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20070917162923.0 |
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980728s1997 ci a m 000 0 hrv |
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|9 (HR-ZaNSK)219034
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|9 (HR-ZaNSK)980728001
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|a (HR-ZaNSK)000218803
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|a 615.9.07
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|a Herak-Kramberger, Carol Mirna
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|a Mehanizam nastanka fosfaturije u štakora trovanih kadmijem :
|b doktorska disertacija /
|c Carol Mirna Herak-Kramberger.
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| 260 |
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|a Zagreb :
|b C. M. Herak-Kramberger,
|c 1997
|e ([s. l. :
|f s. n.])
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| 300 |
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|a 100 str. :
|b ilustr., table, graf. prikazi ;
|c 30 cm.
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|a Mentor: Ivan Sabolić; Komisija za ocjenu: Franjo Plavšić, Krista Kostial, Tihana Žanić-Grubišić, Ivan Sabolić; Komisija za obranu: Franjo Plavšić, Krista Kostial, Tihana Žanić-Grubišić, Ivan Sabolić; datum obrane: 14. 07.1997.
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|a Sveučilište u Zagrebu, Farmaceutsko-biokemijski fakultet, Zagreb, 1997
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| 504 |
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|a Bibliografija: str. 88-97
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| 504 |
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|a Sažetak
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| 520 |
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|a Sažetak: Toksični učinci kadmija na staničnoj razini slabo su poznati. Nefrotoksični učinak kadmija očituje se uglavnom u odsječcima S1 i S2 stanica PK tako što oštećuje reapsorpciju i sekreciju tvari i tekućina u ovom dijelu nefrona. Posljedice dugotrajne izloženosti kadmiju u subkronično trovanim ljudima i pokusnim životinjama su izražena proteinurija, glukozurija, aminoacidurija, poliurija i fosfaturija. U ovom radu ispitala sam stanične mehanizme koji dovode do poremećaja reapsorpcije Pi u PK i fosfaturije u štakora trovanih kadmijem. Razvila sam eksperimentalni model nefrotoksičnosti u štakora In vivo s poremećajem funkcije PK koji odgovara kliničkoj slici nefropatije uzrokovane dugotrajnoj izloženosti kadmiju u ljudi.
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|a Ovakav eksperimentalni model stvorila sam ubrizgavanjem CdCl2 (2 mg Cd/kg dnevno, s.c.) tijekom 14 dana. U VČM izoliranim iz kore bubrega kontrolnih štakora i štakora trovanih kadmijem izmjerila sam o Na+ ovisan prijenos glukoze, Pi i sukfata obilježenih radioizotopima. Primitak glukoze i Pi bio je smanjen u VČM iz štakora obrađenih kadmijem, dok primitak sulfata u istim VČM nije bio promijenjen u poredbi sa kontrolnim VČM. Rabeći poliklonsko protutijelo na prijenosnik Pi tipa II(NaPi-2), imunoblotiranjem i imunohistokemijski sam pokazala da je sadržaj NaPi-2 u četkastoj membrani stanica PK u štakora trovanih kadmijem izrazito smanjen. Nasuprot tomu, sadržaj pora za vodu (AQP 1), u četkastoj membrani nije bio bitno promijenjen uslijed obrade štakora kadmijem.
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|a Da bih razjasnila mehanizme djelovanja kadmija u stanici, koji su doveli do smanjenja broja prijenosnika Pi u četkastoj membrani stanica PK, ispitala sam učinak kadmija na endocitozu i aktivnost V-ATPaze In vivo i In vitro. Endocitoza u stanicama PK, koju sam ispitala nakon ubrizgavanja fluorescein-izotiocijanat-dekstrana (FITC-dekstran) i.v. štakorima In vivo, bila je znatno smanjena u štakora obrađenih kadmijem. Aktivnos V-ATPaze (ATPaze osjetljive na bafilomicin), koja je neophodna za zakiseljavanje unutrašnjosti organela endosomsko-lizosomskog odjeljka i time osigurava normalne procese unutarstanične reciklaže membranskih proteina, u VČM iz štakora obrađenih kadmijem, bila je jako smanjena. Pokusi imunoblotiranja i imunohistokemije pokazali su da je sadržaj podjedinicva V-ATPaze od 31 kDa i 70 kDa u
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| 520 |
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|a četkastoj membrani štakora obrađenih kadmijem također drastično smanjen. U In vitro pokusima, aktivnost V-ATPaze osjetljive na bafilomicin u VČM i EV smanjivala se s porastom koncentracije kadmija i s trajanjem izloženosti kadmiju. Na isti je način kadmijem bio inhibiran In vitro proces zakiseljavanja EV. Pored toga, kadmij In vitro povećao je propusnost membrane EV za H+ i tako narušio gradijent koncentracije H+, kojeg stvara V-ATPaza (H+-pumpa). Posljedica ovakvog djelovanja kadmija na aktivnost V-ATPaze može dovesti do oslabljenog zakiseljavanja endosoma, a potom i do poremećaja reciklaže prijenosnika četkaste membrane i smanjenja reapsobcije u stanicama PK.
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|a Nadalje imunoblotiranje i imunohistokemijski pokusi sa monoklonskim protutijelom na [alfa]-tubulin, pokazali su da je kadmij poremetio pravilno ustrojstvo mikrotubula u stanicama PK, i da je sadržaj [alfa]-tubulina (monomera) bio veći u tkivu bubrega štakora trovanih kadmijem nego u tkivu kontronih štakora. Ovi nalazi ukazuju da kadmij uzrokuje depolimerizaciju mikrotubula i/ili sprečava polimerizaciju tubulina. Poremećaj ustrojstva mikrotubula u stanici mogao bi za posljedicu imati ometen proces egzocitoze i sotga smanjen broj molekula NaPi-2 i drugih prijenosnika u četkastoj membrani. Metodom northern blota pokazala sam da je sadržaj specifične mRNA za NaPi-2 u kori bubrega štakora trovanih kadmijem znatno smanjen, dok u sadržaju mRNA za [beta]-aktin i GAPDH u tkivu kontrolnih štakora i štakora trovanih kadmijem nema bitne razlike.
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|a Abstract: The cellular mechanisms of cadmium (Cd) nephrotoxicity are poorly undrstood. The main sites of Cdnephrotoxicity are S1 and S2 segments of proximal tubules (PT). The signs of Cd-induced nephrotoxicity in humans and experimental animals reflect imparied reabsorption of filtered solutes and water and are manifested by proteinuria, aminoaciduria, glucosuria, polyuria and phosphaturia. In this study the cellular machanisms of phosphaturia caused by Cd poisonong in rats were investigated. An appropriate rat model of Cd-nephrotoxicity was developed by injecting CdCl2 for 2 weeks (2 mg Cd/kg body weight daily, s.c.). As studied by rapid filtration technique in isolated renal cortical brush-border membrane vesicles (BBMV), Na+-gradient-driven uptakes of phosphate 32P and 3H-D-glucose were markedly decreased in Cd-treated rats,
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| 520 |
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|a whereas the uptake of 35S remained unchanged. Western blot analysis of BBMV and indirect immunocytochemistry in frozen kidny section were performed using a polyclonal antibody against the type II Na/Pi-cotransporter (NaPi-2). The results obtained showed a strongly diminished expression of NaPi-2 in zhe brush-border membrane of Cd-treated rats. However, expression of the water channel aquaporin 1 (AQP1), estimated by Immunoblotting of BBMV proteins with a specific polyclonal antibody, was not affected by Cd treatment. In order to elucidate the cellular mechanisms that lead to the reduced number of NaPi-2 molecules in the brush-border membrane in Cd poisoning, the effect of Cd on endocytosis and vacuolar (V)-ATPase in PT cells In vivo and In vitro was studied.
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|a Following i.v. injection of fluorescein-isothiocyanate-dextran (FITC-dextran) into the rats In vivo, the endocytosis of this fluorophore in Pt cells from Cd-intoxicated rats was strongly impaired. The bafilomycin-sensitive ATPase (V-ATPase) is considered crucial in acidifying the organelles of endosomal comparment, and therefore very important in intracellular protein trafficking and membrane protein recycling. The activity of this ATPase was strongy reduced in BBMV isolated from Cd-intoxicated rats. As demonstrated by immunohistochemical and western blot data, the brush-border from Cd-treated rats exhibited a dramatically diminished content of the 31 kDa and 70 kDa V-ATPase subunits.
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| 520 |
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|a In vitro experiments, Cd inhibited bafilomycin-sensive ATPase in BBMV and isolated renal cortical endocytic vesicles (EV) in a concentration-and time-dependet manner. Additionally, Cd In vitro inhibited the V-ATPase-driven acidification of EV in a concentracion-and time-dependent manner and accelerated the dissipation of the transmembrane pH gradient by increasing H+ conductance of the vesicle membrane. A disruption of intravesicular acidification in intracellular organelles by Cd mayimpair vrsicle-mediate recycling of brush-border mambrane transporters, leading to a diminished reabsorptive capacty of the membrane and may inhibit the reapsorbtion of filtered proteins in PT.
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| 520 |
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|a Furthermore, indirect immunocytochemistry in frozen kidney sections with a monoclonal antibody against [alfa]-tubulin demonstrated a mssive disruption of the mcrotubular network of PT cells in Cd-intoxicated rats; western blots of rat kidney cortex homogenates showed a much stronger staining of the 55 kDa protein band related to [alfa]-tubulin in samples from Cd-trated rats, indicating depolimerization of microtubules induced by Cd. A disruptive effect of Cd upon microtubules may cause impaired exocytosis in PT cells, and lead to adiminished number of NaPi-2 and other transporters in the brush-border membrane. Finally, northern blot analysis showed a strong reduction of the NaPi-2 related mRNA in the corticaltissue of Cd-affected kidneys, whereas the expression of [beta]-actin and GAPDH related mRNAs remained unchanged.
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| 520 |
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|a Conclusion: A long-term Cd tratment in rats causes an impaired transport of Pi across the brush-border mambrans of PT cells, mainly due to a decreased content of NaPi-2 transporters. A loss of NaPi-2 transporters in the brush-border mambrane may result from the impaired intracellular trafficking of membrane proteins via endo-andexocytosis caused by a) Cd-inhibitons of V-ATPase and intravesicular acidification. and b) Cd-induced depolimerization of microtubules. In addition, Cd may reduce mRNA and protein synthesis.
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| 650 |
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7 |
|a Otrovanja
|x Kadmij
|x Mehanizmi nefrotoksičnosti
|2 nskps
|
| 700 |
1 |
|
|a Sabolić, Ivan,
|c liječnik
|4 cns
|
| 700 |
1 |
|
|a Plavšić, Franjo
|4 oth
|
| 700 |
1 |
|
|a Kostial-Šimonović, Krista
|4 oth
|
| 700 |
1 |
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|a Žanić-Grubišić, Tihana
|4 oth
|
| 981 |
|
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|p CRO
|r HRB1997
|
| 998 |
|
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|n DCD/97
|c sbno9808
|c rjkp9901
|
| 852 |
4 |
|
|j DCD-ZG-180/98
|
| 876 |
|
|
|e DCD
|a 180/1998
|
| 886 |
0 |
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|2 unimarc
|b 09689nam0 2200433 450
|