Guanylyl Cyclase/natriuretic Peptide receptor-A Research Articles

Insulin and glucose tolerance assessment in guanylyl cyclase/natriuretic peptide receptor-A gene-targeted mutant mice

Atrial natriuretic peptide (ANP), acting through guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) regulates blood pressure and cardiac homeostasis. The disruption of Npr1 (encoding NPRA) in mice exhibits hypertension and provokes renal and cardiac disorder. However, the underlying mechanisms are not yet precisely determined. The objective of this study was to determine whether Npr1 plays a role in regulating glucose homeostasis in global Npr1 gene-disrupted mice. The adult male (14-18 wks) Npr1 haplotype gene-knockout ( Npr1+/-, 1-copy), wild-type ( Npr1+/+, 2-copy), and gene-duplicated ( Npr1+ +/+ +, 4-copy) mice were fasted overnight (16 h) and given free access to water. The mice were administered with glucose both orally and intraperitoneally (2 g/kg body weight) to determine the oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT). For the insulin tolerance test (ITT), mice were also fasted for 16 h and then received an ip-injection of insulin (1.0 UI/kg body weight). Blood glucose levels were determined by performing tail bleeds at 0, 15, 30, 60, 90, and 120 min using the AlphaTRAK blood glucose monitoring system. The results showed that administration of glucose resulted in a greater increases in blood glucose levels at 120 mins in 1-copy mice (OGTT: 237 ± 5 mg/dL, IPGT: 246 ± 6 mg/dL, ITT; 239 ± 7 mg/dL) than 2-copy male (OGTT: 131 ± 3 mg/dL, IPGT: 126 ± 6 mg/dL, ITT; 127 ± 6 mg/dL), respectively. The blood glucose was also significantly lower in 4-copy mice (OGTT: 113 ± 5 mg/dL, IPGT: 108 ± 7 mg/dL, ITT; 107 ± 3 mg/dL) than in 2-copy mice. Systolic blood pressure (SBP) was determined by non-invasive tail-cuff method (Visitech 2000). SBP was significantly greater in 1-copy mice (130 ± 4 mmHg) than 2-copy mice (100 ± 3 mmHg). Similarly, SBP was also significantly lower in 4-copy mice (90 ± 2) than 2-copy mice. The increase in plasma glucose levels was significantly lower in OGTT than in IPGTT. The present results suggest that decreased Npr1 gene copy number predisposes to hyperglycemia, insulin resistance, and hypertension. These results indicate that the Npr1 gene might regulate blood glucose homeostasis. This work was supported by the NIH grant (DK133833). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DNA methylation inhibitor regulates guanylyl cyclase/natriuretic peptide receptor-A gene expression via 5'-untranslated region in mesangial and smooth muscle cells

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is activated by cardiac hormones atrial and brain natriuretic peptides (ANP, BNP) and produces intracellular second messenger cGMP. The physiological actions of ANP/NPRA/cGMP signaling cascade largely include diuresis, natriuresis, vasorelaxation, and organ protection, which maintains blood pressure and opposed renal, cardiac, and vascular injury and remodeling. However, the mechanisms that contribute to the activation of Npr1 (coding for GC-A/NPRA) expression is not well understood. The present study was aimed at understanding the epigenetic mechanisms of DNA methyltransferase (DNMT) inhibitor, 5-Azacytidine (5-Aza)-mediated Npr1 gene transcription. The studies were carried out in mouse mesangial cells (MMCs) and rat thoracic aortic smooth muscle (RTASM) cells, cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with 5-Aza for 24 h. Western blot analysis showed that 5-Aza enhanced NPRA protein levels by 5-fold and 7-fold in a dose-dependent manner in MMCs and RTASM cells, respectively, and greatly stimulated intracellular accumulation of cGMP in ANP-treated cells. Treatment with 5-Aza attenuated DNMT activity and repressed DNMT1 protein levels in cells. The MethPrimer search result showed the presence of three cytosine-phosphate-guanine (CpG) islands in the Npr1 full length promoter and 5'-untranslated region (UTR), namely island 1 (-886 to -752, 134 bp), island 2 (-310 to -158, 152 bp), and island 3 (-154 to +310, 464 bp) from the transcription start site. Addition of 5'-UTR encompassing +56 to +359 bp region to the Npr1 promoter constructs, (-1982/+55) and proximal promoter (-356/+55), was inhibitory to Npr1 transcriptional activity, independent of the cell type. Furthermore, treatment with 5-Aza distinctly induced Npr1 promoter activity of the construct -356/5'-UTR by 10-fold in MMCs suggesting that the repressive effect of DNA methylation on Npr1 gene transcription might be via 5'-UTR. The results demonstrate that 5-Aza acts as a potent inducer of Npr1 expression and signaling and provides new insights in the epigenetic control of Npr1, an important player in the control of hypertension and cardiorenal axis. Supported by the NIH grants (HL062147 and DK133833). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Abstract 068: Genetic Mechanisms Of Cardiac Hypertrophy And Fibrosis In Global Npr1 Gene-ablated Male And Female Mice

Genetic ablation of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) in mice triggers cardiac remodeling associated with hypertrophy, fibrosis, and heart failure. The objective of this study was to determine the mechanisms regulating the developments of cardiac hypertrophy and fibrosis in global Npr1 (encoding NPRA) gene-knockout (KO) mice and whether transforming growth factor-beta 1 (TGF-β1) plays a critical role in this process. Both male and female Npr1 KO ( Npr1 -/- , 0-copy), heterozygous ( Npr1 +/- , 1-copy), and wild-type (WT; Npr1 +/+ , 2-copy) mice were administered with TGF-β1 receptor antagonist GW788388 (2 mg/kg/day) by intraperitoneal (ip) injection for 28 days. The heart weight/body weight (HW/BW) and tibia length (TL) to BW (TL/BW) ratios were determined. Echocardiography was used to determine the cardiac structure and function. Quantitative polymerase chain reaction (qPCR) and Western blot were used to quantify the hypertrophic and fibrotic markers. HW/BW and TL/BW ratios were significantly higher in Npr1 KO mice and significantly greater in males than females. The Npr1 -/- mice showed significant induction of cardiac hypertrophic marker collagen-1α (3.5-fold) and fibrotic markers, monocyte chemoattractant protein (MCP, 4-fold), connective tissue growth factor (CTGF, 5-fold), alpha-smooth muscle actin (α-SMA, 4-fold) compared with WT mice. The expression of phosphorylated extracellular regulated kinase 1/2 (pERK1/2) was also upregulated in Npr1 -/- mice. Left ventricular end-systolic and diastolic dimensions (LVED-s, LEVD-d) and posterior wall thickness (PWT) were significantly increased in male KO mice than female KO mice compared with WT mice. The fractional shortening (FS) was compromised in male KO mice than female KO mice. The Treatment of Npr1 -/- mice with GW788388, prevented the development of cardiac hypertrophy and fibrosis compared to vehicle-treatment. The results suggest that the cardiac hypertrophy and fibrosis in Npr1 -/- mice is regulated through TGF-β1- SMAD-pathways.

Vitamin D Regulates Natriuretic Peptide Receptor‐A Gene Expression via Modulation of Histone Deacetylases and DNA Methyltransferase Activity

Cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces the second messenger cGMP. cGMP activates downstream signaling and biological consequences of ANP/NPRA such as natriuresis, diuresis, vasorelaxation, antimitogenic responses, and cardiac and renal anti‐hypertrophic effects. The activity and expression of Npr1 gene (coding for GC‐A/NPRA) assessed primarily through ANP‐stimulated cGMP production, are mediated by agents, including autoregulation involving natriuretic peptides, osmotic sensors, and other external and internal stimuli, but the hormonal and epigenetic mechanisms mediating Npr1 regulation are not well understood. The objective of present study was to study the effect of Vitamin D (vitD) in the regulation of Npr1 gene transcription and expression via modulation of epigenetic factors. Our bioinformatics study with the murine Npr1promoter has shown presence of 4 vitD response elements (VDRE) in the region ‐583 to ‐495 from the transcription start site having perfect and VDRE‐like consensus sequence. Npr1promoter activity was determined by transient transfection of Npr1 promoter deletion constructs in cultured rat thoracic aortic smooth muscle cells (RTASMCs) and mouse mesangial cells (MMCs) treated with Vitamin D₃ (1α, 25‐dihydroxy; VD3) using dual luciferase assay kit. Luciferase assay demonstrated that VD3 enhances Npr1 promoter activity by more than 5‐6‐fold in RTASMCs and MMCs in a dose‐dependent manner. Western blot and densitometry analysis showed increasing concentrations of VD3 significantly induced NPRA protein levels by 3.5‐fold in MMCs and 4.5‐fold in RTASMCs and maximum effect was observed at 100 nM. There was 45%‐50% inhibition of histone deacetylase (HDAC) activity in presence of VD3 in both cell lines as measured by HDAC activity/inhibition ELISA kit. Moreover, treatment with VD3 reduced class I HDAC enzymes, HDAC1 and HDAC3 protein levels and dose‐dependently enhanced acetylation level of histones, H3 at lysine residues 9 and 14 (H3‐K9/14 ac) and H4 at lysine residue 12 (H4‐K14ac). Treatment of RTASMCs and MMCs with VD3 attenuated DNA methyltransferase (DNMT) activity by 40%‐55% as measured by DNMT activity/inhibition ELISA kit. The results demonstrate that VD3 regulate Npr1 gene expression by modulation of histone deacetylases and DNMT activity. The identification of epigenetic targets of vitamin D signaling as a regulator of Npr1gene transcription and protein expression will have important implications in hypertension and cardiovascular regulation.

Glucose homeostasis is impaired in Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene‐Knockout and Gene‐Duplication Mutant Mice

Atrial and brain natriuretic peptides (ANP and BNP) are produced in the heart, regulate blood pressure (BP) and fluid homeostasis through vasodilatory and diuretic actions. Both ANP and BNP act by binding to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPR‐A). Systemic disruption of Npr1(encoding GC‐A/NPRA) leads to volume overload, high BP, and congestive heart failure. However, the underlying mechanisms are not yet precisely determined. The aim of this study was to investigate whether Npr1plays a critical role in regulating glucose homeostasis in Npr1 gene‐disrupted mice. The adult male and female (16‐18 wk) Npr1 gene‐knockout haplotype (Npr1+/‐, 1‐copy), wild‐type (Npr1+/+, 2‐copy), and gene‐duplicated (Npr1+ +/+ +, 4‐copy) mice were fasted for 16 h and given free access to water. The glucose administration was done both orally and intraperitoneally (IP: 2 g/kg body weight) in mice to determine oral glucose tolerance test (OGTT) and IP glucose tolerance test (IPGTT). The glucose levels in blood were determined by performing tail bleeds at 0, 15, 30, 60, 90, and 120 min using the AlphaTRAK blood glucose monitoring system (Zoetis Inc, Kalamazoo, MI). Systolic BP (SBP) was determined by non‐invasive computerized tail‐cuff method (Visitech 2000). The results showed that the blood glucose level was elevated to the maximum value at 15 min after glucose administration. Glucose levels were declined to near base line at 120 min in 2 copy mice (OGTT: 102 ± 3 mg/dL in male and 97 ± 4 mg/dL in female, IPGT: 101 ± 4 mg/dL in male and 96 ± 3 mg/dL in female). However, in 1‐copy mice, the blood glucose levels remained elevated even after 120 min (OGTT: 248 ± 5 mg/dL in male and 226 ± 3 mg/dL in female, IPGT: 254 ± 4 mg/dL in male and 229 ± 5 mg/dL in female) compared with 2‐copy mice. The blood glucose levels were also significantly lower at 120 min in 4‐copy mice (OGTT: 77 ± 4 mg/dL in male and 74 ± 3 mg/dL in female, IPGT: 75 ± 3 mg/dL in male and 71 ± 2 mg/dL in female) compared with 2‐copy mice. SBP was significantly greater in 1‐copy mice (137 + 4 mmHg in male and 126 + 3 mmHg in female) than 2‐copy mice (100 + 2 mmHg in male and 91 + 2 mmHg in female). SBP was significantly lower in 4‐copy mice (83 + 2 mmHg in male and 77 + 2 mmHg in female) than 2‐copy mice. OGTT showed a significantly lower level of maximal blood glucose compared with IPGTT. The findings of the present study suggest that Npr1 plays a critical role in the regulation of glucose homeostasis and the loss of Npr1 significantly increases the glucose levels in mutant animals.

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.

Class I HDAC Differentially Regulates Blood Pressure and Renal Injury in Npr1 Gene‐targeted Male and Female Mutant Mice

Atrial and brain natriuretic peptides (ANP, BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which plays a critical role in the regulation of blood pressure (BP) and fluid volume homeostasis, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit pathophysiology of hypertension, kidney disease, and heart failure; however, the mechanisms regulating Npr1 expression are not well understood. The objective of the present study was to gain insight into the effect of class I‐specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on Npr1 gene expression and regulation of BP and renal injury in gene‐targeted mutant mice. We utilized 16‐ to 20‐weeks old male and female Npr1 gene‐knockout haplotype (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+), and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice. All mice genotypes were injected intraperitoneally with MGCD0103 (2 mg/kg) at an alternate day for 2‐weeks. The Western blot results showed that MGCD0103 significantly increased the renal NPRA protein levels in male and female mice compared with vehicle‐treated (olive oil) controls. After MGCD treatment, the renal GC‐A activity was significantly increased in both male and female mice. Male mice exhibited significantly higher systolic BP (SBP) than female mice and treatment with MGCD0103 decreased SBP in both 1‐copy male (106 ± 0.6 vs. vehicle‐treated, 129 ± 1.9; mmHg) and female (97 ± 2.2 vs. vehicle‐treated, 110 ± 2.1 mmHg, p < 0.001) mice. Renal HDAC activity was considerably higher (p < 0.05) in male mice than female mice; however, treatment with MGCD0103 attenuated (p < 0.05) HDAC activity by 40% in male and ~50% in female animals. Significantly higher urinary albumin to creatinine ratio was detected in male mice compared with female mice, which was attenuated by MGCD0103 treatment in male mice. The present results demonstrate that class I HDAC inhibitor, MGCD0103 upregulates NPRA expression in vivo, lowers SBP, and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a gender‐related manner.Support or Funding InformationThis work was supported by NIH grant HL062147.

Abstract P2045: Genetic Disruption of Npr1 Depletes T Regulatory Cells and Provokes High Levels of Proinflammatory Cytokines and Fibrosis in the Kidneys of Female Mutant Mice

The aim of the present study was 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 2/4 (TLR2/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 T regulatory 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.

SAT-046 Modulation of Histone 3 Acetylation and Methylation Enhances the Expression and Function of Npr1 in Haplotype Mice

Atrial and brain natriuretic peptides (ANP, BNP) activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which plays a critical role in the regulation of blood pressure and fluid volume homeostasis, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 (coding for GC-A/NPRA) gene exhibit hypertension and renal disorders; however, the mechanisms regulating Npr1 expression and function are not well understood. The objective of the present study was to gain insight into the epigenetic regulatory mechanisms involved in Npr1 gene expression modulated by class I specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103). We utilized 16- to 20-weeks old male and female Npr1 gene-knockout haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+) and gene-duplicated heterozygous (3-copy; Npr1++/+) mice. Mice were injected intraperitoneally with MGCD0103 (2 mg/kg) at an alternate days for 2-weeks. The Western blot analysis revealed that MGCD0103 significantly increased the renal NPRA protein levels in all male and female mice groups compared with vehicle-treated controls. After MGCD0103 treatment, the renal Npr1 mRNA expression increased in Npr1 haplotype male and female mice. Renal HDAC activity was significantly higher (p < 0.05) in all genotypes of male mice than female mice; however, treatment with MGCD0103 markedly attenuated HDAC activity in both male (~40%, p < 0.05) and female (~50%, p < 0.05) animals. The histone acetyltransferase (HAT) activity was lower in untreated 1-copy male mice compared with wild type mice and MGCD0103 treatment significantly increased the HAT activity whereas no significant differences were observed in drug- or vehicle-treated female mice. The drug treatment significantly enhanced the acetylation levels of H3-K9 and H3-K27 in 1-copy male mice and H3-K18 in 1-copy female mice. There was substantial increase in protein levels of active gene transcription mark H3-K4 tri-methylation and decrease in suppressive marks H3-K9 and H3-K27 methylation in MGCD0103-treated 1-copy male and female mice. The present results suggest that MGCD0103 epigenetically regulates Npr1 expression in vivo in 1-copy haplotype male and female mice, via inhibition of HDAC activity, upregulation of the H3 acetylation and attenuation of H3 methylation levels. These present findings will have important implications in prevention and treatment of hypertension-related renal pathophysiological conditions under reduced NPRA signaling. This work was supported by NIH grants (HL057531 and HL062147) and a partial support from Tulane Carol Lavin Bernick grant award.

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.

Inhibition of DNA Methylation Regulates Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression

Cardiac hormone atrial natriuretic peptide binds to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. The molecular and epigenetic mechanisms that contribute to the activation of Npr1 (coding for GC‐A/NPRA) gene expression are not well understood. The present study was aimed at understanding the mechanisms involved in DNA methylation inhibitor, 5‐Azacytidine (5‐aza)‐mediated Npr1 (coding for GC‐A/NPRA) gene transcription. The studies were carried out in rat thoracic aortic vascular smooth muscle cells (RTASMC), cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and treated with 5‐aza for 24 h. The MethPrimer search result showed the presence of three cytosine‐phosphate‐guanine (CpG) islands on the Npr1 full length promoter, namely island 1 (−886 to −752, 134 bp), island 2 (−310 to −58, 152 bp), and island 3 (−514 to +310, 464 bp) from the transcription start site. 5‐azacytidine significantly induced mRNA levels of Npr1 gene by 4‐fold and NPRA protein expression by 3‐fold in RTASMCs in a dose‐dependent manner. Treatment of cells with 5‐aza significantly (p&lt;0.05) attenuated DNA methyltransferase 1 (DNMT1) protein expression in a dose‐dependent manner. However, there was no noticeable change in the DNMT3a and DNMT3b protein levels. There was a significant reduction in the DNMT activity in 5‐aza‐treated RTASMCs compared with vehicle–treated cells as quantified by EpiQuik DNMT activity assay. The present findings provide a novel regulatory mechanism for Npr1 gene transcription, an important player in the control of hypertension and cardiovascular homeostasis.Support or Funding InformationThis work was supported by an Institutional Development Award (IDeA) from the NIGMS and a partial support from Tulane Carol Lavin Bernick grant award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

Atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor‐a (GC‐A/NPRA), which regulates blood pressure through inhibition of renin‐angiotensin‐aldosterone system (RAAS). The aim of the present study was to determine whether targeted‐disruption of Npr1 (encoding GC‐A/NPRA) upregulates pro(renin) receptor (P)RR expression and leads to 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 homozygous (Npr1++/++; 4‐copy) mice were utilized. The renal expression of (P)RR, ACE‐1, and AT1R were analyzed. To identify the canonical pathway of (P)RR, we administered ACE‐1 inhibitor (captopril), AT1R inhibitor (losartan), and MAPKs inhibitors (U0126 and SB203580) to all Npr1 mice. The renal expression of (P)RR was increased by 3‐fold in 0‐copy and 2‐fold in 1‐copy mice compared with 2‐copy mice, which was also associated with increased expression of ACE‐1 and AT1R genes. Similarly, the renal expression of phosphorylated MAPKs (p‐Erk1/2 and p‐p38) were enhanced by 3.5‐fold and 3‐fold, respectively, in 0‐copy mice with significant increases in 1‐copy mice compared with 2‐copy mice. Proinflammatory cytokines were also significantly elevated in Npr1 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 expression of p‐ERK1/2 and p‐p38 in the Npr1 mice genotypes. The present findings suggest that ablation of Npr1 upregulates (P)RR, MAPKs (p‐Erk1/2 and p‐p38), and proinflammatory cytokines in 0‐copy and 1‐copy mice. In contrast, the duplication of Npr1 gene copy exhibited anti‐inflammatory and antihypertensive effects by reducing the expression of RAAS components, MAPKs, and proinflammatory cytokines.Support or Funding InformationThis work was supported by NIH grants (HL057531 and HL062147).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P161: Effect of Rapamycin on the Expression of T Regulatory Cells, Foxp3, and Toll-Like Receptors in the Kidneys of Npr1 Gene-Knockout Mice

The alterations in the inflammatory system contribute to the pathogenesis of hypertension leading to end-organ damage. Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) disruption elevates blood pressure and activates the immunogenic responses in mice. The goal of this study was to determine the effect of rapamycin on the expression of T regulatory (Treg) cells, Foxp3, and Toll-like receptors (TLRs) in Npr1 gene-disrupted and -duplicated mice. The adult 0-copy ( Npr1 -/- ), 2-copy ( Npr1 +/+ ), and 4-copy ( Npr1 ++/++ ) female mice were pre-treated with rapamycin (1mg/kg/day). Kidneys, spleen, and blood were collected, qRT-PCR, Western blot, immunofluorescence, and flow cytometry analyses were done to assess the expression of Tregs, Foxp3, and TLRs. The disruption of Npr1 led to significant reductions in Foxp3 + mRNA expression in 0-copy (77.5%) and 1-copy (71.5%) mice but not in 2-copy and 4-copy mice. Similarly, CD25 + expression was reduced in 0-copy mice by 75% and in 1-copy by 60% compared to 2-copy and 4-copy mice. In contrast, the total CD4 + count was up-regulated by 40% in 0-copy and 31% in 1-copy mice. Treatment with rapamycin up-regulated Foxp3 + cells in 0-copy (17.38%, P &lt;0.001) and 1-copy (8%, P &lt;0.05) mice, and CD25 + T cell levels were increased by 62.4% ( P &lt;0.001) in 0-copy mice and 38.6% ( P &lt;0.05) in 1-copy mice compared to vehicle-treated control mice. In contrast, CD4 + cell expression was significantly reduced by 15.4% ( P &lt;0.001) in 0-copy and 12.2% ( P &lt;0.05) in 1-copy mice. Interestingly, rapamycin significantly ( P &lt; 0.001) increased renal Foxp3 + protein expression to 85% in 0-copy mice and 68% in 1-copy mice compared to untreated controls. The expression of TLR2/TLR4 was significantly increased by 3.4- to 4.5-fold in 1-copy and 0-copy mice than 2-copy and 4-copy mice; however, rapamycin greatly decreased the expression of both TLRs in both 1-copy and 0-copy mice. In summary, rapamycin treatment upregulated the expression of Foxp3 + , CD25 + cells, and TLRs while suppressing pathogenic CD4 + T cell expression in 0-copy and 1-copy mice kidneys. The present results implicate that Npr1 and immune cell interactions could provide new therapeutic targets for the treatment and prevention of hypertension and kidney dysfunction in humans.

Abstract 101: Transforming Growth Factor-Beta-Receptor Antagonist Blocks the Cardiac Fibrosis and Remodeling in Guanylyl Cyclase/Natriuretic Peptide Receptor-A Gene-Disrupted Mice

Targeted-disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) exhibits hypertension and provokes congestive heart failure in mice; however, the underlying mechanisms are not well clear. The objective of this study was to determine whether transforming growth factor-beta receptor (TGF-βR) antagonist, GW788388 inhibits the development of cardiac fibrosis and remodeling in Npr1 gene-disrupted mice. The adult male (16-20 weeks) Npr1 null mutant ( Npr1 -/- , 0-copy), heterozygous ( Npr1 +/- , 1-copy), and wild-type ( Npr1 +/+ , 2-copy) mice were orally administered with TGF-βR antagonist, GW788388 (1 mg/kg/day) for 28 days. The expression of cardiac fibrotic markers was analyzed using real-time PCR and Western blot. Heart weight-to-body weight (HW/BW) ratios were determined and heart functions were measured by echocardiographic analysis. The Npr1 -/- mice showed markedly increased cardiac fibrosis and HW/BW ratio with increased expression of collagen-1α (3.5-fold), monocyte chemoattractant protein (4-fold), connective tissue growth factor (CTGF, 5-fold), α-smooth muscle actin (α-SMA, 4-fold), TGF-βRI (4-fold), TGF-βRII (3.5-fold) and SMAD proteins (SMAD-2, 5-fold; SMAD-3, 3-fold) compared with Npr1 +/+ mice. The expression of phosphorylated extracellular-regulated kinase (pERK1/2) was also up-regulated by 68% ( P &lt;0.001) in Npr1 -/- mice. The treatment of Npr1 -/- mice with GW788388 prevented the development of cardiac fibrosis and down-regulated the expression of fibrotic markers and SMAD proteins by 70-75% ( P &lt;0.001) compared to vehicle-treated mice. In contrast, the expression of pERK1/2 proteins was unaffected in GW7885388-treated mice excluding the involvement of non-genomic pathway. The left ventricular dimensions (systole and diastole) and fractional shortening were significantly ( P &lt;0.001) improved in the drug-treated Npr1 -/- mice. The results suggest that the cardiac fibrosis, remodeling, and dysfunction in Npr1 -/- mice are regulated through TGF-βR-mediated SMAD-dependent canonical pathway. The findings will be important for the development of new molecular therapeutic targets for the treatment of cardiac fibrosis, remodeling, and dysfunction in humans.

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.

Enhanced expression of pro‐renin receptor and proinflammatory cytokines in Npr1 gene disrupted mice

Atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor‐a (GC‐A/NPRA), which regulates blood pressure through inhibition of renin‐angiotensin‐aldosterone system (RAAS). The present study was aimed to determine whether targeted‐disruption of Npr1 gene (coding for GC‐A/NPRA) upregulates pro(renin) receptor (P)RR expression and leads to activation of mitogen activated protein kinases (MAPKs) in Npr1 gene‐knockout mice. The results showed that Npr1 gene disruption enhanced renal (pro)renin receptor expression by 3‐fold in 0‐copy (−/−) mice compared with 2‐copy (+/+) control mice, which in turn was associated with increased expression of angiotensin converting enzyme (ACE) and angiotensin receptor type 1 (AT1R) gene by 2‐fold and 3‐fold respectively. To identify the angiotensin II‐ (Ang II) dependent and ‐independent mechanism of (P)RR, ACE inhibitor captopril and AT1R antagonist losartan were administered to Npr1 mice genotypes. Both drugs did not alter the expression of (pro)renin receptor in all Npr1 mice genotypes. In parallel, renal expression of (MAPKs) p‐Erk1/2 and p‐p‐38 were increased by 2.5‐fold and 3‐fold in 0‐copy mice compared with 2‐copy control mice. Treatment with captopril did not alter the expression of p‐Erk1/2 and p‐p38 in Npr1 mice genotypes compared with their untreated mice counterparts. Interestingly, losartan reduced the expression of p‐ERK1/2 and p‐p38 in 1‐copy mice compared with vehicle‐treated control groups. The present findings suggest that disruption of Npr1 gene upregulates (P)RR and activates MAPKs p‐Erk1/2 and p‐p38 in 0‐copy mice, independent of Ang II generation by enhancing pro‐inflammatory cytokines in a gene dose‐dependent manner.Support or Funding InformationNIH/NHLBI: HL57531NIH/NHLBI: HL62147This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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 5min 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.

Abstract 12920: Transforming Growth Factor-Beta-Dependent Pathway Induces the Cardiac Fibrosis and Remodeling in Guanylyl Cyclase/Natriuretic Peptide Receptor-A Gene-Disrupted Mice

Introduction: Targeted-ablation of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) in mice exhibits hypertension and provokes congestive heart failure; however, the underlying mechanisms are not well understood. Hypothesis: To determine whether transforming growth factor-beta 1 (TGF)-β1 receptor antagonist, GW788388 inhibits the development of cardiac fibrosis and remodeling in GC-A/NPRA gene-disrupted mice. Methods: The adult male (16-20 weeks) Npr1 null mutant ( Npr1 -/- , 0-copy), heterozygous ( Npr1 +/- , 1-copy), and wild-type ( Npr1 +/+ , 2-copy) mice were orally administered with TGF-β1 receptor antagonist, GW788388 (2 mg/kg/day) for 28 days. The expression of cardiac fibrotic markers was analyzed using real-time PCR and Western blot. Heart weight-to-body weight (HW/BW) ratios were determined and heart functions were measured by echocardiographic analysis. Results: The Npr1 -/- mice showed markedly increased cardiac fibrosis and HW/BW ratio with increased expression of collagen-1α (3.5-fold), monocyte chemoattractant protein (4-fold), connective tissue growth factor (CTGF, 5-fold), α-smooth muscle actin (α-SMA, 4-fold), TGF-βRI (4-fold), TGF-βRII (3.5-fold) and SMAD proteins (SMAD-2, 5-fold; SMAD-3, 3-fold) compared with Npr1 +/+ mice. The expression of phosphorylated extracellular-regulated kinase (pERK1/2) was also up-regulated in Npr1 -/- mice. The treatment of Npr1 -/- mice with GW788388 significantly prevented the development of cardiac fibrosis and down-regulated the expression of fibrotic markers and SMAD proteins compared to vehicle-treated mice. In contrast, the expression of pERK1/2 proteins was unaffected in GW7885388-treated mice excluding the involvement of non-genomic pathway. The left ventricular dimensions (systole and diastole) and fractional shortening were significantly improved in the drug-treated Npr1 -/- mice. Conclusions: The results suggest that the cardiac fibrosis, remodeling, and dysfunction in Npr1 -/- mice are regulated through TGF-β-mediated SMAD-dependent canonical pathway. The findings will be important for the development of new molecular therapeutic targets for the treatment of cardiac fibrosis, remodeling, and dysfunction in human patients.