In the present study, we confirmed previous findings demonstrating the benefits of MP on chronic kidney graft outcome . Considering the importance of the solution bias when measuring the impact of machine preservation  and the fact that we previously demonstrated the superiority of MP over CS, all other parameters being equal [12, 19], we did not include in the present study a CS group using standard static preservation solution (for instance University of Wisconsin) as we felt a repetition of these experiments would represent an unethical use of animals, in addition, an non-randomized prospective multi-center study showed that in clinical practice IGL-1 solution has the same effectiveness as UW solution in cold storage . However, in previous studies using the same DCD model with static University of Wisconsin preservation, we found serum creatinine levels of approximately 400 μmol/L at 3 months , which when compared to the data of the present suggests superiority of MP versus CS when these modes are used in clinic-like conditions. Nevertheless, use of MPS for both CS and MP permitted the measurement of the benefits of the RM3 without interference from solution components, and indeed when using MPS, recommended solution for the RM3 machine, we show that MP increases graft survival and preserves chronic function parameters such as proteinuria compared to static storage. However, other typical parameters such as serum creatinine and fibrosis were not changed by the machine. In depth analysis of the phenotypes within the kidney graft by RTqPCR showed decreased levels in the expression of markers typical of chronic lesion such as Notch 4, which has been shown to inhibit angiogenesis , thus likely impeding the proper reparation mechanisms within the graft. We also detected lower p47phox expression in MPS-MP group, a subunit of NADPH oxidase. NADPH oxidase is a protein complex located in the plasma membrane as well as in the membrane of phagosome, which variation in expression have been linked to the level of oxidative stress in the tissue and to chronic injury . Thus, MP appears beneficial in regards to chronic stress on the parenchyma.
Another important mechanism of chronic kidney graft lesion is epithelial to mesenchymal transition, during which epithelial cells differentiate and alter their phenotype towards that of a mesenchymal cell, thus going from a polarized, anchored and non dividing cell to an unpolarized, mobile and fast proliferating cell. This regenerative process is believed to be deregulated and promote fibrosis in the context of chronic graft rejection . We investigated this phenomenon by first using RTqPCR to observe expression of Vimentin, typical marker of EMT, and CTGF, an important downstream effector of TGF-β involved in fibrosis  and EMT . In our study, MPS-MP grafts had decreased expression of these markers compared to MPS-CS graft, a result which was then confirmed by immunohistofluorescence for Vimentin.
Hence, although interstitial fibrosis measurement using standard method (Sirius Red Staining) did not reveal a difference between these groups, mechanistic analysis showed that this process was slowed down in the MP group compared to the CS group. These results suggest that uncovering MP benefits may be difficult using standard methods of evaluation. We previously came to a similar conclusion during the evaluation of the Lifeport machine , when the use of KPS, of identical composition than MPS, made comparison between MP and CS more difficult as there was only slight differences in survival and typical biochemical evaluation. There also, proteinuria was one of the most discriminating markers.
In this previous study as well as the present one, the bias of the perfusion solution was very apparent. In both studies, multivariate analyses revealed both an individual effect of the machine and the solution on outcome markers, but more importantly showed a significant effect of the combination of factors. Our results herein show that the evidence for MP benefits is difficult to find when using IGL1 in CS and MP.
We previously reported little differences between IGL1 and MPS in MP when follow up was limited to 30 days, with a slight advantage for the use of MPS in terms of histological injury. Herein, longer follow up highlighted differences between both IGL groups and MPS-CS as evidenced by several parameters such as survival and proteinuria, as well as lesional markers detected by RTqPCR. Both IGL groups displayed reduced expression of TGFβ and Vimentin, which was confirmed by immunostaining, suggesting a better level of protection obtained through the use of the PEG-containing solution.
The use of PEG involves the concept of immunocamouflage , we thus investigated the immune response within the grafts. Interestingly, classical evaluation of immune cell invasion did not reveal differences between the groups (data not shown), however RTqPCR evaluation of immune markers determine some effects of IGL compared to MPS. IGL preserved grafts presented reduced levels of IL-10 and IL-1Rn, typical of Th2 phenotype. As Th2 immunity is linked to the development of fibrosis , the protection provided by IGL appears to be directed against fibrotic pathways.
Further investigation of innate immunity by RTqPCR analysis revealed that expression of TNFα, TLRs and Pselectin was the lowest in IGL grafts. TNF-α is one of the earliest cytokine produced in response to stresses, particularly by resident dendritic cells . TLRs are well described effectors of ischemia reperfusion injury  and TLR4 as recently been shown to have a central role in kidney graft injury, both on the short and long term . Pselectin is a marker of endothelial activation. Hence, the use of IGL appears to lower the immunogenicity of the graft, confirming our previous findings in an allotransplantation model .
The benefits from combining IGL and MP were more difficult to determine. Indeed, the only statistical difference between the IGL-CS and IGL-MP groups was in the recorded level of TNFα expression, which was significantly lower than controls, suggesting a negative regulation taking place within the kidney at the time of analysis. More data points would be necessary to describe in further detail the dynamics of immune regulation in this context. However, the difficulty in assessing the benefits of the combined use of IGL1 and MPS is concordant with retrospective and prospective studies published in the literature which have shown the benefit of MP  but did not clearly demonstrate the independent effect of MP with different preservation solutions. However, IGL-MP group results were more consistently reaching statistically significant difference to MPS-CS compared to the other groups. Thus, combining MP and IGL increases the protective potential of each technique to better preserve the graft against chronic adverse outcomes.
The difficulty in discriminating between groups with classical approached such as histology and biochemistry underlines the need for tools providing in depth analysis of the tissue phenotypes. Herein we demonstrate that although two tissues show similar levels of fibrosis, one group is much further along on the path towards fibrosis development according to the transcripts expressed by the cells. These results highlight the need for further research on in depth biomarkers, particularly using highly reproducible models to identify specific reporters of injury linked to variations in a single parameter.
In conclusion, this study demonstrates that the evaluation of machine perfusion is highly dependant on the solution used. Multivariate analysis of variance confirmed that the perfusion solution was significantly associated with the result, and moreover that combination of solution and machine could have additive effects. IGL-1 used in either machine or static preservation offers similar levels of protection than standard MP. Importantly, our data is in contradiction with a previous report from our team with a shorter follow up time. This highlight the needs for proper follow up durations in preclinical studies, particularly in research areas where chronic outcome is of crucial importance. The compatibility of IGL-1 with both machine perfusion and static storage could represent an advantage for clinical teams when choosing the correct solution to use for multi-organ collection. The path towards improving machine perfusion, and organ quality, may thus involve the optimization of the solution and the correct use of colloids.