ELISA for urinary trehalase with monoclonal antibodies: a technique for assessment of renal tubular damage.

BACKGROUND: alpha,alpha-Trehalase, located on renal proximal tubules, is a glycoprotein that hydrolyses alpha,alpha-trehalose to two glucose molecules. Urinary trehalase reflects damage to renal proximal tubules, but its activity has not been measured routinely because measurement of catalytic activity is rather complicated and because conventional assays for enzyme activity might not reflect all of the trehalase protein because of enzyme inactivation in urinary samples. METHODS: We established novel monoclonal antibodies for human trehalase and a sandwich ELISA for quantification of urinary trehalase. We determined the urinary trehalase protein concentration with this ELISA and trehalase catalytic activity, and the results of these two methods were compared. RESULTS: The ELISA system was more sensitive than the detection of enzyme activity and could detect a subtle difference in the amount of trehalase present in renal diseases. The within- and between-assay CVs in the ELISA were 6.7-7.6% and 6.2-8.2%, respectively. Highly significant increases in both the quantity and activity were seen in patients with nephrotic syndrome (acute phase), Lowe syndrome, and Dent disease. The quantities were 70- to 200-fold greater, whereas enzyme activities were, at most, 10-fold higher than those of control subjects. In the detection of small amounts of trehalase in patients with chronic glomerulonephritis and renal anomalies, quantities were better than enzyme activities. CONCLUSIONS: We have established an ELISA system for quantification of urinary trehalase that uses novel monoclonal antibodies. Our ELISA system is simpler and more sensitive than a conventional activity assay and reflects trehalase protein. This ELISA can be a useful as a common tool for clinical assessment of renal proximal tubular damage.

Reduction of Staphylococcus aureus in atopic skin lesions with acid electrolytic water--a new therapeutic strategy for atopic dermatitis.

The subjects studied were 22 pediatric patients newly diagnosed with atopic dermatitis (AD); 11 were treated with acid electrolytic water (AEW), which has a strong bactericidal activity (AEW group), and the other 11 with tap water (placebo group). AEW or tap water, 1 ml/cm2 (body surface area), was sprayed on their skin lesions with a spray gun each twice a day for a week. There were no significant differences between the two groups in regard to sex, age, serum IgE, peripheral eosinophil counts, grading scores of AD, and duration of AD. The study was designed as a randomized, placebo-controlled, double-blind clinical trial. Colony counts of Staphylococcus aureus on skin lesions in the AEW group, both 3 min after spraying (P < 0.05) and after 1 week of skin treatment (P < 0.01), were significantly decreased as compared with colony counts before treatment, while there was no significant difference in the placebo group before and after treatment. Grading scores of AD also decreased in the AEW group (P < 0.01), but not in the placebo group. Both the subjects' guardians' evaluation and a referee physician's evaluation of treatment effect were significantly higher in the AEW group than in the placebo group (P < 0.01). AEW may be potentially effective in preventing a staphylococcal chronic inflammation in AD because of its strong bactericidal activity.

Molecular cloning, sequencing and expression of cDNA encoding human trehalase.

A complete cDNA clone encoding human trehalase, a glycoprotein of brush-border membranes, has been isolated from a human kidney library. The cDNA encodes a protein of 583 amino acids with a calculated molecular weight of 66,595. Human enzyme contains a typical cleavable signal peptide at amino terminus, five potential glycosylation sites, and a hydrophobic region at carboxyl terminus where the protein is anchored to plasma membranes via glycosylphosphatidylinositol. The deduced amino acid sequence of the human enzyme showed similarity to sequences of the enzyme from rabbit, silk worm, Tenebrio molitor, Escherichia coli and yeast. Northern blots revealed that human trehalase mRNA of approx. 2.0 kb was found mainly in the kidney, liver and small intestine. Expression of the recombinant trehalase in E. coli provided a high level of the enzyme activity. The isolation and expression of cDNA for human trehalase should facilitate studies of the structure of the gene, as well as a basis for a better understanding of the catalytic mechanism.

Endothelin-1 has a priming effect on production of superoxide anion by alveolar macrophages: its possible correlation with bronchopulmonary dysplasia.

The purpose of this study was to clarify the role of endothelin (ET)-1 in the development of bronchopulmonary dysplasia (BPD). Tracheal aspirates were obtained from 27 newborn babies with respiratory distress (13 with BPD and 14 without BPD) who were mechanically ventilated. Production of superoxide anion (O2-) by rabbit alveolar macrophages was determined by preincubation with the tracheal aspirate supernatant (TAS) and stimulation with phorbol myristate acetate (PMA). O2- production was demonstrated only when PMA was added to the experimental system and was enhanced with TAS of infants who later developed BPD compared with TAS from infants without BPD. The effects of ET-1 and ET-3 on O2- production and the blockade by anti-ET-1 antibody and BQ123 (ET A receptor antagonist) were also examined. The enhancing effect was blocked by either anti-ET-1 antibody or BQ123. PMA-stimulated production of O2- increased when cells were preincubated with several doses of ET-1 (5 x 10(-13) to 2 x 10(-12) M), whereas ET-3 was without effect. TAS contained significant amounts of immunoreactive ET-1, and there was a close positive correlation (r = 0.764) between the activity of O2- production and immunoreactive ET-1 levels in TAS samples. These results may be interpreted to indicate that ET-1 synthesized by and secreted from tracheal epithelial cells and/or alveolar macrophages has a priming effect on alveolar macrophages to produce O2-, thus possibly contributing to the development of BPD.

Human trehalase: characterization, localization, and its increase in urine by renal proximal tubular damage.

By using polymerase chain reaction, cDNA encoding human renal trehalase has been isolated. The partial amino acid sequence deduced by the cDNA showed homologies in rabbit, Tenebrio molitor and silkworm trehalase. Northern blots showed renal trehalase mRNA to be about 2.0 kb. To examine the properties of renal and urinary human trehalase, the trehalase cDNA was inserted in the pMAL-cRI vector downstream from the malE gene, which encodes maltose-binding protein. Transfection of the recombinant pMAL-cRI in Escherichia coli provided high levels of expression of the maltose binding protein-trehalase fusion protein. A rabbit was immunized with purified fusion protein, and antihuman trehalase antibodies were obtained. Immunoblot analysis disclosed that renal and urinary trehalase exhibited a molecular mass of about 75 kDa. Analysis by indirect fluorescent microscopy demonstrated that the enzyme located in only proximal tubular cells. Urinary trehalase activity was low in the healthy infants and elevated in patients with asphyxia. Markedly high activity was observed in a patient with Lowe syndrome. The immunoreactive urinary trehalase with 75 kDa was increased dependent on the elevation of the activity. On the basis of these findings, we conclude that the increase of urinary trehalase reflects the extent of renal tubular damage, and we propose that urinary trehalase can be a specific marker of renal tubular damage.

Urinary trehalase activity is a useful marker of renal proximal tubular damage in newborn infants.

To clarify the reliability of urinary trehalase activity as a marker of cellular proliferation and/or damage of renal proximal tubules, the activity was examined in healthy newborn infants or infants treated with tobramycin, a drug known as causing tubular cell damage. Eighty-one newborn infants (56 mature infants and 25 premature infants) were enrolled in the study. Urinary trehalase was examined using a spot urine sample during the first 7 days of age and on the 10th day of age. A good positive correlation was observed between urinary trehalase activity/creatinine ratio (T/Cr) on the 10th day of age and conceptional age or body weight (n = 46, r = 0.58, p < 0.001). Urinary trehalase of 29 healthy mature infants was higher during the first few days of age, after which it decreased to an almost steady level. Urinary trehalase of 6 premature infants during the first few days of age was significantly lower than that of mature infants, after which it increased and became equal to that of the mature infants on the 7th day of age. Treatment with ampicillin (100 mg/kg) and tobramycin (5 mg/kg) of 6 mature infants with pneumonia for 6 days resulted in a significant elevation of the urinary T/Cr. The extent of this elevation was greater than that of the urinary N-acetyl-beta-D-glucosaminidase (NAG) activity/creatinine ratio (NAG/Cr). A significant correlation was observed between the urinary T/Cr and the urinary NAG/Cr (r = 0.67, p < 0.01) or gamma-glutamyl transpeptidase/creatinine ratio (r = 0.48, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of renal tubular damage in preterm infants with renal failure.

This study attempts to clarify the characteristics of renal tubular damage in preterm infants with renal failure. Sixty-one neonates (17 term and 44 preterm infants) were divided into three groups: 15 infants with intrinsic renal failure (IRF), five term and 10 preterm; 19 with pre-renal renal failure (PRF), five term and 14 preterm; and 27 without renal dysfunction (control), seven term and 20 preterm. Urine was collected for an 8 h period on the 2nd or 3rd day of life to determine the following parameters: creatinine clearance (Ccr), fractional excretion of sodium (FENa), urinary N-acetyl-beta-D-glucosaminidase (NAG) index and endothelin-1 (ET-1) excretion. Parameters of renal tubular function and/or renal tubular damage such as FENa, NAG index and ET-1 excretion were considered as a useful marker to differentiate IRF from PRF in preterm infants. However, these parameters were significantly elevated in preterm infants with PRF. These findings led us to make the following speculations: (i) renal tubular damage may easily occur in preterm infants; and (ii) there still remains difficulty in differentiation between IRF and PRF using Ccr instead of the fluid challenge test.

Production of endothelin-1 induced by blood transfusion in premature infants.

We demonstrated a relationship between blood transfusion and plasma ET-1 levels in 10 premature infants with anemia. Changes in plasma ET-1 and lipoperoxide levels, blood pressure and available oxygen before and after the blood transfusion were determined. Plasma ET-1 levels were significantly elevated after blood transfusion (before: 8.0 +/- 2.8; after: 15.7 +/- 5.4 pg/ml, p < 0.01). A positive correlation was observed between plasma ET-1 levels and lipoperoxide levels (r = 0.887, p < 0.01). There was a significant correlation between plasma ET-1 levels and transfused blood volumes (r = 0.758, p < 0.01). No positive correlation was observed between plasma ET-1 levels and available oxygen. These results suggest that blood transfusion may be a stimulator of ET-1 overproduction and that it may lead to tissue injury through an endothelin-induced oxygen radical formation system.

Meconium-induced lung injury mediated by activation of alveolar macrophages.

To clarify the mechanism of meconium-induced cellular injury, we examined the effect of meconium on the alveolar macrophages (AM) and bronchial epithelial cells (AK-D). Meconium obtained from healthy newborns was added to culture medium of AM and/or AK-D, which were cultured for 1 hour. Superoxide anion production of AM and the expression of intercellular adhesion molecule-1 (ICAM-1) on AK-D stimulated by meconium or oxygen radicals were determined. As a result, superoxide anion production of AM significantly increased when AM was cultured with meconium. Expression of ICAM-1 on AK-D appeared when AK-D was stimulated by hydroxyl radical but did not when AK-D was cultured with meconium. These results suggest that meconium-induced lung injury may occur through an activation of alveolar macrophages and the macrophage-epithelial cell axis may be important for the pathogenesis of meconium aspiration syndrome.