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.

Neutral trehalase Nth1p of Saccharomyces cerevisiae encoded by the NTH1 gene is a multiple stress responsive protein.

We have shown previously that expression of the NTH1 gene is increased at heat stress (40 degrees C) both at the mRNA and enzymatic activity levels. This increased expression was correlated to the requirement of the NTH1 gene for recovery after heat shock at 50 degrees C and the presence of stress responsive elements STRE (CCCCT) 3 times in its promoter region [S. Nwaka et al., FEBS Lett. 360 (1995) 286-290; S. Nwaka et al., J. Biol. Chem. 270 (1995) 10193-10198]. We show here that expression of the NTH1 gene and its product, neutral trehalase (Nthlp), are also induced by other stressors such as H2O2, CuSO4, NaAsO2, and cycloheximide (CHX). Heat-induced expression of the NTH1 gene is shown to be accompanied by accumulation of trehalose. In contrast, the chemical stressors which also induce the expression of NTH1 did not lead to accumulation of trehalose under similar conditions. Our data suggest that: (1) heat- and chemical stress-induced expression of neutral trehalase is largely due to de novo protein synthesis, and (2) different mechanisms may control the heat- and chemical stress-induced expression of NTH1 at the transcriptional level. Participation of neutral trehalase (Nth1p) in multiple stress response dependent and independent on trehalose is discussed.

Apical trehalase expression associated with cell patterning after inducer treatment of LLC-PK1 monolayers.

Trehalase, a differentiation-specific marker of renal proximal tubule brush border membrane, is expressed in confluent long-term cultures of the renal epithelial cell line LLC-PK1. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membrane-associated differentiated functions (Yoneyama and Lever: J. Cell. Physiol. 121: 64-73, 1984). In the present study, we utilize a polyclonal antibody specific for renal trehalase to demonstrate that trehalase expression induced in LLC-PK1 cultures after HMBA treatment is localized in cells forming a three-dimensional network of strands across the confluent monolayer. The antitrehalase antibody recognized an apical membrane antigen of apparent molecular weight 100-110 kD both in LLC-PK1 cultures and in the corresponding pig renal brush border membranes. Strand formation and total trehalase activity increased in parallel as a function of inducer concentration and duration of exposure. Strand formation and trehalase expression were also greatly enhanced in monolayers grown on a Nuclepore filter support even in the absence of inducer. Strand formation was not a prerequisite for induced trehalase expression in culture, since strands did not develop in cultures treated with N, N'-dimethylformamide (DMF) and equally potent inducer of trehalase expression. In this case, cells which expressed increased levels of trehalase were dispersed at random over the monolayer. Induction of strand formation and trehalase expression by HMBA required a minimum exposure period of 48 hr and persisted up to a week after removal of inducer. By contrast, the response to DMF required continuous presence of inducer. Levels of trehalase declined even in the continuous presence of inducer in local regions of low cell density created by wound-repair of the monolayer. In addition to the membrane-bound form, trehalase activity was also recoverable from the culture medium, but release of trehalase was not affected by inducers. These observations are consistent with the view that a cell type committed to express a program of differentiation after HMBA treatment or growth on a permeable support is organized in specific cell patterns visible as strands over the confluent cell monolayer.

Isolation and characterization of four forms of trehalase from rabbit kidney cortex.

Four forms of renal trehalase were isolated and purified to homogeneity. Hydrophobic interaction chromatography separated two forms; A-form and B-form. Both forms were subdivided further on Con A-Sepharose and were stained with periodic acid-Schiff reagent, indicating that they are glycoproteins. The four forms of renal trehalase showed no significant difference in Km values for trehalose and K1 values for various inhibitors. The optimum pH of the four forms was pH 6.0 in phosphate buffer. Apparent molecular weights on gel filtration of the four forms were the same, 175,000. Furthermore, the four forms showed the same antigenicity on double immunodiffusion. However, isoelectric point (pI), susceptibility to HgCl2, stability at -80 degrees C and Na+ activation behavior were different. Glycoprotein forms were more susceptible to HgCl2 and showed lower Na+ activation than nonglycoprotein forms. The pI of less hydrophobic forms (A1, A2) was more acidic than that of more hydrophobic forms (B1, B2). On the basis of these results, it is likely that four forms of renal trehalase are "isozymes."

Localization of renal and intestinal trehalase with immunofluorescence- and enzyme-labeled antibody techniques.

The localization of trehalase with fluorescein isothiocyanate-conjugated and peroxidase-conjugated antibody techniques was examined. Antiserum against purified rabbit renal trehalase was produced against guinea pigs. Anti-renal trehalase immunoglobulin (Ig)G was isolated from the serum and used for the immunohistochemical localization of intestinal and renal trehalases. Specific fluorescence and peroxidase staining were observed in the brush borders of proximal tubules and of intestinal epithelial cells. These results are in good agreement with the biochemical results. Thus, it is concluded that trehalase is specifically localized in the renal and intestinal brush borders. Sections of rabbit intestine and of rabbit kidney treated with anti-rabbit renal trehalase IgG were observed to have a specific fluorescence at the brush borders. Sections of rat intestine treated with the same antibody, however, showed no specific fluorescence at the brush borders. From these results, it is strongly suggested that renal trehalase and intestinal trehalase from the rabbit have common antigenic determinants and that these differ from those in rat intestinal trehalase.

Topographical relationship between sucrase and leucine beta-naphthylamidase on the microvilli membrane of rabbit intestinal mucosal cells.

The topographical relationship between sucrase [EC 3.2.1.26] and leucine beta-naphthylamidase (LNAase) on the microvilli membrane of rabbit small-intestinal mucosal cells was studied assuming that where enzymes with different antigenicities, A and B, are situated in close proximity on the surface of microvilli vesicles, the agglutination of vesicles by anti-A antibody is inhibited by the previous binding of monovalent fragments of anti-B antibody to enzyme B on the surface of vesicles. Like anti-sucrase antibody, anti-LNAase antibody quantitatively agglutinated microvilli vesicles. It inhibited the membrane-bound LNAase activity in the same manner as the detergent-solubilized activity. This inhibitory effect of anti-LNAase antibody was not interfered with by monovalent fragments of anti-sucrase antibody. However, the monovalent fragments inhibited vesicle agglutination by anti-LNAase antibody as well as by anti-sucrase antibody. These results indicate that LNAase is located on the outer surface of microvilli vesicles and suggest that LNAase and sucrase are situated in close proximity on the membrane surface of microvilli vesicles.