Diagnostic Value Of Molecular Pathology And Functional MRI In Acute Renal Rejection

IntroductionKidney transplantation has become the best replacement therapy for patients with chronic renal failure,however,allograft dysfunction after transplantation has negative impact on graft and patient survival.Graft failure from immunologic factors and death from opportunistic infection in the first year after transplantation are no longer common clinical outcomes other than 30 years ago.However,how to detect rejection and distinguish it from other causes of renal dysfunctionn is still an challenging topic in the transplantation field.With the application of new immunosuppressive agents,some patients with typical clinical manifestation of acute rejection presented with atypical pathological change; Meanwhile,some patients with stable renal function were detected with abnormal pathological presentation in protocol biopsies.Current Banff criteria is not sufficient for clinical practice in some atypical cases.To target individualized immunosuppressive regemin,it is important to evaluate the immune state of this special population.In recent years,researchers have been dedicated to explore more sensitive and timely indicators for the diagnosis of early renal allograft dysfunction.Although several non-invasive monitoring indicator were confirmed to be effective,none of them was recognized and applied regularly in clinical practice as an independent indicator.A rise in the level of serum creatinine is used as the indicator of allograft dysfunction regularly in clinical practice,to aid in the resolution of the difficult differential diagnosis of allograft dysfunction,invasive renal biopsy and noninvasive imaging technqiue are often performed regularly in clinic.Until now,pathological diagnosis is still the golden criteria to evaluate the function of renal allograft irrespective of its limitations.Infiltration of mononuclear cells, especially T lymphocytes has predomindant status in the Banff criteria.In this study, we analyze the immune phenotype of T lymphocyte infiltration in allograft biopsies, explore the immue state evaluation value of FOXP3(functional marker of regulatory T cells)and Granzyme B(functional marker of cytotoxic T cells)in combination with clinical data.We hope the molecular pathological study will be of supplemented value for present pathological diagnosis criteria.Meanwhile,we prospectively study the diagnosis value of blood oxygen level-dependent magnetic resonance imaging(BOLD-MR/)by assessing the oxygenation state of renal transplants in acute rejection,find it is a non-invasive diadynamic imaging technique with good sensitivity and specificity in the diagnosis of allograft dysfunction early after kidney transplantation.It has dignostic value of acute renal rejection in pathophysiological respect.Our topic is expected to provide new dignostic methods for acute renal rejection in terms of molecular immunology and pathophysiology on the basis of histological and functional method which are now used regularly in clinical practice,and to provide some clinical basis for future research. PartⅠImmune phenotye analysis of T lymphocytes infiltration in renal biopsies for the diagnosis of acute renal rejectionObjectiveTo analyze the immune phenotype of T lymphocytes infiltration in renal biopsies with typical clinical manifestation of acute rejection but atypical pathological change (clinical rejection),compared with normal group and biopsy-proved acute rejection,to judge the immune state of this special population.Methods175 specimens were enrolled,including 67 with biopsy-proved acute T cell mediated rejection,63 with clinical rejection,consisted of 51 with borderline change and 12 with only inflammatory cell infiltrations but no obvious pathological abnormalities, and 45 with normal pathological presentations but inflammatory cell infiltrations in protocal biopsies with stable renal function in our follow-up time.Detect the expression of FOXP3(functional marker of regulatory T cells)and Granzyme B (functional marker of cytotoxic T cells)by the means of immunohistochemical methods in serial slices of the same biopsy.Quantitation of positive cell number and the ratio of FOXP3-positive T cells/Granzyme B-positive T cells were recorded simultaneously.Results100%(45/45)of normal group presented with regulatory phenotype dominated by FOXP3-positive T lymphocytes,94.03%(63/67)of biopsy-proved acute T cell mediated rejection group presented with cytotoxic phenotype dominated by Granzyme B-positive T lymphocytes;Borderline change group presented with similar phenotype compared with BPAR group(86.27%vs 94.03%,P=0.204);only inflammatory cell infiltrations but no obvious pathological abnormalities group also presented with similar phenotype compared with BPAR group(83.33%vs 94.03%,P=0.224).ConclusionsRenal biopsies with typical clinical manifestation of acute rejection but atypical pathological change(clinical rejection)have similar immune phenotype of T cell infiltration compared with biopsy-proved T cell mediated acute rejection.The ratio of FOXP3(functional marker of regulatory T cells)-positive T cells to Granzyme B (functional marker of cytotoxic T cells)-positive T cells could be a valuble diagnostic molecular indicator in pathological criteria. PartⅡImmune phenotye analysis of T lymphocytes infiltration in protocol renal biopsies for the evaluation of immune stateObjectiveTo analyze the immune phenotype of T lymphocytes infiltration in protocol renal biopsies with stable renal function within 2 months after transplantation,and to explore the evaluation value of related immune markers.MethodsAll specimens were obtained in protocal biopsies,100 specimens were enrolled, including 10 with subclinical rejection(SCR group),29 with borderline change (Pro-BL group)and 61 with only inflammatory cell infiltrations but no obvious pathological abnormalities(Pro-N group).Detect the expression of FOXP3(functional marker of regulatory T cells)and Granzyme B(functional marker of cytotoxic T cells) by the means of immunohistochemical methods in serial slices of the same biopsy. Quantitation of positive cell number and the ratio of FOXP3-positive T cells/Granzyme B-positive T cells were recorded simultaneously.Clinical data during the follow-up time were also recorded.ResultsCompared with indicated biopsies,the majority cases presented with regulatory phenotype dominated by FOXP3-positive T lymphocytes in protocal biopsies; however,20%(2/10)in SCR group,17.2%(5/29)in Pro-BL group and 13.1%(8/61) in Pro-N group presented with cytotoxic phenotype dominated by Granzyme B-positive T lymphocytes;All these 15 allografts developed different type of abnormalities in functon within 1 year after biopsy,including biopsy-proved acute rejection,clinical diagnostic acute rejection,and fluctuation of serum creatinine level which was improved by adding amount of immunosuppressive agents.ConclusionsAlthough with abnormal pathological changes according to Banff criteria,the majority cases in protocol biopsies presented with regulatory phenotype dominated by FOXP3-positive T lymphocytes;The ratio of FOXP3(functional marker of regulatory T cells)-positive T cells to Granzyme B(functional marker of cytotoxic T cells) -positive T cells could evaluate the immune state of allograft in protocol biopsy and predict the clinical outcome,it could be an early-warning indicator to guide the adjustment of immunosuppressive regimens. PartⅢDiagnosis and prediction of early acute renal transplant rejection with blood oxygen level-dependent MR imagingObjectiveTo prospectively assess the oxygenation state of renal transplants using blood oxygen level-dependent magnetic resonance imaging(BOLD-MRI)to diagnose and predict early acute renal rejection,and differentiate between acute rejection and acute tubular necrosis.MethodsBOLD MR imaging was performed in a cohort of 110 patients undergoing cadaver renal transplants between Dec 2005 and March 2007,82 had clinically normal functioning transplants,21 had biopsy-proved acute rejection and 7 had biopsy-proved acute rejection.Cortical R2*(CR2*)and medullary R2*(MR2*)levels of transplanted kidneys were measured.ResultsThe mean CR2~* level was significantly higher in the ATN group(15.25±1.03/s) compared to the normal group(13.35±2.31/s,P=0.028)and AR group(12.02±1.72/s,P=0.001).There was a significant difference also between the AR group and normal group on CR2~* levels(P=0.013).The mean M R2~* level was significantly lower in the AR group(14.02±2.68/s)compared to the normal group(16.66±2.82/s,P＜0.001)andATN group(19.47±1.62/s,P＜0.001).There was also a significant difference between the ATN group and normal group on M R2~* levels(P= 0.011).There were no correlations between characteristics such as patient age, post-operation time,post-biopsy time,Scr level,HB level,urine output volume,MAP level,CNI trough concentration and R2~* levels,except between MAP level and CR2~* level(P=0.029);ROC curve analyses suggested that cortical and medullary R2~* values could accurately discriminate acute rejection and acute tubular necrosis from normal function in the early posttransplant period;In the normal functioning transplants group,those with lower medullary R2~* values(MR2~*＜14.9/sec,n=23)had higher acute rejection rates than those with higher medullary R2~* values (MR2~*＞14.9/sec,n=59)in the first 6 months posttransplant,but the difference between two groups was not significant(17.39%vs 8.47%,P=0.259).ConclusionsThere were siginificant tissue oxygen bioavailability change during the episodes of acute rejection and acute tubular necrosis.BOLD-MRI could be a non-invasive dynamic imaging technique with good sensitivity and specificity,and may have the forecast value;It could also be a valuable method to discriminate between acute rejection and acute tubular necrosis.