Aaron Haig

RIPK3-Mediated Necroptosis Promotes Donor Kidney Inflammatory Injury and Reduces Allograft Survival

Kidney transplant injury occurs with ischemia and alloimmunity. Members of the receptor interacting protein kinase family (RIPK1,3) are key regulators of “necroptosis,” a newly recognized, regulated form of necrosis. Necroptosis and apoptosis death appear to be counterbalanced as caspase‐8 inhibition can divert death from apoptosis to necrosis. Inhibition of necroptosis in donor organs to limit injury has not been studied in transplant models. In this study, necroptosis was triggered in caspase inhibited tubular epithelial cells (TEC) exposed to tumor necrosis factor alpha in vitro, while RIPK1 inhibition with necrostatin‐1 or use of RIPK3−/− TEC, prevented necroptosis. In vivo, short hairpin RNA silencing of caspase‐8 in donor B6 mouse kidneys increased necroptosis, enhanced high‐mobility group box 1 release, reduced renal function and accelerated rejection when transplanted into BALB/c recipients. Using ethidium homodimer perfusion to assess necrosis in vivo, necrosis was abrogated in RIPK3−/− kidneys postischemia. Following transplantation, recipients receiving RIPK3−/− kidneys had longer survival (p = 0.002) and improved renal function (p = 0.03) when compared to controls. In summary, we show for the first time that RIPK3‐mediated necroptosis in donor kidneys can promote inflammatory injury, and has a major impact on renal ischemia–reperfusion injury and transplant survival. We suggest inhibition of necroptosis in donor organs may similarly provide a major clinical benefit.

IL-37 inhibits IL-18-induced tubular epithelial cell expression of pro-inflammatory cytokines and renal ischemia-reperfusion injury.

Cytokines and chemokines produced by tubular epithelial and infiltrating cells are critical to inflammation in renal ischemia-reperfusion injury. IL-37, a newly described IL-1 family member, inhibits IL-18-dependent pro-inflammatory cytokine production by its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia-reperfusion injury is unknown. Here we found that exposure of tubular epithelial cells to exogenous IL-37 downregulated hypoxia and the IL-18-induced expression of TNFα, IL-6, and IL-1β. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Moreover, pro-inflammatory cytokine expression was augmented in IL-37 mRNA-silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL-37. In a mouse ischemic injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IL-6, and IL-1β and improved mononuclear cell infiltration, kidney injury, and function. Thus, human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 as an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses and promote kidney function after renal ischemic injury and transplantation.

RIPK3‐Mediated Necroptosis Regulates Cardiac Allograft Rejection

Cell death results in tissue damage and ultimately donor graft rejection and can occur as an active molecular process through apoptotic, necrotic and newly identified receptor interacting protein 1 and 3 kinase (RIPK1/3)‐mediated necroptotic pathways. Necroptosis leads to the release of inflammatory molecules which can activate host immune cells. This pathway has yet to be studied in heart transplantation. We have found that necroptosis was induced in murine cardiac microvascular endothelial cell (MVEC) under anti‐apoptotic condition following tumor necrosis factor alpha treatment. Necroptotic cell death and release of the danger molecule high mobility group box 1 (HMGB1) were inhibited by the RIPK1 inhibiting molecule necrostatin‐1 and by genetic deletion of RIPK3. In addition, tissue necrosis, release of HMGB1 and graft cell infiltrate were attenuated in RIPK3 null heart allografts following transplantation. Finally, a brief sirolimus treatment markedly prolonged RIPK3 null cardiac allograft survival in allogeneic BALB/c recipients as compared to WT C57BL/6 donor grafts (95 ± 5.8 vs. 24 ± 2.6 days, p < 0.05). This study has demonstrated that RIPK1/3 contributes to MVEC death and cardiac allograft survival through necroptotic death and the release of danger molecules. Our results suggest that targeting RIPK‐mediated necroptosis may be an important therapeutic strategy in transplantation.

Hydrogen sulfide treatment improves long-term renal dysfunction resulting from prolonged warm renal ischemia-reperfusion injury

INTRODUCTION The incidence of renal cell carcinoma (RCC) continues to rise concurrently with the increased prevalence of end-stage renal disease worldwide. Treatment for small renal masses continues to be partial nephrectomy mostly involving the clamping of renal blood vessels. Although necessary, this technique results in warm renal ischemia and reperfusion injury (IRI) to the afflicted kidney. We have recently demonstrated that hydrogen sulfide (H2S), a novel endogenous gaseous molecule, protects against prolonged cold and short-term warm renal IRI. In the current study, we examined whether exogenous H2S has long-term protective effects against warm renal IRI associated with renal surgical procedures. METHODS Uni-nephrectomized Lewis rats underwent 1 hour of warm ischemia induced by clamping of the renal pelvis. Animals underwent either intraperitoneal treatment with phosphate buffered saline (PBS; IRI group) or PBS supplemented with 150 μM NaHS (H2S group), and were compared against Sham-operated rats. RESULTS H2S treatment improved long-term renal function as serum creatinine at day 7 was significantly decreased in the H2S group compared to IRI animals (p < 0.05). H2S treatment decreased the expression of pro-inflammatory markers TLR-4, TNF-α, IFNγ, IL-2 and ICAM-1, increased the expression of pro-survival molecule Bcl-2 and decreased the expression of pro-apoptotic marker BID at postoperative day 1. H2S-treated kidneys also showed a significant decrease (p < 0.05) in infiltration of macrophages at day 7 post-IRI compared to no treatment. CONCLUSION H2S treatment improved long-term renal function and decreased long-term inflammation associated with warm IRI, and may offer a novel therapeutic approach to preventing warm IRI-induced renal injury associated with renal surgical procedures.

MicroRNA and mRNA Signatures in Ischemia Reperfusion Injury in Heart Transplantation

Ischemia reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation, leading to graft failures and lower long-term survival rate of the recipient. Several studies have demonstrated that microRNAs (miRNAs) are vital regulators of signalling pathways involved in I/R injury. The present study aims to quantify the altered expression levels of miRNA and mRNA upon I/R injury in a mouse heart transplantation model, and to investigate whether these miRNA can regulate genes involved in I/R injury. We performed heterotopic heart transplantation on mouse models to generate heart tissue samples with I/R and non-I/R (control). The expression levels of miRNAs as well as genes were measured in heart grafts by microarray and real time RT-PCR. miRNA alteration in cardiomyocytes exposed to hypoxia was also detected by qRT-PCR. We observed significant alterations in miRNA and gene expression profile after I/R injury. There were 39 miRNAs significantly downregulated and 20 upregulated up to 1.5 fold in heart grafts with I/R injury compared with the grafts without I/R. 48 genes were observed with 3 fold change and p<0.05 and 18 signalling pathways were enriched using Keggs pathway library. Additionally, hypoxia/reperfusion induced primary cardiomyocyte apoptosis and altered miRNA expression profiles. In conclusion, this is the first report on miRNA expression profile for heart transplantation associated with I/R injury. These findings provide us with an insight into the role of miRNA in I/R injury in heart transplantation.

Natural Killer Cells Mediate Long-term Kidney Allograft Injury.

Background Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell–mediated rejection. We have recently demonstrated that natural killer (NK) cells are cytotoxic to tubular epithelial cells and contribute to acute kidney ischemia-reperfusion injury. The role of NK cells in kidney allograft rejection has not been studied. Methods A “parent to F1” kidney transplant model was used to study NK cell–mediated transplant rejection. Results The C57BL/6 kidneys were transplanted into fully nephrectomized CB6F1 (C57BL/6 x BALB/c) mice. Serum creatinine levels increased from baseline (18.8 ± 5.0 &mgr;mol/L to 37.2 ± 5.9 &mgr;mol/L, P < 0.001) at 60 days after transplantation. B6Rag−/−-to-CB6F1Rag−/− (B6Rag−/−xBALB/cRag−/−) recipients, which lack T and B cells but retain NK cells, showed similar levels of kidney dysfunction 65 days after transplantation (creatinine, 33.8 ± 7.9 &mgr;mol/L vs 17.5 ± 5.1 &mgr;mol/L in nontransplant Rag−/− mice, P < 0.05). Importantly, depletion of NK cells in Rag1−/− recipients inhibited kidney injury (24.6 ± 5.5 &mgr;mol/L, P < 0.05). Osteopontin, which can activate NK cells to mediate tubular epithelial cell death in vitro, was highly expressed in 60 days kidney grafts. Osteopontin null kidney grafts had reduced injury after transplantation into CB6F1 mice (17.7 ± 3.1 &mgr;mol/L, P < 0.001). Conclusions Collectively, these data demonstrate for the first time that independent of T and B cells, NK cells have a critical role in mediating long-term transplant kidney injury. Specific therapeutic strategies that target NK cells in addition to conventional immunosuppression may be required to attenuate chronic kidney transplant injury.

Glycyrrhizic Acid Ameliorates HMGB1-Mediated Cell Death and Inflammation after Renal Ischemia Reperfusion Injury

Background: Renal ischemia reperfusion injury (IRI) leads to acute kidney injury (AKI) and the death of tubular epithelial cells (TEC). The release of high-mobility group box-1 (HMGB1) and other damage-associated molecular pattern moieties from dying cells may promote organ dysfunction and inflammation by effects on TEC. Glycyrrhizic acid (GZA) is a functional inhibitor of HMGB1, but its ability to attenuate the HMGB1-mediated injury of TEC has not been tested. Methods/Results: In vitro, hypoxia and cytokine treatment killed TEC and resulted in the progressive release of HMGB1 into the supernatant. GZA reduced the hypoxia-induced TEC death as measured by annexin-V and propidium iodide. Hypoxia increased the expression of MCP-1 and CXCL1 in TEC, which was reduced by GZA in a dose-dependent manner. Similarly, the HMGB1 activation of effector NK cells was inhibited by GZA. To test the effect of HMGB1 neutralization by GZA in vivo, mice were subjected to renal IRI. HMGB1 protein expression increased progressively in kidneys from 4 to 24 h after ischemia and was detected in tubular cells by 4 h using immunohistochemistry. GZA preserved renal function after IRI and reduced tubular necrosis and neutrophil infiltration by histological analyses and ethidium homodimer staining. Conclusions: Importantly, these data demonstrate for the first time that AKI following hypoxia and renal IRI may be promoted by HMGB1 release, which can reduce the survival of TEC and augment inflammation. Inhibition of the interaction of HMGB1 with TEC through GZA may represent a therapeutic strategy for the attenuation of renal injury following IRI and transplantation.

Hydrogen Sulfide Treatment Mitigates Renal Allograft Ischemia-Reperfusion Injury during Cold Storage and Improves Early Transplant Kidney Function and Survival Following Allogeneic Renal Transplantation

PURPOSE Ischemia-reperfusion injury is unavoidable during organ transplantation. Prolonged ischemia-reperfusion injury is detrimental to short-term and long-term graft function and survival. H2S is a recently characterized, endogenously produced gaseous molecule with important physiological roles that has been shown to be cytoprotective during tissue ischemia-reperfusion injury. The current study aimed to determine whether H2S could mitigate cold renal ischemia-reperfusion injury in the clinically relevant context of allogeneic renal transplantation. MATERIALS AND METHODS Following bilateral native nephrectomy Lewis rats underwent renal transplantation with kidneys from Brown Norway donor rats that were flushed with cold (4C) standard University of Wisconsin preservation solution (University of Wisconsin preservation solution group) or cold University of Wisconsin preservation solution plus 150 μM NaHS (H2S group) solution. Kidneys were stored for 6 hours at 4C in the same solution. Recipient animals were monitored for 14 days or until sacrifice using metabolic cages to assess various parameters of renal graft function. RESULTS H2S treatment improved early allograft survival and function, and decreased early levels of necrosis, apoptosis and Kim-1 compared to University of Wisconsin preservation solution alone. H2S treatment did not affect allograft rejection. Rather, it modulated the early allograft transcriptome to decrease the expression of renal injury, coagulation and cellular stress response genes, and increase the expression of cellular proliferation and Ifn-γ induced genes compared to University of Wisconsin preservation solution alone. CONCLUSIONS To our knowledge our findings are the first to show that H2S protects donor kidneys against cold ischemia-reperfusion injury in the context of allogeneic renal transplantation. This potentially represents a novel cost-effective therapeutic solution to mitigate ischemia-reperfusion injury and improve the clinical outcomes of renal transplantation.

Natural killer cells play a critical role in cardiac allograft vasculopathy in an interleukin-6--dependent manner.

Background Approximately 50% of cardiac transplants fail in the long term, and currently, there are no specific treatments to prevent chronic rejection. In the clinic, donor cardiac graft ischemia time is limited to within a few hours and correlates with delayed graft function and organ failure. It is still unknown how ischemic injury negatively influences allograft function over the long term despite advances in immunosuppression therapy. Methods Allogeneic cardiac grafts were stored at 4°C for 4 hr before being transplanted into T/B cell–deficient Rag−/− mice or T/B/natural killer (NK) cell–deficient &ggr;c−/−Rag−/− mice. Grafts were harvested 60 days after transplantation and indicators of chronic allograft vasculopathy (CAV) were quantified. Results We have found that cold ischemia of cardiac grafts induces CAV after transplantation into Rag1−/− mice. Interestingly, cold ischemia–induced CAV posttransplantation was not seen in T/B/NK cell–deficient &ggr;c−/−Rag−/− mice. However, cardiac grafts in &ggr;c−/−Rag−/− mice that received an adoptive transfer of NK cells developed CAV, supporting the role of NK cells in CAV development. Analysis of various cytokines that contribute to NK cell function revealed high interleukin (IL)-6 expression in cardiac grafts with CAV. In addition, IL-6–deficient cardiac grafts did not develop CAV after transplantation into allogeneic Rag−/− mice. Conclusion These data demonstrate that cold ischemia and NK cells play critical roles in the development CAV. Natural killer cells and injured grafts may play a reciprocal role for CAV development in an IL-6–independent manner. Specific therapeutic strategies may be required to attenuate NK cell contribution to chronic cardiac rejection.

Kidney Injury Molecule-1 Protects against Gα12 Activation and Tissue Damage in Renal Ischemia-Reperfusion Injury

Ischemic acute kidney injury is a serious untreatable condition. Activation of the G protein α12 (Gα12) subunit by reactive oxygen species is a major cause of tissue damage during renal ischemia-reperfusion injury. Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein that is highly up-regulated during acute kidney injury, but the physiologic significance of this up-regulation is unclear. Here, we report for the first time that Kim-1 inhibits Gα12 activation and protects mice against renal ischemia-reperfusion injury. We reveal that Kim-1 physically interacts with and inhibits cellular Gα12 activation after inflammatory stimuli, including reactive oxygen species, by blocking GTP binding to Gα12. Compared with Kim-1(+/+) mice, Kim-1(-/-) mice exhibited greater Gα12 and downstream Src activation both in primary tubular epithelial cells after in vitro stimulation with H2O2 and in whole kidneys after unilateral renal artery clamping. Finally, we show that Kim-1-deficient mice had more severe kidney dysfunction and tissue damage after bilateral renal artery clamping, compared with wild-type mice. Our results suggest that KIM-1 is an endogenous protective mechanism against renal ischemia-reperfusion injury through inhibition of Gα12.