Inhibition of NF-κB-induced inflammatory responses by angiotensin II antagonists in aged rat kidney

Abstract

In this study, we explored the mechanisms by which the angiotensin converting enzyme inhibitor (ACEI), enalapril, and the Ang II receptor blocker (ARB), losartan suppress oxidative stress and NF-κB activation-induced inflammatory responses in aged rat kidney. The experimentations were carried out utilizing aged (24-month-old) Brown Norway×Fischer 344 (F1) male rats which were randomized into 3 groups and administered enalapril (40 mg/kg), losartan (30 mg/kg) or placebo for 6 months (daily p.o.). The level of reactive species (RS), peroxynitrite (ONOO(-)), GSH/GSSG and lipid peroxidation were measured. The activity of the pro-inflammatory transcription factor NF-κB, and gene expression of proteins in upstream signaling cascades were measured by electro-mobility shift assay (EMSA) and Western blotting. Enalapril and losartan differentially attenuated redox imbalance and the redox-sensitive transcription factor, the NF-κB pathway. Furthermore, stimulation of the NF-κB activation pathway by phosphorylation of p65 was attenuated by both compounds. Moreover, mediation of phosphorylation of p65 by phosphorylation of IκB kinase αβ (IKKαβ) and mitogen- and stress-activated protein kinase-1 (MSK-1), were also inhibited by enalapril and losartan. Finally, both compounds also lowered expression of NF-κB-dependent inflammatory genes, such as cyclooxygenase-2 (COX-2), and inducible NO synthase (iNOS). Only losartan lowered levels of 5-lipoxygenase (5-LOX). These findings indicate that enalapril and losartan differentially suppress inflammatory responses via inhibition of oxidative stress-induced NF-κB activation in aged rat kidney.

Figure 1. Chemical structure of angiotensin II antagonists

Angiotensin converting enzyme inhibitor, enalapril (A) and angiotensin II receptor blocker, losartan (B)

Figure 2. Effect of enalapril and losartan on redox imbalance in the aged rat

Kidney homogenates were prepared to determine relative levels of oxidative stress. RS levels were determined by DCF-DA with a fluorescent probe (A). ONOO⁻ was measured by DHR-123 with a fluorescent probe (B). GSH/GSSH was measured by o-phthaldehyde (C). MDA+HAE was determined using a Bioxytech LPO-586 Assay Kit (D). Each value is expressed as mean ± S.E.M. of three determinations and normalized mg protein. *P<0.01, **P<0.001 vs. normal aged rat by ANOVA. RS, reactive species; ONOO^–, peroxynitrite; MDA, malondialdehyde; HAE, hydroxyalkene; GSH, glutathione; GSSG, glutathione disulfide ENA, enalapril; LOS, losartan.

Figure 3. Effect of enalapril and losartan on MAPKs activation in the aged rat

Cytosolic proteins were prepared to determine the phosphorylation levels of MAPKs. p-ERK, p-p38, and p-JNK levels were detected by Western blotting using anti p-p38-, anti p-ERK- and anti p-JNK-specific monoclonal antibodies in the cytosol. One representative blot of each protein is shown from 3 experiments that yielded similar results. Levels were normalized to total ERK, p38, JNK and β-actin. Values are the relative optical intensity of each band normalized as a percentage of the untreated control. **P<0.01, ***P<0.001 vs. normal aged rat by ANOVA. MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; ENA, enalapril; LOS, losartan.

Figure 4. Effect of enalapril and losartan on NF-κB activation in the aged rat

Nuclear proteins were prepared to determine the transcriptional activity of NF-κB. NF-κB binding activity was detected by EMSA in nuclear proteins (A). Phosphorylation of IκB and nuclear translocation of p65/p50 subunit were detected by Western blotting using anti p-IκB-, anti p65- and anti p50-specific polyclonal antibodies. (B). Levels were normalized to β-actin in the cytosolic fraction and histone H1 in the nuclear fraction. One representative blot is shown from 3 experiments that yielded similar results. * P<0.05, **P<0.01, ***P<0.001 vs. normal aged rat. BL, probe without nuclear protein sample; Cold, competition assay using 100-fold excess of unlabeled NF-κB oligonucleotide; ENA, enalapril; LOS, losartan.

Figure 5. Effect of enalapril and losartan on phosphorylation of p65 in the aged rat

Cytosolic and nuclear proteins were prepared to determine the phosphorylation levels of p65. Ser 536 phosphorylation of p65 and IKKαβ phosphorylation were detected by Western blotting using anti p-p65- (Ser 536) and anti p-IKKαβ-specific polyclonal antibodies (A). Ser 276 phosphorylation of p65 and MSK-1 phosphorylation were detected by Western blotting using anti p-p65- (Ser 276) and anti p-MSK-1-specific polyclonal antibodies (B). Levels were normalized to β-actin in the cytosolic fraction and histone H1 in the nuclear fraction. One representative blot is shown from 3 experiments that yielded similar results. **P<0.01, ***P<0.001 vs. normal aged rat. IKKαβ, IκB kinaseαβ; MSK-1. mitogen- and stress-activated protein kinase-1; ENA, enalapril; LOS, losartan.

Figure 6. Effect of enalapril and losartan on NF-κB-dependent pro-inflammatory genes and adhesion molecules expression in the aged rats

Cytosolic proteins were prepared to determine the expression levels of NF-κB-dependent pro-inflammatory gene and adhesion molecules. Western blotting was performed to detect renal COX-2, iNOS, and 5-LOX levels in cytoplasmic extracts from aged rats (A). NF-κB-dependent Adhesion molecules levels were detected by Western blot using anti VCAM-1 and anti ICAM-1-specific polyclonal antibodies. Levels were normalized to β-actin. One representative blot is shown from 3 experiments that yielded similar results. **P<0.01, ***P<0.001 vs. normal aged rat by ANOVA. COX-2; cyclooxygenase 2, 5-LOX; 5-lipoxygenase, iNOS; inducible NO synthase; VCAM-1; vascular cell adhesion molecule 1, ICAM-1; Inter-Cellular Adhesion Molecule 1, ENA, enalapril; LOS, losartan