Clinical Chemistry 46: 636-643, 2000;
(Clinical Chemistry. 2000;46:636-643.)
© 2000 American Association for Clinical Chemistry, Inc.
ELISA for Urinary Trehalase with Monoclonal Antibodies: A Technique for Assessment of Renal Tubular Damage
Reiko Ishihara1,a,
Shigeru Taketani2,
Misa Sasai-Takedatsu1,
Yasushi Adachi3,
Minoru Kino1,
Akiko Furuya4,
Nobuo Hanai4,
Rikio Tokunaga2 and
Yohnosuke Kobayashi1
1
Department of Pediatrics, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8506, Japan.
2
Department of Hygiene, Kansai Medical University, Fumizonocho 10-15, Moriguchi,
Osaka 570-8506, Japan.
3
First Department of
Pathology, Kansai Medical University, Fumizonocho 10-15, Moriguchi,
Osaka 570-8506, Japan.
4
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co.,
Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194-8533, Japan
a Author for correspondence. Fax 81-6-6992-3522; e-mail ishihara{at}takii.kmu.ac.jp
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Abstract
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Background: 
,-Trehalase, located on renal proximal tubules,
is a glycoprotein that hydrolyses ,-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.
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Introduction
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,-Trehalase (EC 3.2.1.28) is an ectoenzyme located on the
brush border membrane of mammalian kidney and small intestine. The
enzyme catalyzes hydrolysis of ,-trehalose
(1--D-glucopyranosyl--D-glucopyranoside)
to form two molecules of glucose, and substrate specificity of the
enzyme is highly restricted. The glucose thus formed in the intestine
probably is absorbed to be utilized as an energy source because
patients deficient in intestinal trehalase have been reported to show
diarrhea after ingestion of trehalose-containing mushrooms
(1). The function of renal trehalase, however, is unknown.
Increased trehalase activity in urine has been reported to be
associated with damage to renal proximal tubules and some kinds of
renal diseases (2)(3)(4).
Mammalian trehalase is linked to membrane by a
glycosylphosphatidylinositol anchor, and
phosphatidylinositol-specific phospholipase C is required for the
trehalase to be completely liberated from the membrane
(5). The enzyme has been purified from mammalian
kidneys and shows a molecular mass of 75 kDa (6)(7)(8).
Trehalase cDNA has been isolated from rabbit (6), human
(7)(9), and rat (10) tissue. The
complete sequence of the human trehalase cDNA showed that the enzyme
consists of 583 amino acid residues and contains a putative leader
peptide of 19 amino acids at the NH2 terminus,
five putative glycosylation sites, and a hydrophobic region at the COOH
terminus, corresponding to the site of the glycosylphosphatidylinositol
anchor (9).
Trehalase activity in urine has been estimated by determination of
formed glucose after the removal of endogenous glucose from urinary
samples by complicated gel filtration (3)(7).
The enzyme activity is also measured spectrophotometrically with
oxidation of glucose by glucose dehydrogenase (11).
Quantification of trehalase is useful in the diagnosis of renal
diseases, but the activity in human urine is quite low. Moreover,
methods for determining the activity usually are time-consuming and
complicated, and their application to routine clinical study is
difficult. Measurement of the enzyme activity also has a disadvantage,
because the activity of the enzyme present in urine can be
different among diseases because of a membrane-anchored protein.
Furthermore, it is unclear whether the urinary enzyme activity reflects
the actual concentration.
Demonstration of functional expression of human trehalase in
Escherichia coli (9) facilitates the isolation of
monoclonal antibody for human trehalase that can be used to develop an
ELISA method. In this study, therefore, we isolated the
recombinant enzyme and obtained monoclonal antibodies against human
trehalase; we then applied them in a rapid and highly sensitive ELISA
method to evaluate urinary trehalase. In addition, we compared urinary
trehalase concentrations and
N-acetyl-ß-D-glucosaminidase
(NAG)1
activity in patients with renal diseases. We also performed
histochemical analysis of renal trehalase and showed extremely high
excretion of trehalase in the urine of patients with tubular damage.
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Materials and Methods
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design and synthesis of peptide for immunization to raise an
antibody
Monoclonal antibodies against human trehalase were raised using
recombinant trehalase and synthesized trehalase peptide. A sequence
corresponding to amino acid residues 291–307 (SKDVELADTLPEGDREA)
(9) was selected. A cysteine residue was introduced into the
C-terminal region of the peptide for conjugation to hemocyanin. The
above peptide was synthesized by the standard
9-fluorenylmethoxycarbonyl solid-phase synthesis method
(12). Peptides were analyzed and purified by reverse-phase
HPLC. Recombinant human trehalase (9) was also used
as an immunogen after partial purification by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Eight-week-old
female BALB/c mice were immunized by intraperitoneal injections of the
recombinant protein or conjugated peptide with the adjuvant. Three or
four consecutive injections of the immunogen were given at weekly
intervals starting 2 weeks after the first immunization. The mice were
sacrificed 3 or 4 days after the last injection. The spleen cells were
fused with P3.X63/Ag8.U1 (P3.U1) by the modified method of Kohler and
Milstein (13). The fused cells were cultured with 96-well
culture plates (Nunc) in RPMI 1640 (Nissui) containing fetal
bovine serum, hypoxanthine, aminopterin, and thymidine. After 8–10
days of culture, the antibody activity of the culture supernatant from
each well of the hybridoma was tested by ELISA. Positive wells were
subcloned twice by a limiting dilution method.
selection of monoclonal antibody by elisa
A 96-well ELISA plate (Greiner) was coated with the recombinant
trehalase purified by SDS-PAGE (9) or obtained conjugated
peptide at 4 °C for 16 h. After the removal of the
protein solutions, the plate was washed three times with
phosphate-buffered saline (PBS) and blocked by PBS containing 10
g/L bovine serum albumin, followed by a 1-h incubation
at room temperature. After the plate was washed with PBS, the
supernatant from the hybridoma culture was added and incubated at room
temperature for 2 h. The plate was washed four times with PBS
containing 0.5 g/L Tween 20 (PBS-Tween 20), after which
horseradish peroxidase (HRP)-conjugated rabbit anti-mouse
immunoglobulin (Dako) was added, and the plate was incubated at room
temperature for 1 h. After the plate was washed four times with
PBS-Tween 20, it was incubated with HRP substrate,
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and
H2O2 at room temperature.
The absorbance was measured with a microplate reader (Model 550;
Bio-Rad) at 415 nm.
sandwich elisa for determination of urinary trehalase
The wells of 96-well microplates (Nunc) were coated overnight with
50 µL of monoclonal antibody for human trehalase KM2287 (10
mg/L in PBS) at 4 °C. After removal of the antibody
solution, plates were washed three times with PBS. For blocking, PBS
containing 10 g/L bovine serum albumin (100 µL) was added to each
well, and the plates were incubated at 4 °C overnight. After
removal of the blocking solution, urine samples diluted 10-fold with
PBS containing 1 g/L SDS (50 µL) and then heated were added
to the wells and incubated at 37 °C for 1 h by shaking at 130
rpm. The purified trehalase (50 µL) in a concentration range of
13–994 µg/L was also incubated and served as a calibrator.
Plates were subsequently washed five times with PBS-Tween 20. A
biotin-conjugated monoclonal antibody KM2275 solution (50 µL of a 1
mg/L solution in PBS containing 10 g/L bovine serum albumin) was added
to the wells, and the plates were incubated at room temperature for
2 h. After the plates were washed five times with PBS-Tween 20, 50
µL of HRP-streptavidin (Dako) was added. The samples were left at
room temperature for 1 h. The plates were washed five times with
PBS-Tween 20, and then H2O2
and 50 µL of the substrate solution,
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), were
added to each well. After a 15-min incubation at room temperature, 50
µL of a 50 g/L SDS solution was added to stop the reaction.
Absorbance was measured with a microplate reader at 415 nm. All
experiments were performed in duplicate.
assay of trehalase activity
The trehalase activity in urine was assayed as described
previously (3)(7). Briefly, a urine sample (0.5
mL) was passed through a Sephadex G-25M column (PD-10; Pharmacia, LKB
Biotechnology) equilibrated with 5 mmol/L phosphate buffer, pH 6.2, to
remove endogenous glucose. The fractions containing proteins were
collected, and the volume was adjusted to 1.5 mL. To 0.9 mL of the
eluted sample was added 0.1 mL of 0.25 mol/L trehalose; the mixture was
then incubated at 37 °C for 120 min. The glucose thus formed was
determined with a glucose determination kit (Kyowa Medics). Trehalase
activity was expressed as micromoles of glucose liberated per hour per
liter of urine.
nag enzyme assay
Enzyme activity of NAG was assayed with the
sodiom-cresolsulfonphthaleinyl
N-acetyl-ß-D-glucosaminidase method
using an NAG determination kit (Shionogi Co. Ltd.).
creatinine assay
Creatinine was assayed by the method of Jaffe with a creatinine
determination kit (Wako Pure Chemicals).
The amount of trehalase (as estimated by ELISA), trehalase activity,
and NAG activity were expressed as their ratios to creatinine.
immunoblot analysis of renal and urinary trehalase
A specimen of a human kidney cortex-mix obtained from a
nephrectomized patient with renal carcinoma and sections of porcine and
bovine kidneys were used for immunoblotting. The tissue was lysed with
Laemmli sample buffer (14); the lysates were then sonicated
and boiled for 1 min. The proteins were separated by SDS-PAGE. After an
electrophoretic run, proteins on the gel were electroblotted onto a
polyvinylidene difluoride membrane (Bio-Rad). The conditions of
immunoblotting and detection of the cross-reactive antigen were as
described previously (7). To assess the amount of urinary
trehalase, urine samples were mixed with Laemmli sample buffer and
boiled for 1 min, and urine samples (10 µL) were loaded in the slots
of the gel. SDS-PAGE was carried out as described previously
(7).
immunohistochemical staining
A piece of the human kidney described above was fixed with
phosphate-buffered formalin, dehydrated, and embedded in paraffin wax.
Tissue sections of 3 µm were cut and mounted onto glass slides and
dried at 50 °C overnight. Sections were dewaxed in xylene,
rehydrated in a gradual alcohol series, fixed in methanol, incubated
for 10 min with 30 mL/L
H2O2 to block the
endogenous peroxidase activity, and subsequently washed with distilled
water. For trehalase staining, sections were incubated for 15 min in a
prewarmed solution of 1 g/L pepsin dissolved with HCl. After
blocking with Blocking Solution 2 (Dako), sections were incubated with
primary monoclonal antibody at 4 °C overnight. After sections were
washed three times with Tris-buffered saline, they were
incubated at room temperature for 90 min with HRP-conjugated anti-mouse
immunoglobulin (EnVision System; Dako). After washing, sections were
developed with diaminobenzidine reagent, rinsed in distilled water,
counterstained with hematoxylin (Merck), dehydrated, and mounted with a
Malinol (Muto Pure Chemicals).
urine samples
Spot urine samples were obtained from 41 healthy children (16
males and 25 females; age range, 0–18 years) and 41 patients (23 males
and 18 females; age range, 0–19 years) with renal diseases, including
nephrotic syndrome (acute and remission phases), chronic
glomerulonephritis such as IgA nephropathy and anaphylactoid purpura
glomerulonephritis, renal anomaly, acute renal failure, and those with
renal proximal tubular damage, including Lowe syndrome and Dent
disease. Urine specimens were centrifuged at 900g at
4 °C, and supernatants were withdrawn and stored at -60 °C until
analysis. All subjects gave their informed consent before participating
in this study. The data were assessed by the Mann–Whitney
U-test.
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Results
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characterization of monoclonal antibodies for human trehalase
Cell fusion gave eight hybridoma clones that produced antibodies
(KM2275, KM2276, and KM2285–KM2290). The monoclonal antibodies KM2275
and KM2276 were raised against recombinant trehalase, and others were
against the constructed peptide. The antibodies KM2275 and KM2276
responded to the recombinant trehalase but not to the constructed
peptide. We previously observed (9) that trehalase mRNA is
expressed predominantly in the kidney and small intestine, a finding
consistent with observations that trehalase is located on the brush
border membrane of kidney and intestine (6)(7).
We then analyzed the localization of trehalase in a human kidney, using
the monoclonal antibody for trehalase. When KM2275 was used, a dense
staining was observed at the brush border membrane of the proximal
tubules, but other regions, such as glomeruli, distal tubules, and
collecting ducts, were not stained (Fig. 1
). Staining patterns similar to those above were observed with
KM2287 (data not shown). Immunoblot analysis with KM2275 disclosed that
a sharp band with molecular mass of 75 kDa was observed with the human
kidney, but not with the porcine and bovine kidneys (Fig. 2
A, lanes 1–3). Moreover, of all the antibodies we obtained,
only KM2287 and 2275 exhibited a good response to both the recombinant
trehalase and the urinary trehalase (Fig. 2A
, lanes 4 and 5).
The immunoblot analysis showed that the amount of 75-kDa protein from a
patient with Lowe syndrome was high, which was consistent with the
previous observation (7) that the urinary trehalase activity
of this disorder was very high (248 µmol of glucose
formed · h-1 · g
creatinine-1). We further examined the
amount of trehalase in patients with other renal diseases. A band of 75
kDa was observed in patients with chronic glomerulonephritis and focal
segmental glomerulosclerosis (FSGS; Fig. 2B
, lanes 5 and 7).
Pathological observations of a renal biopsy specimen of the FSGS
patient showed progressively extended glomerulosclerosis and widespread
destruction of proximal tubules (data not shown). Bands of
~30 kDa as well as 75 kDa were shown in urine of a patient with acute
renal failure (Fig. 2B
, lane 3), whereas trehalase was either absent or
minimally present in urine specimens of healthy subjects (Fig. 2B
, lanes 1, 2, 4, and 6).
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Figure 1. Expression of trehalase in human kidney cortex-mix.
A and B, only the brush-border membrane
remains positive, whereas glomeruli and distal tubules are negative.
Magnification: (A), x50; (B), x100.
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Figure 2. Immunoblot analysis of trehalase.
(A), porcine (lane 1), bovine
(lane 2), and human (lane 3) kidney
cortex-mix tissues and urine from a patient with Lowe syndrome
(lanes 4 and 5) were treated as described
in the text. Proteins were analyzed by SDS-PAGE and immunoblotted using
monoclonal antibodies KM2275 (lanes 1–4) and KM2287
(lane 5). (B), urinary trehalase from
healthy subjects (lanes 1, 2,
4, and 6) and patients with acute renal
failure (lane 3), chronic glomerulonephritis
(lane 5), and FSGS (lane 7) was also
examined using monoclonal antibody KM2275. The mobilities of molecular
markers are indicated on the left.
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assay characteristics
We developed a sandwich ELISA to assess the amount of urinary
trehalase, using KM2287 and biotinylated 2275 antibodies. As shown in
Fig. 3
A, the absorbance at 415 nm against calibrator exhibited a
linear relation for trehalase concentrations of 0–1000 µg/L.
To determine the optimal condition for the ELISA, we examined whether
treatment of urine with SDS solution was suitable. Fig. 3B
shows the
effect of dilution of urinary samples with SDS. Compared with a native
urine specimen from a healthy subject (Fig. 3B
, panel a, column 1),
when SDS was added at a final concentration of 1 g/L, the
relative value increased (Fig. 3B
, panel a, column 2). When
the urine was diluted with 10 volumes of PBS containing 1 g/L SDS (Fig. 3B
, panel a, column 3), the relative trehalase value was
twofold higher than that when 1 g/L SDS was added (Fig. 3B
, panel a,
column 2). When the urine was diluted 10 volumes of PBS without SDS
(Fig. 3B
, panel a, column 4), the relative trehalase value increased,
but the extent was less than the value for 1 g/L SDS (Fig. 3B
, panel a,
column 2). In all urine samples of patients and healthy subjects, high
values were obtained by 10-fold dilution with PBS containing 1 g/L SDS.
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Figure 3. Characterization of ELISA for urinary trehalase.
(A), calibration curve. The absorbance at 415 nm plotted
against the calibrator exhibited a linear relationship for trehalase at
concentrations of 0–1000 µg/L. (B), effect of SDS on
the determination of urinary trehalase. Urine samples were untreated
(column 1), heated at 95 °C for 1 min in the presence
of 1 g/L SDS (column 2), diluted with 10 volumes of PBS
containing 1 g/L SDS and heated at 95 °C for 1 min (column
3), or diluted with 10 volumes of PBS (column
4). Samples were from a healthy-10-year-old boy
(a), a 15-year-old boy with chronic glomerulonephritis
(b), a 1-year-old girl with hydronephrosis
(c), a 12-year-old boy with nephrotic syndrome (acute
phase; d), and a 10-year-old girl with FSGS
(e). Relative values were expressed by calculations
based on the absorbance values for the diluted samples.
(C), effect of pH on the ELISA. The ELISA was carried
out as described in Materials and Methods except that
various pHs were used. (D), urine dilution curve. Urine
samples were diluted with PBS containing 1 g/L SDS.
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We next examined the effect of pH on this ELISA system, and optimal
values pH of 6.0–8.0 were obtained (Fig. 3C
). A dilution curve showed
that the values estimated by this ELISA were correlated with 10- to
20-fold diluted samples (Fig. 3D
). When the urine was diluted with PBS,
the pH of the samples adjusted to ~7.4, and thus a good response to
this ELISA was obtained.
When urine values obtained with this ELISA for gel-filtered urines were
compared with those obtained for unfiltered samples, the trehalase
values obtained after gel filtration were 110–226 µg/L, whereas
those obtained before gel filtration were 20–50 µg/L.
The precision of the ELISA was estimated with different urine samples
containing trehalase enzyme. The within- and between-assay CVs were
6.7–7.6% (n = 8) and 6.2–8.2% (n = 14), respectively
(Table 1
). Exogenously added trehalase was well recovered from urine
samples containing different concentrations of endogenous trehalase
(Table 2
).
When urine samples were stored at -60 °C, -30 °C, or 4 °C
for 30 days, the enzyme amount did not decrease. Possible interfering
substances, such as ascorbic acid (10 g/L), glucose (100 g/L), albumin
(10 g/L), or creatinine (1 g/L), did not affect this ELISA.
concentration and activity of trehalase in human urine samples
The urinary trehalase activity of the healthy group was 26.6
± 14 µmol · h-1 · g
creatinine-1, and the trehalase concentration
measured by the ELISA was 466 ± 343 ng/g creatinine. These values
showed no sex- or age-related differences (data not shown). This new
ELISA detected, as protein, a small amount of urinary
trehalase whose activity was not detected.
There was a positive correlation in healthy subjects between the
urinary trehalase activity and the trehalase concentration
(P <0.01; r = 0.15; Fig. 4
). The
upper limits of trehalase activity and concentration were 56
µmol · h-1 · g
creatinine-1 and 906 ng/g creatinine,
respectively. As shown in Fig. 5
B, urinary trehalase concentrations in nephrotic syndrome
patients (acute phase) were extremely high, i.e., ~200-fold higher
than the control group (Fig. 5B
, group B; P <0.05), but
serum trehalase in patients was not detected. The concentrations in
patients in remission from nephrotic syndrome were similar those in the
controls (Fig. 5B
, group C). The variability in activity in groups B
and C was similar to that measured by the ELISA (Fig. 5A
, groups B and
C). The patients with Lowe syndrome and Dent disease showed very high
concentrations, and they were 200- and 70-fold higher, respectively,
than in the controls (Fig. 5B
, a and b of group F). The trehalase
activity of a patient with acute renal failure caused by
Salmonella infection was low (15.7
µmol · h-1 · g
creatinine-1), whereas the trehalase
concentrations estimated by the ELISA was markedly high (13 642 ng/g
creatinine), and it was 27-fold higher than in the controls (Fig. 5
, c of group F). Compared with the controls, patients with
chronic glomerulonephritis (Fig. 5
, group D) had higher trehalase
activities (1.5-fold higher) and concentrations (6.2-fold
higher). The trehalase concentrations in patients with renal
anomalies (Fig. 5
, group E) were also very high compared with the
controls (P <0.05), although the activities in these
patients were not high compared with the controls (P =
0.88). Patients with chronic glomerulonephritis and acute phase
nephrotic syndrome exhibited 1.5- to 5-fold higher NAG activity (acute
phase of nephrotic syndrome, 11.0–16.4 U/g creatinine; chronic
glomerulonephritis, 4.5–10.4 U/g creatinine) than did healthy controls
(3.2 ± 2.4 U/g creatinine). Thus, the extent to which the
trehalase concentration was increased, as estimated by the ELISA, was
much higher than the measured increases in trehalase activity and NAG
activity.
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Figure 4. Relationship between trehalase activity and trehalase
concentration in healthy subjects.
Dotted lines show the upper limits (95th percentiles).
Cr, creatinine.
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Figure 5. Urinary trehalase values in patients with renal diseases.
(A), urinary trehalase activity; (B),
urinary trehalase concentration estimated by ELISA. Group
A, controls (n = 41; M/F = 16/25); group
B, patients with nephrotic syndrome (acute phase; n = 4;
M/F = 2/2); group C, patients with nephrotic
syndrome (remission phase; n = 7; M/F = 4/3); group
D, patients with chronic glomerulonephritis (total, n =
16; M/F = 9/7; IgA nephropathy, n = 8; anaphylactoid purpura
glomerulonephritis, n = 3; others, n = 5); group
E, patients with renal anomaly (n = 11; M/F = 5/6);
group F, others (a, 8-year-old boy with
Lowe syndrome; b, 16-year-old boy with Dent disease;
c, 7-year-old boy with acute renal failure attributable
to Salmonella infection). Cr,
creatinine.
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Discussion
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Using monoclonal antibodies, we developed an ELISA for
determination of urinary trehalase. Urinary trehalase comes from the
apical membranes of the renal proximal tubular cells:
immunohistochemical staining of the renal cortex-mix shows the
existence of this enzyme on the brush border of the renal proximal
tubules (Fig. 1
). Trehalase is an ectoenzyme that is linked to membrane
by a glycosylphosphatidylinositol anchor, and excretion of the enzyme
can be influenced by renal injuries. Other investigators have reported
increased urinary trehalase activity in mercuric chloride-induced
nephrotoxic rabbits as well as in patients with Itai-Itai disease and
inhabitants of a cadmium-polluted area (15)(16).
Sasai-Takedatsu et al. (3) also reported that the urinary
trehalase activity is a better indicator of renal proximal tubular
damage than NAG activity because of its early detection. However, in
the measurement of the activity, the conditions needed to assess the
urinary trehalase accurately are complicated
(3)(7) because gel filtration is necessary for
each sample. Although analytical methods for urinary trehalase that use
electrophoresis and immunoblots can provide interesting information on
the underlying diseases, they are time-consuming and expensive for
clinical examination. On the other hand, our ELISA system overcame
these drawbacks as to its facility and cost.
In this ELISA, the treatment of urine with SDS solution led to an
increase in binding of the antibodies. The obtained antibodies for the
recombinant trehalase were raised by injection of trehalase purified by
SDS-PAGE, indicating not only that the antibody preferably recognizes
SDS-treated trehalase but also that SDS can solubilize trehalase
protein in urine samples. Moreover, using the samples after gel
filtration, we obtained trehalase values higher than those obtained
with untreated samples, which indicated that low-molecular weight
substances interfere with the binding of the antibody to the antigen.
Thus, the dilution of urine samples with PBS was essential for
obtaining reproducible values. This treatment contributes not only to
neutralization of urine samples but also to dilution of interfering
factors that may decrease the binding of antibodies to antigens.
Although we could not identify the interfering substances, they may be
present in some patients, as indicated by the increased binding of
antibodies when samples were diluted 10-fold with PBS containing 1 g/L
SDS.
The antibodies that we used for the ELISA exhibited a good response to
human urine as well as human renal cortex-mix, and a 75-kDa band in
urine may generally reflect the urinary trehalase activity (Fig. 2
).
There were apparent discrepancies in urinary trehalase values when
compared between the ELISA and activity assay, especially in acute
renal failure and renal anomalies. These discrepancies may be
attributable to proteolytic degradation of trehalase in urine, although
the stability of trehalase in the renal brush border membrane or urine
is not clear. We found that the antibody reacted with the ~75-kDa and
30-kDa bands in urine from a patient with acute renal failure (Fig. 2B
, lane 3). The 30-kDa band may be one of the proteolytic degradation
products of intact trehalase. In this patient, trehalase activity was
as same as in the control, although the 75-kDa band was obtained by
urine SDS-PAGE. The reason for this is unclear, but it may be that the
active center of trehalase is broken in fulminating infections. Because
our designed ELISA can detect the truncated trehalase whose activity
had been lost, it has the advantage of measuring the actual trehalase
concentration even in urine samples that have been stored a long
time. Actually, the enzyme concentrations in patients with
acute phase nephrotic syndrome, chronic glomerulonephritis, and renal
anomaly were much higher than the values estimated by the increased
activities. These data suggest that our ELISA system can detect the
whole trehalase enzyme better than the activity assay and can also
detect renal tubular damage by reflecting the release of trehalase from
apical membranes as a result of renal injury.
Glomerular disorders such as acute phase nephrotic syndrome show
markedly increased trehalase concentrations, which indicates that the
tubular cells are highly damaged. It has been reported that urinary NAG
activity is increased in acute nephrotic syndrome
(17)(18). These authors concluded that patients
with acute nephrotic syndrome might have tubular damage. Other
investigators have suggested a relationship between increased NAG
activity and the relapse of nephrotic syndrome (19). Our
study confirmed previous observations (17)(18)
and also described a new sensitive assay for determination of urinary
trehalase to evaluate tubular damage. In this connection, although the
urinary NAG activities of patients with renal damage was 1.5- to 5-fold
higher than in controls, the magnitude of the increase in trehalase
concentrations estimated by this ELISA was much higher (6.2- to
200-fold). Sasai-Takedatsu et al. (3) reported that
trehalase activity increased earlier than NAG activity in the patients
with renal proximal tubular damage. These results suggest that
measurement of trehalase in urine contributes to the identification of
microdamage to proximal tubules.
In conclusion, this study describes for the first time a new
sandwich ELISA for urinary trehalase that uses monoclonal antibodies.
Our ELISA method is superior to conventional activity assays because of
its ability to detect the whole enzyme. Although further studies are
necessary to demonstrate the clinical validity of the present ELISA
system, it may be a potent technique to assess damage to renal proximal
tubules and may be useful in daily clinical assessment of renal
diseases.
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Acknowledgments
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This study was supported in part by a grant from the Osaka Kidney
Foundation (OKF99-0010) and the Mami Mizutani Foundation. We thank Dr.
Y. Komiyama for valuable advice.
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Footnotes
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1 Nonstandard abbreviations: NAG, N-acetyl-ß-D-glucosaminidase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; and FSGS, focal segmental glomerulosclerosis. 
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Abstract
Introduction
