Detection of C3d-binding donor-specific anti-HLA antibodies at diagnosis of humoral rejection predicts renal graft loss.

Antibody-mediated rejection (AMR) is a major cause of kidney graft loss, yet assessment of individual risk at diagnosis is impeded by the lack of a reliable prognosis assay. Here, we tested whether the capacity of anti-HLA antibodies to bind complement components allows accurate risk stratification at the time of AMR diagnosis. Among 938 kidney transplant recipients for whom a graft biopsy was performed between 2004 and 2012 at the Lyon University Hospitals, 69 fulfilled the diagnosis criteria for AMR and were enrolled. Sera banked at the time of the biopsy were screened for the presence of donor-specific anti-HLA antibodies (DSAs) and their ability to bind C1q and C3d using flow bead assays. In contrast with C4d graft deposition, the presence of C3d-binding DSA was associated with a higher risk of graft loss (P<0.001). Despite similar trend, the difference did not reach significance with a C1q-binding assay (P=0.06). The prognostic value of a C3d-binding assay was further confirmed in an independent cohort of 39 patients with AMR (P=0.04). Patients with C3d-binding antibodies had worse eGFR and higher DSA mean fluorescence intensity. In a multivariate analysis, only eGFR <30 ml/min per 1.73 m(2) (hazard ratio [HR], 3.56; 95% confidence interval [CI], 1.46 to 8.70; P=0.005) and the presence of circulating C3d-binding DSA (HR, 2.80; 95% CI, 1.12 to 6.95; P=0.03) were independent predictors for allograft loss at AMR diagnosis. We conclude that assessment of the C3d-binding capacity of DSA at the time of AMR diagnosis allows for identification of patients at risk for allograft loss.

Comparison of a Bayesian network with a logistic regression model to forecast IgA nephropathy.

Models are increasingly used in clinical practice to improve the accuracy of diagnosis. The aim of our work was to compare a Bayesian network to logistic regression to forecast IgA nephropathy (IgAN) from simple clinical and biological criteria. Retrospectively, we pooled the results of all biopsies (n = 155) performed by nephrologists in a specialist clinical facility between 2002 and 2009. Two groups were constituted at random. The first subgroup was used to determine the parameters of the models adjusted to data by logistic regression or Bayesian network, and the second was used to compare the performances of the models using receiver operating characteristics (ROC) curves. IgAN was found (on pathology) in 44 patients. Areas under the ROC curves provided by both methods were highly significant but not different from each other. Based on the highest Youden indices, sensitivity reached (100% versus 67%) and specificity (73% versus 95%) using the Bayesian network and logistic regression, respectively. A Bayesian network is at least as efficient as logistic regression to estimate the probability of a patient suffering IgAN, using simple clinical and biological data obtained during consultation.

Long-term kidney disease outcomes in fibrillary glomerulonephritis: a case series of 27 patients.

BACKGROUND: Fibrillary glomerulonephritis (GN) is a rare disorder with poor renal prognosis. Therapeutic strategies, particularly the use of immunosuppressive drugs, are debated. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 27 adults with fibrillary GN referred to 15 nephrology departments in France between 1990 and 2011 were included. All patients were given renin-angiotensin system blockers and 13 received immunosuppressive therapy, including rituximab (7 patients) and cyclophosphamide (3 patients). OUTCOMES & MEASUREMENTS: Clinical and histologic features of patients and kidney disease outcome. Renal response was defined as a >50% decrease in 24-hour proteinuria with <15% decline in estimated glomerular filtration rate (eGFR). RESULTS: All patients presented with proteinuria, associated with nephrotic syndrome (41%), hematuria (73%), and hypertension (70%). Baseline median eGFR was 49 mL/min/1.73 m(2). Eight patients had a history of autoimmune disease and none had evidence of hematologic malignancy during follow-up. Light microscopic studies showed mesangial GN (70%), predominant pattern of membranous GN (19%), or membranoproliferative GN (11%). By immunofluorescence, immunoglobulin G (IgG) deposits (IgG4, 15/15; IgG1, 9/15) were polyclonal in 25 cases. Serum IgG subclass distribution was normal in the 6 patients tested. After a median 46-month follow-up, renal response occurred in 6 of 13 patients who received immunosuppressive therapy with rituximab (5 patients) or cyclophosphamide (1 patient). Of these, 5 had a mesangial or membranous light microscopic pattern, and median eGFR before therapy was 76 mL/min/1.73 m(2). In contrast, chronic kidney disease progressed in 12 of 14 patients who were not given immunosuppressive therapy, 10 of whom reached end-stage renal disease. LIMITATIONS: Number of patients, retrospective study, use of multiple immunosuppressive regimens. CONCLUSIONS: The therapeutic approach in fibrillary GN remains challenging. The place of immunosuppressive therapy, particularly anti-B-cell agents, needs to be assessed in larger collaborative studies.

Intragraft Th17 infiltrate promotes lymphoid neogenesis and hastens clinical chronic rejection.

To evaluate the influence of intragraft inflammatory infiltrate on the course of chronic rejection, 11 human renal grafts, detransplanted for terminal failure, were analyzed. Samples were divided into two groups according to their graft survival (> or < or = 8 y). In both groups, the main cell population infiltrating the graft interstitia was T lymphocytes. The extent of the lymphocytic infiltration and the distribution of naive and memory, CD4(+) and CD8(+) T cells, were similar in both groups. Although all types of Th polarization profiles can lead to terminal chronic rejection, a correlation between shorter graft survival and the presence of Th17 cells that produce IL-17 and IL-21 was observed. In contrast, grafts infiltrated by regulatory T cells survived significantly longer. The correlation between the expressions of activation-induced cytidine deaminase (the key enzyme of the germinal center reaction) and IL-21 suggests that Th17 could exert their deleterious effect by promoting lymphoid neogenesis, namely, the organization of inflammatory effectors into ectopic germinal centers in which a local humoral immune response is elicited. Further studies will determine whether Th17 infiltration can be used as a prognosis tool and whether the Th17 subset constitutes a therapeutic target for slowing down chronic rejection.

Chronic humoral rejection mediated by anti-HLA-DP alloantibodies: insights into the role of epitope sharing in donor-specific and non-donor specific alloantibodies generation.

We report the case of a renal transplanted patient, in whom the detection of a unique anti HLA-DP antibody response preceded the development of chronic humoral rejection. In addition to donor-specific anti-DP alloantibodies, the patient displayed reactions against several non-donor-specific DP antigens (NDSA). Interestingly, we found that all the DP molecules recognized by the alloantibodies displayed the same amino-acid sequence suggesting that epitope sharing between unrelated HLA molecules was the mechanism underlying NDSA generation. This case highlights the pathogenicity of anti-DP alloantibodies and suggests that it could be more meaningful to match the epitopes than the HLA antigens for the prevention of rejection.

[Lymphoid neogenesis and lymphangiogenesis: two newcomers in the pathophysiology of chronic rejection]. / Néogenèse lymphoïde et lymphangiogenèse: deux nouveaux mécanismes impliqués dans la physiopathologie du rejet chronique.

Chronic rejection is one of the main causes of late allograft failure and no therapy is currently available to prevent efficiently its development. Improving the comprehension of the mechanisms involved in the pathophysiology of chronic rejection is a mandatory step to propose innovative therapies that would prolong grafts' survival. Using the rat aortic interposition model of chronic vascular rejection, we have demonstrated that the intragraft inflammatory infiltrate progressively organized itself into a functional ectopic lymphoid tissue (tertiary lymphoid organ) supporting the local synthesis of alloantibody. Thus, during chronic rejection the graft is at the same time the target and the site of elaboration of the humoral allo-immune response. This hypothesis has been confirmed in the clinical setting by the analysis of human grafts (kidneys, hearts and lungs) removed for terminal failure due to chronic rejection. This lymphoid neogenesis process, previously identified in other chronic inflammatory diseases, occurs with a strikingly high frequency in chronically rejected grafts, suggesting that an additional mechanism synergizes to initiate the development of tertiary lymphoid organs during chronic rejection. We propose that the defective lymphatic drainage of chronically rejected organs triggers lymphoid neogenesis and we discuss the complex crosstalk between lymphoid neogenesis and lymphangiogenesis that takes place during chronic rejection.

Absence of c4d deposition in human composite tissue (hands and face) allograft biopsies: an immunoperoxidase study.

Deposition of the C4d complement degradation product has been shown to be a marker of antibody-mediated rejection of solid organ allografts, including kidney, heart, liver, and lung. We investigated whether C4d deposition also would be useful in monitoring rejection in human composite tissue allografts. A total of 60 mucocutaneous formalin-fixed, paraffin-embedded and four frozen biopsy specimens from four patients with composite tissue allografts (three hands, one face) taken during a period of 7 days to 7 years after graft were immunostained for C4d by an immunoperoxidase and an immunofluorescence technique, respectively. C4d deposition was not found in any of the specimens studied, even though several of them showed pathological signs of rejection. No human leukocyte antigen alloantibodies were detected in any of the patients during the study period. These results suggest that humoral rejection occurs rarely, if at all, in the setting of human composite tissue allografts.

Expression of VE-Cadherin in Peritubular Endothelial Cells during Acute Rejection after Human Renal Transplantation.

Genes involved in acute rejection (AR) after organ transplantation remain to be further elucidated. In a previous work we have demonstrated the under-expression of VE-Cadherin by endothelial cells (EC) in AR following murine and human heart transplantation. Serial sections from 15 human kidney Banff-graded transplant biopsies were examined for the presence of VE-Cadherin and CD34 staining by immunohistochemistry (no AR (n = 5), AR grade IA (n = 5), or AR grade IIA (n = 5)). Quantification of peritubular EC staining were evaluated and results were expressed by the percentage of stained cells per surface analysed. There was no difference in CD34 staining between the 3 groups. VE-Cadherin expression was significantly reduced in AR Grade IIA when compared to no AR (P = .01) and to AR grade IA (P = .02). This study demonstrates a reduced VE-Cadherin expression by EC in AR after renal transplantation. The down-regulation of VE-Cadherin may strongly participate in human AR.

[Evaluation of IGL-1, a new organ preservation solution: preclinical results in renal transplantation]. / Evaluation de IGL-1, une nouvelle solution de conservation d'organes: résultats pré-cliniques en transplantation rénale.

INTRODUCTION: Renal ischaemia and reperfusion lesions partly determine short-term and long-term graft survival. Organ preservation conditions appear to play a decisive role. This article presents the preclinical experimental results obtained in renal transplantation with an extracellular organ preservation solution, in which polyethylene glycol (PEG) is used as colloid. METHODS AND RESULTS: The effects of inversion of Na+ and K+ gradients in the IGL-1 preservation solution compared to UW and replacement of hydroxylethyl starch (HES) by PEG were evaluated in an ex vivo isolated, perfused rat kidney model and then in a pig renal autotransplantation model. In these experimental models, after 24 hours of static storage, the sodium reabsorption fraction correlated with the quality of tubular function of the kidney and the glomerular filtration rate were constantly better in the IGL-1 group than in the UW group. In vivo, in the pig, resumption of renal function was significantly better in the IGL-1 group and histological examination demonstrated a significant reduction of expression of Major Histocompatibility Complex (MHC) type II, an indirect marker of inflammation, but also a reduction of markers of apoptosis and fibrosis for kidneys preserved in IGL-1. CONCLUSION: In animal renal transplantation, IGL-1 ensures better resumption of renal function than UW, which currently remains the "gold standard"preservation solution. Further studies must be conducted to determine whether this new generation solution can replace UW as the reference solution.

Effect of IGL-1, a new preservation solution, on kidney grafts (a pre-clinical study).

Ischemia-reperfusion injury conditions short-term and long-term graft function. The effects of the inversion of K+ and Na+ concentrations and substitution with polyethylene glycol for hydroxyethyl starch in University of Wisconsin (K-UW) solution were evaluated in isolated perfused rat kidneys and in autotransplanted pig kidneys. In the rat model kidneys were cold-stored for 24 h in K-UW or Na-UW or Na-PEG UW solutions (IGL-1 solution). Fractional sodium reabsorption and glomerular filtration rate was better in kidneys preserved in Na-UW and IGL-1 solution than those preserved in K-UW solution. In the pig model the left kidney was harvested and preserved in K-UW or IGL-1 solution for 24 h and then transplanted. In the autotransplanted pig model, kidneys preserved in IGL-1 solution showed a better function and a significant reduction of MHC class II expression, cellular apoptosis and interstitial fibrosis. In conclusion, kidneys preserved in IGL-1 solution tolerated ischemia/reperfusion injury better than those preserved in K-UW solution.

Ischemia induces early expression of a new transcription factor (6A3-5) in kidney vascular smooth muscle cells: studies in rat and human renal pathology.

Acute renal failure, characterized by rapid decline in glomerular filtration rate, is a major cause of morbidity and mortality. During the evolution of renal diseases chronic ischemia develops. Indeed, acute or chronic renal failure may occur as a result of renal ischemia, which induces cells to dedifferentiate, proliferate, or become apoptotic. In this study, we have investigated the expression of a newly identified transcription factor, 6A3-5, under in vitro and in vivo conditions. Proliferating vascular smooth muscle were investigated in response to different mitogenic agents. The 6A3-5 expression was then studied in ischemic rat kidney, induced by renal pedicle clamping, followed, or not, by reperfusion. Subsequently human renal biopsies from early kidney grafts and chronic renal diseases were also investigated for 6A3-5 protein expression by immunohistochemistry. In vitro study shows an over-expression of 6A3-5 following 2 to 4 hours stimulation by serum or Angiotensin II, of rat proliferating aortic smooth muscle cell. Moreover, in vivo study shows that this new protein is over expressed in rat kidney submitted to 45 minutes ischemia. An anti-6A3-5 antibody shows the protein to be expressed in smooth muscle cells of the arterioles and intermediate size arteries, in mesangial cells and interstitial myofibroblasts. In human biopsies of early kidney grafts and renal disease, the same up-regulation of 6A3-5, as in acute ischemic situation, is observed. This 6A3-5 expression is intimately associated with alpha-smooth muscle cell actin expression in mesangial cells, arteriolar smooth muscle cells as well as interstitial myofibroblasts. Transcription factor 6A3-5 could potentially be a novel early vascular marker of acute and chronic renal ischemic stress implicated in tissue remodeling.

Interstitial expression of alpha-SMA: an early marker of chronic renal allograft dysfunction.

BACKGROUND: Renal myofibroblast infiltration has been shown to be strongly associated with renal function decline in several chronic renal diseases. The purpose of the present study was to investigate whether early detection of myofibroblast infiltration using alpha-smooth-muscle actin (alpha-SMA) expression in time-zero biopsies predicts renal allograft dysfunction. METHODS: We studied renal tissue from 38 renal transplant patients from whom biopsies had been taken after vascular anastomosis during transplantation to ascertain whether myofibroblasts infiltration predicts renal graft survival. Immunohistochemistry was performed on time-zero biopsies to determine alpha-SMA expression, and this was compared to annual glomerular filtration rate (GFR) variation and other parameters including cold ischaemic time (CIT), donor and recipient age, number of acute rejections, and delayed graft function (DGF). GFR was measured by inulin clearance during of 3 years of follow-up after the transplantation. Progressors were defined as patients with an annual GFR decline >5 ml/min/year. RESULTS: We found a significant correlation between interstitial alpha-SMA expression in time-zero biopsies and GFR evolution during the post-transplantation course (r=0.60, P<0.001). Although progressors had greater interstitial alpha-SMA expression than non progressors (7.9+/-0.7 vs 4.3+/-0.4%), they showed only a tendency towards higher glomerular alpha-SMA expression. In addition, progressors had more interstitial fibrosis in time-zero biopsies than non-progressors. There was no relationship between alpha-SMA expression and CIT, donor and recipient ages, number of acute rejections, and occurrence of DGF. CONCLUSION: This study suggests that alpha-SMA evaluation in time-zero biopsies, especially the combination of alpha-SMA expression and interstitial fibrosis, can strongly predict chronic renal allograft dysfunctions.