Ramu Periyasamy’s research while affiliated with Tulane University and other places

Targeted-deletion of Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor-A, GC-A/NPRA) exhibits hypertrophic and proliferative effects in target organs of Npr 1 gene-knockout mice. Fibrosis and hypertrophy are regulated by p21 Cip1 and p27 Kip1 , cell-cycle regulatory proteins that inhibit target cyclin and cyclin-dependent kinase (cyclin-CDK) complex. We examined the activation of CDK blocker (p21 Cip1 /p27 Kip1 ) in Npr1 gene-knockout (0-copy; Npr 1 -/- ) mice and guanylyl cyclase (GC) inhibitor, A71915-treated and cGMP-dependent protein kinase (cGK) inhibitor, Rp-8-Br-cGMPS (Rp)-treated wild-type 2-copy ( Npr 1 +/+ ) and gene-duplicated 4-copy ( Npr 1 ++/++ ) mice. Blood pressure (BP) was significantly higher in 0-copy mice (138.6 ± 3.1 mmHg) and lower in 4-copy mice (86.0 ± 2.8 mmHg) than 2-copy mice (102.2 ± 1.7 mmHg). Treatment with A71915 and Rp showed significant changes in BP in 2-copy mice but caused only small increase in 4-copy mice. We found a significant decrease in renal cGMP levels with diminished cGK activity in 0-copy mice (p<0.0001) as well as A71915-treated (p<0.001) and Rp-treated (p<0.05) 2-copy and 4-copy mice as compared with controls animals. While significant activation of p-Erk1/2 (3-fold), p-p38MAPK (4-fold), p21 Cip1 (6-fold), and p27 Kip1 (5-fold) occurred in 0-copy, A71915-treated 2-copy, and A71915-treated 4-copy mice but Rp treatment caused minimal changes compared to control mice. There were significant increases in the proinflammatory cytokines, including TNF-α (6-fold), and IL-6 (3-fold) and profibrotic cytokine TGF-β1 (4-fold) in plasma and kidneys of 0-copy and A791915-treated 2-copy mice, but less in A71915-treated 4-copy mice than controls. Progressive renal pathology, including fibrosis, mesangial matrix expansion, tubular hypertrophy, and perivascular infiltration were significantly scored in 0-copy and A71915-treated 2-copy mice, but did so minimally in 4-copy mice compared with controls. The present results suggest that Npr1 has a pivotal role in inhibiting the renal fibrosis and pathology and exerts renal protective effects through the cGMP/cGK axis by repressing the CDK inhibitors, p21 Cip1 and p27 Kip1 . This work was supported by NIH grant (HL062147).

Cell‐cycle regulatory proteins (p21Cip1/p27Kip1) inhibit cyclin and cyclin‐dependent kinase (CDK) complex that promotes fibrosis and hypertrophy. The present study examined the role of CDK blockers, p21Cip1/p27Kip1 in the progression of renal fibrosis and dysfunction using Npr1 (encoding guanylyl cyclase/natriuretic peptide receptor‐A, GC‐A/NPRA) gene‐knockout (0‐copy; Npr1−/−), 2‐copy (Npr1+/+), and 4‐copy (Npr1++/++) mice treated with GC inhibitor, A71915 and cGMP‐dependent protein kinase (cGK) inhibitor, (Rp‐8‐Br‐cGMPS). A significant decrease in renal cGMP levels and cGK activity was observed in 0‐copy mice and A71915‐ and Rp‐treated 2‐copy and 4‐copy mice compared with controls. An increased phosphorylation of Erk1/2, p38, p21Cip1, and p27Kip1 occurred in 0‐copy and A71915‐treated 2‐copy and 4‐copy mice, while Rp treatment caused minimal changes than controls. Pro‐inflammatory (TNF‐α, IL‐6) and pro‐fibrotic (TGF‐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 p21Cip1 and p27Kip1.

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

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 Information This 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 .

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 Information NIH/NHLBI: HL57531 NIH/NHLBI: HL62147 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Introduction: Mice lacking functional guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) exhibit hypertension, kidney disease, and heart failure. In this study, we investigated the effect of butyric acid, a histone deacetylase (HDAC) inhibitor and all-trans retinoic acid (ATRA) hybrid drug (ATRA-BA) on the attenuation of renal inflammation and fibrosis in Npr1 haplotype mice. Methods and results: Adult (18-20 week old) male Npr1 haplotype (1-copy; Npr1 +/-; n=10), wild-type (2-copy; Npr1 +/+ ; n=10), and gene-duplicated (3-copy; Npr1 ++/+ ; n=10) mice were treated with ATRA-BA (1.0 mg/kg/day) by intraperitoneal injections for 2-weeks. A significant decrease in systolic blood pressure was observed in ATRA-BA-treated Npr1 +/- mice (treated, 113.3 ± 1.5 vs. control, 130.4 ± 1.9; p < 0.01; n=10). Treatments with ATRA-BA showed a marked reduction in tubulo-interstitial fibrosis (50%, p < 0.001) and a decrease in renal col 1α levels (treated, 25.9 ± 1.2 vs. control, 57.2 ± 2.9, p < 0.001) in Npr1 +/- mice. Significant reduction was observed in renal IL-6 (55%, p < 0.001) and MCP-1 (61%, p < 0.01) protein levels in ATRA-BA-treated Npr1 +/- mice compared with untreated controls. There was significantly increased renal NF-κB (p65) protein expression and activity in Npr1 +/- mice; however, reduced levels were observed in Npr1 +/++ mice compared with wild-type mice. Renal NF-κB (p65) activity decreased (52%, p < 0.01) in drug-treated Npr1 +/- mice compared with controls. Treatment with ATRA-BA facilitated dissociation of HDAC1/2 from STAT1 protein complex and enhanced STAT1 acetylation. Furthermore, acetylated STAT1 formed complex with NF-κB (p65), thereby inhibiting its activity. Higher urinary albumin creatinine ratio and urinary protein was detected in Npr1 +/- mice compared with Npr1 +/+ mice and a complete reversal was observed in drug-treated Npr1 +/- animals. Conclusions: The present results demonstrate that ATRA-BA reduces blood pressure and repairs the abnormal renal pathology in haplotype Npr1 +/- mice by modulating STAT1, HDAC1/2, NF-κB (p65) interactions. The findings will be important in developing the strategies for treating the hypertension and renal pathological conditions.

The objective of the present study was to determine the effect of a hybrid drug of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all-trans retinoic acid (ATRA) on the attenuation of renal inflammation and fibrosis in Npr1 gene-disrupted mutant mice. Adult (18-20 week old) male Npr1 gene-disrupted heterozygous (1-copy; Npr1 +/- ) wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were treated with ATRA-NaBu hybrid drug (1.0 mg/kg/day) by intraperitoneal injections for 2-weeks. A significant decrease in systolic blood pressure was observed in ATRA-NaBu-treated haplotype Npr1 +/- mice compared with untreated controls (treated, 113.3 ± 1.5 vs. control, 130.4 ± 1.9; p < 0.01). After treatment with ATRA-NaBu, a marked reduction in tubulo-interstitial fibrosis (50%, p < 0.001) and decreased renal collagen type I alpha 2 expression by 55% (treated, 25.9 ± 1.2 vs. control, 57.2 ± 2.9, p < 0.001) was observed in Npr1 +/- mice. Treatment with ATRA-NaBu also increased creatinine clearance (ml/24 h) in Npr1 +/- mice (245.7 ± 24.3 vs. control, 83.8 ± 3.9). Similarly, higher urinary albumin to creatinine ratio was detected in Npr1 +/- mice (0.84 ± 0.03) vs. control (0.35 ± 0.03; p < 0.01) and a complete reversal was observed in drug-treated Npr1 +/- mice (0.39 ± 0.05). Significant decreases were observed in renal (pg/mg protein) tumor necrosis factor-alpha (TNF-α) (4.1 ± 0.7 vs. control, 27.2 ± 2.6; p < 0.01) and interleukin (IL)-6 (11.8 ± 0.8 vs. control, 59.9 ± 3.6; p < 0.01) in ATRA-NaBu-treated Npr1 +/- mice. Western blot analyses showed significant reduction in renal TNF-α and IL-6 protein expression by 54%, (p < 0.001) and 61%, (p < 0.01), respectively, in ATRA-NaBu-treated Npr1 +/- mice. There was 49% increase in renal NF-κB (p65) DNA binding activity in Npr1 +/- mice and 51% lower activity in Npr1 +/++ mice compared with wild-type mice. Western blot analysis revealed distinctly higher levels of renal NF-κB (p65) protein expression in Npr1 +/- mice and reduced levels in Npr1 +/++ mice compared with wild-type controls. The present results provide direct evidence that ATRA-NaBu hybrid drug acts as a potent anti-inflammatory agent, which will have important implications in the pathophysiology of renal injury and hypertension.

Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA)/cGMP signaling plays a well‐defined role in the regulation of blood pressure and blood volume. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit hypertension, kidney disease, and heart failure. The objective of the present study was to determine the combined effect of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all‐trans retinoic acid (ATRA) on enhanced renal functions and attenuation of renal fibrosis in Npr1 gene‐disrupted mutant mice. Adult (18–20 week old) male Npr1 gene‐disrupted heterozygous (1‐copy; Npr1 +/− ), wild‐type (2‐copy; Npr1 +/+ ), and gene‐duplicated (3‐copy; Npr1 ++/+ ) mice were treated by injecting ATRA‐NaBu hybrid drug (1.0 mg/kg/day) intraperitoneally for 2‐weeks. A marked attenuation in tubulo‐interstitial fibrosis was observed in Npr1 +/− mice after treatment with ATRA‐NaBu (50%, p < 0.001). Western blot analyses exhibited reduction in renal expression of collagen type I alpha 2 and transforming growth factor‐beta in ATRA‐NaBu‐treated Npr1 +/− mice compared with vehicle‐treated control animals. A significant decrease in systolic blood pressure was observed in ATRA‐NaBu‐treated Npr1 +/− mice. The ATRA‐NaBu treatment enhanced plasma cGMP levels in Npr1 +/ , Npr1 +/+ , and Npr1 ++/+ mice. Creatinine clearance (CCr) was significantly reduced by 62% in Npr1 +/− mice and 2‐fold higher in Npr1 +/++ mice compared with wild‐type control mice. Treatment with ATRA‐NaBu hybrid markedly enhanced CCr in Npr1 +/− mice comparable with vehicle‐treated group. Proteinuria was detected in Npr1 +/− mice as evidenced by significantly (p < 0.01) high urinary albumin content compared with Npr1 +/+ mice. A complete reversal of proteinuria was observed in ATRA‐NaBu hybrid‐treated Npr1 +/− mice group compared with Npr1 +/− control mice. Moreover, the increased histone deacetylase activity in Npr1 +/− mice was significantly reduced by ATRA‐NaBu treatment compared with untreated Npr1 +/− control mice. The present results provide direct evidence that ATRA‐NaBu acts as a potent antifibrotic agent and repairs the renal pathology in Npr1 +/− mice, which will have important implications in prevention of hypertension‐related renal pathophysiological conditions. Support or Funding Information This work was supported by the NIH grants R01HL057531 and R01HL062147.

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

The objective of the present study was to delineate the mechanisms of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) expression in vivo. We utilized 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 Ets-1, retinoic acid receptor α, and HATs (p300 and p300/CBP 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 (SBP) and renal expression of α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) compared with 2-copy and 3-copy mice. Treatment with ATRA and NaBu synergistically attenuated the expression of α-SMA and PCNA and reduced SBP 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 SBP in mice with reduced Npr1 gene copy number, which will have important implications in prevention and treatment of hypertension-related renal pathophysiological conditions.