Investigation of the transcriptional profile of human kidneys during machine perfusion reveals potential benefits of haemoadsorption

Abstract

Transplantation is the optimal treatment for most patients with end stage kidney disease but organ shortage is a major challenge. Normothermic machine perfusion (NMP) has been used to recondition marginal organs but the mechanisms by which NMP might benefit transplant kidneys are not fully understood. Furthermore, the question of whether removal of pro-inflammatory mediators from the perfusate might offer additional benefits in optimising kidneys prior to transplantation has not been addressed. Using pairs of human kidneys obtained from the same donor, we compared the effect of NMP with that of cold storage on the global transcriptome of kidneys, and then went on to investigate the impact of adding a haemoadsorption device to the NMP circuit. We found that cold storage significantly reduced the expression of inflammatory genes, but also of genes required for energy generation such as those encoding oxidative phosphorylation (OXPHOS) enzymes. In contrast, during NMP, there was marked upregulation OXPHOS genes, as well as a number of immune and inflammatory pathway genes. The induction of inflammatory genes during NMP was substantially attenuated by the addition of a haemoadsorber to the perfusion circuit, which also further increased OXPHOS pathway gene expression. Together, our data suggest that absorption of pro-inflammatory mediators from the perfusate represents a useful intervention that may further improve organ viability and should be tested in clinical practice. 4C, D). Oxygen consumption and acid-base homeostasis were also similar between kidney pairs and were not significantly impacted by the addition of the HA (Table 3). Thus, over 4 hours of NMP, the HA had no impact on the parameters currently used clinically to generate quality assessment scores. Addition of a haemoadsorber to the perfusion circuit has a substantial impact on gene expression, reducing inflammatory pathway genes and increasing OXPHOS pathway genesDespite the lack of effect on perfusion parameters, we found a substantial effect of the HA on gene expression. At 2 hours post-perfusion, n=1794 genes were upregulated in NMP kidneys but only half this number (n=898) were increased when the HA was present (Figure 5A). Similarly, at 4 hours n=4026 genes were up-regulated in the NMP group and n=2606 in the NMP+HA group ( Figure 5A). The number of genes down-regulated was also reduced by the addition of HA (Figure 5A). Of note, n=1702 genes were upregulated at both 2 and 4 hour timepoints ( Figure 5A), including TNF and IL6 (Figure 5B). The addition of a HA reduced the up-regulation of these pro-inflammatory genes (Figure 5B), supporting the thesis that re-circulating small molecules can exacerbate inflammation in perfused organs. Further differences were observed between the groups for many members of the cytokine and chemokine gene families (Figure S3A and B). To address difference in gene expression as a result of the addition of a HA to NMP, we directly compared the experimental groups after 4 hours of NMP and found 46 genes were signific...