MicroRNA antagonist therapy during normothermic machine perfusion of donor kidneys.

Normothermic machine perfusion (NMP) is a novel clinical approach to overcome the limitations of traditional hypothermic organ preservation. NMP can be used to assess and recondition organs prior to transplant and is the subject of clinical trials in solid organ transplantation. In addition, NMP provides an opportunity to deliver therapeutic agents directly to the organ, thus avoiding many limitations associated with systemic treatment of the recipient. We report the delivery of oligonucleotide-based therapy to human kidneys during NMP, in this case to target microRNA function (antagomir). An antagomir targeting mir-24-3p localized to the endothelium and proximal tubular epithelium. Endosomal uptake during NMP conditions facilitated antagomir co-localization with proteins involved in the RNA-induced silencing complex (RISC) and demonstrated engagement of the miRNA target. This pattern of uptake was not seen during cold perfusion. Targeting mir-24-3p action increased expression of genes controlled by this microRNA, including heme oxygenase-1 and sphingosine-1-phosphate receptor 1. The expression of genes not under the control of mir-24-3p was unchanged, indicating specificity of the antagomir effect. In summary, this is the first report of ex vivo gymnotic delivery of oligonucleotide to the human kidney and demonstrates that NMP provides the platform to bind and block detrimental microRNAs in donor kidneys prior to transplantation.

miR-21 antagonism reprograms macrophage metabolism and abrogates chronic allograft vasculopathy.

Despite much progress in improving graft outcome during cardiac transplantation, chronic allograft vasculopathy (CAV) remains an impediment to long-term graft survival. MicroRNAs (miRNAs) emerged as regulators of the immune response. Here, we aimed to examine the miRNA network involved in CAV. miRNA profiling of heart samples obtained from a murine model of CAV and from cardiac-transplanted patients with CAV demonstrated that miR-21 was most significantly expressed and was primarily localized to macrophages. Interestingly, macrophage depletion with clodronate did not significantly prolong allograft survival in mice, while conditional deletion of miR-21 in macrophages or the use of a specific miR-21 antagomir resulted in indefinite cardiac allograft survival and abrogated CAV. The immunophenotype, secretome, ability to phagocytose, migration, and antigen presentation of macrophages were unaffected by miR-21 targeting, while macrophage metabolism was reprogrammed, with a shift toward oxidative phosphorylation in naïve macrophages and with an inhibition of glycolysis in pro-inflammatory macrophages. The aforementioned effects resulted in an increase in M2-like macrophages, which could be reverted by the addition of L-arginine. RNA-seq analysis confirmed alterations in arginase-associated pathways associated with miR-21 antagonism. In conclusion, miR-21 is overexpressed in murine and human CAV, and its targeting delays CAV onset by reprogramming macrophages metabolism.

Dominant regulation of long-term allograft survival is mediated by microRNA-142.

Organ transplantation is often lifesaving, but the long-term deleterious effects of combinatorial immunosuppression regimens and allograft failure cause significant morbidity and mortality. Long-term graft survival in the absence of continuing immunosuppression, defined as operational tolerance, has never been described in the context of multiple major histocompatibility complex (MHC) mismatches. Here, we show that miR-142 deficiency leads to indefinite allograft survival in a fully MHC mismatched murine cardiac transplant model in the absence of exogenous immunosuppression. We demonstrate that the cause of indefinite allograft survival in the absence of miR-142 maps specifically to the T cell compartment. Of therapeutic relevance, temporal deletion of miR-142 in adult mice prior to transplantation of a fully MHC mismatched skin allograft resulted in prolonged allograft survival. Mechanistically, miR-142 directly targets Tgfbr1 for repression in regulatory T cells (TREG ). This leads to increased TREG sensitivity to transforming growth factor - beta and promotes transplant tolerance via an augmented peripheral TREG response in the absence of miR-142. These data identify manipulation of miR-142 as a promising approach for the induction of tolerance in human transplantation.

Circulating exosomal miR-92b: Its role for cancer immunoediting and clinical value for prediction of posttransplant hepatocellular carcinoma recurrence.

A recurrence of hepatocellular carcinoma (HCC) after living donor liver transplantation (LDLT) is one of the major concerns reflecting the higher mortality of HCC. This study aimed to explore the impact of circulating exosomes on HCC development and recurrence. One-shot transfusion of hepatoma serum to naïve rats induced liver cancer development with gradual elevation of alpha-fetoprotein (AFP), but exosome-free hepatoma serum failed to induce AFP elevation. The microarray analysis revealed miR-92b as one of the highly expressing microribonucleic acids in hepatoma serum exosomes. Overexpression of miR-92b enhanced the migration ability of liver cancer cell lines with active release of exosomal miR-92b. The hepatoma-derived exosomal miR-92b transferred to natural killer (NK) cells, resulting in the downregulation of CD69 and NK cell-mediated cytotoxicity. Furthermore, higher expression of miR-92b in serum exosomes was confirmed in HCC patients before LDLT, and its value at 1 month after LDLT was maintained at a higher level in the patients with posttransplant HCC recurrence. In summary, we demonstrated the impact of circulating exosomes on liver cancer development, partly through the suppression of CD69 on NK cells by hepatoma-derived exosomal miR-92b. The value of circulating exosomal miR-92b may predict the risk of posttransplant HCC recurrence.

Defining a microRNA-mRNA interaction map for calcineurin inhibitor induced nephrotoxicity.

Calcineurin inhibitors induce nephrotoxicity through poorly understood mechanisms thereby limiting their use in transplantation and other diseases. Here we define a microRNA (miRNA)-messenger RNA (mRNA) interaction map that facilitates exploration into the role of miRNAs in cyclosporine-induced nephrotoxicity (CIN) and the gene pathways they regulate. Using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we isolated RNAs associated with Argonaute 2 in the RNA-induced silencing complex (RISC) of cyclosporine A (CsA) treated and control human proximal tubule cells and identified mRNAs undergoing active targeting by miRNAs. CsA causes specific changes in miRNAs and mRNAs associated with RISC, thereby altering post-transcriptional regulation of gene expression. Pathway enrichment analysis identified canonical pathways regulated by miRNAs specifically following CsA treatment. RNA-seq performed on total RNA indicated that only a fraction of total miRNAs and mRNAs are actively targeted in the RISC, indicating that PAR-CLIP more accurately defines meaningful targeting interactions. Our data also revealed a role for miRNAs in calcineurin-independent regulation of JNK and p38 MAPKs caused by targeting of MAP3K1. Together, our data provide a novel resource and unique insights into molecular pathways regulated by miRNAs in CIN. The gene pathways and miRNAs defined may represent novel targets to reduce calcineurin induced nephrotoxicity.

Successful Treatment of Kaposi Sarcoma-Associated Herpesvirus Inflammatory Cytokine Syndrome After Kidney-Liver Transplant: Correlations With the Human Herpesvirus 8 miRNome and Specific T Cell Response.

After transplant, patient infection with human herpesvirus 8 (HHV-8) and Kaposi sarcoma-associated herpesvirus (KSHV) is known to cause aggressive tumors and severe nonneoplastic complications. These latter syndromes are driven by HHV-8/KSHV lytic reactivations and related hyperinflammatory host responses typically characterized by high viral loads, elevated levels of cytokines and other inflammation biomarkers, cytopenia, organ failure, high fever, and worsening conditions (with no evidence of B cell neoplasias). These disorders are associated with a high mortality rate, often due to lack of prompt diagnosis, effective therapeutic approaches, and adequate follow-up. These features resemble most of those defining the so-called KSHV-associated inflammatory cytokine syndrome (KICS), which was recently recognized in patients positive for human immunodeficiency virus (HIV). In this report, we describe-for the first time-a case of a KICS-like nonneoplastic recurrent complication occurring after transplant in an HIV-negative patient that was successfully treated by a combination of anti-CD20 monoclonal therapy, antivirals, and modification of the immunosuppressive regimen. In addition to clinical and laboratory findings collected during 3-year follow-up, we report novel experimental data on HHV-8-specific T cell dynamics and circulating microRNA profile, showing correlations with clinical course and other laboratory markers (including viral load, C-reactive protein, and cytokine levels), providing useful information about abnormal cellular and cytokine dynamics underlying HHV-8-associated inflammatory disorders in posttransplant patients.

Inducing Hepatitis C Virus Resistance After Pig Liver Transplantation-A Proof of Concept of Liver Graft Modification Using Warm Ex Vivo Perfusion.

Normothermic ex vivo liver perfusion (NEVLP) offers the potential to optimize graft function prior to liver transplantation (LT). Hepatitis C virus (HCV) is dependent on the presence of miRNA(microRNA)-122. Miravirsen, a locked-nucleic acid oligonucleotide, sequesters miR-122 and inhibits HCV replication. The aim of this study was to assess the efficacy of delivering miravirsen during NEVLP to inhibit miR-122 function in a pig LT model. Pig livers were treated with miravirsen during NEVLP or cold storage (CS). Miravirsen absorption, miR-122 sequestration, and miR-122 target gene derepression were determined before and after LT. The effect of miravirsen treatment on HCV infection of hepatoma cells was also assessed. NEVLP improved miravirsen uptake versus CS. Significant miR-122 sequestration and miR-122 target gene derepression were seen with NEVLP but not with CS. In vitro data confirmed miravirsen suppression of HCV replication after established infection and prevented HCV infection with pretreatment of cells, analogous to the pretreatment of grafts in the transplant setting. In conclusion, miravirsen delivery during NEVLP is a potential strategy to prevent HCV reinfection after LT. This is the first large-animal study to provide "proof of concept" for using NEVLP to modify and optimize liver grafts for transplantation.

RNA Profiling in Human and Murine Transplanted Hearts: Identification and Validation of Therapeutic Targets for Acute Cardiac and Renal Allograft Rejection.

Acute cellular rejection (ACR) is the adverse response of the recipient's immune system against the allogeneic graft. Using human surveillance endomyocardial biopsies (EMBs) manifesting ACR and murine allogeneic grafts, we profiled implicated microRNAs (miRs) and mRNAs. MiR profiling showed that miR-21, -142-3p, -142-5p, -146a, -146b, -155, -222, -223, and -494 increased during ACR in humans and mice, whereas miR-149-5p decreased. mRNA profiling revealed 70 common differentially regulated transcripts, all involved in immune signaling and immune-related diseases. Interestingly, 33 of 70 transcripts function downstream of IL-6 and its transcription factor spleen focus forming virus proviral integration oncogene (SPI1), an established target of miR-155, the most upregulated miR in human EMBs manifesting rejection. In a mouse model of cardiac transplantation, miR-155 absence and pharmacological inhibition attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRs. Finally, we corroborated our miR signature in acute cellular renal allograft rejection, suggesting a nonorgan specific signature of acute rejection. We concluded that miR and mRNA profiling in human and murine ACR revealed the shared significant dysregulation of immune genes. Inflammatory miRs, for example miR-155, and transcripts, in particular those related to the IL-6 pathway, are promising therapeutic targets to prevent acute allograft rejection.

MicroRNA-494 promotes cyclosporine-induced nephrotoxicity and epithelial to mesenchymal transition by inhibiting PTEN.

A major complication associated with cyclosporine (CsA) treatment is nephrotoxicity. In this study, we examined whether microRNAs play a role in cyclosporine-induced nephrotoxicity. Treatment of mice with CsA resulted in nephrotoxicity that was associated with an early increase in expression of microRNA mmu-miR-494 (miR-494). Similarly, tubular epithelial cell epithelial-mesenchymal transition (EMT) induced by CsA toxicity resulted in the upregulation of microRNA-494 and a decrease in PTEN levels in vitro. miR-494 directly targeted Pten and negatively regulated its expression. Preventing Pten targeting by miR-494 was sufficient to prevent CsA induced EMT. Knockdown of miR-494 prevented the downregulation of PTEN in tubular epithelial cells following CsA treatment and also prevented CsA induced EMT. Thus, miR-494 plays a major role in promoting CsA induced nephrotoxicity through its ability to target Pten thereby contributing to EMT. We suggest that manipulating miR-494 expression may represent a novel approach to preventing EMT associated with CsA induced nephrotoxicity.

Hcmv-miR-UL22A-5p: A Biomarker in Transplantation With Broad Impact on Host Gene Expression and Potential Immunological Implications.

Cytomegalovirus (CMV) encodes multiple microRNAs. While these have been partially characterized in vitro, their relevance to clinical CMV infection has not been evaluated. We analyzed samples from a cohort of solid organ transplant patients with CMV disease (n = 245) for viral microRNA expression. Several CMV microRNAs were readily detectable in patients with CMV disease in variable relative abundance. Expression level generally correlated with DNA viral load and the absence of viral microRNA was associated with faster viral clearance. Detection of hcmv-miR-UL22A-5p at baseline independently predicted the recurrence of CMV viremia upon discontinuation of antiviral therapy (OR 3.024, 95% CI: 1.35-6.8; p = 0.007). A combination of direct mRNA targeting by the microRNA and indirect modulation of gene expression involving isoforms of the transcriptional regulator C-MYC may be responsible for the broad effects seen in the association of gene transcripts with the RNA-induced silencing complex and in global protein expression upon hcmv-miR-UL22A-5p transfection. This novel study of in vivo viral microRNA expression profiles provides unique insight into the complexity of clinical CMV infection following transplantation. We provide evidence that viral microRNAs may have complex effects on gene expression and be associated with specific virologic and clinical outcomes, and thus could be further evaluated as biomarkers.

Class-specific histone/protein deacetylase inhibition protects against renal ischemia reperfusion injury and fibrosis formation.

Renal ischemia-reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure. Histone/protein deacetylases (HDACs) regulate gene accessibility and higher order protein structures and may alter cellular responses to a variety of stresses. We investigated whether use of pan- and class-specific HDAC inhibitors (HDACi) could improve IRI tolerance in the kidney. Using a model of unilateral renal IRI, we investigated early renal function after IRI, and calculated fibrosis after IRI using an automated scoring system. We found that pan-HDAC inhibition using trichostatin (TSA) yielded significant renal functional benefit at 24-96 hours (p < 0.001). Treated mice developed significantly less fibrosis at 30 days (p < 0.0004). Class I HDAC inhibition with MS-275 yielded similar effects. Protection from fibrosis formation was also noted in a cold ischemia transplant model (p < 0.008) with a trend toward improved cold ischemic survival in TSA-treated mice. These effects were not accompanied by induction of typical ischemic tolerance pathways or by priming of heat shock protein expression. In fact, heat shock protein 70 deletion or overexpression did not alter renal ischemia tolerance. Micro-RNA 21, known to be enhanced in vitro in renal tubular cells that survive stress, was enhanced by treatment with HDACi, pointing to possible mechanism.

Circulating microRNAs associate with diabetic nephropathy and systemic microvascular damage and normalize after simultaneous pancreas-kidney transplantation.

Because microvascular disease is one of the most important drivers of diabetic complications, early monitoring of microvascular integrity may be of clinical value. By assessing profiles of circulating microRNAs (miRNAs), known regulators of microvascular pathophysiology, in healthy controls and diabetic nephropathy (DN) patients before and after simultaneous pancreas-kidney transplantation (SPK), we aimed to identify differentially expressed miRNAs that associate with microvascular impairment. Following a pilot study, we selected 13 candidate miRNAs and determined their circulating levels in DN (n = 21), SPK-patients (n = 37), healthy controls (n = 19), type 1 diabetes mellitus patients (n = 15) and DN patients with a kidney transplant (n = 15). For validation of selected miRNAs, 14 DN patients were studied longitudinally up to 12 months after SPK. We demonstrated a direct association of miR-25, -27a, -126, -130b, -132, -152, -181a, -223, -320, -326, -340, -574-3p and -660 with DN. Of those, miR-25, -27a, -130b, -132, -152, -320, -326, -340, -574-3p and -660 normalized after SPK. Importantly, circulating levels of some of these miRNAs tightly associate with microvascular impairment as they relate to aberrant capillary tortuosity, angiopoietin-2/angiopoietin-1 ratios, circulating levels of soluble-thrombomodulin and insulin-like growth factor. Taken together, circulating miRNA profiles associate with DN and systemic microvascular damage, and might serve to identify individuals at risk of experiencing microvascular complications, as well as give insight into underlying pathologies.

A comprehensive analysis of the cellular and EBV-specific microRNAome in primary CNS PTLD identifies different patterns among EBV-associated tumors.

Primary central nervous system (pCNS) posttransplant lymphoproliferative disorder (PTLD) is a complication of solid organ transplantation characterized by poor outcome. In contrast to systemic PTLD, Epstein-Barr virus (EBV)-association of pCNS PTLD is almost universal, yet viral and cellular data are limited. To identify differences in the pattern of EBV-association of pCNS and systemic PTLD, we analyzed the expression of latent and lytic EBV transcripts and the viral and cellular microRNAome in nine pCNS (eight EBV-associated) and in 16 systemic PTLD samples (eight EBV-associated). Notably although 15/16 EBV-associated samples exhibited a viral type III latency pattern, lytic transcripts were also strongly expressed. Members of the ebv-miR-BHRF1 and ebv-miR-BART clusters were expressed in virtually all EBV-associated PTLD samples. There were 28 cellular microRNAs differentially expressed between systemic and pCNS PTLD. pCNS PTLD expressed lower hsa-miR-199a-5p/3p and hsa-miR-143/145 (implicated in nuclear factor kappa beta and c-myc signaling) as compared to systemic PTLD. Unsupervised nonhierarchical clustering of the viral and cellular microRNAome distinguished non-EBV-associated from EBV-associated samples and identified a separate group of EBV-associated pCNS PTLD that displayed reduced levels of B cell lymphoma associated oncomiRs such as hsa-miR-155, -21, -221 and the hsa-miR-17-92 cluster. EBV has a major impact on viral and cellular microRNA expression in EBV-associated pCNS PTLD.