Dynamic conditioning of porcine kidney grafts with extracellular vesicles derived from urine progenitor cells: A proof-of-concept study

Abstract

: Among strategies to limit ischemia/reperfusion (IR) injuries in transplantation, cell therapy using stem cells to condition/repair transplanted organs appears promising. We hypothesized that using a cell therapy based on extracellular vesicles (EVs) derived from urine progenitor cells (UPCs) during hypothermic and normothermic machine perfusion can prevent IR-related kidney damage. We isolated and characterized porcine UPCs and their extracellular vesicles (EVs). Then these were used in an ex vivo porcine kidney preservation model. Kidneys were subjected to warm ischemia (32 min) and then preserved by hypothermic machine perfusion (HMP) for 24 h before 5 h of normothermic machine perfusion (NMP). Three groups were performed (n = 5-6): Group 1 (G1): HMP/vehicle + NMP/vehicle, Group 2 (G2): HMP/EVs + NMP/vehicle, Group 3 (G3): HMP/EVs + NMP/EVs. Porcine UPCs were successfully isolated from urine and fully characterized as well as their EVs which were found of expected size/phenotype. EVs injection during HMP alone, NMP alone, or both was feasible and safe and did not impact perfusion parameters. However, cell damage markers (LDH, ASAT) were decreased in G3 compared with G1, and G3 kidneys displayed a preserved tissue integrity with reduced tubular dilatation and inflammation notably. However, renal function indicators such as creatinine clearance measured for 5 h of normothermic perfusion or NGAL perfusate's level were not modified by EVs injection. Regarding perfusate analysis, metabolomic analyses and cytokine quantification showed an immunomodulation signature in G3 compared with G1 and highlighted potential metabolic targets. In vitro, EVs as well as perfusates from G3 partially recovered endothelial cell metabolic activity after hypoxia. Finally, RNA-seq performed on kidney biopsies showed different profiles between G1 and G3 with regulation of potential IR targets of EVs therapy. We showed the feasibility/efficacy of UPC-EVs for hypothermic/normothermic kidney conditioning before transplantation, paving the way for combining machine perfusion with EVs-based cell therapy for organ conditioning. HIGHLIGHTS: ·UPCs from porcine urine can be used to generate a cell therapy product based on extracellular vesicles (pUPC-EVs). ·pUPC-EVs injection during HMP and NMP decreases cell damage markers and has an immunomodulatory effect. ·pUPC-EVs-treated kidneys have distinct biochemical, metabolic, and transcriptomic profiles highlighting targets of interest. ·Our results pave the way for combining machine perfusion with EV-based cell therapy for kidney conditioning.

Keywords: cell therapy; exosomes; extracellular vesicles; kidney preservation; kidney transplantation; machine perfusion; preclinical porcine model; urine progenitor/stem cells.

FIGURE 1

Porcine urine progenitor cells (pUPCs) and EVs isolation/characterization and study design. (A) Isolation of pUPCs from a porcine urine sample. (B) pUPC morphology (left picture: 40×; right picture: 100×). (C) Isolation of EVs from porcine urine progenitor cells. (D) EVs visualization using scanning electronic microscopy (left pictures) and transmission electronic microscopy (right pictures). (E) EVs analysis using nanosight tracking analysis (black line: averaged data; red shape: standard deviation). Averaged size (mean size of all nanoparticles population (full sample)) = 115 nm. Mode size (mean size of the more concentrated nanoparticle population) = 79 nm; averaged concentration = 3.6 × 10¹⁰ ± .5 × 10¹⁰ particles/mL.

FIGURE 2

Experimental groups and design. (A) Experimental groups: Group 1 (G1 – Vhl) received only PBS (Vhl) during hypothermic machine perfusion (HMP) and normothermic machine perfusion (NMP) (n = 5 kidneys for G1); Group 2 (G2 – EVs‐H) received pUPC‐EVs during HMP and PBS during NMP (n = 6 kidneys for G2): Group 3 (G3 – EVs‐HN) received pUPC‐EVs during both HMP and NMP (n = 6 kidneys for G3). (B) Experimental design and samplings at different timings (T for timing) (detailed Material and Methods for details).

FIGURE 3

Safety and efficacy of pUPC‐EVs during 24 h of HMP. (A) Perfusion flow expressed in ml/min/100 g of kidney weight during the 24 h of HMP; time is represented in h; data were monitored continuously on the LifePort software; left panel: kinetics; right panel: AUC analysis. (B) Kidney resistance in mmHg/mL/min/100 g of kidney weight during the 24 h of HMP; time is represented in hours; data were monitored continuously on the LifePort software; left panel: kinetics; right panel: AUC analysis. (C) Concentration of cell damage markers in the HMP perfusates: ASAT, LDH, and lactate; left panel: kinetics; right panel: AUC analysis. (ns if p > .05, * if p ≤.05, ** if p ≤  .01, *** if p ≤  .001.).

FIGURE 4

Safety and efficacy of pUPC‐EVs during 5 h of NMP (part 1). (A) Perfusion flow expressed in ml/min/100 g of kidney weight during the 5 h of NMP; time is represented in minutes; left panel: kinetics; right panel: Area under the curve (AUC) analysis. (B) Kidney resistance in mmHg/mL/min/100 g of kidney weight during the 5 h of NMP; time is represented in minutes; left panel: kinetics; right panel: AUC analysis. (C) Oxygen consumption in µmol/min/100 g of kidney weight during the 5 h of NMP; time is represented in minutes; left panel: kinetics; right panel: AUC analysis. (D) Kidney weight gain during the whole procedure (HMP + NMP) in grams. (A) Urine production; left panel: cumulative urine volume (mL); middle panel; urine production in ml/min/100 g of kidney weight; right panel: AUC analysis. (ns if p > .05, * if p ≤  .05, ** if p ≤  .01 and *** if p ≤  .001.).

FIGURE 5

Safety and efficacy of pUPC‐EVs during 5 h of NMP (part 2). (A) Concentration of cell damage markers in NMP perfusates: aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), and lactate; left panel: kinetics; right panel: AUC analysis. (B) Renal function markers: Creatinine in µmol/L/100 g of kidney weight (left panel: kinetics; right panel: AUC analysis), Creatinine clearance (mL/5 h/100 g of kidney weight), and sodium excretion. (ns if p > .05, * if p ≤  .05, ** if p ≤  .01 and *** if p ≤  .001.).

FIGURE 6

Histological analysis of kidney biopsies: CD, cell detachment; DTL, dilatated tubule; CD10 St: CD10 staining; II, inflammated interstitium. Scores were determined using the following scale, analyzing 10 fields per slide. For cell detachment and tubular dilatation: 0: normal; 1: lesions <10%; 2: lesions 11−25%; 3: lesions 26−50%; 4: lesions 51−75%; 5: lesions > 75% (extensive necrosis). For inflammation: 0: no infiltrate; 1: light localized, 2 light diffuse; 3: intense localized; 4: intense diffuse and 5: extensive. For CD10 staining: number of positive cells per field (10 fields). Meaning for statistical differences. Within each group, T0 versus T5: *p < .05, **p < .01, ***p < .001 (Mann–Whitney test). Between groups, T0 vs. T0: §p < .05, §§p < .01, §§§p < .001 (Kruskal–Wallis + Dunn's multiple. comparisons test), #p < .05, ##p < .01, ###p < .001 (Kruskal–Wallis + Dunn's multiple comparison test.

FIGURE 7

Analysis of normothermic perfusate content: Concentration of cytokines/growth factors in the NMP perfusates: IL1α, IL1β, IL8, IL1ra, ANG, IGF1, PGE2, and IL18. Left panel: kinetics; right panel: AUC analysis. (ns if p > .05, * if p ≤ .05, ** if p ≤ .01 and *** if p ≤ .001.).

FIGURE 8

In vitro impact of perfusates and pUPC‐EVs on pAOECs (A–G) and hRGECs (H). (A,B) Mitochondrial activity (absorbance value after XTT assay) of pAOECs (porcine aortic endothelial cells) in basal condition cultured for 48 h with (A) a mix of their regular medium and ex vivo perfusates (diluted perfusates); (B) diluted perfusates from a “blank” NMP procedure when EVs were injected but without kidney on the perfusion circuit. T0: no EVs (before the injection occurring at t = 60 min), T2: 2 h of NMP (1 h after EVs injection), T5: 5 h of NMP (4 h after EVs injection). (C–G) Seahorse analysis of pAOECs in basal condition cultured for 48 h with diluted perfusates; data are representative of the metabolic activity from 10 000 cells; (C) Oxygen consumption rate: (D) ATP‐linked respiration; (E) Basal respiration; (F) Maximal respiration; (G) Spare respiration capacity; (H) Mitochondrial activity (absorbance value after XTT assay) of hRGECs (human renal glomerular endothelial cells) in hypoxia‐reoxygenation treated with pUPC‐EVs (or PBS as vehicle, group Vhl/Vhl) either in the hypoxia phase (group EVs/Vhl), during both hypoxia and normothermic reoxygenation (EVs/EVs) or during normothermic reoxygenation only (Vhl/EVs). See the Materials and Methods section for details regarding cell culture protocols and metabolic analyses with the Seahorse analyzer. (ns if p > .05, * if p ≤ .05, ** if p ≤ .01 and *** if p ≤ .001.).

FIGURE 9

Transcriptomic analysis of kidney biopsies. (A) Principal component analysis of the RNAseq data for G1, G2, and G3 groups. (B) Principal component analysis of the RNA‐seq data for G1 and G3 groups only. (C) Volcano‐plot analysis showing differential gene expression between G1 and G3. (D) Heat map showing most differentially regulated gene expression between samples from G1 and G3; data are deposited on GEO (number GSE255005).