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.

guanylyl cyclase/natriuretic peptide receptor-a (GC-A/NPRA) is primarily responsible for the regulatory actions of atrial and brain natriuretic peptides (ANP and BNP) (13, 18, 27, 49). Earlier studies using functional expression and targeted disruption of Npr1 coding for GC-A/NPRA in mice demonstrated its hallmark significance in protecting against renal and cardiac hypertrophic and fibrotic growth, inflammation, extracellular matrix deposition, and cell proliferative responses (12, 35, 44, 46, 64, 65). The endogenous ANP/NPRA system inhibits renal fibrosis; exogenous ANP treatment reduces the expression of proinflammatory cytokines and prevents acute kidney injury (43, 44). Nonetheless, the roles of hormonal and epigenetic modulators in attenuation of renal remodeling, inflammation, and fibrosis, as shown under reduced NPRA signaling, have not been defined. Npr1 is a highly regulated gene, and its expression in both in vitro and in vivo conditions is modulated by various factors, including hormones, growth factors, extracellular osmolality, and certain physiological and pathophysiological conditions (5, 19).

Our previous studies have indicated that all-trans-retinoic acid (ATRA) enhances Npr1 gene transcription and expression in cultured mouse mesangial cells (30). The natural retinoids are derivatives of vitamin A and exist in different forms, including ATRA, 9-cis retinoic acid, and 4-oxo-retinoic acid. They have multiple functions, including inhibition of cell proliferation, induction of cell differentiation, regulation of apoptosis, and attenuation of inflammation (6, 38, 59). Retinoic acid acts as a ligand to activate retinoid nuclear receptors, which include retinoic acid receptors and/or retinoid X receptors, each having three isotypes, α, β, and γ (3). These retinoic acid receptors are expressed in the kidney and play roles in renal development, particularly formation of the ureteral bud, and codetermine the final number of glomeruli per kidney (10, 20). Numerous studies have shown that ATRA and its agonists have preventive and therapeutic effects in various experimental kidney diseases by interfering with factors that contribute to renal damage (6, 25, 40, 54). The physiological effects of ATRA and its receptors are mediated by modulating gene expression, which is a dynamic and orchestrated process involving association of receptors with a multitude of coregulators and epigenetic modulators (3, 9, 61).

Previous evidence has indicated that histone deacetylase (HDAC) inhibitors (HDACi), including sodium butyrate (NaBu), trichostatin A, and valproic acid, have produced beneficial effects in preclinical models of renal and cardiovascular diseases (4, 26). Both in vitro and in vivo studies have also indicated synergism between ATRA and HDACi when they are used to treat renal cell carcinomas (8, 55). It has been suggested that hyaluronan mixed ester of butyric and retinoic acid enhances cardiac repair in infarcted rat and pig hearts (58, 66). A considerable number of studies have shown that HDACs play critical roles in the development and progression of chronic diseases, including kidney disease, cardiac hypertrophy, cancer, and inflammation (13, 26, 36, 67). In the present study, we examined the effect of ATRA and NaBu on renal fibrosis, proinflammatory cytokines, and blood pressure in haplotype mice carrying only one copy of the Npr1 gene.

MATERIALS AND METHODS

Animals and treatments.

Npr1 gene-disrupted and gene-duplicated mice were produced by homologous recombination using embryonic stem cells as previously described (46, 47, 51). All mice were littermate progeny of the C57/BL6 genetic background. The mice were bred and maintained at the Tulane University Health Sciences Center animal facility and handled under protocols approved by the Institutional Animal Care and Use Committee. Animals were maintained in a 12:12-h light–dark cycle (0600 to 1800 h) at 25°C and fed regular chow (Purina Laboratory) and tap water ad libitum. Male mice genotypes were used in all groups: Npr1 gene-disrupted haplotype (Npr1^+/−, 1-copy), wild-type (Npr1^+/+, 2-copy), and gene-duplicated heterozygous (Npr1^++/+, 3-copy) mice. During the 2-wk study period, 18- to 22-wk old male Npr1^+/− (n = 40), Npr1^+/+ (n = 40), and Npr1^++/+ (n = 40) mice were divided into following four treatment groups: Group I (n = 10), vehicle treated (control); Group II (n = 10), all-trans-retinoic acid (ATRA)-treated (0.5 mg·kg⁻¹·day⁻¹); Group III (n = 10), sodium butyrate (NaBu)-treated (0.5 mg·kg⁻¹·day⁻¹); Group IV (n = 10), ATRA-NaBu-treated (1 mg·kg⁻¹·day⁻¹). ATRA and NaBu were obtained from Sigma-Aldrich (St. Louis, MO), and ATRA-NaBu was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). ATRA and ATRA-NaBu stock solutions were prepared at 20 and 10 mg/ml concentrations in DMSO, respectively, diluted with olive oil to appropriate concentrations, vortexed for 2 min at room temperature, and administered intraperitoneally. NaBu was prepared at 20 mg/ml in PBS (pH 7.4) and injected intraperitoneally. Control groups were injected with vehicle (DMSO and olive oil). All protocols were approved by the Institutional Animal Care and Use Committee at Tulane University Health Sciences Center.

Blood pressure measurements.

The systolic blood pressure of Npr1 gene-targeted mice was measured by a noninvasive computerized tail-cuff method using Visitech BP2000 (Visitech Systems, Apex, NC) as previously described (56). Initially all mice were trained for blood pressure measurement (acclimatization) for 7 days, and the actual blood pressure was calculated as the average of three to five sessions from 12th day till 15th day of the treatment.

Blood and tissue collection.

Blood was collected by cardiac puncture under CO₂ anesthesia in prechilled tubes containing 10 µl of heparin (1,000 USP units/ml). Plasma was separated by centrifugation at 3,000 rpm for 10 min at 4°C and stored at −80°C until use. Animals were euthanized by administration of a high concentration of CO₂. Kidney tissues were dissected, frozen in liquid nitrogen, and stored at −80°C. One slice from each kidney tissue sample was kept into 10% buffered formalin overnight and processed for histological studies.

Cell transfection and luciferase assay.

Mouse mesangial cells (MMCs) were isolated and cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and insulin-transferrin-sodium selenite as previously described (50). The cells were transfected with Npr1 promoter construct using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). Luciferase activity was measured using the dual luciferase assay system (Promega, Wisconsin, MD) as previously reported (30). Twenty-four hours after transfection, cells were kept in DMEM containing 0.1% BSA for 12 h and treated with varying concentrations of ATRA, NaBu, and ATRA-NaBu for another 24 h in DMEM containing 0.1% BSA. All cultures were maintained at 37°C under an atmosphere of 5% CO₂-95% O₂.

Histological studies.

Kidney tissues from each group were fixed in 10% buffered paraformaldehyde solution. Paraffin-embedded tissue sections (5 µm) were stained with hematoxylin and eosin to study the changes in renal morphology. Kidney sections were stained with Masson’s trichrome to observe the presence of interstitial collagen fiber accumulation (blue color) as a marker of renal fibrosis. The percentage of the mesangial matrix expansion (MME) and fibrosis relative to the total kidney area was determined by calculations made in 20 randomly selected microscopic fields in five sections per kidney for renal injury using ImagePro Plus image analysis software (Media Cybernetics, Silver Spring, MD).

Plasma cGMP assay.

Frozen plasma samples were used to analyze cGMP levels using a direct cGMP immunoassay kit (Enzo Lifesciences, Farmingdale, NY) according to the manufacturer’s protocol. The results are expressed as picomoles of cGMP per milliliter for plasma.

Measurement of albumin, creatinine, and total protein in urine and plasma.

Urinary albumin levels were measured in a 24-h urine sample collected from mice kept in metabolic cages, using enzyme-linked immunosorbent assay (ELISA) kit from Bethyl Laboratories (Montgomery, TX). Plasma albumin levels were measured utilizing quantichrom BCG albumin assay kit, and total protein was assayed using quantichrom total protein assay kit from Bioassay Systems (Hayward, CA) following manufacturer’s instructions. Plasma and urine creatinine concentrations were determined using creatinine assay kit (Bioassay Systems) following manufacturer’s protocol. Creatinine clearance (CCr) was calculated from the urinary creatinine concentration, plasma creatinine concentration, and urine volume (V) and expressed as microliters per minute.

Cytosolic and nuclear extract preparation and immunoblot assay.

Cytosolic and nuclear proteins were prepared from frozen kidney tissues as described previously (11). The protein concentration of the extracts was measured with Bradford protein detection kit (Bio-Rad, Hercules, CA). Cytoplasmic fraction (40–60 µg) was mixed with sample loading buffer and separated by using 10% SDS-PAGE essentially as described earlier (30). Proteins were electrotransferred onto a PVDF membrane, and the membrane was blocked with 1× Tris-buffered saline-Tween 20 (TBST; 25 mM Tris, 500 mM NaCl, and 0.05% Tween 20, pH 7.5) containing 5% fat-free milk for 1 h and incubated overnight in TBST containing 3% fat-free milk at 4°C with primary antibodies (1:250 dilution) and the corresponding secondary anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated antibodies (1:5,000 dilutions). The primary antibodies directed against collagen-1α (Col 1α) (sc-8788), plasminogen activator inhibitor-1 (PAI-1) (sc-8979), transforming growth factor-β1 (TGF-β1) (sc-146), connective tissue growth factor (CTGF) (sc-25440), tumor necrosis factor-α (TNF-α) (sc-1350), interleukin (IL)-6 (sc-1265), IL-10 (sc-73309), macrophage chemoattractant protein-1 (MCP-1) (sc-288879), NF-κB (p65) (sc-372), STAT-1 p84/p91 (sc-346), HDAC1 (sc-8410), HDAC2 (sc-7899), Ac-lysine (sc-32268), and H3 (sc-10809) were purchased from Santa Cruz Biotechnology. The antibody against β-actin (A5316) was obtained from Sigma-Aldrich. Protein bands were visualized with a SuperSignal West Femto Chemiluminescent kit (Thermo Scientific, Rockford, IL) using an Alpha Innotech detection system (ProteinSimple, San Jose, CA), and densitometry analysis was done by AlphaView software (ProteinSimple).

Real-Time RT-PCR assay.

Total RNA was extracted from vehicle- and drug-treated mice kidneys using RNeasy plus Mini Kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized from 1 µg of total RNA using an RT² First Strand Kit (Qiagen). PCR amplification was done in triplicate in a 20-µl reaction volume using RT² Real-Time SYBR Green/ROX PCR Master Mix (Qiagen) using the Mx3000P QPCR System as previously described (30). Primers for amplification of Npr1 and β-actin were purchased from Qiagen. β-Actin was amplified from all samples as a housekeeping gene to normalize expression levels of target genes between different samples. The reaction mixture without template cDNA was used as a negative control. Relative expression of Npr1 was determined by the comparative Ct value as described earlier (30).

Plasma and renal levels of proinflammatory cytokines.

The concentrations of proinflammatory cytokines and chemokine, including TNF-α, IL-6, IL-10, and MCP-1, were measured in both plasma and kidney tissue homogenates with a Milliplex MAP kit, which are multiplex bead-based assays and utilize Luminex xMAP technology according to the manufacturer’s instructions (EMD Millipore, Billerica, MA). Fifty-milligrams of kidney tissues were homogenized in a 20-fold excess (wt/vol) ice-cold low salt solution A {50 mM HEPES, pH 7.0, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, protease inhibitor cocktail [Sigma P8340, containing 240 μg/ml 4-(2-aminomethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 5 μg/ml aprotinin, 10 μg/ml leupeptin, 14 μg/ml bestatin, 10 μg/ml pepstatin A, and 5 μg/ml E-64], phosphatase inhibitors (1 mM orthovanadate and 30 mM sodium fluoride), 1 mM DTT, and 0.5 mM PMSF}. The homogenate was centrifuged for 1 min at 100 g and 4°C to remove tissue debris, the supernatant was transferred to a fresh tube, 10% NP-40 was added for a final concentration of 0.05%, and tubes were mixed by gentle inversion and immediately centrifuged for 10 min at 1,000 g and 4°C. The supernatant was used as cytosolic extract for analyzing cytokines level. Spectrally addressed polystyrene beads coated with cytokine-specific monoclonal antibodies were used to capture the cytokine of interest using a Bio-Plex Instrument (Bio-Rad) according to the manufacturer’s guidelines. The instrument sorted out and measured the fluorescent signal from each bead by dual excitation sources.

NF-κB (p65) transcription factor binding assay.

NF-κB (p65) DNA binding activity in kidney nuclear extracts was determined with a transcription factor binding assay colorimetric ELISA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s protocol. Briefly, NF-κB (p65) present in the nuclear extract binds specifically to the NF-κB response element (a double-stranded DNA sequence containing the NF-κB response elements immobilized to the wells of a 96-well plate) and the DNA:protein complex was detected by addition of specific primary antibody directed against NF-κB (p65). A secondary antibody conjugated to HRP was added, and the absorbance was read at 450 nm.

Total HDAC and histone acetyltransferase activity assay.

Total HDAC and histone acetyltransferase (HAT) enzyme activities were measured in kidney nuclear extracts of drug- and vehicle-treated mice using a fluorescent-based kit from Cayman Chemical (Ann Arbor, MI) and colorimetric ELISA assay kit from Epigentek (Farmingdale, NY), respectively. The HDAC activity was calculated by measuring the amount of deacetylated fluorescent product, which was directly proportional to the HDAC enzyme activity and is expressed as nanomoles per minute per milliliter. The HAT enzyme activity assay measures the amount of acetylated product. Absorbance was read at 450 nm, and results were calculated using a standard curve following manufacturer’s instructions and expressed as ng/h/mg protein.

Immunoprecipitation of acetylated STAT.

Kidney tissue homogenates (100 µg of protein) from vehicle- and ATRA-NaBu-treated Npr1^+/−, Npr1^+/+, and Npr1^++/+ mice were incubated with 2 µg of polyclonal STAT1 or Ac-lys antibody for 2 h at 4°C, and protein A/G agarose beads were added and kept for overnight incubation at 4°C. After the beads were washed, proteins were eluted by being boiled in 2× SDS loading buffer and electrophoresed. For detection of acetylated STAT1, membranes were incubated with anti-Ac-lys (AKL5C1) antibody and treated with anti-mouse HRP-conjugated secondary antibody. Immunoprecipitation with rabbit IgG was taken as negative control. The antibodies used in immunoprecipitation assay were same as used for immunoblot assay.

Statistical analysis.

The results are expressed as means ± SE. Statistical analyses were conducted using GraphPad PRISM 5.0 software (GraphPad Software, San Diego, CA). The statistical significance of the differences in multiple-group comparisons was evaluated by one-way ANOVA, followed by a Dunnett’s test to compare responses to ATRA, NaBu, or ATRA-NaBu treatments with vehicle-treated controls. Comparison between any two groups (vehicle-treated, Npr1^+/−, and Npr1^+/+ or vehicle-treated, Npr1^+/+, and Npr1^++/+ mice) was analyzed by two-tailed unpaired Student’s t-test. P = 0.05 was considered statistically significant.

RESULTS

ATRA and NaBu treatments reduced the plasma and renal proinflammatory cytokines in Npr1^+/− mice.

We determined the effect of ATRA and NaBu treatments on proinflammatory cytokine levels in gene-disrupted haplotype Npr1^+/−, wild-type Npr1^+/+, and gene-duplicated heterozygous Npr1^++/+ mice. Plasma and renal levels of IL-6 were increased by 2.0- and 2.5-fold, respectively, in Npr1^+/− mice compared with Npr1^+/+ control mice; however, the levels were significantly lower by almost 45% in Npr1^++/+ mice. Treatments with ATRA and NaBu significantly reduced the plasma and kidney levels of IL-6 by 69 and 60%, respectively, in Npr1^+/−mice compared with untreated groups (Fig. 1, A and B). Both the plasma and renal levels of TNF-α were also increased by two- and threefold, respectively, in Npr1^+/− mice compared with Npr1^+/+ and Npr1^++/+ mice; however, treatment with ATRA-NaBu drastically reduced these levels by 65–72% in treated animals compared with vehicle-treated control groups (Fig. 1, C and D).

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.

Conversely, the IL-10 level was decreased in the plasma and kidneys (40 and 55%, respectively) of Npr1^+/− mice as compared with Npr1^+/+ mice. This level increased significantly in ATRA-NaBu-treated groups compared with untreated groups (Fig. 1, E and F). On the other hand, MCP-1 was augmented in plasma and kidney tissues of Npr1^+/− mice but substantially reduced in ATRA-NaBu-treated mice compared with vehicle-treated controls (Fig. 1, G and H). As detected by Western blot analysis, there were also significantly decreased levels of renal protein expression of IL-6, TNFα, and MCP-1 and an increased level of IL-10 in ATRA-NaBu-treated Npr1^+/− mice compared with vehicle-treated controls (Fig. 2, A–E).

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.

ATRA and NaBu treatments enhanced STAT1 and NF-κB (p65) interactions and reduced NF-κB (p65) activity in Npr1^+/− mice.

To examine whether the increase in levels of proinflammatory cytokines in Npr1^+/− mice was due to enhanced NF-κB signaling, we analyzed the DNA-binding activity of NF-κB (p65) in renal nuclear extract. Renal NF-κB (p65) DNA-binding activity was enhanced by 49% in Npr1^+/− mice as compared with Npr1^+/+ mice (Fig. 3A). However, treatment with ATRA-NaBu significantly attenuated DNA-binding activity of NF-κB (p65) in Npr1^+/− mice as compared with vehicle-treated controls. Western blot analysis revealed distinctly higher levels of renal NF-κB (p65) protein expression in Npr1^+/− mice; reduced levels were observed in Npr1^++/+ mice as compared with Npr1^+/+ controls (Fig. 3B). Interestingly, ATRA-NaBu treatment led to significant increases in STAT1 protein levels. Coimmunoprecipitation assays indicated interaction of STAT1 with NF-κB (p65) and HDAC1/2 proteins in vehicle- and ATRA-NaBu-treated mice (Fig. 3, C and D). Western blot analysis of the renal anti-STAT1-immunoprecipitate fractions from vehicle-treated Npr1^+/− mice showed weak interaction of STAT1 with NF-κB (p65) and increased association with HDAC1/2 as compared with vehicle-treated Npr1^+/+ and Npr1^++/+ mice. Treatment with ATRA-NaBu induced significant interactions between STAT1 and NF-κB (p65), as well as dissociation of HDAC1/2 from STAT1, in treated groups as compared with vehicle-treated controls.

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.

ATRA and NaBu treatments decreased HDAC activity and increased HAT activity with acetylation of STAT1 in Npr1^+/− mice.

To determine the involvement of HDAC activity, we further investigated the effect of ATRA-NaBu on renal HDAC and HAT activity and its effect on STAT1 acetylation levels in Npr1^+/− mice. Figure 4A shows that renal HDAC activity was 2.7-fold higher in Npr1^+/− mice and 49% lower in Npr1^++/+ mice as compared with Npr1^+/+ mice. However, a significant reduction in HDAC activity was observed in ATRA (23.8%, P < 0.05)-, NaBu (39.3%, P < 0.01)-, and ATRA-NaBu (60.4%, P < 0.001)-treated Npr1^+/− mice compared with vehicle-treated controls. In contrast, Npr1^+/− mice exhibited 48% attenuated renal HAT activity as compared with Npr1^+/+ mice (Fig. 4B). Treatment with ATRA-NaBu markedly increased HAT activity in Npr1^+/− mice; however, coimmunoprecipitation analysis showed enhanced acetylated STAT1 protein in STAT1-immunoprecipitated fractions as detected by acetyl-lysine antibody in ATRA-NaBu-treated mice (Fig. 4C). Conversely, the presence of STAT1 protein in acetyl-lysine-immunoprecipitate fractions confirmed an increase in STAT1 acetylation with ATRA-NaBu treatment (Fig. 4D). However, no acetylation of NF-κB (p65) was detected in ATRA-NaBu-treated mice groups compared with vehicle-treated controls. To determine the effect of increasing concentrations of ATRA, NaBu, and ATRA-NaBu on Npr1 gene transcription, we performed in vitro studies utilizing MMCs transiently transfected with Npr1 promoter and then treated with the drugs. There was a dose-dependent increase in luciferase activity of the Npr1 promoter treated with ATRA (5-fold, P < 0.01; 0.5 µM) and NaBu (2.5-fold, P < 0.05; 0.5 µM) independently, whereas ATRA-NaBu (12.5-fold, P < 0.001; 1.0 µM) together exhibited synergistic effect compared with control cells (Fig. 4E). The efficacy of ATRA and NaBu on Npr1 transcription was further confirmed by analyzing Npr1 mRNA expression in the kidneys of drug-treated mice. Treatment with ATRA and NaBu significantly induced renal Npr1 mRNA expression in Npr1^+/− (ATRA, 2.2-fold, P < 0.05; NaBu, 1.9-fold, P < 0.5; ATRA-NaBu, 4.4-fold, P < 0.001), Npr1^+/+ (ATRA, 2.1-fold, P < 0.05; NaBu, 1.9-fold, P < 0.5; ATRA-NaBu, 3.9-fold, P < 0.01), and Npr1^++/+ (ATRA, 1.7-fold, P < 0.05; NaBu, 1.4-fold, P < 0.5; ATRA-NaBu, 3.4-fold, P < 0.01) mice compared with their vehicle-treated controls (Fig. 4F).

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.

ATRA- and NaBu-mediated renal morphological changes and attenuation of renal fibrosis in Npr1^+/− mice.

To investigate the beneficial effect of ATRA and NaBu treatments on renal morphology and fibrosis, we used Masson’s trichrome and hematoxylin and eosin staining of kidney sections from treated and control mice. Masson’s trichrome staining of kidney sections showed increased collagen deposition in the interstitial spaces and glomerulus in haplotype Npr1^+/− mice as compared with Npr1^+/+ and Npr1^++/+ mice (Fig. 5A). The percentage of blue-stained fibrotic area, which indicates tubulointerstitial and glomerular fibrosis, was significantly reduced after treatment with ATRA (25%, P < 0.001), NaBu (15%, P < 0.001), and ATRA-NaBu (72%, P < 0.001) (Fig. 5B). Kidney sections stained with hematoxylin and eosin showed progressive expansion of interstitial spaces, referred to as mesangial MME, in Npr1^+/− mice as compared with Npr1^+/+ and Npr1^++/+ mice (Fig. 5C). Haplotype Npr1^+/− mice also exhibited an increase in tubular damage, characterized by tubular dilation with flattened epithelium, as compared with Npr1^+/+ and Npr1^++/+ mice. As shown in Fig. 5D, the percentage of MME scores were reduced in Npr1^+/− mice treated with ATRA (25%, P < 0.001) and NaBu (15%, P < 0.001), whereas ATRA-NaBu synergistically attenuated the MME score (60%, P < 0.001) as compared with Npr1^+/− control mice. In Npr1^+/− mice as compared with Npr1^+/+ and Npr1^++/+ mice, there was considerably high expression of renal fibrotic markers (Fig. 6, A–E). In addition, Col 1α, PAI-1, TGF-β1, and CTGF proteins were observed. Treatments with ATRA-NaBu significantly reduced expression levels of fibrotic markers in Npr1^+/− mice as compared with untreated Npr1^+/− control mice.

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.

ATRA- and NaBu-mediated reduction in blood pressure and renal functional changes in Npr1^+/− mice.

We further determined the effect of ATRA and NaBu treatments on blood pressure and renal functions. Treatments with ATRA, NaBu, and ATRA-NaBu all significantly reduced the systolic blood pressure in Npr1^+/− mice (ATRA, 118.7 ± 1.3, P < 0.05; NaBu, 121.9 ± 1.5, P < 0.05; and ATRA-NaBu, 113.3 ± 1.6, P < 0.01) as opposed to systolic blood pressure in vehicle-treated controls (130.4 ± 1.9) (Fig. 7A). Substantial decreases in plasma cGMP levels were also observed in Npr1^+/− mice (69%, P < 0.01) compared with Npr1^+/+ mice. However, after treatment with ATRA-NaBu, an additive effect on plasma cGMP levels was observed in Npr1^+/− (3.7-fold; P < 0.001), Npr1^+/+ (2.4-fold; P < 0.001), and Npr1^++/+ (2.1-fold; P < 0.01) mice (Fig. 7B). CCr was significantly reduced in Npr1^+/− mice (62%; 58.2 ± 2.8 µl/min) but was 1.8-fold higher in Npr1^++/+ mice (344.7 ± 30.0 µl/min). However, treatment with ATRA-NaBu significantly increased CCr in Npr1^+/− mice (170.6 ± 10.3, P < 0.001) compared with Npr1^+/+ mice (197.3 ± 19.9 µl/min) (Fig. 7C).

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.

Proteinuria occurred in Npr1^+/− mice, as shown by their considerably (P < 0.01) higher urinary albumin content than that in Npr1^+/+ mice (Fig. 7D). A complete reversal of proteinuria was observed in ATRA-NaBu-treated Npr1^+/− mice compared with Npr1^+/− control groups. A marked increase in kidney weight-to-body weight ratio was observed in Npr1^+/− mice (9.5%; P < 0.05) compared with Npr1^+/+ mice. However, kidney weight-to-body weight ratio was reduced significantly (14%; P < 0.01) in ATRA-NaBu-treated Npr1^+/− mice compared with vehicle-treated controls (Table 1). Urinary total protein in Npr1^+/− mice was significantly increased (P < 0.01), but it was reversed in ATRA-NaBu-treated Npr1^+/− mice compared with untreated controls. On the other hand, Npr1^+/− mice exhibited significantly lower plasma levels of total protein, albumin, and creatinine than did Npr1^+/+ mice; however, treatment with ATRA-NaBu resulted in increased levels comparable with those in Npr1^+/+ mice (Table 2).

Table 1.

Analyses of kidney weight/body weight ratio and total urinary mg protein/mg creatinine in Npr1 mice genotypes treated with ATRA and NaBu

	Npr1 Mice Genotypes
Parameters/Treatments	Npr1+/−	Npr1+/+	Npr1++/+
Kidney wt/body wt, mg/g
Vehicle	7.68 ± 0.2#	7.01 ± 0.04	6.92 ± 0.1
ATRA	7.11 ± 0.1	6.79 ± 0.03	6.85 ± 0.4
NaBu	7.18 ± 0.6	6.83 ± 0.03	6.89 ± 0.2
ATRA-NaBu	6.60 ± 0.2**	6.42 ± 0.01	6.68 ± 0.3
Total urinary, mg protein/mg creatinine
Vehicle	6.06 ± 0.4##	3.26 ± 0.4	2.41 ± 0.3
ATRA	4.10 ± 0.4*	2.86 ± 0.4	2.36 ± 0.3
NaBu	4.55 ± 0.2*	2.9 ± 0.3	2.39 ± 0.2
ATRA-NaBu	2.94 ± 0.1***	2.32 ± 0.3	2.11 ± 0.2

Values represent the means ± SE of 3 independent experiments in triplicate; n = 6 in each group. The urine total protein and creatinine levels were determined as described in materials and methods. ATRA, all-trans-retinoic acid (ATRA); NaBu, sodium butyrate (NaBu).

^*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^+/+).

Table 2.

Analyses of plasma protein, albumin, and creatinine levels among Npr1 mice genotypes treated with ATRA and NaBu

	Npr1 Mice Genotypes
Parameters/Treatments	Npr1+/−	Npr1+/+	Npr1++/+
Plasma creatinine, mg/dl
Vehicle	0.44 ± 0.05#	0.22 ± 0.04	0.15 ± 0.03
ATRA	0.32 ± 0.03	0.2 ± 0.03	0.14 ± 0.03
NaBu	0.36 ± 0.02	0.17 ± 0.03	0.14 ± 0.02
ATRA-NaBu	0.23 ± 0.03**	0.16 ± 0.01	0.11 ± 0.02
Plasma total protein, g/dl
Vehicle	4.60 ± 0.2##	5.56 ± 0.2	6.09 ± 0.2
ATRA	5.13 ± 0.2	5.55 ± 0.3	5.80 ± 0.3
NaBu	5.35 ± 0.1	5.53 ± 0.3	5.87 ± 0.4
ATRA-NaBu	5.48 ± 0.3*	5.97 ± 0.3	6.18 ± 0.3
Plasma albumin, g/dl
Vehicle	2.40 ± 0.1#	3.04 ± 0.1	3.18 ± 0.1
ATRA	2.69 ± 0.2	3.10 ± 0.4	3.19 ± 0.2
NaBu	2.62 ± 0.2	3.20 ± 0.3	3.22 ± 0.2
ATRA-NaBu	2.98 ± 0.3	3.38 ± 0.3	3.66 ± 0.3

Values represent the means ± SE of 3 independent experiments in triplicate; n = 10 in each group. The plasma creatinine, total protein, and albumin levels were determined as described in materials and methods.

^*P < 0.05;

^**P < 0.01 (vehicle- vs. drug-treated groups).

^#P < 0.05;

^##P < 0.01 (vehicle-treated Npr1^+/−  or Npr1^++/+ vs. Npr1^+/+).

DISCUSSION

The present results demonstrate that ATRA and NaBu, a pharmacological inhibitor of HDACs, significantly reduced the expression of proinflammatory cytokines, as well as blood pressure and kidney fibrosis, and enhanced renal function in Npr1^+/− haplotype mice. We analyzed the role of ATRA-NaBu signaling, which leads to enhanced acetylation of STAT1, thereby inhibiting HDAC and NF-κB activity in the kidneys of Npr1^+/− haplotype mice, thus preventing inflammation and fibrosis in these animals. Previous studies have suggested that ATRA exerts beneficial effects against renal injury in experimental models of kidney diseases (6, 10, 71). Also, treatment with ATRA has been shown to decrease protein levels of fibrotic markers in animal models of nephrectomy and glomerulonephritis in vivo and in cultured glomerular mesangial cells and renal tubular epithelial cells in vitro (37, 38, 48, 76). Retinoids have also been found to preserve renal function and to decrease albuminuria, glomerular and tubular damage and blood pressure in models of acute and chronic mesangioproliferative glomerulonephritis disease and nephrectomized rats (37, 54, 68). Studies have indicated that ATRA treatment lowered systolic blood pressure in nephrectomized rats and in the genetic model of spontaneously hypertensive rats (37, 69, 74). Lowering of blood pressure has been demonstrated to have beneficial effects on attenuation of renal inflammation and injury (53, 72). Recently, it has been shown that inhibition of endoplasmic reticulum stress for 6 wk in hypertensive-diabetic kidneys reduced blood pressure, albumin excretion, and glomerular injury, while increasing glomerular filtration rate in Wistar rats (70). A defect in the endogenous ATRA system has been shown to contribute to the loss of protection against the development of HIV-associated nephropathy in mice (52), and ATRA receptor agonists have exhibited reduced proteinuria and kidney injury (75). Those earlier findings are consistent with our current findings that ATRA and NaBu treatments not only significantly reduced renal injury, proteinuria, and blood pressure but led to improved kidney function in Npr1^+/− haplotype mice.

We found that ATRA and NaBu substantially attenuated protein expression of proinflammatory cytokines (TNF-α, IL-6, and chemokine MCP-1) in the kidneys of Npr1^+/− haplotype mice. Also, treatment with ATRA-NaBu decreased relative quantities of TNF-α, IL-6, and MCP-1 in plasma and renal tissues. Previous studies have indicated that proinflammatory cytokines play pivotal roles in promoting hypertension and renal and vascular injury and dysfunction (18, 27, 57). Retinoic acid modulates immune and inflammatory responses by blocking the expression of proinflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-6, IL-12, IL-2, and MCP-1. However, retinoic acid also enhances the production of IL-10 under various pathophysiological conditions such as multiple sclerosis and the development of mesangial proliferative glomerulonephritis in IL-6 transgenic mice (15, 39, 57). Inhibition of HDAC activity has been shown to reduce production of proinflammatory cytokines, including IL-2, TNF-α, IL-6, IL-1β, and IFN-γ, under in vivo and in vitro conditions, which were triggered by respiratory syncytial virus, mitogenic anti-CD3, lipopolysaccharide, and phorbol myristate acetate (16, 22, 33, 73). Evidence suggests that HDACi also modulates proinflammatory changes in renal tubulointerstitial and glomerular injury and attenuates proteinuria and renal fibrosis (41, 62, 63). Previous studies have suggested that NaBu decreases gentamicin-induced nephrotoxicity by enhancing the activity of renal antioxidant enzymes and the expression of prohibitin protein (60). In this study, ATRA-NaBu suppressed the expression of inflammation-related genes in Npr1^+/− haplotype mice. Moreover, treatment with ATRA-NaBu significantly attenuated NF-κB (p65) DNA-binding activity, thus modulating its downstream signaling.

The ANP/NPRA system has been found to inhibit fibrosis in mice and to attenuate injuries in kidney, lung, and heart in a rat model of renal ischemia-reperfusion by anti-inflammatory effects (42, 44). Studies have indicated that systemic disruption of Npr1 in mice activates NF-κB pathways and enhances the inflammatory responses in the kidneys; however, the inhibition of NF-κB signaling attenuated abnormal renal pathology in these animals (12, 32, 44). Previously, we have reported that in gene-disrupted Npr1^−/− (0-copy) and Npr1^+/− mice, NF-kB (p65) expression was significantly higher as compared with wild-type (Npr1^+/+) mice (11, 12). Those earlier studies have also shown that in Npr1^−/− and Npr1^+/− mice the phosphorylation levels of IkBα and p65 were markedly higher as compared with Npr1^+/+ mice. However, the present study details a novel counterregulatory signaling mechanism of ATRA- and NaBu-mediated attenuation of NF-kB (p65) activity that is regulated via modulation of STAT1 and HDAC interactions in Npr1 gene-targeted haplotype mice. Our results demonstrate that treatment with ATRA and NaBu enhances the STAT1 acetylation and increases the complex formation between acetylated STAT1 and NF-kB (p65) leading to a decreased p65 DNA-binding capability and henceforth significantly diminished the NF-kB (p65)-dependent expression of inflammatory molecules in the kidneys of drug-treated Npr1^+/− mice compared with untreated control groups. Our previous studies showed that ATRA markedly induced Npr1 expression and receptor signaling in mouse mesangial cells in vitro and in kidneys of intact mice in vivo (30, 31). ATRA signaling has been shown to protect cardiomyocytes from high-glucose-induced apoptosis through suppression of the NF-κB signaling pathway and to inhibit lipopolysaccharide-induced NF-κB activity (45). NF-κB activity is significantly increased in vitamin A-deficient mice; however, antioxidant vitamin therapy (A, C, and E) has provided cardioprotection in burn-trauma-mediated cardiac NF-κB activation and interruption of cardiomyocyte cytokine secretion in rats (1, 24). Several studies have found that ATRA induces STAT1 expression and activation. STAT1 has also been shown to repress NF-κB-mediated signaling (2, 14, 28). Our present findings demonstrate that treatment with ATRA-NaBu attenuated STAT1.HDAC1/2 complex formation and induced both STAT1 acetylation and its physical association with NF-κB (p65), thereby attenuating downstream NF-κB (p65) signaling.

HDAC inhibitors have emerged as anti-inflammatory drugs because of their suppressive effects on proinflammatory molecules. Earlier, it was suggested that not only is HDAC activity required for the efficient initiation and/or elongation of inflammatory gene transcription mediated by NF-κB but that HDACi directly attenuates NF-κB-induced gene expression (17). Recently, it has been shown that HDACi blocks NF-κB-dependent transcription of proinflammatory molecules under various pathological conditions (16, 34). Studies have indicated that STAT1 is an acetylated protein and that its acetylation depends on the balance between STAT1-associated HDACs and histone acetyltransferases (21, 23, 28, 29). It has been suggested that acetylated STAT1 interacts with NF-κB (p65) and consequently inhibits not only its activity but the expression of NF-κB target genes such as IL-6 and MCP-1 (2, 23, 28). Henceforth, the anti-inflammatory properties of ATRA and NaBu can be explained as a consequence of modulation of NF-κB pathways by increased STAT1 acetylation and decreased HDAC activity in a haplotype Npr1^+/− mouse model. The current findings demonstrate that ATRA-NaBu significantly inhibits renal inflammation and fibrosis and activates NPRA/cGMP signaling in Npr1^+/− mice, which in turn suppresses the NF-κB-mediated activation of inflammatory pathways (Fig. 8, A and B).

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.

In conclusion, the present results directly demonstrate that ATRA and NaBu, independently and in combination, markedly reduced the expression levels of proinflammatory cytokines and renal fibrosis and dysfunction by enhancing NPRA/cGMP signaling in haplotype Npr1^+/− mice. Pharmacological inhibition of HDAC activity by ATRA-NaBu enhanced STAT1 expression and attenuated its interaction with HDAC1/2, thereby increasing STAT1 acetylation. In addition, our findings demonstrate that acetylated STAT1 forms a complex with NF-κB (p65) and inhibits its DNA-binding ability.

GRANTS

This research was supported by National Heart, Lung, and Blood Institute Grants HL-057531 and HL-062147 and partial support from the National Institute of General Medical Sciences IDeA Program (COBRE Hypertension Pilot Project and COBRE Aging Pilot Project).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.K. and K.N.P. conceived and designed research; P.K., V.R.G., R.P., G.R., U.S., and K.N.P. performed experiments; P.K., V.R.G., R.P., G.R., U.S., and K.N.P. analyzed data; P.K., V.R.G., R.P., and K.N.P. interpreted results of experiments; P.K. and K.N.P. prepared figures; P.K. and K.N.P. drafted manuscript; P.K. and K.N.P. edited and revised manuscript; P.K., V.R.G., R.P., G.R., U.S., and K.N.P. approved final version of manuscript.