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Angiotensin II induced inflammation in the kidney and in the heart of double transgenic rats.

Theuer J, Dechend R, Muller DN, Park JK, Fiebeler A, Barta P, Ganten D, Haller H, Dietz R, Luft FC - BMC Cardiovasc Disord (2002)

Bottom Line: Cardiac hypertrophy index was significantly reduced (4.90 plus minus 0.1 vs. 5.77 plus minus 0.1 mg/g, p < 0.001) compared to dTGR.PDTC markedly reduced the immunoreactivity of p65.Our data show that inhibition of NF-kappaB by PDTC markedly reduces inflammation, iNOS expression in the dTGR most likely leading to decreased cytotoxicity, and cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Franz Volhard Clinic, Charite' and Max Delbrück Center for Molecular Medicine, Berlin, Humboldt University of Berlin, Germany. theuer@fvk-berlin.de

ABSTRACT

Background: 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, iNOS and Tissue Factor expression. Furthermore we show evidence that Ang II causes the upregulation of NF-kB in our model.

Methods: We started PDTC-treatment on four weeks old dTGR (200 mg/kg sc) and age-matched SD rats. Blood-pressure- and albuminuria- measurements were monitored during the treatment period (four weeks). The seven weeks old animals were killed, hearts and kidneys were isolated and used for immunohistochemical-and electromobility shift assay analysis.

Results: Chronic treatment with the antioxidant PDTC decreased blood pressure (162 plus minus 8 vs. 190 plus minus 7 mm Hg, p = 0.02). Cardiac hypertrophy index was significantly reduced (4.90 plus minus 0.1 vs. 5.77 plus minus 0.1 mg/g, p < 0.001) compared to dTGR. PDTC reduced 24 h albuminuria by 85 % (2.7 plus minus 0.5 vs. 18.0 plus minus 3.4 mg/d, p < 0.001) and prevented death significantly. Vascular injury was ameliorated in small renal and cardiac vessels. PDTC inhibited NF-kappaB binding activity in heart and kidney. Immunohistochemical analysis shows increased expression of the p65 NF-kappaB subunit in the endothelium, smooth muscles cells of damaged small vessels, infiltrated cells, glomeruli, tubuli and collecting ducts of dTGR. PDTC markedly reduced the immunoreactivity of p65.

Conclusion: Our data show that inhibition of NF-kappaB by PDTC markedly reduces inflammation, iNOS expression in the dTGR most likely leading to decreased cytotoxicity, and cell proliferation. Thus, NF-kappaB activation plays an important role in ANG II-induced end-organ damage.

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Immunohistochemical analysis of the subunit p65 of NF-κB in the heart of dTGR show a increased expression in the endothelial cells and smooth muscles cells in the vessel wall, which was partially reduced by PDTC.
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Figure 22: Immunohistochemical analysis of the subunit p65 of NF-κB in the heart of dTGR show a increased expression in the endothelial cells and smooth muscles cells in the vessel wall, which was partially reduced by PDTC.

Mentions: Next, we investigated the effects of PDTC on iNOS expression. We observed a strong increase in iNOS expression in the glomeruli in the vessel walls of renal arterioles (Fig. 21d,e,f). Treatment with PDTC greatly reduced the iNOS immunoreactivity both in the blood vessels and the glomeruli. Furthermore, we investigated the activation of the transcription factor NF-κB, which is one of the main regulator of ICAM-1 and iNOS gene expression. Immunohistochemistry (phase contrast resolution) shows the localization of the subunit p65 of NF-κB in a cardiac vessel. The p65 expression was increased in the endothelium, smooth muscle cells (Fig. 22), as well as in the vessel wall, infiltrated cells, glomeruli, and tubules (data not shown) of dTGR kidneys. PDTC markedly reduced p65 expression (Fig. 22). No immunoreaction was observed in non-transgenic SD rats (Fig. 22). The antibody recognizes an epitope overlapping the nuclear location signal of p65 subunit and therefore selectively stains released, activated NF-κB after dissociation from its inhibitor IκBa [24]. We used EMSA for the detection of NF-κB DNA binding activity in the kidney (Fig. 23A) and heart (Fig. 23D). NF-κB DNA binding activity in the kidney was markedly reduced by PDTC treatment and even more in the heart. Each lane represents a separate animal. Renal homogenates were incubated with antibodies against the NF-κB subunits anti-p50, anti-p65, anti-c-Rel, and anti-Rel B (23 B). Binding specificity was demonstrated by competition of excess unlabeled oligonucleotides containing the kB site from the MHC-enhancer (H2K) (23 C). AP-1 activity was increased in dTGR kidneys compared with SD (data not shown). However, PDTC treatment did not reduce AP-1 binding activity (data not shown).


Angiotensin II induced inflammation in the kidney and in the heart of double transgenic rats.

Theuer J, Dechend R, Muller DN, Park JK, Fiebeler A, Barta P, Ganten D, Haller H, Dietz R, Luft FC - BMC Cardiovasc Disord (2002)

Immunohistochemical analysis of the subunit p65 of NF-κB in the heart of dTGR show a increased expression in the endothelial cells and smooth muscles cells in the vessel wall, which was partially reduced by PDTC.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC65512&req=5

Figure 22: Immunohistochemical analysis of the subunit p65 of NF-κB in the heart of dTGR show a increased expression in the endothelial cells and smooth muscles cells in the vessel wall, which was partially reduced by PDTC.
Mentions: Next, we investigated the effects of PDTC on iNOS expression. We observed a strong increase in iNOS expression in the glomeruli in the vessel walls of renal arterioles (Fig. 21d,e,f). Treatment with PDTC greatly reduced the iNOS immunoreactivity both in the blood vessels and the glomeruli. Furthermore, we investigated the activation of the transcription factor NF-κB, which is one of the main regulator of ICAM-1 and iNOS gene expression. Immunohistochemistry (phase contrast resolution) shows the localization of the subunit p65 of NF-κB in a cardiac vessel. The p65 expression was increased in the endothelium, smooth muscle cells (Fig. 22), as well as in the vessel wall, infiltrated cells, glomeruli, and tubules (data not shown) of dTGR kidneys. PDTC markedly reduced p65 expression (Fig. 22). No immunoreaction was observed in non-transgenic SD rats (Fig. 22). The antibody recognizes an epitope overlapping the nuclear location signal of p65 subunit and therefore selectively stains released, activated NF-κB after dissociation from its inhibitor IκBa [24]. We used EMSA for the detection of NF-κB DNA binding activity in the kidney (Fig. 23A) and heart (Fig. 23D). NF-κB DNA binding activity in the kidney was markedly reduced by PDTC treatment and even more in the heart. Each lane represents a separate animal. Renal homogenates were incubated with antibodies against the NF-κB subunits anti-p50, anti-p65, anti-c-Rel, and anti-Rel B (23 B). Binding specificity was demonstrated by competition of excess unlabeled oligonucleotides containing the kB site from the MHC-enhancer (H2K) (23 C). AP-1 activity was increased in dTGR kidneys compared with SD (data not shown). However, PDTC treatment did not reduce AP-1 binding activity (data not shown).

Bottom Line: Cardiac hypertrophy index was significantly reduced (4.90 plus minus 0.1 vs. 5.77 plus minus 0.1 mg/g, p < 0.001) compared to dTGR.PDTC markedly reduced the immunoreactivity of p65.Our data show that inhibition of NF-kappaB by PDTC markedly reduces inflammation, iNOS expression in the dTGR most likely leading to decreased cytotoxicity, and cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Franz Volhard Clinic, Charite' and Max Delbrück Center for Molecular Medicine, Berlin, Humboldt University of Berlin, Germany. theuer@fvk-berlin.de

ABSTRACT

Background: 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, iNOS and Tissue Factor expression. Furthermore we show evidence that Ang II causes the upregulation of NF-kB in our model.

Methods: We started PDTC-treatment on four weeks old dTGR (200 mg/kg sc) and age-matched SD rats. Blood-pressure- and albuminuria- measurements were monitored during the treatment period (four weeks). The seven weeks old animals were killed, hearts and kidneys were isolated and used for immunohistochemical-and electromobility shift assay analysis.

Results: Chronic treatment with the antioxidant PDTC decreased blood pressure (162 plus minus 8 vs. 190 plus minus 7 mm Hg, p = 0.02). Cardiac hypertrophy index was significantly reduced (4.90 plus minus 0.1 vs. 5.77 plus minus 0.1 mg/g, p < 0.001) compared to dTGR. PDTC reduced 24 h albuminuria by 85 % (2.7 plus minus 0.5 vs. 18.0 plus minus 3.4 mg/d, p < 0.001) and prevented death significantly. Vascular injury was ameliorated in small renal and cardiac vessels. PDTC inhibited NF-kappaB binding activity in heart and kidney. Immunohistochemical analysis shows increased expression of the p65 NF-kappaB subunit in the endothelium, smooth muscles cells of damaged small vessels, infiltrated cells, glomeruli, tubuli and collecting ducts of dTGR. PDTC markedly reduced the immunoreactivity of p65.

Conclusion: Our data show that inhibition of NF-kappaB by PDTC markedly reduces inflammation, iNOS expression in the dTGR most likely leading to decreased cytotoxicity, and cell proliferation. Thus, NF-kappaB activation plays an important role in ANG II-induced end-organ damage.

Show MeSH
Related in: MedlinePlus