Ian Lobb

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.

Detrimental effects of prolonged warm renal ischaemia-reperfusion injury are abrogated by supplemental hydrogen sulphide: an analysis using real-time intravital microscopy and polymerase chain reaction.

Whats known on the subject? and What does the study add?

Supplemental hydrogen sulphide protects transplant kidney function and prolongs recipient survival after prolonged cold ischaemia-reperfusion injury by mitigating renal graft apoptosis and inflammation.

Whats known on the subject? and What does the study add?

Hydrogen sulphide and the kidney: important roles in renal physiology and pathogenesis and treatment of kidney injury and disease.

The kidney is an essential mammalian organ that serves to filter toxins and metabolic by-products out of the blood, which are then excreted through urine. Hydrogen sulphide (H2S) is a recently characterized, endogenous gaseous molecule with important physiological roles. Many interesting roles continue to be identified for H2S related specifically to the kidney. The current review discusses how production and action of H2S influences normal physiology of the kidney. We investigate as well the many roles H2S plays in the pathogenesis and treatment of kidney injury and disease, such as chronic kidney disease (CKD), ureteral obstruction (UO), hyperhomocysteinaemia (HHcy), drug-induced nephrotoxicity (DIN) and renal ischaemia reperfusion injury (IRI). We suggest that H2S plays a complex and essential role in the normal function of the kidney and dysregulation of H2S production can directly or indirectly contribute to the pathogenesis of renal disease and injury. Also, H2S could be a promising potential therapeutic treatment to decrease the severity of several renal diseases. Further research will identify increasingly important and complex roles for H2S in renal physiology and how H2S can be effectively utilized to improve clinical outcomes of renal disease.

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.

Hydrogen sulfide protects renal grafts against prolonged cold ischemia‐reperfusion injury via specific mitochondrial actions

Ischemia–reperfusion injury is unavoidably caused by loss and subsequent restoration of blood flow during organ procurement, and prolonged ischemia–reperfusion injury IRI results in increased rates of delayed graft function and early graft loss. The endogenously produced gasotransmitter, hydrogen sulfide (H2S), is a novel molecule that mitigates hypoxic tissue injury. The current study investigates the protective mitochondrial effects of H2S during in vivo cold storage and subsequent renal transplantation (RTx) and in vitro cold hypoxic renal injury. Donor allografts from Brown Norway rats treated with University of Wisconsin (UW) solution + H2S (150 μM NaSH) during prolonged (24‐h) cold (4°C) storage exhibited significantly (p < 0.05) decreased acute necrotic/apoptotic injury and significantly (p < 0.05) improved function and recipient Lewis rat survival compared to UW solution alone. Treatment of rat kidney epithelial cells (NRK‐52E) with the mitochondrial‐targeted H2S donor, AP39, during in vitro cold hypoxic injury improved the protective capacity of H2S >1000‐fold compared to similar levels of the nonspecific H2S donor, GYY4137 and also improved syngraft function and survival following prolonged cold storage compared to UW solution. H2S treatment mitigates cold IRI–associated renal injury via mitochondrial actions and could represent a novel therapeutic strategy to minimize the detrimental clinical outcomes of prolonged cold IRI during RTx.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel-Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease.

Hydrogen Sulfide Induced Erythropoietin Synthesis is Regulated by HIF Proteins

PURPOSE Anemia of end stage renal disease affects 90% of patients on hemodialysis and it is a tremendous concern of patients and health care providers. Renal disease creates a state of renal hypoxia, which may contribute to a lack of erythropoietin production from the kidney when low oxygen levels are sensed. This necessitates the use of exogenous erythropoietin preparations. MATERIALS AND METHODS Recent evidence suggests that endogenously derived hydrogen sulfide may mediate oxygen sensing in tissues. Given the known involvement of other small molecules such as nitric oxide in erythropoietin production and the observation of decreased urinary H2S levels in patients with renal failure, we postulated that H2S may be the primary mediator of erythropoietin production during hypoxia. PK1, 786-O and Hep3B cells were incubated in hypoxia (1% O2) for 24 hours. Hypoxic cells were treated with the H2S donor GYY 4137 and the H2S inhibitor hydroxylamine. Following hypoxia erythropoietin, HIF-1α, HIF-2α and CBS expression was measured by quantitative real-time polymerase chain reaction and Western blot. RESULTS Hydroxylamine administration led to a significant decrease in erythropoietin, HIF-1α, HIF-2α and CBS protein levels during hypoxia. This was rescued by administration of GYY 4137 for erythropoietin, CBS and HIF-2α. Additionally, CSE -/- mice placed in hypoxia for 72 hours showed decreased renal erythropoietin production compared to wild-type mice. CONCLUSIONS These data suggest previously undocumented interplay of the production and action of H2S during hypoxia with subsequent erythropoietin production. The use of novel hydrogen sulfide donors could represent an alternative to standard therapies of anemia of renal failure.

A Hibernation-Like State for Transplantable Organs: Is Hydrogen Sulfide Therapy the Future of Organ Preservation?

SIGNIFICANCE Renal transplantation is the treatment of choice for end-stage renal disease, during which renal grafts from deceased donors are routinely cold stored to suppress metabolic demand and thereby limit ischemic injury. However, prolonged cold storage, followed by reperfusion, induces extensive tissue damage termed cold ischemia/reperfusion injury (IRI) and puts the graft at risk of both early and late rejection. Recent Advances: Deep hibernators constitute a natural model of coping with cold IRI as they regularly alternate between 4°C and 37°C. Recently, endogenous hydrogen sulfide (H2S), a gas with a characteristic rotten egg smell, has been implicated in organ protection in hibernation. CRITICAL ISSUES In renal transplantation, H2S also seems to confer cytoprotection by lowering metabolism, thereby creating a hibernation-like environment, and increasing preservation time while allowing cellular processes of preservation of homeostasis and tissue remodeling to take place, thus increasing renal graft survival. FUTURE DIRECTIONS Although the underlying cellular and molecular mechanisms of organ protection during hibernation have not been fully explored, mammalian hibernation may offer a great clinical promise to safely cold store and reperfuse donor organs. In this review, we first discuss mammalian hibernation as a natural model of cold organ preservation with reference to the kidney and highlight the involvement of H2S during hibernation. Next, we present recent developments on the protective effects and mechanisms of exogenous and endogenous H2S in preclinical models of transplant IRI and evaluate the potential of H2S therapy in organ preservation as great promise for renal transplant recipients in the future. Antioxid. Redox Signal. 28, 1503-1515.

Daily therapy with a slow-releasing H 2 S donor GYY4137 enables early functional recovery and ameliorates renal injury associated with urinary obstruction

OBJECTIVES To assess the effects of slow-releasing H2S donor GYY4137 on post-obstructive renal function and injury following unilateral ureteral obstruction (UUO) by using the UUO and reimplantation (UUO-R) model in rats and to elucidate potential mechanisms by using an in vitro model of epithelial-mesenchymal transition (EMT). METHODS Male Lewis rats underwent UUO at the left ureterovesical junction. From post-operative day (POD) 1-13, rats received daily intraperitoneal (IP) injection of phosphate buffered saline (PBS, 1 mL) or GYY4137 (200 μmol/kg/day in 1 mL PBS, IP). On POD 14, the ureter was reimplanted back into the bladder, followed by a right nephrectomy. Urine and serum samples were collected to monitor renal function. On POD 30, the left kidney was removed and tissue sections were stained with H&E, TUNEL, CD68, CD206, myeloperoxidase, and Massons trichrome to determine cortical thickness, apoptosis, inflammation, and fibrosis. In our in vitro model of EMT, NRK52E cells were treated with 10 ng/mL TGF-β1, 10 μM GYY4137 and/or 50 μM GYY4137. Western blot analysis was performed to determine the expression of E-cadherin, vimentin, Smad7 and TGF-β1 receptor II (TβRII). RESULTS GYY4137 led to a moderate decrease in post-obstructive serum creatinine, cystatin C and FENa. We also observed a trend towards a decrease in post-obstructive proteinuria following GYY4137 treatment. Histologically, we observed a significant decrease in apoptosis, inflammation, and fibrosis. Furthermore, our in vitro studies demonstrate that in the presence of TGF-β1, GYY4137 significantly decreases vimentin and TβRII and significantly increases E-cadherin and Smad7. CONCLUSIONS H2S may help to accelerate the recovery of renal function post-obstruction and attenuates renal injury associated with UUO. It is possible that H2S mitigates fibrosis by regulating the TGF-β1-mediated EMT pathway. Taken together, our data suggest that H2S may be a potential novel therapy for improving renal function and limiting renal injury associated with obstructive uropathy.