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KIM-1 mediates the uptake of exosomes and transfer of MHC II

(2016) Kramers, B.J.

Urinary exosomes (EXO) are lipid membrane bound structures that mediate intercellular signaling through the transfer of proteins, miRNA and other factors. Kidney injury molecule 1 (KIM-1) acts as a phosphatidylserine (PS) receptor, inducing the uptake of apoptotic cells and necrotic debris. We hypothesize that KIM-1 acts as an endocytosis receptor, taking up EXO containing PS and mediating the intercellular exchange of signaling molecules, such as MHC II. LLC-PK1 cells expressing KIM-1 (PK1-KIM1) and cells expressing empty vector (PK1-pcDNA) were incubated with liposomes composed of PS and fluorescently labeled phosphatidylcholine (PC) or EXO isolated from LLC-PK1 cells, mouse dendritic cells (DC) or human urine by ultracentrifugation. EXO were fluorescently labeled with CytoTracker dye or MHC II-GFP. Uptake was quantified by measuring fluorescence intensity and flow cytometry. EXO were characterized by western blot and electron microscopy. MHC-II levels in urinary EXO from healthy subjects and CKD patients were determined by western blot. PK1-KIM-1 took up significantly more liposomes than PK1-pcDNA. EXO derived from LLC-PK1 cells, DCs and urine were positive for EXO markers HSP70, Flot-1 and TSG101. The diameter of EXO were between 30-150 nm. KIM-1 expressing cells took up significantly more EXO than empty vector expressing cells independent of the source of EXO. Urinary EXO from CKD patients were found to carry MHC II while EXO from healthy subjects were MHC II negative. We found the transfer of MHC II to primary PTCs to be greater in cells expressing KIM-1 after incubation with DC-derived EXO. We conclude that KIM-1 is a PTC receptor for EXO. MHC-II on EXO can be transferred to and presented by KIM-1 positive cells, suggesting EXO may serve to link dendritic cell activation to PTC MHC II expression and antigen presentation cells in inflammatory settings.





ID 3401
Moeder ID 3084
Volgorde Kramers, B.J.
Naam KramersBJ
Publiceren yes
OAI-naam Student_thesis
Path root/geneeskunde/2016/KramersBJ/
Gemaakt op: 2017-05-22 07:10:22
Gemodificeerd op: 2017-05-22 07:10:52
Digitaal ID 59228ede24e14
Afstudeerrichting opleiding/afstudeerrichting 1
Studierichting Studierichting 1
Titel KIM-1 mediates the uptake of exosomes and transfer of MHC II
Ruilverkeer mogelijk no
Printen in opdracht no
Aantal pagina's 22
Publicatiejaar 2016
Taal en
Engelse samenvatting Urinary exosomes (EXO) are lipid membrane bound structures that mediate intercellular signaling through the transfer of proteins, miRNA and other factors. Kidney injury molecule 1 (KIM-1) acts as a phosphatidylserine (PS) receptor, inducing the uptake of apoptotic cells and necrotic debris. We hypothesize that KIM-1 acts as an endocytosis receptor, taking up EXO containing PS and mediating the intercellular exchange of signaling molecules, such as MHC II. LLC-PK1 cells expressing KIM-1 (PK1-KIM1) and cells expressing empty vector (PK1-pcDNA) were incubated with liposomes composed of PS and fluorescently labeled phosphatidylcholine (PC) or EXO isolated from LLC-PK1 cells, mouse dendritic cells (DC) or human urine by ultracentrifugation. EXO were fluorescently labeled with CytoTracker dye or MHC II-GFP. Uptake was quantified by measuring fluorescence intensity and flow cytometry. EXO were characterized by western blot and electron microscopy. MHC-II levels in urinary EXO from healthy subjects and CKD patients were determined by western blot. PK1-KIM-1 took up significantly more liposomes than PK1-pcDNA. EXO derived from LLC-PK1 cells, DCs and urine were positive for EXO markers HSP70, Flot-1 and TSG101. The diameter of EXO were between 30-150 nm. KIM-1 expressing cells took up significantly more EXO than empty vector expressing cells independent of the source of EXO. Urinary EXO from CKD patients were found to carry MHC II while EXO from healthy subjects were MHC II negative. We found the transfer of MHC II to primary PTCs to be greater in cells expressing KIM-1 after incubation with DC-derived EXO. We conclude that KIM-1 is a PTC receptor for EXO. MHC-II on EXO can be transferred to and presented by KIM-1 positive cells, suggesting EXO may serve to link dendritic cell activation to PTC MHC II expression and antigen presentation cells in inflammatory settings.
Nederlandse samenvatting Exosomen (EXO) in de urine zijn vesikels met lipide membranen die intercellulair contact mediëren door het overbrengen van eiwitten, miRNA en andere lading. Kidney injury molecule 1 (KIM-1) fungeert als een fosfatidylserine (PS) receptor, als zodanig induceert het de opname van apoptotische cellen en cel afbraakproducten. Wij stellen de hypothese dat KIM-1 als endocytose receptor EXO opneemt en hiermee intercellulaire uitwisseling van signaalmioleculen zoals MHC-II induceert. LLC-PK1 cellen die KIM-1 presenteren (PK1-KIM1) en cellen die een lege vector presenteren (PK1-pcDNA) werden geïncubeerd met liposomen bestaande uit PS en fluorescent gelabelde fosfatidylcholine of EXO middels ultracentrifugatie geisoleerd uit LLC-PK1 cellen, murine dendritische cellen (DC) of humane urine. EXO waren fluorescent gelabeld met CytoTracker dye of MHC-II-GFP. Opname werd gekwantificeerd middels fluorescentie sterkte of flow cytometrie. EXO werden gekarakteriseerd door middel van western blot en elektronenmicroscopie. MHC-II in EXO uit urine van gezonde personen en CKD-patiënten werd vastgesteld middels western blot. PK1-KIM-1 cellen namen significant meer liposomen op dan PK1-pcDNA cellen. EXO afkomstig van LLC-PK1 cellen, DC en urine waren positief voor EXO geassocieerde eiwitten HSP70, Flot-1 en TSG101. De diameters van de EXO lagen tussen 30-150nm. KIM-1 presenterende cellen namen significant meer EXO op dan lege vector presenterende cellen, onafhankelijk van het type EXO. EXO uit de urine van CKD-patiënten presenteerde MHC-II, EXO uit de urine van gezonde personen was negatief voor MHC-II. MHC-II werd meer overgebracht naar proximale tubuluscellen (PTC) met KIM-1 dan naar PTC zonder KIM-1.
Concluderend isKIM-1 is een PTC-receptor voor EXO. MHC-II op EXO kan overbracht worden op en gepresenteerd worden door KIM-1 positieve cellen, dit suggereert dat EXO de link kunnen zijn tussen dendritische cel activatie, MHC-II expressie op PTC en antigeen presentatie in een inflammatoire setting.
Onderwijsinstelling Medical Sciences
Type embargo abstract openbaar, scriptie op aanvraag
Auteur(s) Kramers, B.J.
UMCG begeleider(s) Supervisor Groningen, The Netherlands; Navis, Prof. Gerjan J. MD PhD Department of internal medicin
Begeleider(s) opleidingsinstelling Supervisors Boston, Massachusetts, United States of America; Brooks, Craig R. PhD; Bonventre, Prof. Joseph V. MD PhD Chief, Renal Division Chie
Auteur(s) Kramers, B.J.
UMCG begeleider(s) Supervisor Groningen, The Netherlands; Navis, Prof. Gerjan J. MD PhD Department of internal medicin


 
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