Chronic Allograft Damage Research Articles

Editorial introductions

Editorial introductions

The Presence of Urinary Renal Progenitor Cells in Stable Kidney Transplant Recipients Anticipates Allograft Deterioration.

Long-term kidney transplant outcomes have reached mild improvements recently. Parietal epithelial cells (PECs) are progenitor cells located along the Bowman’s capsule that can be isolated in urine, and display the capability to replace podocytes, but in certain situations cause glomerulosclerosis. In this study, a cohort of stable kidney transplant recipients with 6 months protocol biopsy was divided in two groups depending on the presence (uPEC+; n = 41) or absence (uPEC-; n = 25) of PECs in urine and followed for 2 years. No differences were found between groups at 6 months after transplantation considering clinical variables, alloimmune response, renal function, albuminuria and graft pathology. However, uPEC+ group showed increased podocyturia and a higher rate of proliferating PECs along the Bowman’s capsule, without concomitant enhancement of the CD44 pro-sclerotic activation marker. Accordingly, 2 years follow up evidenced poorer outcomes in the uPEC+ group with worse renal function, increased albuminuria, wider mesangial expansion and more severe IFTA. In summary, chronic allograft damage can progress in certain stable-supposed grafts by podocyte detachment and reactive PECs proliferation, being the uPEC presence a biomarker of this process. This damage-response regenerative process, if sustained in time, might fail in preserve the allograft function and histology. Our study raises new prospects to overcome current limits on long-term allograft results.

The value of diffusion tensor imaging in early diagnosis of chronic allograft nephropathy and monitoring graft fibrosis in rats

Objective To estimate the value of diffusion tensor imaging (DTI) in early diagnosis of chronic allograft nephropathy (CAN) and monitoring of graft fibrosis in rat models. Methods Thirty CAN rat models were established as experimental group by transplanting Fisher donor kidneys into Lewis recipients. Thirty Lewis rats that received Lewis kidneys served as control group. Serum creatinine (SCr) was monitored regularly every two weeks from 14 days after transplantation. Eight rats were randomly selected by random number table method and underwent DTI examination at 4, 12, 20 weeks after modeling. DTI scans were performed on the renal cortex and medulla to measure apparent diffusion coefficient (ADC) and fractional anisotropy (FA). From the remaining 22 rats in each group, 6 rats were randomly selected and underwent pathological analysis at 4, 12, 20 weeks after modeling. Histological changes in the kidney were evaluated by chronic allograft damage index (CADI) scores. The expression of alpha-smooth muscle actin (α-SMA) and Vimentin were quantitatively measured. The differences in creatinine, DTI parameters, CADI score, α-SMA, Vimentin expression level were analyzed by two independent samples t test in two groups, the differences among CADI score, α-SMA, Vimentin expression level of the experimental group were compared using ANOVA. The correlations among DTI parameters and CADI score, α-SMA and Vimentin expression level were analyzed using Pearson analysis. Results The creatinine in the experimental group increased continuously, and the creatinine in the control group showed no significant increase. The difference in creatinine between the two groups was statistically significant from 8th week after operation (P<0.01). There was no obvious difference in the size and signal intensity of transplanted kidneys in control group at different time points. Compared with the control group, the graft kidney in the experimental group at the 4 weeks demonstrated increased signal intensity with mild increased volume of kidney, and the boundaries between cortex and medulla were not clear. The cortex and medulla showed gradually increased signal intensity, heterogeneous signal distribution and marginal haziness over time. The ADC and FA value of renal cortex and medulla in experimental group were significantly lower than those in control group at 4, 12, 20 weeks (P<0.05). The ADC and FA values of the cortex and medulla gradually decreased in the experimental group over time, while the values of the parameters in the control group did not show a significant decrease. The ADC and FA values of the cortex and medulla were negatively correlated with the scores of CADI, and the expression level of α-SMA, Vimentin in the experimental group(r=-0.50 to-0.85, P<0.01). Conclusion DTI can be an effective technique for early diagnosis of CAN and monitoring of graft fibrosis process. Key words: Kidney transplantation; Chronic allograft nephropathy; Diffusion tensor imaging; Comparative study

Non-invasive staging of chronic kidney allograft damage using urine metabolomic profiling.

Chronic kidney allograft damage is characterized by IFTA and GS. We sought to identify urinary metabolite signatures associated with severity of IFTA and GS in pediatric kidney transplant recipients. Urine samples (n=396) from 60 pediatric transplant recipients were obtained at the time of kidney biopsy and assayed for 133 metabolites by mass spectrometry. Metabolite profiles were quantified via PLS-DA. We used mixed-effects regression to identify laboratory and clinical predictors of histopathology. Urine samples (n=174) without rejection or AKI were divided into training/validation sets (75:25%). Metabolite classifiers trained on IFTA severity and %GS showed strong statistical correlation (r=.73, P<.001 and r=.72; P<.001, respectively) and remained significant on the validation sets. Regression analysis identified additional clinical features that improved prediction: months post-transplant (GS, IFTA); and proteinuria, GFR, and age (GS only). Addition of clinical variables improved performance of the %GS classifier (AUC=0.9; 95% CI=0.85-0.96) but not for IFTA (AUC=0.82; 95% CI=0.71-0.92). Despite the presence of potentially confounding phenotypes, these findings were further validated in samples withheld for rejection or AKI. We identify urine metabolite classifiers for IFTA and GS, which may prove useful for non-invasive assessment of histopathological damage.

Novel Therapeutics Identification for Fibrosis in Renal Allograft Using Integrative Informatics Approach

Chronic allograft damage, defined by interstitial fibrosis and tubular atrophy (IF/TA), is a leading cause of allograft failure. Few effective therapeutic options are available to prevent the progression of IF/TA. We applied a meta-analysis approach on IF/TA molecular datasets in Gene Expression Omnibus to identify a robust 85-gene signature, which was used for computational drug repurposing analysis. Among the top ranked compounds predicted to be therapeutic for IF/TA were azathioprine, a drug to prevent acute rejection in renal transplantation, and kaempferol and esculetin, two drugs not previously described to have efficacy for IF/TA. We experimentally validated the anti-fibrosis effects of kaempferol and esculetin using renal tubular cells in vitro and in vivo in a mouse Unilateral Ureteric Obstruction (UUO) model. Kaempferol significantly attenuated TGF-β1-mediated profibrotic pathways in vitro and in vivo, while esculetin significantly inhibited Wnt/β-catenin pathway in vitro and in vivo. Histology confirmed significantly abrogated fibrosis by kaempferol and esculetin in vivo. We developed an integrative computational framework to identify kaempferol and esculetin as putatively novel therapies for IF/TA and provided experimental evidence for their therapeutic activities in vitro and in vivo using preclinical models. The findings suggest that both drugs might serve as therapeutic options for IF/TA.

Biopsy transcriptome expression profiling to identify kidney transplants at risk of chronic injury: a multicentre, prospective study

Chronic injury in kidney transplants remains a major cause of allograft loss. The aim of this study was to identify a gene set capable of predicting renal allografts at risk of progressive injury due to fibrosis. This Genomics of Chronic Allograft Rejection (GoCAR) study is a prospective, multicentre study. We prospectively collected biopsies from renal allograft recipients (n=204) with stable renal function 3 months after transplantation. We used microarray analysis to investigate gene expression in 159 of these tissue samples. We aimed to identify genes that correlated with the Chronic Allograft Damage Index (CADI) score at 12 months, but not fibrosis at the time of the biopsy. We applied a penalised regression model in combination with permutation-based approach to derive an optimal gene set to predict allograft fibrosis. The GoCAR study is registered with ClinicalTrials.gov, number NCT00611702. We identified a set of 13 genes that was independently predictive for the development of fibrosis at 1 year (ie, CADI-12 ≥2). The gene set had high predictive capacity (area under the curve [AUC] 0·967), which was superior to that of baseline clinical variables (AUC 0·706) and clinical and pathological variables (AUC 0·806). Furthermore routine pathological variables were unable to identify which histologically normal allografts would progress to fibrosis (AUC 0·754), whereas the predictive gene set accurately discriminated between transplants at high and low risk of progression (AUC 0·916). The 13 genes also accurately predicted early allograft loss (AUC 0·842 at 2 years and 0·844 at 3 years). We validated the predictive value of this gene set in an independent cohort from the GoCAR study (n=45, AUC 0·866) and two independent, publically available expression datasets (n=282, AUC 0·831 and n=24, AUC 0·972). Our results suggest that this set of 13 genes could be used to identify kidney transplant recipients at risk of allograft loss before the development of irreversible damage, thus allowing therapy to be modified to prevent progression to fibrosis. National Institutes of Health.

Acute Renal Failure - A Serious Complication in Patients After Kidney Transplantation.

Free radical-mediated injury releases proinflammatory cytokines and activates innate immunity. It has been suggested that the early innate response and the ischemic tissue damage play roles in the development of adaptive responses, which may lead to acute kidney rejection. Various durations of hypothermic kidney storage before transplantation add to ischemic tissue damage. The final stage of ischemic injury occurs during reperfusion that develops hours or days after the initial insult. Repair and regeneration processes occur together with cellular apoptosis, autophagy and necrosis and a favorable outcome is expected if regeneration prevails. Along the entire transplantation time course, there is a great demand for novel immune and nonimmune injury biomarkers. The use of these markers can be of great help in the monitoring of kidney injury in potential kidney donors, where acute kidney damage can be overlooked, in predicting acute transplant dysfunction during the early post-transplant periods, or in predicting chronic changes in long term followup. Numerous investigations have demonstrated that biomarkers that have the highest predictive value in acute kidney injury include NGAL, Cystatin C, KIM-1, IL-18, and L-FABP. Most investigations show that the ideal biomarker to fulfill all the needs in renal transplant has not been identified yet. Although, in many animal models, new biomarkers are emerging for predicting acute and chronic allograft damage, in human allograft analysis they are still not routinely accepted and renal biopsy still remains the gold standard.

Use of Contrast-Enhanced Ultrasonography to Evaluate Chronic Allograft Nephropathy in Rats and Correlations between Time-Intensity Curve Parameters and Allograft Fibrosis

This study quantitatively analyzed changes in the hemodynamic characteristics of renal allografts at different stages in a rat chronic allograft nephropathy (CAN) model as well as the relationship between hemodynamic parameters and renal allograft fibrosis using contrast-enhanced ultrasonography (CEUS). The experimental group used a CAN rat model (n = 30), and the control group used an orthotopic syngeneic renal transplant model (n = 30). After surgery, creatinine clearance rates were regularly monitored every 2 wk. The checking times were set at 4, 12 and 24 wk after surgery, which represent early, middle and late stage of CAN, respectively. At different stages of CAN, eight rats from each group were randomly selected for CEUS examination. Time-intensity curve (TIC) parameters, including rise time, peak intensity, mean transit time, area under the curve, wash-in slope, time-to-peak and α-smooth muscle actin (α-SMA) expression; Vimentin expression; and chronic allograft damage index scores were evaluated by linear correlation analysis. Before the creatinine clearance rate showed significant abnormalities, the renal allografts in the experimental group had already presented pathologic changes associated with CAN. In the early stage after surgery, compared to the TIC curve of the control group, the experimental group showed increased rise time, mean transit time, area under the curve and time-to-peak, and decreased wash-in slope (p < 0.05). Chronic allograft damage index scores and the expression levels of α-SMA and Vimentin proteins in renal allografts were correlated with TIC parameters (p < 0.05). Compared to creatinine clearance rate, CEUS can detect CAN at earlier stages. The correlations between TIC-related parameters and the expression levels of α-SMA and Vimentin in renal allografts indicate that CEUS is a feasible way to assess the degree of renal allograft fibrosis quantitatively.

CXC Chemokine Receptor 4 (CXCR4) Antagonist, a Novel Pathway to Prevent Chronic Allograft Nephropathy.

BACKGROUND Chronic allograft nephropathy (CAN) remains a major problem for long-term graft survival and different pathways participate in its development. CXC chemokine receptor 4 (CXCR4) is significantly upregulated following renal injury and fibrotic response. We investigated the effect of AMD3100, a CXCR4 antagonist, on the development of CAN in rat models. MATERIAL AND METHODS CAN rat models (n=20) were established using male Fisher 344 to Lewis rats. Rats in the experimental group (n=10) were treated with AMD3100 (1 mg/kg/day subcutaneously, 0-12 weeks), rats in the control group (n=10) were treated with saline. The serum creatinine levels were monitored every week. Kidney grafts were harvested 12 weeks after modeling for histological analysis. We used chronic allograft damage index (CADI) scores to evaluate each group. Q-PCR and Western blotting were used to measure CXCR4, TGF-β1/Smad3 signaling pathway and α-smooth muscle actin (α-SMA) expression in renal allograft tissue. RESULTS CXCR4 expression was increased significantly in the control group which developed intense chronic changes after 12 weeks. Histological changes of CAN in the experimental group were ameliorated by AMD3100 which also had better graft function compare to the control group. AMD3100 significantly blunted the increase in the mRNA expression level of CXCR4, TGF-β1/Smad3, and α-SMA. A significant reduction in TGF-β1 and α-SMA protein content was observed only in the experimental group as shown in a representative Western blot. CONCLUSIONS Based on these findings, CXCR4 expression may mediate in part the development of CAN. AMD3100 may ameliorate histological changes of CAN and maintain better allograft function. It blunts downstream effects of TGF-β1 signaling and fibroblast activation. Therefore, antagonism of CXCR4 may provide a novel way to prevent the development of CAN.

Intrarenal B Cell Cytokines Promote Transplant Fibrosis and Tubular Atrophy.

Renal transplantation is the optimum treatment for end-stage renal failure. B cells have been identified in chronic allograft damage (CAD) and associated with the development of tertiary lymphoid tissue within the human renal allograft. We performed renal transplantation in mice to model CAD and identified B cells forming tertiary lymphoid tissue with germinal centers. Intra-allograft B220(+) B cells comprised of IgM(high) CD23(-) B cells, IgM(lo) CD23(+) B cells, and IgM(lo) CD23(-) B cells with elevated expression of CD86. Depletion of B cells with anti-CD20 was associated with an improvement in CAD but only when administered after transplantation and not before. Isolated intra-allograft B cells were cultured and shown to synthesize multiple cytokines, the most abundant of these were GRO-α (CXCL1), RANTES (CCL5), IL-6 and MCP-1 (CCL2). Tubular loss was observed with T cell accumulation within the allograft and development of interstitial fibrosis, whilst type III collagen deposition was observed in areas of F4/80(+) macrophages and PDGFR-β(+) and transgelin(+) fibroblasts, all of which were reduced by B cell depletion. We have shown that intra-allograft B cells are key mediators of CAD. B cells possibly contribute to CAD by intra-allograft secretion of cytokines and chemokines.

Nanoparticle enhanced MRI scanning to detect cellular inflammation in experimental chronic renal allograft rejection.

Objectives. We investigated whether ultrasmall paramagnetic particles of iron oxide- (USPIO-) enhanced magnetic resonance imaging (MRI) can detect experimental chronic allograft damage in a murine renal allograft model. Materials and Methods. Two cohorts of mice underwent renal transplantation with either a syngeneic isograft or allograft kidney. MRI scanning was performed prior to and 48 hours after USPIO infusion using T2∗-weighted protocols. R2∗ values were calculated to indicate the degree of USPIO uptake. Native kidneys and skeletal muscle were imaged as reference tissues and renal explants analysed by histology and electron microscopy. Results. R2∗ values in the allograft group were higher compared to the isograft group when indexed to native kidney (median 1.24 (interquartile range: 1.12 to 1.36) versus 0.96 (0.92 to 1.04), P < 0.01). R2∗ values were also higher in the allograft transplant when indexed to skeletal muscle (6.24 (5.63 to 13.51)) compared to native kidney (2.91 (1.11 to 6.46) P < 0.05). Increased R2∗ signal in kidney allograft was associated with macrophage and iron staining on histology. USPIO were identified within tissue resident macrophages on electron microscopy. Conclusion. USPIO-enhanced MRI identifies macrophage.

Identification of a Molecular Signature to Predict the Progression of Kidney Fibrosis Post Transplantation.

Chronic injury in kidney transplants remains a major cause of graft loss. The aim of the study was to identify a predictive gene set capable of classifying renal grafts at risk for progressive injury due to fibrosis. The Genomics of Chronic Allograft Rejection (GoCAR) study is a prospective, multicenter study. Biopsies obtained prospectively from renal allograft recipients (n=159) with stable renal function 3 months after transplantation were analyzed for gene expression by microarray. Genes correlating with 12-month Chronic Allograft Damage Index (CADI) but not 3 month CADI were identified, and analysis performed on 101 recipients with 12-month CADI to determine the predictive capacity of this gene set relative to current clinical and histopathology parameters. We identified a 13 gene set that was independently predictive for the development of chronic allograft damage at 1 year. The high predictive capacity of the gene set (0.947) was superior to clinical indicators (AUC 0.73). While histological parameters failed to discriminate between grafts in which the CADI progressed from non-progressors, the predictive gene set accurately differentiated between these biopsies (AUC=0.987). Testing on an independent cohort within GoCAR (n=45) by qPCR and three independent publically available microarray expression gene sets validated our gene sets ability to predict renal allograft injury. A gene set obtained at 3 months from stably functioning renal allografts correlated with the progression of established markers for chronic allograft damage at 12 months and proved superior to clinico-pathological variables currently used in clinical practice to in identifying renal transplant recipients at risk of graft loss.

Proteomic Analysis Identified Urine Proteins Predictive of Chronic Allograft Injury.

Aim: To correlate early urine protein biomarkers with long term kidney transplant histology and function; to assess biomarker potential to predict subsequent allograft dysfunction and chronic interstitial fibrosis. Method: Patient cohort was selected from a single center within the GoCAR study who had urine samples at 3 and 12 months and protocol biopsies at 0, 3, and 12 months post transplant. Patients were grouped by chronic damage scores using Banff and Chronic Allograft Damage Index (CADI) from 12 month biopsies or change in estimated glomerular filtration rate (eGFR). Urine proteins at 3 and 12 months associated with chronic damage at 12 months were identified by two-dimensional differential in gel electrophoresis (2D-DIGE) and tandem mass spectrometry. Differential protein abundance was calculated using DeCyder software and further interrogated using the STRING network program. Results: Delayed graft function occurred in 25% and biopsy proven acute rejection in 17% of patients. Acute and chronic damage scores were low at 3 months. Proteinuria or eGFR were not significantly different between chronic damage analysis groups. From the 3 month urine, 9 proteins including kininogen, MASP2 and cystatin M correlated to fibrosis at 12 months (5 with Banff, 4 with CADI) and 2 proteins correlated with change in eGFR. The top KEGG pathway in the STRING network were coagulation and complement cascades. Follow up 12 month urine analysis based on the same chronic damage groups revealed 11 proteins, 4 identified in the previous 3 month analysis and 7 new proteins, including CD14 and β2 microglobulin. The top KEGG pathway at 12 months was antigen processing and presentation. Conclusion: Using traditional proteomic techniques, we have identified potential non-invasive biomarkers that are predictive of chronic allograft damage. The dominant urine proteome alters between 3 and 12 months expressing a different panel of proteins. While early urine protein signatures predict subsequent damage, validation in larger and more diverse cohorts is required.

Early Subclinical Alloantibody-Associated Injury in Positive Crossmatch Kidney Transplantation (+XMKTx) Despite Terminal Complement Blockade.

Introduction. Terminal complement blockade with Eculizumab (EC) prevents early clinical antibody-mediated rejection (AMR) in +XMKTx. The aim of this study was to investigate subclinical microvascular inflammation and endothelial cell activation early after +XMKTx. Methods. +XMKTx recipients (baseline B flow XM 200-450) received: 1) plasma exchange (PE) alone (N=48); or 2) PE + post-transplant EC (N=30). EC was continued for at least 1 month or until the BFXM was <200 (11 patients received EC for >3 months). A -XMKTx control group was age and sex matched. Protocol biopsy specimens were analyzed using light (LM) and electron microscopy (EM). Intragraft mRNA expression was assessed using Illumina Ht12v4 microarrays. Gene set scores were calculated using the mean Log2 value of all detectable transcripts. Results: AMR in the first 3 months was lower in the EC group than the PE-alone group (6.7% vs. 43.8%, p<0.01). During EC treatment, well-functioning +XMKTx showed LM evidence of microvascular inflammation (PTC score >1) at 1 month (10%) and 3 months (26.7%). Gene expression analysis demonstrated microvascular inflammation not detected by LM at these time points (T-cell, macrophage, NK-cell and activated dendritic cell transcripts). Inflammatory transcripts were similar in the EC and PE-alone groups and were generally absent in -XMKTx (Figure 1). Paired biopsy specimens (7-10 days vs 3 months) showed stable inflammatory gene expression in both EC and PE-only groups suggesting that EC had little impact on inflammation. Reactive endothelial cells were identified by EM (28% at 1 month and 58% at 3 months), but generally were absent on LM.Figure: No Caption available.Conclusions: While EC decreases early clinical AMR, evidence of subclinical antibody-associated injury is pervasive early after +XMKTx and LM underestimates this condition. These data suggest that microvascular inflammation and endothelial cell activation are independent of C5 and that additional therapies likely will be needed to prevent chronic antibody-associated allograft damage. DISCLOSURE:Stegall, M.: Grant/Research Support, Alexion Pharmaceuticals.

Role of Carbon Monoxide Releasing Molecules(CORMs) in Toll-Like Receptor Mediated Immune Responses in Renal Transplantation.

Introduction: In spite of recent advances in early renal transplant survival, chronic allograft damage remains a serious obstacle to the long term success of kidney transplantation. Dendritic cells (DCs) and macrophages play a crucial role in both innate and adaptive immune responses provoked by Toll-like receptors (TLRs) that lead to sterile inflammation in ischemia-reperfusion injury. Based on our novel findings of the anti-inflammatory action of CORMs, we studied TLR mediated immune activation in allograft damage to unveil possible TLR/CORMs based therapeutic intervention in renal transplantation. Method: TLR expression in DCs, Macrophage, and human monocytes by RT-PCR, Western Blot and immunostaining. TLR manipulation by over-expressing dominant negative form of specific TLR; blocking by inhibitory peptides; knocking down by siRNA, shRNA; Inhibition with TLR antagonist or inhibitors; Stimulation of TLR with natural ligands or DAMPs; Treatment of cells with CORM401 & inactive CORM401. Detection of IL6, TNFα, IL10, IL12 etc by ELISA. Characterization of the DCs by TLR4, 9, MHCclassII, CD83, CD80, CD86, IL12, IL10 by flow cytometry. Result: When CORM-3 was infused directly into the donor kidney, an improvement in survival, function and histology vs. controls (p<0.05) was observed. In addition, Brown Norway rats were pre-treated with CORM-2 6hr prior to procurement and transplantation into allogeneic Lewis rats. Histologic examination (day10) showed lymphocytic infiltrate, glomerular congestion, and acute tubular necrosis in control animals whereas CORM-2 treated animals had completely normal histology. TLRs were differentially expressed by bone marrow DCs, macrophages and human monocyte-derived cells. Gene expression analysis of 43,000 mRNAs demonstrated that CORM-401 modulated many genes which include markers of DC maturation (CD80, CD83, CD86), diverse immune responses (TLR9, NFkB, TNFa, IL6, IL1b, IL10) and pro-to-anti-inflammatory conversion (Regnase1, TRIB1). Random Forest algorithm analysis on immune functions also concluded wide spectrum of intervention of CORM-401 from immune activation to leukocyte activation.Conclusion: TLRs on DCs and macrophages promote inflammation subsequent to kidney IRI, the leading cause of rejection. CORMs have potential to protect kidney allograft function and survival.

Calcineurin Free Protocol Is Associated With Increase in FOXP3 Expression in the Peripheral Blood of Recipients of Extended Criteria Donor Kidneys.

Background: The significance of the factor forkhead box (FOXP3) expression in the peripheral blood of renal transplant recipients (RTx) of extended (ECD) or standard (SCD) criteria donor kidneys is yet to be determined. Aim: To investigate the clinical significance of FOXP3 level in relation to the type of kidney donor, immunosuppressive protocol (ISS) and graft function in stable ECD RTx. Methods: Prospective, open label study with 36 stable RTx randomized according to the type of donor to receive tacrolimus (TAC; n= 19) or everolimus (EVL; n=17) based protocols combined to mycophenolate sodium, prednisone and induction therapy with basiliximab. Using qRT-PCR we measured FOXP3 expression in the peripheral blood of patients (pts) at the 3rd month after Tx. Results: Greater expression of FOXP3 was observed in recipients of ECD when compared to SCD (ECD=1.98 ± 2.8 vs SCD= 0.63 ± 0.71; p=0.02). EVL based immunosuppression (EVL= 1.49 ± 2.1 vs TAC= 0.51 ± 0.57; p=0.03) and absence of DGF post-Tx (DGF- = 1.94 ± 2.5 vs DGF+ = 0.54 ± 0.6; p=0.014) were also associated with high levels of FOXP3 expression. Cold ischemia time, acute rejection episodes and serum creatinine (sCr) at 3rd month were not associated with changes in FOXP3 expression. Multivariate logistic regression analysis showed that ECD (p= 0.025) absence of DGF (0.015) were independently associated with high levels of FOXP3. Correlation between sCr and FOXP3 was inverse in TAC treated pts and positive in those taking EVL (r= 0.74, P = 0.007). FOXP3 expression was low in kidneys with mild or no chronic lesions (IFTA score < 2) regardless the ISS. Patients treated with EVL presenting IFTA score ≥ 2 had increased expression of FOXP3 (p=NS vs TAC). Conclusions: Results suggest that recipients of ECD kidneys without having undergone DGF, treated with EVL presented an increased expression of FOXP3 in their peripheral blood. The higher FOXP3 levels associated with more chronic histological lesions may represent an anti-inflammatory response elicited by ongoing chronic allograft damage. DISCLOSURES:Abbud-Filho, M.: Grant/Research Support, Novartis. Dias, C.: Grant/Research Support, Novartis. Caldas, H.: Grant/Research Support, Novartis. Nunes, R.: Grant/Research Support, Novartis. Mazeti, C.: Grant/Research Support, Novartis. Fernandes-Charpiot, I.: Grant/Research Support, Novartis. Baptista, M.: Grant/Research Support, Novartis. Abbud-Filho, M.: Grant/Research Support, Novartis.

Pathophysiology and treatment options of chronic renal allograft damage

Chronic rejection is a poorly understood entity albeit a frequent cause of graft failure. Despite the advent of new immunosuppressive agents, neither the slope of graft destruction nor the frequency is ameliorated. There are a number of hypothesis which try to explain the conundrum of chronic graft destruction: ongoing rejection, antibody-mediated rejection, poor choice of organs, hyperfiltration, calcineurin inhibitors (CNI) nephrotoxicity and non-compliance among them. None of these hypotheses can explain all features of the process, thus, it is likely that they act in combination. What seems to be clear is a beneficial effect of early angiotensin-converting enzyme (ACE)/AT1 blocker treatment. It is less clear, however, whether a reduction or a switch from CNIs to other immunosuppressants prolongs graft survival. This review highlights the pathophysiological aspects that are important for the development of chronic allograft damage in the context of possible treatment options.

Investigation of the role of B lymphocytes and tertiary lymphoid tissue in a murine model of renal chronic allograft damage

BackgroundNodular B-lymphocyte rich infiltrates have been identified in chronically rejected renal allografts and biopsies of acute transplant rejection, and this has been associated with the development of tertiary lymphoid tissue (TLT). However their significance is unclear, with conflicting published data. We aim to investigate the role and significance of B-lymphocyte infiltrates and the development of TLT in a murine model of renal chronic allograft damage. MethodsWe used congenic strains with donor C57bl/6-BM12 mice kidneys transplanted into C57bl/6 recipients, this being a single MHC-II mismatch. Using immunohistochemistry we investigated the presence of B lymphocytes within the allograft. In addition we investigated other markers of chronic allograft damage in this model including lymphatic expansion, microvessel rarefication, and fibrosis. FindingsNodular aggregates of B cells appearing to be TLT developed over 12 weeks; however, in some allografts we observed a scattered B-cell pattern. The B-cell infiltrate of the allograft cortex increased progressively with a significant increased density by 12 weeks compared with 5 days after transplantation. Microvessels were counted with a 25-point graticule, and there was a significant difference between allograft and native kidney cortex (p<0·01) with both time and transplant kidney being responsible for the effect. There was a significant difference in the number of lymphatic vessels at 12 and 8 weeks compared with 5 days (p<0·05). Similar to findings in chronic allograft nephropathy, the expanded lymphatics were seen both in the tubulointerstitium and in the perivascular regions. The B-lymphocyte phenotype was explored and shown by immunofluoresence to form germinal centres and IgG-positive plasma cell (CD138+) differentiation. InterpretationWe have shown that this strain combination closely models that of chronic rejection of the renal allograft. Furthermore, we have identified the progressive infiltration and expansion of the B-lymphocytes compartment within the allograft cortex. This work has provided the basis for further investigation of B-lymphocyte depletion and the prospects of identifying a regulatory B lymphocyte. FundingKidney Research UK.

Editorial introductions

Editorial introductions

Immune mechanisms of ischemia-reperfusion injury in transplantation

All allografts suffer a number of unavoidable ischemic insults. These, starting with brain death and ending with reperfusion, are very troublesome, as ischemia-reperfusion injury (IRI) is demonstrated to be a major cause of allograft damage in various types of transplantations. To counter the threat this poses to allograft function, investigators have worked diligently over the past decades in clinical settings and in the laboratory to understand the pathophysiology and immune mechanism underlying IRI hoping to ultimately devise strategies that lessen its detrimental effects on allografts. Herein, we review the major immune components of the IRI dynamic process. Better understanding of the cellular pathophysiological processes underlying IRI will hopefully result in the design of more targeted therapies to prevent the injury, hasten repair, and minimize chronic progressive allograft damage.