H2S-Enriched Flush out Does Not Increase Donor Organ Quality in a Porcine Kidney Perfusion Model

Abstract

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.

Keywords: H2S; brain death; cytokines; ex vivo kidney perfusion; hydrogen sulphide; ischemia reperfusion injury; kidney graft preservation; transplantation.

Figure 1

Experimental overview. Group 1 Sham control, group 2 Sham + H₂S treatment, group 3 DBD control, group 4 DBD + H₂S treatment. Abbreviations: DBD = donation after brain death. H₂S = hydrogen sulphide. NMP = normothermic machine perfusion. SCS = static cold storage.

Figure 2

Cytokine release. (A–E): respectively IL-1b, IL-8, TNF IL-6, and IL-10 levels (pg/mL) in the perfusate during NMP. Data presented as median + interquartile range. * p < 0.05. Abbreviations: AUC = area under the curve. TNF = tumor necrosis factor. NMP = normothermic machine perfusion.

Figure 3

Complement activation. (A): C3a levels after the flushes and after SCS (ng/mL). (B): TCC in the perfusate during NMP (CAU/mL). (C): C3a in the perfusate during NMP (ng/mL). (D): C3a excretion in the urine (ng/h). (E): Fractional sodium excretion after 4 h NMP in the lowest TCC kidneys and the highest TCC kidneys. Data presented as median + interquartile range. * p < 0.05. Abbreviations: TCC = terminal complement complex. NMP = normothermic machine perfusion. SCS = Static cold storage.

Figure 4

Metabolic activity and ROS. (A): Oxygen consumption (mlO₂/min/100 gr). (B): Fractional sodium excretion (%). (C): Metabolic coupling (mmol/min/100 gr/mlO₂). (D): ATP values during preservation and after NMP (µmol/gr protein). (E): MDA after NMP, measured in tissue (µmol MDA/gram protein). (F): Arterial blood flow during NMP (mL/min). Data presented as median + interquartile range. * p < 0.05. Abbreviations: AUC = area under the curve. MDA = malondialdehyde.

Figure 5

Renal function and injury markers. (A): Creatinine clearance (mL/min/100 gr). (B): Proteinuria (mg/h). (C): LDH (relative change from baseline). (D): ASAT (relative change from baseline). (E). BAX/BCL2 ratios, measured in tissue after NMP. (F): NGAL secretion in urine (mg/h). Data presented as median + interquartile range. * p < 0.05. Abbreviations: AUC = area under the curve. LDH = lactate dehydrogenase. ASAT = aspartate aminotransferase. NGAL = urinary neutrophil gelatinase-associated lipocalin. BAX = bcl-2-like protein 4. BCL2 = B-cell lymphoma 2.

Figure 6

Representative histopathological images.