Institute of Medicinal Chemistry, Hoshi University, 2-4-41, Ebara, Shinagawaku Tokyo 142-8501, Japan
Received on August 12, 2003; revised on September 17, 2003; accepted on September 21, 2003
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Abstract |
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Key words: diabetes / hyaluronidase / serum / urine / zymography
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Introduction |
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Hyaluronidases (HAases), a family of enzymes involved in the degradation of HA, have also begun to attract attention. In humans, six HAase genes have been identified: HYAL1, HYAL2, and HYAL3 are clustered on chromosome 3p21.3, and PH20, HYAL4, and HYALP1 are clustered on chromosome 7q31.3. With the possible exception of HYAL4 and HYALP1, the other four are recognized as HAases that degrade HA (Csóka et al., 2001). Hyal-1, originally identified as a serum enzyme, was the first HAase to be purified to homogeneity from plasma (Frost et al., 1997
). The highest levels of mRNA of HYAL1 are found in the major parenchymal tissues, such as liver, kidney, spleen, and heart. mRNA of HYAL2 is also present in most tissues, except adult brain, and the protein product Hyal-2 is considered to be a typical lysosomal enzyme originally. More recent evidence has shown that Hyal-2 can also be exposed on the cell surface bound to the plasma membrane via a glycosylphosphatidylinositol anchor (Lepperdinger et al., 1998
; Rai et al., 2001
). PH-20, or testicular HAase, is primarily involved in the degradation of the HA-rich cumulus layer surrounding the egg by sperm cells (Cherr et al., 2001
). HYAL3 transcripts have been detected in brain and liver tissues, but the protein product Hyal3 is uncharacterized (Triggs-Raine et al., 1999
). Among the three characterized HAases, Hyal-1 and Hyal-2 are acidic enzymes having an optimum pH of 3.94.3 and PH-20 is neutral HAase having a broad optimum pH of 56.
Recently, a patient with a mutation of HYAL1 termed mucopolysaccharidosis IX has been described. The clinical phenotype is surprisingly mild, indicating that other HAases such as Hyal-2 and Hyal-3 compensate for the disorder of Hyal-1 (Triggs-Raine et al., 1999). Several investigators have also demonstrated that the levels or expression of HAases are changed in tissues, sera, or urine in diseases such as cancer (Lokeshwar et al., 1996
; 1999
; Bertrand et al., 1997
; Tamakoshi et al., 1997
; Laudat et al., 2000
), suggesting an important role of the enzymes.
Diabetes mellitus is a metabolic disease characterized by hyperglycemia secondary to relative or absolute insulin deficiency. Chajara et al. (1999, 2000a
) have demonstrated that HA participates in the development of diabetic angiopathy. However, the involvement of HAase in diabetic situations has scarcely been reported. Therefore, we attempted to investigate the changes in serum and urinary HAase levels with the progress of diabetes, using experimental models of two types of diabetes mellitus, streptozotocin (STZ)-induced diabetic Wistar and Goto-Kakizaki (GK) rats.
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Results |
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Discussion |
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Urinary total HAase activity also was significantly increased in the STZ group compared to that in the control group (Table I, Figure 5c). Leakage of blood HAase due to renal basement membrane impairment and deviation of HAase due to destruction of ureter or bladder cells were considered to be the causes. Of these, the most likely cause of increased urinary HAase activity in the STZ group may be leakage of blood HAase. It is known that a minute amount of urinary protein, that is, microalbuminuria, appears in the early stage of diabetic nephropathy. Phillips et al. (1999) reported that in STZ-induced daibetic rats, prominent mesangial cell proliferation and glomerular macrophage infiltration were detected from 3 days after induction. Cadaval et al. (2000)
showed that the increase of 24-h urine volume as well as albuminuria remained significant from the second week. Therefore, it is quite possible that these findings support leakage of blood HAase from the early stage, resulting in an increase in urinary HAase activity.
This assumption was strongly supported by the present finding that there was a good correlation between total urinary HAase activity and albumin excretion in the urine samples from STZ, GK, and control rats (Table I), although only in single measurements at the 18th week (age 26 weeks). Namely, only the STZ group had significant increases in both urinary HAase activity and albumin excretion compared with those of the control group. Furthermore, a 120-kDa HAase band that may have been derived from blood was detected only in the STZ group urine. The measurement of total urinary HAase activity was performed only in the 8th, 15th, and 18th week in this study, but it is necessary to extensively investigate when urinary HAase starts to increase during the progression of diabetes, including the relationship with the development of nephropathy. In contrast, there were no significant differences not only in urinary HAase activity but also in albumin excretion between the GK and control groups, implying that blood HAase as well as albumin did not leak in the GK group. This is compatible with the report that although slight abnormality was noted in the glomeruli in 26-week-old GK rats, it had not reached renal dysfunction (Phillips et al., 1999). The findings of the present study suggest that total urinary HAase activity, as well as the appearance of 120-kDa HAase band, may be used as a good marker reflecting progression of nephropathy in diabetes.
Rat HAase is considered to consist of six genes, by analogy to human and mouse HAases that have been most extensively characterized among mammalian enzymes, but only rat PH20 (Hou et al., 1996) and HYAL2 (Liu et al., 2003
) genes have been identified. On the other hand, the presence of HAase activity in the rat has long been known in various tissues and body fluids including serum. Recently Fiszer-Szafarz et al. (1990)
, using native PAGE zymography, demonstrated the polymorphism of HAase in sera from several mammalian species, including humans and rats. Fiszer-Szafarz et al. (2000)
further demonstrated that human HAases from somatic tissues and body fluids were present in multiple forms and that some of the multiple forms were changed by sialidase treatment to desialylated forms, including their dimer forms. In the present work, sodium doceyl sulfate (SDS)PAGE zymography was used, not the native PAGE zymography used by Fiszer-Szafarz et al. (1990)
, revealing that two of the four rat serum HAase bands at 0 time incubation were changed to the two other bands on incubation in the presence of SDS (Figure 6). These conversions of rat serum isomers, however, seemed not to be due to desialylation because sialidase activity, if present in rat serum, could scarcely act in the presence of 2% SDS. Furthermore, the possibility of desialylation was unlikely because desialylation would result, rather in an increase in electrophoretic mobility owing to the decreased molecular weight. The most probable explanation is that rat serum HAase isomers are not as easily SDS-denatured as human serum HAase (Hyal-1, 59 kDa) and they take longer to be completely SDS-denatured to finally give 132- and 73-kDa bands. This explanation is not contradictory to the smearing of the smaller bands up to the larger bands, because a completely denatured, extended form of protein moves more slowly than a native compact form of protein on SDSPAGE gels. Thus, rat serum seemed to contain only two, but not four, forms of HAase, consisting of the major one of 73 kDa (Hyal-1 type) and the minor one of 132 kDa. Similarly, rat urine seemed to contain only one, but not two, form of HAase. Rat urinary HAase is considered to be Hyal-1 type HAase, analogous to human urine that contains Hyal-l (57 kDa) as a major HAase and its proteolytically processed smaller isoform (45 kDa) (Csóka et al., 1997
). Both the 71- and 46-kDa bands of rat urine, when examined carefully, appeared not to be a simple broad band but to be a doublet-shaped broad band. This strongly suggested that these doublet-shaped broad bands consisted of two very similar but not identical sized HAases, the larger of which being Hyal-1 type HAase (73 kDa) and the smaller being a processed form (a little less than 73 kDa) of Hyal-1 type enzyme, possibly with kidney proteases. It is noteworthy that only STZ rats, but not GK rats or control rats, displayed a urinary HAase band of 120 kDa in addition to the doublet-shaped broad band centered at 71 kDa, which was commonly exhibited by every group of rats. Although the nature of the urinary HAase band of 120 kDa is unknown, it possibly results from circulating HAase isomer of 132 kDa, due to enhanced glomerular permeability in STZ rats, with concomitant processing by kidney proteases.
In the present work, we demonstrated for the first time the enhancement of circulating HAase from the early process of diabetes development. Furthermore, the increase in total urinary HAase activity, together with the appearance of urinary 120-kDa HAase, may be a useful marker for diabetic nephropathy.
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Materials and methods |
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Experimental animals
Male Wistar and GK rats (Japan Laboratory Animals, Tokyo) of 8 weeks' age were used. Wistar rats were separated into two groups, control and STZ groups. To induce type 1 diabetes mellitus, a single injection of STZ (60 mg/kg body weight) dissolved in normal saline buffered with 0.1 M sodium citrate (pH 4.5) was carried out via the tail vein of STZ group rats. Control group rats received an equivalent volume of the dissolving buffer alone. Spontaneously diabetic GK rats were used as a model of type 2 diabetes mellitus. The three groups of rats were housed three or four per cage and fed ad libitum with regular chow and tap water from 8 to 26 weeks old. All experiments were performed in accordance with the Guidelines for Animal Experiments in Hoshi University.
Blood was sampled from the jugular vein and, after standing for 30 min at 4°C, centrifuged at 500 x g for 15 min to obtain serum. Serum glucose level was determined by a glucose oxidase method with a kit (Glucose CII-Test Wako) supplied by Wako.
For 24-h urine collection, each rat was placed in a metabolic cage for 24 h. The collected urine was centrifuged to remove debris and frozen until use. Urinary albumin was measured by using a rat albumin enzyme-linked immunosorbent assay kit (Shibayagi, Gunma, Japan) with rat albumin as a standard.
Determination of HAase activity
Serum HAase activity was determined by the fluorimetric Morgan-Elson assay method as described recently (Takahashi et al., 2003). Briefly, 125 µl of the substrate solution (1.5 mg/ml HA in 0.1 M formate buffer, pH 3.9, containing 0.1 M NaCl and 1.5 mM saccharic acid 1,4-lactone) was mixed with 5 µl serum and digested at 37°C for 15 min. After heating in a boiling water bath for 5 min, the Morgan-Elson reaction was started by the addition of 25 µl 0.8 M potassium tetraborate reagent (pH 10.4) and subsequent heating for 3 min in a boiling water bath. After cooling to room temperature, 0.75 ml DMAB reagent was added and incubated at 37°C for 20 min. After centrifugation at 18,000 x g at 4°C for 10 min, the fluorescence (excitation, 545 nm; emission, 604 nm) of the clear supernatant was measured against that of a blank test, which was carried out in the same way except that the enzyme reaction mixture was incubated for 0 min. One unit of HAase activity was defined as the amount of enzyme required to produce 1 µmol of reducing terminal GlcNAc per min under the specified conditions.
Zymography of HAase
HAase was detected by HA zymography (Miura et al., 1995; Podyma et al., 1997
) with slight modifications. Rat serum was diluted with nine volumes of 0.15 M NaCl and mixed with an equivalent volume of Laemmli's sample buffer (Laemmli, 1970
) containing 4% SDS and no reducing reagent. After incubation for 1 h at 37°C without heating, 3 µl of the mixture (serum: 0.15 µl) were applied to 7% SDSpolyacrylamide gels containing 0.17 mg/ml HA. Rat urine was directly mixed with an equivalent volume of sample buffer and, after incubation for 1 h at 37°C, 520 µl of the mixture (urine: 2.510 µl) were applied. After electrophoretic run at 25 mA for approximately 70 min at 4°C, gels was rinsed with 2.5% Triton X-100 for 80 min at room temperature and incubated with 0.1 M formate buffer (pH 3.5) containing 0.03 M NaCl for 18 h at 37°C on an orbital shaker. Gels were then treated with 0.1 mg/ml Actinase E in 20 mM TrisHCl buffer (pH 8.0) for 2 h at 37°C. To visualize digestion of the HA, gels were stained with 0.5% Alcian blue in 25% ethanol10% acetic acid. After destaining, gels were counterstained with Coomassie brilliant blue R-250. For the determination of HAase activity, the stained gel was scanned on an Epi-Light 2000 image analyzer with Luminous Imager version 2.0 (Aisin Cosmos, Japan). The relative band intensities of rat serum and urinary HAases were calculated from the ratios to the band intensity of HAase from 0.5 µl of a control human serum, as a standard on the same gel.
PAGE analysis of HA digest mixtures
The procedures employed for electrophoresis using 15% polyacrylamide gel were essentially as described by Ikegami-Kawai and Takahashi (2002). HA digest samples to be examined were prepared as follows. One hundred twenty five microliters of a 1.5 mg/ml HA solution was digested with 5 µl of serum as described except that an incubation time of 30 min was used. After centrifugation at 18,000 x g for 10 min, 10 µl of the clear supernatant was mixed with 2 µl 2 M sucrose in Tris/borate/ethylenediamine tetra-acetic acid buffer and 2 µl of the mixture was applied directly to the gels. The electrophoretic run was carried out at 4°C first at 250 V for 20 min, then at 580 V for 10 min, and additionally at 450 V for approximately 15 min. After electrophoresis, oligosaccharides were fixed in the gel matrix by soaking the gel in 0.05% Alcian blue dissolved in water for 0.5 h in the dark. After destaining in water, the gel was subjected to silver staining, beginning from the oxidation step. The stained gel was scanned on an Epi-Light 2000 image analyzer with Luminous Imager version 2.0. The HAase activity was measured as a total of the relative band intensities for oligosaccharides of n18 to n24.
Statistical analysis
Results are expressed as means ± SD. For comparison of multiple means, analysis of variance was used, followed by Fisher's protected least significant difference test. The significance level was indicated in each experiment.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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