20.500.12556/DKUM-93044 Izolacija in priprava celičnih linij ledvic z namenom razvoja novih in vitro modelov nefrotoksičnosti Isolation and Preparation of Kidney Cell Lines for the Development of Novel In Vitro Nephrotoxicity Models Ledvice so ključne za številne homeostatske mehanizme in so odgovorne za velik delež izločanja snovi iz telesa, zaradi česar so posebej dovzetne za delovanje nefrotoksičnih spojin. Med različnimi vrstami ledvičnih celic so proksimalne tubulne epitelijske celice (PTEC), ki sodelujejo pri tubularni sekreciji snovi, najbolj izpostavljene in zato ključnega pomena za razumevanje nefrotoksičnosti ter interakcij med zdravili v predkliničnih raziskavah. Omejena razpoložljivost in funkcionalna variabilnost komercialno dostopnih PTEC predstavlja ozko grlo pri teh raziskavah, zato je razvoj zanesljivih protokolov za njihovo izolacijo ključna za nadaljnji razvoj področja. V tej disertaciji sta predstavljena dva optimizirana protokola za izolacijo primarnih PTEC iz rutinskih biopsij ledvičnega tkiva, pridobljenih od 13 bolnikov. Rezultat tega sta dve celični liniji, poimenovani MFUM-RPTEC-1 in MFUM-RPTEC-2. Obe celični liniji izražata za PTEC celice ključne označevalce, vključno s SGLT2, MRP4, OAT1, OAT3, OCT2, P-gp, MATE1, N-kadherinom, ZO-1 in CK-18. Ob gojenju na Transwell ploščah so celice pokazale ustrezno polarizacijo, saj so bili transportni proteini lokalizirani na predvideni (apikalni ali bazolateralni) strani, kar je bilo potrjeno tudi s konfokalno mikroskopijo. Potencial ustvarjenih celičnih linij za uporabo v predkliničnih raziskavah smo preverjali s pripravo enostavnega in vitro modela za raziskovanje nefrotoksičnosti in interakcij med izbranimi zdravili. Celice so ohranile svojo viabilnost tudi po izpostavitvi nefrotoksičnim spojinam, kot so ciklosporin, omeprazol in njuni kombinaciji, kar dokazuje potencial celičnih linij za nadaljnji razvoj orodij in vzpostavitev raziskav na področju farmakologije ledvic. The kidneys play a key role in numerous homeostatic mechanisms and are responsible for a large proportion of substance excretion from the body, making them particularly susceptible to the effects of nephrotoxic compounds. Among the various types of renal cells, proximal tubular epithelial cells (PTECs), which are involved in tubular secretion, are the most exposed and therefore critical for understanding nephrotoxicity and drug–drug interactions in preclinical studies. The limited availability and functional variability of commercially available PTECs represent a bottleneck in such research, highlighting the need for reliable protocols for their isolation to advance the field. This dissertation presents two optimized protocols for isolating primary PTECs from routine kidney tissue biopsies obtained from 13 patients. As a result, two cell lines were established, named MFUM-RPTEC-1 and MFUM-RPTEC-2. Both cell lines express key PTEC markers, including SGLT2, MRP4, OAT1, OAT3, OCT2, P-gp, MATE1, N-cadherin, ZO-1, and CK-18. When cultured on Transwell inserts, the cells exhibited appropriate polarization, with transport proteins localized to the expected (apical or basolateral) membrane, as confirmed by confocal microscopy. The potential of the established cell lines for use in preclinical studies was evaluated through the development of a simple in vitro model for investigating nephrotoxicity and drug interactions. The cells retained their viability even after exposure to possibly nephrotoxic compounds such as cyclosporine, omeprazole, and their combination, demonstrating the potential of these cell lines for further development of tools and research in the field of renal pharmacology. proksimalne tubulne epitelijske celice ledvična biopsija izolacija celic nefrotoksičnost in vitro model proximal tubular epithelial cells kidney biopsy cell isolation nephrotoxicity in vitro model true true false [T. Petreski] Slovenski jezik Angleški jezik Doktorska disertacija 2025-06-02 23:30:41 2025-11-05 13:47:26 2025-11-06 03:08:34 0000-00-00 00:00:00 2025 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 576/577:601.2:616.61(043.3) 255998467 DOK_Petreski_Tadej_2025.pdf DOK_Petreski_Tadej_2025.pdf 1 0443068D5ACC7B6B7C0457F1C7458598 6da457ff863b7e2dce9d36df2c72e4f2ba4283e0b3a24846d74e547c7273cf30 815f4487-a83e-11f0-9756-00155d000105 https://dk.um.si/Dokument.php?lang=slv&id=199814 Medicinska fakulteta 0 0 0