[Urinary trehalase activity as an indicator of renal dysfunction in lead smelters]. / Stezenie trehalazy w moczu jako wskaznik dysfunkcji nerek osób zawodowo narazonych na dzialanie olowiu.

Epidemiological and experimental studies have demonstrated that lead and cadmium are responsible for renal dysfunction. Urinary trehalase is known as a good marker of proximal tubular renal brush border destruction in the population environmentally exposed to cadmium. The aim of this study was to determine the impact of occupational exposure to lead on the renal function and urinary trehalase activity. The study was carried out in 68 workers, aged 46 +/- 6 years, employed in a copper foundry. Blood lead, cadmium, copper and manganese concentrations were measured by atomic absorption spectrophotometry. Urinary trehalase activity was determined by the method of Nakano and Itoh. Trehalase activity was increased in copper smelters as compared to controls. There also was a positive linear correlation between blood lead level and urinary trehalase activity (r = 0.44; p < 0.05). Negative correlations between blood lead and copper concentrations (r = -0.30; p < 0.05) and between serum copper and trehalase level (r = 0.68; p < 0.001) were found. The results show that urinary trehalase activity could be a good indicator of the renal brush border dysfunction in copper smelters. This marker could be useful in the early diagnosis of nephrotoxic effect of lead.

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.

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)

Urinary trehalase activity in chronic glomerulonephritis.

To determine the diagnostic role of urinary trehalase in chronic glomerular disease, urinary trehalase activity and other urinary markers such as N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (gamma-GTP), lactate dehydrogenase (LDH), lysozyme and beta 2-microglobulin (BMG) were measured in patients with chronic glomerulonephritis, nephrotic syndrome and chronic renal failure. Urinary trehalase activity was significantly increased in chronic glomerular disease, especially nephrotic syndrome, as compared with that in the healthy subjects. The highest incidence of elevated excretion was observed for trehalase with 52% in chronic glomerular disease, followed by NAG. Urinary trehalase activities in the patients were significantly correlated with the urinary levels of protein, NAG and AAP and total score of tubular damage, but not correlated with urinary levels of BMG or lysozyme. In patients with chronic glomerulonephritis and nephrotic syndrome, there was no significant difference in urinary trehalase activities between with and without hematuria. These results indicate that in some patients with chronic glomerular disease, there is tubular involvement as substantiated by elevation of the other urinary enzymes and BMG. Urinary trehalase is elevated more often in these types of disease than other markers of tubular damage.

[Acute nephrotoxicity by CDDP (on tubular damage)].

Cisplatin (CDDP) is known to cause a nephrotoxic side effect. By using urinary trehalase which is localized renal tubular brush border, we examined an acute cisplatin nephrotoxicity. 1) With less than 300 mg of CDDP, urinary trehalase activity in the 1st day was significantly increased than preadministration. And urinary beta 2-MG was significantly increased in the 2nd day. But urinary NAG was not significantly increased. 2) With 300 mg or more of CDDP, urinary trehalase, beta 2-MG and NAG were more increased than preadministration. Urinary trehalase and beta 2-MG were inclined to increment. But urinary NAG was not inclined to decrement. Above all, renal tubular damage is reversible with less than 300 mg of CDDP, but is irreversible with 300 mg or more of CDDP.

Urinary trehalase activity as an indicator of kidney injury due to environmental cadmium exposure.

One hundred and seventy-eight subjects, patients with Itai-itai disease and their family members, aged 12-87 years living in a cadmium (Cd)-polluted area in the Jinzu River basin (Cd-exposed group) and 176 controls (control group) were examined. In the Cd-exposed group urinary trehalase increased with increasing age, urinary beta 2-microglobulin (beta 2-m) and retinol-binding protein. Although urinary cadmium was higher in the Cd-exposed group, no particular correlation was found between urinary trehalase and urinary cadmium. Seventeen men and 11 women showed raised urinary trehalase activities despite normal values of urinary beta 2-m (less than 300 micrograms/g.creatinine), suggesting that urinary trehalase increases earlier than urinary beta 2-m. In 19 patients with Itai-itai disease included in the Cd-exposed group, urinary trehalase decreased with decreasing reciprocal of serum creatinine, suggesting that urinary trehalase decreases in the most advanced cases of chronic cadmium nephropathy due to reduced tubular cell mass.

Severity of tubular brush border damage in cadmium-polluted area (Jinzu river basin): clinical role of urinary trehalase.

Urinary trehalase activity and leucine aminopeptidase activity were parabolically correlated with urinary beta 2-microglobulin, and these enzymes were observed to be biphasic in relation to urinary beta 2-microglobulin when the study populations included patients of Itai-itai disease and inhabitants of a cadmium-polluted area. Furthermore, urinary trehalase activity was parabolically correlated with urinary total protein and urinary glucose. From these results, it is inferred that by measuring both urinary trehalase and urinary beta 2-microglobulin, one can elucidate the degree of tubular damage.

Elevation of urinary trehalase activity in patients of itai-itai disease.

The elevation of urinary trehalase activity in patients of itai-itai disease was examined. Urinary trehalase was correlated with tubular reabsorption of phosphorus (%TRP): the lower the trehalase activity, the worse was %TRP. Furthermore, this activity was inversely correlated with urinary glucose and urinary total protein. In itai-itai disease, the excretion of beta 2-microglobulin seems to be maximal, and urinary trehalase activity was low in the latter stages of the disease. It is inferred that itai-itai disease produces extremely severe tubular damage as well as glomerular dysfunction.

[Tubular damage in toxemia of pregnancy using urinary trehalase as a marker].

We proved reversible tubular damage in edema and in toxemia of pregnancy using urinary trehalase as a marker. 1. Urinary trehalase activity in mild toxemia (edema: more than 0.5 kg body weight gain per week) was significantly increased as compared with normal pregnancy (less than 0.5 kg body weight gain per week) (p less than 0.02). Urinary albumin content, however, was not significantly changed with edema. 2. Toxemia of pregnancy showed significantly high urinary trehalase activity, NAG activity and beta 2-MG content as compared with the 3rd trimester of pregnancy. Urinary trehalase activity of severe toxemia was significantly higher than that of mild toxemia. Urinary NAG and beta 2-MG showed similar results to urinary trehalase. On the 5th and 30th puerperal days there was significantly lower trehalase activity than in the 3rd trimester. Urinary beta 2-MG in toxemia was significantly decreased at the 30th puerperal day as compared with the 3rd trimester and 5th puerperal day. However, no significant decrease was observed in urinary NAG. 3. Urinary trehalase activity in superimposed toxemia of pregnancy was significantly increased as compared with the 3rd trimester of pregnancy. However, urinary trehalase activity on the 5th puerperal day was significantly decreased, but was still significantly high. These results show that pregnancy with edema and pure toxemia of pregnancy cause renal tubular damage and this damage is reversible. In the stage of edema, no remarkable glomerular damage, but tubular damage could occur.

Urinary trehalase as an early indicator of cadmium-induced renal tubular damage in rabbit.

The significance of urinary trehalase as a possible early indicator of renal disorder was examined using Cd-treated rabbits, which received 1 mg/kg Cd thrice weekly for 3 months subcutaneously. The results showed that urinary trehalase increased significantly from 1 week after treatment, earlier than LAP, ALP, proteinuria and glucosuria, with no changes in plasma trehalase level. A marked decrease in trehalase activity in renal brush border membranes prepared from Cd-treated rabbits was observed. It was also confirmed by immunohistological techniques that Cd treatment resulted in a marked decrease in specific fluorescence compared with controls. Ouchterlony double diffusion analysis demonstrated that urine and renal brush border extracts formed precipitation lines against anti-renal trehalase IgG, indicating that urinary trehalase and renal trehalase had the same antigenicity. Therefore, the facts presented here would suggest that urinary trehalase originated from the renal brush border, indicating its superiority as a diagnostic tool over other indicative indicating its superiority as a diagnostic tool over other indicative enzymes like LAP and ALP in detecting injury to renal proximal tubular cells in the early stage.

[Urinary trehalase activity during normal pregnancy: tubular dysfunctions].

By measuring urinary trehalase which is localized in renal brush borders, we examined whether tubular dysfunction occurred or not during and after normal pregnancy. Urinary trehalase activity in 1st trimester, 2nd trimester, and 3rd trimester was increased significantly as compared with the controls (=non-pregnant women) (p less than 0.01). After delivery, urinary trehalase activity in puerperal 5th day was decreased to the level of the 1st trimester, and the activity of puerperal on the 30th day was in the range of the control value. Urinary beta 2-microglobulin (beta 2-MG) in the 1st trimester and 2nd trimester was not increased significantly as compared with the controls. Urinary beta 2-MG in the 3rd trimester, however, was significantly higher than that in the controls. After delivery, the value for beta 2-MG in puerperal on the 5th day was not decreased significantly as compared with the 3rd trimester, and in puerperal on the 30th day, urinary beta 2-MG was decreased to the control value. Urinary N-acetyl-beta-D-glucosaminidase (NAG) in the 3rd trimester was significantly higher than that in the controls (p less than 0.01) and in the 1st trimester (p less than 0.02). After delivery, a significant decrease was observed in urinary NAG between 5th puerperal day and the 3rd trimester. Moreover, urinary NAG on the 30th puerperal day was the level of the controls. On the basis of these results, it is inferred that tubular dysfunction is observed during normal pregnancy.

Detection of renal tubular lesions after abdominal aortography and selective renal arteriography by quantitative measurements of brush-border enzymes in the urine.

Quantitative determination of brush-border enzyme excretion in the 24-hour urine is a much more sensitive index of renal tubular damage after aortography and selective renal arteriography than the conventional renal function tests such as serum creatinine and clearance determinations. Among the five brush-border enzymes which we investigated, alkaline phosphatase (AP) was the most sensitive diagnostic pointer. In 90% of hypertensive patients without detectable pre-existing renal parenchymal damage, abnormal levels of AP excretion in the urine were found on the same day as or on the day after the intra-arterial injection of contrast medium. Measurement of other brush-border enzymes does not provide any further diagnostic information. Provided there is no pre-existing renal parenchymal damage, the lesion caused by the contrast medium is transient and is usually reversed within 48 h. For the early detection of tubular lesions caused by tri-iodinated benzoic acid derivatives, AP excretion in the 24-hour urine should be measured at least twice--on the day of the contrast medium injection and on the following day.

Elevation of urinary trehalase in mercuric chloride-induced nephrotoxic rabbits: urinary trehalase as a specific indicator of renal brush border damage.

The origin of urinary trehalase in mercuric chloride-induced nephrotoxic rabbits was demonstrated with biochemical and immunochemical techniques. Urinary trehalase was dramatically increased with HgCl2-induced nephrotoxicity. The nephrotoxic kidney showed an extreme decrease in specific fluorescence with fluorescein isothiocyanate (FITC)-conjugated antibody technique. Moreover, trehalase activity in the membrane fraction was remarkably decreased in the nephrotic kidney compared with the control. Judging from the results of immunodiffusion, urinary trehalase and renal trehalase exhibit the same antigenicity. From the data of a time course analysis of nephrotoxicity, the excretion of urinary trehalase was earlier than that of urinary sugar. Previous results show that renal trehalase is localized in the renal tubular brush borders. From these results, it is suggested that urinary trehalase is originated in the renal brush borders. In consideration of the results described in previous papers and in this paper, it is proposed that urinary trehalase is a good indicator of renal brush border damage.

Evaluation of urinary enzyme patterns in patients with kidney diseases and primary benign hypertension.

The urinary excretion of lactate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, arylsulphatase A, alpha-glucosidase, beta-galactosidase, trehalase, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and leucine arylamidase was studied in 68 patients with biopsy-proved glomerular, 54 with interstitial renal disease and in 97 patients suffering from primary hypertension. The enzyme output of these 219 patients was compared to that of a reference population of 100 thoroughly selected healthy subjects. The highest incidence of elevated enzyme excretion was observed for N-acetyl-beta-glucosaminidase with 88% in glomerulopathies and 78% in interstitial disease, followed by beta-galactosidase. 94% of the patients with glomerular kidney disease, 90% of those with interstitial disease and about 60% of the subjects with primary benign hypertension revealed an output of at least one enzyme above upper reference limit. The highest average enzymuria occured in glomerulopathies, particularly high values in patients with the nephrotic syndrome. Application of discriminant analysis to the urinary enzyme pattern of glomerular and interstitial renal diseases resulted in an overall correct classification into the appropriate group of 89% of all patients. The discrimination between glomerular and interstitial disease was better in patients with normal renal function than in those with reduced function. Results show, that the analysis of urinary enzyme patterns may be a helpful adjunct for differential diagnosis of kidney diseases.

Normal limits of urinary excretion of eleven enzymes.

Urinary excretion of lactate dehydrogenase, hydroxybutyrate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, arylsulphatase A, alpha-glucosidase, beta-galactosidase, trehalase, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and leucinearylamidase was studies in a carefully selected group of 100 healthy subjects, 50 women and 50 men. Enzyme activities were assayed in 3-h morning samples after gel filtration of the urine. Activities were related to time volume, and to urinary creatinine concentration. Several transforming functions had to be applied to enzyme output data to obtain an approximation to gaussian frequency distribution. Men showed a significantly higher excretion of gamma-glutamyltransferase, alpha-glucosidase, trehalase, N-acetyl-beta-glucosaminidase,beta-glucuronidase, and leucine arylamidase activity than did women if enzyme activity was related to urinary time volume. Women excreted more lactate dehydrogenase, hydroxybutyrate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, alpha-glucosidase, trehalase, and N-acetyl-beta-glucosaminidase activity than did men, if urinary creatinine was used as the basis of reference. Reference intervals were calculated as 2.5 and 97.5 percentiles for both sexes.

[Influence of parenterally injected trehalose in mammals having trehalase activity at different sites]. / Influence de l'injection parentérale de tréhalose chez des mammifères possédant une activité tréhalasique diversement localisée

When trehalose is injected via parenteral pathway into animals lacking kidney trehalase (rat), more than 75 per cent of this disaccharide is eliminated in urine. When the injected animals possess an active kidney trehalase (guinea-pig, rabbit), there is only a low urinary trehalose excretion. Moreover, in rabbit, a marked hyperglycaemia is observed which is due to the rapid hydrolysis of trehalose by kidney trehalase.