(Hypertension. 2000;35:360.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From the Franz Volhard Clinic (E.M., D.N.M., J.-K.P., R.D., F.S., A.F., M.B., H.H., F.C.L.), Medical Faculty of the Charité, Humboldt University of Berlin; Institute of Biomedicine (E.M.), University of Helsinki, Finland; Max Delbrück Center for Molecular Medicine (D.G.), Berlin, Germany; Institute for Clinical Pharmacology (D.G.), Universitätsklinikum-Benjamin Franklin, Free University of Berlin, Germany; and Hoffmann-La Roche (V.B.), Basel, Switzerland.
Correspondence to Dr Friedrich C. Luft, Franz Volhard Clinic, Wiltberg Strasse 50, 13125 Berlin, Germany. E-mail luft{at}fvk-berlin.de
| Abstract |
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B (NF-
B). 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 proliferationassociated 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-
B 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 IIinduced 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-
B transcriptional pathway.
Key Words: angiotensin II cyclosporin A albuminuria monocytes interleukins nitric oxide
| Introduction |
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B (NF-
B) and activator protein-1 are involved with the initiation of chemokine and cytokine overexpression in dTGR.13 14 We now test whether CsA prevents perivascular inflammation in the kidney and heart and thereby ameliorates the development of Ang IIinduced end-organ damage. We also 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 NF-
B. | Methods |
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24 hours after the last drug dose starting at the age of 5 weeks. Urine samples were collected over a 24-hour period by metabolic cages at 5, 6, and 7 weeks. Rats were sacrificed while under thiopental anesthesia (150 mg/kg IP) at the age of 7 weeks. Blood samples for CsA determination were drawn via aortic puncture into prechilled tubes containing EDTA (6.25 mmol/L) as an anticoagulant. Tissue samples for an electrophoretic mobility shift assay (EMSA) of NF-
B were snap-frozen in liquid nitrogen, and samples for immunohistochemistry were snap-frozen in isopentane (-35°C). All samples were stored at -80°C until assayed.
Immunohistochemistry
Frozen kidneys and hearts were cryosectioned at 6 µm and air dried as described previously.15 The sections were fixed with cold acetone, air dried, and washed with Tris-buffered saline (0.05 mol/L Tris buffer and 0.15 mol/L NaCl, pH 7.6). The sections were incubated for 60 minutes in a humid chamber at room temperature with primary monoclonal antibodies against rat monocytes/macrophages (ED1; Serotec); Ki-67, a nuclear cell proliferationassociated antigen expressed in all active stages of the cell cycle (MIB-5; Dianova); and IL-6 (R and D Systems Europe) and with a polyclonal antibody against iNOS (Affinity BioReagents Inc). Semiquantitative scoring of ED-1 and MIB-5positive cells in the heart and kidney was performed with the use of a computerized cell count program (KS 300 3.0; Zeiss). Fifteen separate areas of each heart and kidney samples (n=5 in both groups) were analyzed. The kidney samples were examined without knowledge of the group of the rat.
EMSA
Tissue extraction and EMSA for the transcription factor NF-
B were performed as described in detail elsewhere.19 20 For EMSA, total kidney and heart homogenates (50 µg) were incubated in binding reaction medium [2 µg poly(dI/dC), 1 µg BSA, 1 mmol/L DTT, 20 mmol/L HEPES, pH 8.4, 60 mmol/L KCl, and 8% Ficoll]with 0.5 ng 32P-dATP end-labeled oligonucleotide containing the NF-
B binding site from the MHC enhancer (H2K; 5'-gatcCAGGGCTGGGGATTCCCC ATCTCCACACG) at 30°C for 30 minutes. The DNA/protein complexes were analyzed in a 5% polyacrylamide gel/0.5% Tris buffer, dried, and autoradiographed. In competition assays, 50 or 100 ng unlabeled H2K oligonucleotides was used.
Concentration Assays
Whole blood CsA concentration was determined with the use of fluorescence polarization immunoassay (Abbott TDX cyclosporine monoclonal whole blood method; Abbott Laboratories) with a monoclonal antibody specific for the parent molecule according to the manufacturers instructions. Urinary rat albumin was measured with a commercially available ELISA with rat albumin as the standard (Celltrend).
Statistical Analysis
Data are presented as mean±SEM. Statistically significant differences in mean values were tested with ANOVA and Tukeys multiple range test. A value of P<0.05 was considered statistically significant. The data were analyzed with the use of SYSTAT statistical software (SYSTAT Inc).
| Results |
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The perivascular monocyte/macrophage infiltration in the kidney (Figure 3A) and heart of untreated dTGR (not shown) was severe. The number of Ki-67positive cells in the kidney (Figure 3D) and heart (not shown) vascular wall was also significantly higher in untreated dTGR than in SD rats. The use of CsA prevented local monocyte/macrophage infiltration and vascular cell proliferation (Figures 3B and 3E). In SD rats, CsA did not influence the number of ED-1 or Ki-67positive cells. Semiquantitative scoring of ED-1 and Ki-67positive cells is shown in Figures 3C and 3F, respectively. IL-6 expression in the vessel wall of renal artery was higher compared with SD rats (Figures 4A to 4C). Interestingly, the IL-6positive cells colocalized with infiltrating neutrophils. iNOS expression in the glomeruli and renal arterial wall was also significantly higher compared with SD rats (Figures 4D to 4F). CsA suppressed the IL-6 and iNOS expression in the kidney (Figures 4B and 4E). EMSA for the detection of NF-
B showed greater DNA binding activity in the kidney (Figure 5) and heart of untreated dTGR compared with SD rats. CsA significantly reduced NF-
B DNA binding activity.
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| Discussion |
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B in the kidney and heart. The most important finding in our study was that CsA completely prevented cardiovascular death, decreased albuminuria by 90%, and markedly attenuated the development of hypertension and cardiac hypertrophy in dTGR. The beneficial effects of CsA were closely related to suppression of monocyte/macrophage infiltration and cell proliferation in the kidneys and heart. We also provide evidence that CsA suppresses IL-6 and iNOS expression through the NF-
B transcriptional pathway. Our findings underscore the central role of vascular inflammation in the pathogenesis of myocardial and renal damage in dTGR and strongly suggest that immunomodulatory drugs might be useful as adjunctive therapy in the prevention of Ang IIinduced cardiovascular complications. Hypertension is a major risk factor for left ventricular hypertrophy and end-stage renal failure. In the present study, CsA slightly but significantly decreased blood pressure in dTGR. Conceivably, the beneficial effects of CsA were mediated in part by lowered blood pressure. However, in our previous study,15 even the complete normalization of blood pressure with nonrenin-angiotensin systemdependent, triple-drug therapy (ie, hydralazine, reserpine, and hydrochlorothiazide) had no major effects on cardiac hypertrophy or albuminuria. Therefore, it is unlikely that the beneficial effects of CsA were mediated by blood pressuredependent mechanisms.
CsA very effectively suppressed perivascular monocyte/macrophage infiltration and vascular smooth muscle cell proliferation in the kidneys and heart. The immunosuppressive effect of CsA is usually explained on the basis of calcineurin inhibition in the T cells. CsA inhibits the transcription of several inflammatory mediators, including IL-2, granulocyte macrophage colonystimulating factor, tumor necrosis factor-
, and interferon-
. Recently, Khanna and Hosenpud21 provided evidence that the antiproliferative effect CsA occurs through the induction of the cell cycle inhibitor p21 and that this induction is dependent on transforming growth factor-ß1. We found in the present study that CsA suppresses iNOS expression in the kidneys and heart. With the use of EMSA, we were able to demonstrate that the CsA-induced suppression of iNOS was associated with decreased DNA binding activity of NF-
B. Thus, our finding is in good agreement with a previous report by McCaffrey et al22 demonstrating the CsA sensitivity of the NF-
B site of the IL-2R
promoter in untransformed murine T cells. Whether CsA interferes directly with NF-
B or indirectly by influencing the cross-talk between transcription factors NFAT and NF-
B23 remains to be determined.
IL-6 is a multifunctional proinflammatory cytokine that has several biological activities, including the induction of B cell differentiation, T cell activation, the induction of acute-phase proteins in the liver, and the production of platelets.24 25 IL-6 is secreted from macrophages, T cells, endothelial cells, mesangial cells, and vascular smooth muscle cells.26 27 28 29 30 Previous studies have demonstrated that vascular smooth muscle cells inducibly secrete IL-6 in response to Ang II.31 32 33 34 Several regulatory DNA cis-elements, such as activator protein-1, cAMP response element, NF-IL-6, and NF-
B, have been found in the promoter region of the IL-6 gene. Recently, Han et al32 showed that Ang II induces IL-6 transcription in vascular smooth muscle cells through the NF-
B transcription pathway. The present study provides the first evidence that Ang II also regulates IL-6 secretion in vivo. Because IL-6 has been shown to have chemotactic properties,35 it is tempting to speculate that within the vascular wall, IL-6 may play an important role in monocyte/macrophage recruitment.
CsA inhibits calcineurin, which is a calcium-dependent phosphatase that activates the NF-AT transcription factors. Transgenic mice expressing activated forms of calcineurin or NF-AT3 in the heart develop cardiac hypertrophy and heart failure. The hypertrophy was blocked with CsA administration.36 However, inhibition of cardiac hypertrophy was not achieved with the use of CsA in rats with constricted abdominal aortas.37 Aortic banding activates the renin-angiotensin system; however, the hypertrophy is principally related to pressure overload. Our model is primarily initiated by the local effects of Ang II. We showed that NF-
B is activated in this model and that possibly NF-AT transcription factors are also involved in cardiac hypertrophy. Signaling in cardiac hypertrophy involves both extracellular and intracellular events.11 Although controversial, the role of calcineurin-related pathways in cardiac hypertrophy deserves additional scrutiny.
In conclusion, our findings indicate that CsA protects against Ang IIinduced 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, through the suppression of IL-6 and iNOS expression via the NF-
B transcriptional pathway.
| Acknowledgments |
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| Footnotes |
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Received September 14, 1999; first decision October 12, 1999; accepted October 19, 1999.
| References |
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B site of the IL2R
promoter in untransformed murine T cells. Nucleic Acid Res. 1994;22:21342142.
B transcription factors. Circ Res. 1999;84:695703.This article has been cited by other articles:
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