Necroptosis-related genes allow novel insights into predicting graft loss and diagnosing delayed graft function in renal transplantation.

Ischemia-reperfusion injury (IRI) is an inevitable pathophysiological phenomenon in kidney transplantation. Necroptosis is an undoubtedly important contributing mechanism in renal IRI. We first screened differentially expressed necroptosis-related genes (DENRGs) from public databases. Eight DENRGs were validated by independent datasets and verified by qRT-PCR in a rat IRI model. We used univariate and multivariate Cox regression analyses to establish a prognostic signature, and graft survival analysis was performed. Immune infiltrating landscape analysis and gene set enrichment analysis (GSEA) were performed to understand the underlying mechanisms of graft loss, which suggested that necroptosis may aggravate the immune response, resulting in graft loss. Subsequently, a delayed graft function (DGF) diagnostic signature was constructed using the Least Absolute Shrinkage and Selection Operator (LASSO) and exhibited robust efficacy in validation datasets. After comprehensively analyzing DENRGs during IRI, we successfully constructed a prognostic signature and DGF predictive signature, which may provide clinical insights for kidney transplant.

Identification of renal ischemia reperfusion injury subtypes and predictive strategies for delayed graft function and graft survival based on neutrophil extracellular trap-related genes.

Background: Ischemia reperfusion injury (IRI) is an inevitable process in renal transplantation, which is closely related to serious postoperative complications such as delayed graft function (DGF), acute rejection and graft failure. Neutrophil extracellular traps (NETs) are extracellular DNA structures decorated with various protein substances released by neutrophils under strong signal stimulation. Recently, NETs have been found to play an important role in the process of IRI. This study aimed to comprehensively analyze the expression landscape of NET-related genes (NRGs) during IRI, identify clusters with different degrees of IRI and construct robust DGF and long-term graft survival predictive strategies. Methods: The microarray and RNA-seq datasets were obtained from the GEO database. Differentially expressed NRGs (DE-NRGs) were identified by the differential expression analysis, and the NMF algorithm was used to conduct a cluster analysis of IRI samples. Machine learning algorithms were performed to screen DGF-related hub NRGs, and DGF and long-term graft survival predictive strategies were constructed based on these hub NRGs. Finally, we verified the expression of Cxcl1 and its effect on IRI and NETs generation in the mouse IRI model. Results: This study revealed two IRI clusters (C1 and C2 clusters) with different molecular features and clinical characteristics. Cluster C1 was characterized by active metabolism, mild inflammation and lower incidence of DGF, while Cluster C2 was inflammation activated subtype with a higher incidence of DGF. Besides, based on DGF-related hub NRGs, we successfully constructed robust DGF and long-term graft survival predictive strategies. The mouse renal IRI model verified that Cxcl1 was significantly upregulated in renal tissues after IRI, and using a CXCL8/CXCL1 inhibitor could significantly improve renal function, alleviate renal tubular necrosis, tissue inflammatory response, and NET formation. Conclusion: This study identified two distinct IRI clusters based on DE-NRGs and constructed robust prediction methods for DGF and graft survival, which can provide references for early prevention and individualized treatment of various postoperative complications after renal transplantation.

VAV1 Gene Polymorphism is Associated With Kidney Allograft Rejection.

BACKGROUND: VAV1 is an intracellular signal transduction protein that plays a significant role in signal transduction in T cells. Several studies suggest that VAV1 signaling plays significant roles in allograft rejection. The aim of this study was to examine the association between VAV1 gene polymorphisms and renal allograft function. METHODS: The study included 270 patients after allograft renal transplantation. We examined the associations between VAV1 gene polymorphisms and complications after transplantation, such as delayed graft function, acute rejection, and chronic allograft dysfunction. RESULTS: There were no statistically significant associations between VAV1 genotypes and delayed graft function and chronic allograft dysfunction. Among patients with acute allograft rejection, we observed decreased frequencies of VAV1 rs2546133 TT and CT genotypes (P = .03) and T allele (P = .02), as well as VAV1 rs2617822 GG and AG genotypes (P = .05) and G allele (P = 0.04). In the multivariate regression analysis, the higher number of VAV1 rs2546133 T alleles showed a protective effect against the acute rejection in kidney allograft recipients. CONCLUSIONS: The results of our study suggest that polymorphisms in the VAV1 gene are associated with kidney allograft rejection.

Cytokine absorption during human kidney perfusion reduces delayed graft function-associated inflammatory gene signature.

Transplantation is the optimal treatment for most patients with end-stage kidney disease but organ shortage is a major challenge. Normothermic machine perfusion (NMP) has been used to recondition marginal organs; however, mechanisms by which NMP might benefit organs are not well understood. Using pairs of human kidneys obtained from the same donor, we compared the effect of NMP with that of cold storage on the global kidney transcriptome. We found that cold storage led to a global reduction in gene expression, including inflammatory pathway genes and those required for energy generation processes, such as oxidative phosphorylation (OXPHOS). In contrast, during NMP, there was marked upregulation OXPHOS genes, but also of a number of immune and inflammatory pathway genes. Using biopsies from kidneys undergoing NMP that were subsequently transplanted, we found that higher inflammatory gene expression occurred in organs with prolonged delayed graft function (DGF). Therefore, we used a hemoadsorber (HA) to remove pro-inflammatory cytokines. This attenuated inflammatory gene expression increased OXPHOS pathway genes and had potentially clinically important effects in reducing the expression of a DGF-associated gene signature. Together, our data suggest that adsorption of pro-inflammatory mediators from the perfusate represents a potential intervention which may improve organ viability.

Recent Advances on Biomarkers of Early and Late Kidney Graft Dysfunction.

New biomarkers of early and late graft dysfunction are needed in renal transplant to improve management of complications and prolong graft survival. A wide range of potential diagnostic and prognostic biomarkers, measured in different biological fluids (serum, plasma, urine) and in renal tissues, have been proposed for post-transplant delayed graft function (DGF), acute rejection (AR), and chronic allograft dysfunction (CAD). This review investigates old and new potential biomarkers for each of these clinical domains, seeking to underline their limits and strengths. OMICs technology has allowed identifying many candidate biomarkers, providing diagnostic and prognostic information at very early stages of pathological processes, such as AR. Donor-derived cell-free DNA (ddcfDNA) and extracellular vesicles (EVs) are further promising tools. Although most of these biomarkers still need to be validated in multiple independent cohorts and standardized, they are paving the way for substantial advances, such as the possibility of accurately predicting risk of DGF before graft is implanted, of making a "molecular" diagnosis of subclinical rejection even before histological lesions develop, or of dissecting etiology of CAD. Identification of "immunoquiescent" or even tolerant patients to guide minimization of immunosuppressive therapy is another area of active research. The parallel progress in imaging techniques, bioinformatics, and artificial intelligence (AI) is helping to fully exploit the wealth of information provided by biomarkers, leading to improved disease nosology of old entities such as transplant glomerulopathy. Prospective studies are needed to assess whether introduction of these new sets of biomarkers into clinical practice could actually reduce the need for renal biopsy, integrate traditional tools, and ultimately improve graft survival compared to current management.

Kidney-intrinsic factors determine the severity of ischemia/reperfusion injury in a mouse model of delayed graft function.

Delayed graft function due to transplant ischemia/reperfusion injury adversely affects up to 50% of deceased-donor kidney transplant recipients. However, key factors contributing to the severity of ischemia/reperfusion injury remain unclear. Here, using a clinically relevant mouse model of delayed graft function, we demonstrated that donor genetic background and kidney-intrinsic MyD88/Trif-dependent innate immunity were key determinants of delayed graft function. Functional deterioration of kidney grafts directly corresponded with the duration of cold ischemia time. The graft dysfunction became irreversible after cold ischemia time exceeded six hours. When cold ischemia time reached four hours, kidney grafts displayed histological features reflective of delayed graft function seen in clinical kidney transplantation. Notably, kidneys of B6 mice exhibited significantly more severe histological and functional impairment than kidneys of C3H or BALB/c mice, regardless of recipient strains or alloreactivities. Furthermore, allografts of B6 mice also showed an upregulation of IL-6, neutrophil gelatinase-associated lipocalin, and endoplasmic reticulum stress genes, as well as an increased influx of host neutrophils and memory CD8 T-cells. In contrast, donor MyD88/Trif deficiency inhibited neutrophil influx and decreased the expression of IL-6 and endoplasmic reticulum stress genes, along with improved graft function and prolonged allograft survival. Thus, kidney-intrinsic factors involving genetic characteristics and innate immunity serve as critical determinants of the severity of delayed graft function. This preclinical murine model allows for further investigations of the mechanisms underlying delayed graft function.

Molecular Fingerprints of Borderline Changes in Kidney Allografts Are Influenced by Donor Category.

The fate of transplanted kidneys is substantially influenced by graft quality, with transplantation of kidneys from elderly and expanded criteria donors (ECDs) associated with higher occurrence of delayed graft function, rejection, and inferior long-term outcomes. However, little is known about early molecular fingerprints of these events in different donor categories. Borderline changes represent the most frequent histological finding early after kidney transplantation. Therefore, we examined outcomes and transcriptomic profiles of early-case biopsies diagnosed as borderline changes in different donor categories. In this single-center, retrospective, observational study, we compared midterm outcomes of kidney transplant recipients with early borderline changes as a first pathology between ECD (n = 109), standard criteria donor (SCDs, n = 109), and living donor (LD, n = 51) cohorts. Intragraft gene expression profiling by microarray was performed in part of these ECD, SCD, and LD cohorts. Although 5 year graft survival in patients with borderline changes in early-case biopsies was not influenced by donor category (log-rank P = 0.293), impaired kidney graft function (estimated glomerular filtration rate by Chronic Kidney Disease Epidemiology Collaboration equation) at M3, 1, 2, and 3 years was observed in the ECD cohort (P < 0.001). Graft biopsies from ECD donors had higher vascular intimal fibrosis and arteriolar hyalinosis compared to SCD and LD (P < 0.001), suggesting chronic vascular changes. Increased transcripts typical for ECD, as compared to both LD and SCD, showed enrichment of the inflammatory, defense, and wounding responses and the ECM-receptor interaction pathway. Additionally, increased transcripts in ECD vs. LD showed activation of complement and coagulation and cytokine-cytokine receptor pathways along with platelet activation and cell cycle regulation. Comparative gene expression overlaps of ECD, SCD, and LD using Venn diagrams found 64 up- and 16 down-regulated genes in ECD compared to both LD and SCD. Shared increased transcripts in ECD vs. both SCD and LD included thrombospondin-2 (THBS2), angiopoietin-like 4 (ANGPTL4), collagens (COL6A3, COL1A1), chemokine CCL13, and interleukin IL11, and most significantly, down-regulated transcripts included proline-rich 35 (PRR35) and fibroblast growth factor 9. Early borderline changes in ECD kidney transplantation are characterized by increased regulation of inflammation, extracellular matrix remodeling, and acute kidney injury transcripts in comparison with both LD and SCD grafts.

Changes in double-strand DNA breaks predict delayed graft function (DGF) in Japanese renal allograft recipients.

BACKGROUND: Although delayed graft function (DGF) is a serious complication following kidney transplantation, a reliable and early diagnostic test is lacking to identify the grade of DGF. METHODS: We investigated changes in double-strand DNA breaks (DSBs), and factors related to DGF, such as ischemic times at transplantation and serum creatinine (sCr) levels. DSBs were detected by phospho-histone H2A.X (γ-H2AX) expression and cellular regeneration by Ki-67 before (0 h) and 1 h after allograft reperfusion (1 h) in each subject. RESULTS: The expression of γ-H2AX or Ki-67 at 0 h showed no difference between the living and deceased donors. γ-H2AX at 1 h decreased in the living donors, but increased in the deceased donors compared with that of 0 h(p = 0.017). Changes (Δ) in γ-H2AX between 0 and 1 h were different among subgroups, i.e., immediate function, slow graft function with dialysis < 7 days, DGF with dialysis < 4 weeks, severe DGF with dialysis > 4 weeks, or primary non-function (PNF) (p = 0.04). Severe DGF and PNF cases showed greater increase in Δγ-H2AX (p = 0.019), and were distinguished by > 12% of Δγ-H2AX at 100% sensitivity and 88.2% specificity (ROC analysis, AUC: 0.922, p = 0.023). In a multivariate regression analysis, donor sCr and Δγ-H2AX were two main predictors of the grade of DGF (p = 0.002). The expression of Ki-67 was very low at both 0 h and 1 h. CONCLUSION: The combination of donor sCr and Δγ-H2AX from 0 to 1 h after reperfusion may predict severe DGF and PNF in the early phase.

Endogenous intronic antisense long non-coding RNA, MGAT3-AS1, and kidney transplantation.

ß-1,4-mannosylglycoprotein 4-ß-N-acetylglucosaminyltransferase (MGAT3) is a key molecule for the innate immune system. We tested the hypothesis that intronic antisense long non-coding RNA, MGAT3-AS1, can predict delayed allograft function after kidney transplantation. We prospectively assessed kidney function and MGAT3-AS1 in 129 incident deceased donor kidney transplant recipients before and after transplantation. MGAT3-AS1 levels were measured in mononuclear cells using qRT-PCR. Delayed graft function was defined by at least one dialysis session within 7 days of transplantation. Delayed graft function occurred in 22 out of 129 transplant recipients (17%). Median MGAT3-AS1 after transplantation was significantly lower in patients with delayed graft function compared to patients with immediate graft function (6.5 × 10-6, IQR 3.0 × 10-6 to 8.4 × 10-6; vs. 8.3 × 10-6, IQR 5.0 × 10-6 to 12.8 × 10-6; p < 0.05). The median preoperative MGAT3-AS1 was significantly lower in kidney recipients with delayed graft function (5.1 × 10-6, IQR, 2.4 × 10-6 to 6.8 × 10-6) compared to recipients with immediate graft function (8.9 × 10-6, IQR, 6.8 × 10-6 to 13.4 × 10-6; p < 0.05). Receiver-operator characteristics showed that preoperative MGAT3-AS1 predicted delayed graft function (area under curve, 0.83; 95% CI, 0.65 to 1.00; p < 0.01). We observed a positive predictive value of 0.57, and a negative predictive value of 0.95. Long non-coding RNA, MGAT3-AS1, indicates short-term outcome in patients with deceased donor kidney transplantation.

Expression Profiling of Exosomal miRNAs Derived from the Peripheral Blood of Kidney Recipients with DGF Using High-Throughput Sequencing.

Delayed graft function (DGF) is one of the major obstacles for graft survival for kidney recipients. It is profound to reduce the incidence of DGF for maintaining long-term graft survival. However, the molecular regulation of DGF is still not adequately explained and the biomarkers for DGF are limited. Exosomes are cell-derived membrane vesicles, contents of which are stable and could be delivered into recipient cells to exert their biological functions. Consequently, exosome-derived proteomic and RNA signature profiles are often used to account for the molecular regulation of diseases or reflect the conditional state of their tissue as biomarkers. Few researches have been done to demonstrate the function of exosomes associated with DGF. In this study, high-throughput sequencing was used to explore the miRNA expression profiling of exosomes in the peripheral blood of kidney recipients with DGF. We identified 52 known and 5 conserved exosomal miRNAs specifically expressed in recipients with DGF. Three coexpressed miRNAs, hsa-miR-33a-5p_R-1, hsa-miR-98-5p, and hsa-miR-151a-5p, were observed to be significantly upregulated in kidney recipients with DGF. Moreover, hsa-miR-151a-5p was positively correlated with the first-week serum CR, BUN, and UA levels of the kidney recipients after transplantation. Furthermore, we also analyzed functions and signaling pathways of the three upregulated miRNAs target genes to uncover putative mechanism of how these exosomal miRNAs functioned in DGF. Overall, these findings identified biomarker candidates for DGF and provided new insights into the important role of the exosomal miRNAs regulation in DGF.

Micro RNA 146a-5p expression in Kidney transplant recipients with delayed graft function / Expressão do Micro RNA 146a-5p em pacientes transplantados renais com disfunção inicial do enxerto

ABSTRACT Introduction: The development of novel non-invasive biomarkers of kidney graft dysfunction, especially in the course of the delayed graft function period would be an important step forward in the clinical practice of kidney transplantation. Methods: We evaluated by RT-PCR the expression of miRNA-146 to -5p ribonucleic micro-acids (miRNAs) in the peripheral blood and renal tissue obtained from kidney transplant recipients who underwent a surveillance graft biopsy during the period of delayed graft function. Results: In biopsy samples, the expression of miR-146a-5p was significantly increased in the group of patients with delayed graft function (DGF) (n = 33) versus stables patients (STA) (n = 13) and patients with acute rejection (AR) (n = 9) (p = 0.008). In peripheral blood samples, a non-significant increase of miR-146a-5p expression was found in the DGF group versus STA and AR groups (p = 0.083). No significant correlation was found between levels of expression in biopsy and plasma. ROC curve analysis revealed an AUC of 0.75 (95% CI: 0.62-0.88) for the renal tissue expression and 0.67 (95% CI 0.52-0.81) for the peripheral blood expression. Conclusion: We conclude that miR-146a-5p expression has a distinct pattern in the renal tissue and perhaps in the peripheral blood in the setting of DGF. Further refinements and strategies for studies should be developed in the field of non-invasive molecular diagnosis of kidney graft dysfunction.

RESUMO Introdução: O desenvolvimento de novos biomarcadores não invasivos para disfunção do enxerto renal, especialmente no decurso da disfunção inicial do enxerto, seria de enorme valia para a prática clínica do transplante renal. Métodos: A técnica de RT-PCR foi utilizada para avaliar a expressão de microRNA 146a-5p no sangue periférico e no tecido renal de receptores de transplante submetidos a biópsia renal de controle no decurso de disfunção inicial do enxerto. Resultados: A expressão de miR-146a-5p estava significativamente aumentada nas amostras de biópsia do grupo de pacientes com disfunção inicial do enxerto (DIE) (n = 33) em relação aos pacientes estáveis (n = 13) e aos com rejeição aguda (RA) (n = 9) (p = 0,008). Foi detectado aumento não significativo da expressão de miR-146a-5p nas amostras de sangue periférico do grupo com DIE em comparação aos pacientes estáveis e com RA (p = 0,083). Não foi identificada correlação significativa entre os níveis de expressão no plasma e na biópsia. A análise da curva COR revelou uma ASC de 0,75 (IC 95%: 0,62-0,88) para a expressão no tecido renal e de 0,67 (IC 95% 0,52-0,81) no sangue periférico. Conclusão: A expressão de miR-146a-5p tem um padrão distinto no tecido renal e talvez no sangue periférico em cenários de DIE. Maiores refinamentos e estratégias adicionais de estudo devem ser desenvolvidos na área do diagnóstico molecular não invasivo da disfunção do enxerto renal.

MicroRNAs in Kidney Machine Perfusion Fluid as Novel Biomarkers for Graft Function. Normalization Methods for miRNAs Profile Analysis.

MicroRNAs (miRNAs) are post-transcriptional regulators that have emerged as promising biomarkers in kidney transplantation. Quantification of miRNAs can be analyzed by means of biological normalization. The purpose of normalization is to remove technical variation in data, which is not related to the biological changes under investigation. Proper normalization is critical for the correct analysis and interpretation of results. MATERIAL AND METHODS: A prospective cohort study was conducted on graft dysfunction in kidney transplantation from expanded criteria donors. After RNA extraction quantitative real-time polymerase chain reaction was performed. The exogenous spike-in normalization was used as technical normalization. Relative expression was calculated using the 2-ΔΔCt method and UniSp2 spike-in was used as reference for normalization. Results obtained were further analyzed by the application of the mean expression value that uses the calculated mean of all miRNAs in a given sample. RESULTS: The mean expression value approach confirmed the significance of a subset of the miRNAs previously identified for delayed graft function development and composed by miRNAs miR-486-5p, miR-144-3p, miR-142-5p, and miR-144-5p. CONCLUSIONS: MicroRNAs are becoming increasingly important as biomarkers in multiple disease processes including kidney transplantation. Perfusion fluid, particularly during hypothermic machine perfusion, provides a valuable pretransplantation source for identification of organ viability biomarkers. Although there is no clear consensus concerning the normalization technique, the mean expression value method shows the better normalization strategy.

Corin Is Downregulated in Renal Ischemia/Reperfusion Injury and Is Associated with Delayed Graft Function after Kidney Transplantation.

Renal ischemia/reperfusion (IR) injury is one of the most important risk factors for the occurrence of delayed graft function (DGF) after kidney transplantation; however, its mechanism remains not fully understood. In the present study, we screened differentially expressed genes in a murine model of renal IR injury by using high-throughput assays. We identified Corin as one of the most significantly downregulated genes among 2218 differentially expressed genes (≥2-fold, P < 0.05). By using a real-time qPCR assay, we observed that the expression of renal Corin in IR-injured mice was reduced to 11.5% of the sham-operated mice and that the protein level of renal Corin in IR-injured mice was also downregulated. Interestingly, renal IR injury in mice induced the downregulation of Corin in heart tissues, suggesting that the overall synthesis of Corin may be suppressed. We recruited 11 recipients complicated with DGF and 16 without DGF, and plasma Corin concentrations were determined by ELISA. We observed that the plasma Corin levels were indeed reduced in recipients complicated with DGF (0.98 vs. 1.95 ng/ml, P < 0.05). These findings demonstrate that Corin may be a potential biomarker of DGF after kidney transplantation and may participate in the regulation of renal IR injury.

Donor Plasma Mitochondrial DNA Is Correlated with Posttransplant Renal Allograft Function.

BACKGROUND: The lack of accurate biomarkers makes it difficult to determine whether organs are suitable for transplantation. Mitochondrial DNA (mtDNA) correlates with tissue damage and kidney disease, making it a potential biomarker in organ evaluation. METHODS: Donors who had experienced cardiac death and successfully donated their kidneys between January 2015 and May 2017 were included this study. We detected the level of mtDNA in the plasma of the donor using quantitative real-time polymerase chain reaction and then statistically analyzed the relationship between the level of mtDNA and the delayed graft function (DGF) of the recipient. RESULTS: The incidence of DGF or slowed graft function (SGF) increased by 4 times (68% versus 16%, P < 0.001) when the donor mtDNA (dmtDNA) level was >0.114. When dmtDNA levels were >0.243, DGF and primary nonfunction were approximately 100% and 44%, respectively. Moreover, dmtDNA was an independent risk factor for slowed graft function and DGF. A prediction model for DGF based on dmtDNA achieved an area under the receiver operating characteristic curve for a prediction score as high as 0.930 (95% confidence interval 0.856-1.000), and the validation cohort results showed that the sensitivity and specificity of the model were 100% and 78%, respectively. dmtDNA levels were correlated with 6-month allograft function (R=0.332, P < 0.001) and 1-year graft survival (79% versus 99%, P < 0.001). CONCLUSIONS: We conclusively demonstrated that plasma dmtDNA was an independent risk factor for DGF, which is valuable in organ evaluation. dmtDNA is a possible first predictive marker for primary nonfunction and worth further evaluation.

MicroRNAs in AKI and Kidney Transplantation.

MicroRNAs are epigenetic regulators of gene expression at the posttranscriptional level. They are involved in intercellular communication and crosstalk between different organs. As key regulators of homeostasis, their dysregulation underlies several morbidities including kidney disease. Moreover, their remarkable stability in plasma and urine makes them attractive biomarkers. Beyond biomarker studies, clinical microRNA research in nephrology in recent decades has focused on the discovery of specific microRNA signatures and the identification of novel targets for therapy and/or disease prevention. However, much of this research has produced equivocal results and there is a need for standardization and confirmation in prospective trials. This review aims to provide an overview of general concepts and available clinical evidence in both the pathophysiology and biomarker fields for the role of microRNA in AKI and kidney transplantation.

Micro RNA 146a-5p expression in Kidney transplant recipients with delayed graft function.

INTRODUCTION: The development of novel non-invasive biomarkers of kidney graft dysfunction, especially in the course of the delayed graft function period would be an important step forward in the clinical practice of kidney transplantation. METHODS: We evaluated by RT-PCR the expression of miRNA-146 to -5p ribonucleic micro-acids (miRNAs) in the peripheral blood and renal tissue obtained from kidney transplant recipients who underwent a surveillance graft biopsy during the period of delayed graft function. RESULTS: In biopsy samples, the expression of miR-146a-5p was significantly increased in the group of patients with delayed graft function (DGF) (n = 33) versus stables patients (STA) (n = 13) and patients with acute rejection (AR) (n = 9) (p = 0.008). In peripheral blood samples, a non-significant increase of miR-146a-5p expression was found in the DGF group versus STA and AR groups (p = 0.083). No significant correlation was found between levels of expression in biopsy and plasma. ROC curve analysis revealed an AUC of 0.75 (95% CI: 0.62-0.88) for the renal tissue expression and 0.67 (95% CI 0.52-0.81) for the peripheral blood expression. CONCLUSION: We conclude that miR-146a-5p expression has a distinct pattern in the renal tissue and perhaps in the peripheral blood in the setting of DGF. Further refinements and strategies for studies should be developed in the field of non-invasive molecular diagnosis of kidney graft dysfunction.

A molecular signature for delayed graft function.

Chronic kidney disease and associated comorbidities (diabetes, cardiovascular diseases) manifest with an accelerated ageing phenotype, leading ultimately to organ failure and renal replacement therapy. This process can be modulated by epigenetic and environmental factors which promote loss of physiological function and resilience to stress earlier, linking biological age with adverse outcomes post-transplantation including delayed graft function (DGF). The molecular features underpinning this have yet to be fully elucidated. We have determined a molecular signature for loss of resilience and impaired physiological function, via a synchronous genome, transcriptome and proteome snapshot, using human renal allografts as a source of healthy tissue as an in vivo model of ageing in humans. This comprises 42 specific transcripts, related through IFNγ signalling, which in allografts displaying clinically impaired physiological function (DGF) exhibited a greater magnitude of change in transcriptional amplitude and elevated expression of noncoding RNAs and pseudogenes, consistent with increased allostatic load. This was accompanied by increased DNA methylation within the promoter and intragenic regions of the DGF panel in preperfusion allografts with immediate graft function. Pathway analysis indicated that an inability to sufficiently resolve inflammatory responses was enabled by decreased resilience to stress and resulted in impaired physiological function in biologically older allografts. Cross-comparison with publically available data sets for renal pathologies identified significant transcriptional commonality for over 20 DGF transcripts. Our data are clinically relevant and important, as they provide a clear molecular signature for the burden of "wear and tear" within the kidney and thus age-related physiological capability and resilience.

Pretransplant Immune Interplay Between Donor and Recipient Influences Posttransplant Kidney Allograft Function.

BACKGROUND: Renal transplant candidates present immune dysregulation caused by chronic uremia, and deceased kidney donors present immune activation induced by brain death. Pretransplant donor and recipient immune-related gene expression were examined in the search for novel predictive biomarkers crosslinking recipient and donor pretransplant immune status with transplant outcome. MATERIALS AND METHODS: This study included 33 low-risk consecutive renal transplant recipients and matched deceased donors. The expression of 29 genes linked to tissue injury, T-cell activation, cell migration, and apoptosis were assessed in postreperfusion kidney biopsies, as well as 14 genes in pretransplant peripheral blood of the kidney recipients. Gene expression was analyzed with real-time polymerase chain reaction on custom-designed low-density arrays. RESULTS: Donor MMP9 expression was related to delayed graft function occurrence (P = .036) and short term kidney allograft function (14th day rs = -0.44, P = .012; 1st month rs = -0.46, P = .013). Donor TGFB1 expression was associated with short- and long-term graft function (14th day rs = -0.47, P = .007; 3rd month rs = -0.63, P = .001; 6th month rs = -0.52, P = .010; 12th month rs = -0.45, P = .028; 24th month rs = -0.64, P = .003). Donor TGFB1 expression was not related to donor age (rs = 0.32, P = .081), which was also an independent factor influencing the outcome. Recipient gene expression was not related to graft function but determined the acute rejection risk. Recipient IFNG and, to a lesser extent, IL18 expression were protective against acute rejection (area under the curve [AUC] 0.84, P < .001, and AUC 0.79, P < .001, respectively). CONCLUSION: Kidney transplant outcome depends on the interplay between donor-related immune factors, which mostly affect allograft function and recipient immune milieu, influencing an alloreactive response.

Cytokine Gene Polymorphism Profiles in Kidney Transplant Patients - Association of +1188A/C RS3212227 SNP in the IL12B Gene Prevents Delayed Graft Function.

BACKGROUND AND AIMS: Transplantation is the best treatment option for end stage kidney disease. The most common early complications in post-transplant period are acute rejection (AR) of the graft and delayed graft function (DGF). The underlying mechanisms in these events are heterogeneous and at least in part involve cytokine genes which regulate immune response to allograft. We have investigated whether functional single nucleotide polymorphisms (SNP) in the genes encoding IFN-γ (IFNG), TNF (TNFA), IL-10 (IL10) and p40 subunit of IL-12/IL-23 (IL12B) could predict risk of AR and DGF in kidney allograft recipients. METHODS: Our study involved 152 kidney transplant recipients on standard immunosuppressive regimen which included calcineurin inhibitors, mycophenolic acid derivatives and corticosteroids. Genotyping of IFNG, TNFA, IL10 and IL12B was performed using commercial TaqMan assays. RESULTS: We found association between the carriers of AA genotype of IL12B +1188A/C polymorphism (rs3212227) and a lower rate of DGF (p = 0.037, OR = 0.45, 95% CI = 0.21-0.96), implying protective role of A allele in the pathogenesis of DGF in kidney transplant recipients, whereas no such association was observed with AR. None of the analyzed SNPs in TNFA (-308G/A), IFNG (+874T/A), IL10 (-1082G/A, -819T/C, -592C/A) were associated with AR or DGF in our patients. CONCLUSIONS: Our study shows a preliminary evidence that the AA genotype of rs3212227 SNP in the IL12B gene might be associated with a lower risk for DGF after kidney transplantation. In the future, additional well-designed large studies are required for the validation of our results.

Ficolin-2 Gene rs7851696 Polymorphism is Associated with Delayed Graft Function and Acute Rejection in Kidney Allograft Recipients.

Ficolin-2 is an activator of the complement system that acts via the lectin pathway. Complement activation plays a substantial role in the renal injury inherent to kidney transplantation. In this study, we examined the associations between ficolin-2 gene polymorphisms in exon 8 and kidney allograft function. This study comprised 270 Caucasian deceased-donor renal transplant recipients. The following parameters were recorded in each case: delayed graft function (DGF), acute rejection (AR), and chronic allograft dysfunction. Among patients with DGF, we observed a significantly increased frequency of rs7851696 GT and TT genotypes as well as T allele (TT + GT vs GG OR 1.98, 95% CI 1.12-3.48, p = 0.02; T vs G OR 2.08, 95% CI 1.27-3.41, p = 0.005). There was also an increased frequency of rs4521835 GG and TG genotypes as well as G alleles; however, these differences were on the borderline of statistical significance (GG + TG vs TT, OR 1.75, 95% CI 0.98-3.12, p = 0.07; G vs T OR 1.45, 95% CI 1.00-2.09, p = 0.050). In addition, we observed an increased frequency of acute allograft rejection in carriers of ficolin-2 rs7851696 T alleles on the borderline of statistical significance (TT + GT vs GG OR 1.75, 95% CI 0.97-3.16, p = 0.08), but the frequency of T allele was significantly higher in patients with AR (T vs G OR 1.71, 95% CI 1.02-2.87, p = 0.048). The results of our study suggest that ficolin-2 rs7851696 gene polymorphism influences kidney allograft functions, with T allele increasing the risk of DGF and AR.