Olive leaf extract in addition to losartan treatment improves albuminuria and progression of focal segmental glomerulosclerosis in hypertensive rats: the role of Klotho

Abstract

Prethodne studije su pokazale da klotho poseduje anti-inflamatornu, antifibrotičku i antioksidativnu ulogu, i da postoji direktna pozitivna korelacija izmedu klotho-a i albuminurije kod nekih animalnih modela hronične bubrežne bolesti (CKD). Fokalno segmentna glomeruloskleroza (FSGS) je oblik CKD, koji se odlikuje prisutnom proteinurijom, hipoalbuminemijom i oksidativnim stresom. Ranije smo pokazali da blokada receptora angiotenzina II tipa 1 losartanom (L) smanjuje proteinuriju, glomerulosklerozu, tubulointersticijsku inflamaciju i fibrozu u animalnom modelu FSGS izazvane adriamicinom (ADR, antitumorski lek) kod spontano hipertenzivnih pacova (SHR), ali ove promene u bubrezima su i dalje bile veće od kontrolnih vrednosti. Ekstrakt lista masline (Olea europaea L.) (OLE), dobro poznat kao prirodni antioksidans, bogat je fenolnim jedinjenjima koja se smatraju odgovornim za snažna antioksidativna i anti-inflamatorna dejstva ekstrakta. Stoga smo ispitivali da li tretman losartanom u kombinaciji sa ekstraktom lista masline može biti efikasniji u usporavanju progresije FSGS. Odrasle ženke SHR su podeljene u četiri grupe. Kontrolni pacovi su primili fiziološki rastvor. SHADR, SHADR+L i SHADR+L+OLE grupe su primile ADR (2 mg/kg telesne mase i.v.) dva puta u intervalu od 3 nedelje. Nakon druge injekcije, SHADR+L i SHADR+L+OLE grupe su primale gavažom L (10 mg/kg/dan), odnosno L+OLE (10+80 mg/kg/dan). Na kraju 6-nedeljnog tretmana analizirali smo albuminuriju, nivo azot monoksida (NO,), 6-nitrotriptofan (marker nitracije proteina), klotho i histopatologiju bubrega. U model grupi uočeno je mačajno povećanje albuminurije i indeksa oitećenja bubrega, ito je bilo praćeno značajno smanjenom ekspresijom klotho proteina i 6-nitortriptofana, kao i nivoa NO, u homogenatu bubrega u poredenju sa ovim vrednostima u kontrolnoj grupi. Tretman losartanom je mačajno smanjio albuminuriju, glomerulosklerozu i tubulointersticijska oštećenja u poredenju sa model grupom, ali su oštećenja bubrega i dalje bila mačajno veća nego u kontrolnoj grupi. Takode, vrednost NO, je vraćena na kontrolni nivo, a proteinska ekspresija 6-nitrotriptofana je mačajno povećana, dok je klotho-a ostala nepromenjena, u poredenju sa model grupom. Interesantno, kada je tretmanu losartanom dodat ekstrakt lista masline primećeno je mačajno i još jače smanjenje albuminurije, glomeruloskleroze, tubulointersticijske inflamacije i fibroze u poredenju sa model grupom. Takode, ovi parametri su sniženi do nivoa koji se nisu mačajno razlikovali od onih u kontroli. Pored toga, ekspresija proteina klotho-a u bubrezima je mačajno povećana u poredenju sa grupama SHADR+L i model Naši rezultati su pokazali da je ekstrakt lista masline kombinovan sa losartanom bio efikasniji od samog tretmana losartanom u usporavanju progresije FSGS kod SHR, a ovaj povoljan efekat uključivao je Klotho signalni put.

Previous studies have shown the anti-inflammatory, antifibrotic and antioxidant properties of klotho, and a direct positive correlation between klotho and albuminuria in some chronic kidney disease (CKD) animal models. Focal segmented glomerulosclerosis (FSGS), a form of CKD, is distinguished by proteinuria, hypoalbuminemia, and oxidative stress. We previously showed that blockade of angiotensin Õ type 1 receptor with losartan (L) decreased proteinuria, glomerulosclerosis, tubulointerstitial inflammation, and fibrosis in animal model of FSGS induced by Adriamycin (ADR, anti-cancer agent) in spontaneously hypertensive rats (SHR), but these renal alterations were still higher than control values. Olive (Olea europaea L.) leaf extract (OLE), well-known as natural antioxidant, is rich in phenolic compounds that are thought to be responsible for its powerful antioxidant and anti-inflammatory properties. Here, we examined if losartan treatment in combination with olive leaf extract consumption could be more effective in slowing down the progression of FSGS. Adult female SHRs were divided in four groups. Control rats received vehicle. SHADR, SHADR+L and SHADR+L+OLE groups received ADR (2 mg/kg body weight i.v.) twice in 3-week-interval. After the second injection, SHADR+L, and SHADR+L+OLE groups received by gavage L (10 mg/kg/day), and L+OLE (10+80 mg/kg/day), respectively. At the end of6-week treatment, we analysed albuminuria, renal nitric oxide level (NO,), 6-nitrotryptophan (marker ofnitration of proteins), klotho, and renal histopathology In model group we found significantly increased albuininuria and renal injury score, followed by significantly decreased klotho protein expressions, NOx level, and 6- nitortryptophan in comparison to these values in control group. Losartan treatment significantly reduced albuminuria, glomerulosclerosis, and tubulointerstitial injuries compared to model group, but renal alterations were still significantly higher than in control group. Furthermore, NO, level reversed to control and 6-nitrotryptophan protein expression significantly increased, but klotho remained unchanged, compared to model group. Interestingly, when olive leaf extract was added to losartan treatment, a significant and even stronger decrease of albuminuria, glomerulosclerosis, tubulointerstitial inflammation and fibrosis were observed in comparison to model group. Also, these parameters were lowered to the levels not significantly different than in control. Furthermore, renal protein expression of klotho was significantly increased compared to SHADR+L and model groups. Our results showed that olive leaf extract in addition to losartan treatment was more effective than losartan alone in slowing down the progression of FSGS in SHR, and that beneficial effect involved the klotho signalling pathway.