Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

Abstract

The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (-/-) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice.

Fig. 1.

Comparative analysis of p65, IκBα phosphorylation, and IκBα degradation in Npr1 gene-disrupted mouse kidneys with or without inhibitor treatment. A: renal expression levels of p-p65 (Ser276), p-p65 (Ser536), p65, p-IκBα (Ser32/36), and IκBα proteins as determined by Western blot analysis. β-Actin was used as a loading control. Densitometric analyses of the respective bands were done by Alpha Innotech phosphoimager and are shown in B–F for each protein. 2-Copy and 0-copy mice were treated with different inhibitors, andrographolide (Andro), pyrrolidine dithiocarbamate (PDTC), and etanercept (Eta), at different doses. Veh, saline-treated group. Bar represents mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001; ♣P < 0.01; n = 7 mice per group.

Fig. 2.

Comparative analysis of IKK activity by in vitro phosphorylation assay and measurements of NF-κB DNA binding activity by EMSA in Npr1 gene-disrupted mice. A: autoradiogram of phosphorylation ability of IKK enzyme complex to phosphorylate IκBα in kidneys of 2-copy and 0-copy mice with or without inhibitor treatment. B: the respective densitometric analyses. C: autoradiogram of the binding activity of NF-κB DNA binding by EMSA. Veh, saline-treated group. Bar represents mean ± SE. **P < 0.01; ♣P < 0.01; n = 6 mice per group.

Fig. 3.

Quantitative analysis of plasma and renal proinflammatory cytokines and growth factor in Npr1 gene-disrupted mice. A, C, E, and H: plasma levels of TNF-α, IL-6, IL-1α, and active TGF-β1, respectively. B, D, F, G, and I: concentrations of TNF-α, IL-6, IL-1α, IL-1β, and active TGF-β1, respectively, in kidney tissues. All analytes were determined with multiplex kits from Millipore according to the manufacturer's guidelines. Veh, saline-treated group. Values are expressed as means ± SE. **P < 0.01; ♣P < 0.01; n = 8 mice per group.

Fig. 4.

Renal expression of proinflammatory cytokines, anti-inflammatory cytokine, and growth factor in Npr1 gene-disrupted mice. A: renal protein expression of proinflammatory cytokines, TNF-α, IL-6, IL-1α, and IL-1β and anti-inflammatory cytokine, IL-10, with growth factor, TGF-β1 by Western blot. B–G: the densitometric analyses for TNF-α, IL-6, TGF-β1, IL-1α, IL-1β, and IL-10, respectively. Veh, saline-treated group. **P < 0.01; ♣P < 0.01; n = 6 mice per group.

Fig. 5.

Comparative analysis of p-p65 immunoexpression and renal histology for mesangial matrix expansion (MME) and renal fibrosis in Npr1 gene-disrupted mice. A: immunofluorescence of p-p65 protein in the kidney sections is shown by arrows. Top: images of p-p65 immunoexpression in glomerular mesangial cells and tubular epithelial cells. Middle: images with DAPI for nucleus. Bottom: respective merged images in 2-copy and 0-copy mice kidney sections. The p-p65 immunofluorescence was reduced in glomeruli, and in tubular epithelial cells (shown by arrowhead) in 0-copy mice kidney after treatment with Andro, PDTC, and Eta compared with saline-treated Npr1 0-copy mice. B: renal kidney sections stained with hematoxylin and eosin (H&E), indicating the MME. Increased MME were observed in 0-copy kidney compared with 2-copy control. C: accumulation of collagen (renal fibrosis) in the kidney sections of 0-copy mice, after staining with Masson's trichrome. D–F: their quantitative analysis, respectively. Veh, saline-treated group; ***P < 0.001; ♣P < 0.01; n = 5 mice per group.

Fig. 6.

Effect of inhibitors on NF-κB targeted and nontargeted genes in the kidneys of Npr1 gene-disrupted mice. A: relative mRNA expression of NF-κB targeted genes (TNF-α, IL-6, IL-1α and IL-1β) normalized to GAPDH mRNA in the kidney tissues with or without inhibitor treatment. B: mRNA expression of NF-κB nontargeted genes (TGF-β1 and Col1α) relative to GAPDH mRNA in the kidney tissues. Veh, saline-treated group. Bar represents mean ± SE. **P < 0.01; ***P < 0.001; ♣P < 0.01; n = 7 mice per group.

Fig. 7.

Effect of inhibitors on the IKK and NF-κB family member genes by qRT-PCR in the kidneys of Npr1 gene-disrupted mice. A: relative mRNA expression of IKK enzyme subtypes (IKK-α, β, γ, ε) normalized to GAPDH mRNA. B and C: mRNA expression of NF-κB family members, IκBα, p65 (RelA), p50 (NFKB1), p52 (NFKB2), relB, and cRel normalized with GAPDH mRNA in the kidney tissues. Veh, saline-treated group. Values are expressed as mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001; ♣P < 0.01; n = 7 mice per group.

Fig. 8.

Diagrammatic representation of the impact of NPRA deficiency in renal remodeling and impairment. Disruption of Npr1 gene leads to impaired renal functions, which triggers specific structural and molecular changes in 0-copy mice kidney. Activated IKK enzymes induce the phosphorylation of IκBα, which then undergoes enzymatic degradation. The reduction of cytosolic IκBα favors the constitutive activation of NF-κB, which would translocate into the nucleus to activate the transcription of proinflammatory cytokines, growth factors, and extracellular matrix protein genes. In the contrary, the increased amount of cytosolic IκBα favors the formation of trimeric complex between p65, p50 and IκBα and thereby withdraws the free p65 molecule to inhibit the transcription process. ANP, atrial natriuretic peptide; KHD, kinase homology domain; GCD, guanylyl cyclase domain; IKK, IκB kinase; IκBα, inhibitory κB α.