Ex vivo conditioning of porcine renal grafts using exosomes derived from Urine Progenitor Cells

L’ischémie-reperfusion est un phénomène physiopathologique complexe, indissociable de la transplantation rénale, et induisant des lésions pouvant conduire à la dysfonction de l’organe à court et moyen terme et potentiellement à sa perte. Les reins issus de donneurs décédés après arrêt circulatoire ainsi que ceux issus de donneurs à critères étendus, dont l’utilisation ne fait qu’augmenter, sont des greffons particulièrement sensibles à ces lésions ; parmi les stratégies visant à contrôler celles-ci, notre étude vise à tester une thérapie cellulaire innovante basée sur des exosomes dérivés de Cellules Progénitrices Urinaires porcines (pUPCs) combinée à une perfusion hypothermique en machine de perfusion (HMP) suivie d’une perfusion normothermique ex vivo en machine de perfusion (NMP).Les pUPCs ont été isolées et caractérisées par cytométrie en flux, immunofluorescence et western blotting. Les exosomes dérivés de pUPCs ont été isolés par ultracentrifugation et caractérisés par western blotting, microscopie électronique et par nanotracking assay. Les reins porcins, prélevés à l’abattoir, ont été soumis à 32 min d’ischémie chaude puis conservés en HMP pendant 24 heures à 4°C. Par la suite, les reins ont été perfusés ex vivo en normothermie oxygénée durant 5h à 37°C. Pour la thérapie cellulaire, des exosomes dérivés de pUPCs (Exo) ont été injectés via l’artère rénale porcine. Trois groupes expérimentaux ont été réalisés (n = 5-6/groupe) : Groupe 1 : HMP/véhicule + NMP/véhicule, Groupe 2 : HMP/Exo + NMP/véhicule, Groupe 3 : HMP/Exo + NMP/Exo. Les analyses effectuées comprennent notamment des paramètres fonctionnels et histologiques, les analyses de l’expression des protéines/cytokines dans les perfusats ainsi que l’effet des perfusats sur des cultures de cellules aortiques porcines in vitro.Tout d’abord, les pUPCs ont été isolées et amplifiées à partir d’échantillons d’urine porcine. Les pUPCs isolées expriment des marqueurs de cellules souches mésenchymateuses comme CD90 et sont négatives pour CD31 (marqueur endothélial) et CD45 (marqueur de cellules souches hématopoïétiques). Les pUPCs ont la capacité d’acquérir un phénotype épithélial (expression d’E-cadhérine) après différenciation en cellules tubulaires avec la présence de transporteurs d’eau (AQP1 et AQP2). Les exosomes dérivés de pUPCs expriment des marqueurs exosomaux Alix et TSG101, ont une taille attendue (30-100 nm de diamètre) et sont délimités par une double membrane. L'injection d'exosomes dérivés de pUPCs pendant 24 heures de HMP est faisable, n’impactant pas négativement les paramètres de perfusion, mais aucune différence significative n'a été montrée (analyse en fin de HMP). L'injection d'exosomes pendant la NMP est également possible et n'a pas d'impact significatif sur le débit de perfusion, la résistance rénale, la consommation d'oxygène, la prise de poids des reins, la production d'urine, la fonction rénale (créatinine et sa clairance) et le niveau d'ATP. Cependant, certains marqueurs de lésions cellulaires sont diminués dans le Groupe 3 (ASAT, LDH) et les analyses des perfusats de NMP montrent que l’injection d’Exo induit une immunomodulation (diminution des cytokines pro-inflammatoires (IL1α, IL1β, IL8) et augmentation de la cytokine anti-inflammatoire IL1RA). In vitro, la présence d’exosomes au sein des perfusats permet d’augmenter l’activité mitochondriale de cellules endothéliales porcines en culture. Des analyses métabolomiques et transcriptomiques sont en cours afin de comprendre le mécanisme d’action de ces vésicules. Cette étude montre la faisabilité et l’intérêt potentiel d’utiliser des exosomes dérivés de pUPCs pour le conditionnement du greffon rénal ex vivo. Ces résultats doivent être évalués sur un modèle de transplantation rénale porcine.Ischemia-reperfusion is a complex pathophysiological phenomenon, indissociable from renal transplantation and inducing lesions that can lead to organ dysfunction at short and medium term and potentially to its loss. Kidneys from deceased donors after circulatory arrest as well as those from extended criteria donors, whose use is increasing, are particularly sensitive to these lesions; among the strategies developed to control these lesions, our study aims at testing an innovative cell therapy based on exosomes derived from porcine Urine Progenitor Cells (pUPCs) combined with hypothermic perfusion machine (HMP) followed by normothermic ex vivo perfusion machine (NMP).pUPCs were isolated and characterized by flow cytometry, immunofluorescence and western blotting. Exosomes derived from pUPCs were isolated by ultracentrifugation and characterized by western blotting, electron microscopy and nanotracking assay. Porcine kidneys were collected at the slaughterhouse and subjected to 32 min of warm ischemia and then stored in HMP for 24 h at 4°C. Subsequently, the kidneys were perfused ex vivo in oxygenated normothermia for 5 h at 37°C. For cell therapy, pUPC-derived exosomes (Exo) were injected via the porcine renal artery. Three experimental groups were performed (n = 5-6/group): Group 1: HMP/Vehicle + NMP/Vehicle, Group 2: HMP/Exo + NMP/Vehicle, Group 3: HMP/Exo + NMP/Exo. Analyses include functional and histological parameters, quantification of protein/cytokine expression in the perfusates as well as analysis of the impact of the perfusates on porcine aortic cell cultures in vitro.First, pUPCs were isolated and amplified from porcine urine samples. Isolated pUPCs express mesenchymal stem cell markers such as CD90 and are negative for CD31 (endothelial marker) and CD45 (hematopoietic stem cell marker). The pUPCs have the ability to acquire an epithelial phenotype (E-cadherin expression) after differentiation into tubular cells with the presence of water transporters (AQP1 and AQP2). Exosomes derived from pUPCs are of expected size (30-100 nm of diameter), express exosomal markers Alix and TSG101 and are delimited by a double membrane. Injection of pUPCs-derived exosomes during 24 hours of HMP is feasible and does not negatively impact perfusion parameters, but no significant difference was shown (analysis at the end of HMP). Exosome injection during NMP is also feasible and does not significantly impact perfusion rate, renal resistance, oxygen consumption, kidney weight gain, urine production, renal function (creatinine and its clearance) and ATP level. However, some markers of cell injury are decreased in Group 3 (AST, LDH) and analyses of NMP perfusates show that Exo injection induces immunomodulation (decrease of pro-inflammatory cytokines (IL1α, IL1β, IL8) and increase of the anti-inflammatory cytokine IL1RA). In vitro, the presence of exosomes within perfusates increases the mitochondrial activity of cultured porcine endothelial cells. Metabolomic and transcriptomic analyses are ongoing to understand the mechanism of action of these vesicles. This study shows the feasibility and potential interest of using pUPC-derived exosomes for ex vivo renal graft conditioning. These results need to be validated in a porcine renal transplantation model

Urine-derived stem/progenitor cells: A focus on their characterization and potential

International audienceCell therapy, i.e., the use of cells to repair an affected tissue or organ, is at the forefront of regenerative and personalized medicine. Among the multiple cell types that have been used for this purpose [including adult stem cells such as mesenchymal stem cells or pluripotent stem cells], urine-derived stem cells (USCs) have aroused interest in the past years. USCs display classical features of mesenchymal stem cells such as differentiation capacity and immunomodulation. Importantly, they have the main advantage of being isolable from one sample of voided urine with a cheap and unpainful procedure, which is broadly applicable, whereas most adult stem cell types require invasive procedure. Moreover, USCs can be differentiated into renal cell types. This is of high interest for renal cell therapy-based regenerative approaches. This review will firstly describe the isolation and characterization of USCs. We will specifically present USC phenotype, which is not an object of consensus in the literature, as well as detail their differentiation capacity. In the second part of this review, we will present and discuss the main applications of USCs. These include use as a substrate to generate human induced pluripotent stem cells, but we will deeply focus on the use of USCs for cell therapy approaches with a detailed analysis depending on the targeted organ or system. Importantly, we will also focus on the applications that rely on the use of USC-derived products such as microvesicles including exosomes, which is a strategy being increasingly employed. In the last section, we will discuss the remaining barriers and challenges in the field of USC-based regenerative medicine

A Sodium Oxalate-Rich Diet Induces Chronic Kidney Disease and Cardiac Dysfunction in Rats

International audienceChronic kidney disease (CKD) is a worldwide public health issue affecting 14% of the general population. However, research focusing on CKD mechanisms/treatment is limited because of a lack of animal models recapitulating the disease physiopathology, including its complications. We analyzed the effects of a three-week diet rich in sodium oxalate (OXA diet) on rats and showed that, compared to controls, rats developed a stable CKD with a 60% reduction in glomerular filtration rate, elevated blood urea levels and proteinuria. Histological analyses revealed massive cortical disorganization, tubular atrophy and fibrosis. Males and females were sensitive to the OXA diet, but decreasing the diet period to one week led to GFR significance but not stable diminution. Rats treated with the OXA diet also displayed classical CKD complications such as elevated blood pressure and reduced hematocrit. Functional cardiac analyses revealed that the OXA diet triggered significant cardiac dysfunction. Altogether, our results showed the feasibility of using a convenient and non-invasive strategy to induce CKD and its classical systemic complications in rats. This model, which avoids kidney mass loss or acute toxicity, has strong potential for research into CKD mechanisms and novel therapies, which could protect and postpone the use of dialysis or transplantation