Mitochondrial Consequences of Organ Preservation Techniques during Liver Transplantation.

Allograft ischemia during liver transplantation (LT) adversely affects the function of mitochondria, resulting in impairment of oxidative phosphorylation and compromised post-transplant recovery of the affected organ. Several preservation methods have been developed to improve donor organ quality; however, their effects on mitochondrial functions have not yet been compared. This study aimed to summarize the available data on mitochondrial effects of graft preservation methods in preclinical models of LT. Furthermore, a network meta-analysis was conducted to determine if any of these treatments provide a superior benefit, suggesting that they might be used on humans. A systematic search was conducted using electronic databases (EMBASE, MEDLINE (via PubMed), the Cochrane Central Register of Controlled Trials (CENTRAL) and Web of Science) for controlled animal studies using preservation methods for LT. The ATP content of the graft was the primary outcome, as this is an indicator overall mitochondrial function. Secondary outcomes were the respiratory activity of mitochondrial complexes, cytochrome c and aspartate aminotransferase (ALT) release. Both a random-effects model and the SYRCLE risk of bias analysis for animal studies were used. After a comprehensive search of the databases, 25 studies were enrolled in the analysis. Treatments that had the most significant protective effect on ATP content included hypothermic and subnormothermic machine perfusion (HMP and SNMP) (MD = -1.0, 95% CI: (-2.3, 0.3) and MD = -1.1, 95% CI: (-3.2, 1.02)), while the effects of warm ischemia (WI) without cold storage (WI) and normothermic machine perfusion (NMP) were less pronounced (MD = -1.8, 95% CI: (-2.9, -0.7) and MD = -2.1 MD; CI: (-4.6; 0.4)). The subgroup of static cold storage (SCS) with shorter preservation time (< 12 h) yielded better results than SCS ≥ 12 h, NMP and WI, in terms of ATP preservation and the respiratory capacity of complexes. HMP and SNMP stand out in terms of mitochondrial protection when compared to other treatments for LT in animals. The shorter storage time at lower temperatures, together with the dynamic preservation, provided superior protection for the grafts in terms of mitochondrial function. Additional clinical studies on human patients including marginal donors and longer ischemia times are needed to confirm any superiority of preservation methods with respect to mitochondrial function.

Circulating CXCL10 and IL-6 in solid organ donors after brain death predict graft outcomes.

We tested the hypothesis that circulating CXCL10 and IL-6 in donor after brain death provide independent additional predictors of graft outcome. From January 1, 2010 to June 30, 2012 all donors after brain death managed by the NITp (n = 1100) were prospectively included in this study. CXCL10 and IL-6 were measured on serum collected for the crossmatch at the beginning of the observation period. Graft outcome in recipients who received kidney (n = 1325, follow-up 4.9 years), liver (n = 815, follow-up 4.3 years) and heart (n = 272, follow-up 5 years) was evaluated. Both CXCL-10 and IL-6 showed increased concentration in donors after brain death. The intensive care unit stay, the hemodynamic instability, the cause of death, the presence of risk factors for cardiovascular disease and the presence of ongoing infection resulted as significant determinants of IL-6 and CXCL10 donor concentrations. Both cytokines resulted as independent predictors of Immediate Graft Function. Donor IL-6 or CXCL10 were associated with graft failure after liver transplant, and acted as predictors of recipient survival after kidney, liver and heart transplantation. Serum donor IL-6 and CXCL10 concentration can provide independent incremental prediction of graft outcome among recipients followed according to standard clinical practice.

FGF15 improves outcomes after brain dead donor liver transplantation with steatotic and non-steatotic grafts in rats.

BACKGROUND & AIMS: Donation after brain death (DBD) grafts are associated with reduced graft quality and function post liver transplantation (LT). We aimed to elucidate i) the impact of FGF15 levels on DBD grafts; ii) whether this impact resulted from altered intestinal FXR-FGF15; iii) whether administration of FGF15 to donors after brain death could confer a benefit on graft function post LT; and iv) whether FGF15 affects bile acid (BA) accumulation. METHODS: Steatotic and non-steatotic grafts from DBD donors and donors without brain death were transplanted in rats. FGF15 was administered alone or combined with either a BA (cholic acid) or a YAP inhibitor. RESULTS: Brain death induced intestinal damage and downregulation of FXR. The resulting reduced intestinal FGF15 was associated with low hepatic FGF15 levels, liver damage and regenerative failure. Hepatic FGFR4-Klb - the receptor for FGF15 - was downregulated whereas CYP7A1 was overexpressed, resulting in BA accumulation. FGF15 administration to DBD donors increased hepatic FGFR4-Klb, reduced CYP7A1 and normalized BA levels. The benefit of FGF15 on liver damage was reversed by cholic acid, whereas its positive effect on regeneration was maintained. YAP signaling in DBD donors was activated after FGF15 treatment. When a YAP inhibitor was administered, the benefits of FGF15 on regeneration were abolished, whereas its positive effect on hepatic damage remained. Neither the Hippo-YAP-BA nor the BA-IQGAP1-YAP axis was involved in the benefits of FGF15. CONCLUSION: Alterations in the gut-liver axis contribute to the reduced quality of DBD grafts and the associated pathophysiology of LT. FGF15 pre-treatment in DBD donors protected against damage and promoted cell proliferation. LAY SUMMARY: After brain death, potential liver donors have reduced intestinal FXR, which is associated with reduced intestinal, circulatory and hepatic levels of FGF15. A similar reduction in the cell-surface receptor complex Fgfr4/Klb is observed, whereas CYP7A1 is overexpressed; together, these molecular events result in the dangerous accumulation of bile acids, leading to damage and regenerative failure in brain dead donor grafts. Herein, we demonstrate that when such donors receive appropriate doses of FGF15, CYP7A1 levels and hepatic bile acid toxicity are reduced, and liver regeneration is promoted.

Normothermic Ex Vivo Kidney Perfusion Improves Early DCD Graft Function Compared With Hypothermic Machine Perfusion and Static Cold Storage.

BACKGROUND: Better preservation strategies for the storage of donation after circulatory death grafts are essential to improve graft function and to increase the kidney donor pool. We compared continuous normothermic ex vivo kidney perfusion (NEVKP) with hypothermic anoxic machine perfusion (HAMP) and static cold storage (SCS) in a porcine kidney autotransplantation model. METHODS: Porcine kidneys were exposed to 30 minutes of warm ischemia and then reimplanted following either 16 hours of either SCS, HAMP (LifePort 1.0), or NEVKP before autotransplantation (n = 5 per group). The contralateral kidney was removed. Animals were followed for 8 days. RESULTS: Grafts preserved by NEVKP demonstrated improved function with more rapid recovery compared with HAMP and SCS (mean peak serum creatinine: 3.66 ± 1.33 mg/dL [postoperative d 1 [(POD1)], 8.82 ± 3.17 mg/dL [POD2], and 12.90 ± 2.19 mg/dL [POD3], respectively). The NEVKP group demonstrated significantly increased creatinine clearance calculated on POD3 (63.6 ± 19.0 mL/min) compared with HAMP (13.5 ± 10.3 mL/min, P = 0.001) and SCS (4.0 ± 2.6 mL/min, P = 0.001). Histopathologic injury scores on POD8 were lower in both perfused groups (NEVKP and HAMP, score: 1-1.5) compared with SCS (score: 1-3, P = 0.3), without reaching statistical significance. CONCLUSIONS: NEVKP storage significantly improved early kidney function compared with both cold preservation strategies, although HAMP also demonstrates improvement over SCS. NEVKP may represent a novel, superior preservation option for donation after circulatory death renal grafts compared with conventional hypothermic methods.

Oxalate deposition in renal allograft biopsies within 3 months after transplantation is associated with allograft dysfunction.

BACKGROUND: Calcium oxalate (CaOx) deposition in the kidney may lead to loss of native renal function but little is known about the prevalence and role of CaOx deposition in transplanted kidneys. METHODS: In patients transplanted in 2014 and 2015, all for-cause renal allograft biopsies obtained within 3 months post-transplantation were retrospectively investigated for CaOx deposition. Additionally, all preimplantation renal biopsies obtained in 2000 and 2001 were studied. RESULTS: In 2014 and 2015, 388 patients were transplanted, of whom 149 had at least one for-cause renal biopsy. Twenty-six (17%) patients had CaOx deposition. In the population with CaOx deposition: Patients had significantly more often been treated with dialysis before transplantation (89 vs. 64%; p = 0.011); delayed graft function occurred more frequently (42 vs. 23%; p = 0.038); and the eGFR at the time of first biopsy was significantly worse (21 vs. 29 ml/min/1.73m2; p = 0.037). In a multivariate logistic regression analysis, eGFR at the time of first biopsy (OR 0.958, 95%-Cl: 0.924-0.993, p = 0.019), dialysis before transplantation (OR 4.868, 95%-Cl: 1.128-21.003, p = 0.034) and the time of first biopsy after transplantation (OR 1.037, 95%-Cl: 1.013-1.062, p = 0.002) were independently associated with CaOx deposition. Graft survival censored for death was significantly worse in patients with CaOx deposition (p = 0.018). In only 1 of 106 preimplantation biopsies CaOx deposition was found (0.94%). CONCLUSION: CaOx deposition appears to be primarily recipient-derived and is frequently observed in for-cause renal allograft biopsies obtained within 3 months post-transplantation. It is associated with inferior renal function at the time of biopsy and worse graft survival.

Histological characteristics of Acute Tubular Injury during Delayed Graft Function predict renal function after renal transplantation.

Acute Tubular Injury (ATI) is the leading cause of Delayed Graft Function (DGF) after renal transplantation (RTX). Biopsies taken 1 week after RTX often show extensive tubular damage, which in most cases resolves due to the high regenerative capacity of the kidney. Not much is known about the relation between histological parameters of renal damage and regeneration immediately after RTX and renal outcome in patients with DGF. We retrospectively evaluated 94 patients with DGF due to ATI only. Biopsies were scored for morphological characteristics of renal damage (edema, casts, vacuolization, and dilatation) by three independent blinded observers. The regenerative potential was quantified by tubular cells expressing markers of proliferation (Ki67) and dedifferentiation (CD133). Parameters were related to renal function after recovery (CKD-EPI 3, 6, and 12 months posttransplantation). Quantification of morphological characteristics was reproducible among observers (Kendall's W ≥ 0.56). In a linear mixed model, edema and casts significantly associated with eGFR within the first year independently of clinical characteristics. Combined with donor age, edema and casts outperformed the Nyberg score, a well-validated clinical score to predict eGFR within the first year after transplantation (R2  = 0.29 vs. R2  = 0.14). Although the number of Ki67+ cells correlated to the extent of acute damage, neither CD133 nor Ki67 correlated with renal functional recovery. In conclusion, the morphological characteristics of ATI immediately after RTX correlate with graft function after DGF. Despite the crucial role of regeneration in recovery after ATI, we did not find a correlation between dedifferentiation marker CD133 or proliferation marker Ki67 and renal recovery after DGF.

The pleural mesothelium and transforming growth factor-ß1 pathways in restrictive allograft syndrome: A pre-clinical investigation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) hampers long-term survival after lung transplantation. Common fibrosis-related mechanisms in idiopathic pulmonary fibrosis and CLAD instigated the consideration of investigating the differential regulation of pleural mesothelium and transforming growth factor-ß1 (TGF-ß1) in restrictive allograft syndrome (RAS). METHODS: TGF-ß1 was assessed in bronchoalveolar lavage (BAL) fluid using enzyme-linked immunoassay and via immune staining of explant biopsies. To assess the role of the pleura, explanted bronchiolitis obliterans syndrome (BOS) and RAS lungs were compared using computed tomography scans, calretinin stainings, Western blot, and quantititative real-time PCR. Last, a pleural mesothelial cell line was used to assess mesothelial-to-mesenchymal transition and its inhibition. RESULTS: TGF-ß1 was increased in BAL of RAS patients (p = 0.035), and was present in the (sub)pleural area of biopsies. Explanted RAS lungs demonstrated an increased volume fraction of pleura (p = 0.0004), a higher proportion of calretinin-positive stainings (p = 0.0032), and decreased E-cadherin (p = 0.019) and increased α-smooth muscle actin (p = 0.0089) mRNA expression and protein levels in isolated pleural tissue. Moreover, TGF-ß1 stimulation of pleural mesothelial cells led to a phenotypical switch to mesenchymal cells, accompanied with an increased migratory capacity. Interleukin-1α was able to accentuate TGF-ß1‒induced mesothelial-to-mesenchymal transition. None of the tested drugs could inhibit mesothelial-to-mesenchymal transition at the used concentrations. CONCLUSIONS: Our results support an interplay between TGF-ß1 and the pleural mesothelium in the pathophysiology of RAS.

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.

Outcomes and challenges of a kidney transplant programme at Groote Schuur Hospital, Cape Town: A South African perspective.

INTRODUCTION: Access to dialysis and transplantation in the developing world remains limited. Therefore, optimising renal allograft survival is essential. This study aimed to evaluate clinical outcomes and identify poor prognostic factors in the renal transplant programme at Groote Schuur Hospital [GSH], Cape Town. . METHOD: Data were collected on all patients who underwent a kidney transplant at GSH from 1st July 2010 to the 30 June 2015. Analyses were performed to assess baseline characteristics, graft and patient survival, as well as predictors of poor outcome. . RESULTS: 198 patients were transplanted. The mean age was 38 +/- 10.5 years, 127 (64.1%) were male, and 86 (43.4%) were of African ethnicity. Deceased donor organs were used for 130 (66.7%) patients and living donors for 65 (33.3%). There were > 5 HLA mismatches in 58.9% of transplants. Sepsis was the commonest cause of death and delayed graft function [DGF] occurred in 41 (21.4%) recipients. Patient survival was 90.4% at 1 year and 83.1% at 5 years. Graft survival was 89.4% at 1 year and 80.0% at 5 years. DGF (HR 2.83 (1.12-7.19), p value = 0.028) and recipient age > 40 years (HR 3.12 (1.26-7.77), p value = 0.014) were predictors of death. CONCLUSION: Despite the high infectious burden, stratified immunosuppression and limited tissue typing this study reports encouraging results from a resource constrained transplant programme in South Africa. Renal transplantation is critical to improve access to treatment of end stage kidney disease where access to dialysis is limited.

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.

Graft Pre-conditioning by Peri-Operative Perfusion of Kidney Allografts With Rabbit Anti-human T-lymphocyte Globulin Results in Improved Kidney Graft Function in the Early Post-transplantation Period-a Prospective, Randomized Placebo-Controlled Trial.

Introduction: Although prone to a higher degree of ischemia reperfusion injury (IRI), the use of extended criteria donor (ECD) organs has become reality in transplantation. We therefore postulated that peri-operative perfusion of renal transplants with anti-human T-lymphocyte globulin (ATLG) ameliorates IRI and results in improved graft function. Methods: We performed a randomized, single-blinded, placebo-controlled trial involving 50 kidneys (KTx). Prior to implantation organs were perfused and incubated with ATLG (AP) (n = 24 kidney). Control organs (CP) were perfused with saline only (n = 26 kidney). Primary endpoint was defined as graft function reflected by serum creatinine at day 7 post transplantation (post-tx). Results: AP-KTx recipients illustrated significantly better graft function at day 7 post-tx as reflected by lower creatinine levels, whereas no treatment effect was observed after 12 months surveillance. During the early hospitalization phase, 16 of the 26 CP-KTx patients required dialysis during the first 7 days post-tx, whereas only 10 of the 24 AP-KTx patients underwent dialysis. No treatment-specific differences were detected for various lymphocytes subsets in the peripheral blood of patients. Additionally, mRNA analysis of 0-h biopsies post incubation with ATLG revealed no changes of intragraft inflammatory expression patterns between AP and CP organs. Conclusion: We here present the first clinical study on peri-operative organ perfusion with ATLG illustrating improved graft function in the early period post kidney transplantation. Clinical Trial Registration: www.ClinicalTrials.gov, NCT03377283.

Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.

Ischemia-reperfusion injury during kidney transplantation predisposes to delayed graft function, rejection, and premature graft failure. Exacerbation of tissue damage and alloimmune responses may be explained by necroinflammation: an autoamplification loop of cell death and inflammation, which is mediated by the release of damage-associated molecular patterns (eg, high-mobility group box-1; HMGB1) from necrotic cells that activate both innate and adaptive immune pathways. Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is upregulated on injured proximal tubular epithelial cells and enables them to clear apoptotic and necrotic cells. Here we show a pivotal role for clearance of dying cells in regulating necroinflammation in a syngeneic murine kidney transplant model. We found persistent KIM-1 expression in KIM-1+/+ kidney grafts posttransplantation. Compared to recipients of KIM-1+/+ kidneys, recipients of KIM-1-/- kidneys exhibited significantly more renal dysfunction, apoptosis and necrosis, tubular obstruction, and graft failure. KIM-1-/- grafts also had more inflammatory cytokines, infiltrating neutrophils, and macrophages compared to KIM-1+/+ grafts. Most significantly, passive release of HMGB1 from apoptotic and necrotic cells led to dramatically higher serum HMGB1 levels and increased proinflammatory macrophages in recipients of KIM-1-/- grafts. Our data identify an endogenous protective mechanism against necroinflammation in kidney grafts that may be of therapeutic relevance in transplantation.

Renal PGC1α May Be Associated with Recovery after Delayed Graft Function.

BACKGROUND: Delayed renal graft function (DGF) contributes to the determination of length of hospitalization, risk of acute rejection, and graft loss. Existing tools aid the diagnosis of specific DGF etiologies such as antibody-mediated rejection, but markers of recovery have been elusive. The peroxisome proliferator gamma co-activator-1-alpha (PGC1α) is highly expressed in the renal tubule, regulates mitochondrial biogenesis, and promotes recovery from experimental acute kidney injury. OBJECTIVES: We aimed to determine the association between renal allograft PGC1α expression and recovery from delayed graft function. METHODS: We retrospectively analyzed patients undergoing renal transplantation at a single center from January 1, 2008 to June 30, 2014. PGC1α expression was assessed by immunostaining and ultrastructural characteristics by transmission electron microscopy. Of 34 patients who underwent renal biopsy for DGF within 30 days of transplant, 21 were included for analysis. RESULTS: Low PGC1α expression was associated with a significantly longer time on dialysis after transplant (median of 35.5 vs. 16 days, p < 0.05) and a significantly higher serum creatinine (sCr) at 4 weeks after transplantation among those who discontinued dialysis (5 vs. 1.65 mg/dL, p < 0.0001). Low PGC1α expression was not associated with higher sCr at 12 weeks after transplantation. Ultrastructural characteristics including apical membrane blebbing and necrotic luminal debris were not informative regarding clinical outcomes. CONCLUSIONS: These data suggest that higher PGC1α expression is associated with faster and more complete recovery from DGF. Mitochondrial biogenesis may be a therapeutic target for DGF. Larger studies are needed to validate these findings.

Kidney injury molecule-1 staining in renal allograft biopsies 10 days after transplantation is inversely correlated with functioning proximal tubular epithelial cells.

BACKGROUND: Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are promising biomarkers for monitoring delayed graft function (DGF) after kidney transplantation. Here we investigated localization and distribution of KIM-1 and NGAL staining in renal allograft biopsies and studied their association with histological features, functional DGF (fDGF) and the tubular function slope (TFS), a functioning proximal tubular epithelial cell (PTEC) marker. METHODS: Day 10 protocol biopsies of 64 donation after circulatory death recipients were stained for KIM-1 and NGAL and the positive area was quantified using ImageJ software. Biopsies were scored according to Banff and acute tubular necrosis (ATN) criteria. A 99mtechnetium-mercaptoacetyltriglycine (99mTc-MAG3)-renography was performed to calculate TFS. RESULTS: KIM-1 staining was located on the brush border of tubular epithelial cells (TECs) and correlated with denudation, while NGAL was present more focally in a cytoplasmic distribution. KIM-1 and NGAL staining were not correlated and no co-localization was observed. Quantitative stainings were not associated with fDGF, but KIM-1 tended to be higher in patients with prolonged fDGF (≥21 days; P = 0.062). No correlation was observed between the quantitative tissue stainings and urinary KIM-1 or NGAL. Quantitative KIM-1 staining was inversely correlated with the TFS (Spearman's ρ = -0.53; P < 0.001), whereas NGAL was not. The latter finding might be because cortical NGAL staining is dependent on filtration and subsequent reabsorption by functioning PTECs. Staining of NGAL was indeed restricted to PTECs, as shown by co-localization with a PTEC-specific lectin. CONCLUSIONS: KIM-1 and NGAL staining showed different localization and distribution. Quantitative KIM-1 staining was inversely correlated with functioning PTECs.

Joint Assessment of Donor and Recipient hTERT Gene Polymorphism Provides Additional Information for Early Kidney Transplantation Outcomes.

BACKGROUND There are several genes and genetic loci affecting telomere length, including hTERT gene and BICD1 gene as well as polymorphisms within chromosome 18. It has been demonstrated that the age of the donor is a negative factor associated with long-term kidney allograft function, and that post-transplant complications accelerate transplanted organ aging, thus contributing to estimated glomerular filtration rate (eGFR) decreases. The aim of this study was a joint assessment of donors' and recipients' hTERT and BICD1 genes as well as chromosome 18 polymorphisms with regard to early kidney transplantation outcomes. MATERIAL AND METHODS The study enrolled 74 pairs of Polish Caucasian kidney allograft cadaveric donors (60% male, mean age 45.99±14.62) and recipients (50.0% male, mean age 48.89±13.50). The transplantation procedure (Tx) was performed between 2001 and 2012. All samples were genotyped in duplicate using Real-Time PCR. RESULTS This study showed that rs2735940 hTERT CX-TT donor-recipient genotype pair was associated with almost five times higher odds (OR=4.82; 95% CI: 1.32-18; p=0.016) of delayed graft function (DGF), and that rs2735940 hTERT, rs2630578 BICD1, and rs7235755 chromosome 18 polymorphisms combined pairs were not associated with acute rejection (AR). CONCLUSIONS In conclusion, both the donor's and the recipient's rs2735940 hTERT gene polymorphism was associated with early graft function after transplantation. The odds of DGF were almost five times higher for a combination of CX (CT or CC) donor genotype and TT recipient genotype. Joint assessment of donor-recipient genotype pairs provides more information for prediction of early kidney transplantation outcomes.

Use of a Targeted Urine Proteome Assay (TUPA) to identify protein biomarkers of delayed recovery after kidney transplant.

PURPOSE: Development of delayed graft function (DGF) following kidney transplant is associated with poor outcomes. An ability to rapidly identify patients with DGF versus those with immediate graft function (IGF) may facilitate the treatment of DGF and the research needed to improve prognosis. The purpose of this study was to use a Targeted Urine Proteome Assay to identify protein biomarkers of delayed recovery from kidney transplant. EXPERIMENTAL DESIGN: Potential biomarkers were identified using the Targeted Urine Proteome (MRM) Assay to interrogate the relative DGF/IGF levels of expression of 167 proteins in urine taken 12-18 h after kidney implantation from 21 DGF, 15 SGF (slow graft function), and 16 IGF patients. An iterative Random Forest analysis approach evaluated the relative importance of each biomarker, which was then used to identify an optimum biomarker panel that provided the maximum sensitivity and specificity with the least number of biomarkers. CONCLUSIONS AND CLINICAL RELEVANCE: Four proteins were identified that together distinguished DGF with a sensitivity of 77.4%, specificity of 82.6%, and AUC of 0.891. This panel represents an important step toward identifying DGF at an early stage so that more effective treatments can be developed to improve long-term graft outcomes.

Hypoxia-Inducible Factor-1α Expression in Kidney Transplant Biopsy Specimens After Reperfusion Is Associated With Early Recovery of Graft Function After Cadaveric Kidney Transplantation.

BACKGROUND: Ischemia/reperfusion injury during kidney transplantation (KTx) delays allograft recovery. Hypoxia-inducible factor-1α (HIF-1α) is the key regulator of the protective response to ischemia/reperfusion injury. We evaluated the impact of the HIF-1α signaling pathway on allograft recovery during cadaveric KTx. METHODS: Between 1996 and 2015, 46 patients underwent cadaveric KTx. The expression levels of HIF-1α-related proteins, including phosphoinositide 3-kinase, phosphorylated (p)-Akt, p-mammalian target of rapamycin, p-Eukaryotic translation initiation factor 4E, p-S6 ribosomal protein, and HIF-1α, were immunohistochemically evaluated and semi-quantitatively scored in graft biopsy specimens after 1 hour of revascularization. Ten kidney biopsy specimens collected during donor nephrectomy for living KTx were used as controls. Delayed graft function (DGF) was defined as the need for dialysis within 1 week of KTx. We compared the staining scores of each protein and several clinical parameters between patients with and those without DGF. RESULTS: Expression levels of all six proteins in specimens after revasculization were elevated compared with those in controls. Thirty-five patients had DGF. Expression levels of PI3K, p-AKT, p-mTOR, p-eIF4E, and HIF-1α were significantly higher in patients without DGF than in those with DGF. Univariate analysis identified expression levels of p-Akt, p-S6, and HIF-1α, in addition to donor type (heart beating/non-heart beating), cold ischemic time, and donor age as significant predictors of DGF. Of these, only expression levels of HIF-1α and donor type were independently associated with DGF in multivariate analysis. CONCLUSIONS: Up-regulation of HIF-1α in allografts after reperfusion may be a predictor of early recovery after cadaveric KTx.

Delayed Graft Function Phenotypes and 12-Month Kidney Transplant Outcomes.

BACKGROUND: Ischemia-reperfusion injury (IRI) leading to delayed graft function (DGF), defined by the United Network for Organ Sharing as dialysis in the first week (UNOS-DGF), associates with poor kidney transplant outcomes. Controversies remain, however, about dialysis initiation thresholds and the utility for other criteria to denote less severe IRI, or slow graft function (SGF). METHODS: Multicenter, prospective study of deceased-donor kidney recipients to compare UNOS-DGF to a definition that combines impaired creatinine reduction in the first 48 hours or greater than 1 dialysis session for predicting 12-month estimated glomerular filtration rate (eGFR). We also assessed 10 creatinine and urine output-based SGF definitions relative to 12-month eGFR. RESULTS: In 560 recipients, 215 (38%) had UNOS-DGF, 330 (59%) met the combined definition, 14 (3%) died, and 23 (4%) had death-censored graft failure by 12 months. Both DGF definitions were associated with lower adjusted 12-month eGFR (95% confidence interval)-by 7.3 (3.6-10.9) and 7.4 (3.8-11.0) mL/min per 1.73 m, respectively. Adjusted relative risks for 12-month eGFR less than 30 mL/min per 1.73 m were 1.9 (1.2-3.1) and 2.1 (1.1-3.7), with unadjusted areas under the curve of 0.618 and 0.627, respectively. For SGF definitions, postoperative day (POD) 7 creatinine had the strongest association with 12-month eGFR, and POD5 creatinine and creatinine reduction between POD1 and POD2 demonstrated modest separations in 12-month eGFR. CONCLUSIONS: Although UNOS-DGF does not adequately predict 12-month function on its own, our findings do not support changing the definition. Postoperative day 7 creatinine is correlated with 12-month eGFR, but large translational studies are needed to understand the biological link between IRI severity at transplant and longer-term outcomes.

Lowering of Messenger Ribonucleic Acid Toll-Like Receptors 2-4,9 in Peripheral Blood Mononuclear Cells in Kidney Allograft Recipients, Relationships With Immunosuppressive Treatment, and Delayed Graft Function Occurrence.

BACKGROUND: Both tacrolimus (Tac) and cyclosporine (CsA) inhibit control peripheral blood mononuclear cells (PBMC) after stimulation of various Toll-like receptors (TLR) at supra-pharmacological concentrations. Earlier studies demonstrated that 24 hours after kidney transplantation (KT), the expression of the TLR4 messenger RNA (mRNA) in PBMC from patients with subsequent delayed graft function (DGF+) was lower than in patients without DGF (DGF-). An assessment was made of the interaction of immunosuppression with TLR mRNA in PBMC and to verify whether the reduced expression of TLR-2,3,4,9 mRNA in PBMC is permanent in DGF+. METHODS: We investigated mRNA expression of TLR in non-stimulated PBMC. All patients were transplanted more than 1 month before PBMC acquisition. Patients were divided into groups with respect to positive or negative history of delayed graft function (DGF+/-). RESULTS: The expression of TLR2, TLR3, and TLR9 in patients was lower than that in the control group. We found an association of Tac C0 with expression of TLR4 only and CsA dose per 1 kg body weight with TLR2 or up to 6 months after KT with TLR9. Mofetil mycophenolate (MMF)contributed to the change of TLR4 expression in the CsA group but not in the Tac group. TLR3 and TLR9 were nearly equally sensitive to both Tac and CsA, with a decrease of expression with respect to control. DGF+ was associated with variable degree of reduction of TLR2, TLR3, TLR4, and TLR 9 expression. CONCLUSIONS: We showed the importance of immunosuppression and delayed graft function as factors that modify the overall expression of mRNA-TLR PBMC for a period of time after KT. Patients with a history of DGF have chronically decreased expression of mRNA TLR2, TLR3, TLR4, and TLR9. This fact is associated with poorer graft function. Measuring the expression of the TLR in the upper range of therapeutic doses of calcineurin inhibitors and MMF gives the opportunity to assess the strength, effectiveness, and toxicity of immunosuppression.