Study of the Role of the Tyrosine Kinase Receptor MerTK in the Development of Kidney Ischemia-Reperfusion Injury in RCS Rats

International audienceRenal ischaemia reperfusion (I/R) triggers a cascade of events including oxidative stress, apoptotic body and microparticle (MP) formation as well as an acute inflammatory process that may contribute to organ failure. Macrophages are recruited to phagocytose cell debris and MPs. The tyrosine kinase receptor MerTK is a major player in the phagocytosis process. Experimental models of renal I/R events are of major importance for identifying I/R key players and for elaborating novel therapeutical approaches. A major aim of our study was to investigate possible involvement of MerTK in renal I/R. We performed our study on both natural mutant rats for MerTK (referred to as RCS) and on wild type rats referred to as WT. I/R was established by of bilateral clamping of the renal pedicles for 30′ followed by three days of reperfusion. Plasma samples were analysed for creatinine, aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), kidney injury molecule -1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) levels and for MPs. Kidney tissue damage and CD68-positive cell requirement were analysed by histochemistry. monocyte chemoattractant protein-1 (MCP-1), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and histone 3A (H3A) levels in kidney tissue lysates were analysed by western blotting. The phagocytic activity of blood-isolated monocytes collected from RCS or WT towards annexin-V positive bodies derived from cultured renal cell was assessed by fluorescence-activated single cell sorting (FACS) and confocal microscopy analyses. The renal I/R model for RCS rat described for the first time here paves the way for further investigations of MerTK-dependent events in renal tissue injury and repair mechanisms

Dynamic transcriptomic analysis of ischemic injury in a porcine pre-clinical model mimicking donors deceased after circulatory death

Présenté au: 18. Annual Congress of the French Speaking Transplantation SocietyToulouse FranceDate: 4 au 7 décembre 2018International audienceIntroduction: Due to organ shortage, clinicians are prone to consider alternative type of organ donors among them donors deceased after circulatory death (DCD). However, especially using these organs which are more prone to graft dysfunction, there is a need to better understand mechanistic events occurring during ischemia phase and leading to ischemia/reperfusion injuries (IRI). The aim of this study is to provide a dynamic transcriptomic analysis of preclinical porcine model kidneys subjected to ischemic stress mimicking DCD donor.Methods: We compared cortex and corticomedullary junction (CMJ) tissues from porcine kidneys submitting to 60 min warm ischemia (WI) followed by 0, 6 or 24 h of cold storage in University of Wisconsin solution versus control non‐ischemic kidneys (n = 5 per group).Results: 29 cortex genes and 113 CMJ genes were significantly up or down‐regulated after WI versus healthy kidneys, and up to 400 genes were regulated after WI and more 6 or 24 h of cold storage (p < 0.05). Home selected gene kinetic classification, Gene‐ontology‐biological processes and Gene‐ontology‐molecular‐function functional enrichment analysis revealed relevant genes implication during WI and cold storage.Conclusion: We uncovered targets which we will further validate as biomarkers and new therapeutic targets to optimize graft kidney quality before transplantation and improve whole transplantation outcome

Dynamic transcriptomic analysis of ischemic injury in a porcine pre-clinical model mimicking donors deceased after circulatory death

Présenté au: 18. Annual Congress of the French Speaking Transplantation SocietyToulouse FranceDate: 4 au 7 décembre 2018Introduction: Due to organ shortage, clinicians are prone to consider alternative type of organ donors among them donors deceased after circulatory death (DCD). However, especially using these organs which are more prone to graft dysfunction, there is a need to better understand mechanistic events occurring during ischemia phase and leading to ischemia/reperfusion injuries (IRI). The aim of this study is to provide a dynamic transcriptomic analysis of preclinical porcine model kidneys subjected to ischemic stress mimicking DCD donor.Methods: We compared cortex and corticomedullary junction (CMJ) tissues from porcine kidneys submitting to 60 min warm ischemia (WI) followed by 0, 6 or 24 h of cold storage in University of Wisconsin solution versus control non‐ischemic kidneys (n = 5 per group).Results: 29 cortex genes and 113 CMJ genes were significantly up or down‐regulated after WI versus healthy kidneys, and up to 400 genes were regulated after WI and more 6 or 24 h of cold storage (p < 0.05). Home selected gene kinetic classification, Gene‐ontology‐biological processes and Gene‐ontology‐molecular‐function functional enrichment analysis revealed relevant genes implication during WI and cold storage.Conclusion: We uncovered targets which we will further validate as biomarkers and new therapeutic targets to optimize graft kidney quality before transplantation and improve whole transplantation outcome

Dynamic transcriptomic analysis of ischemic injury in a porcine pre-clinical model mimicking donors deceased after circulatory death

Due to organ shortage, clinicians are prone to consider alternative type of organ donors among them donors deceased after circulatory death (DCD). However, especially using these organs which are more prone to graft dysfunction, there is a need to better understand mechanistic events ocuring during ischemia phase and leading to ischemia/reperfusion injuries (IRI). The aim of this study is to provide a dynamic transcriptomic analysis of preclinical porcine model kidneys subjected to ischemic stress mimicking DCD donor. We compared cortex and corticomedullary junction (CMJ) tissues from porcine kidneys submitted to 60 min warm ischemia (WI) followed by 0, 6 or 24 hours of cold storage in University of Wisconsin solution versus control non-ischemic kidneys (n=5 per group). 29 cortex genes and 113 CMJ genes were significantly up or down-regulated after WI versus healthy kidneys, and up to 400 genes were regulated after WI followed by 6 or 24 hours of cold storage (p < 0.05). Functionnal enrichment analysis (home selected gene kinetic classification, Gene-ontology-biological processes and Gene-ontology-molecular-function) revealed relevant genes implication during WI and cold storage. We uncovered targets which we will further validate as biomarkers and new therapeutic targets to optimize graft kidney quality before transplantation and improve whole transplantation outcome

The alarmin concept applied to human renal transplantation: evidence for a differential implication of HMGB1 and IL-33.

The endogenous molecules high mobility group box 1 (HMGB1) and interleukin-33 (IL-33) have been identified as alarmins, capable of mediating danger signals during tissue damage. Here, we address their possible role as innate-immune mediators in ischemia-reperfusion injury (IRI) following human kidney transplantation. We analysed serum and urinary HMGB1 and IL-33 levels, all determined by enzyme-linked immunosorbent assay, in a cohort of 26 deceased renal transplant recipients. Urinary HMGB1 and IL-33 levels were significantly increased as soon as 30 min after reperfusion, as compared to those before treatment. Moreover, both serum and urinary IL-33 (but not HMGB1) increase was positively correlated with cold ischemia time, from 30 min to 3 days post-transplantation. In vitro, human umbilical vein endothelial cells subjected to hypoxia conditions released both HMGB-1 and IL-33, while only the latter was further increased upon subsequent re-oxygenation. Finally, we postulate that leukocytes from renal recipient patients are targeted by both HMGB1 and IL-33, as suggested by increased transcription of their respective receptors (TLR2/4 and ST2L) shortly after transplantation. Consistent with this view, we found that iNKT cells, an innate-like T cell subset involved in IRI and targeted by IL-33 but not by HMGB1 was activated 1 hour post-transplantation. Altogether, these results are in keeping with a potential role of IL-33 as an innate-immune mediator during kidney IRI in humans

Defining the optimal duration for normothermic regional perfusion in the kidney donor: A porcine preclinical study

International audienceKidneys from donation after circulatory death (DCD) are highly sensitive to ischemia‐reperfusion injury and thus require careful reconditioning, such as normothermic regional perfusion (NRP). However, the optimal NRP protocol remains to be characterized. NRP was modeled in a DCD porcine model (30 minutes of cardiac arrest) for 2, 4, or 6 hours compared to a control group (No‐NRP); kidneys were machine‐preserved and allotransplanted. NRP appeared to permit recovery from warm ischemia, possibly due to an increased expression of HIF1α‐dependent survival pathway. At 2 hours, blood levels of ischemic injury biomarkers increased: creatinine, lactate/pyruvate ratio, LDH, AST, NGAL, KIM‐1, CD40 ligand, and soluble‐tissue‐factor. All these markers then decreased with time; however, AST, NGAL, and KIM‐1 increased again at 6 hours. Hemoglobin and platelets decreased at 6 hours, after which the procedure became difficult to maintain. Regarding inflammation, active tissue‐factor, cleaved PAR‐2 and MCP‐1 increased by 4‐6 hours, but not TNF‐α and iNOS. Compared to No‐NRP, NRP kidneys showed lower resistance during hypothermic machine perfusion (HMP), likely associated with pe‐NRP eNOS activation. Kidneys transplanted after 4 and 6 hours of NRP showed better function and outcome, compared to No‐NRP. In conclusion, our results confirm the mechanistic benefits of NRP and highlight 4 hours as its optimal duration, after which injury markers appear

The Alarmin Concept Applied to Human Renal Transplantation: Evidence for a Differential Implication of HMGB1 and IL-33

<div><p>The endogenous molecules high mobility group box 1 (HMGB1) and interleukin-33 (IL-33) have been identified as alarmins, capable of mediating danger signals during tissue damage. Here, we address their possible role as innate-immune mediators in ischemia-reperfusion injury (IRI) following human kidney transplantation. We analysed serum and urinary HMGB1 and IL-33 levels, all determined by enzyme-linked immunosorbent assay, in a cohort of 26 deceased renal transplant recipients. Urinary HMGB1 and IL-33 levels were significantly increased as soon as 30 min after reperfusion, as compared to those before treatment. Moreover, both serum and urinary IL-33 (but not HMGB1) increase was positively correlated with cold ischemia time, from 30 min to 3 days post-transplantation. <i>In vitro</i>, human umbilical vein endothelial cells subjected to hypoxia conditions released both HMGB-1 and IL-33, while only the latter was further increased upon subsequent re-oxygenation. Finally, we postulate that leukocytes from renal recipient patients are targeted by both HMGB1 and IL-33, as suggested by increased transcription of their respective receptors (TLR2/4 and ST2L) shortly after transplantation. Consistent with this view, we found that iNKT cells, an innate-like T cell subset involved in IRI and targeted by IL-33 but not by HMGB1 was activated 1 hour post-transplantation. Altogether, these results are in keeping with a potential role of IL-33 as an innate-immune mediator during kidney IRI in humans.</p></div

Hypoxia/re-oxygenation-induced release <i>in vitro</i> of HMGB1 and IL-33.

<p>Confluent (≈95%) monolayer HUVEC were exposed to sixteen hours hypothermia/hypoxia in UW solution (H16h) followed by 1 hour (R1h), or 3 hours (R3h) of re-oxygenation in a new culture medium (Medium 200) at 37°C in 20% O₂. Confluent (≈95%) monolayer HUVEC were used as controls (Ctl). Early release of HMGB1 and IL-33 by HUVEC in response to in vitro hypoxia/re-oxygenation (A–B). HMGB1 (A) and IL-33 (B) in cell culture supernatants were quantified by ELISA. Increase of IL-33 but not HMGB1 mRNAs in HUVEC in response to <i>in vitro</i> hypoxia/re-oxygenation (C–D). Total RNA was extracted from monolayer HUVEC at the indicated time points and expression of HMGB1 (C) and IL-33 (D) mRNAs was quantified by RT-qPCR. Data are expressed as means ± SEM or of fold change relative to D0 and are representative of three separate experiments. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> Ctl by Mann-Whitney test.</p

Correlation of serum and urinary alarmin levels with cold ischemia time.

<p>POD: Post Operative Day. The correlation coefficient (<i>r</i>) is calculated by the non-parametric Spearman’s rank correlation test. A p-value<0.05 was considered significant.</p

Increased levels of HMGB1, IL-33 and sST2 in serum and urine shortly after renal IRI.

<p>HMGB1, IL-33 and sST2 levels were quantified by ELISA in serum and urine of kidney graft recipients (n = 26) before transplantation (D0) as control time, and 30 minutes (H0.5), 3 hours (H3), day 1 (POD1) and day 3 (POD3) after transplantation. Serum, urine and urinary molecule/creatinine ratio levels for HMGB1 (A–C), IL-33 (D–F) and sST2 (G–I). Note that serum samples from only 6 out of 26 transplanted patients contained measurable amounts of IL-33. Data are expressed as means ± SEM. *p<0.05, **p<0.01, ***p<0.001 by Wilcoxon or Mann-Whitney test, as appropriate. ns, no significant.</p