Kailash N. Pandey

BACKGROUND: Atrial and brain natriuretic peptides activate GC-A/NPRA (guanylyl cyclase-A/natriuretic peptide receptor-A) and regulate blood pressure and electrolyte homeostasis. Renal tubule (RT) dysfunction results in decreased kidney function and increased blood pressure. We determined the sex-specific consequences of RT cell-specific deletion of Npr1 (encoding NPRA) on blood pressure and renal hemodynamics. METHODS: Mice were generated with inducible RT knockout by breeding lox-flanked (flox/flox: f/f ) exons 1 to 2 in Npr1 with mice expressing the Pax8-rtTA-LC-1- Cre transgene. Doxycycline-treated RT cell-specific Npr1 knockout ( Npr1 f/ −), heterozygous (HT; Npr1 f/+ ), and wild-type ( Npr1 f/f ) male and female mice were used. Proximal tubule, distal tubule, and cortical collecting duct were isolated from RT- Npr1 knockout mice and did not express Npr1 mRNA or protein. RESULTS: RT cell-specific male knockout and HT mice showed significantly lower glomerular filtration rate, creatinine clearance, and urinary sodium excretion than female mice, compared with wild-type mice. The effect of Npr1 deletion was more severe on high-salt diets than their normal-diet counterparts. Loss of Npr1 in RT segments significantly increased systolic blood pressure and mean arterial pressure in a sex-specific manner. Mutant male mice showed higher total urinary protein and albumin-creatinine ratios than female mice. On a high-salt diet, male knockout and HT mice showed greater salt sensitivity than female mice. CONCLUSIONS: Loss of Npr1 along the nephron tubules leads to arterial hypertension and abnormal renal functional hemodynamic changes that are more pronounced in male mice compared with female mice.

Our results demonstrate that the podocyte-specific deletion of Npr1 showed increased blood pressure (BP) and altered biomarkers of renal functions, with greater magnitudes in animals fed a high-salt diet in a sex-dependent manner. The results suggest a direct and sex-dependent effect of Npr1 ablation in podocytes on the regulation of BP and renal function and reveal that podocytes may be considered an important target for the ANP-BNP/NPRA/cGMP signaling cascade.

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.

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.

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.

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.

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)-β1 receptor (TGF-β1R) 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-βRI (4.3-fold), TGF-βRII (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-β1-mediated SMAD-dependent pathway.

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.

Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP) bind and activate cognate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and play pivotal roles in the regulation of blood pressure, renal functions and cardiac and vascular homeostasis. The disruption of the Npr1 gene (coding for GC‐A/NPRA) in mice exhibits hypertension and provokes, renal dysfunction, and congestive heart failure; however, the underlying mechanisms are not yet precisely determined. The objective of present study was to investigate whether Npr1 gene plays a critical role in regulating glucose homeostasis in Npr1 gene‐disrupted hypertensive mice. The adult male and female (14‐18 weeks) Npr1 gene‐disrupted haplotype ( Npr 1 ^+/‐ , 1‐copy), wild‐type ( Npr1 ^+/+ , 2‐copy), and gene‐duplicated homozygous ( Npr1 ^{+ +/+ +} , 4‐copy) mice were fasted overnight (16 h) and given free access to water. The mice were administrated both orally and intraperitoneally with glucose (2 g/kg body weight) to determine the oral glucose tolerance test (OGTT) and the intraperitoneal glucose tolerance test (IPGTT). 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 2 g/kg body weight of glucose resulted in a significantly greater levels of increased in glucose in Npr1 ^+/‐ (1‐copy) mice than Npr1 ^+/+ (2‐copy), and Npr1 ^{+ +/+ +} (4‐copy) male and female mice. Systolic blood pressure (SBP) was determined by non‐invasive tail‐cuff method using Visitech 2000 computerized system. SBP was significantly greater in 1‐copy male mice (130 + 4 mmHg) and 1‐copy female mice (122 + 3 mmHg) than 2‐copy male (100 + 3 mmHg) and 2‐copy female mice (95 + 2 mmHg), respectively. Similarly, SBP was also significantly lower in 4‐copy male mice (93 + 2) and 4‐copy female mice (88 + 2 mmHg) than 2‐copy male and female mice, respectively. We observed that after glucose administration with OGTT, the increase in plasma glucose levels were significantly lower than the glucose administration by IPGTT. These results revealed that Npr1 gene markedly prevented a steep rise of blood glucose levels after glucose challenge and ameliorated glucose intolerance in wild‐type and gene‐duplicated mice, suggesting that glucose homeostasis may be regulated by Npr1 gene and the loss of NPRA exerts detrimental effects on renal and cardiac functions in mutant mice.

Cell‐cycle regulatory proteins (p21 ^Cip1 /p27 ^Kip1 ) inhibit cyclin and cyclin‐dependent kinase (CDK) complex that promotes fibrosis and hypertrophy. The present study examined the role of CDK blockers, p21 ^Cip1 /p27 ^Kip1 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, p21 ^Cip1 , and p27 ^Kip1 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‐β1) 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 p21 ^Cip1 and p27 ^Kip1 .