Complement Blockade in Recipients Prevents Delayed Graft Function and Delays Antibody-mediated Rejection in a Nonhuman Primate Model of Kidney Transplantation.

BACKGROUND: Complement activation in kidney transplantation is implicated in the pathogenesis of delayed graft function (DGF). This study evaluated the therapeutic efficacy of high-dose recombinant human C1 esterase inhibitor (rhC1INH) to prevent DGF in a nonhuman primate model of kidney transplantation after brain death and prolonged cold ischemia. METHODS: Brain death donors underwent 20 h of conventional management. Procured kidneys were stored on ice for 44-48 h, then transplanted into ABO-compatible major histocompatibility complex-mismatched recipients. Recipients were treated with vehicle (n = 5) or rhC1INH 500 U/kg plus heparin 40 U/kg (n = 8) before reperfusion, 12 h, and 24 h posttransplant. Recipients were followed up for 120 d. RESULTS: Of vehicle-treated recipients, 80% (4 of 5) developed DGF versus 12.5% (1 of 8) rhC1INH-treated recipients (P = 0.015). rhC1INH-treated recipients had faster creatinine recovery, superior urinary output, and reduced urinary neutrophil gelatinase-associated lipocalin and tissue inhibitor of metalloproteinases 2-insulin-like growth factor-binding protein 7 throughout the first week, indicating reduced allograft injury. Treated recipients presented lower postreperfusion plasma interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, and IL-18, lower day 4 monocyte chemoattractant protein 1, and trended toward lower C5. Treated recipients exhibited less C3b/C5b-9 deposition on day 7 biopsies. rhC1INH-treated animals also trended toward prolonged mediated rejection-free survival. CONCLUSIONS: Our results recommend high-dose C1INH complement blockade in transplant recipients as an effective strategy to reduce kidney injury and inflammation, prevent DGF, delay antibody-mediated rejection development, and improve transplant outcomes.

Targeted donor complement blockade after brain death prevents delayed graft function in a nonhuman primate model of kidney transplantation.

Delayed graft function (DGF) in renal transplant is associated with reduced graft survival and increased immunogenicity. The complement-driven inflammatory response after brain death (BD) and posttransplant reperfusion injury play significant roles in the pathogenesis of DGF. In a nonhuman primate model, we tested complement-blockade in BD donors to prevent DGF and improve graft survival. BD donors were maintained for 20 hours; kidneys were procured and stored at 4°C for 43-48 hours prior to implantation into ABO-compatible, nonsensitized, MHC-mismatched recipients. Animals were divided into 3 donor-treatment groups: G1 - vehicle, G2 - rhC1INH+heparin, and G3 - heparin. G2 donors showed significant reduction in classical complement pathway activation and decreased levels of tumor necrosis factor α and monocyte chemoattractant protein 1. DGF was diagnosed in 4/6 (67%) G1 recipients, 3/3 (100%) G3 recipients, and 0/6 (0%) G2 recipients (P = .008). In addition, G2 recipients showed superior renal function, reduced sC5b-9, and reduced urinary neutrophil gelatinase-associated lipocalin in the first week posttransplant. We observed no differences in incidence or severity of graft rejection between groups. Collectively, the data indicate that donor-management targeting complement activation prevents the development of DGF. Our results suggest a pivotal role for complement activation in BD-induced renal injury and postulate complement blockade as a promising strategy for the prevention of DGF after transplantation.

Kidney After Liver Transplantation Matched-pair Analysis: Are Kidneys Allocated to Appropriate Patients to Maximize Their Survival?

BACKGROUND: Kidney after liver transplantation (KALT) is the best therapeutic option for patients with end-stage renal disease after orthotopic liver transplantation (OLT). New allocation policies prioritize kidneys to patients in renal failure within the first year following OLT. There is little data on how kidney quality, measured by kidney donor profile index (KDPI), impacts KALT survival outcomes. METHODS: The United Network for Organ Sharing database was queried for adult KALT recipients from 1988 to 2015 and compared to their paired kidney transplant alone (KTA) recipients. Seven hundred forty-five pairs were stratified into 3 KDPI subgroups and compared patient survival, graft survival, and death-censored graft survival among matched-paired recipients. RESULTS: Overall, KTA recipients had superior patient and graft survival compared with the KALT group. KTA patient survival was superior for all 3 KDPI subgroups analysis. KTA graft survival was superior compared with KALT recipients of KDPI 21%-85% kidneys. Inferior graft half-life was observed in KALT versus KTA recipients with KDPI 21%-85% and >85%. CONCLUSIONS: From a utilitarian perspective, it is important that kidneys are allocated to recipients that are able to maximize their benefit from the full life of the organ. In KTA recipients, graft quality correlates directly to graft survival. However, in KALT patients receiving the matched-pair kidneys of the KTA recipients, patient mortality, rather than kidney quality, dictates graft survival significantly. As allocation practices continue developing, utilization of expanded criteria kidneys that better match anticipated patient and graft survival should be strongly considered to maximize the benefits of limited resources for the greatest number of patients.

Brain Death Enhances Activation of the Innate Immune System and Leads to Reduced Renal Metabolic Gene Expression.

BACKGROUND: Brain death (BD)-associated inflammation has been implicated in decreased kidney allograft function and survival, but the underlying mechanisms have not been well distinguished from the conditions of critical care itself. We have developed a clinically translatable model to separate and investigate strategies to improve donor management and critical care. METHODS: Brain-dead (n = 12) and sham (n = 5) rhesus macaques were maintained for 20 hours under intensive care unit-level conditions. Samples were collected for immunophenotyping, analysis of plasma proteins, coagulation studies, and gene analysis for changes in immune and metabolic profile with comparison to naive samples (n = 10). RESULTS: We observed an increase in circulating leukocytes and cytokines, activation of complement and coagulation pathways, and upregulation of genes associated with inflammation in both brain-dead and sham subjects relative to naïve controls. Sham demonstrated an intermediate phenotype of inflammation compared to BD. Analysis of gene expression in kidneys from BD kidneys revealed a similar upregulation of inflammatory profile in both BD and sham subjects, but BD presented a distinct reduction in metabolic and respiratory processes compared to sham and naïve kidneys. CONCLUSION: BD is associated with activation of specific pathways of the innate immune system and changes to metabolic gene expression in renal tissue itself; however, sham donors presented an intermediate inflammatory response attributable to the critical care environment. The early onset and penetrating impact of this inflammatory response underscores the need for early intervention to prevent perioperative tissue injury to transplantable organs.

The impact of kidney donor profile index on delayed graft function and transplant outcomes: A single-center analysis.

INTRODUCTION: Renal transplant outcomes result from a combination of factors. Traditionally, donor factors were summarized by classifying kidneys as extended criteria or standard criteria. In 2014, the nomenclature changed to describe donor factors with the kidney donor profile index (KDPI). We aim to evaluate the relationship between KDPI and delayed graft function (DGF), and the impact KDPI on transplant outcomes for both donor after cardiac death (DCD) and donor after brain death (DBD). METHODS: An IRB-approved single-center retrospective chart review was performed from January 1999 to July 2013. The patients were divided into six groups: DBD KDPI ≤60, DBD KPDI 61-84, DBD KDPI ≥85, DCD KDPI ≤60, DCD KPDI 61-84, and DCD KDPI ≥85. Rates of DGF, patient survival, and graft survival were examined among groups. RESULTS: A total of 2161 kidney transplants were included. DGF rates increased, and graft and patient survival decreased with increasing KDPI (P < .001). DCD kidneys had higher DGF rates than their DBD counterparts (P < .001). In DCD kidneys, a higher KDPI score did not significantly affect the DGF rates (P > .302). There was no significant difference in graft or patient survival in all-comers when comparing DCD and DBD kidneys with equivalent KDPIs (P > .317). Patients with DGF across all categories demonstrated worse graft half-lives. CONCLUSION: The KDPI system is an accurate predictor of donor contributions to transplant outcomes. Recipients of DBD kidneys experience an increase in the rate of DGF as their KDPI increases. DCD kidneys have higher DGF rates than their DBD counterparts with similar KDPIs. Patients with documented post-transplant DGF had between 3- and 5-year shorter graft half-lives when compared to recipients that did not experience DGF. Initiatives to reduce the rate of DGF could provide a significant impact on graft survival and result in a reduction in the number of patients requiring retransplant.

Guidelines for the management of a brain death donor in the rhesus macaque: A translational transplant model.

INTRODUCTION: The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature. METHODS: Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed. RESULTS: A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal's temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model. CONCLUSION: We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials.

Complement inhibition attenuates acute kidney injury after ischemia-reperfusion and limits progression to renal fibrosis in mice.

The complement system is an essential component of innate immunity and plays a major role in the pathogenesis of ischemia-reperfusion injury (IRI). In this study, we investigated the impact of human C1-inhibitor (C1INH) on the early inflammatory response to IRI and the subsequent progression to fibrosis in mice. We evaluated structural damage, renal function, acute inflammatory response, progression to fibrosis and overall survival at 90-days post-injury. Animals receiving C1INH prior to reperfusion had a significant improvement in survival rate along with superior renal function when compared to vehicle (PBS) treated counterparts. Pre-treatment with C1INH also prevented acute IL-6, CXCL1 and MCP-1 up-regulation, C5a release, C3b deposition and infiltration by neutrophils and macrophages into renal tissue. This anti-inflammatory effect correlated with a significant reduction in the expression of markers of fibrosis alpha smooth muscle actin, desmin and picrosirius red at 30 and 90 days post-IRI and reduced renal levels of TGF-ß1 when compared to untreated controls. Our findings indicate that intravenous delivery of C1INH prior to ischemic injury protects kidneys from inflammatory injury and subsequent progression to fibrosis. We conclude that early complement blockade in the context of IRI constitutes an effective strategy in the prevention of fibrosis after ischemic acute kidney injury.

Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion.

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ∼50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion.