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Supplemental Methods Kidney Retrieval and Transplantation

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Supplemental Methods Kidney Retrieval and Transplantation Supplemental Methods Kidney Retrieval and Transplantation Porcine kidney retrieval and heterotopic autotransplantation was performed as previously described elsewhere by our laboratory.1 In brief, animals were given intramuscular injections of ketamine (20mg/kg; Bimeda-MTC Animal Health Inc., Cambridge, Canada), midazolam (0.3mg/kg; Pharmaceutical Partners of Canada Inc, Richmond Hill, Canada), and atropine (0.04mg/kg; Rafter 8 Products, Calgary, Canada) for anestheisa. Isoflurane (1.5%; Pharmaceutical Partners of Canada Inc; Richmond Hill, Canada) was administered after intubation. Sterile technique with a midline incision was used to dissect the right kidney. Heparin (2,000 IU; Sandoz Canada Inc., Toronto, Canada) was administered intravenous (IV) 2 min prior to inducing 30min WI through vascular clamping. Following resection, grafts were immediately flushed with 300mL of histidine-tryptophan-ketoglutarate (HTK) solution (Metapharm Inc., Brantford, Canada) at 4oC prior to placement on NEVKP circuit or cold storage. During storage, pigs recovered from anesthesia after the abdomen was closed. Pigs were re-anesthestized before transplantation with 5mL Propofol IV (PharmaScience Inc., Montreal, Canada). A 150mg/h continuous infusion of Propofol IV was given with 1.5% isofluorane after intubation. A contralateral nephrectomy was performed and this kidney was discarded. The stored kidney was then flushed with 300mL of HTK solution prior to performing the following renal anastomoses: renal vein end-to-side to vena cava, renal artery end-to-side to aorta, and donor ureter side-to-side to recipient ureter. Perioperative care, drug administration, serum sample collection, 24h urine collection, and follow-up were performed as previously described by our group.1 Normothermic Ex Vivo Kidney Perfusion. We reported our group’s Normothermic Ex-Vivo Kidney Perfusion previously2,3. In short, kidneys were perfused for 8 hours in a circuit consisting of a neonatal cardiopulmonary bypass, a S3 heart-lung machine and a centrifugal pump in continuous flow (Sorin Group Inc., Markham, Canada). A custom-made double walled organ chamber was used to house the kidney and to maintain sterility and normothermic temperatures. The perfusate was created to represent near physiological conditions. This consisted of Ringer’s lactate, Steen solution (XVIVO Perfusion AB, Goteborg, Sweden), washed erythrocytes, double reverse osomosis water, sodium bicarbonate (8.4%), calcium gluconate (10%, 100mg/mL), and heparin. Verapamil at a continues flow rate of 0.25mg/h was provided to the arterial portion of the circuit and a continuous infusion of amino acids, glucose, and insulin (Dextrose+Travasol+Humulin R, 1mL/h) was added to the venous reservoir and titrated to achieve a target glucose concentration of 5-15mmol/L. Oxygen and carbon dioxide gas (95%/5%; 2L/min) was also administered continuously. Through the course of perfusion, additional Ringer’s lactate (10mL/h) was provided to replace urine and other insensible losses from the circuit. Static Cold Storage Following retrieval and cold flush described above, the donor kidney was placed in a sterile organ bag (CardioMed Supplies Inc., Lindsay, Canada) containing cold HTK solution, stored around ice to maintain temperatures at 4oC and stored for 8 hours. No Storage Control Group The no storage (NS) control animals were described before by our group4. Kidney retrieval and induction of WI were performed as above. Grafts were flushed with 300mL of HTK following retrieval and grafts were immediately re-transplanted as above without any additional storage time. Animals were only recovered from anaesthesia following completion of transplantation. Additional Statistical Analysis Unless otherwise specified, significance was defined as p<0.05 between NEVKP and SCS groups. For multiple comparisons, one-way analysis of variance (ANOVA) was used with Tukey’s post-hoc test to identify significant differences between >2 groups. 1. Kaths, J.M., et al. Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol. J Vis Exp, 53765 (2016). 2. Kaths, J.M., et al. Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation. J Vis Exp, e52909 (2015). 3. Kaths, J.M., et al. Continuous Normothermic Ex Vivo Kidney Perfusion Improves Graft Function in Donation After Circulatory Death Pig Kidney Transplantation. Transplantation 101, 754-763 (2017). 4. Hamar, M., et al. Normothermic Ex Vivo Kidney Perfusion Reduces Warm Ischemic Injury of Porcine Kidney Grafts Retrieved After Circulatory Death. Transplantation 102, 1262-1270 (2018). 50 14 * * 1200 45 12 * * 1000 40 10 35 800 8 30 * 600 6 * 25 400 20 4 * 15 Serum Creatinine (mg/dL)Serum Creatinine * 200 (µmol/L) Serum Creatinine 2 10 0 0 5 1 2 3 4 5 6 7 8 Creatinine Clearance (ml/min) Clearance Creatinine 0 Post Operative Day Baseline POD4 DCD30min + 8h NEVKP (n=5) DCD30min + 8h SCS (n=5) DCD30min + 8h NEVKP (n=5) Supplemental Figure 2. 8-day post-operative renal graft function measured through A. serum creatinine expressed as mean +/- standard deviation and B. creatinine clearance obtained following 24hr urine collection also expressed as mean +/- standard deviation. *p<0.05. DCD: Donation-after-cardiac death. POD: post-operative day. SCS: static cold storage. NEVKP: Normothermic Ex-Vivo Kidney Perfusion 50 14 * 1200 40 12 * 1000 10 30 800 8 20 600 6 * 400 4 10 Serum Creatinine (mg/dL)Serum Creatinine 200 (µmol/L) Serum Creatinine 2 0 Creatinine Clearance (ml/min) Clearance Creatinine 0 0 POD 3 Baseline 1 2 3 Post Operative Day DCD30min + 8h NEVKP (n=3) DCD30min + 8h NEVKP (n=3) DCD30min + 8h SCS (n=3) DCD30min + NS (n=3) DCD30min + NS (n=3) DCD30min + 8h SCS (n=3) Supplemental Figure 3. 3-day post-operative renal graft function of specimens used for microarray analysis measured through A. serum creatinine expressed as mean +/- standard deviation and B. creatinine clearance obtained following 24hr urine collection also expressed as mean +/- standard deviation. *p<0.05 compared to either NEVKP or NS group. DCD: Donation-after-cardiac death. POD: post-operative day. SCS: static cold storage. NEVKP: Normothermic Ex-Vivo Kidney Perfusion. NS: No Storage Gene Forward Primer 5'-3' Reverse Primer 5"-3" TBP1 AACAGTTCAGTAGTTATGAGCCAGA AGATGTTCTCAAACGCTTCG UMOD CAAGTCGGATAATGGCAAATGG CACTTGCTCAGGGACATCTT RBP4 CCTCCAGAAAGG AAACGATGA GGGCAAACACGAAGGAGTA MPC2 CCTCTCAGCTCTTCCGTATTTG CAAACTAGGTCCCAGTGGTTATG GALNT11 TTCAACGTGGAAGGCTCTATC ATCAGGGTCACCGTAGTCA PROM1 GCTAGAGCACGCCTACAATATC CCTTCCTCATCCACCTTCATTAC Supplementary Table 1: List of primers utilized for internal validation of microarray results utilizing quantitative reverse-transcriptase polymerase chain reaction. Supplementary Table 2: Differentially Expressed Genes between Static Cold Storage and No-Storage of Kidneys Probe ID HGNC Identifier Additional Annotation logFC in SCS wAveExpr P.Value adj.P.Val 15274170 UMOD uromodulin -2.2525602 11.0595161 0.00187892 0.04320137 15330275 PLET1 placenta expressed transcript 1 -2.1505172 7.75301223 0.00108196 0.04093857 15294918 SLC5A8 solute carrier family 5 member 8 -1.9965187 4.76605691 0.00272561 0.04822607 15217592 KNG1 kininogen 1 -1.933088 9.45121031 0.00118137 0.04093857 15283532 SLC26A7 solute carrier family 26 member 7 -1.8612124 8.42039964 0.00058876 0.03931424 15281713 SLC16A4 solute carrier family 16 member 4 -1.854482 9.31489005 0.00067451 0.03982804 15334658 VSIG1 V-set and immunoglobulin domain co-1.8334947 5.50330168 0.00136065 0.04110148 15330824 SIAE sialic acid acetylesterase -1.7555774 7.84192217 9.01E-05 0.03850992 15286875 LRRC8B leucine rich repeat containing 8 VRAC -1.7029981 6.81658788 0.00230506 0.04519769 15223630 AADAT aminoadipate aminotransferase -1.6997545 9.62865478 0.00011445 0.03850992 15291169 CYP2D6 vitamin D3 25-Hydroxylase -1.6711564 7.46539085 0.00015403 0.03850992 15218761 KCNJ15 potassium voltage-gated channel sub-1.636074 10.4645971 0.00024448 0.03850992 15193527 DDO D-aspartate oxidase -1.610899 8.67598064 0.00018102 0.03850992 15281348 CASQ2 calsequestrin 2 -1.6024776 7.65930123 9.61E-06 0.03850992 15265909 BHMT betaine--homocysteine S-methyltrans -1.5643616 9.53824641 0.00200785 0.04399721 15292973 AVPR1A arginine vasopressin receptor 1A -1.5634325 5.79405825 0.00022971 0.03850992 15329730 RAB30 PREDICTED: Sus scrofa RAB30, memb-1.5490232 7.04271254 0.0001523 0.03850992 15252166 ATP6V0A4 ATPase H+ transporting V0 subunit a4 -1.54733 8.44029403 0.00062813 0.03967531 15316824 RHCG Rh family C glycoprotein -1.5414203 7.67684581 0.00128199 0.04093857 15239643 SLC38A11 PREDICTED: Sus scrofa putative sodiu-1.4942519 5.13682631 2.91E-05 0.03850992 15326771 GLB1L2 beta-galactosidase-1-like protein 2 -1.4761513 7.04977438 0.00026845 0.03850992 15232054 MTR 5-methyltetrahydrofolate-homocyste-1.468887 8.05121837 3.21E-05 0.03850992 15258518 BHMT betaine--homocysteine S-methyltrans -1.4659728 10.2126542 0.00174443 0.04255231 15332793 DDC dopa decarboxylase -1.4655742 9.47102618 0.00209151 0.04399721 15232327 SLC16A9 solute carrier family 16 member 9 -1.459386 9.34535284 0.00207319 0.04399721 15191953 SLC22A1 solute carrier family 22 member 1 -1.4537212 9.76384652 0.00051821 0.03931424 15285077 RHBG Rh family B glycoprotein -1.4464682 6.3105154 0.0004284 0.03850992 15250517 ATP6V0A4 ATPase H+ transporting V0 subunit a4 -1.4450954 7.60859836 0.00058147 0.03931424 15321532 ETNPPL ethanolamine-phosphate phospho-ly -1.4361644 5.04400296 0.00081567 0.04093857 15184490 RRAGD Ras related GTP binding D -1.4150207
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