Expression of neural adhesion molecules (NCAM) in healthy and diseased renal tissue

Abstract

Introduction: Kidney performs essential physiological roles that include metabolic waste excretion and maintenance of fluid and electrolyte balance. Different factors may lead to renal dysfunction. Kidney diseases are currently a global public health problem. Fortunately, dialysis and transplantation provide life-saving treatments, but these therapies are rife with limitations. Thus, new ways of therapies, such as stem cell treatment are more than required as an alternative and new glimmer of hope to all nephrology patients. Neural cell adhesion molecule (NCAM) is widely expressed during kidney development and it presents potential marker for renal stem/progenitor cells. Aim: Aim of this dissertation was evaluation of NCAM presence in different types of renal tissue (normal fetal or adult, and pathologically changed), and comparison and correlation of NCAM expression with already defined stem cell markers in order to more closely characterize renal fetal progenitor cells. Method: Specifically designed primers were used for reverse transcriptase PCR, after mRNA isolation from fetal, neonatal, adult normal, adult tissues with interstitial fibrosis and tumor tissues, to clarify presence of different NCAM on nucleotide level. Also, Western Blot tests were performed in order to identify expression of different NCAM protein isoforms in healthy and kidney with disease. Further, FACS analysis was done on renal cell lines to correlate NCAM expression with adult renal stem cell markers CD24 and CD133. Co-expression and co-localization of NCAM with other cell surface markers was also examined using immunohistochemistry and double immunofluorescent staining. Results: Our results showed aberrant NCAM expression in renal tumor tissues as well as in tissue with interstitial fibrosis. Interestingly in both types of mentioned tissues NCAM had co-expression with FGFR1, tyrosine kinase receptor, responsible for cell proliferation and aggressive behavior in some epithelial tumors. Considering NCAM expression on fetal samples possible renal progenitor population cell pool of NCAM+CD24+ cells was found, which was not the case with NCAM+CD133+ cell pool. Also, we had detected that NCAM molecules were post-trasnaltionally modified...

UVOD: Bubreg ima značajnu fiziološku ulogu u izlučivanju štetnih produkata metabolizma putem urina, kao i ulogu u održanju ravnoteže vode i elektorlita. Različiti faktori mogu dovesti do poremećaja funkcije bubrega. Bolesti bubrega su trenutno globalni problem javnog zdravlja. Dijaliza i transplantacija su trenutno dominantni vidovi terapije koje se primenjuju u lečenju bubrežne isuficijencije, ali uz puno ograničenja. Novi načini terapije, kao što je tretman matičnim ćelijama, su više nego potrebni kao alternativa standardnim metodama lečenja i predstavljaju novi tračak nade za sve pacijente koji imaju neki oblik bubrežne disfunkcije. Neuralni ćelijski adhezioni molekul (NCAM) je široko ispoljen tokom razvoja bubrega i predstavlja potencijalni marker renalnih stem/progenitorskih ćelija. CILJ: Cilj ove disertacije je evaluacija NCAM ekspresije u različitim tipovima bubrežnog tkiva (fetalno, adultno normalno i adultno tkivo sa intersticijskom fibrozom i tumorsko tkivo), kao i njena korelacija sa ekspresijom već definisanih markera renalnih stem ćelija kako bi se preciznije definisale fetalne renalne progenitorske ćelije. METOD: Detekcija različitih NCAM izoformi na nivou nukleotida je vršena primenom RT-PCR metode, nakon izolacije iRNK iz fetalnog i normalnog adultnog tkiva, kao i tumorskog i tkiva sa intersticijskom fibrozom. Prisustvo NCAM isoformi na proteinskom nivou, u pomenutim tkivima detektovano je upotrebom Western blot-a i Imunoprecipitacije. Ko-ekspresija NCAM-a sa CD24 i CD133, markerima adultnih renalnih progenitora ispitivana je upotrebom protočne citomertije (FACS) na renalnim ćelijskim linijama. Imunohistohemijskim i dvostrukim imunofluorescentnim bojenjem ispitivana je koekspresija i kolokalizaciji NCAM-a sa drugim ćelijskim markerima. REZULTATI: RT-PCR rezultati ove doktorske disertacije su pokazale da sve ćelijske linije i ispitivana tkiva, osim tumorskog tkiva eksprimiraju tri glavne NCAM isoforme NCAM-120, NCAM-140 i NCAM-180. Takođe, detektovana je aberentnaprekomerna NCAM ekspresija u tumorskom, kao i u tkivu sa intersticijskom fibrozom. Interesantan nalaz čini pokazana koekspresija NCAM-a sa FGFR1, tirozin kinaznim receptorom, koji ima ulogu u ćelijskoj proliferaciji i agresivnom ponašanju pojedinih 7 epitelnih tumora.