**1. Introduction**

Organ damage resulting from preservation or reperfusion still represents a major issue in transplantation medicine. Extended preservation times or graft donation after cardiocirculatory standstill in the donor are often conflicted with a reduced recovery after transplantation affecting early graft function, as well as long-term survival.

Although the underlying mechanistic pathways of preservation injury are not yet fully understood, altered mitochondrial integrity upon reperfusion has been deciphered as a major culprit for graft dysfunction after transplantation [1,2]. During cold ischemic preservation, opening of the mitochondrial transition pore (MTP) could be observed along with mitochondrial swelling [3,4]. However, mitochondrial respiratory capacity has been shown to remain stable for up to 24 h of cold storage and only deteriorates after unusual prolongation of cold ischemia or upon subsequent warm reperfusion [5].

**Citation:** Zlatev, H.; von Horn, C.; Minor, T. Preservation of Mitochondrial Coupling and Renal Function by Controlled Oxygenated Rewarming of Porcine Kidney Grafts. *Biomolecules* **2021**, *11*, 1880. https:// doi.org/10.3390/biom11121880

Academic Editor: Liang-Jun Yan

Received: 10 November 2021 Accepted: 9 December 2021 Published: 14 December 2021

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Likewise, cytosolic release of cytochrome c and sequential induction of apoptosis are only observed after subsequent rewarming [4,6,7]. In line with these observations, main parts of organ preservation injury substantiate not during cold storage but upon and in consequence of abrupt rewarming [8,9]. Hence, significant alleviation of functional impairment of kidney grafts after transplantation could be achieved by modifying the rise in temperature during reoxygenation using a controlled oxygenated rewarming (COR) protocol subsequent to cold storage by thermoregulated machine perfusion [10,11]. The use of an incremental temperature rise instead of an abrupt return to normothermia improved post-ischemic recovery of discarded human donor livers [12] and resulted in superior results after experimental [10] and clinical [13] kidney transplantation. COR was also found to be operative in significantly reducing the initiation of the mitochondrial apoptotic pathway upon reperfusion along, with a better preservation of mitochondrial content of NAD+ [14]. Likewise, COR improved global recovery of tissue energetics [9] and largely preserved oxygen utilization efficiency at the whole organ level that was otherwise found significantly disturbed [11].

Controlled oxygenated rewarming during machine perfusion classically comprises three phases. A first period of oxygenated cold perfusion is undertaken to fuel residual aerobic metabolism to ameliorate subcellular homeostasis while avoiding major temperature shifts [15,16]. Then follows a thermal transition phase of slow and adapted increase in the perfusion temperature [9] and a final steady state period of perfusion at the final temperature for a variable time span [7].

Although the positive effects of a brief hypothermic perfusion period alone have been thoroughly established in that the cellular aerobic energetic homeostasis could be improved in the cold and that the tissue is better prepared for the following abrupt warm reperfusion [15–17], the relevance of an extended cold perfusion phase versus an immediate start of the gentle warming up protocol in the setting of COR has not yet been addressed.

Thus, the aim of the present study was to scrutinize the relative impacts of a hypothermic equilibration phase in the setting of a controlled rewarming protocol with a special focus on mitochondrial function and coupling status.

#### **2. Materials and Methods**

All experiments were performed in accordance with the federal law regarding the protection of animals. The principles of laboratory animal care (NIH publication no. 85-23, revised 1985) were followed.

Kidneys were removed from dead German Landrace pigs weighing between 25 and 30 kg. Then, 15 min after cardio-circulatory standstill, the renal artery was cannulated and the kidneys were flushed by 100 cm gravity with 100 mL of HTK solution (Köhler Chemie, Bensheim, Germany) on the back-table at 4 ◦C. No heparin was given at any time. After 18 h of static cold preservation in HTK solution, the grafts were randomly assigned to one of the following groups (n = 6, resp.):


**Figure 1.** Representation of the thermal kinetic during controlled oxygenated rewarming by machine perfusion of porcine kidney grafts. Gentle rewarming of kidneys is either performed after an initial 30 min hypothermic equilibration period (COR, hatched line) or by starting the rewarming protocol immediately after connection to the machine (iCOR, continuous line).

Perfusion with Aqix-RS-I (Life Science Group, Bedford, UK) was started at a temperature of 8 ◦C but instant controlled oxygenated rewarming (iCOR) was started increasing temperature up to 35 ◦C during the first 60 min in a hyperbolic pattern comparable to the middle period in group 2. The rise in temperature was accompanied by an adapted increase of the perfusion pressure from 30 to 75 mmHg. The last 30 min of perfusion was kept constant at 35 ◦C.

#### *2.1. Reperfusion Model*

Prior to reperfusion all grafts were flushed with 100 mL of cold saline solution and exposed to no flow conditions at room temperature for 20 min in order to imitate the time of surgical engraftment.

The functional recovery of the grafts was tested using an established in vitro model, as previously described [19], which was modified regarding the replacement of fluid loss by urine production during ongoing reperfusion [20].

In brief, kidneys were put into a moist chamber and perfused at 37 ◦C with 1000 mL Krebs–Henseleit buffer to which were added 2.2% bovine serum albumin (PAN-Biotech, Aidenbach, Germany) and 20 mL of concentrated amino acid solution (RPMI 1640 Amino Acids Solution, 50×, PAN-Biotech, Aidenbach, Germany).

Perfusate was oxygenated with a mixture of 95% oxygen and 5% carbon dioxide by a hollow fiber oxygenator (Hilite LT 1000, Medos, Stolberg, Germany) and supplemented with 0.05 g/L of creatinine to allow for calculation of renal clearances. Cannulation of the ureter was performed with PE tubing for urine collection throughout the reperfusion period.

Urine produced during reperfusion was collected and reinfused after filtration (13 μm) at 100 mL intervals to the reservoir in order to prevent alterations in the composition of the perfusate over time, which would be encountered when replacing urine loss by adding balanced salt solution [21]. The volume of urine production was measured for each individual 30 min interval and representative aliquots from each fraction were pooled for the respective intervals for later analysis of metabolites.

Kidney perfusion pressure was set at 90 mmHg and automatically maintained by a servo-controlled roller pump connected to a pressure sensor placed in the inflow line immediately prior to the renal artery.

At the end of the perfusion, kidneys were removed from the perfusion chamber and samples were taken for biochemical analysis and for isolation of mitochondria.
