Artificial Mitochondria Transfer to Mitigate Ischemic Damage in an In Vitro Model of Tubular Injury and in an Ex Vivo Porcine Model of DCD Renal Transplantation
1Un. of Turin, Turin, Italy, 2Children Hospital, Los Angeles, CA, 3Un. of L'Aquila, L'Aquila, Italy, 4VA Tech, Blacksburg, VA, 5Duke, Durham, NC, 6WFIRM, Winston Salem, NC, 7University of Florida, Gainesville, FL, 8Wake Forest Baptist, Winston-Salem, NC
Meeting: 2022 American Transplant Congress
Abstract number: 366
Keywords: Machine preservation, Preservation, Renal function, Renal ischemia
Topic: Basic Science » Basic Science » 14 - Ischemia Reperfusion
Session Information
Session Name: Machine Perfusion and Organ Rehabililtation - Basic
Session Type: Rapid Fire Oral Abstract
Date: Monday, June 6, 2022
Session Time: 5:30pm-7:00pm
Presentation Time: 6:30pm-6:40pm
Location: Hynes Ballroom A
*Purpose: We aimed at assessing whether artificial mitochondrial transfer (AMT) mitigates renal injury in an in vitro model of tubular damage and in an ex vivo porcine model of DCD renal transplantation.
*Methods: In vitro: human conditional immortalized proximal tubular cells (ciPTECs) were damaged and then treated with AMT or placebo. Endpoints were proliferative capacity, intracellular toxicity, ROS, mitochondria polarization, ATP concentration and oxidative phosphorylation.Ex vivo: 3 pairs of porcine kidneys were used. After procurement, organs were immerged in a 36°C bath for 30′ and then benched. One kidney was treated with AMT, while the mate organ received placebo. Organs were placed in a perfusion system for 24-h. Perfusate was collected at 0, 6, 12, 18 and 24-h and analyzed with Raman spectroscopy (RS). Biopsies taken at baseline and 24-h were analyzed with standard pathology, TUNEL, immunohistochemistry (IHC) and RNAseq.
*Results: In vitro, AMT reverted the proliferative impairment generated by damage as demonstrated by the increase of ATP production and BRDU proliferation assay. Decrease of cell cytotoxicity was demonstrated by accumulation and cleavage of GF-AFC peptide. AMT restored the reduction of mitochondrial membrane potential, together with the coherent reduction of oxidative stress, as demonstrated by the decrease of ROS generation and TBARS production. AMT reverted the detrimental effect on the expression of important enzymes correlated with TCA cycles (citrate synthase, alpha-ketoglutarate, succinate, malate) and oxidative phosphorylation, suggesting an improvement of mitochondrial function. Recovery of glutaminase expression was also observed.
Ex vivo, longitudinal comparison of the molecular signature of perfusate composition at RS showed that AMT-treated kidneys shed fewer molecular species over 24-h, indicating greater stability/viability. In these kidneys, pathology showed less damage. RNAseq analysis showed activation of genes and pathways involved in kidney damage at 24-h, including proinflammatory cytokine signaling (IL-1, IL-6, IL-8, IL-17) and increased lipid metabolism. AMT ameliorated the inflammatory response, which was reflected by downregulation of genes involved in neutrophil recruitment, including IL1A, CXCL8, and PIK3R1. AMT regulated lipid transport and biosynthesis by decreasing the gene activity, including ATP binding cassette subfamily A member 1 (ABCA1), epiregulin (EREG), stearoyl-CoA desaturase (SCD), fatty acid binding protein 3 (FABP3).
*Conclusions: AMT mitigates damage both in vitro and ex vivo.
To cite this abstract in AMA style:
Rossi A, Sedrakyan S, Montali F, Aardena C, Abraham N, Barbas A, Byers LN, Palles C, Asthana A, Robertson J, Perin L, Senger R, Bussolati B, Orlando G. Artificial Mitochondria Transfer to Mitigate Ischemic Damage in an In Vitro Model of Tubular Injury and in an Ex Vivo Porcine Model of DCD Renal Transplantation [abstract]. Am J Transplant. 2022; 22 (suppl 3). https://atcmeetingabstracts.com/abstract/artificial-mitochondria-transfer-to-mitigate-ischemic-damage-in-an-in-vitro-model-of-tubular-injury-and-in-an-ex-vivo-porcine-model-of-dcd-renal-transplantation/. Accessed September 7, 2024.« Back to 2022 American Transplant Congress