Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion. Porcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology. Hydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment. In conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation.

Background: Ex vivo normothermic machine perfusion (NMP) is a promising tool for assessing an isolated kidney prior to transplantation. However, there is no consensus on the perfusate's optimal oxygen‐carrying capacity to support renal function. To investigate the association of hemoglobin levels with renal function parameters, a retrospective analysis of isolated, normothermically, perfused porcine kidneys was performed.Methods: Between 2015 and 2021, a total of 228 kidneys underwent 4 h of NMP with perfusates that varied in hemoglobin levels. A generalized linear model was used to determine the association of hemoglobin levels with time‐weighted means of renal function markers, such as fractional sodium excretion (FENa) and creatinine clearance (CrCl). Stratified by baseline hemoglobin level (6 mmol/L), these markers were modeled over time using a generalized linear mixed‐effects model. All models were adjusted for potential confounders.Results: Until a hemoglobin level of around 5 mmol/L was reached, increasing hemoglobin levels were associated with superior FENa and CrCl. Thereafter, this association plateaued. When hemoglobin levels were categorized, hemoglobin 6 mmol/L showed no additional benefits in renal function.Conclusion: In conclusion, we found an association between baseline hemoglobin levels and superior renal function parameters, but not injury, during NMP of porcine kidneys. Furthermore, we show that performing a retrospective cohort study of preclinical data is feasible and able to answer additional questions, reducing the potential use of laboratory animals.

Impaired endogenous nitric oxide (NO) production may contribute to graft failure and premature mortality in kidney transplant recipients (KTR). We investigated potential associations of 24-h urinary NOx (NO₃^- + NO₂^-) excretion (uNOx) with long-term outcomes. uNOx was determined by HPLC and GC-MS in 698 KTR and in 132 kidney donors before and after donation. Additionally, we measured urinary nitroso species (RXNO) by gas-phase chemiluminescence. Median uNOx was lower in KTR compared to kidney donors (688 [393-1076] vs. 1301 [868-1863] before donation and 1312 [982-1853] μmol/24 h after donation, P < 0.001). During median follow-up of 5.4 [4.8-6.1] years, 150 KTR died (61 due to cardiovascular disease) and 83 experienced graft failure. uNOx was inversely associated with all-cause mortality (HR per doubling of uNOx: 0.84 [95% CI 0.75-0.93], P < 0.001) and cardiovascular mortality (HR 0.78 [95% CI 0.67-0.92], P = 0.002). The association of uNOx with graft failure was lost when adjusted for renal function (HR per doubling of uNOx: 0.89 [95% CI 0.76-1.05], P = 0.17). There were no significant associations of urinary RXNO with outcomes. Our study suggests that KTR have lower NO production than healthy subjects and that lower uNOx is associated with a higher risk of all-cause and cardiovascular mortality.

Kidney extraction time has a detrimental effect on post-transplantation outcome. This study aims to improve the flush-out and potentially decrease ischemic injury by the addition of hydrogen sulphide (H₂S) to the flush medium. Porcine kidneys (n = 22) were extracted during organ recovery surgery. Pigs underwent brain death induction or a Sham operation, resulting in four groups: donation after brain death (DBD) control, DBD H₂S, non-DBD control, and non-DBD H₂S. Directly after the abdominal flush, kidneys were extracted and flushed with or without H₂S and stored for 13 h via static cold storage (SCS) +/− H₂S before reperfusion on normothermic machine perfusion. Pro-inflammatory cytokines IL-1b and IL-8 were significantly lower in H₂S treated DBD kidneys during NMP (p = 0.03). The non-DBD kidneys show superiority in renal function (creatinine clearance and FENa) compared to the DBD control group (p = 0.03 and p = 0.004). No differences were seen in perfusion parameters, injury markers and histological appearance. We found an overall trend of better renal function in the non-DBD kidneys compared to the DBD kidneys. The addition of H₂S during the flush out and SCS resulted in a reduction in pro-inflammatory cytokines without affecting renal function or injury markers.

Main Problem: Following cold aortic flush in a deceased organ donation procedure, kidneys never reach the intended 0–4°C and stay ischemic at around 20°C in the donor’s body until actual surgical retrieval. Therefore, organ extraction time could have a detrimental influence on kidney transplant outcome.Materials and Methods: We analyzed the association between extraction time and kidney transplant outcome in multicenter data of 5,426 transplant procedures from the Dutch Organ Transplantation Registry (NOTR) and 15,849 transplant procedures from the United Network for Organ Sharing (UNOS).Results: Extraction time was grouped per 10-min increment. In the NOTR database, extraction time was independently associated with graft loss [HR 1.027 (1.004–1.050); p = 0.022] and with DGF [OR 1.043 (1.021–1.066); p < 0.005]. An extraction time >80 min was associated with a 27.4% higher hazard rate of graft failure [HR 1.274 (1.080–1.502); p = 0.004] and such kidneys had 43.8% higher odds of developing DGF [OR 1.438, (1.236–1.673); p < 0.005]. In the UNOS database, increasing extraction times in DCD donors were associated with DGF [OR 1.036 (1.016–1.055); p < 0.005]. An extraction time >30 min was associated with 14.5% higher odds of developing DGF [OR 1.145 (1.063–1.233); p < 0.005].Discussion: Prolonged kidney extraction time negatively influenced graft survival in Dutch donors and increased DGF risk in all deceased donor recipients.