Pamela Harding

Cyclooxygenase-2 Mediates Increased Renal Renin Content Induced by Low-Sodium Diet

We hypothesized that neuronal nitric oxide synthase and cyclooxygenase-2, which both exist in the renal cortex, predominantly in the macula densa, play a role in the control of renal renin tissue content. We studied the possible role of neuronal nitric oxide synthase in regulating renal renin content by using mice in which the neuronal nitric oxide synthase gene has been disrupted (nNOS-/-) compared with its two progenitor strains, the 129/SvEv and the C57BL/6, to determine if the absence of neuronal nitric oxide synthase would result in decreased renal renin content or blunt the increase observed during low sodium intake. Renal renin content from cortical slices was determined in adult mice from all three strains maintained on a normal sodium diet. Renal renin content was significantly reduced in the nNOS-/- mice compared with the 129/SvEv and the C57BL/6 mice (3.11 +/- 0.23 versus 5.66 +/- 0.50 and 7.55 +/- 1.17 micrograms angiotensin l/mg dry weight, respectively; P < .005), suggesting that neuronal nitric oxide synthase may stimulate renal renin content under basal conditions. Neither selective pharmacological inhibition of neuronal nitric oxide synthase using 7-nitroindazole or disruption of the neuronal nitric oxide synthase gene affected the increase in renal content observed during dietary sodium restriction. The influence of cyclooxygenase-2 on renal renin content through a macula densa-mediated pathway was studied using a selective cyclooxygenase-2 inhibitor, NS398, in 129/SvEv mice. A low-sodium diet increased renal renin content from 6.97 +/- 0.52 to 11.59 +/- 0.79 micrograms angiotensin l/mg dry weight (P < .005); but this increase was blocked by NS398. In addition, treatment with NS398 reduced renin mRNA in response to a low-sodium diet. Thus, increased renal renin content in response to dietary sodium restriction appears to require the induction of cyclooxygenase-2, while neuronal nitric oxide synthase appears to affect basal but not stimulated renal renin content.

Effect of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline on DNA and Collagen Synthesis in Rat Cardiac Fibroblasts

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell entry into the S phase of the cell cycle and is normally present in human plasma. Ac-SDKP is exclusively hydrolyzed by ACE, and its plasma concentration is increased 5-fold after ACE inhibition in humans. We examined the effect of 0.05 to 100 nmol/L Ac-SDKP on 24-hour 3H-thymidine incorporation (DNA synthesis) by cardiac fibroblasts both in the absence and presence of 5% FCS. Captopril (1 &mgr;mol/L) was added in all cases to prevent the degradation of Ac-SDKP. Treatment of cardiac fibroblasts with 5% FCS increased thymidine incorporation from a control value of 12 469±594 to 24 598±1051 cpm (P <0.001). Cotreatment with 1 nmol/L Ac-SDKP reduced stimulation to control levels (10 373±200 cpm, P <0.001). We measured hydroxyproline content and incorporation of 3H-proline into collagenous fibroblast proteins and found that Ac-SDKP blocked endothelin-1 (10−8 mol/L)–induced collagen synthesis in a biphasic and dose-dependent manner, causing inhibition at low doses, whereas high doses had little or no effect. It also blunted the activity of p44/p42 mitogen-activated protein kinase in a biphasic and dose-dependent manner in serum-stimulated fibroblasts, suggesting that the inhibitory effect of DNA and collagen synthesis may depend in part on blocking mitogen-activated protein kinase activity. Participation of p44/p42 in collagen synthesis was confirmed, because a specific inhibitor for p44/p42 activation (PD 98059, 25 &mgr;mol/L) was able to block endothelin-1–induced collagen synthesis, similar to the effect of Ac-SDKP. The fact that Ac-SDKP inhibits DNA and collagen synthesis in cardiac fibroblasts suggests that it may be an important endogenous regulator of fibroblast proliferation and collagen synthesis in the heart. Ac-SDKP may participate in the cardioprotective effect of ACE inhibitors by limiting fibroblast proliferation (and hence collagen production), and therefore it would reduce fibrosis in patients with hypertension.

Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-induced target organ damage

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-alpha by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-alpha release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 +/- 1.06 mean fluorescent intensity for vehicle and 10.63 +/- 0.35 for Ac-SDKP; P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-alpha by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension.

Decreased intracellular calcium stimulates renin release via calcium-inhibitable adenylyl cyclase

Intracellular calcium and cAMP are the 2 second messengers that regulate renin release; cAMP stimulates renin release from juxtaglomerular (JG) cells, whereas increased intracellular calcium inhibits it. We hypothesized that decreased intracellular calcium acts by activating calcium-inhibitable isoforms of adenylyl cyclase, increasing cAMP, and stimulating renin secretion. We used a primary culture of JG cells isolated from C-57/B6 mice. Cells were plated to a density of 70% in serum-free medium and incubated for 2 hours with or without 100 &mgr;mol/L of the cytosolic calcium chelator 5′5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Intracellular cAMP content was 4.04±0.92 pM/mL per milligram of protein, and it increased by125±33% (P<0.01) with BAPTA-AM. Basal renin was 1.28±0.40 &mgr;g of angiotensin I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 182±62% (P<0.025). Western blots using an antibody that recognizes adenylyl cyclase types V and VI yielded a characteristic band of ≈135 kDa. When primary cultures of isolated JG cells were tested for the calcium-inhibitable isoforms of adenylyl cyclase, they showed intense focal cytoplasmic staining. Cells stained for both renin and adenylyl cyclase V/VI showed colocalization in the cytoplasm, primarily on the granules. An adenylyl cyclase inhibitor (SQ 22,536) completely blocked BAPTA-AM–stimulated renin release and JG cell cAMP content. We conclude that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Thus, decreased intracellular calcium stimulates adenylyl cyclase, resulting in cAMP synthesis and, consequently, renin release.

Adenylyl Cyclase Isoform V Mediates Renin Release From Juxtaglomerular Cells

We have shown previously that decreasing intracellular calcium in the juxtaglomerular cells increases both cAMP formation and renin release. We hypothesized that this is because of an interaction between intracellular calcium and the calcium-inhibitable isoform of adenylyl cyclase, type-V. We used primary cultures of juxtaglomerular cells isolated from C-57/B6 mice at 70% to 80% confluence. Western blots were performed on isolated juxtaglomerular cells using antibodies against either of the 2 calcium inhibitable isoforms of adenylyl cyclase, types-V and -VI. Only the antibody against adenylyl cyclase-V gave us a strong band at 120 kDa as expected. Immunolabeling in juxtaglomerular cells with confocal microscopy found immunofluorescence for the adenylyl cyclase-V–specific antibody compared with either negative controls or cells stained with the adenylyl cyclase-VI antibody. Reducing isolated juxtaglomerular intracellular calcium with 100 &mgr;mol/L of the cytosolic calcium chelator BAPTA-AM stimulated both cAMP (3.49±0.70 to 10.09±0.81 pmol/mL per milligram of protein; P<0.002) and renin release (1001.8±81.5 to 1648.0±139.1 ng of angiotensin I per milliliter per hour per milligram of protein; P<0.01). The selective adenylyl cyclase-V inhibitor NKY80 completely blocked both BAPTA-AM–stimulated cAMP formation and renin release. We conclude that lowering intracellular calcium is permissive, allowing an increased activity of the calcium-inhibitable isoform adenylyl cyclase-V (but not adenylyl cyclase-VI) in the juxtaglomerular cell, producing cAMP, which stimulates renin secretion.

Expression and Function of the Calcium-Sensing Receptor in Juxtaglomerular Cells

Calcium-sensing receptors sense and translate micromolar changes of extracellular calcium into changes in intracellular calcium. Renin, a component of the renin-angiotensin system, is synthesized by, stored in, and released from the juxtaglomerular cells through a cAMP-dependent pathway. Increased intracellular calcium inhibits the adenylyl cyclase isoform type V, cAMP formation, and renin release from juxtaglomerular cells. We hypothesized that calcium-sensing receptors are expressed in juxtaglomerular cells and mediate changes in intracellular calcium and renin release. To test this we used primary cultures of isolated mouse juxtaglomerular cells in which we ran RT-PCR, Western blots, and immunofluorescence. RT-PCR showed a positive band at the expected 151 bp consistent with calcium-sensing receptor. Western blots showed a 130- to 150-kDa band confirming the calcium-sensing receptor in juxtaglomerular cells. Immunofluorescence and confocal microscopy using 2 different antibodies against the calcium-sensing receptor in juxtaglomerular cells showed positive fluorescence in the juxtaglomerular cells, which also had positive labeling for renin. To test whether calcium-sensing receptors regulate renin release, juxtaglomerular cells were incubated with a calcium-sensing receptor agonist, the calcimimetic cinacalcet-HCl, at concentrations of 50 and 1000 nmol/L in 0.25 mmol/L of calcium medium. Cinacalcet-HCl decreased juxtaglomerular cell cAMP formation to 47.3±6.8% and 44.2±9.7% of basal, respectively (P<0.001), and decreased renin release from 541.9±86.2 to 364.6±64.1 (P<0.05) and 279.6±56.9 (P<0.005) ng of angiotensin I per milliliter per hour per milligram of protein, respectively. We conclude that juxtaglomerular cells express the calcium-sensing receptor and that their activation leads to inhibition of adenylyl cyclase-V activity, decreasing cAMP formation and suppressing renin release.

Chronic cyclooxygenase-2 inhibition blunts low sodium-stimulated renin without changing renal haemodynamics.

Background Cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, is found in the macula densa of the renal cortex and is upregulated by dietary sodium restriction. Because of this discrete cortical localization, we hypothesized that COX-2 plays a role in the chronic stimulation of renin via the macula densa pathway. Methods We examined the effect of the selective COX-2 inhibitor NS 398 in male Sprague–Dawley rats. Results A low sodium diet (0.02% NaCl) for 14 days elevated plasma–renin activity (PRA) nine-fold, from 6.1 ± 2.0 to 54.9 ± 6.5 ng angiotensin I (Ang I)/ml per h (P < 0.0001). Selective COX-2 inhibition with NS 398 had no effect on PRA in animals on normal sodium (5.1 ± 1.3 ng Ang I/ml per h), but decreased PRA by 41% in sodium-restricted rats, to 33.3 ± 3.6 ng Ang I/ml per h (P < 0.05). Chronic treatment with NS 398 did not decrease renal renin content (31.8 ± 1.8 versus 33.5 ± 2.6 ng Ang I/mg per h, with NS 398 versus controls), nor did it influence systemic blood pressure or renal haemodynamics. Neither urinary sodium excretion nor prostaglandin (PG)E2 excretion was altered in rats given NS 398. Chronic treatment with the non-selective COX inhibitor indomethacin during sodium restriction over 5 days reduced PRA by 35%, from 29.36 ± 4.81, to 19.13 ± 2.88 ng Ang I/ml per h (P < 0.05). Indomethacin had no effect on blood pressure or renal blood flow but reduced urinary PGE2 excretion by 70%. Conclusions One component of the chronic stimulation of PRA by dietary sodium restriction via the macula densa pathway appears to involve the induction of COX–2.

Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa

Macula densa cells produce superoxide (O2−) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa and the role of each one in NaCl-induced O2− production. To identify which isoforms are expressed, we applied single-cell RT-PCR to macula densa cells isolated by laser capture microdissection and to MMDD1 cells (a macula densa-like cell line). The captured cells expressed neuronal NOS (marker of macula densa), NOX2, and NOX4 but not NOX1. Expression of the NOXs and neuronal NOS was essentially identical in the MMDD1 cells. Thus, we used MMDD1 cells to investigate which isoform is responsible for NaCl-induced O2− production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O2− exposed to low-salt solution (LS; 70 mmol/L of NaCl) or high-salt solution (HS; 140 mmol/L of NaCl). Exposing control cells (scrambled small-interfering RNA) to HS increased O2− concentrations from 0.75±0.28 to 1.48±0.46 U/min per 105 cells in LS and HS, respectively (P<0.001). Inhibiting NOX2 blocked the HS-induced increase in O2− (0.62±0.39 versus 0.76±0.31 U/min per 105 cells in LS and HS groups, respectively). Blocking NOX4 did not affect HS-induced O2− levels. O2− levels in the control cells during LS and HS were 0.80±0.30 and 1.56±0.49 U/min per 105 cells, respectively (P<0.001); whereas O2− levels in NOX4-small-interfering RNA–treated cells during LS and HS were 0.40±0.25 and 1.26±0.51 U/min per 105 cells, respectively (P<0.001). We conclude that, whereas macula densa cells express the NOX2 and NOX4 isoforms, NOX2 is primarily responsible for NaCl-induced O2− generation.

Role of prolylcarboxypeptidase in angiotensin II type 2 receptor-mediated bradykinin release in mouse coronary artery endothelial cells.

Activation of angiotensin II type 2 receptors (AT2R) causes the release of kinins, which have beneficial effects on the cardiovascular system. However, it is not clear how AT2R interact with the kallikrein-kinin system to generate kinins. Prolylcarboxypeptidase is an endothelial membrane-bound plasma prekallikrein activator that converts plasma prekallikrein to kallikrein, leading to generation of bradykinin from high-molecular-weight kininogen. We hypothesized that AT2R-induced bradykinin release is at least in part mediated by activation of prolylcarboxypeptidase. Cultures of mouse coronary artery endothelial cells were transfected with an adenoviral vector containing the AT2R gene (Ad-AT2R) or green fluorescent protein only (Ad-GFP) as control. We found that overexpression of AT2R increased prolylcarboxypeptidase mRNA by 1.7-fold and protein 2.5-fold compared with Ad-GFP controls. AT2R overexpression had no effect on angiotensin II type 1 receptor mRNA. Bradykinin release was increased 2.2-fold in AT2R-transfected cells. Activation of AT2R by CGP42112A, a specific AT2R agonist, increased bradykinin further in AT2R-transfected cells. These effects were diminished or abolished by AT2R blockade or a plasma kallikrein inhibitor. Furthermore, blocking prolylcarboxypeptidase with a small interfering RNA partially but significantly reduced bradykinin release by transfected AT2R cells either at the basal condition or when stimulated by the AT2R agonist CGP42112A. These findings suggest that overexpression of AT2R in mouse coronary artery endothelial cells increases expression of prolylcarboxypeptidase, which may contribute to kinin release.

Reduced Cardiac Remodeling and Function in Cardiac-Specific EP4 Receptor Knockout Mice With Myocardial Infarction

We have shown previously that cyclooxygenase-2 inhibition reduces cardiac hypertrophy and fibrosis postmyocardial infarction (MI) in a mouse model and that prostaglandin E2 stimulates cardiomyocyte hypertrophy in vitro through its EP4 receptor. Because the role of cardiac myocyte EP4 in cardiac function and hypertrophy in vivo is unknown, we generated mice lacking EP4 only in cardiomyocytes (CM- EP4 knockout [KO]). Twelve- to 14-week–old mice were evaluated using echocardiography and histology. There were no differences in ejection fraction, myocyte cross-sectional area, and interstitial collagen fraction between KO mice and littermate controls. To test the hypothesis that EP4 is involved in cardiac remodeling after MI, we induced MI by ligating the left anterior descending coronary artery. Two weeks later, the mice were subjected to echocardiography, and hearts were removed for histology and Western blot. There was no difference in infarct size between KO mice and controls; however, KO mice showed less myocyte cross-sectional area and interstitial collagen fraction than controls. Also, CM-EP4 KO mice had reduced ejection fraction. Because the transcription factor Stat-3 is involved in hypertrophy and protection from ischemic injury, we tested whether it was activated in control and KO mouse hearts after MI. Western blot indicated that Stat-3 was activated in control hearts after MI but not in KO hearts. Thus, CM-EP4 deletion decreased hypertrophy, fibrosis, and activation of Stat-3. However, cardiac function was unexpectedly worsened in these mice. We conclude that cardiac myocyte EP4 plays a role in hypertrophy via activation of Stat-3, a process that seems to be cardioprotective.