Plasmalyte solution

The ex vivo perfusion setup used in this study is identical to the setup used in previous studies [18 (link),19 (link),20 (link)] by our center (Figure 7). Mean perfusion pressure was maintained at 60 mmHg through adjusting the flow of the perfusate. Fresh perfusate (1 L) was prepared for preservation and reperfusion by mixing 250 mL of Hemopure (generously provided by HbO2 Therapeutics, Souderton, PA, USA) with 750 mL of PlasmaLyte solution (Baxter International Inc., Deerfield, IL, USA). The perfusate was supplemented with the following: heparin (5000 U), sodium bicarbonate (8.4%, 10 mL) and Ancef (1 g). Pairs of kidneys were perfused together due to having a single pulsatile pump. However, each kidney was considered as one replicate because its urine output was collected individually.

The ex vivo pulsatile perfusion apparatus used in the present study is illustrated in Figure 1B and identical to the setup used in our previous studies [13 (link)]. Following 4 h of SCS at 4 °C, all kidneys were reperfused with stressed autologous blood for 4 h at 37 °C to simulate reperfusion after kidney transplantation. Blood in the perfusion circuit from the donor pig was oxygenated using an external oxygen gas supply, and 5000 units of heparin per 1 L bag was added to prevent clotting [55 (link)]. By adjusting the flow, the mean perfusion pressure was maintained at 60 mmHg after an initial 5 min period of gradual increase. Urine and arterial blood samples were collected hourly, and tissue oxygenation (measured with a monitor) and the volume of urine production during reperfusion were recorded. PlasmaLyte solution (Baxter, Deerfield, IL, USA) was used to compensate for the fluid volume loss. In addition, blood parameters (pH, pO₂, and lactate) were also measured hourly using iSTAT Handheld Blood Analyzer (Abbott Laboratories, Chicago, IL, USA). After 4 h of reperfusion, the kidney samples were collected, cut into sections (including cortex and medulla), and stored in 10% neutral-buffered formalin for histopathological analysis.

Fluid challenge consisted of a minimum volume of 500 ml crystalloid (NaCl 0.9 %, Hartman’s or Plasmalyte solution (Baxter, Lessines, Belgium) or hydroxyethyl starch 6 % (Voluven, Fresenius, Bad Homburg, Germany)) infused at a minimum rate of 1,000 ml/hour under arterial pressure guidance [15 (link)]. Fluid administration was stopped when MAP reached a predetermined goal (generally MAP >65 mmHg, and/or MAP and/or stroke volume increase >10–15 % compared to baseline) and/or central venous pressure increased >15 mmHg. Systemic hemodynamic variables were recorded and renal Doppler measurements were performed before and at the end of the fluid challenge. The urine output volumes over the 3 h preceding and following the fluid challenge were recorded (Fig. 1).Fig. 1

Summary of the study design. The intervention was a fluid challenge that consisted of infusion of a minimum volume of 500 ml at a minimum rate of 1,000 ml/hour. Interlobar artery Doppler variables, blood pressure, heart rate and urine output were recorded before and after the fluid challenge and stabilization of hemodynamic variables. UO/3H: urine output volumes measured over 3 hours

The human placenta was procured from uncomplicated full-term cesarean resection from healthy mothers. The placenta was received from Karolinska Institute Hospital, Stockholm under ethical permit number: 2015/419-34/4. Signed, informed consent was provided by the mother to use the sample for research purposes. All the pathogen-positive deliveries (including HBV, HCV, syphilis, and HIV) were excluded, and current revised exclusion criteria includes positiveness for COVID-19. No information regarding human donors (mother and newborn child) was transmitted or diffused, and the details were de-personalized, in order to respect privacy. Placentae were delivered and processed within 3 h post-partum. A protocol for hAEC isolation was previously described [27 (link)]. Briefly, the amnion membrane was removed from the inner surface of the placenta and washed multiple times with Ringers solution (Baxter, Sweden) and plasmalyte solution (Baxter, Norfolk, UK) to remove blood. The amnion membrane was digested using TrypLE 10× (Gibco, Grand land, NY, USA) for 30 min at 37 °C to primarily release epithelial cells from the amnion membrane. The dispersed hAEC were collected by centrifugation and filtered through a 100 µm cell strainer. Cell viability was determined by the Trypan Blue exclusion (TBE) method (ThermoFisher, Waltham, MA, USA).

This study was approved by Héma-Québec’s Research Ethics Committee (CER#2020-010), and all participants signed an informed consent form. Whole blood (450 mL) was collected using the Leukotrap^® WB system (Haemonetics, Braintree, MA, USA) according to the manufacturer’s instructions. Immediately after the blood donation, PBMCs were isolated using gradient centrifugation with a Ficoll-Paque solution (Cytiva, Vancouver, BC, Canada) and Leucosep tubes (Greiner Bio-One; Monroe, NC, USA) according to the manufacturer’s instructions. PBMCs were collected and washed with DPBS (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.25% human albumin (CLS Behring, Ottawa, ON, Canada). The PBMCs were then suspended in a Plasma-Lyte solution (Baxter, Mississauga, ON, Canada) containing 5% human albumin and 18% CryoSure-Dex40 (WAK-Chemi medical, Steinbach, Germany), aliquoted, and frozen in liquid nitrogen for subsequent use.