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Technical Briefs |
uzhan Deyneli1
1
Marmara University School of Medicine, Section of Endocrinology and Metabolism and
2
Department of Cardiology, Sakizgulu sok. No. 1-3, D:15, Kadikoy Istanbul, Turkey 81030;
3
Hipokrat Research Laboratories, Istanbul, Turkey;
a author for correspondence: fax 90-216-428-0013, e-mail dyavuz{at}turk.net
Glycosaminoglycans (GAGs) are major components of the basement membranes and play a key role in their molecular organization and function (1)(2)(3). Some authors have proposed that increased loss of proteoglycan from glomerular basement membrane (GBM) alters glomerular charge selectivity, which contributes to urinary loss of albumin (4)(5)(6)(7)(8).
Several clinical studies have shown that the GAG content of human GBM is significantly decreased and that urinary loss of GAG is markedly increased in diabetic patients (7)(8)(9). Among the possible mechanisms of diabetic microalbuminuria are decreased synthesis (10) and/or increased loss of GAGs (9)(11) from the GBM.
Although the importance of microalbuminuria is unclear in hypertensive patients, it may be an early marker of glomerular functional and structural changes (12)(13). Because hypertension is considered a systemic disease, basement membrane changes should be widespread. Whether basement membranes from other tissues are also affected and whether these changes are the result of hypertension remains to be determined. Therefore, in our study, we examined the effects of hypertension on basement membrane anionic charges in newly diagnosed, untreated essential hypertensive patients.
Twelve nonsmoking, essential hypertensive patients, stage I or II according to the sixth report of Joint National Committee (14), and 12 age- and sex-matched healthy normotensive controls were included in the study. All patients gave informed consent, and the institutional local ethics committee approved the study.
Blood pressure was measured after a 30-min rest on three different occasions at the sitting position, and Korotkov sounds phase V were estimated as diastolic blood pressure. None of the patients had ever received any antihypertensive medication before participation in the study. The duration of the hypertension was 6 ± 4 months according to the medical records. Secondary hypertension was excluded. Diabetes and impaired glucose tolerance were excluded with an oral glucose tolerance test.
A 24-h urine sample was collected, and creatinine, microalbumin, total GAG, and GAG subfractions were measured. Blood samples were also collected to measure red blood cell anionic charge (RBCCh) and creatinine concentrations.
RBCCh was evaluated with a cationic dye, Alcian Blue 8Gx (cat. no. A5268; Sigma) according to a previously described method (15)(16), with minor modifications. Platelets and leukocytes were removed by the method of Beutler et al. (17). The intra- and interassay CVs for Alcian Blue binding were 5.8% and 7.6%, respectively.
The urine total GAG concentration was measured in 24-h urine samples with a colorimetric method described by Jong et al. (18), using 1,9-dimethylene blue (Aldrich) and bovine kidney heparan sulfate as the calibrator (cat. no. H 7640; Sigma) with a Shimadzu 2000 UV spectrophotometer at a wavelength of 520 nm. The intra- and interassay CVs were 2.4% and 15%, respectively.
Urinary GAG subfractions were separated with a method described by Heickendorff et al. (19) with minor modifications. In untreated urine samples, GAGs were precipitated with cetylpyridinium chloride after an overnight incubation at 4 °C and then centrifuged. GAG complexes were then dissolved in n-propanolol. Ethanol containing 20 g/L potassium acetate was used for further precipitation; distilled water was added to dissolve the complexes. Isolated GAGs were separated by electrophoresis on cellulose acetate (Titan III; Helena Laboratory). The samples were analyzed in duplicate using 0.1 mol/L barium acetate (pH 5.0) buffer and 0.3 mol/L cadmium acetate (pH 4.1) buffer systems. The distribution of GAGs was quantified by densitometry at a wavelength of 610 nm after staining with 1 g/L Alcian Blue. The following were used as calibrators: heparan sulfate from bovine kidney (cat. no. H 7640; Sigma), chondroitin sulfate A from bovine trachea (cat. no. C 8529; Sigma), and dermatan sulfate from shark cartilage (cat. no. C 4384; Sigma). The distribution of GAG types was expressed as fraction of the total GAG content.
Urinary albumin was measured by the nephelometric method, using a kit from Behring Diagnostics. The inter- and intraassay CVs were 4.4% and 4.3%, respectively. The urine serum and creatinine concentrations were measured by the automated Jaffé method with a Boehringer kit.
Statistical analysis was performed with an IBM-compatible PC using the Instat II program. Kruskal-Wallis ANOVA, Mann–Whitney U-tests, and Student t-tests were used as appropriate for comparisons, and the Spearman rank test was used for correlation analysis. The results were expressed as mean ± SE.
The clinical and biochemical findings in both groups are shown
in Table 1
. The urinary total GAG excretion was significantly higher in
the hypertensive patients (P <0.05). Although the heparan
sulfate subfraction was higher in hypertensive patients compared with
the normotensive group, the dermatan sulfate subfraction of GAG was
higher in the normotensives. Urinary chondroitin sulfate subfractions
were similar in both groups. However, when absolute urinary excretion
rates were considered, the excretion rates for heparan, chondroitin,
and dermatan sulfate in urine were 3.68, 1.22, and 4.58 mg/day for the
hypertensive group, and 0.35, 2.06, and 2.8 mg/day for the control
group, respectively.
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The binding of Alcian Blue to RBCs was 448 ± 6.3 ng Alcian Blue/106 RBC in hypertensive patients and 468 ± 1.5 ng Alcian Blue/106 RBC in the normotensive group (P <0.001). Although the urinary albumin excretion rate was negatively correlated with RBCCh (r = -0.35; P <0.05), it did not reach statistical significance with urinary total GAG excretion. RBCCh was slightly correlated with urinary total GAG excretion (r = -0.43; P <0.05). Diastolic blood pressure was positively correlated with albuminuria (r = 0.59; P <0.005) and urinary total GAG excretion (r = 0.55; P <0.01).
Similar increases of 24-h urinary total GAG excretion have been reported in diabetic patients. Previous studies have found that an increased urinary heparan sulfate excretion rate is associated with the loss of basement membrane anionic charge in diabetic nephropathy (7)(8)(9)(10). If hypertension is considered a systemic disease that exhibits vascular dysfunctional changes and end-organ complications similar to those seen in diabetes (12)(13), increased urinary GAG excretion could be attributed to the renal effects of hypertension.
Because RBCCh, which is a crude reflection of GBM anionic charge (15)(20), was correlated with urinary GAG excretion in our study, we propose an association between increased urinary GAG excretion and the loss of GBM anionic content in essential hypertensive patients. Heintz et al. (21) documented a significantly decreased urinary small heparan sulfate excretion in hypertensive patients that is clearly distinct from basement membrane-associated large heparan sulfate proteoglycan. Taken together, these findings suggest a complex rearrangement of GAG metabolism in hypertension, with both decreased synthesis and/or increased urinary loss of local heparan sulfate-containing molecules that may contribute to the former.
Our study showed that all GAG components other than heparan sulfate were affected in the early stages of essential hypertension.
The correlation between diastolic blood pressure and albuminuria has been shown previously (22)(23). In our study, diastolic blood pressure values were positively correlated with 24-h urinary albumin and GAG excretion, suggesting a direct effect of increased arterial pressure on GBM. We conclude that hypertension alters urinary GAG excretion and that loss of glomerular anionic content may be associated with increased urinary GAG excretion.
References
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