(Hypertension. 2000;35:800.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From the Department of Cardiovascular Medicine (T.A., N.I., J.-i.T., R.N., M.O.), University of Tokyo Graduate School of Medicine (Japan); Department of Pathology (I.M.), Tokai University School of Medicine, Kanagawa, Japan; and Pediatric Nephrology Laboratory (S.-S.T., J.R.I.), Massachusetts General Hospital, Boston, Mass.
Correspondence to Nobukazu Ishizaka, MD, PhD, Department of Cardiovascular Medicine, University of Tokyo, Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail nobuishizka-tky{at}umin.ac.jp
| Abstract |
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Key Words: hypertension angiotensin II proteinuria oxidative stress kidney
| Introduction |
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Heme oxygenase (HO) is a rate-limiting enzyme of heme catabolism that has 2 isoforms: HO-1, an inducible form,9 10 and HO-2, a constitutive form. Induced HO-1 is thought to act as an antioxidative and anti-inflammatory defense mechanism through the degradation of cellular heme (pro-oxidant) and increase in biliverdin (antioxidant11 ). The carbon monoxide (CO) that is produced also has physiological functions, such as vascular relaxation12 and inhibition of platelet aggregation,13 through the activation of soluble guanylyl cyclase. The recent findings that HO-1 gene transfer ameliorated oxidative tissue injury14 and that oxidant-induced cellular injury was increased in HO-1 knockout mice15 and in human HO-1 deficiency16 provide further direct evidence that HO-1 acts favorably against oxidative stress. In the kidney, both HO-1 and HO-2 are present in the tubular epithelial cells,17 18 suggesting that the HO system also plays a role in the kidney. In fact, HO-1 induction exerts a protective effect on renal function in animal models of rhabdomyolysis,19 cisplatin nephrotoxicity,20 and nephrotoxic nephritis.21 In previous reports, we demonstrated that HO-1 expression was regulated by Ang II in vascular smooth muscle in both pressor-dependent22 and pressor-independent23 manners.
Renal damage occurs via pressor-dependent and -independent mechanisms. The present study was designed to examine both such mechanisms in the angiotensin II (Ang II) model of hypertension in the rat. We first assessed the pressor-dependent and -independent effects of Ang II on renal function. We next examined the effect of Ang II on renal HO-1 expression. We also used immortalized rat proximal tubule cells (IRPTCs) to investigate the effect of Ang II on HO-1 expression in the proximal tubule in vitro.
| Methods |
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Some rats were subjected to a daily intraperitoneal injection of the HO-1 inducer hemin (50 µmol · kg-1 · d-1; Sigma Chemical Co) or the HO-1 inhibitor zinc-protoporphyrin (ZnPP) (50 µmol · kg-1 · d-1; Porphyrin Products), which was started 2 days before pump implantation and continued until sacrifice. In addition, some rats were subjected to a daily intraperitoneal injection of the same amount of hemin or ZnPP for 9 consecutive days without Ang II infusion.
Assay of Plasma and Urine Samples
A 24-hour urine sample was collected before sacrifice. Plasma
and urine creatinine concentrations were measured with the
Jaffe reaction, and urinary protein was measured with the pyrogallol
redmolybdate protein dye-binding method (SRL).
Culture of Transformed Rat Proximal Tubular Cells
Culture of established IRPTCs was performed as described
previously.24 Briefly, cells were cultured in DMEM with
5% FCS. Cultures were supplemented with 3.8 mg/mL
NaHCO3, 25 mmol/L HEPES buffer (pH 7.5),
0.1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 µg/mL
streptomycin, and 0.01 mmol/L nonessential amino acids. For the
Ang II stimulation, cells were cultured in DMEM supplemented with 0.1%
FCS for 48 hours before and throughout the stimulation.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from homogenized heart
according to the acid guanidinium thiocyanatephenolchloroform
method with Isogen (Wako Pure Chemicals). Rat HO-1 cDNA (a kind gift
from Dr S. Shibahara, Tohoku [Japan] University School of Medicine)
was labeled with
-32P-dCTP (DuPont-New England
Nuclear) with commercial kits (Nippon Gene). Hybridization was
performed as described previously.23 Hybridized bands were
visualized and quantified with a bioimaging analyzer (BAS 2000;
Fuji Photo Film), and band density was normalized to the intensity of
ethidium bromidestained 28S and 18S ribosomal RNAs.
Protein Purification and Western Blot Analysis
Protein was isolated through homogenization
of samples in the lysis buffer (50 mmol/L HEPES, 5 mmol/L
EDTA, and 50 mmol/L NaCl, pH 7.5) containing protease
inhibitors (10 µg/mL aprotinin, 1 mmol/L PMSF, and
10 µg/mL leupeptin). Antibodies against rat HO-1 and rat HO-2
(StressGen) were used at a 1:1000 dilution, and horseradish
peroxidaseconjugated secondary antibody (Jackson
ImmunoResearch) was used at a 1:2000 dilution. The ECL Western blotting
system (Amersham Life Sciences) was used for detection. Bands were
visualized with a luminoanalyzer (LAS-1000; Fuji Photo Film).
Band intensity was calculated with Image software (NIH, Research
Service Branch) and expressed as a percentage of control.
Immunohistochemistry
Immunohistochemistry of HO-1 was performed as described
previously.25 Briefly, deparaffinized sections were
preincubated with 10% horse serum. Sections were then incubated with
the antiHO-1 antibody at a 1:200 dilution at 37°C for 1 hour.
Slides were washed and incubated with biotinylated secondary antibody.
After treatment of the slides with Elite ABC kit (Vector Laboratories),
antigens were visualized with the 3,3-diaminobenzidine
tetrahydrochloride (DAKO) system. Counterstaining was performed with
methyl green (DAKO).
Assay of HO Activity
Biliverdin reductase was crudely purified according to the
method of Tenhunen et al.26 The assay of HO activity was
performed as described previously.23
Statistical Analysis
Data are expressed as mean±SEM with ANOVA, followed by a
multiple comparisons test for comparisons of initial data before
expression as a percentage of the control. A value of
P<0.05 was considered to be statistically significant.
| Results |
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Effect of Ang II and NE on Renal Function and Proteinuria
An infusion of Ang II at the dosage of 0.7 mg ·
kg-1 · d-1 for 7
consecutive days resulted in a significant decrease in the
glomerular filtration rate (GFR) determined through
creatinine clearance (Table 2). Ang II also increased proteinuria to
3 times the control level. All of these laboratory values returned
to normal levels after the administration of the specific
AT1 receptor blocker losartan. The
nonspecific vasodilator hydralazine also normalized proteinuria
but did not reverse the Ang IIinduced decrease in GFR. In rats
receiving either a subpressor dose of Ang II (0.25 mg ·
kg-1 · d-1) or NE,
neither GFR nor the degree of proteinuria was different from that in
normotensive rats.
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Effect of Ang II Infusion on HO-1 Expression in the Kidney
We then investigated HO-1 regulation in the kidney of rats with
Ang IIinduced hypertension. HO-1 mRNA was significantly increased as
early as 1 day after Ang II infusion and increased further for up to 7
days (Figures 1A and 1B). This increase
in HO-1 mRNA expression was accompanied by an increase in HO-1 protein
(Figures 1C and 1D). HO activity in the microsomal fraction was
significantly elevated in the kidney of Ang IIinfused rats compared
with the control animals (5.4±0.8 versus 2.9±0.4 nmol bilirubin
· mg-1 · h-1,
n=4, P<0.05).
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Effect of Vasodilators and NE on HO-1 Expression in the
Kidney
Both hydralazine and losartan completely blocked
the Ang IIinduced HO-1 protein upregulation in the kidney (Figures 2A and 2B), which suggests that Ang
IIinduced HO-1 upregulation is a pressor-dependent event. This notion
is supported by the fact that the subpressor dose of Ang II infusion
did not increase HO-1 protein expression (Figures 2C and 2D). In
contrast, however, NE infusion did not upregulate HO-1 at all (Figures 2C and 2D). These findings suggest that the presence of both
hypertension and high levels of circulating Ang II is necessary for the
upregulation of renal HO-1 expression. Ang II infusion did not alter
HO-2 expression in the kidney (Figure 2E). To examine whether
Ang II has a direct effect on tubular HO-1 expression, IRPTCs were
stimulated with 100 nmol/L Ang II. As shown in Figure 2F, Ang II
stimulation significantly increased the HO-1 expression in IRPTCs
(compared with control, 2 h 337±58% and 4 h 340±25%; n=4,
P<0.05.)
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HO-1 Immunohistochemistry
Immunohistochemistry of HO-1 revealed that HO-1 was present in
both proximal and distal tubules in the kidney of normotensive rats.
HO-1 staining in the tubular epithelial cells was more extensively
distributed after Ang II infusion (Figures 3A and 3B). Under higher magnification, a
distinct staining for HO-1 along the basal side of tubular epithelial
cells was evident, whereas the luminal side of these cells was poorly
stained in the kidney of normotensive rats (Figure 3C). In
contrast, HO-1 staining was more extensive and was seen throughout the
epithelial cells (Figure 3D) in the kidney of Ang IIinfused
rats. HO-1 expression was also increased in the arterial
wall at the renal hilus in response to Ang II infusion (Figures 3E and 3F).
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Effects of Inducer and Inhibitor of HO on HO-1
Expression, Hemodynamics, Renal Function, and
Proteinuria
To investigate the possible
physiological relevance of Ang IIinduced HO-1
upregulation in the kidney, either hemin or ZnPP was
intraperitoneally administered to rats. As
expected, hemin injection, but not ZnPP injection, upregulated renal
HO-1 protein expression in the kidney (Figure 4A). HO-1 was also markedly upregulated
by hemin injection in the other tested organs (Figure 4B).
Immunohistochemistry revealed that hemin-induced HO-1 upregulation in
the kidney was mainly seen in the tubular region (Figure 4C).
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Hemin injection slightly decreased blood pressure in control rats, although it was not statistically significant. In addition, hemin normalized the Ang IIinduced blood pressure elevation (Table 3). In contrast, ZnPP did not affect blood pressure in either control rats or these receiving Ang II (Table 3). Hemin or ZnPP injection did not significantly change GFR or proteinuria in normotensive rats. However, hemin increased GFR and decreased proteinuria in Ang IIinfused rats. On the contrary, ZnPP augmented the Ang IIinduced decrease in GFR and increase in proteinuria (Table 3).
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| Discussion |
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Our first observation was that Ang II infusion resulted in an increase in proteinuria and a decrease in GFR. The former was blocked by either losartan or hydralazine, but the latter was blocked only by losartan. Because NE infusion affected neither GFR nor proteinuria, it appears that in the Ang IIinfused rat, AT1 receptormediated action of Ang II is responsible for the decreased GFR, whereas the synergistic action of Ang II and pressor overload is critical for the increased proteinuria.
We then found that HO-1 was upregulated in the kidney of rats rendered hypertensive with Ang II infusion but not in rats receiving the NE infusion. In addition, Ang IIinduced HO-1 upregulation was blocked by either losartan or hydralazine. These data suggest that the synergistic action of circulating high levels of Ang II and pressor overload is critical for HO-1 upregulation. This pattern of HO-1 regulation in the kidney differs from that in the aorta, where HO-1 has been reported to be upregulated in response to hypertension per se.22 The precise mechanisms of HO-1 upregulation remain unknown. Other investigators have reported elevated intrarenal Ang II levels in the Ang IIinfused rat.27 Because AT1 receptor is abundantly expressed in the proximal and distal tubules,28 it is possible that HO-1 upregulation was due to the direct effect of intraluminal Ang II. In the present study, the finding that HO-1 was upregulated by Ang II in vitro in IRPTCs, in which AT1 receptor is expressed,24 may support this hypothesis. It was of note that Ang IIinduced HO-1 upregulation occurred in parallel with increased proteinuria. It has been reported that tubular protein overload upregulates proinflammatory mediators in the renal tubules.29 30 Therefore, it is possible that HO-1 was upregulated in response to increased proteinuria as an anti-inflammatory defense.
Occasionally, when HO-1 was upregulated, a 30-kDa immunoreactive band appeared on immunoblot analysis (Figures 1C, 2A, 2C, 4A, and 4B). Because that 30-kDa band emerged when HO-1 protein was markedly increased in IRPTCs with the infection of an adenoviral vector containing the rat HO-1 gene (N. Ishizaka, unpublished data, 1999), this band most likely is HO-1 protein that has undergone posttranslational modification or degradation.
The possible physiological importance of renal HO-1 upregulation in the Ang IIinfused rat is 3-fold. First, like nitric oxide synthase,31 HO-1 may partially counteract the vasoconstrictor influence of elevated circulating Ang II via the CO-mediated activation of soluble guanylate cyclase. Second, HO may activate 70-pS potassium channel in the rat thick ascending limb32 via CO production. Potassium recycling via the activation of potassium channel plays an important role in the provision of an adequate supply of potassium to the Na+,K+,Cl- cotransporter. Therefore, Ang II may play a role in sodium reabsorption through the activation of potassium channel as well as the activation of the Na+,K+,Cl- cotransporter.33 Third, the induction of HO-1 may be an adaptive response that protects the kidney against renal insults. HO-1 induction exerts a renoprotective effect in animal models of rhabdomyolysis19 and nephrotoxic nephritis.21 In the present study, the administration of ZnPP to Ang IIinfused rats further decreased GFR and increased proteinuria. In contrast, the administration of hemin to Ang IIinfused rats ameliorated GFR decrease and lessened proteinuria. These findings support the hypothesis that Ang IIinduced HO-1 upregulation in the kidney provides renoprotection. Because hemin administration completely suppressed Ang IIinduced blood pressure elevation, its antiproteinuric effect may also be attributed to its antihypertensive effect, as in the case with hydralazine. This antihypertensive effect of hemin appears to be consistent with previous reports showing that hemin or HO substrates normalized blood pressure in other types of hypertension.34 35 The mechanisms of the antihypertensive effect of HO-1 upregulation may involve CO-mediated activation of soluble guanylate cyclase36 and the activation of calcium-activated potassium channels.37
In summary, the continuous infusion of Ang II induced a renal injury in both a pressor-independent (decreased GFR) and a pressor-dependent (proteinuria) manner. HO-1 was upregulated in the kidney of rats with Ang IIinduced hypertension. The findings that HO-1 induction ameliorated and HO inhibition augmented Ang IIinduced renal injury suggest that Ang IIinduced upregulation of HO-1 may act renoprotectively against renal injury evoked by chronic Ang II infusion.
| Acknowledgments |
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| Footnotes |
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Received August 24, 1999; first decision October 19, 1999; accepted October 26, 1999.
| References |
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