3. Discussion
mitochondrial dysfunction, Alzheimer’s disease, hypertensive, renal injury, sepsis, kidney injury
RENAL, GRAFT DYSFUNCTION, SEPSIS, HAND ISCHEMIA, RENAL DISEASE, EVENTS, OXIDATIVE STRESS, MITOCHONDRIAL DYSFUNCTION
In this post hoc analysis of the VAPOR-1 trial in LDKT, we observed differences in the dynamics of plasma mtDNA genes D-loop, ND1, and ND6, which changed over time and differed between vascular compartments. Additionally, we observed that baseline characteristics were able to explain the variability of mtDNA dynamics between subjects. In addition, both absolute and categorical mtDNA variables positively predicted the baseline of the eGFR model. The prediction for acute rejection episodes with mtDNA variables was ambiguous, and the odds of acute rejection were both higher and lower with absolute and categorical mtDNA variables. Our results show that mtDNA can be measured in the systemic venous, systemic arterial, and renal venous compartment of recipients. Donor pre-transplantation mtDNA levels were significantly lower compared to pre-transplantation recipient levels. This is most likely due to the fact that recipients are exposed to long-lasting continuous injurious events as a consequence of their renal disease, dialysis, and other related comorbidities. Eirin and colleagues reinforced this observation by demonstrating that in hypertensive patients urinary mtDNA levels are higher compared to healthy controls and that urinary mtDNA is associated with renal injury markers and graft dysfunction [Systemic arterial levels of mtDNA genes showed similar patterns of release, with stable mtDNA levels up to 30 min after reperfusion, followed by a significant increase at 2 h post-transplantation. This time window of the reperfusion period is a known feature of the extensive amount of oxidative stress associated with IRI [To our knowledge, this is the first study to have investigated mtDNA release derived directly from the kidney after reperfusion. Renal venous mtDNA levels were highest 30 s after reperfusion. As IRI inevitably occurs in transplantation, and as mitochondrial dysfunction has a central role in IRI, mtDNA is expected to be released from the transplanted kidney into the renal circulation of the recipient. In combination with a more local representation of injury and likely urinary excretion of mtDNA, this might explain the observation of lower concentrations of mtDNA in renal venous samples. It is interesting that renal venous mtDNA, reflecting local renal IRI, occurs in the early reperfusion period, whereas the systemic arterial increase occurs at 2 h post transplantation. This might reflect the capacity of mtDNA to propagate downstream activation of injurious pathways [It is important to account for variable patterns of mtDNA levels between patients. Our study showed that a female recipient had an increased baseline of systemic arterial and renal venous D-loop. Several studies have demonstrated that biological sex differences between donor and recipient impacts graft outcome [To our knowledge, to date, no study has investigated the predictive qualities of plasma mtDNA in a living donor kidney transplantation cohort. Accumulating evidence has identified plasma mtDNA as a pathogenic amplifier of injury in various clinical conditions, including in Alzheimer’s disease and sepsis [Although kidneys derived from deceased donors are exposed to a far greater extent of IRI, kidneys derived from living donors also experience IRI that impacts outcomes. In line with previous studies, our results demonstrated that systemic arterial ND1 and ND6 levels measured after reperfusion positively predicted acute rejection episodes, with higher levels at 5 min after reperfusion increasing the odds of an acute rejection episode. Contrary to these results, we demonstrated that higher mtDNA variables resulted in a higher baseline of the eGFR model. Therefore, it remains unclear to what extent mtDNA levels are predictive of optimal or suboptimal graft outcomes. It is of importance to mention that mtDNA increased the baseline of the eGFR model, which is representative of 1-month eGFR. The outcome of acute rejection episodes in a two-year follow-up period is a categorical variable, for which we did not account for a time factor. Factors of transplantation (e.g., biological sex and ischemia times), but also differences between the function of the three mtDNA genes, may have an influence on the predictive capacity. It might also be the case that a certain threshold of mtDNA is needed for physiological functioning, and beneficial for repair and mitochondrial genome stability, but when exceeded, cellular injury and pro-inflammatory immune activation occurs. As our cohort consisted of high-quality organs, with minimal IRI, this range of mtDNA levels may not have been observed in our study. A phenotypic switch from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages could explain this dual role of mtDNA [The strengths of our study include the measurement of donor mtDNA levels and the sequential measurements over time in recipients, providing valuable insights into the dynamics of mtDNA. Furthermore, we measured mtDNA in different vascular compartments (systemic venous, renal venous, systemic arterial) collectively covering samples that would be easily collectable in a routine clinical setting. However, similar timepoints of collection may be more beneficial for comparison between vascular compartments. This post hoc analysis of the VAPOR-1 study was limited by a small cohort of 56 patients receiving a high-quality kidney with a low incidence of inferior post-transplantation outcomes, due to limited IRI during LDKT. However, we did find interesting significant predictive functions of mtDNA for graft outcome. An important limitation to acknowledge is that we did not correct for multiple testing. Our study was of explorative design, and we considered that multiple testing correction was not warranted. Furthermore, in this study, mtDNA damage was not assessed to gain knowledge about the quality of mtDNA. In addition, excretion of mtDNA was not studied, and therefore we did not elaborate on the kinetics of mtDNA. In conclusion, this study is the first to explore plasma mtDNA in a LDKT cohort and in the renal graft effluent. Our findings indicate that plasma mtDNA has the potential to be of added value for the current screening methods of graft potency. mtDNA variables were predictive for optimal and suboptimal graft outcome parameters, and additional research in a validation cohort is warranted. Additional research is also needed into the function of the different mtDNA genes and the variability between patients. Furthermore, urinary mtDNA also has great potential to be a non-invasive injury marker of kidney injury and repair, and therefore we will proceed by studying urinary mtDNA in recipients of the VAPOR-1 trial. 