Dosadašnja istraživanja pokazala su da renin angiotenzin sistem (RAS) ima važnu
ulogu u patogenezi i progresiji fokalno segmentne glomeruloskleroze (FSGS).
Oksidativni stres je prisutan u hroničnoj bubrežnoj slabosti i doprinosi progresiji bolesti.
Povezanost oksidativnog stresa i RAS u procesima progresije FSGS još je nedovoljno
razjašnjena. Stoga su istraživanja u okviru ove disertacije bila usmerena ka ispitivanju
efekata hronične primene tempola (sakupljač slobodnih radikala, SOD mimetik),
losartana (blokator receptora za angiotenzin II tipa 1, AT1R), kao i njihove kombinacije,
na usporavanje progresije FSGS izazvane adrijamicinom (ADR) kod spontano
hipertenzivnih (SH) pacova.
Životinje su inicijalno deljene u dve eksperimentalne grupe, kontrolnu (SHC) i
grupu koja je primila ADR 2 mg/kg i.v. dva puta u intervalu od 21 dan. Nakon druge
injekcije ADR, životinje su dobijale vodu (SHADR), losartan (SHADR+L), tempol
(SHADR+T) i kombinovani tretman (SHADR+T+L) gavažom. Na kraju šeste nedelje
tretmana vršena su hemodinamska merenja i uzorkovanje krvi, urina i bubrega. Na
osnovu biohemijskih parametara vršena je procena lipidnog statusa i bubrežne funkcije.
Urađena je histopatološka analiza bubrega. Imunihistohemijskom metodom ispitivani su
proteini citosketeta, nestin i vimentin, čije izmenjene ekspresije su pokazatelj oštećenja
bubrežnog tkiva. Matriksna metaloproteinaza-1 (MMP-1), koja učestvuje u degradaciji
komponenti vanćelijskog mariksa i očuvanju integriteta glomerula, određivana je ELISA
metodom. Ispitivani su parametri oksidativnog stresa: lipidna peroksidacija i nivo
karbonilovanih proteina (PCOs); aktivnosti enzima antioksidativnog sistema: superoksid
dismutaze (SOD), katalaze (CAT), glutation peroksidaze (GPx) i glutation reduktaze
(GR); kao i antioksidativni kapacitet. Primenom imunohistohemijske, Western blot i
ELISA metode u bubrezima su određivane ekspresije Nox2 i Nox4 izoforme katalitičke
subjedinice NADPH oksidaze, glavni izvor reaktivnih kiseoničnih vrsta u ćeliji, uključene
u proces inflamacije. Ekspresija proteina tri izoforme azot-monoksid sintaze, inducibilna
(iNOS), endotelna (eNOS) i neuronska (nNOS), određivane su Western blot i
imunohistohemijskom metodom u tkivu bubrega. Određivani su ukupni metaboliti NO-a
(nitriti, nitrati) u urinu i bubrezima...Previous studies have shown that renin angiotensin system (RAS) plays an
important role in the pathogenesis and progression of focal segmental glomerulosclerosis
(FSGS). Oxidative stress is involved in the development and progression of chronic
kidney disease. However, the association of oxidative stress and RAS in the progression
of FSGS has not been completely elucidated. In this study we investigated the effects of
chronic tempol (free radical scavenger, SOD mimetic), losartan (selective angiotensin II
type 1 receptor (AT1R) blocker), and their combined treatment in slowing down the
progression of FSGS, in spontaneously hypertensive (SH) rats with adriamycin (ADR)
nephropathy.
Animals were initially divided into two experimental groups: control (SHC) and
group that received ADR 2 mg/kg i.v. twice in an interval of 21 days. After the second
injection of ADR, the animals were given tap water (SHADR), losartan (SHADR+L),
tempol (SHADR+T) or combined treatment (SHADR+T+L) by gavage. Hemodynamic
measurements were performed, blood, urine, and kidney samples were taken for
biochemical and histopathological analysis. Immunohistochemical method was used for
protein expression and localization of the nestin and vimentin, proteins of cytoskeleton
network, and change in their protein expression is a marker of cell injury. Matrix
metalloproteinase-1 (MMP-1), which participates in the degradation of the extracellular
matrix components and preserving the integrity of the glomerulus, was determined by the
ELISA method. The parameters of oxidative stress: lipid peroxidation, protein carbonyl
content (PCOs); antioxidant defense: superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPx), glutathione reductase (GR) and antioxidant capacity were
analysed. Immunohistochemical, Western blot and ELISA method were used for analysis
of the Nox2 and Nox4 protein expression, isoforms of the catalytic subunit of NADPH
oxidase, the main source of reactive oxygen species in the cell, involved in the
inflammation process. Protein expression of three isoforms of azote monoxide synthase,
inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) were determined by
Western blot and immunohistochemical method in the kidney. Total metabolites of NO
(nitrites, nitrates) were measured in the kidney and urine..
