Normothermic ex vivo kidney perfusion preserves mitochondrial and graft function after warm ischemia and is further enhanced by AP39

Abstract

We previously reported that normothermic ex vivo kidney perfusion (NEVKP) is superior in terms of organ protection compared to static cold storage (SCS), which is still the standard method of organ preservation, but the mechanisms are incompletely understood. We used a large animal kidney autotransplant model to evaluate mitochondrial function during organ preservation and after kidney transplantation, utilizing live cells extracted from fresh kidney tissue. Male porcine kidneys stored under normothermic perfusion showed preserved mitochondrial function and higher ATP levels compared to kidneys stored at 4 °C (SCS). Mitochondrial respiration and ATP levels were further enhanced when AP39, a mitochondria-targeted hydrogen sulfide donor, was administered during warm perfusion. Correspondingly, the combination of NEVKP and AP39 was associated with decreased oxidative stress and inflammation, and with improved graft function after transplantation. In conclusion, our findings suggest that the organ-protective effects of normothermic perfusion are mediated by maintenance of mitochondrial function and enhanced by AP39 administration. Activation of mitochondrial function through the combination of AP39 and normothermic perfusion could represent a new therapeutic strategy for long-term renal preservation.

Fig. 1. Five-hour normothermic ex vivo perfusion ameliorates ischemia-reperfusion injury compared to renal preservation with static cold storage.

A Schema of the study design: kidneys retrieved  from 30-kg pigs were subjected to 60 min of warm ischemia, randomized to 5 h of ex vivo warm perfusion or static cold storage, and followed for 3 days after autotransplantation and contralateral nephrectomy. The no warm ischemia group that underwent transplantation after minimal storage was used as sham control (n = 5 pigs/No WI, 6 pigs/SCS, and 6 pigs/NEVKP). B Plasma creatinine levels and urea nitrogen from preoperative to day 3 (n = 5 pigs No WI, 6 pigs SCS, and 6 pigs NEVKP). C 24-h urine storage test from day 2 to day 3. 24-hour urine output, creatinine clearance, and fractional excretion of sodium (n = 5 pigs No WI, 6 pigs SCS, and 5 pigs NEVKP). In one animal in the NEVKP group, the urine sample could not be collected postoperatively due to technical error and was excluded from the analysis. D PAS stained image at the border between renal cortex and medulla obtained on day 3, comparing tubular injury score and inflammation score (n = 5 pigs No WI, 6 pigs SCS, and 6 pigs NEVKP). Scale bar, 100 μm. Results are presented as means ± SD. Comparisons between two groups were made using the two-tailed unpaired t test, and comparisons between three groups were made using ANOVA and the Tukey post hoc test. Panel (A), created with BioRender.com, was released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. WI, ischemia; SCS, static cold storage; NEVKP, normothermic ex vivo kidney perfusion.

Fig. 2. Differences in mitochondrial function and ATP levels based on the method of organ preservation.

A Oxygen consumption rate in cells extracted from renal cortex tissue for analysis of mitochondrial respiration after organ preservation (n = 3 pigs/No WI, 3 pigs/SCS, and 3 pigs/NEVKP; n = 17 replicates/No WI, n = 17 replicates/SCS, and 18 replicates/NEVKP). Changes from baseline to after metabolic stress. B Differences in baseline oxygen consumption rates, calculated ATP-linked respiration, maximal respiration capacity, and reserve capacity (n = 3 pigs Healthy Kidney, 3 pigs SCS, and 3 pigs NEVKP; n = 17 replicates Healthy Kidney, n = 17 replicates SCS, and 18 replicates NEVKP). C ATP concentration per 100000 cells obtained from renal cortical tissue after organ storage and before implantation and at 30 min after implantation (n = 5 pigs No WI, 5 pigs SCS, and 5 pigs NEVKP). D Immunofluorescence co-staining of Hsp60 (green) and DAPI (blue) within porcine kidneys after organ storage and before implantation and at 30 min after implantation. Scale bar, 10 μm (n = 5 pigs No WI, 6 pigs SCS, and 6 pigs NEVKP). E Circulating cell-free miDNA at 3 h and 3 days after reperfusion (n = 5 pigs No WI, 6 pigs SCS, and 6 pigs NEVKP). Results are presented as means ± SD. Comparisons between two groups were made using the two-tailed unpaired t test, and comparisons between three groups were made using ANOVA and the Tukey post hoc test. WI, ischemia; SCS, static cold storage; NEVKP, normothermic ex vivo kidney perfusion; OCR, oxygen consumption rate; ATP, adenosine triphosphate; mtDNA, mitochondrial DNA.

Fig. 3. Evaluation of the effect of NEVKP on oxidative stress and inflammation markers associated with mitochondrial injury in ischemia-reperfusion injury.

A Graphical representation of the biological response triggered by mitochondrial injury in ischemia-reperfusion injury. Mitochondrial dysfunction releases reactive oxygen species and mtDNA, which in turn activate inflammatory pathways. B Comparison of plasma levels of TBARS, a marker of oxidative stress, from preoperative to day 3 post-implantation (n = 5 pigs/No WI, 6 pigs/SCS, and 6 pigs/NEVKP). C Plasma concentration of myeloperoxidase, a marker of inflammation, at 1–3 days post-implantation (n = 5 pigs No WI, 6 pigs SCS, and 6 pigs NEVKP). Results are presented as means ± SD. Comparisons between two groups were made using the two-tailed unpaired t test, and comparisons between three groups were made using ANOVA and the Tukey post hoc test. Panel (A), created with BioRender.com, was released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. WI, ischemia; SCS, static cold storage; NEVKP, normothermic ex vivo kidney perfusion; ATP, adenosine triphosphate; mtDNA, mitochondrial DNA; TBARS, 2-thiobarbituric acid reactive substances; MPO, myeloperoxidase.

Fig. 4. Addition of AP39 during NEVKP following WI has beneficial effects on renal blood flow and urine output.

A Schema of the study design: kidneys retrieved  from 30-kg pigs were subjected to 60 min of warm inhibition, randomized to 5 h of ex vivo warm perfusion with or without AP39 mitochondria-targeted drug, and followed for 3 days after autotransplantation and contralateral nephrectomy (n = 6 pigs/NEVKP and 5 pigs/NEVKP + AP39). B Renal blood flow, intravascular resistance, urine volume and total urine output, urine glucose, lactate, and glucose levels during 5 h of warm perfusion (n = 5–6 pigs NEVKP and 5 pigs NEVKP + AP39). In one animal in the NEVKP group, the urine sample could not be collected postoperatively due to technical error and was excluded from the analysis. Results are presented as means ± SD. Comparisons between the two groups were made using the two-tailed unpaired t test. Panel (A), created with BioRender.com, was released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. NEVKP, normothermic ex vivo kidney perfusion.

Fig. 5. Administration of AP39 during perfusion reduces ischemia-reperfusion injury and enhances graft function after transplantation.

A PAS stained image at the border between the renal cortex and medulla obtained on day 3, comparing tubular injury score and inflammation score (n = 6 pigs/NEVKP and 5 pigs/NEVKP + AP39). Scale bar, 100 μm. B Results of renal function trends. Plasma creatinine levels and urea nitrogen from preoperative to day 3 (n = 6 pigs NEVKP, 5 pigs NEVKP + AP39, and 3 pigs SCS + AP39). C 24-h urine storage test from day 2 to day 3. 24-hour urine output, creatinine clearance, and fractional excretion of sodium (n = 5 pigs NEVKP and 5 pigs NEVKP + AP39). In one animal in the NEVKP group, the urine sample could not be collected postoperatively due to technical error and was excluded from the analysis. Results are presented as means ± SD. Comparisons between the two groups were made using the two-tailed unpaired t test. NEVKP, normothermic ex vivo kidney perfusion.

Fig. 6. AP39 administration further activates mitochondrial function in the kidney during NEVKP.

A ATP concentration per 100000 cells was obtained from renal cortical tissue at time points after organ storage and before implantation, and at 30 min after implantation (n = 5 pigs/NEVKP and 5 pigs/NEVKP + AP39). B Oxygen consumption rate in cells extracted from renal cortex tissue for analysis of mitochondrial respiration after organ preservation. Changes from baseline to after metabolic stress (n = 3 pigs NEVKP and 3 pigs NEVKP + AP39; n = 18 replicates NEVKP and 15 replicates NEVKP + AP39). C Differences in baseline oxygen consumption rates, calculated ATP-linked respiration, maximal respiration capacity, and reserve capacity (n = 3 pigs NEVKP and 3 pigs NEVKP + AP39; n = 18 replicates NEVKP and 15 replicates NEVKP + AP39). D Immunofluorescence co-staining of Hsp60 (green) and DAPI (blue) within porcine kidneys at time points after organ storage and before implantation and at 30 min after implantation (n = 6 pigs NEVKP and 5 pigs NEVKP + AP39). Scale bar, 10 μm. E Circulating cell-free miDNA at 3 h and 3 days after reperfusion (n = 6 pigs NEVKP and 5 pigs NEVKP + AP39). Results are presented as means ± SD. Comparisons between the two groups were made using the two-tailed unpaired t test. NEVKP, normothermic ex vivo kidney perfusion; OCR, oxygen consumption rate; ATP, adenosine triphosphate; mtDNA, mitochondrial DNA.

Fig. 7. Addition of AP39 during NEVKP reduces oxidative stress and inflammation.

A Comparison of plasma and perfusate levels of TBARS, a marker of oxidative stress, from preoperative to day 3 post-implantation (n = 6 pigs/NEVKP and 5 pigs/NEVKP + AP39). B Plasma concentration of myeloperoxidase, a marker of inflammation, at 1–3 days post-implantation (n = 6 pigs NEVKP and 5 pigs NEVKP + AP39). Results are presented as means ± SD. Comparisons between the two groups were made using the two-tailed unpaired t test. NEVKP, normothermic ex vivo kidney perfusion; TBARS, 2-thiobarbituric acid reactive substances; MPO, myeloperoxidase.