Juan S. Danobeitia

The role of complement in the pathogenesis of renal ischemia-reperfusion injury and fibrosis

The complement system is a major component of innate immunity and has been commonly identified as a central element in host defense, clearance of immune complexes, and tissue homeostasis. After ischemia-reperfusion injury (IRI), the complement system is activated by endogenous ligands that trigger proteolytic cleavage of complement components via the classical, lectin and/or alternative pathway. The result is the formation of terminal complement components C3a, C5a, and the membrane attack complex (C5b-9 or MAC), all of which play pivotal roles in the amplification of the inflammatory response, chemotaxis, neutrophil/monocyte recruitment and activation, and direct tubular cell injury. However, recent evidence suggests that complement activity transcends innate host defense and there is increasing data suggesting complement as a regulator in processes such as allo-immunity, stem cell differentiation, tissue repair, and progression to fibrosis. In this review, we discuss recent advances addressing the role of complement as a regulator of IRI and renal fibrosis after organ donation for transplantation. We will also briefly discuss currently approved therapies that target complement activity in kidney ischemia-reperfusion and transplantation.

IL-1β Receptor Blockade Protects Islets Against Pro-inflammatory Cytokine Induced Necrosis and Apoptosis

Pro‐inflammatory cytokines (PIC) impair islet viability and function by activating inflammatory pathways that induce both necrosis and apoptosis. The aim of this study was to utilize an in vitro rat islet model to evaluate the efficacy of a clinically approved IL‐1 receptor antagonist (Anakinra) in blocking PIC induced islet impairment. Isolated rat islets were cultured for 48 h ± PIC (IL‐1β, IFNγ, and TNFα) and ±IL‐1ra then assayed for cellular integrity by flow cytometry, MAPK phosphorylation by proteome array, and gene expression by RT‐PCR. Nitric oxide (NO) release into the culture media was measured by Griess reaction. Islet functional potency was tested by glucose stimulated insulin secretion (GSIS) and by transplantation into streptozotocin‐induced diabetic NOD.scid mice. Rat islets cultured with PIC upregulated genes for NOS2a, COX2, IL6, IL1b, TNFa, and HMOX1. IL‐1ra prevented the PIC induced upregulation of all of these genes except for TNFa. Inhibition of PIC induced iNOS by NG‐monomethyl‐L‐arginine (NMMA) only blocked the increased expression of HMOX1. IL‐1ra completely abrogated the effects of PIC with respect to NO production, necrosis, apoptosis, mitochondrial dysfunction, GSIS, and in vivo potency. IL‐1ra was not effective at preventing the induction of necrosis or apoptosis by exogenous NO. These data demonstrate that Anakinra is an effective agent to inhibit the activation of IL‐1β dependent inflammatory pathways in cultured rat islets and support the extension of its application to human islets in vitro and potentially as a post transplant therapy. J. Cell. Physiol. 220: 341–347, 2009.

A Simplified Approach to Human Islet Quality Assessment

Background. The successful treatment of patients with type 1 diabetes by islet transplantation is affected by a multitude of factors of which infusion of the highest quality tissue is essential. The current standard pretransplant quality assessments lack sensitivity, accuracy, and objectivity in the determination of islet viability and potency. We hypothesized that a multiparametric approach focused on islet cell metabolic state, mitochondrial integrity, and in vitro glucose-stimulated insulin secretion (GSIS) could provide data predictive of in vivo function. The objective of this study was to validate a novel set of islet quality assays and develop a simplified islet quality scoring system for both basic research and clinical applications. Methods. A series of 42 human islet preparations were screened using standard and novel methods, which included determination of yield, viability by fluorescent microscopy, GSIS, percentage of islet loss in culture, quantification of adenine nucleotides, flow cytometric measurement of viability, apoptosis, and mitochondrial membrane potential (MMP). In vivo functional potency was tested by minimal model transplant in streptozotocin-induced diabetic NOD.scid mice. Results. Functionally potent islet preparations showed significantly greater numbers of cells with polarized MMP, higher ATP-to-ADP ratios, and increased glucose-induced insulin secretion. The MMP, ATP-to-ADP ratio, and GSIS data were combined into a single islet scoring formula that showed more than 86% accuracy in predicting in vivo functional potency. Conclusions. Our study demonstrates that a multiparametric approach using objective assessments focused on islet cell mitochondrial integrity and in vitro function can provide data predictive of in vivo function.

Early Activation of the Inflammatory Response in the Liver of Brain-Dead Non-Human Primates

BACKGROUND Donor brain death (BD) triggers a systemic inflammatory response that reduces organ quality and increases immunogenicity of the graft. We characterized the early innate immune response induced by BD in the liver and peripheral blood of hemodinamically stable non-human primates (NHP). METHODS Rhesus macaques were assigned to either brain death or control group. BD was induced by inflation of a subdurally placed catheter and confirmed clinically and by cerebral angiography. Animals were monitored for 6 h after BD and managed to maintain hemodynamic stability. RESULTS Cortisol, epinephrine, nor-epinephrine, and IL-6 levels were elevated immediately after BD induction. Neutrophils and monocytes significantly increased in circulation following BD induction, while dendritic cells were decreased at 6 h post-induction. Flow cytometry revealed increased expression of chemokine receptors CxCR1, CxCR2, CCR2, and CCR5 in peripheral blood leukocytes from NHP subjected to BD. Microarray analysis demonstrated a significant up-regulation of genes related to innate inflammatory responses, toll-like receptor signaling, stress pathways, and apoptosis/cell death in BD subjects. Conversely, pathways related to glucose, lipid, and protein metabolism were down-regulated. In addition, increased expression of SOCS3, S100A8/A9, ICAM-1, MHC class II, neutrophil accumulation, and oxidative stress markers (carboxy-methyl-lysine and hydroxynonenal) were detected by immunoblot and immunohistochemistry. CONCLUSIONS Activation of the innate immune response after BD in association with a down-regulation of genes associated with cell metabolism pathways in the liver. These findings may provide a potential explanation for the reduced post-transplant function of organs from brain dead donors. In addition, this work suggests potential novel targets to improve donor management strategies.

Correction of insulin sensitivity and glucose disposal after pancreatic islet transplantation: preliminary results.

Aims: Pancreatic islet transplantation (PIT) represents a potential curative treatment for patients with type 1 diabetes, but only 10–15% of patients remain insulin independent 5 years post‐transplant. It is not known whether intrinsic insulin resistance exacerbated by immunosuppression plays a pivotal role in low graft survival. The study objective was to understand the changes in insulin resistance, glucose effectiveness (Sg) and free fatty acid dynamics (FFAd) before and after PIT.

A multicenter study: North american islet donor score in donor pancreas selection for human islet isolation for transplantation

Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents). With the aid of univariate logistic regression analyses, we developed the North American Islet Donor Score (NAIDS) ranging from 0 to 100 points. The c index in the development cohort was 0.73 (95% confidence interval 0.70–0.76). The success rate increased proportionally as the NAIDS increased, from 6.8% success in the NAIDS < 50 points to 53.7% success in the NAIDS ≥ 80 points. We further validated the NAIDS using a separate set of data consisting of 179 islet isolations. A comparable outcome of the NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in clinical practice. Apart from its utility in clinical decision making, the NAIDS may also be used in a research setting as a standardized measurement of pancreas quality.

Early metabolic markers that anticipate loss of insulin independence in type 1 diabetic islet allograft recipients

The objective of this study was to identify predictors of insulin independence and to establish the best clinical tools to follow patients after pancreatic islet transplantation (PIT). Sequential metabolic responses to intravenous (I.V.) glucose (I.V. glucose tolerance test [IVGTT]), arginine and glucose‐potentiated arginine (glucose‐potentiated arginine‐induced insulin secretion [GPAIS]) were obtained from 30 patients. We determined the correlation between transplanted islet mass and islet engraftment and tested the ability of each assay to predict return to exogenous insulin therapy. We found transplanted islet mass within an average of 16 709 islet equivalents per kg body weight (IEQ/kg BW; range between 6602 and 29 614 IEQ/kg BW) to be a poor predictor of insulin independence at 1 year, having a poor correlation between transplanted islet mass and islet engraftment. Acute insulin response to IVGTT (AIRGLU) and GPAIS (AIRmax) were the most accurate methods to determine suboptimal islet mass engraftment. AIRGLU performed 3 months after transplant also proved to be a robust early metabolic marker to predict return to insulin therapy and its value was positively correlated with duration of insulin independence. In conclusion, AIRGLU is an early metabolic assay capable of anticipating loss of insulin independence at 1 year in T1D patients undergoing PIT and constitutes a valuable, simple and reliable method to follow patients after transplant.

Flow cytometric quantification of glucose-stimulated beta-cell metabolic flux can reveal impaired islet functional potency.

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet β-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+i) (Fura Red), stimulated with glucose, and analyzed on a FACS VantageTM flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet β-cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+i. Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and β-cell glucose responsiveness as indicators of functional potency.

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-β1 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.

Donor Pretreatment With IL-1 Receptor Antagonist Attenuates Inflammation and Improves Functional Potency in Islets From Brain-Dead Nonhuman Primates.

Most pancreas and islet grafts are recovered from brain-dead (BD) donors. In this study we characterized the early inflammatory response induced by brain death in pancreata and islets from nonhuman primate donors and evaluated the effect of targeted anti-inflammatory intervention in the protection of pancreatic islets prior to transplantation. BD donors were monitored for 6 h and assigned to three experimental groups: group 1: BD-untreated donors (BD-UT) (n = 7), group 2: BD + donor pretreatment with IL-1ra (n = 6), and group 3: non-BD animals serving as controls (n = 7). We observed an IL-1ra-dependent reduction in the mobilization and activation of neutrophils from bone marrow and a significantly reduced accumulation of CD68+ leukocytes in the pancreas and islets after brain death induction. Donor treatment with IL-1ra significantly decreased chemokine mRNA expression (MCP-1, IL-8, and MIP-1a) and attenuated the activation of circulating neutrophils and intraislet macrophages as demonstrated by a reduction in intracellular IL-1β, IL-6, MCP-1, and MIP-1α expression. As a result, IL-1ra dramatically improved viability, mitochondrial membrane polarity, and islet engraftment in mice transplanted using a minimal islet mass. These results suggest that early immunomodulation targeting inflammation in the BD donor may represent an effective therapeutic strategy to improve islet quality and function prior to transplantation.