(Hypertension. 2001;37:433.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
From the Institute of Biomedicine (Z.J.C., T.V., K.N., H.V., E.M.A.M.), Department of Pharmacology and Toxicology, University of Helsinki, Helsinki, Finland; the Department of Medicine (I.T.), Helsinki University Central Hospital and Minerva Institute for Medical Research, Helsinki, Finland; the Department of Pharmacology and Toxicology (H.R., D.M., J.-K.P., F.C.L.), University of Oulu, Oulu, Finland; and the Franz Volhard Clinic, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany.
Correspondence to Eero Mervaala, MD, PhD, Assistant Professor, Institute of Biomedicine, Department of Pharmacology and Toxicology, University of Helsinki, PO Box 8, FIN-00014 Helsinki, Finland. E-mail eero.mervaala{at}helsinki.fi
| Abstract |
|---|
|
|
|---|
, a
vasoconstrictor and antinatriuretic
arachidonic acid metabolite produced by oxidative
stress, was increased 400% in GK rats on a high-sodium diet. Valsartan
decreased blood pressure in rats fed a low-sodium diet and prevented
the inflammatory response. In rats fed a high-sodium diet, valsartan
did not decrease blood pressure or improve endothelial
dysfunction but protected against albuminuria,
inflammation, and oxidative stress. As measured by quantitative
autoradiography, AT1 receptor
expression in the medulla was decreased in GK compared with Wistar
rats, whereas cortical AT1 receptor expression,
medullary and cortical angiotensin type 2
(AT2) receptor expressions, and adrenal ACE and
neutral endopeptidase expressions were unchanged. A
high-sodium diet did not influence renal AT1,
AT2, ACE, or neutral
endopeptidase expressions. In valsartan-treated GK
rats, the cortical and medullary AT1 receptor
expressions were decreased in the presence and absence of a high-sodium
diet. A high-sodium diet increased plasma brain natriuretic
peptide concentrations in presence and absence of valsartan treatment.
We conclude that hypertension in GK rats is salt sensitive and
associated with endothelial dysfunction and
perivascular inflammation. AT1 receptor blockade
ameliorates inflammation during a low-sodium diet and partially
protects against salt-induced vascular damage by blood
pressureindependent
mechanisms.
Key Words: acetylcholine nitroprusside receptors, angiotensin angiotensin-converting enzyme peptides
| Introduction |
|---|
|
|
|---|
80% of patients with NIDDM
are hypertensive at the time of the
diagnosis.2 At present,
the mechanisms involved in the pathogenesis of hypertension and
vascular complications in NIDDM are poorly understood.
Several lines of evidence suggest a crucial role of
oxidative stress and angiotensin II (Ang II) in the
pathogenesis of hypertension and endothelial
dysfunction.3 4
Endothelial dysfunction in hypertension may be due to
impaired NO synthesis and/or inactivation of
endothelium-derived NO by reactive oxygen species, such
as superoxide, hydrogen peroxide, and
peroxynitrite.3 The balance
between NO and superoxide might be even more important than the
absolute levels of either
alone.3 We have recently shown
in double transgenic rats harboring human renin and human
angiotensinogen genes that Ang II induces profound
inflammation and activates redox-sensitive genes via nuclear
factor-
B activation, independent of blood
pressure.5 6 7
Endothelial dysfunction and oxidative stress
have also been linked to the pathogenesis of
diabetes.8 9 Impaired
vascular relaxation in response to acetylcholine (ACh) has been
reported in diabetic animals and
humans.8 9 The
mechanisms of endothelial dysfunction are incompletely
understood; increased vascular production of
superoxide8 and inactivation
of endothelium-derived relaxing factors by glycosylated
hemoglobin8 10 have
been suggested. Interestingly, high glucose concentration and
hyperglycemia promote leukocyte adhesion to the
endothelium through upregulation of adhesion molecule
expression in a nuclear factor-
Bdependent
fashion.11 Whether increased
adhesion of leukocytes to the endothelium causes
endothelial dysfunction in NIDDM is not known. The
pathogenesis of NIDDM in Goto-Kakizaki (GK) rats, a nonobese type II
diabetic rat model originally derived by repeated inbreeding of
glucose-intolerant Wistar
rats,12 includes impaired
ontogenetic development of islet cells, abnormal insulin release after
a glucose load, insulin resistance, a basal
hyperinsulinemia, and abnormal glucose
metabolism.13 14
Recent studies have demonstrated that GK rats showed several structural
changes in the kidney similar to those observed in NIDDM patients
without overt kidney disease. These changes consisted of thickening of
the glomerular basement membrane and tubular basement
membrane, glomerular hypertrophy, and early
podocyte
damage.13 14 15
However, the kidneys of GK rats do not typically show
glomerulosclerosis or interstitial
fibrosis.13 14 15
We used GK rats to test whether endothelial dysfunction
and inflammation participate in the pathogenesis of hypertension and
diabetic nephropathy in GK rats. We also evaluated the
cardiovascular and renal effects of a high-sodium diet
and chronic angiotensin type 1 (AT1)
receptor blockade in GK rats because of the potential importance of
this
system.16
| Methods |
|---|
|
|
|---|
measurements. The heart and kidneys were excised, washed with ice-cold
saline, blotted dry, and weighed. Tissue samples for
immunohistochemistry and autoradiography were
snap-frozen in isopentane (-35°C). All samples were stored at
-80°C until they were assayed. For measurements of vascular
responses, superior mesenteric arteries were carefully excised and
cleaned of the adherent connective tissue. Two successive sections
(3 mm), 5 mm distal from the mesenteric arteryaorta
junction, were used. For morphological analysis, tissue samples
were fixed in 4% buffered paraformaldehyde at room
temperature, dehydrated in graded alcohol, and embedded in
paraffin.20 Sections (2 to 3
µm) were cut with the use of a microtome (Leitz 1512). The sections
were deparaffinized and rehydrated before being stained with
hematoxylin-eosin and Massons trichrome. The tissues were examined
without knowledge of the rat group from which they were
taken. Frozen kidneys were processed, and semiquantitative scoring was performed as described in detail previously.5 20 Briefly, ice-cold acetone-fixed cryosections (6 µm) were air-dried and immersed in TBS (0.05 mol/L Tris buffer and 0.15 mol/L NaCl, pH 7.6). All incubations were performed in a humid chamber at room temperature. At first, the sections were incubated in 10% normal donkey serum (Dianova) for 30 minutes to block any nonspecific binding. The sections were incubated for 60 minutes with the following monoclonal antibodies: antiED-1 (Serotec) and intercellular adhesion molecule-1 (ICAM-1, 1A29, R&D Systems). After they were washed with TBS, the sections were incubated with Cy3-conjugated secondary antibodies (donkey anti-mouse IgG-Cy3, Dianova) for 60 minutes. After a final washing with TBS, slides were mounted in Vectashield mounting medium (Vector Laboratories). In control sections, in which a primary antibody was substituted by isotype control antibody CBL 600 mouse IgG1-ve control (Cymbus Biotechnology) at the same final concentration, no specific immunolabeling was observed. The nonspecific binding of secondary antibodies was excluded by omitting the primary antibody. Preparations were analyzed under a Zeiss Axioplan-2 microscope (Carl Zeiss) and digitally photographed with use of the AxioVision 2 multichannel image processing system (Carl Zeiss Vison).
The relaxation curves to cumulative ACh and sodium nitroprusside (SNP) were determined in mesenteric artery rings precontracted with 1 µmol/L norepinephrine (FTO3C transducer, model 7 C8 polygraph, Grass Instrument Co) as described earlier.21 To analyze the components of endothelium-dependent vascular relaxation, cyclooxygenase and/or NO synthase (NOS) were inhibited in some experiments with diclofenac (3 µmol/L) and NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L), respectively. For autoradiographic studies, frozen kidney sections (20 µm thick) were cut on a cryostat at -17°C, thaw-mounted onto Super Frost Plus slides, dried in a desiccator under reduced pressure at 4°C overnight, and stored at -80°C with silica gel until further processing for autoradiographic studies. ACE, angiotensin receptors types 1 and 2 (AT1 and AT2 receptors, respectively), and neutral endopeptidase (NEP) were quantified by in vitro autoradiography as described earlier.22 23 24 25 26 27
Urinary albumin was measured by ELISA with rat
albumin used as a standard (Immun Diagnostik), and urinary
nitrate+nitrite (NOX) concentration was assessed
with a commercially available NOX
colorimetric assay kit (Cayman). Serum
8-isoprostaglandin F2
concentration was determined by ELISA (Cayman), and serum insulin was
determined by radioimmunoassay (Linco Research) according the
instructions of the manufacturer. Blood glucose was measured with an
automatic analyzer (Reflotron, Boehringer-Mannheim).
Plasma ANP and BNP levels were measured by radioimmunoassay as
described in detail
earlier.28
Data are presented as mean±SEM. Statistically significant differences in mean values were tested by ANOVA and the Fisher protected least significant difference test. ANOVA for repeated measurements was applied for data consisting of repeated observations at successive time points. The differences were considered significant at P<0.05. The data were analyzed by use of SYSTAT statistical software (SYSTAT Inc).
| Results |
|---|
|
|
|---|
|
Endothelium-mediated vascular relaxation of
norepinephrine-precontracted mesenteric
arterial rings in response to ACh was markedly impaired in
GK rats compared with Wistar rats, but the
endothelium-independent relaxations to SNP were similar
in both strains
(Figure 2A and 2B). Preincubation of the arterial
rings with the NOS inhibitor L-NAME and the
cyclooxygenase inhibitor diclofenac
inhibited relaxations to ACh almost completely in GK rats but not in
Wistar rats
(Figure 3). The high-sodium diet impaired vascular relaxation
in response to ACh and also to SNP
(Figure 2A and 2B). Valsartan treatment improved
endothelium-dependent vascular relaxation during the
low-sodium diet but had no effect on
endothelium-independent vascular relaxation
(Figure 2A and 2B). The improvement of
endothelium-dependent vascular relaxation by valsartan
was also found after preincubation of the arterial rings
with L-NAME and diclofenac
(Figure 3). Serum 8-isoprostaglandin
F2
levels tended
(P<0.1>0.05) to be higher in
GK rats than in Wistar rats
(Figure 2C). The high-sodium diet increased serum
8-isoprostaglandin F2
levels by
400% in GK rats; the increase was prevented by valsartan
(Figure 2C). Twenty-fourhour urinary
NOX excretion, a marker of total body NO
generation, was similar in both rat strains but was markedly increased
in GK rats on the high-sodium diet
(Figure 2D). NOX excretion was
normalized in valsartan-treated GK rats on the high-sodium diet
(Figure 2D).
|
|
The kidneys of GK rats on the high-sodium diet showed some mesangial thickening and slight thickening of the media of the afferent arterioles. There were no clear signs of glomerulosclerosis or interstitial fibrosis (Figure 4A). In GK rats on the low-sodium diet, there was already a significant monocyte/macrophage infiltration (ED-1positive cells) in the renal perivascular space and increased expression of ICAM-1 in the interstitium, intima, and adventitia of the small renal vessels (Figure 4D and 4E). The high-sodium diet aggravated the inflammatory response in GK rats (Figure B to 4E). Valsartan treatment decreased the number of ED-1positive cells and ICAM-1 expression during both low-sodium and high-sodium diets.
|
AT1 receptor expression in the medulla was decreased in GK rats compared with Wistar rats, whereas cortical AT1 receptor expression, medullary and cortical AT2 receptor expressions, and renal ACE and NEP expressions were unchanged (Table). The high-sodium diet did not influence renal AT1, AT2, ACE, or NEP expressions. In valsartan-treated GK rats, the cortical and medullary AT1 receptor expressions were decreased in the presence and absence of the high-sodium diet. There were no differences between the groups in plasma ANP concentrations (Table). Plasma BNP levels were increased by the high-sodium diet in the presence and absence of valsartan treatment (Table). Blood glucose and serum insulin levels were higher in GK rats compared with Wistar rats but were not markedly influenced by the high-sodium diet or valsartan treatment (Table).
|
| Discussion |
|---|
|
|
|---|
Previous studies have revealed that unlike several other rodent models of NIDDM, GK rats are not hypertensive, hyperlipemic, or obese.13 Furthermore, the prolonged hyperglycemia and hyperinsulinemia in GK rats are not associated with any marked renal functional changes, although structural changes such as thickening of the glomerular and tubular basement membranes as well as glomerular hypertrophy are detected.13 14 15 In good accordance with these findings, we found only a 1.5-fold increase in 24-hour albuminuria in GK rats on the low-sodium diet. However, with radiotelemetry, we were able to detect a 15 mm Hg difference in MAP between GK and Wistar rats. Although hypertension is commonly associated with endothelial dysfunction, it is likely that factors other than hypertension are also involved in the pathogenesis of endothelial dysfunction in GK rats on a low-sodium diet. The endothelium-independent vascular relaxation in response to SNP was unchanged in GK rats, indicating that the sensitivity of arterial smooth muscle cells to NO is unaltered.
We also found a profound perivascular monocyte/macrophage infiltration and ICAM-1 overexpression in the kidneys of GK rats on a low-sodium diet. Our finding supports the previous notion that oxygen-derived free radicals generated by the inflammatory cells are able to reduce the bioavailability of endothelium-derived NO.3 4 There was no difference in the NOX excretion, suggesting that endothelial NO production is unaltered in GK rats. The efficacy of valsartan to ameliorate inflammatory response as well as endothelial dysfunction indicates the involvement of increased renin-angiotensin system activity in the pathogenesis. Our findings agree with earlier studies demonstrating that Ang II stimulates superoxide generation by increasing the activity of NAD(P)H oxidase in vitro29 and in vivo.30 However, we would like to emphasize that the vascular responses and the degree were analyzed from different tissues, ie, from the mesenteric artery and the kidney, and in fact, the serum 8-isoprostaglandin level tended to be increased only in GK rats on a low-sodium diet. Therefore, we cannot completely conclude that the endothelial dysfunction found in the present study was due to increased oxidative stress.
The endothelium-mediated relaxation that remains resistant to NOS and cyclooxygenase inhibition is thought to be mediated by endothelium-derived hyperpolarizing factor, which appears to be a cytochrome P450derived arachidonic acid epoxide. Current data support the concept that the vascular relaxation elicited by endothelium-derived hyperpolarizing factor is mediated by the opening of Ca2+-derived K+ channels in the vascular smooth muscle cells. Our findings suggest that endothelial dysfunction in GK rats could be attributed, at least in part, to reduced relaxation via arterial K+ channels. Furthermore, our finding that valsartan was able to improve the endothelium-dependent vascular relaxation also after preincubation of the arterial rings with the NOS inhibitor L-NAME and the cyclooxygenase inhibitor diclofenac suggests that valsartan is able to improve vascular relaxation via arterial K+ channels. However, the improvement of endothelium-dependent vascular relaxation by valsartan might also have been due, at least in part, to stimulation of AT2 receptors, leading to increased production of bradykinin and NO as demonstrated by Siragy et al.31
High-sodium intake may increase blood pressure, induce
cardiac hypertrophy, and deteriorate the therapeutic
effects of most antihypertensive
drugs.32 The effects are
mediated primarily by volume overload as well as by increased
sympathetic nervous system
activity.33 Previous studies
have demonstrated that hypertension in patients with diabetes is often
volume dependent and salt
sensitive.1 We found that a
high-sodium diet causes a marked increase in blood pressure associated
with the development of cardiac hypertrophy. A high-sodium
diet also induces renal damage and aggravates inflammation and
endothelial dysfunction. Furthermore, a high-sodium
diet impairs the sensitivity of arterial smooth muscle
cells to NO. The mechanism of the impaired sensitivity remains
unsolved. The detrimental effects of a high-sodium diet are mediated
partly by volume overload, inasmuch as plasma BNP concentration was
markedly increased by the high-sodium diet. The 4-fold increase in
serum 8-isoprostaglandin
F2
levels indicates that the detrimental
effects of dietary sodium are associated with increased oxidative
stress. In contrast, we were unable to detect any salt-induced changes
in renal AT1, AT2, or ACE
expressions. The renal expression of NEP, the major metabolizing enzyme
for ANP and BNP, was also unaltered by the high-sodium diet.
Interestingly, valsartan effectively protected against the development
of salt-induced renal damage, inflammatory response, and oxidative
stress. Our findings indicate that hypertension in diabetic GK rats is
salt sensitive and associated with endothelial
dysfunction and perivascular inflammation. AT1
receptor blockade partially protects against salt-induced vascular
damage by blood pressureindependent
mechanisms.
| Acknowledgments |
|---|
Received October 26, 2000; first decision November 27, 2000; accepted December 14, 2000.
| References |
|---|
|
|
|---|
B inhibition ameliorates
angiotensin IIinduced inflammatory damage in rats.
Hypertension. 2000;35:193201.This article has been cited by other articles:
![]() |
J. H. Lee, S. Xia, and L. Ragolia Upregulation of AT2 receptor and iNOS impairs angiotensin II-induced contraction without endothelium influence in young normotensive diabetic rats Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2008; 295(1): R144 - R154. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. K. Harris, M. M. Elgebaly, W. Li, K. Sachidanandam, and A. Ergul Effect of chronic endothelin receptor antagonism on cerebrovascular function in type 2 diabetes Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2008; 294(4): R1213 - R1219. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. P. Chandler, E. E. Morgan, T. A. McElfresh, T. A. Kung, J. H. Rennison, B. D. Hoit, and M. E. Young Heart failure progression is accelerated following myocardial infarction in type 2 diabetic rats Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1609 - H1616. [Abstract] [Full Text] [PDF] |
||||
![]() |
F. Lang, C. Bohmer, M. Palmada, G. Seebohm, N. Strutz-Seebohm, and V. Vallon (Patho)physiological Significance of the Serum- and Glucocorticoid-Inducible Kinase Isoforms. Physiol Rev, October 1, 2006; 86(4): 1151 - 1178. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Azuma, R. Kawamori, Y. Toyofuku, Y. Kitahara, F. Sato, T. Shimizu, K. Miura, T. Mine, Y. Tanaka, M. Mitsumata, et al. Repetitive Fluctuations in Blood Glucose Enhance Monocyte Adhesion to the Endothelium of Rat Thoracic Aorta Arterioscler. Thromb. Vasc. Biol., October 1, 2006; 26(10): 2275 - 2280. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. M. Boini, A. M. Hennige, D. Y. Huang, B. Friedrich, M. Palmada, C. Boehmer, F. Grahammer, F. Artunc, S. Ullrich, D. Avram, et al. Serum- and glucocorticoid-inducible kinase 1 mediates salt sensitivity of glucose tolerance. Diabetes, July 1, 2006; 55(7): 2059 - 2066. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Satofuka, A. Ichihara, N. Nagai, K. Yamashiro, T. Koto, H. Shinoda, K. Noda, Y. Ozawa, M. Inoue, K. Tsubota, et al. Suppression of ocular inflammation in endotoxin-induced uveitis by inhibiting nonproteolytic activation of prorenin. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2686 - 2692. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Sachidanandam, A. Harris, J. Hutchinson, and A. Ergul Microvascular versus macrovascular dysfunction in type 2 diabetes: differences in contractile responses to endothelin-1. Experimental Biology and Medicine, June 1, 2006; 231(6): 1016 - 1021. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Ogawa, N. Hirawa, T. Tsuchida, N. Eguchi, Y. Kawabata, A. Numabe, H. Negoro, R. Hakamada-Taguchi, K. Seiki, S. Umemura, et al. Urinary excretions of lipocalin-type prostaglandin D2 synthase predict the development of proteinuria and renal injury in OLETF rats Nephrol. Dial. Transplant., April 1, 2006; 21(4): 924 - 934. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. K. Harris, J. R. Hutchinson, K. Sachidanandam, M. H. Johnson, A. M. Dorrance, D. W. Stepp, S. C. Fagan, and A. Ergul Type 2 Diabetes Causes Remodeling of Cerebrovasculature via Differential Regulation of Matrix Metalloproteinases and Collagen Synthesis: Role of Endothelin-1 Diabetes, September 1, 2005; 54(9): 2638 - 2644. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Nagai, Y. Oike, K. Noda, T. Urano, Y. Kubota, Y. Ozawa, H. Shinoda, T. Koto, K. Shinoda, M. Inoue, et al. Suppression of Ocular Inflammation in Endotoxin-Induced Uveitis by Blocking the Angiotensin II Type 1 Receptor Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 2925 - 2931. [Abstract] [Full Text] [PDF] |
||||
![]() |
L. Ragolia, T. Palaia, T. B. Koutrouby, and J. K. Maesaka Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1273 - C1281. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Yasunari, K. Maeda, T. Watanabe, M. Nakamura, J. Yoshikawa, and A. Asada Comparative effects of valsartan versus amlodipine on left ventricular mass and reactive oxygen species formation by monocytes in hypertensive patients with left ventricular hypertrophy J. Am. Coll. Cardiol., June 2, 2004; 43(11): 2116 - 2123. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. R. Sowers Insulin resistance and hypertension Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1597 - H1602. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. D. Dobrian, S. D. Schriver, T. Lynch, and R. L. Prewitt Effect of salt on hypertension and oxidative stress in a rat model of diet-induced obesity Am J Physiol Renal Physiol, October 1, 2003; 285(4): F619 - F628. [Abstract] [Full Text] [PDF] |
||||
![]() |
X. Zhao, D. M. Pollock, E. W. Inscho, D. C. Zeldin, and J. D. Imig Decreased Renal Cytochrome P450 2C Enzymes and Impaired Vasodilation Are Associated With Angiotensin Salt-Sensitive Hypertension Hypertension, March 1, 2003; 41(3): 709 - 714. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||