Effectiveness of pure argon for renal transplant preservation in a preclinical pig model of heterotopic autotransplantation

Abstract

Background: In kidney transplantation, the conditions of organ preservation following removal influence function recovery. Current static preservation procedures are generally based on immersion in a cold-storage solution used under atmospheric air (approximately 78 kPa N2, 21 kPa O2, 1 kPa Ar). Research on static cold-preservation solutions has stalled, and modifying the gas composition of the storage medium for improving preservation was considered. Organoprotective strategies successfully used noble gases and we addressed here the effects of argon and xenon on graft preservation in an established preclinical pig model of autotransplantation.

Methods: The preservation solution Celsior saturated with pure argon (Argon-Celsior) or xenon (Xenon-Celsior) at atmospheric pressure was tested versus Celsior saturated with atmospheric air (Air-Celsior). The left kidney was removed, and Air-Celsior (n = 8 pigs), Argon-Celsior (n = 8) or Xenon-Celsior (n = 6) was used at 4 °C to flush and store the transplant for 30 h, a duration that induced ischemic injury in our model when Air-Celsior was used. Heterotopic autotransplantation and contralateral nephrectomy were performed. Animals were followed for 21 days.

Results: The use of Argon-Celsior vs. Air-Celsior: (1) improved function recovery as monitored via creatinine clearance, the fraction of excreted sodium and tubulopathy duration; (2) enabled diuresis recovery 2-3 days earlier; (3) improved survival (7/8 vs. 3/8 pigs survived at postoperative day-21); (4) decreased tubular necrosis, interstitial fibrosis, apoptosis and inflammation, and preserved tissue structures as observed after the natural death/euthanasia; (5) stimulated plasma antioxidant defences during the days following transplantation as shown by monitoring the "reduced ascorbic acid/thiobarbituric acid reactive substances" ratio and Hsp27 expression; (6) limited the inflammatory response as shown by expression of TNF-alpha, IL1-beta and IL6 as observed after the natural death/euthanasia. Conversely, Xenon-Celsior was detrimental, no animal surviving by day-8 in a context where functional recovery, renal tissue properties and the antioxidant and inflammation responses were significantly altered. Thus, the positive effects of argon were not attributable to the noble gases as a group.

Conclusions: The saturation of Celsior with argon improved early functional recovery, graft quality and survival. Manipulating the gas composition of a preservation medium constitutes therefore a promising approach to improve preservation.

Fig. 1

Saturation of Celsior with argon, xenon or atmospheric air. Celsior was saturated with argon, xenon or atmospheric air in a glass bottle (part a; picture A). The kidney graft was flushed using the cold solution of interest (250 ml) via a catheter inserted into the renal artery (b; B). The remaining solution (250 ml) was transferred in a plastic pocket (c; C) prior to an additional saturation step (d). The graft was then immersed in the solution of interest, and the pocket was sealed by welding (e; D). A final saturation step was performed prior to verifying that the atmosphere was appropriate. The graft was then stored for 30 h at 4 °C before transplantation (f)

Fig. 2

Argon-Celsior improved early graft functional recovery and survival. After transplantation, renal function was monitored using creatinine clearance (a; ml/min), plasma urea (b; mmol/l), diuresis (c; ml/24 h; diuresis before surgery: 1500–2000 ml/24 h), fraction of excreted sodium (FE Na + ; d) and the urine urea/plasma urea ratio (Uu/Pu; e). The data are reported as the means ± SDs; p < 0.050 (asterisks): Argon-Celsior vs. Air-Celsior. The survival data are plotted (f; dashed line Air-Celsior; solid line Argon-Celsior; dotted line Xenon-Celsior)

Fig. 3

Argon-Celsior preserved transplant appearance. Per-operative pictures of the transplants 30 min after reperfusion (1) and at autopsy (2) are shown. A right kidney is shown and represents the authentic situation (control kidney; a, e). After reperfusion (1), macroscopic examination revealed major changes that included an impaired recolouration of the transplant in the Air-Celsior group (b) and further alterations in the Xenon-Celsior group (c) in addition to large necrotic areas (asterisks). The transplants stored in Argon-Celsior (d) exhibited appearances that were similar to those of the control kidneys. At autopsy (2), the Air-Celsior (f) and Xenon-Celsior (g) transplants exhibited significant cortical injuries compared with the Argon-Celsior transplants (h)

Fig. 4

Argon-Celsior preserved tissue integrity. Light microscopy images of kidney tissue samples (×40). The contralateral kidney (see “Methods” section) was considered to represent the authentic renal architecture with normal tubules (CL; a). The kidney taken from the Air-Celsior animals (Air; b) exhibited tubule damage with tubular atrophy, interstitial fibrosis and lymphocyte infiltration (x). The kidney taken from the Xenon-Celsior animals (Xe; c) exhibited major lesions, including strong tubular atrophy and fibrosis. In contrast, the kidney taken from the Argon-Celsior animals (Ar; d) exhibited less tubule damage, weak tubular atrophy and satisfactory trophic signals. Immunostaining of caspase-3 expression associated with kidney samples taken at autopsy is shown (Air-Celsior group: Air; e; Xenon-Celsior group: Xe; f; ×40; Argon-Celsior group: Ar; g): labelling was weaker in Argon-Celsior transplants vs. Air-Celsior and no labelling was found in Xenon-Celsior transplants

Fig. 5

Argon-Celsior stimulated antioxidant defences and limited inflammation. Before left nephrectomy and at postoperative day 1, the antioxidant status was assessed via plasma RAA/TBARS ratio (a; RAA reduced acorbic acid; TBARS thiobarbituric acid reactive substances) and plasma expression of Hsp27 (b). Tissue expression of TNF-alpha, Interleukin-1 beta and Interleukin-6 was determined (c). The data are reported as the means ± SDs; p < 0.050 (asterisks): Argon-Celsior versus Air-Celsior and p < 0.050 (bash): Argon-Celsior versus Xenon-Celsior