*Article* **Preservation of Mitochondrial Coupling and Renal Function by Controlled Oxygenated Rewarming of Porcine Kidney Grafts**

**Hristo Zlatev, Charlotte von Horn and Thomas Minor \***

Surgical Research Department, Clinic for General, Visceral and Transplantation Surgery, University Hospital Essen, Hufelandstr. 55, D-45147 Essen, Germany; chirfor@uk-essen.de (H.Z.); charlotte.von-horn@uk-essen.de (C.v.H.)

**\*** Correspondence: thomas.minor@uk-essen.de; Tel.: +49-201-723-2713; Fax: +49-201-723-6859

**Abstract:** Background: Warm reperfusion after previous cold storage has been shown to have a negative impact on mitochondrial function of organ grafts. Here, we wanted to investigate whether a more controlled warming up of the cold graft by ex vivo machine perfusion with gradually elevated temperature from cold to normothermia (including comparison of two warming up protocols) prior to implantation would be effective in preventing mitochondrial dysfunction upon reperfusion. Methods: All experiments were conducted on porcine kidneys retrieved 15 min after cardiac arrest. After 18 h of cold storage in HTK solution (CS, n = 6), kidneys (n = 6) were subjected to 2 h of reconditioning machine perfusion starting with a hypothermic period followed by a gradual increase in perfusion temperature up to 35 ◦C (controlled oxygenated rewarming—COR). For a second group (n = 6), the slow warming up was begun instantly after connecting the graft onto the machine (iCOR). Functional recovery of all grafts was then observed upon normothermic reperfusion in vitro. At the conclusion of the experiments, tissue specimens were taken for immediate isolation and analysis of renal mitochondria. Results: COR resulted in a significantly and more than 3-fold increased glomerular filtration rate upon reperfusion, along with a significant higher tubular sodium reabsorption and lesser loss of glucose in comparison to the controls. Enzyme release (AST) was also massively reduced during the reperfusion period. Specific analysis at the mitochondrial level revealed significantly better coupling efficiency and spare respiratory capacity in the COR group compared to the cold storage group. Interestingly, additional experiments revealed that the omission of a hypothermic perfusion period did not deteriorate any of the results after COR, provided that the instant temperature increase from 10 to 35 ◦C was effectuated in the same controlled manner. Conclusion: Controlled rewarming after extended cold preservation effectively improves mitochondrial recovery upon reperfusion and early functional outcome of kidney grafts.

**Keywords:** controlled oxygenated rewarming; mitochondrial uncoupling; rewarming injury; temperature paradox
