Routine cold storage leads to hyperacute graft loss in pig-to-primate kidney xenotransplantation; hypothermic machine perfusion may be preferred preservation modality in xenotransplantation

Abstract

Xenotransplantation (XTx) is an increasingly realistic solution to the organ shortage. Clinical XTx may require off-site procurement in a designated pathogen free (DPF) facility necessitating a period of cold ischemic time during transportation. This study evaluates the impact of different kidney preservation strategies on early graft function in pig-to-baboon XTx in a series of eight cases of pig-to-baboon xenotransplantation performed after five hours of cold ischemic time and compares these results to six cases of pig-to-baboon xenotransplantation performed with minimal ischemic time. Our data indicates that porcine kidneys appear to be particularly sensitive to IRI after cold preservation, especially across xenogeneic barriers, and routine static cold storage leads to hyperacute graft loss even in recipients with low levels of preformed antibodies. Hypothermic machine perfusion minimizes IRI and may prevent early xenograft loss.

Figure 1. Representative images of kidney xenografts with 5-hours CIT after reperfusion with low immunological risk (A and B) and high risk (C and D).

(A) SCS-preserved kidney with low risk looked healthy immediately following reperfusion but subsequently exhibited mottling that progressed to diffuse discoloration in 1 hour post-reperfusion (B) The graft preserved using HMP were reperfused without clinically apparent changes. (C) SCS-preserved graft with high risk showed severe discoloration in 1 hour post-reperfusion. (D) A graft preserved using HMP looked healthy immediately following reperfusion but lost function in 2 hours.

Figure 2. Histopathological findings of the kidney xenografts with 5-hours CIT.

(A) SCS-preserved kidney (low risk) showed extensive hemorrhage and hemostasis one hour after reperfusion (hematoxylin and eosin (H&E) staining). (B) HMP-preserved kidney (H&E) did not show significant pathological changes one hour after reperfusion. (C and D) In high-risk cases, severe interstitial hemorrhages were observed in both SCS- and HMP-preserved xenografts.

Figure 3. Complement (C3) deposition on immunofluorescence staining.

Notable deposition of C3, indicating complement activation, was observed in SCS-preserved kidneys, but not in HMP-preserved kidneys.

Figure 4. Cell infiltration immunohistochemistry in the kidney xenografts with 5-hours CIT.

SCS-preserved kidneys in 2 hours post-reperfusion had significantly higher infiltration of myeloperoxidase (MPO) positive neutrophils compared with HMP-preserved kidneys. There was no significant difference in CD56-positive cells (NK cells) and CD68-positive cells (macrophages) between SCS- and HMP-preserved kidneys in 1 hour after transplantation.

Figure 5. Transcriptional landscape of reperfused kidney xenografts by preservation strategy.

Differential expression (x-axis) and significance (y-axis) of DSP genes between HMP (right) and SCS (left) regions of interest. P values were computed using the Wald test from the DESeq2 standard workflow and adjusted for multiple testing with the Benjamini-Hochberg approach.

Figure 6. Spatially resolved B-HOT gene set enrichment of reperfused kidney xenografts by preservation strategy.

Scaled single sample gene set enrichment scores for nine pathways defined by the Banff 2019 Meeting Report. Comparisons between HMP and SCS preservation are shown for combined (pooled), glomerular, and tubulointerstitial regions of interest for each pathway. P values were computed using the two-sided Wilcoxon rank-sum (Mann-Whitney U) test and adjusted for multiple testing with the Benjamini-Hochberg approach (***, P < 0.001; **, P < 0.01; *, P < 0.05; not significant, n.s.).