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Plasma Brain Natriuretic Peptide Levels in Normotensive Noninsulin-Dependent Diabetic Patients with Microalbuminuria

Third Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie 514-8507, Japan

Address all correspondence and requests for reprints to Dr. Yutaka Yano, Third Department of Internal Medicine, Mie University School of Medicine, Edobashi 2–174, Tsu, Mie 514-8507, Japan. E-mail: yanoyuta{at}clin.medic.mie-u.ac.jp

	Abstract

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Brain natriuretic peptide (BNP), a member of the natriuretic peptide family, is produced and released from cardiac ventricles. BNP regulates the body fluid volume, blood pressure, and vascular tones through the A-type guanylate cyclase-coupled receptor. The presence of renal dysfunction in patients with diabetes affects the plasma levels of atrial natriuretic peptide (ANP). In the present study, we investigated the plasma levels of BNP and ANP and their relationship in normotensive diabetic patients with normoalbuminuria and microalbuminuria. Forty-seven normotensive lean noninsulin-dependent diabetic patients (31 with normoalbuminuria, 16 with microalbuminuria), with normal cardiac function, and 30 age-matched control subjects were enrolled in this study. The plasma levels of BNP in diabetic patients with microalbuminuria were significantly higher than those in diabetic patients with normoalbuminuria (16.7 ± 2.4 vs. 9.6 ± 1.3 pg/mL, P < 0.01) or normal subjects (16.7 ± 2.4 vs. 7.0 ± 0.6 pg/mL, P < 0.01). There was a significant positive correlation between plasma BNP levels and urinary albumin excretion rate in all diabetic patients (r = 0.58, P < 0.0001). There was also a significantly positive correlation between plasma BNP and ANP levels in diabetic patients (r = 0.62, P < 0.0001). The increased plasma level of BNP in patients with microalbuminuria and its significant correlation with urinary albumin excretion rate suggest that the elevated circulating levels of BNP are caused by the presence of diabetic nephropathy. Down-regulation of A-type guanylate cyclase-coupled receptor of renal tubules may explain the increased plasma levels of both BNP and ANP in normotensive diabetic patients with microalbuminuria.

	Introduction

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BRAIN natriuretic peptide (BNP) plays an important role in the regulation of body fluid and blood pressure (1, 2). BNP secretion from cardiac ventricles is stimulated by volume or pressure overload, and its vasodilating effect is mediated by A-type guanylate cyclase-coupled receptor (NPR-A) (1, 2). The plasma levels of the atrial natriuretic peptide (ANP) were previously reported to increase in normotensive diabetic patients with microalbuminuria (3, 4, 5, 6). The secretion of ANP is stimulated by increased atrial pressure associated with volume or pressure overload (1, 2). However, the elevation in the plasma level of ANP in diabetic patients can not be completely explained by increased plasma volume or elevation of the right atrial pressure (7, 8). The natriuretic response to volume expansion during saline infusion or water immersion is blunted in diabetic patients (9, 10). It was previously reported that elevation of ANP is caused by down-regulation of NPR-A in target tissues, such as renal tubules (4, 11). The biological activity of BNP, which resembles that of ANP, is also mediated by NPR-A (1, 2). The plasma BNP levels may be influenced by the total number of NPR-A. No study has been carried out regarding the plasma levels of BNP in diabetic patients.

In the present study, we assessed the plasma levels of BNP in normotensive, noninsulin-dependent diabetes mellitus (NIDDM) patients, with or without microalbuminuria, and its relationship with the plasma level of ANP.

	Subjects and Methods

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Subjects

This study comprised 47 nonobese (body mass index = 22.9 ± 0.8) patients (34 men and 13 women) with NIDDM diagnosed according to the criteria of the World Health Organization (12). Data obtained in 30 age-matched, healthy volunteers (20 men, 10 women) with normal glucose tolerance were available for comparison. Informed consent was obtained from all subjects. Diabetic patients presented no history of ischemic heart disease, cerebral vascular accident, or intermittent claudication. None of the patients had arterial hypertension (<140/90 mm Hg), severe nephropathy (serum creatinine, 100 µmol/L), or orthostatic hypotension. All patients had normal cardiothoractic ratio (on plain radiography of the chest) and normal cardiovascular function and no myocardial hypertrophy (on ultrasonography examination). None of the patients was receiving antihypertensive drugs, including angiotensin-converting enzyme inhibitor, before beginning the study. Thirty patients were being treated with diet therapy alone and 17 with oral hypoglycemic agents. Albuminuria was measured in urine collected during 24 h, and repeated measurements (3 times) were done to evaluate the mean value of the urinary albumin excretion rate (UAE). According to the criteria of Mogensen (13), 31 patients showed normoalbuminuria (20 µg/min) and 16 patients showed microalbuminuria (20–200 µg/min). Twenty-four-hour urinary excretion of sodium was also measured in all patients. Fundi of the patients were examined by an ophthalmologist. The grade of retinopathy was determined by using a binocular indirect ophthalmoscope after applying a mydriatic agent (tropicamide). The findings were classified as follows: no change; or with background- or proliferative diabetic retinopathy. One patient had proliferative retinopathy, 9 had background retinopathy, and 37 had normal findings. Patients were categorized as having symptomatic symmetrical diabetic neuropathy based on the presence of paresthesia or numbness, reduced or absent patellar tendon reflex, or reduced vibratory perception. Based on these criteria, diabetic peripheral neuropathy was found in 8 patients (2 with normoalbuminuria, 6 with microalbuminuria). Cardiovascular autonomic neuropathy was assessed by the coefficient of variation of R-R intervals during deep breathing (6 times/min) with 100 beats. No patient had abnormal values of coefficient of variation of R-R intervals, showing that none of our patients had autonomic dysfunction.

Laboratory measurements

The HbA_1C levels (normal range: 4.3–5.8%) were measured by high-performance liquid chromatography. The serum levels of insulin were estimated by RIA kit (Dainabot, Tokyo, Japan). Urinary albumin concentration was measured, at least three times, by RIA kit (Pharmacia, Uppsala, Sweden) in urine collected during 24 h. Plasma glucose levels were measured by a glucose oxidative method. The serum levels of lipids, sodium, and potassium were measured by automated enzymatic methods. Blood sampling was done in all subjects, from an antecubital vein, after fasting overnight and resting over 30 min, in a supine position, after admission. Salt intake was restricted to 153 mmol/day NaCl, around 3 weeks before taking the blood samples. Serum and plasma were separated by centrifuging (1500 g), at 4 C for 20 min, and then stored at -70 C until use. The glomerular filtration rate was estimated by creatinine clearance, calculated using urine collected for 24 h, and the mean value of three measurements was given.

Plasma ANP levels were measured using a commercially available immunoradiometric assay kit (Shionoria ANP kit, Shionogi, Osaka, Japan) (14, 15). The intra- and interassay coefficients of variation were 4.7% and 5.8%, respectively. The detection limit of the assay was 5 pg/mL. Plasma BNP levels were measured using a immunoradiometric assay kit (Shionoria BNP kit, Shionogi), following methods previously described (15). For assaying BNP levels, two monoclonal antibodies against human BNP were used: one recognizing the carboxylterminal sequence of BNP; and the other, its ring structure. Briefly, 100 µL of sample or standard was incubated with 200 µL of mouse ¹²⁵I-labeled antihuman BNP antibody and with beads coated with mouse antihuman BNP antibody, at 4 C for 24 h. The beads were then washed twice using 0.01 M phosphate buffer saline containing 0.02% Tween 20. The remaining radioactivity in the beads was then determined using a -counter. The intra- and interassay coefficients of variation were 5.3% and 5.9%, respectively. The detection limit of the assay was 2.0 pg/mL.

Statistical analysis

Data are expressed as the mean ± SEM. Statistical analyses were performed using the Stat View software package (Abacus Concepts, Berkeley, CA) for the Macintosh computer. The statistical difference among three groups was calculated by the Kruskal-Wallis test; and the difference between the mean of two variables, by the Mann-Whitney U test. The relationship between variables was investigated using the Pearson’s product-moment correlation. The distribution of variables was assessed by the Kolmogorov-Smirnov two-sample test. P < 0.05 was considered statistically significant.

	Results

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The clinical profile of normal subjects and diabetic patients is described in Table 1. Diabetic patients with hypertension, cardiac, or renal dysfunction were excluded from the study. The arterial blood pressure was not significantly different among diabetic patients with normoalbuminuria, those with microalbuminuria, and normal subjects. There was no significant difference in the plasma levels of fasting glucose, HbA1c, and fasting insulin between diabetic patients with normoalbuminuria and those with microalbuminuria (Table 1). The values of hematocrit (43.6 ± 0.8 vs. 43.0 ± 1.0%), the serum levels of sodium (141.8 ± 0.4 vs. 141.2 ± 0.6 mmol/L), potassium (4.2 ± 0.1 vs. 4.1 ± 0.1 mmol/L), creatinine clearance levels (1.60 ± 0.04 vs. 1.56 ± 0.04 mL/sec), 24-h urinary sodium excretion (125 ± 6 vs. 132 ± 9 mmol/24 h), and cardiothoractic ratio (47.8 ± 0.6 vs. 47.5 ± 0.7%) were not significantly different between patients with normoalbuminuria and those with microalbuminuria.

View larger version (29K): [in this window] [in a new window]   Figure 1. Plasma BNP levels in normal subjects and normotensive noninsulin-dependent diabetic patients with normoalbuminuria and microalbuminuria. Data are mean ± SEM.

View larger version (18K): [in this window] [in a new window]   Figure 2. Relationship between plasma BNP and UAE in all normotensive noninsulin-dependent diabetic patients.

View larger version (18K): [in this window] [in a new window]   Figure 3. Relationship between plasma BNP and ANP levels in all normotensive noninsulin-dependent diabetic patients.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

However, the plasma levels of BNP, as yet, have not been reported in diabetic patients. In this study, we evaluated the plasma levels of BNP in NIDDM patients with early-stage diabetic nephropathy. Because the plasma level of BNP is influenced by cardiac or renal function and by blood pressure (19, 20, 21, 22), patients with cardiac or renal dysfunction or arterial hypertension were excluded from the study. The plasma levels of BNP significantly increased in diabetic patients with microalbuminuria, as compared with those with normoalbuminuria and with normal subjects. The plasma levels of BNP were significantly correlated with UAE in diabetic patients. These results suggest that the plasma levels of BNP increase in early-stage diabetic nephropathy.

The mechanism by which the plasma levels of BNP increase in diabetic patients with nephropathy is not clear. It was previously reported that vascular volume expansion is not significantly different between diabetic patients with normoalbuminuria and microalbuminuria (23) and that impairment of ANP response to volume expansion occurs in normotensive diabetic patients (9, 10). These observations may be explained by down-regulation of NPR-A in renal tubules. In this connection, elevation in the plasma levels of ANP was found associated with decreased mRNA levels in the kidneys of rats with hyperglycemia (24). Moreover, low renal response to exogenous ANP was observed in streptozotocin-induced diabetic rats with a decreased number of biological active NPR-A (11). In the present study, the plasma levels of BNP were found to be significantly correlated with the plasma levels of ANP. Because the biological activity of BNP is also mediated by NPR-A, it is conceivable that the elevation in the plasma levels of BNP also occurs because of down-regulation of renal NPR-A in diabetic patients with nephropathy.

Both BNP and ANP are known to decrease vascular tone by acting on the muscular layer of vessels (1, 2). It was previously reported that vascular diastolic response to ANP is decreased in diabetic patients (25), probably because of vascular structural alteration associated with diabetic microangiopathy (26, 27). This abnormal vascular response may also explain the elevation in the plasma levels of BNP. Thus, the increased plasma levels of BNP may occur not only by reduction of sodium excretion in renal tubules but also by reduced vascular diastolic response.

The relationship found between the levels of BNP and microalbuminuria remains unclear. It was previously reported that infusion of ANP increases the urinary excretion of albumin in diabetic patients but not in normal subjects (4, 28). ANP is known to exert a vasodilatory effect on afferent arteries and a vasoconstrictive activity on efferent arteries of glomeruli and thereby increase the glomerular hydraulic pressure (29, 30). Diabetic nephropathy-associated structural abnormalities of glomeruli alter the charge and size selectivity in the glomerular protein filtration. Elevation of urinary albumin excretion is induced by ANP infusion in diabetic patients, probably because of the occurrence of both structural and pressure abnormalities. Because both BNP and ANP bind to the same receptor and have the same biological activity, it is conceivable that abnormally elevated levels of BNP also cause increased glomerular hydraulic pressure and thus induce increased albumin excretion in diabetic patients. However, we believe that the primary cause of the increased circulating levels of BNP and ANP is probably the decrease in the number of its receptors in renal tissue of patients with diabetic nephropathy.

In brief, the present study showed that the plasma levels of BNP are significantly correlated with the degree of microalbuminuria in patients with diabetic nephropathy. Measurement of the plasma level of BNP may be an important adjuvant tool for the assessment of renal complications in patients with diabetes mellitus.

	Acknowledgments

 
We thank Naomi Hashimoto for her helpful secretarial assistance in the preparation of this manuscript.

Received August 7, 1998.

Revised March 16, 1999.

Accepted March 19, 1999.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yano, Y. Articles by Adachi, Y. Search for Related Content PubMed PubMed Citation Articles by Yano, Y. Articles by Adachi, Y.