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Excretion profile and detection of human adipose derived mesenchymal stem cells during and after isolated normothermic machine perfusion of ischemically damaged porcine kidneys

(2017) Eertman, T.B. (Tim)

Objectives: End-stage renal failure is a growing disease worldwide. The renal replacement therapy of choice is a kidney transplantation. Due to a shortage of suitable kidney grafts, the mean waiting time for a transplantation in the Netherlands is over 3.5 years. During this period morbidity and mortality increase. To reduce the shortage, more vulnerable grafts are transplanted. Machine perfusion (MP) of kidney grafts is used increasingly to improve graft quality. There is growing consensus on the beneficial immunomodulatoiry and anti-inflammatory features of mesenchymal stem cells (MSCs) in organ transplantation. In this study our aim was to gain more insights in the excretion products and detection of fluorescent-labeled MSCs during normothermic machine perfusion of an ischemically damaged porcine kidney. Methods: Two experimental perfusion groups consisted of a kidney with 0 (N=6) or 107 (N=5) MSCs added. In the third group solely 107 MSCs were added to the perfusion circuit in absence of a kidney (N=3). Cytokine release and graft function were measured and compared at multiple time points in every experimental group. Homing and migration of MSCs were analysed in tissue, perfusate and urine by flow-cytometry and fluorescence microscopy. Results: Not-significant trends were found in reduced urine output, lower injury marker AST and more suitable pH-regulation in kidneys exposed to MSCs. MSCs are found to be viable in glomeruli and perfusate during 6 hrs. of NMP. Degraded MCSs may have been found in collecting ducts and urine. Cytokines were detected in all 3 groups, indicating cross-reactivity and excretion through solely NMP-exposure occurred. Significant differences were found in IL-6 and IL-8 excretion. Conclusion: We succeeded in measuring functional parameters, cytokine analysis and detection of MSCs using fluorescence microscopy and flow-cytometry. Using Luminex, insights in excretion profile were obtained. Most findings were not significant, but provide sufficient evidence for conducting further research.





ID 3562
Moeder ID 3463
Volgorde Eertman, T.B.
Naam EertmanTB
Publiceren yes
OAI-naam Student_thesis
Path root/geneeskunde/2017/EertmanTB/
Gemaakt op: 2017-08-28 14:12:49
Gemodificeerd op: 2017-08-28 14:12:49
Digitaal ID 59a424e22def9
Afstudeerrichting opleiding/afstudeerrichting 1
Studierichting Studierichting 1
Titel Excretion profile and detection of human adipose derived mesenchymal stem cells during and after isolated normothermic machine perfusion of ischemically damaged porcine kidneys
Ruilverkeer mogelijk no
Printen in opdracht no
Publicatiejaar 2017
Taal nl
Engelse samenvatting Objectives: End-stage renal failure is a growing disease worldwide. The renal replacement therapy of choice is a kidney transplantation. Due to a shortage of suitable kidney grafts, the mean waiting time for a transplantation in the Netherlands is over 3.5 years. During this period morbidity and mortality increase. To reduce the shortage, more vulnerable grafts are transplanted. Machine perfusion (MP) of kidney grafts is used increasingly to improve graft quality. There is growing consensus on the beneficial immunomodulatoiry and anti-inflammatory features of mesenchymal stem cells (MSCs) in organ transplantation. In this study our aim was to gain more insights in the excretion products and detection of fluorescent-labeled MSCs during normothermic machine perfusion of an ischemically damaged porcine kidney. Methods: Two experimental perfusion groups consisted of a kidney with 0 (N=6) or 107 (N=5) MSCs added. In the third group solely 107 MSCs were added to the perfusion circuit in absence of a kidney (N=3). Cytokine release and graft function were measured and compared at multiple time points in every experimental group. Homing and migration of MSCs were analysed in tissue, perfusate and urine by flow-cytometry and fluorescence microscopy. Results: Not-significant trends were found in reduced urine output, lower injury marker AST and more suitable pH-regulation in kidneys exposed to MSCs. MSCs are found to be viable in glomeruli and perfusate during 6 hrs. of NMP. Degraded MCSs may have been found in collecting ducts and urine. Cytokines were detected in all 3 groups, indicating cross-reactivity and excretion through solely NMP-exposure occurred. Significant differences were found in IL-6 and IL-8 excretion. Conclusion: We succeeded in measuring functional parameters, cytokine analysis and detection of MSCs using fluorescence microscopy and flow-cytometry. Using Luminex, insights in excretion profile were obtained. Most findings were not significant, but provide sufficient evidence for conducting further research.
Nederlandse samenvatting Doelstellingen: Wereldwijd neemt de frequentie van eindstadium nierfalen toe. De therapie van eerste keus is niertransplantatie. Echter, door de schaarste aan deze organen is de gemiddelde wachttijd op een niertransplantaat in Nederland meer dan 3.5 jaar. Tijdens deze wachttijd nemen de kwaliteit van leven en overlevingskansen af. Om het tekort te verkleinen, worden steeds vaker kwalitatief slechtere organen getransplanteerd. Machinaal perfunderen (MP) van transplantatienieren t.b.v. orgaankwaliteit wordt in toenemende mate uitgevoerd. Er ontstaat toenemend bewijs over immuunmodulatoire en anti-inflammatoire effecten van mesenchymale stamcellen (MSC) die de uitkomst na orgaantransplantatie bevorderen. In deze studie worden excretieproducten en detectie van fluorescent gelabelde MSC’s tijdens normotherme MP van ischemisch beschadigde varkensnier beschreven. Methode: Perfusies werden uitgevoerd met nier en zonder MSC’s (N=6), met nier in additie van 107 MSC’s (N=5) en zonder nier met 107 MSC’s (N=3). Cytokine uitscheiding en nierfunctie werden bepaald op diverse tijdpunten en vergeleken in alle groepen. Daarnaast werd bekeken of en waar deze MSC’s terecht komen middels flow-cytometrie en fluorescentie microscopie in nierweefsel, perfusaat en urine. Resultaten: In nieren die blootgesteld werden aan MSC’s zijn niet-significante trends gevonden in verlaagde urine productie, verminderde schademarker AST en een betere pH-regulatie. Levende MSC’s zijn teruggevonden in glomeruli en perfusaat tijdens 6 uur NMP. Kapotte celmembranen zijn wellicht gevonden in de verzamelbuizen en urine. Cytokine excretie werd waargenomen in alle groepen, wat betekent dat kruis-reactiviteit en excretie door blootstelling aan NMP voorkomt. Significante verschillen werden aangetroffen in de excretie van IL-6 en IL-8. Conclusie: We zijn geslaagd in het meten van nierfunctie en het detecteren van MSC’s met behulp van flow-cytometrie en fluorescentie microscopie. Door middel van Luminex hebben we meer inzichten in het excretieprofiel van MSC’s verkregen. De meeste bevindingen waren niet significant, maar geven voldoende aanwijzingen voor het doen van verder onderzoek.
Onderwijsinstelling Medical Sciences
Type embargo abstract openbaar, scriptie op aanvraag
Auteur(s) Eertman, T.B. (Tim)
UMCG begeleider(s) Leuvenink, Prof. dr. Henri G.D.; Faculty supervisor; Moers, Dr. Cyril; Daily supervisor; Surgical research laboratory, Department of Surgery.; University Medical Center Groningen
Auteur(s) Eertman, T.B. (Tim)
UMCG begeleider(s) Leuvenink, Prof. dr. Henri G.D.; Faculty supervisor; Moers, Dr. Cyril; Daily supervisor; Surgical research laboratory, Department of Surgery.; University Medical Center Groningen


 
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