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Histomorfološke, imunohistohemijske i biohemijske karakteristike oštećenja bubrega kod miševa u modelu toksične nefropatije izazvane aristolohičnom kiselinom I / Histolomorphological, immunohistochemical and biochemical characteristics of kidney injury in mouse model of aristolochic acid nephropathy

Uvod: Aristolohična kiselina I je nefrotoksična i kancerogena supstanca koja je odgovorna za nefropatiju koja nastaje usled kori&scaron;ćenja herbalnih preparata i čajeva za mr&scaron;avljenje. S obzirom da se ova supstanca može naći u korovskim biljkama, smatra se jednim od glavnih ekotoksikolo&scaron;kih uzroka za nastanak balkanske endemske nefropatije čiji definitivan uzrok jo&scaron; uvek nije otkriven. Toksičnost ove supstance je dokazana na brojnim animalnim modelima, međutim mehanizmi koji dovode do o&scaron;tećenja bubrežnog parenhima jo&scaron; u potpunosti nisu razja&scaron;njeni.  Cilj: Doktorska disertacija je koncipirana sa ciljem da se utvrdi uticaj toksičnog jedinjenja aristolohične kiseline I na histopatolo&scaron;ke i imunohistohemijske karakteristike tubulointersticijuma i glomerula bubrega kod mi&scaron;eva, kao i na biohemijske parametre krvi i urina koji ukazuju na o&scaron;tećenje bubrega. Materijal i metode: U ekperimentu je kori&scaron;ćeno 64 mi&scaron;a soja NMRI koji su podeljeni u tri grupe: eksperimentalna grupa (n=32) koja je dobijala aristolohičnu kiselinu I rastvorenu u polietilen glikolu (2,5% PEG 400) u dozi od 10 mg/kg telesne mase, negativna kontrolna grupa koja je dobijala 2,5% PEG 400 (n=16) i kontrolna grupa koja je dobijala fiziolo&scaron;ki rastovor (n=16). Sve životinje su tretirane intraperitonealno svakodnevno tokom sedam dana. Tokom eksperimenta 8., 17., 29. i 59. dana sakupljan je dvadesetčetvoročasovni urin 8 životinja iz eksperimentalne grupe, 4 životinje iz negativne kontrolne i 4 životinje iz kontrolne grupe. Životinje su žrtvovane 9., 18., 30. i 60. dana, uzeta im je krv, dok su bubrezi posebno odvojeni radi histopatolo&scaron;ke analize. Na bubrežnom tkivu sprovedene su histohemijske, imunohistohemijske i morfometrijske analize, dok su na uzorcima seruma i urina sprovedene biohemijske analize. Dobijeni rezultati su testirani adekvatnim statističkim metodama i prikazani su tabelarno i grafički. Rezultati: Nefrotoksin aristolohična kiselina I nakon 7 dana aplikacije izaziva značajno o&scaron;tećenje bubrežnog parenhima. Pri aplikaciji 2,5% PEG 400 i fiziolo&scaron;kog rastvora ne dolazi do vidljivog o&scaron;tećenja bubrežnog parenhima. Histopatolo&scaron;ku sliku u ranoj fazi eksperimenta (9. i 18. dan) karakteri&scaron;e akutna tubulska nekroza proksimalnih tubula. U kasnijoj fazi (30. i 60. dana) uočava se histopatolo&scaron;ka slika hroničnog intersticijalnog nefritisa sa obilnim mononuklearnim ćelijskim infiltratima limfocitnog porekla kao i postojanje blage intersticijalne fibroze. Kod eksperimentalnih životinja je morfometrijskim metodama utvrđen veći stepen bubrežnog o&scaron;tećenja tubulointersticijuma i smanjen broj podocita u glomerulu u odnosu na kontrolne grupe. Biohemijske analize kod većine eksperimentalnih životinja su pokazale veće koncentracije serumske uree nego kod kontrolnih grupa. Takođe je dokazana albuminurija u kasnijoj fazi eksperimenta koja je veća kod životinja izloženih aristolohičnoj kiselini I nego kod životinja iz kontrolnih grupa. Zaključak: Kori&scaron;ćenjem morfometrijskih metoda u okviru histopatolo&scaron;kih i imunohistohemijskih ispitivanja, uz adekvatne biohemijske analize, može se zaključiti da je aristolohična kiselina I izuzetno nefrotoksično jedinjenje koje izaziva izrazite  promene tubulointersticijuma i glomerula. Podaci ovog istraživanja predstavljaju polaznu osnovu za dalja istraživanja dijagnostike u ranoj fazi nefropatija izazvanih aristolohičnim kiselinama.  / Introduction: Aristolochic acid I is a nephrotoxic and carcinogenic substance responsible for nephropathy caused by the use of herbal preparations and teas for slimminng regimen. Since this substance can be found in plants, it is considered one of the major ecotoxicological causes for the emergence of balkan endemic nephropathy whose definitive cause has not yet been revealed. The toxicity of this substance has been proven on numerous animal models, but pathophysiological mechanisms of kidney injury still remain unclear. Aim: The doctoral dissertation was designed to determine the influence of aristolochic acid on the histopathological and immunohistochemical characteristics of tubulointerstitium and glomerulus in mice, as well as the biochemical parameters of blood and urine that indicate kidney injury. Material and methods: For this study, 64 mouse of NMRI strain is used. They are divided into three groups: an experimental group (n=32) that received aristolochic acid I dissolved in polyethylene glycol (2.5% PEG 400) at a dose of 10 mg/kg of body weight, a negative control group that received 2.5% PEG 400 (n=16) and a control group that received only saline (n=16). All animals were treated intraperitoneally daily for seven days. During the experiment on the 8th, 17th, 29th and 59th day, twenty-four-hour urine was collected from 8 animals from the experimental group, 4 animals from the negative control and 4 animals from the control group. Animals were sacrificed on the 9th, 18th, 30th and 60th days, their blood was taken, while the kidneys were taken for histopathological analysis. Histochemical, immunohistochemical and morphometric analyzes were performed on renal tissue, while biochemical analyzes were performed on serum and urine samples. Obtained results were tested with adequate statistical methods and presented in a tables and graphs. Results: After 7 days of application nefrotoxin aristolochic acid I causes significant kidney injury. After application of 2.5% PEG 400 and saline, there was no visible damage to kidney parenchyma. Histopathological changes at the early stage of the experiment (9th and 18th day) were characterized by acute tubular necrosis of proximal tubules. At a later stage (30th and 60th day), chronic interstitial nephritis was observed in kidneys, with abundant mononuclear cell infiltrates in interstitium and presence of mild interstitial fibrosis. In experimental animals, a higher tubulointerstitial score of kidney injury and a decrease in the number of the podocytes in glomerulus were determined by morphometric methods, compared to the control groups. Biochemical analyzes in most experimental animals showed higher blood urea nitrogen concentrations than in control groups. High concentration of albumin in urine can be found in later stages of the experiment, and those concentrations were higher in animals exposed to aristolochic acid I than in animals from control groups.  Conclusion: Using morphometric, histopathological and immunohistochemical methods, with adequate biochemical analysis, aristolochic acid I is proven to be an extremely nephrotoxic compound that causes drastic changes in tubulointerstitium and glomeruli of kidney parenhyma. Data from this study can be used for further research into early diagnosis of aristolochic acid nephropathy.

Identifer	oai:union.ndltd.org:uns.ac.rs/oai:CRISUNS:(BISIS)108220
Date	18 February 2019
Creators	Miljković Dejan
Contributors	Lalošević Dušan, Đolai Matilda, Andrejić Višnjić Bojana, Trivunić Dajko Sandra, Grujić Milanović Jelica, Mitić Igor
Publisher	Univerzitet u Novom Sadu, Medicinski fakultet u Novom Sadu, University of Novi Sad, Faculty of Medicine at Novi Sad
Source Sets	University of Novi Sad
Language	Serbian
Detected Language	French
Type	PhD thesis

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