Epigenetic mechanisms differentially regulate blood pressure and renal dysfunction in male and female Npr1 haplotype mice.

We determined the epigenetic mechanisms regulating mean arterial pressure (MAP) and renal dysfunction in guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene-targeted mice. The Npr1 (encoding NPRA) gene-targeted mice were treated with class 1 specific histone deacetylase inhibitor (HDACi) mocetinostat (MGCD) to determine the epigenetic changes in a sex-specific manner. Adult male and female Npr1 haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+), and gene-duplicated heterozygous (3-copy; Npr1++/+) mice were intraperitoneally injected with MGCD (2 mg/kg) for 14 days. BP, renal function, histopathology, and epigenetic changes were measured. One-copy male mice showed significantly increased MAP, renal dysfunction, and fibrosis than 2-copy and 3-copy mice. Furthermore, HDAC1/2, collagen1alpha-2 (Col1α-2), and alpha smooth muscle actin (α-SMA) were significantly increased in 1-copy mice compared with 2-copy controls. The expression of antifibrotic microRNA-133a was attenuated in 1-copy mice but to a greater extent in males than females. NF-κB was localized at significantly lower levels in cytoplasm than in the nucleus with stronger DNA binding activity in 1-copy mice. MGCD significantly lowered BP, improved creatinine clearance, and repaired renal histopathology. The inhibition of class I HDACs led to a sex-dependent distinctive stimulation of acetylated positive histone marks and inhibition of methylated repressive histone marks in Npr1 1-copy mice; however, it epigenetically lowered MAP, repaired renal fibrosis, and proteinuria and suppressed NF-kB differentially in males versus females. Our results suggest a role for epigenetic targets affecting hypertension and renal dysfunction in a sex-specific manner.

Podocyte Cell-Specific Npr1 is Required for Blood Pressure and Renal Homeostasis in Male and Female Mice: Role of Sex-Specific Differences.

Atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA), stimulating natriuresis and diuresis and reducing blood pressure (BP), but the role of ANP/NPRA signaling in podocytes (highly specialized epithelial cells covering the outer surfaces of renal glomerular capillaries) remains unclear. This study aimed to determine the effect of conditional deletion of podocyte (PD)-specific Npr1 (encoding NPRA) gene knockout (KO) in male and female mice. Tamoxifen-treated wild-type control (PD Npr1 f/f; WT), heterozygous (PD-Cre-Npr1 f/+; HT), and knockout (PD-Cre-Npr1 f/-; KO) mice were fed a normal-, low-, or high-salt diet for 4 weeks. Podocytes isolated from HT and KO male and female mice showed complete absence of Npr1 mRNA and NPRA protein compared to WT mice. BP, plasma creatinine, plasma sodium, urinary protein, and albumin/creatinine ratio were significantly increased, while plasma total protein, albumin, creatinine clearance, and urinary sodium levels were significantly reduced in the HT and KO male and female mice compared to WT mice. These changes were significantly greater in males than females. On a normal-salt diet, glomerular filtration rate (GFR) was significantly decreased in PD Npr1 HT and KO male and female mice compared with WT mice. Immunofluorescence of podocin and synaptopodin were also significantly reduced in HT and KO mice compared to WT mice. These observations suggest that in podocytes, ANP/NPRA signaling may be crucial in the maintenance and regulation of glomerular filtration and BP and serve as a biomarker of renal function in a sex-dependent manner.

Genetic and Epigenetic Mechanisms Regulating Blood Pressure and Kidney Dysfunction.

The pioneering work of Dr Lewis K. Dahl established a relationship between kidney, salt, and high blood pressure (BP), which led to the major genetic-based experimental model of hypertension. BP, a heritable quantitative trait affected by numerous biological and environmental stimuli, is a major cause of morbidity and mortality worldwide and is considered to be a primary modifiable factor in renal, cardiovascular, and cerebrovascular diseases. Genome-wide association studies have identified monogenic and polygenic variants affecting BP in humans. Single nucleotide polymorphisms identified in genome-wide association studies have quantified the heritability of BP and the effect of genetics on hypertensive phenotype. Changes in the transcriptional program of genes may represent consequential determinants of BP, so understanding the mechanisms of the disease process has become a priority in the field. At the molecular level, the onset of hypertension is associated with reprogramming of gene expression influenced by epigenomics. This review highlights the specific genetic variants, mutations, and epigenetic factors associated with high BP and how these mechanisms affect the regulation of hypertension and kidney dysfunction.

Ligand-Dependent Downregulation of Guanylyl Cyclase/Natriuretic Peptide Receptor-A: Role of miR-128 and miR-195.

Cardiac hormones act on the regulation of blood pressure (BP) and cardiovascular homeostasis. These hormones include atrial and brain natriuretic peptides (ANP, BNP) and activate natriuretic peptide receptor-A (NPRA), which enhance natriuresis, diuresis, and vasorelaxation. In this study, we established the ANP-dependent homologous downregulation of NPRA using human embryonic kidney-293 (HEK-293) cells expressing recombinant receptor and MA-10 cells harboring native endogenous NPRA. The prolonged pretreatment of cells with ANP caused a time- and dose-dependent decrease in 125I-ANP binding, Guanylyl cyclase (GC) activity of receptor, and intracellular accumulation of cGMP leading to downregulation of NPRA. Treatment with ANP (100 nM) for 12 h led to an 80% decrease in 125I-ANP binding to its receptor, and BNP decreased it by 62%. Neither 100 nM c-ANF (truncated ANF) nor C-type natriuretic peptide (CNP) had any effect. ANP (100 nM) treatment also decreased GC activity by 68% and intracellular accumulation cGMP levels by 45%, while the NPRA antagonist A71915 (1 µM) almost completely blocked ANP-dependent downregulation of NPRA. Treatment with the protein kinase G (PKG) stimulator 8-(4-chlorophenylthio)-cGMP (CPT-cGMP) (1 µM) caused a significant increase in 125I-ANP binding, whereas the PKG inhibitor KT 5823 (1 µM) potentiated the effect of ANP on the downregulation of NPRA. The transfection of miR-128 significantly reduced NPRA protein levels by threefold compared to control cells. These results suggest that ligand-dependent mechanisms play important roles in the downregulation of NPRA in target cells.

Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Triggers Differential Cardiac Fibrosis and Disorders in Male and Female Mutant Mice: Role of TGF-ß1/SMAD Signaling Pathway.

The global targeted disruption of the natriuretic peptide receptor-A (NPRA) gene (Npr1) in mice provokes hypertension and cardiovascular dysfunction. The objective of this study was to determine the mechanisms regulating the development of cardiac fibrosis and dysfunction in Npr1 mutant mice. Npr1 knockout (Npr1-/-, 0-copy), heterozygous (Npr1+/-, 1-copy), and wild-type (Npr1+/+, 2-copy) mice were treated with the transforming growth factor (TGF)-ß1 receptor (TGF-ß1R) antagonist GW788388 (2 µg/g body weight/day; ip) for 28 days. Hearts were isolated and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analyses. The Npr1-/- (0-copy) mice showed a 6-fold induction of cardiac fibrosis and dysfunction with markedly induced expressions of collagen-1α (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), α-smooth muscle actin (α-SMA, 6.1-fold), TGF-ßRI (4.3-fold), TGF-ßRII (4.7-fold), and phosphorylated small mothers against decapentaplegic (pSMAD) proteins, including pSMAD-2 (3.2-fold) and pSMAD-3 (3.7-fold), compared with wild-type mice. The expressions of phosphorylated extracellular-regulated kinase ERK1/2 (pERK1/2), matrix metalloproteinases-2, -9, (MMP-2, -9), and proliferating cell nuclear antigen (PCNA) were also significantly upregulated in Npr1 0-copy mice. The treatment of mutant mice with GW788388 significantly blocked the expression of fibrotic markers, SMAD proteins, MMPs, and PCNA compared with the vehicle-treated control mice. The treatment with GW788388 significantly prevented cardiac dysfunctions in a sex-dependent manner in Npr1 0-copy and 1-copy mutant mice. The results suggest that the development of cardiac fibrosis and dysfunction in mutant mice is predominantly regulated through the TGF-ß1-mediated SMAD-dependent pathway.

Guanylyl cyclase/natriuretic peptide receptor-A: Identification, molecular characterization, and physiological genomics.

The natriuretic peptides (NPs) hormone family, which consists mainly of atrial, brain, and C-type NPs (ANP, BNP, and CNP), play diverse roles in mammalian species, ranging from renal, cardiac, endocrine, neural, and vascular hemodynamics to metabolic regulations, immune responsiveness, and energy distributions. Over the last four decades, new data has transpired regarding the biochemical and molecular compositions, signaling mechanisms, and physiological and pathophysiological functions of NPs and their receptors. NPs are incremented mainly in eliciting natriuretic, diuretic, endocrine, vasodilatory, and neurological activities, along with antiproliferative, antimitogenic, antiinflammatory, and antifibrotic responses. The main locus responsible in the biological and physiological regulatory actions of NPs (ANP and BNP) is the plasma membrane guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), a member of the growing multi-limbed GC family of receptors. Advances in this field have provided tremendous insights into the critical role of Npr1 (encoding GC-A/NPRA) in the reduction of fluid volume and blood pressure homeostasis, protection against renal and cardiac remodeling, and moderation and mediation of neurological disorders. The generation and use of genetically engineered animals, including gene-targeted (gene-knockout and gene-duplication) and transgenic mutant mouse models has revealed and clarified the varied roles and pleiotropic functions of GC-A/NPRA in vivo in intact animals. This review provides a chronological development of the biochemical, molecular, physiological, and pathophysiological functions of GC-A/NPRA, including signaling pathways, genomics, and gene regulation in both normal and disease states.

Molecular Signaling Mechanisms and Function of Natriuretic Peptide Receptor-A in the Pathophysiology of Cardiovascular Homeostasis.

The discovery of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP) and their cognate receptors has greatly increased our knowledge of the control of hypertension and cardiovascular homeostasis. ANP and BNP are potent endogenous hypotensive hormones that elicit natriuretic, diuretic, vasorelaxant, antihypertrophic, antiproliferative, and antiinflammatory effects, largely directed toward the reduction of blood pressure (BP) and cardiovascular diseases (CVDs). The principal receptor involved in the regulatory actions of ANP and BNP is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which produces the intracellular second messenger cGMP. Cellular, biochemical, molecular, genetic, and clinical studies have facilitated understanding of the functional roles of natriuretic peptides (NPs), as well as the functions of their receptors, and signaling mechanisms in CVDs. Transgenic and gene-targeting (gene-knockout and gene-duplication) strategies have produced genetically altered novel mouse models and have advanced our knowledge of the importance of NPs and their receptors at physiological and pathophysiological levels in both normal and disease states. The current review describes the past and recent research on the cellular, molecular, genetic mechanisms and functional roles of the ANP-BNP/NPRA system in the physiology and pathophysiology of cardiovascular homeostasis as well as clinical and diagnostic markers of cardiac disorders and heart failure. However, the therapeutic potentials of NPs and their receptors for the diagnosis and treatment of cardiovascular diseases, including hypertension, heart failure, and stroke have just begun to be expanded. More in-depth investigations are needed in this field to extend the therapeutic use of NPs and their receptors to treat and prevent CVDs.

Depletion of cyclic-GMP levels and inhibition of cGMP-dependent protein kinase activate p21Cip1 /p27Kip1 pathways and lead to renal fibrosis and dysfunction.

Cell-cycle regulatory proteins (p21Cip1 /p27Kip1 ) inhibit cyclin and cyclin-dependent kinase (CDK) complex that promotes fibrosis and hypertrophy. The present study examined the role of CDK blockers, p21Cip1 /p27Kip1 in the progression of renal fibrosis and dysfunction using Npr1 (encoding guanylyl cyclase/natriuretic peptide receptor-A, GC-A/NPRA) gene-knockout (0-copy; Npr1-/- ), 2-copy (Npr1+/+ ), and 4-copy (Npr1++/++ ) mice treated with GC inhibitor, A71915 and cGMP-dependent protein kinase (cGK) inhibitor, (Rp-8-Br-cGMPS). A significant decrease in renal cGMP levels and cGK activity was observed in 0-copy mice and A71915- and Rp-treated 2-copy and 4-copy mice compared with controls. An increased phosphorylation of Erk1/2, p38, p21Cip1 , and p27Kip1 occurred in 0-copy and A71915-treated 2-copy and 4-copy mice, while Rp treatment caused minimal changes than controls. Pro-inflammatory (TNF-α, IL-6) and pro-fibrotic (TGF-ß1) cytokines were significantly increased in plasma and kidneys of 0-copy and A71915-treated 2-copy mice, but to lesser extent in 4-copy mice. Progressive renal pathologies, including fibrosis, mesangial matrix expansion, and tubular hypertrophy were observed in 0-copy and A71915-treated 2-copy and 4-copy mice, but minimally occurred in Rp-treated mice compared with controls. These results indicate that Npr1 has pivotal roles in inhibiting renal fibrosis and hypertrophy and exerts protective effects involving cGMP/cGK axis by repressing CDK blockers p21Cip1 and p27Kip1 .

Angiotensin II represses Npr1 expression and receptor function by recruitment of transcription factors CREB and HSF-4a and activation of HDACs.

The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. ANP acting through natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. We tested hypothesis that ANG II plays critical roles in the transcriptional repression of Npr1 (encoding NPRA) and receptor function. ANG II significantly decreased NPRA mRNA and protein levels and cGMP accumulation in cultured mesangial cells and attenuated ANP-mediated relaxation of aortic rings ex vivo. The transcription factors, cAMP-response element-binding protein (CREB) and heat-shock factor-4a (HSF-4a) facilitated the ANG II-mediated repressive effects on Npr1 transcription. Tyrosine kinase (TK) inhibitor, genistein and phosphatidylinositol 3-kinase (PI-3K) inhibitor, wortmannin reversed the ANG II-dependent repression of Npr1 transcription and receptor function. ANG II enhanced the activities of Class I histone deacetylases (HDACs 1/2), thereby decreased histone acetylation of H3K9/14ac and H4K8ac. The repressive effect of ANG II on Npr1 transcription and receptor signaling seems to be transduced by TK and PI-3K pathways and modulated by CREB, HSF-4a, HDACs, and modified histones. The current findings suggest that ANG II-mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for treatment and prevention of hypertension and cardiovascular diseases.

Genetic Ablation and Guanylyl Cyclase/Natriuretic Peptide Receptor-A: Impact on the Pathophysiology of Cardiovascular Dysfunction.

Mice bearing targeted gene mutations that affect the functions of natriuretic peptides (NPs) and natriuretic peptide receptors (NPRs) have contributed important information on the pathogenesis of hypertension, kidney disease, and cardiovascular dysfunction. Studies of mice having both complete gene disruption and tissue-specific gene ablation have contributed to our understanding of hypertension and cardiovascular disorders. These phenomena are consistent with an oligogenic inheritance in which interactions among a few alleles may account for genetic susceptibility to hypertension, renal insufficiency, and congestive heart failure. In addition to gene knockouts conferring increased risks of hypertension, kidney disorders, and cardiovascular dysfunction, studies of gene duplications have identified mutations that protect against high blood pressure and cardiovascular events, thus generating the notion that certain alleles can confer resistance to hypertension and heart disease. This review focuses on the intriguing phenotypes of Npr1 gene disruption and gene duplication in mice, with emphasis on hypertension and cardiovascular events using mouse models carrying Npr1 gene knockout and/or gene duplication. It also describes how Npr1 gene targeting in mice has contributed to our knowledge of the roles of NPs and NPRs in dose-dependently regulating hypertension and cardiovascular events.

Emerging concepts of receptor endocytosis and concurrent intracellular signaling: Mechanisms of guanylyl cyclase/natriuretic peptide receptor-A activation and trafficking.

Endocytosis is a prominent clathrin-mediated mechanism for concentrated uptake and internalization of ligand-receptor complexes, also known as cargo. Internalization of cargo is the fundamental mechanism for receptor-dependent regulation of cell membrane function, intracellular signal transduction, and neurotransmission, as well as other biological and physiological activities. However, the intrinsic mechanisms of receptor endocytosis and contemporaneous intracellular signaling are not well understood. We review emerging concepts of receptor endocytosis with concurrent intracellular signaling, using a typical example of guanylyl cyclase/natriuretic peptide receptor-A (NPRA) internalization, subcellular trafficking, and simultaneous generation of second-messenger cGMP and signaling in intact cells. We highlight the role of short-signal motifs located in the carboxyl-terminal regions of membrane receptors during their internalization and subsequent receptor trafficking in organelles that are not traditionally studied in this context, including nuclei and mitochondria. This review sheds light on the importance of future investigations of receptor endocytosis and trafficking in live cells and intact animals in vivo in physiological context.

Genetic disruption of guanylyl cyclase/natriuretic peptide receptor-A upregulates renal (pro) renin receptor expression in Npr1 null mutant mice.

The objective of the present study was to determine whether targeted-disruption of Npr1 gene (encoding for guanylyl cyclase/natriuretic peptide receptor-A; GC-A/NPRA) upregulates pro(renin) receptor (P)RR expression and leads to the activation of MAPKs in Npr1 gene-knockout mice. The Npr1 homozygous (Npr1-/-; 0-copy), heterozygous (Npr1+/-; 1-copy), wild-type (Npr1+/+; 2-copy), and gene-duplicated (Npr1++/++; 4-copy) mice were utilized. To identify the canonical pathway of (P)RR, we administered ACE-1 inhibitor (captopril), AT1R blocker (losartan), and MAPKs inhibitors (U0126 and SB203580) to all Npr1 mice genotypes. The renal expression of (P)RR mRNA was increased by 3-fold in 0-copy mice and 2-fold in 1-copy mice compared with 2-copy mice, which was also associated with significantly increased expression of ACE-1 and AT1R mRNA levels. Similarly, the phosphorylation of MAPKs (Erk1/2 and p-p38) was enhanced by 3.5-fold and 3.2-fold, respectively, in 0-copy mice with significant increases in 1-copy mice compared with 2-copy mice. The kidney and plasma levels of proinflammatory cytokines were significantly elevated in 0-copy and 1-copy mice. Treatment with captopril and losartan did not alter the expression of (P)RR in any of the Npr1 mice genotypes. Interestingly, losartan significantly reduced the phosphorylation of Erk1/2 and p38 in Npr1 mice. The present results suggest that the ablation of Npr1 upregulates (P)RR, MAPKs (Erk1/2 and p38), and proinflammatory cytokines in 0-copy and 1-copy mice. In contrast, the duplication of Npr1 exhibits the anti-inflammatory and antihypertensive effects by reducing the activation of MAPKs and inhibiting the expression levels of RAAS components and proinflammatory cytokines.

Genetic disruption of Npr1 depletes regulatory T cells and provokes high levels of proinflammatory cytokines and fibrosis in the kidneys of female mutant mice.

The present study was designed to determine the effects of gene knockout of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) on immunogenic responses affecting kidney function and blood pressure (BP) in Npr1 (coding for GC-A/NPRA)-null mutant mice. We used female Npr1 gene-disrupted (Npr1-/-, 0 copy), heterozygous (Npr1+/-, 1 copy), wild-type (Npr1+/+, 2 copy), and gene-duplicated (Npr1++/++, 4 copy) mice. Expression levels of Toll-like receptor (TLR)2/TLR4 mRNA were increased 4- to 5-fold in 1-copy mice and 6- to 10-fold in 0-copy mice; protein levels were increased 2.5- to 3-fold in 1-copy mice and 4- to 5-fold in 0-copy mice. Expression of proinflammatory cytokines and BP was significantly elevated in 1-copy and 0-copy mice compared with 2-copy and 4-copy mice. In addition, 0-copy and 1-copy mice exhibited drastic reductions in regulatory T cells (Tregs). After rapamycin treatment, Tregs were increased by 17% (P < 0.001) in 0-copy mice and 8% (P < 0.001) in 1-copy mice. Renal mRNA and protein levels of TLR2 and TLR4 were decreased by 70% in 0-copy mice and 50% in 1-copy mice. There were significantly higher levels of Tregs and very low levels of TLR2/TLR4 expression in 4-copy mice (P < 0.001). These findings indicate that the disruption of Npr1 in female mice triggers renal immunogenic pathways, which transactivate the expression of proinflammatory cytokines and renal fibrosis with elevated BP in mutant animals. The data suggest that rapamycin treatment attenuates proinflammatory cytokine expression, dramatically increases anti-inflammatory cytokines, and substantially reduces BP and renal fibrosis in mutant animals.

Molecular and genetic aspects of guanylyl cyclase natriuretic peptide receptor-A in regulation of blood pressure and renal function.

Natriuretic peptides (NPs) exert diverse effects on several biological and physiological systems, such as kidney function, neural and endocrine signaling, energy metabolism, and cardiovascular function, playing pivotal roles in the regulation of blood pressure (BP) and cardiac and vascular homeostasis. NPs are collectively known as anti-hypertensive hormones and their main functions are directed toward eliciting natriuretic/diuretic, vasorelaxant, anti-proliferative, anti-inflammatory, and anti-hypertrophic effects, thereby, regulating the fluid volume, BP, and renal and cardiovascular conditions. Interactions of NPs with their cognate receptors display a central role in all aspects of cellular, biochemical, and molecular mechanisms that govern physiology and pathophysiology of BP and cardiovascular events. Among the NPs atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and initiate intracellular signaling. The genetic disruption of Npr1 (encoding GC-A/NPRA) in mice exhibits high BP and hypertensive heart disease that is seen in untreated hypertensive subjects, including high BP and heart failure. There has been a surge of interest in the NPs and their receptors and a wealth of information have emerged in the last four decades, including molecular structure, signaling mechanisms, altered phenotypic characterization of transgenic and gene-targeted animal models, and genetic analyses in humans. The major goal of the present review is to emphasize and summarize the critical findings and recent discoveries regarding the molecular and genetic regulation of NPs, physiological metabolic functions, and the signaling of receptor GC-A/NPRA with emphasis on the BP regulation and renal and cardiovascular disorders.

Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A.

Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using 125I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

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

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.

Retinoic acid and sodium butyrate suppress the cardiac expression of hypertrophic markers and proinflammatory mediators in Npr1 gene-disrupted haplotype mice.

The objective of the present study was to examine the genetically determined differences in the natriuretic peptide receptor-A (NPRA) gene (Npr1) copies affecting the expression of cardiac hypertrophic markers, proinflammatory mediators, and matrix metalloproteinases (MMPs) in a gene-dose-dependent manner. We determined whether stimulation of Npr1 by all-trans retinoic acid (RA) and histone deacetylase (HDAC) inhibitor sodium butyric acid (SB) suppress the expression of cardiac disease markers. In the present study, we utilized Npr1 gene-disrupted heterozygous (Npr1(+/-), 1-copy), wild-type (Npr1(+/+), 2-copy), gene-duplicated (Npr1(++/+), 3-copy) mice, which were treated intraperitoneally with RA, SB, and a combination of RA/SB, a hybrid drug (HB) for 2 wk. Untreated 1-copy mice showed significantly increased heart weight-body weight (HW/BW) ratio, blood pressure, hypertrophic markers, including beta-myosin heavy chain (ß-MHC) and proto-oncogenes (c-fos and c-jun), proinflammatory mediator nuclear factor kappa B (NF-κB), and MMPs (MMP-2, MMP-9) compared with 2-copy and 3-copy mice. The heterozygous (haplotype) 1-copy mice treated with RA, SB, or HB, exhibited significant reduction in the expression of ß-MHC, c-fos, c-jun, NF-κB, MMP-2, and MMP-9. In drug-treated animals, the activity and expression levels of HDAC were significantly reduced and histone acetyltransferase activity and expression levels were increased. The drug treatments significantly increased the fractional shortening and reduced the systolic and diastolic parameters of the Npr1(+/-) mice hearts. Together, the present results demonstrate that a decreased Npr1 copy number enhanced the expression of hypertrophic markers, proinflammatory mediators, and MMPs, whereas an increased Npr1 repressed the cardiac disease markers in a gene-dose-dependent manner.

Transforming growth factor ß1 antagonizes the transcription, expression and vascular signaling of guanylyl cyclase/natriuretic peptide receptor A - role of δEF1.

The objective of this study was to determine the role of transforming growth factor ß1 (TGF-ß1) in transcriptional regulation and function of the guanylyl cyclase A/natriuretic peptide receptor A gene (Npr1) and whether cross-talk exists between these two hormonal systems in target cells. After treatment of primary cultured rat thoracic aortic vascular smooth muscle cells and mouse mesangial cells with TGF-ß1, the Npr1 promoter construct containing a δ-crystallin enhancer binding factor 1 (δEF1) site showed 85% reduction in luciferase activity in a time- and dose-dependent manner. TGF-ß1 also significantly attenuated luciferase activity of the Npr1 promoter by 62%, and decreased atrial natriuretic peptide-mediated relaxation of mouse denuded aortic rings ex vivo. Treatment of cells with TGF-ß1 increased the protein levels of δEF1 by 2.4-2.8-fold, and also significantly enhanced the phosphorylation of Smad 2/3, but markedly reduced Npr1 mRNA and receptor protein levels. Over-expression of δEF1 showed a reduction in Npr1 promoter activity by 75%, while deletion or site-directed mutagenesis of δEF1 sites in the Npr1 promoter eliminated the TGF-ß1-mediated repression of Npr1 transcription. TGF-ß1 significantly increased the expression of α-smooth muscle actin and collagen type I α2 in rat thoracic aortic vascular smooth muscle cells, which was markedly attenuated by atrial natriuretic peptide in cells over-expressing natriuretic peptide receptor A. Together, the present results suggest that an antagonistic cascade exists between the TGF-ß1/Smad/δEF1 pathways and Npr1 expression and receptor signaling that is relevant to renal and vascular remodeling, and may be critical in the regulation of blood pressure and cardiovascular homeostasis.

Role of FQQI motif in the internalization, trafficking, and signaling of guanylyl-cyclase/natriuretic peptide receptor-A in cultured murine mesangial cells.

Binding of the cardiac hormone atrial natriuretic peptide (ANP) to transmembrane guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), produces the intracellular second messenger cGMP in target cells. To delineate the critical role of an endocytic signal in intracellular sorting of the receptor, we have identified a FQQI (Phe(790), Gln(791), Gln(792), and Ile(793)) motif in the carboxyl-terminal region of NPRA. Mouse mesangial cells (MMCs) were transiently transfected with the enhanced green fluorescence protein (eGFP)-tagged wild-type (WT) and mutant constructs of eGFP-NPRA. The mutation FQQI/AAAA, in the eGFP-NPRA cDNA sequence, markedly attenuated the internalization of mutant receptors by almost 49% compared with the WT receptor. Interestingly, we show that the µ1B subunit of adaptor protein-1 binds directly to a phenylalanine-based FQQI motif in the cytoplasmic tail of the receptor. However, subcellular trafficking indicated that immunofluorescence colocalization of the mutated receptor with early endosome antigen-1 (EEA-1), lysosome-associated membrane protein-1 (LAMP-1), and Rab 11 marker was decreased by 57% in early endosomes, 48% in lysosomes, and 42% in recycling endosomes, respectively, compared with the WT receptor in MMCs. The receptor containing the mutated motif (FQQI/AAAA) also produced a significantly decreased level of intracellular cGMP during subcellular trafficking than the WT receptor. The coimmunoprecipitation assay confirmed a decreased level of colocalization of the mutant receptor with subcellular compartments during endocytic processes. The results suggest that the FQQI motif is essential for the internalization and subcellular trafficking of NPRA during the hormone signaling process in intact MMCs.