NPR-A Expression Research Articles

Corin Deficiency Diminishes Intestinal Sodium Excretion in Mice.

Sodium excretion, a critical process in sodium homeostasis, occurs in many tissues, including the kidney and intestine. Unlike in the kidney, the hormonal regulation of intestinal sodium excretion remains unclear. Atrial natriuretic peptide (ANP) is a crucial hormone in renal natriuresis. Corin is a protease critical for ANP activation. Corin and ANP are expressed mainly in the heart. In this study, we investigated corin, ANP, and natriuretic peptide receptor A (Npra) expression in mouse intestines. Corin and ANP expression was co-localized in enteroendocrine cells, whereas Npra expression was on the luminal epithelial cells. In Corin knockout (KO) mice, fecal Na+ and Cl- excretion decreased compared with that in wild-type (WT) mice. Such a decrease was not found in conditional Corin KO mice lacking cardiac corin selectively. In kidney conditional Corin KO mice lacking renal corin, fecal Na+ and Cl- excretion increased, compared to that in WT mice. When WT, Corin KO, and the kidney conditional KO mice were treated with aldosterone, the differences in fecal Na+ and Cl- levels disappeared. These results suggest that intestinal corin may promote fecal sodium excretion in a paracrine mechanism independent of the cardiac corin function. The increased fecal sodium excretion in the kidney conditional Corin KO mice likely reflected an intestinal compensatory response to renal corin deficiency. Our results also suggest that intestinal corin activity may antagonize aldosterone action in the promotion of fecal sodium excretion. These findings help us understand the hormonal mechanism controlling sodium excretion the intestinal tract.

Hawthorn leaves flavonoids attenuate cardiac remodeling induced by simulated microgravity

Context Hawthorn leaves are a kind of widely used medicinal plant in China. The major ingredient, hawthorn leaves flavonoids (HLF), have cardiotonic, cardioprotective, and vascular protective effects. Objective The study evaluated the protective role of HLF in cardiac remodelling and the underlying mechanisms under simulated microgravity by hindlimb unloading rats. Materials and methods Adult male Sprague-Dawley rats were divided into control, HLF, HU (hindlimb unloading) and HU + HLF groups (n = 8). After HU and daily intragastric administration at the dose of 100 mg/kg/d for 8 weeks, cardiac function and structure were evaluated by biochemical indices and histopathology. We identified the main active compounds and mechanisms involved in the cardioprotective effects of HLF via bioinformatics and molecular docking analysis, and relative signalling pathway activity was verified by Western blot. Results HLF treatment could reverse the HU-induced decline in LV-EF (HU, 55.13% ± 0.98% vs. HU + HLF, 71.16% ± 5.08%), LV-FS (HU, 29.44% ± 0.67% vs. HU + HLF, 41.62% ± 4.34%) and LV mass (HU, 667.99 ± 65.69 mg vs. HU + HLF, 840.02 ± 73.00 mg). Furthermore, HLF treatment significantly increased NPRA expression by 135.39%, PKG by 51.27%, decreased PDE5A by 20.03%, NFATc1 by 41.68% and Rcan1.4 by 54.22%. Conclusions HLF plays a protective effect on HU-induced cardiac remodelling by enhancing NPRA-cGMP-PKG pathway and suppressing the calcineurin-NFAT pathway, which provides a theoretical basis for use in clinical therapies.

S-27-5: DELETION OF AT1A RECEPTORS IN THE PROXIMAL TUBULES AUGMENTS NATRIURETIC RESPONSE TO ATRIAL NATRIURETIC PEPTIDE VIA NPR/CGMP/NO SIGNALING

Objective: The proximal tubules of the kidney play a critical role in maintaining normal body salt and fluid balance and blood pressure homeostasis in part by interactions between two key vasoactive hormone receptors. In the proximal tubules, angiotensin II (Ang II) binds and activate AT1a receptors to stimulate proximal tubule Na+ reabsorption, whereas atrial natriuretic peptide (ANP) binds and activates NPRA receptors to inhibit Ang II-induced proximal tubule Na+ reabsorption. These two vasoactive systems play important counteracting roles in the proximal tubules to control proximal tubule Na+ reabsorption in the kidney and maintain blood pressure homeostasis. However, it is not known whether the hypotensive and natriuretic responses to ANP may be augmented by deletion of AT1 (AT1a) receptors selectively in the proximal tubules of the kidney. Design and Methods: The present study used a novel mouse model with proximal tubule-specific knockout of AT1a receptors in the kidney, PT-Agtr1a -/-, to test the hypothesis that deletion of AT1a receptors selectively in the proximal tubules indeed augments the hypotensive and natriuretic responses to ANP. Four groups (n = 9–12 per group) of adult male wild-type and PT-Agtr1a -/- mice were infused without (served as time controls) or with ANP via osmotic minipump (10 ng/min, i.p.) continuously for 2 weeks. Results: Basal blood pressure was about 16 ± 3 mmHg lower (P < 0.01), fractional proximal tubule Na+ reabsorption was significantly lower (P < 0.05), whereas 24 h urinary Na+ excretion was significantly higher in PT-Agtr1a -/- mice (P < 0.01), compared with age-matched wild-type mice. ANP infusion significantly decreased blood pressure and increased the natriuretic response in PT-Agtr1a -/- mice further by inhibiting proximal tubule Na+ reabsorption, compared with wild-type controls (P < 0.01). The augmented hypotensive and natriuretic responses to ANP infusion in PT-Agtr1a -/- mice was associated with increased plasma and kidney cGMP levels (P < 0.01), kidney cortical NPRA and NPRC mRNA expression (P < 0.05), total endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS proteins (P < 0.01), and urinary nitric oxide (NO) excretion (P < 0.01), respectively. Conclusions: Taken together, the results of the present study provide further new insights into the important physiological roles for intratubular Ang II/AT1a and ANP/NPRA in the proximal tubules to regulate proximal tubule Na+ reabsorption and help maintain blood pressure homeostasis. Supported by 2R01DK067299-10A1, 2R01DK102429-03A1, and 1 R01DK123144-01.

Chronological attenuation of NPRA/PKG/AMPK signaling promotes vascular aging and elevates blood pressure.

Hypertension is common in elderly population. We designed to search comprehensively for genes that are chronologically shifted in their expressions and to define their contributions to vascular aging and hypertension. RNA sequencing was conducted to search for senescence‐shifted transcripts in human umbilical vein endothelial cells (HUVECs). Small interfering RNA (siRNA), small‐molecule drugs, CRISPR/Cas9 techniques, and imaging were used to determine genes' function and contributions to age‐related phenotypes of the endothelial cell and blood vessel. Of 25 genes enriched in the term of “regulation of blood pressure,” NPRA was changed most significantly. The decreased NPRA expression was replicated in aortas of aged mice. The knockdown of NPRA promoted HUVEC senescence and it decreased expressions of protein kinase cGMP‐dependent 1 (PKG), sirtuin 1 (SIRT1), and endothelial nitric oxide synthase (eNOS). Suppression of NPRA also decreased the phosphorylation of AMP‐activated protein kinase (AMPK) as well as the ratio of oxidized nicotinamide adenine dinucleotide (NAD+)/reduced nicotinamide adenine dinucleotide (NADH) but increased the production of reactive oxygen species (ROS). 8‐Br‐cGMP (analog of cGMP), or AICAR (AMPK activator), counteracted the observed changes in HUVECs. The Npr1 +/− mice presented an elevated systolic blood pressure and their vessels became insensitive to endothelial‐dependent vasodilators. Further, vessels from Npr1 +/− mice increased Cdkn1a but decreased eNos expressions. These phenotypes were rescued by intravenously administrated 8‐Br‐cGMP and viral overexpression of human PKG, respectively. In conclusion, we demonstrate NPRA/PKG/AMPK as a novel and critical signaling axis in the modulation of endothelial cell senescence, vascular aging, and hypertension.

Proximal Tubule‐Specific Deletion of Angiotensin II Type 1a Receptors Augments Natriuretic Response to Atrial Natriuretic Peptide via NPRA/cGMP/NO Signaling Pathways in the Kidney

We and others have previously shown that the proximal tubules of the kidney expressed high levels of angiotensin II (Ang II) type 1 (AT1) receptors and atrial natriuretic peptide (ANP) type A receptors (NPRA). In the proximal tubules, Ang II binds and activate AT1 (AT1a) receptors to stimulate proximal tubule Na+reabsorption, whereas ANP binds and activates NPRA receptors to inhibit Ang II‐induced proximal tubule Na+reabsorption. Interactions between Ang II and ANP systems play important counteracting roles in the proximal tubules to control proximal tubule Na+ reabsorption in the kidney and maintain blood pressure homeostasis. It is not known, however, whether the hypotensive and natriuretic responses to ANP are altered by the absence of AT1 (AT1a) receptor‐mediated antinatriuretic action of Ang II in the proximal tubules. The present study used a novel mouse model with proximal tubule‐specific knockout of AT1a receptors in the kidney, PT‐Agtr1a−/−, to test the hypothesis that deletion of AT1a receptors selectively in the proximal tubules augments the hypotensive and natriuretic responses to ANP. Two groups (n=9‐12 per group) of adult male wild‐type and PT‐Agtr1a−/− mice were infused without (time controls) or with ANP via osmotic minipump (10 ng/min, i.p.) continuously for 2 weeks. Basal blood pressure was about 16 ± 3 mmHg lower (P<0.01), fractional proximal tubule Na+ reabsorption was lower (P<0.05), and 24 h urinary Na+ excretion was higher in PT‐Agtr1a−/− mice (P<0.01), compared with wild‐type mice. ANP infusion decreased blood pressure and increased the natriuretic response in PT‐Agtr1a−/− mice further by inhibiting proximal tubule Na+ reabsorption, compared with wild‐type controls (P<0.01). The augmented hypotensive and natriuretic responses to ANP infusion in PT‐Agtr1a−/− mice was associated with increased plasma and kidney cGMP levels (P<0.01), kidney NPRA and NPRC expression (P<0.05), total eNOS and phosphorylated eNOS proteins (P<0.01), and urinary NO excretion (P<0.01), respectively. Taken together, the results of the present study provide further new insights into the important physiological roles for intratubular Ang II/AT1a and ANP/NPRA in the proximal tubules to regulate proximal tubule Na+ reabsorption and help maintain blood pressure homeostasis.

Regulation of Blood Pressure and Renal Fibrosis by Class I Specific HDAC Inhibitor, Mocetinostat, in Npr1 Gene‐targeted Male and Female Mice

Mice lacking Npr1 (encoding guanylyl cyclase/natriuretic peptide receptor-A; GC-A/NPRA) exhibit hypertension, kidney disease, and heart failure; however, the epigenetic determinants regulating Npr1 expression and renal function are not well understood. The objective of this study was to investigate the effect of class I-specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103; MGCD) on NPRA expression and regulation of mean arterial pressure (MAP) and renal pathology. Adult male and female Npr1 haplotype (1-copy, Npr1+/-), wild-type (2-copy, Npr1+/+), and gene-duplicated (3-copy, Npr1++/+) mice were injected intraperitoneally with MGCD (2 mg/kg) at alternate days for 2-weeks. Treatment with MGCD significantly enhanced NPRA protein levels and GC activity in male and female mice in all three genotypes. MAP was monitored by telemetry recording. Sex-differences were observed in MAP as female mice had lower MAP (p < 0.01) than male mice, in all genotypes; whereas, 1-copy mice exhibited higher MAP than 2-copy mice. Treatments with MGCD distinctly reduced MAP in 1-copy and 2-copy male mice (untreated 1-copy, 125 ± 3 vs. treated 1-copy, 108 ± 3 mmHg; p < 0.001; untreated 2-copy, 98 ± 2 vs. treated 2-copy, 90 ± 2 mmHg; p < 0.01) and 1-copy and 2-copy female mice (untreated 1-copy, 117 ± 3 vs. treated 1-copy, 105 ± 2 mmHg; p < 0.001; untreated 2-copy, 90 ± 2 vs. treated 2-copy, 84 ± 2 mmHg; p < 0.01). Treatment with MGCD attenuated HDAC activity by 30% in male and 40% in female animals (p < 0.05). The Western blot analyses indicated that 1-copy male and female mice showed upregulation of alpha-smooth muscle actin (α-SMA; 39% and 29%, p < 0.05) and collagen 1 alpha 2 (COL1α2; 49% and 20%, p < 0.05) proteins, respectively, compared with 2-copy mice. Picrosirius red staining in renal sections showed significant collagen deposition in 1-copy mice compared with 2-copy animals of both sexes. MGCD reduced fibrosis by 30%-45% (p < 0.05) in treated 1-copy mice compared with untreated control mice in both sexes. The present results indicate that MGCD lowers MAP, and repairs renal fibrosis in male and female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in both genders.

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 &lt; 0.001) mice. Renal HDAC activity was considerably higher (p &lt; 0.05) in male mice than female mice; however, treatment with MGCD0103 attenuated (p &lt; 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.

Inhibition of HDAC1 and 2 Modulates the Expression and Signaling of Natriuretic Peptide Receptor A in Male and Female Gene‐targeted Mutant Mice

Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signaling activates natriuresis, diuresis, and vasodilatation, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit hypertension and kidney disorders, however, the mechanisms regulating Npr1 expression and function are not well understood. The objective of the present study was to elucidate the mechanisms of Npr1 expression and receptor function involving epigenetic regulatory mechanisms activated by class I histone deacetylase (HDAC) inhibitor, Mocetinostat (MGCD0103). Male and female (16–20 weeks old) Npr1 gene‐disrupted heterozygous (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+) and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice were injected intraperitoneally with MGCD0103 (2 mg/kg) at alternate days for 2‐weeks. The Western blot analysis showed that MGCD0103 significantly increased the renal NPRA protein levels in Npr1+/−, Npr1+/+, and Npr1++/+ mice groups compared with vehicle‐treated controls. Female mice showed increased levels of plasma cGMP compared with male mice and treatment with MGCD0103 significantly induced cGMP levels in the drug‐treated mice groups. The renal HDAC activity and HDAC1 and 2 protein levels in male mice were higher than the corresponding female mice in all the three genotypes. MGCD0103 treatment attenuated HDAC activity by 30%‐40% and HDAC1/2 protein levels in the treated mice groups. Male and female mice showed differential acetylation levels of H3‐K9 and H3‐K27 in the renal tissues and treatment with MGCD0103 significantly enhanced the acetylation levels in the treated mice groups. Both male and female Npr1+/− mice exhibited higher systolic blood pressure (SBP) compared with the corresponding wild type Npr1+/+ mice, whereas female Npr1+/− mice exhibited significantly lower SBP than male Npr1+/− mice. A significant decrease in SBP was observed in MGCD0103‐treated male and female Npr1+/− mice. The present results suggest that MGCD0103 epigenetically regulates NPRA expression in vivo in male and female Npr1+/− mice, via inhibition of HDAC1 and 2 protein expression and histone modifications, which will have important implications in the hypertension and renal disease states.Support or Funding InformationThis work was supported by NIH grants (HL057531 and HL062147) and by an Institutional Development Award (IDeA) from the NIGMS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of natriuretic peptides postprandially invivo and of their receptors in adipocytes by fatty acids invitro.

Natriuretic peptides (NPs) and their receptors gain attention regarding adipocyte function. It was the aim to investigate the expression of natriuretic peptide receptors NPR-A, NPR-B and NPR-C during adipocyte differentiation (AD), upon stimulation with fatty acids (FA), and in murine and human adipose tissue depots (AT) of patients undergoing bariatric surgery (n = 44). The postprandial regulation of NT-proANP and NT-proBNP levels was measured by ELISA and was studied in two cohorts of healthy individuals undergoing an oral glucose tolerance test (OGTT) (n = 100) and an oral lipid tolerance test (OLTT) (n = 100). Adipocyte mRNA expression was investigated by quantitative real-time PCR. During AD, an early expression pattern could be described for NPR-C, a bimodal expression for NPR-B and a late expression pattern for NPR-A. NPR-A and NPR-B expression was high in epididymal and subcutaneous AT but low in peri-renal AT of mice. NPR-C showed a differential expression profile. FA stimulation caused a significant and differential regulation of NPRs in adipocytes. Serum NT-proANP and NT-proBNP concentrations did not change during OGTT, whereas NT-proANP significantly declined during OLTT. Basal NT-proANP and NT-proBNP concentrations were positively correlated with each other and with FGF-19 and FGF-21 levels. Adipocytes and AT show a characteristic expression of NPRs. FA are able to regulate NPR expression differentially. There is a postprandial and negative regulation of serum NT-proANP concentrations after OLTT and of NPR-A after FA stimulation. Both effects could represent a novel hypothetical negative feedback mechanism on adipocyte lipolysis.

Aberrant pro-atrial natriuretic peptide/corin/natriuretic peptide receptor signaling is present in maternal vascular endothelium in preeclampsia.

Corin is a serine protease that converts pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), a cardiac hormone that regulates salt-water balance and blood pressure. ANP is degraded by natriuretic peptide receptor (NPR). This study was to determine if aberrant pro-ANP/corin/NPR signaling is present in maternal vascular system in preeclampsia. Maternal venous blood was obtained from 197 pregnant women (84 normotensive, 16 complicated with chronic hypertension (CHT), 11 mild and 86 severe preeclampsia). Plasma corin and pro-ANP concentrations were measured by enzyme-linked immunosorbent assay. Maternal subcutaneous fat tissue was obtained from 12 pregnant women with cesarean section delivery (6 normotensive and 6 preeclampsia). Vascular ANP and its receptors NPR-A, NPR-B, and NPR-C expression were examined by immunostaining of paraffin embedded subcutaneous fat tissue sections. Corin concentrations were significantly higher in mild (2.78 ± 0.67 ng/ml, p < .05) and severe (2.53 ± 0.18 ng/ml, p < .01) preeclampsia than in normotensive (1.58 ± 0.08 ng/ml) and CHT (1.55 ± 0.20 ng/ml) groups. Pro-ANP concentrations were significantly higher in CHT (1.59 ± 0.53 ng/ml, p < .05) and severe preeclampsia (1.42 ± 0.24 ng/ml, p < .01) than in normotensive (0.48 ± 0.06 ng/ml) and mild preeclampsia (0.52 ± 0.09 ng/ml) groups. ANP and NPR-B expression was undetectable in maternal vessels from normotensive and preeclamptic pregnancies, but reduced NPR-A expression and increased NPR-C expression was found in maternal vessel endothelium in preeclampsia. ANP is a vasodilator and NPR-C is a clearance receptor for ANP. The finding of upregulation of NPR-C expression suggests that circulating ANP clearance or degradation is increased in preeclampsia. These results also suggest that pro-ANP/corin/NPR signaling is dominant in the vascular system in preeclampsia.

PP.22.09] RENAL NATRIURETRIC SYSTEMS ALTERATION IN FRUCTOSE-OVERLOADED HYPERTENSIVE RATS

Objective: Metabolic and hemodynamic changes induced by a high fructose diet could be associated with an impairment of renal natriuretic systems, which contributes to sodium retention and hypertension. There is little evidence about renal dopamine (DA) and type-A natriuretic peptide (ANP) interaction on blood pressure elevation in this model. The aim of this study was to evaluate both natriuretic systems in fructose-overloaded rats. Design and method: Male Sprague Dawley rats were randomly assigned: a) Controls (C, tap water to drink) and b) Fructose overloaded: (F, 10% w/v of fructose to drink), treated during 4, 8 and 12 weeks (n = 8 rats/group/period). Urinary L-dopa and DA (HPLC); diuresis, creatinine and sodium were measured in 24 hour urine samples; and ANP (ELISA), triglycerides, glycemia, cholesterol, insulinemia, creatinine and sodium in plasma. Systolic blood pressure (SBP) was determined by tail-cuff and renal tissue protein expression of L-dopa transporter (LAT-2), D1R and NPR-A receptors by Western blot. Results: Fructose overload increased SBP (mmHg, C4: 121 ± 8 vs. F4: 145 ± 1*; C8: 130 ± 4 vs. F8: 161 ± 10#; C12: 133 ± 5 vs. F12: 163 ± 4#), which positively correlated (R2 = 0.78; p < 0.002) to urinary L-dopa/DA index (C4: 0.49 ± 0.05 vs. F4: 1.9 ± 0.09#; C8: 0.53 ± 0.06 vs. F8: 2.35 ± 0.10#; C12: 0.54 ± 0.07 vs. F12: 2.57 ± 0.20#). An elevation in cortex LAT-2 expression (arbitrary units) (C4: 1.00 ± 0.02 vs. F4: 1.30 ± 0.04*; C8: 1.00 ± 0.05 vs. F8: 1.23 ± 0.09*; C12: 1.00 ± 0.06 vs. F12: 1.40 ± 0.01*) and medulla (C8: 1.00 ± 0.01 vs. F8: 1.42 ± 0.02*; C12: 1.00 ± 0.01 vs. F12: 1.46 ± 0.04*); and a decrease D1R in cortex (C4: 1.00 ± 0.05 vs. F4: 0.72 ± 0.08*; C8: 1.00 ± 0.05 vs. F8: 0.75 ± 0.07*) were found. Plasma ANP decrease (ng/ml: C8: 1.61 ± 0.10 vs. F8: 1.30 ± 0.08*; C12: 1.65 ± 0.32 vs. F12: 1.04 ± 0.16*) along with a lower NPR-A expression in medulla (C4: 1.00 ± 0.05 vs. F4: 0.81 ± 0.02*; C8: 1.00 ± 0.04 vs. F8: 0.80 ± 0.03*; C12: 1.00 ± 0.03 vs. F12: 0.81 ± 0.01*) were also observed (*p < 0.05, #p < 0.01 vs. C). Conclusions: Fructose overload leads to an increase in the urinary L-dopa/DA index from 4th week of treatment that correlates positively with blood pressure elevation. Furthermore the decrease in the natriuretic systems DA and ANP seems to be related to the physiopathology of hypertension in this model.

Klf10 deficiency in mice exacerbates pulmonary inflammation by increasing expression of the proinflammatory molecule NPRA

KLF10 is a transforming growth factor (TGF)-β/Smad downstream regulated gene. KLF10 binds to the promoter of target genes and mimics the effects of TGF-β as a transcriptional factor. In our laboratory, we noted that Klf10 deficiency in mice is associated with significant inflammation of the lungs. However, the precise mechanism of this association remains unknown. We previously identified NPRA as a target gene potentially regulated by KLF10 through direct binding; NPRA knockout have known that prevented lung inflammation in a mouse model of allergic asthma. Here, we further explored the regulatory association between KLF10 and NPRA on the basis of the aforementioned findings. Our results demonstrated that KLF10 acts as a transcriptional repressor of NPRA and that KLF10 binding reduces NPRA expression in vitro. Compared with wild-type mice, Klf10-deficient mice were more sensitive to lipopolysaccharide or ovalbumin challenge and showed more severe inflammatory histological changes in the lungs. Moreover, Klf10-deficient mice showed pulmonary neutrophil accumulation. These findings collectively reveal the precise site where KLF10 signaling affects pulmonary inflammation by attenuating NPRA expression. They also verify the importance of KLF10 and atrial natriuretic peptide/NPRA in exerting influences on chronic pulmonary disease pathogenesis.

Diurnal gene expression of lipolytic natriuretic peptide receptors in white adipose tissue.

Disruption of the circadian rhythm can lead to obesity and cardiovascular disease. In white adipose tissue, activation of the natriuretic peptide receptors (NPRs) stimulates lipolysis. We have previously shown that natriuretic peptides are expressed in a circadian manner in the heart, but the temporal expression profile of their cognate receptors has not been examined in white adipose tissue. We therefore collected peri-renal white adipose tissue and serum from WT mice. Tissue mRNA contents of NPRs - NPR-A and NPR-C, the clock genes Per1 and Bmal1, and transcripts involved in lipid metabolism were quantified at 4-h intervals: in the diurnal study, mice were exposed to a period of 12 h light followed by 12 h darkness (n=52). In the circadian study, mice were kept in darkness for 24 h (n=47). Concomitant serum concentrations of free fatty acids, glycerol, triglycerides (TGs), and insulin were measured. Per1 and Bmal1 mRNA contents showed reciprocal circadian profiles (P<0.0001). NPR-A mRNA contents followed a temporal pattern (P=0.01), peaking in the dark (active) period. In contrast, NPR-C mRNA was expressed in an antiphase manner with nadir in the active period (P=0.007). TG concentrations in serum peaked in the active dark period (P=0.003). In conclusion, NPR-A and NPR-C gene expression is associated with the expression of clock genes in white adipose tissue. The reciprocal expression may thus contribute to regulate lipolysis and energy homeostasis in a diurnal manner.

Diurnal gene expression of lipolytic natriuretic peptide receptors in white adipose tissue

Disruption of the circadian rhythm can lead to obesity and cardiovascular disease. In white adipose tissue, activation of the natriuretic peptide receptors (NPRs) stimulates lipolysis. We have previously shown that natriuretic peptides are expressed in a circadian manner in the heart, but the temporal expression profile of their cognate receptors has not been examined in white adipose tissue. We therefore collected peri-renal white adipose tissue and serum from WT mice. Tissue mRNA contents of NPRs – NPR-A and NPR-C, the clock genes Per1 and Bmal1, and transcripts involved in lipid metabolism were quantified at 4-h intervals: in the diurnal study, mice were exposed to a period of 12 h light followed by 12 h darkness (n=52). In the circadian study, mice were kept in darkness for 24 h (n=47). Concomitant serum concentrations of free fatty acids, glycerol, triglycerides (TGs), and insulin were measured. Per1 and Bmal1 mRNA contents showed reciprocal circadian profiles (P<0.0001). NPR-A mRNA contents followed a temporal pattern (P=0.01), peaking in the dark (active) period. In contrast, NPR-C mRNA was expressed in an antiphase manner with nadir in the active period (P=0.007). TG concentrations in serum peaked in the active dark period (P=0.003). In conclusion, NPR-A and NPR-C gene expression is associated with the expression of clock genes in white adipose tissue. The reciprocal expression may thus contribute to regulate lipolysis and energy homeostasis in a diurnal manner.

Nandrolone decanoate induces cardiac and renal remodeling in female rats, without modification in physiological parameters: The role of ANP system.

Anabolic-androgenic steroids are misused, including women, but little is known about the cardiovascular effects of these drugs on females. Evaluated the effects of nandrolone decanoate (ND), physical exercise and estrogen deficiency on female rats. Female Wistar rats were divided into 8 groups: S and OVX: (SHAM: sham surgery; OVX: ovariectomy, vehicle), SE and OVXE (resistance exercise 5 times a week, vehicle), SD and OVXD (treated with ND, 20 mg/kg/week for 4 weeks); SDE and OVXDE. Treatments were initiated 21 days after surgery. The Bezold–Jarisch reflex was assessed by Phenylbiguanide administration. The right atrium, kidney, and serum were collected for molecular analyses by RT-PCR of atrial natriuretic peptide (ANP), A-type natriuretic peptide receptor (NPR-A) and NPR-C. ELISA assay to estradiol and testosterone concentrations. The gastrocnemius muscle, heart and kidney weights/tibia length were measured.Morphometric analysis of heart was made (H/E) and collagen content of heart and kidney were evaluated using Pirossirius Red. ND treatment increased ANP expression on atrium and decreased NPR-A expression in kidney. Physical exercise and ovariectomy did not alter this parameter. NPR-C level was reduced in the SDE and OVXDE. Renal and cardiac hypertrophy was observed after ND treatment, with collagen deposition. Plasma estrogen concentrations were reduced and serum testosterone concentrations were increased after ND treatment. ANP has an important role in modulating the cardiovascular effects of ND in females. Thismodulating may have occurred by the increasing ANP expression, reducing NPR-A and NPR-C expression levels, and changing sex hormone levels.

No intention to cheat: Coping with unintentional Anti-Doping Rule Violations

Anabolic-androgenic steroids are misused, including women, but little is known about the cardiovascular effects of these drugs on females.Evaluated the effects of nandrolone decanoate (ND), physical exercise and estrogen deficiency on female rats.Female Wistar rats were divided into 8 groups: S and OVX: (SHAM: sham surgery; OVX: ovariectomy, vehicle), SE and OVXE (resistance exercise 5 times a week, vehicle), SD and OVXD (treated with ND, 20 mg/kg/week for 4 weeks); SDE and OVXDE. Treatments were initiated 21 days after surgery. The Bezold–Jarisch reflex was assessed by Phenylbiguanide administration. The right atrium, kidney, and serum were collected for molecular analyses by RT-PCR of atrial natriuretic peptide (ANP), A-type natriuretic peptide receptor (NPR-A) and NPR-C. ELISA assay to estradiol and testosterone concentrations. The gastrocnemius muscle, heart and kidney weights/tibia length were measured. Morphometric analysis of heart was made (H/E) and collagen content of heart and kidney were evaluated using Pirossirius Red.ND treatment increased ANP expression on atrium and decreased NPR-A expression in kidney. Physical exercise and ovariectomy did not alter this parameter. NPR-C level was reduced in the SDE and OVXDE. Renal and cardiac hypertrophy was observed after ND treatment, with collagen deposition. Plasma estrogen concentrations were reduced and serum testosterone concentrations were increased after ND treatment.ANP has an important role in modulating the cardiovascular effects of ND in females. This modulating may have occurred by the increasing ANP expression, reducing NPR-A and NPR-C expression levels, and changing sex hormone levels.

All-Trans Retinoic Acid and Sodium Butyrate Enhance Natriuretic Peptide Receptor A Gene Transcription: Role of Histone Modification

The objective of the present study was to delineate the mechanisms of GC-A/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) expression in vivo. We used all-trans retinoic acid (ATRA) and histone deacetylase (HDAC) inhibitor, sodium butyrate (NaBu) to examine the expression and function of Npr1 using gene-disrupted heterozygous (1-copy; +/-), wild-type (2-copy; +/+), and gene-duplicated heterozygous (3-copy; ++/+) mice. Npr1(+/-) mice exhibited increased renal HDAC and reduced histone acetyltransferase (HAT) activity; on the contrary, Npr1(++/+) mice showed decreased HDAC and enhanced HAT activity compared with Npr1(+)(/+) mice. ATRA and NaBu promoted global acetylation of histones H3-K9/14 and H4-K12, reduced methylation of H3-K9 and H3-K27, and enriched accumulation of active chromatin marks at the Npr1 promoter. A combination of ATRA-NaBu promoted recruitment of activator-complex containing E26 transformation-specific 1, retinoic acid receptor α, and HATs (p300 and p300/cAMP response element-binding protein-binding protein-associated factor) at the Npr1 promoter, and significantly increased renal NPRA expression, GC activity, and cGMP levels. Untreated 1-copy mice showed significantly increased systolic blood pressure and renal expression of α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) compared with 2- and 3-copy mice. Treatment with ATRA and NaBu synergistically attenuated the expression of α-SMA and PCNA and reduced systolic blood pressure in Npr1(+/-) mice. Our findings demonstrate that epigenetic upregulation of Npr1 gene transcription by ATRA and NaBu leads to attenuation of renal fibrotic markers and systolic blood pressure in mice with reduced Npr1 gene copy number, which will have important implications in prevention and treatment of hypertension-related renal pathophysiological conditions.

Natriuretic Peptide Receptor A Signaling Regulates Stem Cell Recruitment and Angiogenesis: A Model to Study Linkage Between Inflammation and Tumorigenesis

Natriuretic peptide receptor A (NPRA), the signaling receptor for the cardiac hormone, atrial natriuretic peptide (ANP), is expressed abundantly in inflamed/injured tissues and tumors. NPRA deficiency substantially decreases tissue inflammation and inhibits tumor growth. However, the precise mechanism of NPRA function and whether it links inflammation and tumorigenesis remains unknown. Since both injury repair and tumor growth require stem cell recruitment and angiogenesis, we examined the role of NPRA signaling in tumor angiogenesis as a model of tissue injury repair in this study. In in vitro cultures, aortas from NPRA-KO mice show significantly lower angiogenic response compared to wild-type counterparts. The NPRA antagonist that decreases NPRA expression, inhibits lipopolysaccharide-induced angiogenesis. The reduction in angiogenesis correlates with decreased expression of vascular endothelial growth factor and chemokine (C-X-C motif) receptor 4 (CXCR4) implicating a cell recruitment defect. To test whether NPRA regulates migration of cells to tumors, mesenchymal stem cells (MSCs) were administered i.v., and the results showed that MSCs fail to migrate to the tumor microenvironment in NPRA-KO mice. However, coimplanting tumor cells with MSCs increases angiogenesis and tumorigenesis in NPRA-KO mice, in part by promoting expression of CXCR4 and its ligand, stromal cell-derived factor 1α. Taken together, these results demonstrate that NPRA signaling regulates stem cell recruitment and angiogenesis leading to tumor growth. Thus, NPRA signaling provides a key linkage between inflammation and tumorigenesis, and NPRA may be a target for drug development against cancers and tissue injury repair.

Abstract A12: Cardiac hormone receptor A, NPRA, as a prognostic marker and target for prostate cancer therapy

Abstract Management of prostate cancer (PCa), the third leading cause of death among American men, is limited by lack of prognostic markers and therapies for targeting androgen-independent PCa. Herein we report that NPRA, the receptor for the cardiac hormone atrial natriuretic peptide, is a potential prognostic marker and therapeutic target for PCa. NPRA is abundantly expressed in the androgen-independent PCa cell lines, PC3 and DU145, but not in normal PrEC cells and only weakly in RWPE and benign prostate hyperplasia (BPH) cells. NPRA is more highly expressed by the tumorigenic TRAMP-C1 and -C2 and PTEN-CaP2 PCa cell lines than the nontumorigenic TRAMP-C3 and PTEN-P2 PCa cell lines. NPRA showed positive correlation with clinical staging in a human PCa tissue microarray (TMA). Prostate tumor cells implanted in mice deficient in atrial natriuretic peptide receptor A (NPRA-KO) failed to grow, and down-regulation of NPRA by siNPRA or NPRA inhibitor, iNPRA, induced apoptosis in PCa cells and reduced tumor burden in mice. The mechanism of iNPRA-induced anti-PCa effects was linked to NPRA-induced expression of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine overexpressed in prostate cancer and associated with tumor survival. SiNPRA significantly reduced MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA expression correlated with MIF expression during PCa progression and treatment of TRAMP-C1 xenografts with iNPRA reduced MIF expression. MIF was also seen in the human PCa TMA. This suggests that NPRA promotes PCa development by regulating MIF and may represent a prognostic marker and target for PCa therapy. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A12.

M1680 Reduced Npra Expression Impairs Somatostatin-Induced Inhibition of Gastric Acid Secretion

Background & Aim: Autoimmune pancreatitis (AIP) is a newly recognized pancreatic disorder. Patients with AIP have several immunologic and histologic abnormalities specific to the disease, including increased levels of serum IgG4 and storiform fibrosis with infiltration of lymphocytes and IgG4-positive plasmacytes in the involved organs. Among the involved organs showing extrapancreatic lesions, bile duct is the most common, exhibiting sclerosing cholangitis (IgG4-SC). However, the role of IgG4 is unclear in the pancreas and involved organs. Recently, it has been reported that regulatory T cells (Tregs) are involved in both the development of various autoimmune diseases and the shift of B-cell toward IgG4 producing plasmacytes. For this report, we analyzed the infiltrated Tregs and IgG4-positive plasma cells in the pancreas and liver by immunohistochemistory and IL-10 producing Tregs by flowcytometry to clarify the role of IgG4 and Tregs in AIP. Subjects and Methods: The 6 AIP patients were obtained from Kansai Medical University. 6 cases of chronic pancreatitis were served as disease control. Hepatic tissue was sectioned from 16 IgG4-SC patients and 26 patients with PSC. All AIP and 14 IgG4-SC patients were diagnosed by Asian criteria. One IgG4-SC patient was diagnosed by Mayo (HISORt) criteria and other was diagnosed by biopsy. We studied infiltrating IgG4-positive cells and Tregs in the pancreas and liver by immunohistochemistry. IL-10 producing Tregs were analyzed from peripheral blood by flow cytometry. Results: In the pancreas, Tregs were increased in the pancreas with AIP (24.6±18.0 cells/HPF) compared with controls (5.1±4.3 cells/HPF). The numbers of IgG4positive plasma cells were significantly higher in AIP (18.6±10.3 cells/HPF) than in controls (1.1±0.7 cells/HPF). However, there were no significant differences in CD3+, CD4+, or CD79+ cells between the AIP (CD3: 63.1±27.1, CD4: 48.2±32.9, CD79: 24.1±9.2 cells/ HPF) and control sections (CD3: 73.6±37.1, CD4: 34.0±23.8, CD79: 14.5±14.6 cells/HPF). In the liver, the numbers of IgG4-positive plasma cells were significantly higher in IgG4SC (16.6±7.7 cells/HPF) than in PSC (4.0±0.7 cells/HPF). Tregs in the patients with IgG4SC (5.4±1.5 cells/HPF) were also significantly increased compared with PSC (2.0±0.3 cells/ HPF). In the patients with IgG4-SC, the numbers of infiltrated Tregs were significantly positively correlated with IgG4-positive plasma cells (R=0.75). In patients with the untreated AIP, IL-10 producing Tregs and IgG4 also positively correlated (R=0.53). Conclusion: In AIP, abundant infiltration of Tregs may affect the switching of B cells to IgG4-producing plasmacytes.