Urtica dioica L. Leaf Extract Dose-Dependently Modulates Oxidative Stress in the Kidney and Exerts Anti-Fibrotic and Anti-Inflammatory Properties by the Molecular Mechanisms Independent of NRF-2 Signalization Mirroring the Effects of Losartan in SHR

Abstract

Previously, we confirmed systemic antihypertensive and antioxidant properties of Urtica dioica L. leaf extract (UE) in spontaneously hypertensive rats (SHR). Here, we aimed to evaluate whether UE can alter the NO and Nrf-2 signaling to prevent local oxidative stress and kidney damage in the model of essential hypertension. SHR were divided into five groups: SHRC-control, received 0.5 mL/day of water, SHR+L received 10 mg/kg/day of losartan, SHR+UE10, SHR+UE50, and SHR+UE200 received 10, 50, and 200 mg/kg/day during next 4 weeks. At the end of the experiment, urine samples were collected for albuminuria and nitrate/nitrite assessment. Mean arterial pressure (MAP) was measured, and blood samples were collected for plasma creatinine evaluation. Kidneys were analyzed for nitrate/nitrite, oxidative stress, and target molecules by biochemical, Western blot, and immunofluorescent techniques. Losartan and UE50 significantly reduced MAP, albuminuria, oxidative stress, fibroinflammatory markers, and NRF-2/CAT/SOD signaling, with a significant increase in 6-nitrotryptophan and eNOS expressions compared to control. The effects of UE showed dose dependence. Beneficial effects of UE and losartan were independent of NRF-2 signalization in SHR. Interestingly, all treatments induced the increase in 6-nitrotryptophan expression, thus further studies are needed to elucidate the mechanisms of such nitrated tryptophan.

Keywords: Nrf-2 signaling; Urtica dioica L.; collagen; fibronectin; hypertension; kidney; nitric oxide; nitro-tryptophan; oxidative stress; plasminogen activator inhibitor 1.

Figure 1

Total (A) nitrate/nitrite (NO_x) and (B) nitrite (NO₂) concentrations in plasma of SHR; total (C) nitrate/nitrite (NO_x) and (D) nitrite (NO₂) concentrations in urine of SHR; total (E) nitrate/nitrite (NO_x); and (F) nitrite (NO₂) concentrations in the kidney of SHR. SHRC received 0.5 mL/day of water, SHR+L received 10 mg/kg/day of losartan, SHR+UE10, SHR+UE50, and SHR+UE200 received 10, 50, and 200 mg/kg/day of Urtica dioica L. leaf extract (UE), respectively. * p < 0.05 and ** p < 0.01 compared to SHRC, # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to SHRL. Data presented as mean ± SEM (one-way ANOVA omnibus test with Fisher least significant difference (LSD) post hoc test, Statistica version 8.0., StatSoft Inc., Tulsa, OK, USA).

Figure 2

Kidney (A) eNOS, (B) nNOS, (C) iNOS, and (D) 6-NO₂Trp protein expressions in SHR with (E) the representative Western blots of NOS isoforms. SHRC received 0.5 mL/day of water, SHR+L received 10 mg/kg/day of losartan, SHR+UE10, SHR+UE50, and SHR+UE200 received 10, 50, and 200 mg/kg/day of Urtica dioica L. leaf extract (UE), respectively, ** p < 0.01 and *** p < 0.001 compared to SHRC, ## p < 0.01 and ### p < 0.001 compared to SHRL. Data presented as mean ± SEM from three independent experiments (one-way ANOVA omnibus test with Fisher least significant difference (LSD) post hoc test, Statistica version 8.0., StatSoft Inc., Tulsa, OK, USA).

Figure 3

Kidney (A) Nrf-2, (B) CAT, and (C) SOD expressions in SHR with (D) representative Western blots. SHRC received 0.5 mL/day of water, SHR+L received 10 mg/kg/day of losartan, SHR+UE10, SHR+UE50, and SHR+UE200 received 10, 50, and 200 mg/kg/day of Urtica dioica L. leaf extract (UE), respectively. * p < 0.05 and ** p < 0.01 compared to SHRC, # p < 0.05 and ## p < 0.01 compared to SHRL. Data presented as mean ± SEM from three independent experiments (one-way ANOVA omnibus test with Fisher least significant difference (LSD) post hoc test, Statistica version 8.0., StatSoft Inc., Tulsa, OK, USA).

Figure 4

(A) antioxidant capacity (kABTS), (B) advanced oxidation protein products (kAOPP), and (C) lipid peroxidation level (kTBARS) in the kidney of SHR. SHRC received 0.5 mL/day of water, SHR+L received 10 mg/kg/day of losartan, SHR+UE10, SHR+UE50, and SHR+UE200 received 10, 50, and 200 mg/kg/day of Urtica dioica L. leaf extract (UE), respectively. * p < 0.05 compared to SHRC, ## p < 0.01 compared to SHRL. Data presented as mean ± SEM (One-way ANOVA omnibus test with Fisher least significant difference (LSD) post hoc test, Statistica version 8.0., StatSoft Inc., Tulsa, OK, USA).

Figure 5

Collagen, fibronectin, α-SMA, and PAI-1 expression in kidneys of all investigated groups (magnification, ×200; α-SMA and PAI-1 of SHRL—magnification, ×100). The SHRC group exhibited slight collagen expressions within the kidney interstitium and mild expression within glomeruli, mostly localized in Bowman’s capsule. The expression of fibronectin was widespread in the interstitial compartment and within the glomeruli. The SHRL group exhibited decreased collagen and fibronectin expression within the kidney interstitium and within glomeruli. SHR+UE10 dose did not significantly reduce fibronectin expression, but collagen was prominently decreased. SHR+UE 50 dose significantly reduced fibronectin and collagen expression in the kidney interstitium, but with slightly preserved collagen expression in Bowman’s capsule. SHR+UE 200 dose completely reduced collagen expression in kidney structures, but without alteration in the expression of fibronectin compared to control and UE10 groups. SHRC and all investigated SHR+UE groups showed slight focal α-SMA expression within the interstitium, along with the normal expression in blood vessels, and also had focal interstitial PAI-1 staining. The SHRL group showed normal α-SMA expression localized to the area of blood vessels, with a reduced PAI-1 immunofluorescent staining. The SHR+UE10 group did not influence the α-SMA expression and PAI-1 expression, while SHR+UE50 and SHR+UE200 reduced expression of both molecules resembling the SHRL group. Nuclei were identified by 4,6-diamino-2-phenylindolyl-dihydrochloride (DAPI). SHRC—the control group, SHRL group—the SHR received losartan 10 mg/kg/day for 4 weeks; SHR+UE10, SHR+UE50, and SHR+UE200 groups—the SHR received 10, 50, and 200 mg/kg/day of UE for 4 weeks.