Effects of chronic losartan and tempol treatments on experimental focal segmental glomerulosclerosis in spontaneously hypertensive rats

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..

Angiotensin 2 type 1 receptor blockade different affects postishemic kidney injury in normotensive and hypertensive rats

Many studies demonstrated that angiotensin 2 type 1 receptor (AT1R) blockade accelerates renal recovery in post-ischaemic kidney but there are many controversies related to its net effect on kidney structure and function. During the past years, our research group was trying to define the pathophysiological significance of the renin-angiotensin system on post-ischemic acute renal failure (ARF) development in normotensive Wistar as well as hypertensive rats (SHR). This review mostly summarizes our experience in that field. Our previous studies in normotensive rats revealed that AT1R blockade, except slightly renal vascular resistance improvement, had no other obvious beneficial effects, and therefore implies angiotensin 2 (Ang-2) overexpression as non-dominant on kidney reperfusion injuries development. Similarly it was observed in Wistar rats with induced mild (L-NAME, 3 mg/kg b.w.) nitric oxide (NO) deficiency. Expectably, in strong induced (L-NAME, 10 mg/kg b.w.) NO deficiency associated with ARF, massive tubular injuries indicate harmful effects of AT1R blockade, implying strongly disturbed glomerular filtration and suggesting special precaution related to AT1R blockers usage. Opposite to previous, by our opinion, AT1R antagonism promises new advance in treatment of essentially hypertensive subjects who develop ARF. Increased glomerular filtration, diminished oxidative stress, and most importantly improved tubular structure in postishemic SHR treated with AT1R blocker losartan, implicate Ang-2 over production as potently agent in the kidney ischemic injury, partly trough generation of reactive oxygen species. These data contribute understanding the pathogenesis of this devastating illness in hypertensive surroundings