Inhibition of HDAC enhances STAT acetylation, blocks NF-κB, and suppresses the renal inflammation and fibrosis in Npr1 haplotype male mice

Abstract

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) plays a critical role in the regulation of blood pressure and fluid volume homeostasis. Mice lacking functional Npr1 (coding for GC-A/NPRA) exhibit hypertension and congestive heart failure. However, the underlying mechanisms remain largely less clear. The objective of the present study was to determine the physiological efficacy and impact of all-trans-retinoic acid (ATRA) and sodium butyrate (NaBu) in ameliorating the renal fibrosis, inflammation, and hypertension in Npr1 gene-disrupted haplotype (1-copy; +/-) mice (50% expression levels of NPRA). Both ATRA and NaBu, either alone or in combination, decreased the elevated levels of renal proinflammatory and profibrotic cytokines and lowered blood pressure in Npr1^+/- mice compared with untreated controls. The treatment with ATRA-NaBu facilitated the dissociation of histone deacetylase (HDAC) 1 and 2 from signal transducer and activator of transcription 1 (STAT1) and enhanced its acetylation in the kidneys of Npr1^+/- mice. The acetylated STAT1 formed a complex with nuclear factor-κB (NF-κB) p65, thereby inhibiting its DNA-binding activity and downstream proinflammatory and profibrotic signaling cascades. The present results demonstrate that the treatment of the haplotype Npr1^+/- mice with ATRA-NaBu significantly lowered blood pressure and reduced the renal inflammation and fibrosis involving the interactive roles of HDAC, NF-κB (p65), and STAT1. The current findings will help in developing the molecular therapeutic targets and new treatment strategies for hypertension and renal dysfunction in humans.

Keywords: Npr1 gene disruption; natriuretic peptide receptor A; proinflammatory cytokines; renal fibrosis; retinoic acid; sodium butyrate.

Fig. 1.

Quantitative analyses of plasma and renal proinflammatory cytokines in all-trans-retinoic acid (ATRA)-, sodium butyrate (NaBu)-, and vehicle-treated Npr1 mice. A, C, E, and G: plasma levels of IL-6, TNF-α, IL-10, and macrophage chemoattractant protein-1 (MCP-1), respectively. B, D, F, and H: renal tissue concentrations of IL-6, TNF-α, IL-10, and MCP-1, respectively. All analytes were determined with multiplex kits from Millipore according to the manufacturer’s guidelines. Values are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 (vehicle- vs. drug-treated groups); #P < 0.05, ##P < 0.01, ###P < 0.001 (vehicle-treated Npr1^+/− or Npr1^++/+ vs. Npr1^+/+); n = 10 mice per group.

Fig. 2.

Effect of ATRA and NaBu on protein levels of proinflammatory and anti-inflammatory cytokines and chemokine in Npr1^+/−, Npr1^+/+, and Npr1^++/+ mice kidneys. A: Western blot (WB) analysis of IL-6, TNF-α, IL-10, and MCP-1 in Npr1^+/−, Npr1^+/+, and Npr1^++/+ mice treated with vehicle, ATRA, NaBu, and ATRA-NaBu. β-Actin was used as loading control. Densitometry analyses of IL-6 (B), TNF-α (C), IL-10 (D), and MCP-1 (E) protein levels in kidney tissues. Values are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 (vehicle- vs. drug-treated group); #P < 0.05, ##P < 0.01, ###P < 0.001 (vehicle-treated Npr1^+/− or Npr1^++/+ vs. Npr1^+/+); n = 9 mice per group.

Fig. 3.

Effect of ATRA and NaBu on NF-κB (p65) binding activity and p65 and signal transducer and activator of transcription 1 (STAT1) protein expression in Npr1^+/−, Npr1^+/+, and Npr1^++/+ mice kidneys. A: NF-κB (p65) binding activity in nuclear extracts of ATRA- and NaBu-treated Npr1 gene-targeted mice kidneys. B: representative Western blots of renal protein expression of NF-κB (p65) and STAT1 in drug- and vehicle-treated mice. C and D: Western blot analysis of histone deacetylase (HDAC) 1/2 and NF-κB (p65) in anti-STAT1-immunoprecipitate (IP) fractions (C) and input (positive control) (D) from ATRA-NaBu-treated and control mice kidneys. *P < 0.05, **P < 0.01 (vehicle- vs. drug-treated groups); #P < 0.0, ##P < 0.01 (Npr1^+/− or Npr1^++/+ vs. Npr1^+/+); n = 10 mice per group.

Fig. 4.

Modulation of HDAC and histone acetyltransferase (HAT) activity and ATRA- and NaBu-dependent acetylation of STAT1 and its interaction with NF-κB (p65) and HDAC1/2 and Npr1 gene expression in Npr1^+/−, Npr1^+/+, and Npr1^++/+ mice kidneys. A: quantification of HDAC activity in nuclear extracts of ATRA-, NaBu-, and ATRA-NaBu treated mice kidneys. B: HAT activity in nuclear extracts of drug- and vehicle-treated mice kidneys. C and D: Western blot analysis of acetylated and total STAT1 and NF-κB (p65) in STAT1 (C)- and D: Ac Lys-(D) immunoprecipitate fractions from ATRA-NaBu-treated mice kidneys. E: luciferase activity of Npr1 proximal promoter construct −356/+55 in MMCs treated with increasing concentrations of ATRA, NaBu, and ATRA-NaBu. F: renal Npr1 mRNA expression in drug-treated and control mice as determined by quantitative real time-RT-PCR, normalized to β-actin mRNA. Bar represents means ± SE of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.01 (vehicle- vs. drug-treated groups); #P < 0.05, ###P < 0.001 (Npr1^+/− or Npr1^++/+ vs. Npr1^+/+); n = 10 mice per group.

Fig. 5.

Comparative analysis of renal histology for fibrosis, mesangial matrix expansion (MME), and protein expression in drug- and vehicle-treated Npr1 gene-targeted mice. A: accumulation of collagen (renal fibrosis) in the kidney sections stained with Masson’s trichrome. B: quantitative analysis of renal fibrosis. C: kidney sections stained with hematoxylin and eosin. D: quantitative analysis of MME in Npr1 gene-targeted drug- and vehicle-treated mice. Bars = 20 µm. Values are expressed as means ± SE. *P < 0.05; ***P < 0.001 (vehicle- vs. drug-treated groups); #P < 0.05, ##P < 0.01 (vehicle-treated Npr1^+/− or Npr1^++/+ vs. Npr1^+/+); n = 10 mice per group.

Fig. 6.

Effect of ATRA and NaBu on protein levels of renal fibrotic markers in Npr1^+/−, Npr1^+/+, and Npr1^++/+ mice. A: representative Western blots. B–E: densitometry analysis of renal fibrotic markers. B: collagen-1α (Col 1α). C: plasminogen activator inhibitor-1 (PAI-1). D: transforming growth factor-β1 (TGF-β1). E: connective tissue growth factor (CTGF) in drug- and vehicle-treated mice. β-actin was used as loading control. Values are expressed as means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001 (vehicle- vs. drug-treated groups); #P < 0.05, ##P < 0.01(vehicle-treated Npr1^+/− or Npr1^++/+ vs. Npr1^+/+); n = 10 mice per group.

Fig. 7.

Effect of ATRA and NaBu on systolic blood pressure, plasma cGMP levels, creatinine clearance, and urinary albumin levels in Npr1^+/−, Npr1^+/+, and Npr1^++/+ mice. A: systolic blood pressure was measured by computerized tail-cuff method in drug-treated and control mice. B: plasma cGMP levels among Npr1 genotypes treated with ATRA and NaBu. C and D: creatinine (C) clearance and urinary albumin levels (D) in Npr1 gene-targeted drug- and vehicle-treated mice. Values are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 (vehicle- vs. drug-treated group); #P < 0.05, ##P < 0.01, ###P < 0.001 (vehicle-treated Npr1^+/− or Npr1^++/+ vs. Npr1^+/+); n = 8 mice per group.

Fig. 8.

Schematic representation of ATRA-NaBu effect, which attenuates renal fibrosis and remodeling by enhanced NPRA/cGMP. A: disruption of Npr1 gene causes the activation of NF-κB cascade, which triggers gene transcription of proinflammatory cytokines and growth factors that promote renal fibrosis and dysfunction. Under these conditions HDAC activity is enhanced and transcription factor STAT1 is associated with HDAC1/2. B: Npr1^+/− mice treated with ATRA-NaBu exhibit higher plasma cGMP levels and reduced NF-κB (p65) protein expression. ATRA-NaBu induces STAT1 protein expression and its dissociation from HDAC1/2, attenuates HDAC activity and increases HAT activity thus enhancing STAT1 acetylation. Acetylated STAT1 associates with NF-κB (p65) and reduces its DNA binding activity that impairs its downstream signaling and suppresses the expression of inflammatory cytokines and fibrotic marker genes, thereby attenuates renal fibrosis and inflammation and improves renal functions.