*2.2. Mitochondria Isolation and Analysis*

The mitochondria were isolated as described in [22]. Mitochondria isolation buffer (MIB) was prepared with final concentrations of 70 mM sucrose, 210 mM mannitol, 53 mM HEPES, and 1 mM EGTA, (pH adjusted to 7.4). Mitochondrial assay buffer (MAS) was composed of 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 2 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and 0.2% *w*/*v* fatty-acid-free BSA (pH 7.4).

The mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed using a Seahorse XFe24 Analyzer (Agient Technologies, Santa Clara, CA, US) and Seahorse XFe24 FluxPack (Agilent Technologies, Santa Clara, CA, US) as described by Rogers et al. (11).

Mitochondria were incubated with substrate (2 mM rotenone and 0.5 M succinate), following by sequential addition of 1M adenosine 5 -diphosphate potassium salt (ADP), 5 mg/mL oligomycin (ATP synthase inhibitor), 10 mM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, mitochondrial uncoupler), and 40 mM antimycin A (complex 3 inhibitor). All substances were purchased from Sigma-Aldrich, Germany. The results were analyzed with Wave Seahorse Software (Agilent Technologies, Santa Clara, CA, USA).

#### *2.3. Analytical Procedures*

Analytical routines were performed as described previously [20]. The activities of aspartate aminotransferase (AST) and concentrations of creatinine were determined in a routine fashion by reflectance photometry on a Reflotron Plus point of care unit (Roche Diagnostics, Mannheim, Germany).

Clearances were calculated for the respective intervals as urinary creatinine x urine flow/perfusate creatinine.

The albumin concentration in urine was measured in a routine fashion at the Laboratory Center of the University Hospital and the amount of protein normalized against the corresponding concentrations of creatinine as the urinary albumin-to-creatinine ratio (mg/mg).

Oxygen partial pressure and perfusate concentrations of sodium were measured in a pH-blood gas analyzer (ABL 815flex acid-base laboratory, Radiometer, Copenhagen).

Oxygen consumption (VO2) was calculated from the differences between arterial and venous sites and expressed as mL min−<sup>1</sup> g−<sup>1</sup> according to the trans-renal flow and kidney mass.

The efficiency of renal O2 utilization was approximated by the ratio of total kidney transport of Na (TNa), accounting for the vast majority of energy consuming processes in the kidney [23], and VO2, with TNa being equal to filtered Na minus excreted Na:

TNa = (GFR × Perfusate Na) − (urinary Na × urine flow)

Fractional excretion of sodium (FE Na+) was calculated according to:

FE Na+= Na+ (urine) <sup>×</sup> Creatinine(perfusate)/Na<sup>+</sup> (perfusate) <sup>×</sup> Creatinine (urine) <sup>×</sup> <sup>100</sup>

Measurement of neutrophil-gelatinase-associated lipocalin (NGAL) was performed with a commercialized ELISA kit (USCN life science, Wuhan, China) according to the instructions of the manufacturer on a fluorescence microplate reader (Tecan, Grailsheim, Germany).

#### *2.4. Histology*

At the conclusion of the experiment, tissue samples were collected for later histological examination. Specimens were cut into 3 mm blocks, fixed by immersion in 4% buffered formalin, and embedded in paraffin. Tissue slides were prepared on a SM 2000R microtome (Leica Instruments, Nußloch, Germany). Light microscopy (20× magnification) of periodic acid Schiff-stained sections were used to demonstrate changes in morphology.

#### *2.5. Statistical Analysis*

Kidneys were randomized withn=6 kidneys per group. Results are expressed as means ± standard deviation. Stochastic significance of differences was assessed using one way analysis of variance and Dunn's multiple comparisons test, if not otherwise indicated.

Data were analyzed with GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Significance was defined as *p* < 0.05.

#### **3. Results**

The glomerular filtration function was evaluated by measurement of the clearance of creatinine during warm reperfusion (cf. Figure 2). Controlled oxygenated rewarming (COR) was followed by a significant enhancement of glomerular filtration rate when compared to cold-stored controls. Of note, omitting the hypothermic equilibration phase in the instant rewarming (iCOR) group did not result in any adverse effect in comparison to the COR group.

**Figure 2.** Clearance of creatinine during reperfusion in vitro after 20 h cold storage (CS) or after 2 h of subsequent controlled oxygenated rewarming with (COR) or without (iCOR) an initial hypothermic perfusion period (\*: *p* < 0.05 COR vs. CS; #: *p* < 0.05 iCOR vs. CS).

Likewise, urinary leakage of albumin (expressed as the albumin/creatinine ratio) was markedly increased to 0.40 ± 0.14 in cold-stored kidneys but significantly (*p* < 0.05) reduced after COR (0.13 ± 0.05), as well as after iCOR treatment (0.12 ± 0.03) prior to reperfusion.

Tubular cell integrity was also found to be protected by both of the rewarming protocols. The concentration of neutrophil-gelatinase-associated lipocalin (NGAL) in the circulating perfusate was used as an indicator of tubular cell stress. It amounted to 6.8 ± 0.6 ng/mL in the cold storage group but was significantly (*p* < 0.05) reduced to 3.4 ± 0.6 and 3.9 ± 1.1 ng/mL in the COR and iCOR groups, respectively.

Differences were even more pronounced regarding the enzyme leakage of AST into the perfusate, which amounted to 659 ± 328 U/L, 140 ± 73 U/L \* and 182 ± 249 U/L \* after CS, COR, and iCOR, resp. (*p* < 0.05).

Tubular cell function was followed by measuring the fractional excretion of sodium from the renal ultrafiltrate (cf. Figure 3). Large amounts of the filtrated sodium could not be re-absorbed and were, thus, excreted with the urine in the cold storage group, although the excretion rate slightly improved during ongoing reperfusion. In contrast, significantly better values were observed after controlled oxygenated rewarming, and this benefit was independent from the inclusion of a hypothermic starting period during the rewarming protocol.

**Figure 3.** Course of fractional excretion of sodium (FENa) during isolated reperfusion in vitro after 20 h cold storage (CS) or after 2 h of subsequent controlled oxygenated rewarming with (COR) or without (iCOR) an initial hypothermic perfusion period (\*: *p* < 0.05 COR vs. CS; #: *p* < 0.05 iCOR vs. CS).

The efficiency of oxygen utilization by the renal tissue is approximated by the ratio between total sodium transport (TNa), accounting for the vast majority of renal energy consumption, and corresponding oxygen consumption (VO2). Cold-stored kidneys showed a markedly reduced TNa/VO2 ratio upon reperfusion, indicating a massively disturbed aerobic efficiency (Figure 4A). However, this impairment of oxygen utilization efficiency was significantly ameliorated by using one of the controlled rewarming protocols prior to reperfusion. Again, the protective effects of both rewarming protocols were virtually identical.

Specific mitochondrial function upon reperfusion was evaluated by respiratory assays with freshly isolated mitochondria from renal tissue at the end of reperfusion. The coupling assay reflects the oxidative phosphorylation efficiency of mitochondria.

In line with the data on oxygen utilization efficiency at the whole organ level, mitochondrial coupling efficiency was notably compromised in the CS group (Figure 4B), whereas controlled rewarming using either the COR or the iCOR protocol resulted in a significantly better preserved oxidative electro-chemical coupling rate at the mitochondrial level.

Spare respiratory capacity (SRC) characterizes mitochondrial reserve to meet an increasing energy demand in response to stress conditions. Mitochondria of only coldstored kidneys showed nearly no functional reserve upon warm reperfusion, whereas mitochondria of COR or iCOR treated kidneys yielded considerably higher SRC values.

Significant differences between the two rewarming protocols regarding mitochondrial function could not be observed in this setting (Figure 4C).

Light microscopy performed on tissue samples obtained after conclusion of the experiment did not disclose any glomerular damage, with only mild alterations in either group. However, some alterations of normal structural appearance were observed, mainly comprising tubular cell vacuolization, which was notably less prominent after COR or iCOR than in the cold storage group. Sporadic signs of necrosis were also observed only after cold storage (Figure 5).

**Figure 4.** Parameters of mitochondrial function upon isolated reperfusion in vitro after 20 h cold storage (CS) or after 2 h of subsequent controlled oxygenated rewarming with (COR) or without (iCOR) an initial hypothermic perfusion period: (**A**) renal aerobic efficiency reflected by the ratio of total tubular sodium transport (TNa) and oxygen consumption (VO2); (**B**) OXPHOS coupling efficiency and (**C**) spare respiratory capacity of isolated mitochondria at the end of the 90 min reperfusion period (\*: *p* < 0.05 vs. CS).

**Figure 5.** Representative sections of kidney tissue after reperfusion subsequent to preservation by either cold storage with controlled oxygenated rewarming (COR), instant controlled oxygenated rewarming (iCOR), or by cold storage without controlled rewarming (CS). PAS staining, 20× original magnification. Note the reduced tubular cell vacuolization and absence of necrosis in the groups COR and iCOR.

#### **4. Discussion**

Gentle elevation of tissue temperature after extended periods of hypothermia during cold preservation has been shown to favor a significantly more thorough restitution of mitochondrial function. Concomitantly, the whole-graft outcome during early reperfusion could be notably improved.

The latter observation is in line with previous reports, indicating controlled oxygenated rewarming to mitigate rewarming injury upon reperfusion in vitro [11], in vivo [10], and in clinical kidney transplantation [13].

Our present study, however, went further. We disclosed a notable reduction in the ratio of total sodium absorption and oxygen consumption (TNa/VO2) after cold storage and reperfusion that was significantly ameliorated by way of gentle warming up of the grafts prior to reperfusion. Thus, oxygen expenditure is only incompletely met by useful endergonic metabolism. At first sight, this seems to be attributable to an increasing uncoupling phenomenon at the mitochondrial level triggered upon warm reperfusion after cold preservation and significantly attenuated upon only slow elevation of temperature.

Another possible explanation for the reduced TNa/VO2 ratio might relate to oxygenconsuming cellular repair processes or resurrection of brush border microvilli [24]. Here, we provide actual direct evidence for mitochondrial uncoupling during the early reperfusion period after cold preservation. In addition, spare respiratory capacity, i.e., the amount of extra ATP that can be produced by oxidative phosphorylation in case of a sudden increase in energy demand, is largely depressed after cold storage. Hence, mitochondrial dysfunction rather than deviant oxygen utilization is responsible for the reduced TNa/VO2 ratio.

Interestingly, controlled oxygenated rewarming is operative in preserving mitochondrial performance upon reperfusion after hypothermic preservation, and this effect was not dependent on a longer hypothermic perfusion phase at the start of the rewarming protocol. This is in ostensible contrast to previous work from others, as well as from us, which consistently shows that a short oxygenated hypothermic perfusion prior to transplantation effectively improves the graft's resilience to reperfusion injury [15,16,25]. In contrast, hypothermic machine perfusion partly restores energetic and metabolic tissue homeostasis and helps to mitigate ischemia or reperfusion injury. However, even satisfactory aerobic conditions in the cold cannot prevent the adverse effects of an abrupt thermal transition to normothermia and a dramatic impairment of the mitochondrial respiratory control ratio [8].

Previous comparative studies have shown that although brief hypothermic machine perfusion improved functional outcome of cold-stored kidney [14] and liver grafts [9], notably better protection was provided by additional controlled oxygenated rewarming. Apparently, even energized tissue remains susceptible to the temperature paradox phenomenon that incurs along with the abrupt rewarming. On the other hand, slowing down of the rewarming process alone appears to be sufficient to allow for a graduated resumption of subcellular, especially mitochondrial homeostasis adapted to physiological demands [7].

In clinical practice, the process of connecting the graft to the machine and starting the ex vivo perfusion is more easily done in the cold, when hypothermia of the perfusate and graft allows for meticulous cannulation of the vessels without danger of normothermic tissue ischemia in the machine. Sometimes, however, limitation of the total perfusion time on the machine may be desirable, e.g., if the recipient is readily prepared or the total preservation time should be kept at a minimum. In these cases, it will, thus, be a suitable measure to start the rewarming protocol in the cold but to initiate the graduated rise in temperature immediately after organ connection has been effectuated and spare up to 30 min of perfusion in the cold.

The well-known limitations of all in vitro models also apply to our study. In contrast to cell culture experiments, which do not allow for functional evaluation of the whole organ, our model strictly mimics the clinical ex vivo graft perfusion and provides functional data on early reperfusion outcome at the whole organ level. Although no long term evaluation of graft function has been possible in the present study, nor could interferences of whole

blood with the post-ischemic tissue be accounted for, close similarities of functional data after COR could be found between the present study and earlier investigations in vivo [10]. Apart from this, the primary goal of the present study, i.e., to decipher the mechanistic role of early mitochondrial dysfunction, triggered upon abrupt warm reperfusion after cold storage and its prevention by controlled oxygenated rewarming could be sensibly addressed by the used model.

In conclusion, our data provide evidence for mitochondrial uncoupling upon abrupt warm reperfusion after extended cold preservation of renal rafts, which can effectively be alleviated by controlling the rewarming process.

**Author Contributions:** H.Z. performed research, analyzed and interpreted data, and wrote the paper. C.v.H. analyzed data and revised the paper. T.M. designed research, performed research, and wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** 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.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available from the authors upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.
