Sodium Thiosulfate-supplemented UW Solution Protects Renal Grafts Against Prolonged Cold Ischemia-reperfusion Injury in A Murine Model Of Syngeneic Kidney Transplantation

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Max Y. Zhang et al

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ABSTRACT

Introduction: Cold ischemia-reperfusion injury (IRI) is an inevitable event that increases post-transplant complications. We have previously demonstrated that supplementation of the University of Wisconsin (UW) solution with non-FDA-approved hydrogen sulfide (H2S) donor molecules minimizes cold IRI and improves renal graft function after transplantation. The present study investigates whether an FDA-approved H2S donor molecule, sodium thiosulfate (STS), will have the same or superior effect in a clinically relevant rat model of syngeneic orthotopic kidney transplantation.

Method: Thirty Lewis rats underwent bilateral nephrectomy followed by syngeneic orthotopic transplantation of the left kidney after 24-hour preservation in either UW or UW+STS solution at 4 ◦C. Rats were monitored to post-transplant day 14 and sacrificed to assess renal function (urine output, serum creatinine, and blood urea nitrogen). Kidney sections were stained with H&E, TUNEL, CD68, and myeloperoxidase (MPO) to detect acute tubular necrosis (ATN), apoptosis, macrophage infiltration, and neutrophil infiltration.

Result: UW+STS grafts showed significantly improved graft function immediately after transplantation, with improved recipient survival compared to UW grafts (p < 0.05). Histopathological examination revealed significantly reduced ATN, apoptosis, macrophage, and neutrophil infiltration and downregulation of pro-inflammatory and pro-apoptotic genes in UW+STS grafts compared to UW grafts (p < 0.05).

Conclusion: We show for the first time that preservation of renal grafts in STS-supplemented UW solution protects against prolonged cold IRI by suppressing apoptotic and inflammatory pathways, and thereby improving graft function and prolonging recipient survival. This could represent a novel clinically applicable therapeutic strategy to minimize the detrimental clinical outcome of prolonged cold IRI in kidney transplantation.

Keywords: Sodium thiosulfate (STS) Ischemia-reperfusion injury (IRI) Static cold storage (SCS) Kidney transplantation Graft and recipient survival

1. Introduction

Kidney transplantation is the optimal treatment for end-stage kidney renal disease (ESRD). Compared to dialysis, kidney transplantation is superior, as it provides better quality of life and confers a significant survival advantage along with its cost-effectiveness [1–3]. However, procurement of donor's kidneys is inherently associated with ischemia-reperfusion injury (IRI), an inevitable consequence of the cessation and subsequent restoration of blood flow during transplantation [4]. The current strategy to mitigate transplant-induced IRI is static cold storage (SCS) of renal grafts in standard preservation solutions such as the University of Wisconsin (UW) solution on the ice at 4 ◦C during the pre-transplant period [5]. However, prolonged SCS has been shown to be associated with increased cell death, inflammation, and other damaging cellular and molecular events, which ultimately result in an increased incidence of delayed graft function (DGF), acute tubular necrosis (ATN), and decreased graft survival [6–10]. In addition, to keep up with the globally rising incidence of ESRD and an ever-increasing number of patients on transplant waiting lists, many transplant centers accept renal grafts with prolonged cold ischemic periods, which further contributes to overall tissue damage. Following SCS is reperfusion, when warm oxygenated blood is restored into the cold ischemic graft. Reperfusion, which is the effector phase of ischemic injury, is characterized by increased tissue injury [11–13].

A potential therapeutic strategy to limit cold IRI during kidney transplantation involves supplementation of standard preservation solution with hydrogen sulfide (H2S), an endogenously produced gasotransmitter that has been shown to play important physiological roles in vasodilation and cellular signaling [14–16]. We have previously shown that prolonged SCS in H2S-supplemented UW solution reduces transplant-induced cold IRI and improves graft survival in murine models of syngeneic and allogeneic kidney transplantation [17–19,41, 42]. However, the H2S donor molecules used in these studies are not clinically viable. This has led to the consideration of using sodium thiosulfate (STS), an H2S donor drug that is approved by the Food and Drug Administration (FDA) to treat calciphylaxis in ESRD patients, cisplatin-induced toxicity in cancer therapy, and as an antidote to cyanide poisoning [20–23]. Recent studies have shown that STS exhibits protective effects in animal models of IRI [24–26]. However, its effect on transplant-induced cold renal IRI is unknown. Therefore, the present study investigates the renoprotective effects of STS in an in vitro model of renal IRI and rat model of syngeneic orthotopic kidney transplantation.

2. Materials and methods

2.1. Experimental in vitro protocol

An in vitro model of cold hypoxia and warm reoxygenation injury that mimics cellular conditions during in vivo cold IRI was used to assess the protective effects of STS during renal IRI. Rat kidney epithelial cells (NRK-52E cell line; ATCC, USA) were used in the in vitro experiments because these cells are susceptible to ischemic injury [27], and their use is consistent with the rat model of transplantation used for the second aim of this study. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) inactivated by heat at 60 ◦C for 20 min and 1% penicillin/streptomycin (P/S). Cells were incubated at normal growth conditions of 37◦C, 21% O2, and 5% CO2. Control cells were in conditions identical to those for pre-experimental cells. Experimental cells were treated with either serum-free media (SF), SF plus 200 nM AP39, or SF with varying concentrations (50 µM, 150 µM, 500 µM, 1 mM) of sodium thiosulfate pentahydrate (STS), which were obtained from a 250 mg/mL injectable solution of STS (Seacalphyx® [Seaford Pharmaceuticals Inc, Mississauga, ON, Canada]). A concentration of 200 nM AP39 was used because we previously showed that AP39 at this concentration is cytoprotective against the same cell line in a similar model of cold IRI [20]. Cells were then incubated at 10 ◦C for 24 h under hypoxic conditions (5% CO2, 0.5% O2, 95% N2) in HypOxystation H85 hypoxia chamber (HYPO2YGEN, USA) to induce cold ischemic injury. A hypothermic temperature of 10 ◦C was used because this was the lowest temperature that could be technologically achieved while maintaining a 0.5% O2 level of hypoxia. Following hypoxia, the media containing experimental cells was replaced with control media, and the cells reoxygenated via incubation under normal growth conditions (37 ◦C, 21% O2, and 5% CO2) for 24 h to simulate reperfusion and its associated injury. Following 24 h reoxygenation, cellular viability was assessed via staining of cells with FITC-conjugated Annexin-V (FITC-Annexin-V; BioLegend, USA) and 7-Aminoactinomycin D (7-AAD; BioLegend, USA), which measures cellular apoptosis and necrosis respectively. Cells were analyzed via flow cytometry using the CytoFLEX S (Beckman Coulter, USA). FlowJo version 11 (FlowJo LLC, USA) was used to appropriately gate the data for statistical analysis.

2.2. Experimental animals

Thirty male Lewis rats weighing 275–300 g and purchased from Charles River (St. Constant, QC, Canada) were housed in the Animal Care and Veterinary Services facility at Western University (London, ON) under standard conditions. Animal studies were approved by the Western University Council on Animal Care and Animal Use with a protocol ID 2018–155.

2.3. Kidney transplant model

Syngeneic kidney transplantation in Lewis rats was performed to eliminate any confounding effects of immunosuppression. Rats were randomized into treatment groups of UW solution alone (UW) or UW+STS, anesthetized with ketamine (30 mg/kg) via intraperitoneal administration, and maintained under anesthesia with isoflurane during surgery. The left donor kidneys were procured under aseptic condition and flushed with 10 mL of either cold (4 ◦C) UW solution (UW group, n = 8) in a 28-G Angiocath Becton-Dickinson, or cold UW solution supplemented with sodium thiosulfate pentahydrate (150 µM Seacalphyx® [Seaford Pharmaceuticals Inc, Mississauga, ON, Canada]; UW+STS group, n = 6) until venous effluent was clear. Grafts were then subjected to SCS in UW solution at 4◦C with or without STS for 24 h to mimic prolonged cold ischemic time as previously described [13]. Following 24 h of SCS and bilateral nephrectomy in recipients, renal grafts were transplanted orthotopically into the left renal fossa of syngeneic recipient rats using 11–0 Prolene sutures as we previously described [22]. Sham-operated rats (mid-line incision only; n = 5), were used to establish a baseline for survival, histological analysis, BUN, and serum creatinine. Additionally, another subset of rats in the UW+STS group had grafts removed pre-emptively on a postoperative day (POD) 3 (n = 5) for histological comparison with UW grafts of recipients that were sacrificed at this time point. All surgeries were performed by the same microsurgeon with the length of surgery for the recipient being approximately 2–3 h for both UW and UW+STS groups. Graft failure was presumed in animals that required premature sacrifice (severe visible distress and/or >20% weight loss) or death. Humane endpoints were checked twice a day and all rats were euthanized by CO2 exposure in a chamber at a flow rate of 40%. At the time of euthanasia, there was no surgical complications that could have resulted in variations in the results.

2.4. Analysis of renal function

Following kidney transplantation, rats were monitored in metabolic cages for 14 days and then sacrificed. Blood and urine samples were collected on POD 3, 5, 7, 10, and 14 to determine parameters of renal function (serum creatinine, blood urea nitrogen [BUN], urine osmolality, and urine output). BUN and serum creatinine were from kidney transplant recipients and Sham-operated rats were measured using IDEXX Catalyst One Chemistry Analyzer machine (Markham, ON). Urine osmolality levels were determined by freezing-point osmometry using the 3320 Osmometer machine (Advanced Instruments, Norwood, MA) and compared to company-provided standards.

2.5. Histopathological and morphometric analysis

Paraffin-embedded kidney tissues were cut into 4 µm-thick sections and mounted onto microscopic slides for histology. The sections were stained with Hematoxylin and Eosin (H&E), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to determine the degree of ATN and apoptosis respectively. H&E sections were assigned a score for ATN by a blinded renal pathologist as per the following scheme: 1 = <11%, 2 = 11–24%, 3 = 25–45%, 4 = 46–75%, 5 = >75% graft ATN. Kidney sections were also stained with the following primary antibodies: kidney injury marker (KIM-1), macrophage surface marker CD68, and neutrophil-specific enzyme myeloperoxidase (MPO; Abcam®, Toronto, Canada) and visualized with secondary antibodies and DAB substrate chromogen using the Dako Envision System (Dako, Glostrup, Denmark) according to the manufacturer protocol followed by analyzed under Eclipse 90i digital light microscope (Nikon® Instruments, New York) at 10x magnification and quantified by ImageJ software v. 1.8 (National Institutes of Health, Bethesda, MD).

2.6. Quantitative PCR analysis

Total RNA was isolated from renal graft tissues obtained at POD 3 using RNeasy® Mini Kit (Qiagen, Toronto, Canada) and reverse transcribed into cDNA using OneScript® Plus cDNA synthesis Kit (ABM, Canada) in conjunction with oligo(dT)12–18 primers according to the manufacturer’s protocol. Isolated RNA and cDNA were analyzed via nanodrop (DeNovix DS-11 Spectrophotometer, Canada) before use, with A260/280 ratings consistently > 1.95 and > 1.8 respectively. The reaction mixture of each qPCR sample had a volume of 20 µL and was made as per Blastaq® Green 2X qPCR Master Mix (ABM, Canada) protocol and analyzed using CFX Connect Real-Time PCR Detection System machine (Bio-Rad, Canada). Primer sequences were designed using Primer-BLAST software (NCBI) against beta-actin, poly (ADP-ribose) polymerase (PARP), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), B-cell lymphoma 2 (Bcl-2), Bcl-2- associated X protein (BAX), caspase 3, BH3 interacting-domain death agonist (BID), c-Jun N-terminal kinase 1/2 (JNK1/2), Pparg coactivator 1 alpha (PGC-1α), mitochondria complex I (NDUFB8), mitochondria complex II (SDHB), mitogen-activated protein kinase 1/2 (ERK1/2), neutrophil gelatinase lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) genes. All genes of interest were normalized against beta-actin. Fold changes of gene expression were compared to Sham-operated rats and were calculated using the ΔΔCt method.

2.7. Statistical analysis

All statistical analyses were conducted using GraphPad (La Jolla, CA) Prism statistical software package, version 9.0. Survival data were analyzed using Kaplan-Meier survival analysis and log rank test while qPCR gene expression data were analyzed using unpaired one-way t-test. All other data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test to determine statistical differences between groups. Statistical significance was accepted at p <0.05. Values are presented as mean ± standard error of mean (SEM).

3. Results

3.1. STS-supplemented serum-free media improves renal tubular epithelial cell survival during cold hypoxia/reoxygenation

Flow cytometry analysis after staining for apoptosis and necrosis showed that NRK-52E cells treated with SF during in vitro cold IRI exhibited significantly decreased cell viability compared to control (normoxic) cells (Fig. 1A; p < 0.05). While all the experimental samples showed significantly lower renal tubular epithelial cell viability than cells grown under normoxic conditions (p < 0.05), cells treated with SF supplemented with 150 µM and 500 µM STS exhibited significantly higher viability than those treated with SF media alone (Fig. 1A; p < 0.05), which corresponded with markedly reduced apoptosis compared to cells treated with SF media alone (Fig. 1B; p < 0.05). Additionally, the increase in cell viability and decrease in apoptosis appears to reach optimum levels with 150 µM and 500 µM STS, as a higher dose reversed these trends (Fig. 1A and B).

3.2. Supplementation of UW solution with STS improves early renal graft survival and function

Preservation of renal grafts in STS-supplemented UW solution significantly improved recipient survival with 83% survival till POD 14 (day of sacrifice) compared to the control group without STS supplementation, which showed 12.5% survival, particularly in the first 3 days (Fig. 2A; p < 0.05). In addition, STS supplementation markedly improved graft function during the early post-transplant period compared to UW treatment alone. Serum creatinine and BUN levels were significantly increased in both UW and UW+STS groups on POD 3, which correlated with decreased urine osmolality compared to Sham (Figs. 2B, C, and 3A; p < 0.05). However, serum creatinine and BUN levels in the UW+STS group significantly decreased on POD 3 with a corresponding increase in urine osmolality compared to the UW group (Fig. 2B, C and 3A; p < 0.05). Interestingly, the levels of serum creatinine and BUN in the UW+STS group decreased steadily from POD 3 to POD 14 with increased urine osmolality and were comparable to those of Sham (Figs. 2B, C, and 3A). Also, urine output in the UW+STS group was significantly higher during the first four postoperative days compared to UW and Sham groups (Fig. 3B; p < 0.05). However, it decreased steadily towards baseline (Sham) value and was comparable to that of Sham on POD 14 while urine output in the surviving rat in the UW group remained higher than baseline value on POD 14 (Fig. 3B).

3.3. Addition of STS to UW solution mitigates transplant-induced cell death after kidney transplantation

Kidney sections obtained on POD 3 and 14 were stained with TUNEL as a measure of apoptotic cell death and scored by a blinded renal pathologist (Fig. 4A). Renal grafts from the UW group exhibited significantly higher apoptotic cell death on POD 3 as indicated by higher TUNNEL score compared to kidneys from UW+STS and Sham groups (Fig. 5A and b; p < 0.05). Grafts from the UW+STS group were not significantly different compared to those of Sham at the same time point and at POD 14 (Fig. 4B). Additionally, while grafts from both UW and UW+STS groups showed significantly increased ATN scores on POD 3 compared to the Sham group (Fig. 5; p < 0.05), UW+STS grafts showed decreased ATN scores on POD 3 compared to UW (Fig. 5; p < 0.05). Also, whereas recipients of UW grafts did not survive to POD 14, and hence their ATN scores could not be determined on POD 14, those of UW+STS grafts survived to POD 14 but showed significantly increased ATN scores compared to the Sham group (Fig. 5; p < 0.05).

3.4. Renal grafts preserved in STS-supplemented UW solution exhibited decreased injury markers and inflammatory infiltrate after kidney transplantation

Kidney sections obtained on POD 3 and 14 were stained with KIM-1 to detect proximal tubular injury as well as CD68 (macrophage marker) and MPO (neutrophil marker) and scored by a blinded renal pathologist (Figs. 6A, 7A, and C). Renal tissue expression of KIM-1, CD68, and MPO were significantly higher in the UW group on POD 3 compared to those of UW+STS and Sham groups (Figs. 6B, 7B and D; p < 0.05) while expression of these markers in UW+STS grafts was not significantly different compared to Sham on POD 14 (Figs. 6B, 7B and D; p > 0.05).

3.5. STS-supplementation to UW solution suppressed renal expression of pro-inflammatory, pro-apoptotic, and mitochondrial genes

Gene expression of pro-inflammatory, pro-apoptotic, mitochondria-targeted, and kidney injury markers were determined via qRT-PCR in transplanted kidneys obtained on POD 3. Expression of the pro-inflammatory genes IFN- γ, TNF-α and IL-6 were markedly increased in UW grafts relative to UW+STS grafts on POD 3 (Fig. 8A; p < 0.05) and followed the same pattern with pro-apoptotic genes PARP, BAX, caspases-3, BID, JNK1 and JNK2 (Fig. 8A; p < 0.05) while expression of anti-apoptotic Bcl-2 was slightly increased in UW+STS group compared to UW group, although this increase did not reach statistical significance (Fig. 8A). In addition, expression of mitochondrial genes PGC-1α, NDUFB8 (complex I), and SDHB (complex II) in UW grafts were significantly decreased compared to UW+STS grafts (Fig. 8B; p < 0.05) while the reverse was observed with ERK1 and ERK2 expressions (Fig. 8B; p < 0.05). Furthermore, KIM-1 gene expression in UW grafts was significantly increased compared to UW+STS grafts (Fig. 8C; p < 0.05) while decreased NGAL expression in UW+STS grafts did not reach statistical significance in comparison with that in UW grafts (Fig. 8C; p > 0.05).

4. Discussion

This study establishes supplementation of standard preservation solution with STS, a clinically viable FDA-approved H2S donor, to mitigate transplant-induced cold renal IRI, improve graft quality and prolong recipient survival. Using an in vitro model of renal IRI and rat model of syngeneic orthotopic kidney transplantation, we demonstrate for the first time that supplementation of UW solution with STS during prolonged SCS is to- and organ protective.

The primary finding in our in vitro model is that STS supplementation to serum-free media protects renal epithelial cells from cold hypoxia and warm reoxygenation-induced apoptosis and increases viability, which is consistent with our previous study where we showed the protective effects of the mitochondria-targeting H2S donor drug, AP39, in an in vitro model of cold renal IRI [20]. Interestingly, a higher STS concentration of 1 mM reversed the beneficial effects, implying that STS exhibits a biphasic dose-response phenomenon referred to as hormesis, in which a lower concentration is cytoprotective while a higher concentration is cytotoxic. Mitochondrial damage is a major consequence of renal IRI, as mitochondrial permeability can inhibit adenosine triphosphate (ATP) production and increase the formation of reactive oxygen species (ROS), a destructive mediator of tissue injury [13]. It has been recently suggested that mitochondria are a primary site of STS activity. Not only is STS known to generate H2S in the mitochondria via glutathione-dependent reduction and vice versa through sulfide oxidation pathway, but also preserves mitochondrial ATP synthesis, decreases ROS production, and improves complex enzyme activities in the electron transport chain (ETC) [24,28–30]. In addition, recent studies showed that STS significantly increased expression of PGC-1α, a positive regulator of mitochondrial biogenesis and ATP production [26], which is in agreement with our in vivo observation. These molecular mechanisms could account for the significantly increased expression of mitochondrial ETC complexes I and II (NDUFB8 and SDHB respectively) in our kidney transplant model with increased survival and function as observed in renal grafts preserved in STS-supplemented UW solution.

Based on our in vitro results, we decided to investigate whether the protective effect of STS is applicable in vivo using a transplant model where cold IRI is a major contributor to graft dysfunction and increased post-transplant complications. Our findings show that prolonged (24 h) SCS of renal grafts in STS-supplemented UW solution significantly improves graft quality and function characterized by decreased serum creatinine and BUN levels, higher urine output, and prolonged recipient survival compared to grafts preserved in UW solution alone. It is important to note that the immediacy of urine output even after clinical kidney transplantation is a critical outcome that determines whether dialysis is required to address delayed graft function (DGF). Therefore, our observation that STS increases urine output immediately after transplantation and is comparable to the Sham group on POD 14 is a promising finding. The observed improvement in renal function after transplantation of grafts preserved in STS-supplemented UW solution also conferred a significant survival advantage. Quantitatively, STS supplementation prolonged the life of the graft such that 83% (5/6) of rats which received UW+STS grafts survived till POD 14 (day of sacrifice) compared to only 12.5% (1/8) of recipient rats of grafts preserved in UW solution without STS supplementation. This finding from the present study also aligns with our previous study, in which only 14% (1 out 7) of recipient rats of grafts were preserved in UW solution for 24 h without H2S (GYY4137) supplementation, survived till POD 14 [20].

In addition to showing improvement in renal function parameters, STS supplementation also inhibited renal graft apoptosis and inflammation by downregulating tissue expression of pro-apoptotic and pro-inflammatory genes while simultaneously upregulating anti-apoptotic genes and decreasing CD68-positive macrophages and MPO-positive neutrophils, which altogether resulted in reduced KIM-1 expression and ATN and ultimately preserved renal morphology. These inflammatory cytokines are known to be mediators of cell death during cold IRI [31], and their reduction in UW+STS grafts is likely due to the well-known characteristics of STS to decrease the endothelial permeability in vascular endothelial monolayer, attenuate cytokine production, and elicit production of anti-inflammatory cytokines [32]. Also, our finding that STS supplementation to UW solution downregulates the expression of pro-inflammatory genes matches that of a previous study on the anti-inflammatory activity of STS by reducing levels of TNF-α and IL-6 in neurological diseases [33,34]. Mechanistically, STS inactivates caspase-3 by attaching to its active site via strong hydrogen bonds and thereby preventing access of natural substrate to the active site, ultimately halting apoptosis [35]. STS also blocks the activation of JNK, a protein that plays a critical role in apoptotic signaling [35], which supports our finding on the downregulating effect of STS on caspase3 and JNK. However, we are unable to determine whether such mechanisms are operational in the present study since we did not perform additional experiments to explore these molecular mechanisms.

A major limitation of our in vitro experiment is our technical challenge of using the current standard preservation temperature 4 ◦C [36], as the 10 ◦C we used was the lowest temperature that could be technologically achieved without jeopardizing the hypoxic environment. This is a real difference in regards to cellular physiological processes and also in preservation techniques. A potential solution is to use chemically induced hypoxia in a plastic bag and place it in a 4 ◦C fridge in order to reflect the clinical settings of SCS. However, these anaerobic atmosphere generation bags were designed to be used at warm temperatures (21–37 ◦C) since the chemical compounds that induce the hypoxic environment function under that condition. Future studies should aim at optimizing the temperature in the hypoxia chamber to mimic clinical SCS settings. Achieving a consistent temperature of 4 ◦C without compromising a hypoxic environment will allow us to determine whether the observed phenotypes in the experimental in vitro cold IRI model are consistently expressed. Apart from the in vitro experiment, our rat transplant model is also not without a drawback. We performed syngeneic kidney transplantation (genetically identical donors and recipients with immunological compatibility), which is only applicable to identical twins in clinical kidney transplantation, whereas most clinical kidney transplantations are allogeneic (genetically different donors and recipients). The allogeneic transplantation forces transplant recipients to undergo immunological tests prior to transplantation to identify the donor’s human leukocyte antigens (HLA; molecules that induce and regulate immune response), determine immunological compatibility between donors and recipients, and avoid organ rejection [37,38]. Future studies using STS should consider allogeneic transplantation. Another limitation kidney transplant model is the fact that we focused on living donors, sub-optimal grafts from donation after cardiac death (DCD) donors are becoming increasingly common as a source of donor's kidneys in many transplant centers globally [39]. DCD exposes donor organs to various periods of warm ischemia in addition to cold ischemic times during SCS, and is associated with increased rates of DGF and decreased graft survival compared to living donors [40]. Therefore, future studies should consider the DCD kidney transplantation model to assess the effect of STS against IRI in this model.

In conclusion, our study demonstrates that supplementation of standard preservation solution with a clinically viable H2S donor drug during prolonged SCS of renal grafts protects against transplant-induced cold renal IRI, improves overall graft quality and graft function, and prolongs transplant recipient survival. Considering that the risk of DGF increases with prolonged cold ischemic time in clinical kidney transplantation, which raises a major clinical concern, the observation that STS protects renal grafts during prolonged SCS and prevents DGF after transplantation provides a great clinical promise that could decrease or prevent the incidence of DGF in clinical kidney transplantation in the near future. Thus, the future benefit of adding STS to preservation solutions may present a potential solution to this ongoing issue. Overall, this study adds to the growing body of literature that supports the cytoprotective effects of STS and other H2S donors against organ IRI, particularly improving graft outcomes in transplant-induced cold renal IRI. These strategies could facilitate the use of more grafts exposed to prolonged cold ischemic times, which could increase the pool of transplantable organs.

CRediT authorship contribution statement

All authors have agreed to the submission of this manuscript. There is no conflict of interest.

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