Title Uloga koštanog morfogenetskog proteina-7 u prevenciji oštećenja tkiva bubrega izazvanog hladnom ishemijom : doktorski rad
Author Tanja Ćelić
Mentor Dragica Bobinac (mentor)
Committee member Sanjin Rački (predsjednik povjerenstva)
Committee member Fran Borovečki (član povjerenstva)
Committee member Natalia Kučić (član povjerenstva) MBZ: 239812
Granter University of Rijeka Faculty of Medicine Rijeka
Defense date and country 2013-03-05, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Clinical Medical Sciences Medical Microbiology
Universal decimal classification (UDC ) 616 - Pathology. Clinical medicine
Abstract CILJ ISTRAŽIVANJA. Ispitati da li je perfuzijom bubrega s rhBMP-7 otopinom bolje očuvana struktura tkiva tijekom hladne ishemije u trajanju do 24 sata u odnosu na komercijalno korištenu UW otopinu. Ispitati mehanizam djelovanja rhBMP-7 na epitelne stanice kanalica bubrega i njegovu učinkovitost ovisno o dužini trajanja hladne ishemije.
MATERIJALI I METODE. Istraživanje je provedeno na eksperimentalnom modelu hladne ishemije bubrega u štakora. Bubrezi su perfundirani sa slijedecim otopinama: fiziološkom, UW, rhBMP-7 i rhBMP-7+UW. Bubrezi su izloženi hladnoj ishemiji kroz 6, 12
i 24 sata. Učinjena su spektrofotometrijska mjerenja razine oštećenja lipida i proteina te razine antioksidativnih enzima. Western blotom izmjerene su razine ekspresije Hsp70, HIF-1alfa i aktivirane kaspaze 3. Metodom PCR-a analizirana je ekspresija mRNA BMP-7, TGF-β1, Smad1, Smad2, Smad3, Smad5 i Smad8. Imunohistokemijskom metodom prikazana je ekspresija i lokalizacija BMP-7, TGF-β1, E-cadherin, α-SMA i PCNA.
REZULTATI. U epitelnim stanicama kanalica bubrega perfundiranih s rhBMP-7 i rhBMP-7+UW otopinom izražena je ekspresija BMP-7 i E-cadherin i nakon 24 sata hladne ishemije. U bubrezima koji nisu perfundirani s rhBMP-7 nalazi se pojačana ekspresija TGF- β1 i α-SMA. Također u tkivu bubrega koji je ispran s rhBMP-7 otopinom povećan je nivo ekspresije mRNA BMP-7. U istom tkivu dokazan je viši nivo ekspresije mRNA Smad1, Smad5 i Smad8, molekula unutarstaničnog signalnog puta za BMP-7. Nivo ekspresije mRNA BMP-7, Smad1, Smad5 i Smad8 podjednako je prisutan kroz cijelo vrijeme trajanja hladne ishemije. Nadalje, u bubrezima perfundiranim s rhBMP-7 otopinom nakon 6 i 12 sati nema promjene razine produkata lipidne peroksidacije i sadržaja karboniliranih proteina u odnosu na bubreg s održanom funkcijom. Razina produkata lipidne peroksidacije i sadržaja karboniliranih proteina je povišena u bubrezima tretiranim s UW otopinom, dok se razina istih povećava u bubrezima tretiranim rhBMP-7 otopinom tek nakon 24 sata hladne ishemije. Ispiranjem tkiva bubrega s rhBMP-7 i rhBMP-7+UW otopinom razina ekspresije Hsp70 i HIF-1alfa viša je u odnosu na bubrege perfundirane samo s UW otopinom. Takoder, u skupini bubrega perfundiranih s rhBMP-7 razina ekspresije aktivirane kaspaze 3 je niža u odnosu na bubrege perfundirane UW otopinom.
ZAKLJUČAK. BMP-7 održava strukturu tkiva bubrega bolje nego UW otopina tijekom 24 sata hladne ishemije. BMP-7 sprječava epitelno-mezenhimalnu transformaciu čime održava epitelni fenotip stanica kanalica, smanjuje apoptozu stanica, potiče ekspresiju protektivnih činitelja i povećava antioksidacijski kapacitet tkiva.
Abstract (english) AIM. To analyze whether the perfusion of kidneys with rhBMP-7 solution can better preserve tissue structure during cold ischemia up to 24 hours compared to commercially used UW solution. To analyze mechanisms of BMP-7 activity in tubular epithelial cells and its proficiency considering the time of cold ischemia.
MATERIAL AND METHODS. The study was conducted on experimental model of cold ischemia in the rat kidneys. Kidneys were perfused with solutions as follows: saline, UW, rhBMP-7 and rhBMP-7 + UW. Kidneys were exposed to cold ischemia during 6, 12 and 24 hours. Spectrophotometric measurements of lipid peroxidation products and protein carbonyl content and levels of antioxidant enzymes activity were performed. Using Western blot were measured levels of expression of Hsp70, HIF-1alpha and activated caspase 3. With PCR method was analyzed the expression of mRNA BMP-7, TGF-β1, Smad1, Smad2, Smad3, Smad5 and Smad8. Immunohistochemical analysis was used to show expression and localization of BMP-7, TGF-β1, E-cadherin, α-SMA and PCNA.
RESULTS. In tubular epithelial cells of the kidneys perfused with rhBMP-7 and rhBMP-7+UW solution the expression of BMP-7 and E-cadherin was observed still after 24 hours of cold ischemia. In the kidneys not perfused with rhBMP-7 high expression of TGF-β1 and α-SMA was found. Also, in the kidneys perfused with rhBMP-7 solution level of mRNA
BMP-7 expression was increased. In the same tissue higher level of mRNA Smad1, Smad5 and Smad8 expression, molecules of intracellular BMP-7 signal pathway, was proved. The levels of mRNA BMP-7, Smad1, Smad5 and Smad8 expression were equally present during whole time of cold ischemia. Furthermore, in the kidneys perfused with rhBMP-7 solution there was no changes of levels of lipid peroxidation products and protein carbonyl content after 6 and 12 hours compared to kidney with sustainable function. The levels of lipid peroxidation products and preotein carbonyl content were higher in the kidneys treated with UW solution, while in the kidneys treated with rhBMP-7 solution their levels increased only
after 24 hours of cold ischemia. After reperfusion with rhBMP-7 and rhBMP-7+UW solutions the levels of Hsp70 and HIF-1alfa expression were higher in relation to the kidneys perfused only with UW solution. Also, in the group of the kidneys perfused with the BMP-7 solution the level of activated caspase 3 expression was lower in relation with the kidneys perfused with UW solution.
CONCLUSION. BMP-7 maintains the morphology of the kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells, reduces cell apoptosis, stimulates the expression of protective factors and increases the antioxidant
capacity of the tissue.
Keywords
hladna ishemija
koštani morfogenetski protein-7
otopine za čuvanje organa
slobodni radikali kisika
transplantacija bubrega
Keywords (english)
bone morphogenetic protein-7
cold ischemia
kidney transplantation
preservation solutions organ
reactive oxygen species
Language croatian
URN:NBN urn:nbn:hr:188:802620
Promotion 2020
Study programme Title: Biomedicine Postgraduate (doctoral) study programme Study programme type: university Study level: postgraduate Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
Catalog URL http://libraries.uniri.hr/cgi-bin/ucat/unilib.cgi?form=D1130410072
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Created on 2017-01-19 17:44:30