Folke Schmidt

NF-κB Inhibition Ameliorates Angiotensin II–Induced Inflammatory Damage in Rats

We recently reported that the activation of nuclear factor-kappaB (NF-kappaB) promotes inflammation in rats harboring both human renin and angiotensinogen genes (double-transgenic rats [dTGR]). We tested the hypothesis that the antioxidant pyrrolidine dithiocarbamate (PDTC) inhibits NF-kappaB and ameliorates renal and cardiac end-organ damage. dTGR feature hypertension, severe renal and cardiac damage, and a 40% mortality rate at 7 weeks. Electrophoretic mobility shift assay showed increased NF-kappaB DNA binding activity in hearts and kidneys of dTGR. Chronic PDTC (200 mg/kg SC) treatment decreased blood pressure (162+/-8 versus 190+/-7 mm Hg; P=0.02) in dTGR compared with dTGR controls. The cardiac hypertrophy index was also significantly reduced (4.90+/-0.1 versus 5.77+/-0.1 mg/g; P 95% (2.5+/-0.8 versus 57. 1+/-8.7 mg/d; P<0.001) and prevented death. Vascular injury was ameliorated in small renal and cardiac vessels. Electrophoretic mobility shift assay showed that PDTC inhibited NF-kappaB binding activity in heart and kidney, whereas AP-1 activity in the kidney was not decreased. dTGR exhibited increased left ventricular c-fos and c-jun mRNA expression. PDTC treatment reduced c-fos but not c-jun mRNA. Immunohistochemistry showed increased p65 NF-kappaB subunit expression in the endothelium and smooth muscle cells of damaged small vessels, as well as infiltrating cells in glomeruli, tubules, and collecting ducts of dTGR. PDTC markedly reduced the immunoreactivity of p65. PDTC also prevented the NF-kappaB-dependent transactivation of the intercellular adhesion molecule ICAM-1 and inducible nitric oxide synthase. Monocyte infiltration was markedly increased in dTGR kidneys and hearts. Chronic treatment reduced monocyte/macrophage infiltration by 72% and 64%, respectively. Thus, these results demonstrate that PDTC inhibits NF-kappaB activity, ameliorates inflammation, and protects against angiotensin II-induced end-organ damage.

Monocyte Infiltration and Adhesion Molecules in a Rat Model of High Human Renin Hypertension

Hypertension and kidney damage in the double transgenic rat (dTGR) harboring both human renin and human angiotensinogen genes are dependent on the human components of the renin angiotensin system. We tested the hypothesis that monocyte infiltration and increased adhesion molecule expression are involved in the pathogenesis of kidney damage in dTGR. We also evaluated the effects of long-term angiotensin-converting enzyme (ACE) inhibition, AT1 blockade, and human renin inhibition on monocyte recruitment and inflammatory response in dTGR. Systolic blood pressure and 24-hour albuminuria were markedly increased in 7-week-old dTGR as compared with age-matched normotensive Sprague Dawley rats. We found a significant monocyte/macrophage infiltration in the renal perivascular space and increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the interstitium, intima, and adventitia of the small renal vessels. alphaLbeta2 integrin and alpha4beta1 integrin, the corresponding ligands for ICAM-1 and VCAM-1, were also found on infiltrating monocytes/macrophages. The expression of plasminogen activator inhibitor-1 and fibronectin in the kidneys of dTGR were increased and distributed similarly to ICAM-1. In 4-week-old dTGR, long-term treatment with ACE inhibition (cilazapril), AT1 receptor blockade (valsartan), and human renin inhibition (RO 65-7219) (each drug 10 mg/kg by gavage once a day for 3 weeks) completely prevented the development of albuminuria. However, only cilazapril and valsartan were able to decrease blood pressure to normotensive levels. Interestingly, the drugs were all equally effective in preventing monocyte/macrophage infiltration and the overexpression of adhesion molecules, plasminogen activator inhibitor-1, and fibronectin in the kidney. Our findings indicate that angiotensin II causes monocyte recruitment and vascular inflammatory response in the kidney by blood pressure-dependent and blood pressure-independent mechanisms. ACE inhibition, AT1 receptor blockade, and human renin inhibition all prevent monocyte/macrophage infiltration and increased adhesion molecule expression in the kidneys of dTGR.

Hypertension-Induced End-Organ Damage: A New Transgenic Approach to an Old Problem

Angiotensin (Ang) II-induced organ damage has fascinated students of hypertension since the work of Wilson and Byrom. We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, and VEGF expression. The expression of TGF-beta and deposition of extracellular matrix proteins follows, which is accompanied by fibrinoid vasculitis in small vessels of the heart and kidneys. Angiotensin-converting enzyme inhibitors and AT1 receptor blockers each lowered blood pressure and shifted pressure natriuresis partially leftward by different mechanisms. When combined, they normalized blood pressure, pressure natriuresis, and protected from vasculopathy completely. Renin inhibition lowered blood pressure partially, but protected from vasculopathy completely. Endothelin receptor blockade had no influence on blood pressure but protected from vasculopathy and improved survival. We show evidence that Ang II stimulates oxidative stress directly or indirectly via endothelin 1 and that NFkappaB is upregulated in this model. We speculate that the transcription factors NFkappaB and AP-1 are involved with initiating chemokine and cytokine expression, leading to the above cascade. The unique model and our pharmacological probes will enable us to test these hypotheses.

Mineralocorticoid Receptor Affects AP-1 and Nuclear Factor-κB Activation in Angiotensin II–Induced Cardiac Injury

Aldosterone is implicated in cardiac hypertrophy and fibrosis. We tested the role of the mineralocorticoid receptor in a model of angiotensin II–induced cardiac injury. We administered spironolactone (SPIRO; 20 mg · kg−1 · d−1), valsartan (VAL; 10 mg · kg−1 · d−1), or vehicle to rats double transgenic for the human renin and angiotensinogen genes (dTGR). We investigated basic fibroblast growth factor (bFGF), platelet-derived growth factor, transforming growth factor-&bgr;1, and the transcription factors AP-1 and nuclear factor (NF)-&kgr;B. We used immunohistochemistry, electrophoretic mobility shift assays, and TaqMan RT-PCR. Untreated dTGR developed hypertension, cardiac hypertrophy, vasculopathy, and fibrosis with a 50% mortality rates at 7 weeks. SPIRO and VAL prevented death and reversed cardiac hypertrophy, while only VAL normalized blood pressure. Both drugs prevented vasculopathy. bFGF was markedly upregulated in dTGR, whereas platelet-derived growth factor-B and transforming growth factor-&bgr;1 were little changed. VAL and SPIRO suppressed this upregulation. Both AP-1 and NF-&kgr;B were activated in dTGR compared with controls. VAL and SPIRO reduced both transcription factors and reduced bFGF, collagen I, fibronectin, and laminin in the interstitium. These findings show that aldosterone promotes hypertrophy, cardiac remodeling, and fibrosis, independent of blood pressure. The effects involve AP-1, NF-&kgr;B, and bFGF. Mineralocorticoid receptor blockade downregulates these effectors and reduces angiotensin II–induced cardiac damage.

Effect of Bosentan on NF-κB, Inflammation, and Tissue Factor in Angiotensin II–Induced End-Organ Damage

Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET)(A/B) receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203+/-8 versus 111+/-2 mm Hg and 67.1+/-8.6 versus 0.3+/-0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-kappaB and AP-1 expression in the kidney and heart; the p65 NF-kappaB subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET(A/B) receptor blockade inhibited NF-kappaB and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET(A/B) receptor blockade inhibits NF-kappaB and AP-1 activation and the NF-kappaB- and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-kappaB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET(A/B) receptors.

Blood Pressure–Independent Effects in Rats With Human Renin and Angiotensinogen Genes

The blood pressure-independent effects of angiotensin II (Ang II) were examined in double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes, in which the end-organ damage is due to the human components of the renin angiotensin system. Triple-drug therapy (hydralazine 80 mg/L, reserpine 5 mg/L, and hydrochlorothiazide 25 mg/L in drinking water) was started immediately after weaning. Triple-drug therapy normalized blood pressure and coronary resistance, only partially prevented cardiac hypertrophy, and had no effect on ratio of renal weight to body weight. Although triple-drug therapy delayed the onset of renal damage, severe albuminuria nevertheless occurred. Semiquantitative scoring of ED-1-positive and MIB-5-positive (nuclear cell proliferation-associated antigen Ki-67) cells showed profound perivascular monocyte/macrophage infiltration and cell proliferation in kidneys and hearts of untreated dTGR. Triple-drug therapy had only a minimal effect on local inflammatory response or vascular cell proliferation. In contrast, a novel orally active human renin inhibitor (HRI), 30 mg/kg by gavage for 4 weeks, normalized blood pressure and coronary resistance and also prevented cardiac hypertrophy and albuminuria. ED-1-positive cells and MIB-5-positive cells were decreased by HRI in hearts and kidneys almost to levels observed in normotensive Sprague-Dawley rats. The renoprotective effects of HRI were at least in part due to improved renal hemodynamics and distal tubular function, since HRI shifted renal pressure-diuresis/natriuresis curves leftward by approximately 35 mm Hg, increased glomerular filtration rate and renal blood flow, and shifted the fractional water and sodium excretion curves leftward. In untreated dTGR, plasma Ang II was increased by 400% and renal Ang II level was increased by 300% compared with Sprague-Dawley rats. HRI decreased plasma human renin activity by 95% and normalized Ang II levels in both plasma and kidney compared with triple-drug therapy. Our findings indicate that in dTGR harboring human renin and angiotensinogen genes, Ang II causes end-organ damage and promotes inflammatory response and cellular growth largely independent of blood pressure.

Angiotensin II (AT1) Receptor Blockade Reduces Vascular Tissue Factor in Angiotensin II-Induced Cardiac Vasculopathy

Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT(1) receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left ventricular hypertrophy with focal areas of necrosis, and die at age 7 weeks. Plasma and cardiac ANG II was three- to fivefold increased compared to Sprague-Dawley rats. Chronic treatment with valsartan normalized blood pressure and coronary resistance completely, and ameliorated cardiac hypertrophy (P < 0.001). Valsartan prevented monocyte/macrophage infiltration, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activation, and c-fos expression in dTGR hearts. NF-kappaB subunit p65 and TF expression was increased in the endothelium and media of cardiac vessels and markedly reduced by valsartan treatment. To analyze the mechanism of TF transcription, we then transfected human coronary artery smooth muscle cells and Chinese hamster ovary cells overexpressing the AT(1) receptor with plasmids containing the human TF promoter and the luciferase reporter gene. ANG II induced the full-length TF promoter in both transfected cell lines. TF transcription was abolished by AT(1) receptor blockade. Deletion of both AP-1 and NF-kappaB sites reduced ANG II-induced TF gene transcription completely, whereas the deletion of AP-1 sites reduced transcription. Thus, the present study clearly shows an aberrant TF expression in the endothelium and media in rats with ANG II-induced vasculopathy. The beneficial effects of AT(1) receptor blockade in this model are mediated via the inhibition of NF-kappaB and AP-1 activation, thereby preventing TF expression, cardiac vasculopathy, and microinfarctions.

Cyclosporin A Protects Against Angiotensin II–Induced End-Organ Damage in Double Transgenic Rats Harboring Human Renin and Angiotensinogen Genes

Leukocyte infiltration and adhesion molecule activation play a central role in the pathogenesis of angiotensin II (Ang II)-induced end-organ damage in double transgenic rats (dTGR) harboring human renin and angiotensinogen genes. We tested the hypothesis that the immunosuppressive agent cyclosporine (CsA) protects against the Ang II-induced myocardial and renal damage in dTGR. Furthermore, we investigated the influence of CsA on interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) expression and the DNA binding activity of transcription factor necrosis factor-kappaB (NF-kappaB). The 4-week-old rats were divided into 4 groups: (1) control dTGR (n=20), (2) dTGR plus CsA (5 mg/kg SC for 3 weeks, n=15), (3) normotensive Sprague-Dawley (SD) rats (n=10), and (4) SD rats plus CsA (n=8). In dTGR, CsA completely prevented cardiovascular death (0 of 15 versus 9 of 20), decreased 24-hour albuminuria by 90% and systolic blood pressure by 35 mm Hg, and protected against the development of cardiac hypertrophy. Whole blood CsA concentrations 24 hours after the last drug treatment were 850+/-15 ng/mL. Semiquantitative ED-1 and Ki-67 (a nuclear cell proliferation-associated antigen) scoring showed that CsA prevented perivascular monocyte/macrophage infiltration and prevented cell proliferation in the kidneys and hearts of dTGR, respectively. The beneficial effects of CsA were, at least in part, mediated by the suppression of IL-6 and iNOS expression. Electrophoretic mobility shift assay revealed that CsA regulated inflammatory response in part through the NF-kappaB transcriptional pathway. In contrast to dTGR, CsA increased blood pressure in normotensive SD rats by 10 mm Hg and had no effect on cardiac mass or 24-hour urinary albumin excretion. Perivascular monocyte/macrophage infiltration, IL-6, and iNOS expression or cell proliferation were not affected by CsA in SD rats. Our findings indicate that CsA protects against Ang II-induced end-organ damage and underscore the central role of vascular inflammatory response in the pathogenesis of myocardial and renal damage in dTGR. The beneficial effects of CsA in the kidney and heart are mediated, at least in part, by suppression of IL-6 and iNOS expression via NF-kappaB transcriptional pathway.

Spironolactone Ameliorates Angiotensin Ii-Induced Renal Damage Via the Inhibition of Ap-1 and Nf-κB

P168 Recently, clinical trials have demonstrated the efficacy of spironolactone (SPIRO) in men. Nevertheless the molecular mechanism of action not completely understood. Locally generated angiotensin II (ANG II) stimulates aldosterone. Therefore, we have tested the hypothesis that SPIRO ameliorates ANG II-induced renal damage. Furthermore, we investigated the effect of SPIRO on the transcription factors AP-1 and NF-κB. We treated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) from week 5 to 7 with SPIRO (20 mg/kg/d). Plasma aldosterone was significantly increased in dTGR vs. SPIRO treated and non-transgenic (SD) rats (p