistically significant differences(P<0.05);All in ARx group have significant difference compared with the control group(P<0.01).4 serum CXCL10 expression after renal transplantation in ratsIn ARx group,expression of serum CXCL10 in 3,5,and 7 days after operation were increasing,there was a significant statistical difference between 5 days and 7 days(P<0.05),there was no significant difference between 3 and 5 days(P>0.05);Compared with the control group,serum CXCL10 expression of 3,5,7 days in ARx group showed significant differences(P<0.01).5 serum CXCL9 expression after renal transplantation in ratsIn ARx group,serum CXCL9 expression of 3,5,7 days significantly increased compared with control group(P<0.01);in ARx group,serum CXCL9 expression of the 7 days was significantly reduced when compared with 3 days and 5 days,respectively(P<0.05),there was no significant difference between 3 and 5 days(P>0.05).CONCLUSION1 Establishment of rat renal acute rejection modelThe method of rat renal artery anastomosis by the end-side continuous suture,renal vein with end-to-end anastomosis,urinary tract reconstruction using donor ureter with bladder flap and receptors’ bladder could successfully establish the rat model of acute rejection in renal transplant recipients.This improved operation is simple,stable,easil2 Expressions of serum CXCL9 and CXCL10 after renal transplantation closely associated with acute rejection episodes,may have diagnostic value for early acute rejection.3 As costimulatory molecules during T cell priming to alloantigen,the expression of CXCL9 and CXCL10 in the process of development of acute rejection may present the change of antagonize each other,the typical trend of balance between CXCL9 and CXCL10,which provides a new train of thought for the noninvasive joint monitoring method in the diagnosis of acute rejection after kidney transplantation.