Ethics declarations
The study complies with all relevant ethical regulations for animal testing and research. Experiments were carried out only on isolated kidneys that were retrieved from dead female pigs (German Landrace, age between 12 and 14 weeks) after euthanasia by i. v. injection of potassium chloride, in deep anesthesia. The procedure of euthanasia for organ retrieval according to § 4 Abs. 3, TSG (German Legislation on animal protection) has been approved by the responsible authority LANUV (Landesamt für Natur, Umwelt, und Verbraucherschutz NRW, Germany).
The principles of laboratory animal care (NIH publication no. 85-23, revised 1985) were followed. No Heparin was given at any time.
Experimental groups
Based on our own experiences and reports from the literature on experimental porcine kidney transplantation, three different groups of investigation were created that not only represent gradual increases in renal injury, but were matched to established graft outcome data after renal transplantation in the pig. Thus, good, acceptable (fair), and poor kidney grafts were investigated according to different times of warm ischemic injury prior to cold storage, that are reported to result in 100% initial graft function, high rates of delayed graft function but ultimate recovery and usual primary non-function, resp9,29,30.
Kidneys were randomly assigned to one of the following groups:
Good kidney grafts (n = 13): Kidneys were removed ~5 min after cardiac standstill of the donor animal. According to own previous experiences, such kidneys do perform virtually identical to kidneys retrieved during intact circulation.
Acceptable kidney grafts (n = 13): The group is intended to represent fair donor organs that might be usable in great majority but usually do already show a reduced initial graft function. These kidneys were removed only 30 min after cardiac standstill of the donor animal. In porcine kidney transplantation, 30 min of warm ischemia prior to cold storage is reported to be afflicted with a high rate of delayed graft function31, but usually associated with 100% ulterior graft survival9.
Critical kidney grafts (n = 13): Aiming to create a group of poor donor organs, these kidneys were removed only 60 min after cardiac standstill of the donor as has been reported to result in 100 % nonfunctioning porcine grafts30,32.
Even if Heparin would have been administered prior to induction of circulatory standstill, which might notably improve the outcome as compared to non-anticoagulated settings, 3-month survival after 60 min of warm ischemia is less than 50 %33.
In line with this, a warm ischemic time of less than 45 min has recently been postulated as acceptance criterion for human kidney donation after cardiac standstill of the donor, according to Maastricht III criteria in a comparable setting10.
In all kidneys, 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 and preserved in a beaker filled with HTK solution kept at 4 °C by the help of a cryo-thermostat.
Normothermic machine perfusion
After 18 h of static storage, kidneys from all groups were put on a machine perfusion circuit and subjected to an end-ischemic machine perfusion of 3 h duration.
According to previous experiences to mitigate tissue damage by abrupt temperature increase (rewarming injury)34, the temperature of the perfusate was slowly elevated from initially 8 °C up to normothermia during the first 30 min with concomitant adjustment of the perfusion pressure from 30 mmHg to 75 mmHg. Perfusate consisted of Aqix RS I solution and was supplemented with bovine serum albumin (40 g/l), sodium bicarbonate 8.4 % (25 ml), and dexamethasone (4 mg). Creatinine was also added at 6 mg/100 ml to allow for measurement of glomerular filtration rate during machine perfusion35.
Perfusion pressure was regulated by a servo-controlled pump that adjusted the renal flow according to the feedback obtained by a connected pressure transducer in the arterial inflow line. Oxygenation was provided by an interposed membrane oxygenator fed at a constant flow rate of 0.6 l/min.
13C-Acetate turnover test
During normothermic end-ischemic machine perfusion, metabolic measurements on citrate cycle throughput were performed by 13C detection in the gas outlet of the oxygenator after bolus injection of 25 mg of 13C-Acetate.
Detection was executed using the novel high-precision 13CORlab device (ArgosMED, Karlsruhe, Germany). The device was connected to the gas-outflow of the oxygenator and directly detected the concentration of 12CO2 as well as13CO2 molecules during the gas transit through the detector, using laser-based spectroscopy along with the measurement of flow, pressure, and temperature of the gas (cf. Fig. 1).
The set-up enables the calculation of the exact amount of substance of 13CO2 at a given time point with a sampling rate of 1/s. The metabolized amount of 13CO2 corresponds to the difference of the detected concentration of 13CO2 and the concentration of 13CO2 at baseline conditions (i.e. before application of the test metabolite). The amount of substance can be derived by multiplying the metabolized concentration of 13CO2 with the total amount of substance of the analyzed gas. The latter can be determined as total volume per time divided by the molar gas volume at standard conditions.
$${eta }^{13}{{{{rm{CO}}}}}_{2}(t)=[{{{{rm{C}}}}}^{13}{{{{rm{CO}}}}}_{2}({{{rm{t}}}})-{{{{rm{C}}}}}^{13}{{{{rm{CO}}}}}_{2}({{{rm{baseline}}}})]times eta ,{{{rm{tot}}}}$$
(1)
$$=[{{{{rm{C}}}}}^{13}{{{{rm{CO}}}}}_{2}({{{rm{t}}}})-{{{{rm{C}}}}}^{13}{{{{rm{CO}}}}}_{2}({{{rm{baseline}}}})]times {{{rm{V}}}}({{{rm{t}}}})/{{{rm{V}}}}({{{rm{st}}}})$$
(2)
η: Amount of substanceC 13CO2: Concentration of 13CO2 at given time pointC 13CO2: Concentration of 13CO2 at baselineV(t): Total gas volume per given timeV(st): Molar gas volume at standard conditions = 22.4 L/mol)
Thus, the metabolic rate can be determined during the linearly rising part of the slope of measured amount of substance over time and is calculated in our study as µmol/min based on a measurement interval of 30 min.
Conventional markers of graft integrity
Renal perfusate flow was detected by using an electromagnetic sensor inserted into the arterial inflow line. The ureter was cannulated and the urine collected during the entire perfusion period. Lactate levels in the perfusate were measured in an acid-base laboratory (ABL800 flex, Radiometer, Copenhagen, Denmark). Creatinine was measured in a routine fashion by reflectance photometry on an Element RC3X point of care unit (scil animal care company, Viernheim, Germany). Urinary alpha glutathione S transferase (GST) was quantified using a porcine ELISA test according to the manufacturer’s instruction (My biosource, San Diego, USA).
Statistics and reproducibility
The hypothesis that the measurement of 13C-acetate metabolism would be valuable to discriminate between the predefined levels of kidney quality should be tested by establishing a respective receiver operating curves (ROC), defining the area under the curve for accurate discrimination by 13C-acetate metabolism measurements between good and acceptable or acceptable and poor grafts. Thereby, an area under the curve of 0.8 or better should be considered as a significant benefit for future use and further investigation of the parameter. The rate of type 2 error has been defined as ß = 0.2. The level of significance for the type 1 error is set to p < 0.05. From these assumptions, it follows that the detection of a discriminative significance compared to the null hypothesis (Area under the ROC curve = 0.5) requires a sample size of n = 13 per group (MedCalc® Statistical Software version 20.009 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2021).
All values were expressed as means ± SD unless otherwise indicated. Differences between groups were tested by one-way ANOVA and parametric comparison of the means using Tukey-Kramer test. Statistical significance was set at P < 0.05. ROC curve data were calculated using Prism 9.01 (GraphPad software Inc., San Diego, CA, USA).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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- Source: https://www.nature.com/articles/s43856-023-00422-6