Decrease in tetrahydrobiopterin as a possible cause
of nephropathy in type II diabetic rats
M Okumura
1, M Masada
2, Y Yoshida
2, H Shintaku
3, M Hosoi
1, N Okada
1, Y Konishi
1, T Morikawa
1, K Miura
4and M Imanishi
11Department of Internal Medicine, Osaka City General Hospital, Osaka, Japan;2Labolatory of Biochemistry, Faculty of Horticulture,
Chiba University, Matsudo, Japan;3Department of Pediatrics, Osaka City University Graduate School of Medicine, Osaka, Japan and
4Department of Applied Pharmacology and Therapeutics, Osaka City University Graduate School of Medicine, Osaka, Japan
A decrease in renal synthesis of nitric oxide (NO) in the progression of diabetic nephropathy has been documented. As (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor of NO synthase, we investigated whether BH4 deficiency is involved in the pathogenesis of nephropathy. Ten-week-old Otsuka Long–Evans Tokushima Fatty (OLETF) rats were used as a type II diabetic model, and Long–Evans Tokushima Otsuka (LETO) rats as the healthy controls. OLETF rats were orally treated with BH4(10 mg/kg daily) or with water from 10 to 61 weeks of age. In another experiment, OLETF rats were treated orally with a calcium channel blocker, benidipine (5 mg/kg daily), or with 0.3%
carboxymethyl cellulose (nontreated) from 10 to 52 weeks of age. Proteinuria was observed periodically, and at the end of the study, BH4level and GTP cyclohydrolase I (GTPCH) activity in the kidney were measured. Proteinuria was observed at 13 weeks of age in the OLETF rats, and deteriorated until 61 weeks of age. Supplemental BH4reduced the proteinuria. At 52 weeks of age, GTPCH activity and the BH4level were decreased in the plasma and kidneys of OLETF rats, whereas they were significantly higher in the benidipine group than in the nontreated group. Proteinuria was milder in the benidipine group than in the nontreated group, without a concomitant decrease in blood pressure. Histologically observed glomerulosclerosis was mild in the BH4and benidipine groups. In type II diabetic rats, renal BH4is considered to play a crucial role in the pathogenesis of diabetic nephropathy. Benidipine was found to preserve BH4 levels, suggesting therapeutic renoprotective effects. Kidney International(2006)70,471–476. doi:10.1038/sj.ki.5000431; published online 14 June 2006
KEYWORDS: biopterin; GTP cyclohydrolase I; benidipine
Nitric oxide (NO) is an important regulator of renal function, including renal microcirculation, renal nerve activity, and tubular reabsorption of fluids and electrolytes.1,2 Although there have been confusing and controversial data regarding whether NO production is beneficial for protection of the kidney in diabetes, a strong body of evidence has supported the contention that, in the diabetic kidney, NO synthesis is decreased,3,4and disorder of NO production is an important cause of nephropathy.5However, the mechanism of impaired NO production in diabetic nephropathy is not fully understood.
In the synthetic pathway of NO, (6R )-5,6,7,8-tetrahydro-biopterin (BH4) is an important allosteric effector of NO
synthase (NOS) through stabilization of the dimer, an active form of NOS.6,7When BH4is severely decreased, NOS does
not synthesize NO but rather O2.8In fact, a decrease in BH4
has been reported in the aorta of rats fed with high fructose (a model of insulin resistance),9and cultured endothelial cells isolated from BioBreeding rats (a model of spontaneous type I diabetes).10 In these studies, a decrease in the activity of GTP cyclohydrolase I (GTPCH), a key enzyme of BH4
synthesis, was suggested to be the cause of the BH4decrease.
However, whether BH4 is decreased in diabetic nephropathy
and to what extent any decrease is involved in the progress of nephropathy are still unclear.
In clinical practice, the progress of diabetic nephropathy is effectively suppressed by blood pressure control with antihypertensive drugs. The blood pressure controlper se is thus considered important to prevent macro- and micro-angiopathy in diabetes.11,12However, it may also be true that inhibition of the renin–angiotensin system yields a renopro-tective effect on chronic kidney disease, in addition to blood pressure reduction. The mechanism of this potential effect is at least one part owing to improvement in glomerular hypertension.13 In addition to this effect, inhibitors of the renin–angiotensin system have been suggested to have protective endothelial function and to increase NO synthe-sis.14 Similar to inhibitors of the renin–angiotensin system, calcium channel blockers (CCBs) have also been demons-trated to enhance NOS in endothelial cells.15Furthermore, benidipine, a long-acting CCB, increases the level of mRNA Received 12 August 2005; revised 16 January 2006; accepted 15
February 2006; published online 14 June 2006
Correspondence: M Imanishi, Department of Internal Medicine, Osaka City General Hospital, 2-13-22 Miyakojimahondori, Miyakojima-ku, Osaka 534-0021, Japan. E-mail: [email protected]
transcript of endothelial NOS in the left ventricular endothelial cells of hypertensive rats16and elevates the level of NO in the ischemic heart of dogs.17 In our preliminary study, we found a decrease in the plasma level of BH4
(a deficiency in BH4) in spontaneous type II diabetic rats
with nephropathy. Upon discovery of this deficiency, we further examined to what extent the deficiency is involved in the etiology of nephropathy. To this end, the effect of supplementation with BH4 was examined, and whether
administration of the CCB, benidipine, is effective for suppression of proteinuria and prevention of BH4deficiency
in this rat model. RESULTS
Changes in body weight, plasma glucose, and blood pressure Table 1 shows the changes in body weight, plasma glucose, and blood pressure measured in 13-, 37-, and 61-week-old rats in protocol 1. On all occasions, the nontreated Otsuka Long–Evans Tokushima Fatty (OLETF) group and the BH4
-treated OLETF group weighed more than the control Long–Evans Tokushima Otsuka (LETO) group. Plasma glucose concentration in the nontreated OLETF group and the BH4-treated OLETF group was significantly higher than
that in the LETO group throughout the study period. Systolic blood pressure was the same between the nontreated OLETF group and the BH4-treated group, both showing significantly
higher levels than in the LETO group. Altogether, BH4had no
effect on body weight, plasma glucose, or blood pressure in this diabetic rat model.
Table 2 shows the changes in body weight, plasma glucose, and blood pressure in protocol 2. Similar to the results in protocol 1, the nontreated OLETF group showed significantly higher levels of body weight, plasma glucose, and systolic blood pressure than those in the LETO group, and the treatment with benidipine resulted in no change in any of these parameters.
Changes in urinary protein excretion
Urinary protein excretion increased with age in the nontreated OLETF group, but not in the LETO group (Figure 1). In contrast, at 37 and 61 weeks of age, urinary protein excretion was significantly lower in the BH4-treated
OLETF group, indicating the amelioration of proteinuria. The effect of benidipine on urinary protein excretion is shown in Figure 2. Benidipine-induced reduction in urinary protein excretion was significant from 28 weeks until the end of the study (52 weeks).
Creatinine clearance
No significant difference in creatinine clearance (CCr) was observed between the OLETF rats and BH4-treated OLETF
rats (4.872.6 and 3.872.0 ml/min/kg body weight, mean7s.d., respectively, P¼0.5759, one-way analysis of variance (ANOVA)). CCr in the LETO rats was 4.072.1 (mean7s.d.). Thus, a decrease in CCr in diabetic nephro-pathy was not observed.
Table 1 | Changes in body weight, plasma glucose, and blood pressure during the BH4supplementation study
Rats 13 weeks 37 weeks 61 weeks
Body weight (g) LETO 360728 518728 579740
OLETF 468747* 711741* 759752* BH4 456731* 688734* 740735*
Plasma glucose (mg/dl) LETO 209732 184728 190730 OLETF 298764* 243741* 247729* BH4 284748* 250750* 250750*
Blood pressure (mm Hg) LETO 11976 11477 114713 OLETF 13676* 13879* 135710* BH4 13079* 139714* 143712* LETO, Long–Evans Tokushima Otsuka rats (controls) (n=12); OLETF, Otsuka Long–-Evans Tokushima Fatty rats that were untreated (n=11); BH4, OLETF rats treated with
BH4(n=9).
Data represent mean7s.d.
*Po0.01 compared with LETO rats of the same age.
Table 2 | Changes in body weight, plasma glucose, and blood pressure during the benidipine treatment study
Rats 16 weeks 28 weeks 40 weeks 52 weeks
Body LETO 384726 478729 519732 539735 weight (g) OLETF 495722* 650737* 701746* 694754* Benidipine 472721* 609740* 655745* 650744* Plasma LETO 198713 187726 191724 179716 glucose OLETF 281732* 287732* 282742* 263736* (mg/dl) Benidipine 259722* 272751* 252745* 245748* Blood LETO 12678 11579 114710 118711 pressure OLETF 148714* 13676* 14178* 145716* (mm Hg) Benidipine 142713* 13379* 135720* 14278* LETO, Long–Evans Tokushima Otsuka rats (controls) (n=12); OLETF, Otsuka Long–-Evans Tokushima Fatty rats that were untreated (n=10); benidipine, OLETF rats treated with benidipine (n=8).
Data represent mean7s.d.
*Po0.01 compared with LETO rats of the same age.
0 100 200 300 400 500 600 13 37 61 Age (weeks) * * * * * # * #
Urinary excretion of protein
(mg/day)
Figure 1|Changes in urinary protein excretion in the three groups of rats.Data represent mean7s.d. Closed bars are for LETO rats (n¼12), open bars are for untreated OLETF rats (n¼11), and stippled bars are for the OLETF rats treated with BH4(n¼9). *Po0.05 compared with LETO rats.#Po0.05 compared with OLETF rats.
Similarly, in protocol 2, no significant difference in CCr was observed between the OLETF rats and BH4-treated
OLETF rats (3.870.9 and 3.970.8 ml/min/kg body weight, mean7s.d., respectively, P¼0.4621, ANOVA). CCr in the LETO rats was 4.270.7 (mean7s.d.). Thus, again no decrease in CCr in diabetic nephropathy was observed. Histologic examination
The histopathological features of the kidneys of the LETO rats, OLETF rats, BH4-treated OLETF rats (protocol 1), and
benidipine-treated OLETF rats (protocol 2) are summarized in Figure 3 and Table 3. Glomerulosclerosis was not seen in the LETO rats, but was evident in both untreated and BH4
-treated OLETF rats (as shown in Table 3). Data were obtained from examination of 12378 glomeruli of each of the untreated OLETF rats (n¼6) and of 12377 glomeruli of each of the BH4-treated rats (n¼6). The glomerular sclerosis
index of BH4-treated rats was less than that of the untreated
rats. As shown in Figure 3c, glomerulosclerosis in BH4
-treated rat kidney was milder than that in un-treated OLETF rat kidney (Figure 3b).
To evaluate glomerulosclerosis in protocol 2, we examined 11475 glomeruli of each of the untreated OLETF rats (n¼6) and 135710 glomeruli of each of the benidipine-treated OLETF rats (n¼6). The glomerular sclerosis index of the benidipine-treated rats was less than that of the untreated rats (Table 3). The glomerulosclerosis in the benidipine-treated rat kidney (Figure 3d) was milder than that in the untreated OLETF rat kidney (Figure 3b) (as shown in Figure 3).
Measurement of plasma and kidney biopterins
Table 4 shows the amounts of total forms of biopterin and BH4 in the plasma and kidneys, as well as the GTPCH
activities in the kidneys. Both total forms of biopterin and BH4 levels were lower in the plasma and kidneys of the
nontreated OLETF group than in the LETO group. In contrast, the amounts of biopterins and BH4were greater in
both the plasma and kidneys in the benidipine-treated OLETF group, indicating a recovery of these biopterins by the treatment with benidipine. The BH4/biopterin ratio indicates
52 40 28 16 Age (weeks) * * * * # * * # * * # 0 400 300 200 100
Urinary excretion of protein (mg/day)
Figure 2|Changes in urinary protein excretion in the three groups of rats.Data represent mean7s.d. Closed bars are for LETO rats (n¼12), open bars are for untreated OLETF rats (n¼10), and stippled bars are for the OLETF rats treated with benidipine (n¼8). *Po0.05 compared with LETO rats.#Po0.05 compared with OLETF rats.
a LETO b OLETF
d Benidipine
c BH4
Figure 3|Light microscopic observation of kidneys.Photographs specifically focused on the glomerulus. (a) LETO at 61 weeks old. (b) OLETF at 61 weeks old. (c) BH4-treated OLETF at 61 weeks old. (d) Benidipine-treated OLETF at 52 weeks old. In (b), enlarged glomerulus and developed nodular diabetic nodules are evident.
Table 3 | The glomerular sclerosis index of OLETF rats and those treated with BH4and benidipine
OLETF BH4 P-value
Glomerular sclerosis index 65.2724.8 34.8715.2 0.0306
OLETF Benidipine P-value
Glomerular sclerosis index 55.0712.4 27.7714.3 0.0104 OLETF, Otsuka Long–Evans Tokushima Fatty rats that were untreated (n=6); BH4,
OLETF rats treated with BH4(n=6); benidipine, OLETF rats treated with benidipine
(n=6).
P-values were analyzed by Mann–WhitneyU-test. Data represent mean7s.d.
Table 4 | Tissue content of biopterin and tetrahydrobiopterin, and GTP cyclohydrolase I activity
LETO OLETF Benidipine Plasma Biop (pmol/ml) 13771 8571* 9971*,# BH4(pmol/ml) 11172 4471* 7671* ,# BH4/Biop 0.8170.01 0.5170.004* 0.7770.009* ,# Kidney
Biop (pmol/g tissue) 32775 20972* 25172*,#
BH4(pmol/g tissue) 26774 13672* 20475*
,#
BH4/Biop 0.8270.003 0.6570.01* 0.8170.02 #
GTPCH (U/mg protein) 0.9970.01 0.5870.01* 0.7470.01*,# LETO, Long–Evans Tokushima Otsuka rats (controls) (n=12); OLETF, Otsuka Long–-Evans Tokushima Fatty rats that were untreated (n=10); benidipine, OLETF rats treated with benidipine (n=8); Biop, amount of total forms of biopterin; BH4,
tetrahydrobiopterin; GTPCH, GTP cyclohydrolase I activity. BH4/Biop indicates the
activity of dihydropteridine reductase. Data represent mean7s.d.
*Po0.01 compared with LETO rats.
#
the activity of dihydropteridine reductase, the recycling enzyme that produces BH4 from BH2. Both in the plasma
and kidney, the ratio of BH4/biopterin was decreased in the
nontreated OLETF rats in comparison with that in the LETO rats. However, the ratio was maintained in the benidipine-treated OLETF rats. Thus, the scavenging activity by BH4in
the OLETF rats was likely recovered by treatment with benidipine. The GTPCH activity in the kidneys was lower in the nontreated OLETF rats than the LETO rats, but was recovered by benidipine.
DISCUSSION
Diabetic nephropathy in OLETF rats is characterized by typical renal lesions such as proteinuria, hyper-trophy, glomerular sclerosis, and microangiopathy.18 The development of nephropathy in the OLETF rats in the present study was evidenced by severe proteinuria and glomerulosclerosis, with concomitant decreases in BH4
and the activity of GTPCH. BH4 supplementation retarded
nephropathy. Thus, BH4and GTPCH may be involved in the
pathogenesis of nephropathy in this diabetic model. In addition, we found that benidipine slowed the progression of nephropathy and impeded the decrease in BH4 and GTPCH
activity.
Although BH4 and benidipine reduced proteinuria and
glomerulosclerosis in these diabetic rats, CCr in the OLETF rats with the treatments did not differ from that in the control OLETF rats. One possible explanation is that the normal level of CCr in the OLETF rats was not affected by these treatments. However, a more likely explanation is that BH4 and benidipine were more effective in ameliorating
morphological changes in this diabetic model (as shown in Figure 3 and Table 3).
The majority of evidence has indicated a beneficial effect of NO in maintaining renal function.19However, NO rapidly reacts with superoxide to form toxic peroxynitrite, and thus peroxynitrite has been considered a crucial factor in deteriorating renal function. This oxidative–nitrosative stress increases and causes diabetic complications.20This idea was recently supported by Chander et al.,21 who demonstrated the therapeutic effect of a peroxynitrite scavenger, ebselen, in Zucker diabetic rats. The present results support the idea that the BH4 depletion and dysregulation of NOS are critically
involved in the pathogenesis of diabetic nephropathy. In diabetics, impaired endothelium-dependent vasodila-tion has been considered to be the result of disorder of the NO system.22 In in vitro studies, BH4 added to bathing
solution ameliorated the endothelium-dependent relaxation of isolated vascular rings from diabetic rats.23 Moreover, impairment of endothelium-dependent relaxation of the aortic strips from fructose-fed rats (insulin-resistant rats) was restored by oral supplementation of BH4.
24
A decrease in BH4
has been demonstrated in the aorta of insulin-resistant rats9,24and in the brain of type I diabetic rats,25where the NO system is known to be impaired. In the kidney, the activity of NOS decreases in the renal cortex of diabetic obese
Zucker rats.26 Chronic NOS inhibition is shown to induce renal structural and functional damage in diabetic nephro-pathy.27,28In the present study, the renal content of BH4 in
OLETF rats at 52 weeks of age was about 50% of that in the healthy control LETO rats at the same age. Taking the biological pathway of NO synthesis into account, a strong contention can be made that, in diabetic kidney, a deficiency in BH4 results in impaired NO synthesis. Impaired NO
synthesis is thus a potential factor of deterioration in diabetic nephropathy.
The activities of GTPCH and dihydropteridine reductase are low in insulin-resistant rats.9,24Consistently, we found in type II diabetic kidney that these two enzyme activities and the content of BH4 were lower than in healthy controls. As
has been understood, a decrease in GTPCH causes a decrease in BH4. In the situation that BH4is depleted, NOS produces
superoxide anion instead of NO.29,30 The superoxide anion decreases the half-life of NO and is also active in forming peroxynitrite. The level of BH4depends on the cellular redox
state; the superoxide anion inhibits the change from BH2
(oxidized form of BH4) to BH4 by suppression of
dihydropteridine reductase.31 The oxidative stress (increase in the superoxide) further decreases the BH4level and
half-life of NO. This vicious cycle appears to occur in diabetics and to cause systemic organ damage, including in the kidney. It is not clear why GTPCH activity was low in the present disease model. However, insulin activates both GTPCH and dihydropteridine reductase, and stimulates BH4 synthesis.32
As the diabetic rats used in the present study have hyperinsulinemia and insulin resistance,18 the insulin-resis-tant state may have caused the decrease in the activity of GTPCH.
In the present study, benidipine impeded the decrease in both GTPCH activity and the BH4 level in the kidney. As
noted above, why GTPCH activity decreased in the diabetic kidney is unclear, and hence the mechanism of the effect of benidipine is likewise unclear. The apparent restoration of BH4 by benidipine might be an indirect effect, as the
therapeutic effect of CCBs on type II diabetic nephropathy has been documented, mostly through improvement in hemodynamics. Moreover, if CCBs are effective in improving insulin resistance, this may explain, at least in part, the mechanism of action of benidipine. Another possibility is that benidipine might activate GTPCH by increasing intracellular Ca in the endothelium, particularly in the glomerular capillaries. Although there is no direct evidence to prove this theory, the following reports support this idea. GTPCH has been shown to be activated by intracellular influx of Ca.33 Nitrendipine has been reported to increase intracellular Ca in aortic endothelial cells in culture.34 Another CCB, felodipine, has been shown to increase endothelial NOS protein mass and nitrate/nitrite generation in rat aortic endothelial cells in culture.15In addition, CCBs have been demonstrated to enhance NO availability in the human forearm vascular bed,35 canine coronary micro-vessels,36and rabbit endothelial cells.37
In the present study, benidipine at a dose of 5 mg/kg did not induce a statistically significant decrease in blood pressure in the diabetic OLETF rats during the observation period from 28 to 52 weeks. In contrast, Jesmin et al.38 reported a significant but minor decrease in blood pressure by oral administration of benidipine (3 mg/kg) to OLETF rats. In clinical cases, when blood pressure is not too high in patients with type II diabetes and nephropathy, the blood pressure-lowering effect of antihypertensive drugs is not always prominent, even if the drugs are effective for renoprotection.39,40In other rats, the blood pressure decrease by benidipine was less when the basal pressure was not very high.41 The blood pressure of the OLETF rats that we used ranged between 135 and 145 mm Hg, whereas that of the LETO rats ranged between 114 and 119 mm Hg on average, indicating a mild hypertensive state in this disease model. Thus, our results indicate that the effect of benidipine to decrease urinary excretion of protein is independent of any antihypertensive effect.
In conclusion, the renal BH4 level was found to be
decreased in the type II diabetic rat model. The decreased level of renal BH4is attributable to the decrease in the activity
of GTPCH in the kidney. The deficiency in BH4is considered
to play a crucial role in the pathogenesis of diabetic nephropathy, probably through impairment of NO synthesis in the kidney. Treatment with benidipine was effective in reducing urinary protein excretion, and maintained renal GTPCH activity and BH4 levels without affecting blood
pressure. This drug is thus considered effective for renopro-tection in this model.
MATERIALS AND METHODS Animals
All procedures were in accordance with our institutional ethical guidelines for animal research. We used spontaneous type II diabetic OLETF rats, and related non-diabetic LETO rats as the healthy controls. Male OLETF and LETO rats were maintained at the animal laboratory of Osaka City General Hospital. All rats were kept in a specific pathogen-free facility with controlled temperature (23721C) and humidity (5575%) with 12-h cycles of light and dark. They were fed standard laboratory chow (CE-2, CLEA Japan Inc., Tokyo, Japan), and tap water was availablead libitum.
Experimental design
Protocol 1 (BH4-supplement study). We prepared three groups of rats: LETO as the control (n¼12), OLETF as the nontreated diabetic rats (n¼11), and OLETF (n¼9) treated with sapropterin hydrochloride (BH4) at a daily dose of 10 mg/kg. BH4was dissolved in water (5 mg/ml) and was administered to the BH4-treated OLETF rats by gastric gavage once a day, starting when the rats were 10 weeks old. The control LETO and the nontreated OLETF rats received water (2 ml/kg) by the same route.
Body weight, systolic blood pressure, and plasma glucose concentration were measured when the rats were 13, 37, or 61 weeks old. At these times, the rats were transferred to metabolic cages, and urine was collected for 24 h at 41C. Blood was sampled from a tail vein.
Protocol 2 (benidipine treatment study). We prepared three groups of rats: LETO as the control (n¼12), a group of nontreated OLETF (n¼10), and a group of OLETF treated with benidipine hydrochloride, at a daily dose of 5 mg/kg (n¼9). Benidipine hydrochloride was suspended in 0.3% carboxymethyl cellulose solution at a concentration of 2.5 mg/ml. The benidipine suspension was administered to benidipine-treated OLETF by the same route as in protocol 1. Both LETO and nontreated OLETF rats received 0.3% carboxymethyl cellulose solution at a daily dose of 2 ml/kg. When the rats were 16, 28, 40, and 52 weeks old, we examined the same parameters as in protocol 1. At 52 weeks of age, the study was stopped and the rats were killed by intraperitoneal injection of pentobarbital. Blood and kidneys were then obtained to determine the biopterin level and GTPCH activity.
Analysis
Urinary protein was measured by the pyrogallol red method. Plasma glucose was measured using a modified hexokinase method. CCr was calculated using a standard formula at the end of the study period with urine collected over a 24-h period. BH4 and each biopterin form were measured using high-performance liquid chromatography as described elsewhere,9,42 and the amount of BH4was estimated from a difference between the total of all the biopterin forms (BH4, BH2, and oxidized biopterin) and alkaline-stable biopterin (BH2and oxidized biopterin). GTPCH activity was assayed using high-performance liquid chromatography to measure neopterin, which was generated from dihydroneopterin tripho-sphate after oxidation and phosphatase treatment.24,42
Measurement of blood pressure
Systolic blood pressure was periodically measured using an electrosphygmomanometer (PS-200A, Riken Kaihatsu, Tokyo, Japan) soon after the day of urine collection. At least three blood pressure readings of each rat were taken over a short period.
Histologic examination
Kidneys were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin, and sections 2–3mm thick were stained with periodic acid-Schiff. All preparations were evaluated by our investigators who had no prior knowledge of the rat groupings. To quantify glomerulosclerosis, the glomerular sclerosis index was used as described previously.43,44 Individual glomeruli were examined, and the severity of glomerulosclerosis was graded from 0 to 4 to express the percentage that the glomerulus in question was affected. Injury scores were then calculated by multiplication of these grades for individual glomeruli by the percentage of glomeruli with the same extent of injury. The extent of injury in each tissue specimen was then obtained by the addition of these injury scores.
Statistical analysis
Data are expressed as the mean7s.d. All differences between groups were analyzed by ANOVA followed by Scheffe’s method (StatView version 5.0), except as noted in ‘Histologic results’. Differences were considered to be statistically significant whenPo0.05.
Materials
LETO and OLETF rats were obtained from the Tokushima Research Institute of Otsuka Pharmaceutical Co. (Tokushima, Japan). BH4 was a kind gift from Suntory Ltd (Osaka, Japan). Benidipine was kindly donated by Kyowa Hakko Co. Ltd (Tokyo, Japan).
ACKNOWLEDGMENTS
We thank Fumihiko Ikemoto, PhD for review of the manuscript. This study was supported by the grants for medical research from Osaka City General Hospital.
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