Donor Hepatic Steatosis Induce Exacerbated Ischemia-Reperfusion Injury Through Activation of Innate Immune Response Molecular Pathways.

BACKGROUND: Severe liver steatosis is a known risk factor for increased ischemia-reperfusion injury (IRI) and poor outcomes after liver transplantation (LT). This study aimed to identify steatosis-related molecular mechanisms associated with IRI exacerbation after LT. METHODS: Paired graft biopsies (n = 60) were collected before implantation (L1) and 90 minutes after reperfusion (L2). The LT recipients (n = 30) were classified by graft macrosteatosis: without steatosis (WS) of 5% or less (n = 13) and with steatosis (S) of 25% or greater (n = 17). Plasma samples were collected at L1, L2, and 1 day after LT (postoperative [POD]1) for cytokines evaluation. Tissue RNA was isolated for gene expression microarrays. Probeset summaries were obtained using robust multiarray average algorithm. Pairwise comparisons were fit using 2-sample t test. P values 0.01 or less were significant (false discovery rate <5%). Molecular pathway analyses were conducted using Ingenuity Pathway Analysis tool. RESULTS: Significantly differentially expressed genes were identified for WS and S grafts after reperfusion. Comprehensive comparison analysis of molecular profiles revealed significant association of S grafts molecular profile with innate immune response activation, macrophage production of nitric oxide and reactive oxygen species, IL-6, IL-8, IL-10 signaling activation, recruitment of granulocytes, and accumulation of myeloid cells. Postreperfusion histological patterns of S grafts revealed neutrophilic infiltration surrounding fat accumulation. Circulating proinflammatory cytokines after reperfusion and 24 hours after LT concurred with intragraft-deregulated molecular pathways. All tested cytokines were significantly increased in plasma of S grafts recipients after reperfusion when compared with WS group at same time. CONCLUSIONS: Increases of graft steatosis exacerbate IRI by exacerbation of innate immune response after LT. Preemptive strategies should consider it for safety usage of steatotic livers.

Liver transplant complications in hepatitis C infected recipients: recurrence versus rejection.

Despite improvement on outcomes post liver transplantation (LT), complications such as HCV recurrence (HCV-rec) and acute cellular rejection (ACR) continue to be a challenge for transplant physicians. Accurate diagnostic tools to better dissect between those complications post-LT are crucial for prompt and correct diagnosis and treatment. It is well known that the overlapping features of clinical and histo-pathological characteristics between these conditions turn difficult the appropriate differential diagnosis. Recently, new technological advances had supported the field of biomarker discovery in many diseases. Disease biomarkers capable to differentiate ACR versus HCV-rec post-LT is a long waited task in the transplant community. This editorial describes and discusses potential biomarkers of disease differentiation including recent reports in the field of genomics, proteomics, immunohistochemistry among other technologies.

The urine microRNA profile may help monitor post-transplant renal graft function.

Noninvasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers, we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation, and the longitudinal validation studies for noninvasive monitoring of graft function. Of 1733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time points after kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early after kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.

Regulation of molecular pathways in ischemia-reperfusion injury after liver transplantation.

BACKGROUND: Ischemia-reperfusion (I/R) injury is a multifactorial phenomenon that occurs during the transplant event and frequently compromises early graft function after liver transplantation (LT). Current comprehension of molecular mechanisms and regulation processes of I/R injury lacks clarity. MicroRNA (miRNA) regulation results critical in several biological processes. METHODS: This study evaluated gene expression and miRNA expression profiles using microarrays in 34 graft biopsies collected at preimplantation (L1) and at 90 min postreperfusion (L2) from consecutives deceased-donor LT recipients. miRNA profiles were first analyzed. Data integration analysis (gene expression/miRNA expression) aimed to identify potential target genes for each identified miRNA from the L1/L2 differential gene expression profile. RESULTS: Pairwise comparison analyses identified 40 miRNAs and 3168 significantly differentially expressed genes at postreperfusion time compared with preimplantation time. Pathway analysis of miRNAs associated these profiles with antiapoptosis, inhibition of cellular proliferation, and proinflammatory processes. Target analysis identified an miRNA-associated molecular profile of 2172 genes involved in cellular growth and proliferation modulation by cell cycle regulation, cell death and survival, and proinflammatory and anti-inflammatory processes. miRNA-independent genes involved proinflammatory molecules. CONCLUSION: We identified a miRNA profile involved in posttranscriptional regulatory mechanisms in I/R injury post-LT. A better understanding of these molecular processes involved in I/R may contribute to develop new strategies to minimize graft injury.

Identification of biomarkers to assess organ quality and predict posttransplantation outcomes.

UNLABELLED: The increased disparity between organ supply and need has led to the use of extended criteria donors and donation after cardiac death donors with other comorbidities. METHODS: We have examined the preimplantation transcriptome of 112 kidney transplant recipient samples from 100 deceased-donor kidneys by microarray profiling. Subject groups were segregated based on estimated glomerular filtration rate (eGFR) at 1 month after transplantation: the GFR-high group (n=74) included patients with eGFR 45 mL/min per 1.73 m(2), whereas the GFR-low group (n=35) included patients with eGFR 45 mL/min or less per 1.73 m(2). RESULTS: Gene expression profiling identified higher expression of 160 probe sets (140 genes) in the GFR-low group, whereas expression of 37 probe sets (33 genes) was higher in the GFR-high group (P<0.01, false discovery rate <0.2). Four genes (CCL5, CXCR4, ITGB2, and EGF) were selected based on fold change and P value and further validated using an independent set of samples. A random forest analysis identified three of these genes (CCL5, CXCR4, and ITGB2) as important predictors of graft function after transplantation. CONCLUSIONS: Inclusion of pretransplantation molecular gene expression profiles in donor quality assessment systems may provide the necessary information for better donor organ selection and function prediction. These biomarkers would further allow a more objective and complete assessment of procured renal allografts at pretransplantation time.

Reduced expression of inflammatory genes in deceased donor kidneys undergoing pulsatile pump preservation.

BACKGROUND: The use of expanded criteria donor kidneys (ECD) had been associated with worse outcomes. Whole gene expression of pre-implantation allograft biopsies from deceased donor kidneys (DDKs) was evaluated to compare the effect of pulsatile pump preservation (PPP) vs. cold storage preservation (CSP) on standard and ECD kidneys. METHODOLOGY/PRINCIPAL FINDINGS: 99 pre-implantation DDK biopsies were studied using gene expression with GeneChips. Kidneys transplant recipients were followed post transplantation for 35.8 months (range = 24-62). The PPP group included 60 biopsies (cold ischemia time (CIT) = 1,367+/-509 minutes) and the CSP group included 39 biopsies (CIT = 1,022+/-485 minutes) (P<0.001). Donor age (42.0±14.6 vs. 34.1±14.2 years, P = 0.009) and the percentage of ECD kidneys (PPP = 35% vs. CSP = 12.8%, P = 0.012) were significantly different between groups. A two-sample t-test was performed, and probe sets having a P<0.001 were considered significant. Probe set level linear models were fit using cold ischemia time and CSP/PPP as independent variables to determine significant probe sets (P<0.001) between groups after adjusting for cold ischemia time. Thus, 43 significant genes were identified (P<0.001). Over-expression of genes associated with inflammation (CD86, CD209, CLEC4, EGFR2, TFF3, among others) was observed in the CSP group. Cell-to-cell signaling and interaction, and antigen presentation were the most important pathways with genes significantly over-expressed in CSP kidneys. When the analysis was restricted to ECD kidneys, genes involved in inflammation were also differentially up-regulated in ECD kidneys undergoing CSP. However, graft survival at the end of the study was similar between groups (P = 0.2). Moreover, the incidence of delayed graft function was not significant between groups. CONCLUSIONS/SIGNIFICANCE: Inflammation was the most important up-regulated pattern associated with pre-implantation biopsies undergoing CSP even when the PPP group has a larger number of ECD kidneys. No significant difference was observed in delayed graft function incidence and graft function post-transplantation. These findings support the use of PPP in ECD donor kidneys.

Pretransplant transcriptome profiles identify among kidneys with delayed graft function those with poorer quality and outcome.

Robust biomarkers are needed to identify donor kidneys with poor quality associated with inferior early and longer-term outcome. The occurrence of delayed graft function (DGF) is most often used as a clinical outcome marker to capture poor kidney quality. Gene expression profiles of 92 preimplantation biopsies were evaluated in relation to DGF and estimated glomerular filtration rate (eGFR) to identify preoperative gene transcript changes associated with short-term function. Patients were stratified into those who required dialysis during the first week (DGF group) versus those without (noDGF group) and subclassified according to 1-month eGFR of >45 mL/min (eGFR(hi)) versus eGFR of ≤45 mL/min (eGFR(lo)). The groups and subgroups were compared in relation to clinical donor and recipient variables and transcriptome-associated biological pathways. A validation set was used to confirm target genes. Donor and recipient characteristics were similar between the DGF versus noDGF groups. A total of 206 probe sets were significant between groups (P < 0.01), but the gene functional analyses failed to identify any significantly affected pathways. However, the subclassification of the DGF and noDGF groups identified 283 probe sets to be significant among groups and associated with biological pathways. Kidneys that developed postoperative DGF and sustained an impaired 1-month function (DGF(lo) group) showed a transcriptome profile of significant immune activation already preimplant. In addition, these kidneys maintained a poorer transplant function throughout the first-year posttransplant. In conclusion, DGF is a poor marker for organ quality and transplant outcome. In contrast, preimplant gene expression profiles identify "poor quality" grafts and may eventually improve organ allocation.

Gene expression changes are associated with loss of kidney graft function and interstitial fibrosis and tubular atrophy: diagnosis versus prediction.

BACKGROUND: Loss of kidney graft function due to interstitial fibrosis (IF) and tubular atrophy (TA) is the most common cause of kidney allograft loss. METHODS: One hundred one allograft tissues (26 samples with IF/TA, 17 normal allografts, and an independent biopsy group collected at 3 month [n=34] posttransplantation) underwent microarray analysis to identify early detection/diagnostic biomarkers of IF/TA. Profiling of 24 allograft biopsies collected at or after 9-month posttransplantation (range 9-18 months) was used for validation. Three-month posttransplantation biopsies were classified as IF/TA nonprogressors (group 1) or progressors (group 2) using graft function and histology at 9-month posttransplantation. RESULTS: We identified 2223 differentially expressed probe sets between IF/TA and normal allograft biopsies using a Bonferroni correction. Genes up-regulated in IF/TA were primarily involved in pathways related to T-cell activation, natural killer cell-mediated cytotoxicity, and programmed cell death. A least absolute shrinkage and selection operator model was derived from the differentially expressed probe sets, resulting in a final model that included 10 probe sets and had 100% training set accuracy. The N-fold crossvalidated error was 2.4% (sensitivity 95.8% and specificity 100%). When 3-month biopsies were tested using the model, all the samples were classified as normal. However, evaluating gene expression of the 3-month biopsies and fitting a new penalized model, 100% sensitivity was observed in classifying the samples as group1 or 2. This model was evaluated in the sample set collected at or after 9-month posttransplantation. CONCLUSIONS: An IF/TA gene expression signature was identified, and it was useful for diagnosis but not prediction. However, gene expression profiles at 3 months might predict IF/TA progression.