Title Izražaj koneksina 37, 40, 43 i 45 i paneksina 1 u bubrezima miševa s isključenim St8sia1 genom
Title (english) Expression of connexins 37, 40, 43 and 45 and pannexin 1 in the kidney of St8sia1 gene knockout mice
Author Diana Meter
Mentor Natalija Filipović (mentor)
Committee member Merica Glavina Durdov (predsjednik povjerenstva)
Committee member Snježana Mardešić (član povjerenstva)
Committee member Josipa Radić (član povjerenstva)
Committee member Janoš Terzić (član povjerenstva)
Granter University of Split School of Medicine Split
Defense date and country 2023-05-16, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Basic Medical Sciences Anatomy
Universal decimal classification (UDC ) 611 - Anatomy
Abstract U miševa s isključenim St8sia1 genom (St8sia1-KO), kojima nedostaje enzim GD3 sintaza, nakupljaju se gangliozidi a-serije, a nedostaju gangliozidi b- i c-serije. Iako se sastav gangliozida značajno mijenja, ukupna koncentracija gangliozida u KO miševa ostaje ista kao u miševa divljeg tipa (WT). Gangliozidi u staničnoj membrani nisu uniformno raspoređeni, nego su grupirani u lipidnim splavima, kao i transmembranski proteini koneksini (Cx), koji su ključni za međustaničnu komunikaciju u mnogim organima, a u nekoliko regulatornih mehanizama u bubrezima igraju funkcionalnu ulogu. S obzirom na to da promijenjeni sastav gangliozida utječe na distribuciju proteina u lipidnim splavima različitih staničnih membrana i tako vjerojatno utječe na distribuciju, transport i funkciju različitih izoformi Cx, cilj ovog istraživanja bio je utvrditi učinke promijenjenog sastava gangliozida na izražaj Cx37, Cx40, Cx43, Cx45 i Panx1 u različitim regijama bubrega St8sia1-KO miševa. Pokusi su provedeni na dvanaest mužjaka miševa u dobi od 6 mjeseci: četiri miša divljeg tipa (C57BL/6-tip, WT) i osam St8sia1-KO miševa. Uzorci mišjih bubrega su obrađeni za imunohistokemiju i potom analizirani. Izražaj Cx37, Cx40, Cx43, Cx45 i Panx1 ispitivan je imunofluorescentnim mikroskopom (BX61, Olympus, Tokio, Japan), dok je kvantitativna procjena imunoreaktivnosti provedena pomoću ImageJ softvera(National Institutes of Health, Bethesda, MD, SAD). Kako bi lokalizirali izražaj koneksina i Panx1 u različitim dijelovima bubrega miša, uradili smo dvostruko imunohistokemijsko bojenje s različitim biljezima tubularnih segmenata. Izražaj koneksina i Panx1 je određen u različitim regijama bubrega: kora (CTX.), vanjski sloj vanjske medule (O.S.), unutarnji sloj vanjske medule (IN.S.) i unutarnja medula (IN.MED. ). Cx37, Cx40, Cx45 i Panx1 značajno su niže izraženi u različitim dijelovima bubrega St8sia1-KO miševa u usporedbi s WT. Svi ovi ranije navedeni koneksini smanjenog su izražaja najkonzistentnije u O.S. u St8sia1-KO miševa, pa možemo zaključiti da su proksimalni tubuli i debeli uzlazni krakovi Henleove petlje dijelovi nefrona koji su najosjetljiviji na promjene u sastavu gangliozida. U CTX. regiji je zabilježen smanjen izražaj Cx37, Cx45 i Panx1, dok su u IN.S. regiji Cx37 i Panx1 bili smanjeno izraženi. Rezultati ovog istraživanja sugeriraju da nedostatak GD3 sintaze u St8sia1-KO miševa dovodi do poremećaja izražaja Cx u bubrezima, što je vjerojatno povezano s promjenom sastava gangliozida. Uočeni poremećaj izražaja Cx u bubrezima St8sia1-KO miševa daje važan uvid u promjene na staničnoj razini koje se mogu vidjeti u hiperglikemijskim stanjima, a time i vrijedne informacije za buduća istraživanja. Izmijenjen sastav gangliozida u bubrezima St8sia1-KO miševa može biti dobar model za proučavanje posljedičnih promjena izražaja Cx u dijabetičkim uvjetima.
Abstract (english) In St8sia1 gene knockout mice (St8sia1-KO) lacking the GD3 synthase enzyme, a-series gangliosides accumulate, and b- and c-series gangliosides are deficient. Total concentration of gangiosides in the KO mice remains the same as in wild-type (WT) mice, while the ganglioside composition markedly changes. In the plasma membrane gangliosides are clustered in lipid rafts, same as connexins (Cx), that are transmembrane proteins critical for intercellular communication in many organs, and play a functional role in several regulatory mechanisms in kidney. Considering that altered ganglioside composition affects the distribution of proteins in lipid rafts of different cell membranes and in this way probably influences the distribution, transport, and function of different Cx isoforms, the aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1-KO mice. Experiments were performed in twelve male 6-month-old mice: four wild-type (C57BL/6-type, WT) and eight St8sia1-KO mice. Mice kidney samples were processed for immunohistochemistry and analyzed. The expression of Cx37, Cx40, Cx43, Cx45 and Panx1 was examined by immunofluorescence microscopy (BX61, Olympus, Tokio, Japan), while quantitative evaluation of immunoreactivity was performed using ImageJ Software (National Institutes of Health, Bethesda, MD, SAD). To localize expression of connexins and Panx1 in different parts of the mice kidney, we performed double fluorescent staining with different tubular segment markers. The expression of connexins and Panx1 was determined in different regions of the kidney: cortex (CTX.), outer stripe of outer medulla (O.S.), inner stripe of outer medulla (IN.S.), and inner medulla (IN.MED.). Cx37, Cx40, Cx45, and Panx1 are significantly lower expressed in different parts of the kidneys of St8sia1-KO mice compared with WT. All of the above mentioned markers are decresased the most consistent in the O.S. in St8sia1-KO mice, so we can conclude that proximal tubules and thick ascending limbs are parts of the nephron most sensitive to changes in ganglioside composition. In the CTX. region reduced expression for Cx37, Cx45 and Panx1 was observed, while in the IN.S. region Cx37 and Panx1 were lower expressed. The results of the present study suggest that deficiency of GD3 synthase in St8sia1-KO mice leads to disturbance in renal Cx expression, which is probably related to alteration of ganglioside composition. The observed disruption of Cx expression in the kidney of St8sia1-KO mice gives important insight in changes on cellular level that can be seen in hyperglycemic conditions, and thus valuable informations for future studies. The disruption of ganglioside composition in the kidney of St8sia1-KO mice may be a good model for the alteration of ganglioside composition and properties of lipid rafts under diabetic conditions.
Keywords
Bubrezi
Koneksini
Gangliozidi
C57BL miševi
Miševi s isključenim genom
Keywords (english)
Kidney
Connexins
Gangliosides
Inbred C57BL Mice
Knockout Mice
Language croatian
URN:NBN urn:nbn:hr:171:781699
Project Number: IP-2014-09-2324 Title: Patofiziološke posljedice promjena sastava lipidnih splavi Title: Pathophysiological consequences of lipid raft changes Acronym: RafTuning Leader: Marija Heffer Jurisdiction: Croatia Funder: HRZZ Funding stream: IP
Study programme Title: Biology of Neoplasms Study programme type: university Study level: postgraduate Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
Type of resource Text
File origin Born digital
Access conditions Open access
Terms of use
Created on 2023-05-22 11:00:22