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α1-Microglobulin in urine as an indicator of renal tubular damage caused by environmental cadmium exposure
63
Toxicology Letters, 22 (1984) 63-68 Elsevier
TOXLett.
1235
ari-MICROGLOBULIN IN URINE AS AN INDICATOR OF RENAL TUBULAR DAMAGE CAUSED BY ENVIRONMENTAL CADMIUM EXPOSURE (cri-Microglobulin;
KoJI
NOGAWA,
DA, MASAO
Pz-microglobulin;
TERIJHIKO
ISHIZAKI
KIDO,
renal damage; cadmium)
YUICHI
and KISAKU
YAMADA,
IKIKO
TSURITANI,
RYUMON
HON-
TERAHATA+
Department of Hygiene and l Department of Clinical Pathology, Kanazawa Medical University, Uchinada. Ishikawa, 920-02 (Japan) (Received
January
9th.
(Revision
received
February
1984)
(Accepted
March
9th,
8th.
1984)
1984)
_-
.-
.._--
-.
_.-
SUMMARY al-Microglobulin (arm) was determined in the urine of both the cadmium-exposed and nonexposed subjects and was compared with those of other urinary parameters such as &-microglobulin (02-m). glucose,
total protein
exposed
subjects.
sensitivity
or amino
acids.
Large amounts
There were significant
and specificity
of both al-m
was similar.
It was concluded
tion caused
by cadmium.
that al-m
correlations
of ur-m between
were detected al-m
in the urine of cadmium-
and other urinary
and &-m as indices of tubular
dysfunction
in urine seemed to be a useful indicator
parameters.
caused
The
by cadmium
of renal tubular
dysfunc-
INTRODUCTION
The kidney is a critical organ in chronic cadmium poisoning. Tubular dysfunction is considered to be an early characteristic in the finding of renal damage caused by cadmium. Low-M, proteins in urine are very sensitive indices of impaired proximal tubular function. In chronic cadmium poisoning large amounts of low-M, proteins such as /3z-microglobulin @z-m), retinol binding protein or lysozyme are detected in urine. Among them, @2-min urine is most widely used as an early sign of renal tubular damage [l], cYi-microglobulin (al-m) is a recently isolated, low-Mr protein Abbreviation:
0378-4274/84/$
APDC-MIBK,
03.00
0
ammonium
Elsevier
Science
pyrollidine
Publishers
dithiocarbamate-methyl
B.V.
isobutyl
ketone.
64 A
lO6-
T
105-
$ ki &
104-
‘2 .-
2 al .f 5
103-
.r c 3 +3 5l f2
lo*r--0.968
0 10 -
:
i8
1 1
10
102
a,- microglobulin
?O3 in urine
104 (pg/g.
4 106
creotlnine)
: t .
r-0.718
cr,-microglobulin
I 105
in urine
oJg/g.creatinine)
Fig. 1, Relationship between orI-microglobulin in urine and µglobuIin (A), glucose (B), total protein (C) and cadmium (D) in urine of the tadmium-exposed (filled circles) and nonexposed (open circles) subjects.
65 C
r-0.863
1
1
1
1
U,-mlcroglobulin
in
10=
lo4
lo3
lo*
10
10”
oJg/g.CreOttnine)
urine
D
106-
z .-E % : ” $
-9
.
. .*
l *
.
104-
.
.
. . .
l
P ‘: ‘5
:*
0
103-
.
0 0 0
.E .z 2
.
. .
.
l
105-
.
.
0
0
0
lo*-
0
0 El
rz0.596 0
P .v C
10
d 1
1 0.5
, 1.0 Cadmium
5.0 concentration
I
I
10.0 in
Urine
50.0 (JJg1g.Creatih-e)
1
100.0
66
(Mr 33 000) initially isolated from the urine of patients with tubular proteinuria [2]. Elevation of this protein in urine and serum was observed in some pathological conditions and particularly in patients with renal dysfunction [3, 41. The present study was undertaken to investigate whether or-m could be used as an early sign of renal tubular dysfunction caused by cadmium. MATERIALS
AND METHODS
Morning urine samples were randomly collected from 27 subjects living in the cadmium-polluted Kakehashi River basin in Ishikawa Prefecture and 10 subjects living in an area free from cadmium pollution. The subjects and controls were all females aged over 50 years. Urine was collected in polyethylene bottles and stored frozen at -20°C until the analysis could be performed. Urinary pH was measured and was neutralized when necessary. (111-mwas analyzed by enzyme-immunoassay using a kit (al-m EIAkit, FujiRebio Co. Tokyo, Japan), 02-m was measured by radioimmunoassay using the phadebas &-microtest (Pharmacia Diagnostic AB, Uppsala, Sweden). Cadmium was determined by flameless atomic absorption spectrophotometry after wet ashing in HNOJ/H~SO~/HC~O~ and extraction with APDC-MIBK. At the same time, total protein, glucose, amino acids and creatinine were measured by standard methods [5]. RESULTS
Table I shows the geometric means and standard deviations of the concentrations of al-m, @z-m, glucose, total protein, amino acid and cadmium in the urine of both the cadmium-exposed and nonexposed subjects. The values were expressed as TABLE
I
GEOMETRIC PARAMETERS
MEANS AND GEOMETRIC IN THE CADMIUM-EXPOSED
STANDARD DEVIATIONS OF THE AND NONEXPOSED SUBJECTS
Cd-exposed
a,-Microglobulin &-Microglobulin
@g/g creatinine) bg/g creatinine)
group
(N = 27)
Nonexposed
G.M.
S.D.
G.M.
1888(r 1 199sp
5.5 7.9
352 33
URINARY
group S.D. 4.2 4.2
Total protein (mg/g creatinine) Glucose (mg/g creatinine)
149.0s 466.5’
6.4 4.3
10.4 99.5
3.8 1.3
Amino acid (mg/g creatinine) Cadmium bg/g creatinine)
272.3” 11.6”
I .7 2.2
157.9 2.4
1.2 1.6
68.8b
9.8
72.3
12.3
Age (year) a P < 0.001. b Arithmetic mean and arithmetic ’ Number
of persons
examined:
standard 26.
deviation
(N = 10)
67
creatinine correction. Cadmium could not be measured in one urine sample of the cadmium-exposed subject because of small urinary volume. Therefore, the total numbers of urine samples analyzed for cadmium were 26 in the cadmium-exposed group and 10 in the nonexposed group. The cadmium-exposed group showed significantly higher values of all urinary parameters than those of the nonexposed group. It should be noted that large amounts of al-m and /32-m were found in the urine of the cadmium-exposed group. In Fig. 1 (a,b,c,d), al-m in urine of both the groups were plotted vs. /32-m, glucose, total protein and cadmium, respectively. All correlation coefficients between crt-m and the other four urinary parameters were statistically significant. As seen in Fig. la, cri-m and /32-m showed a similar trend in both the cadmium-exposed and nonexposed subjects. The correlation coefficient between al-m and @z-mwas 0.968. al-m was less correlated with protein, glucose and cadmium than with ,&-m. Correlation coefficients between al-m and protein, glucose and cadmium were 0.863, 0.718 and 0.596, respectively.
al-m, a low-M, protein, was initially isolated by Ekstrom et al. [2]. This protein is produced in lymphocytes and probably in hepatocytes [6, 71. It is expected that cyl-m is handled in a similar manner by the kidney as those of other low-M, proteins such as &m, retinol binding protein or lysozyme. They are freely filtered through the renal glomeruli and absorbed by the proximal tubular cells where they are degraded. Only trace amounts of these proteins are excreted into the urine because the reabsorption of the substances is almost complete. As shown in Table I, the cadmium-exposed subjects used in the present study were proven to have had renal damage caused by cadmium. They showed significant excretions of low-M, protein, glucose, amino acid, total protein and cadmium in the urine. It was demonstrated that large amounts of ai-m were also excreted in the urine of the cadmium-exposed subjects. As shown in Fig. 1, the concentrations of urinary al-m were significantly correlated with those of the other urinary parameters. al-m was highly correlated with &-m. A correlation coefficient between al-m and &-m was 0.968. Lyl-m was less correlated with all other parameters, e.g., protein, glucose and cadmium, than with 02-m. Therefore it is reasonable to say that urinary amounts of al-m increase significantly in the case of cadmiuminduced renal damage and the usefulness of al-m as an indicator in determining the extent of renal dysfunction caused by cadmium is almost equal to that of urinary P2-m.
There has been considerable discussion on the biological significance of fi2microglobulinuria. Increased Pa-m in urine in cadmium-polluted areas is considered to be caused by a decreased reabsorption capacity of the renal tubules [8, 91. However, Nomiyama et al., reported that the remarkable increase in urinary &-m
68
is associated with renal dysfunction, while the slight increase is caused by mechanisms other than renal dysfunctions [lo]. Tsuchiya et al., also reported that an increase of 02-m in blood and urine in an early stage of cadmium exposure is caused by the increased level of Pz-m in blood, which may be a result of stimulation due to cadmium, but not necessarily by the clinical dysfunction of the reabsorption of f12-m in the renal tubules [ 111. The present study strongly indicates that renal handling of al-m was quite similar to that of 02-m. A study on the mechanism of urinary excretion of al-m would be of great assistance in explaining the biological significance of increased 02-m in urine in cadmium intoxication. REFERENCES 1 T. Kjellstom and M. Piscator, Quantitative Analysis of &Microglobulin as an Indicator of Renal Tubular Function, Pharmacia Diagnostics, Uppsala, Sweden, 1977. 2 B. Ekstrom, P.A. Peterson and I. Berggard, A urinary and plasama al-glycoprotein of low molecular weight: isolation and some properties, Biochem. Biophys. Res. Commun., 65 (1975) 1427-1433. 3 L. Svensson and U. Ravnskov, err-Microglobulin, a new low molecular weight plasma protein, Clin. Chim. Acta, 73 (1976) 415-422. 4 K. Takagi, K. Kin, K. Itoh, H. Enomoto and T. Kawai, Human or-microglobulin levels in various body fluids, J. Clin. Pathol., 33 (1980) 786-791. 5 K. Nogawa, E. Kobayashi, R. Honda, A. Ishizaki, S. Kawano and H. Matsuda, Renal dysfunctions of inhabitants in a cadmium-polluted area, Environ. Res., 23 (1980) 13-23. 6 K. Takagi, K. Kin, Y. Itoh, Y. Kawano, T. Kasahara, T. Shimada and T. Shikata, Tissue distribution of human cYr-microglobulin, J. Clin. Invest., 63 (1979) 318-325. 7 H. Enomoto, Y. Itoh, K. Takagi, H. Harada, T. Kawai, T. Kasahara and F. Sasaki, Studies on human or-microglobulin, VII. Production of or-microglobulin by hepatoma cell line, Physicochem. Biol., 24 (1981) 327-333. 8 K. Shitomi, H. Saito, A. Nakano, H. Unakami, K. Takada, T. Sato, T. Furuyama, K. Yoshinaga, T. Arikawa and K. Nagai, Urinary &-microglobulin for residents in an environmentally cadmiumpolluted area. Studies of generational and sexual differences, and a comparison with the results of proximal renal tubular function tests, Jap. J. Nephrol., 23 (1981) 45-62. 9 K. Nogawa, Biologic indicators of cadmium nephrotoxicity in persons with low-level cadmium exposure, Environ. Health Perspect., (1984) (in press). 10 K. Nomiyama, M. Yotoriyama and H. Nomiyama, Mechanism of flz-microglobulinuria in experimental chronic cadmium intoxication, Jpn. J. Ind. Health, 25 (1983) 23-27. 11 K. Tsuchiya, S. Iwao, M. Sugita and H. Sakurai, Increased &-microglobulin in cadmium exposure: dose-effect relationship and biological significance of a;?-microglobulin, Environ. Health Perspect., 28 (1979) 147-153.
Toxicology Letters, 22 (1984) 63-68 Elsevier
TOXLett.
1235
ari-MICROGLOBULIN IN URINE AS AN INDICATOR OF RENAL TUBULAR DAMAGE CAUSED BY ENVIRONMENTAL CADMIUM EXPOSURE (cri-Microglobulin;
KoJI
NOGAWA,
DA, MASAO
Pz-microglobulin;
TERIJHIKO
ISHIZAKI
KIDO,
renal damage; cadmium)
YUICHI
and KISAKU
YAMADA,
IKIKO
TSURITANI,
RYUMON
HON-
TERAHATA+
Department of Hygiene and l Department of Clinical Pathology, Kanazawa Medical University, Uchinada. Ishikawa, 920-02 (Japan) (Received
January
9th.
(Revision
received
February
1984)
(Accepted
March
9th,
8th.
1984)
1984)
_-
.-
.._--
-.
_.-
SUMMARY al-Microglobulin (arm) was determined in the urine of both the cadmium-exposed and nonexposed subjects and was compared with those of other urinary parameters such as &-microglobulin (02-m). glucose,
total protein
exposed
subjects.
sensitivity
or amino
acids.
Large amounts
There were significant
and specificity
of both al-m
was similar.
It was concluded
tion caused
by cadmium.
that al-m
correlations
of ur-m between
were detected al-m
in the urine of cadmium-
and other urinary
and &-m as indices of tubular
dysfunction
in urine seemed to be a useful indicator
parameters.
caused
The
by cadmium
of renal tubular
dysfunc-
INTRODUCTION
The kidney is a critical organ in chronic cadmium poisoning. Tubular dysfunction is considered to be an early characteristic in the finding of renal damage caused by cadmium. Low-M, proteins in urine are very sensitive indices of impaired proximal tubular function. In chronic cadmium poisoning large amounts of low-M, proteins such as /3z-microglobulin @z-m), retinol binding protein or lysozyme are detected in urine. Among them, @2-min urine is most widely used as an early sign of renal tubular damage [l], cYi-microglobulin (al-m) is a recently isolated, low-Mr protein Abbreviation:
0378-4274/84/$
APDC-MIBK,
03.00
0
ammonium
Elsevier
Science
pyrollidine
Publishers
dithiocarbamate-methyl
B.V.
isobutyl
ketone.
64 A
lO6-
T
105-
$ ki &
104-
‘2 .-
2 al .f 5
103-
.r c 3 +3 5l f2
lo*r--0.968
0 10 -
:
i8
1 1
10
102
a,- microglobulin
?O3 in urine
104 (pg/g.
4 106
creotlnine)
: t .
r-0.718
cr,-microglobulin
I 105
in urine
oJg/g.creatinine)
Fig. 1, Relationship between orI-microglobulin in urine and µglobuIin (A), glucose (B), total protein (C) and cadmium (D) in urine of the tadmium-exposed (filled circles) and nonexposed (open circles) subjects.
65 C
r-0.863
1
1
1
1
U,-mlcroglobulin
in
10=
lo4
lo3
lo*
10
10”
oJg/g.CreOttnine)
urine
D
106-
z .-E % : ” $
-9
.
. .*
l *
.
104-
.
.
. . .
l
P ‘: ‘5
:*
0
103-
.
0 0 0
.E .z 2
.
. .
.
l
105-
.
.
0
0
0
lo*-
0
0 El
rz0.596 0
P .v C
10
d 1
1 0.5
, 1.0 Cadmium
5.0 concentration
I
I
10.0 in
Urine
50.0 (JJg1g.Creatih-e)
1
100.0
66
(Mr 33 000) initially isolated from the urine of patients with tubular proteinuria [2]. Elevation of this protein in urine and serum was observed in some pathological conditions and particularly in patients with renal dysfunction [3, 41. The present study was undertaken to investigate whether or-m could be used as an early sign of renal tubular dysfunction caused by cadmium. MATERIALS
AND METHODS
Morning urine samples were randomly collected from 27 subjects living in the cadmium-polluted Kakehashi River basin in Ishikawa Prefecture and 10 subjects living in an area free from cadmium pollution. The subjects and controls were all females aged over 50 years. Urine was collected in polyethylene bottles and stored frozen at -20°C until the analysis could be performed. Urinary pH was measured and was neutralized when necessary. (111-mwas analyzed by enzyme-immunoassay using a kit (al-m EIAkit, FujiRebio Co. Tokyo, Japan), 02-m was measured by radioimmunoassay using the phadebas &-microtest (Pharmacia Diagnostic AB, Uppsala, Sweden). Cadmium was determined by flameless atomic absorption spectrophotometry after wet ashing in HNOJ/H~SO~/HC~O~ and extraction with APDC-MIBK. At the same time, total protein, glucose, amino acids and creatinine were measured by standard methods [5]. RESULTS
Table I shows the geometric means and standard deviations of the concentrations of al-m, @z-m, glucose, total protein, amino acid and cadmium in the urine of both the cadmium-exposed and nonexposed subjects. The values were expressed as TABLE
I
GEOMETRIC PARAMETERS
MEANS AND GEOMETRIC IN THE CADMIUM-EXPOSED
STANDARD DEVIATIONS OF THE AND NONEXPOSED SUBJECTS
Cd-exposed
a,-Microglobulin &-Microglobulin
@g/g creatinine) bg/g creatinine)
group
(N = 27)
Nonexposed
G.M.
S.D.
G.M.
1888(r 1 199sp
5.5 7.9
352 33
URINARY
group S.D. 4.2 4.2
Total protein (mg/g creatinine) Glucose (mg/g creatinine)
149.0s 466.5’
6.4 4.3
10.4 99.5
3.8 1.3
Amino acid (mg/g creatinine) Cadmium bg/g creatinine)
272.3” 11.6”
I .7 2.2
157.9 2.4
1.2 1.6
68.8b
9.8
72.3
12.3
Age (year) a P < 0.001. b Arithmetic mean and arithmetic ’ Number
of persons
examined:
standard 26.
deviation
(N = 10)
67
creatinine correction. Cadmium could not be measured in one urine sample of the cadmium-exposed subject because of small urinary volume. Therefore, the total numbers of urine samples analyzed for cadmium were 26 in the cadmium-exposed group and 10 in the nonexposed group. The cadmium-exposed group showed significantly higher values of all urinary parameters than those of the nonexposed group. It should be noted that large amounts of al-m and /32-m were found in the urine of the cadmium-exposed group. In Fig. 1 (a,b,c,d), al-m in urine of both the groups were plotted vs. /32-m, glucose, total protein and cadmium, respectively. All correlation coefficients between crt-m and the other four urinary parameters were statistically significant. As seen in Fig. la, cri-m and /32-m showed a similar trend in both the cadmium-exposed and nonexposed subjects. The correlation coefficient between al-m and @z-mwas 0.968. al-m was less correlated with protein, glucose and cadmium than with ,&-m. Correlation coefficients between al-m and protein, glucose and cadmium were 0.863, 0.718 and 0.596, respectively.
al-m, a low-M, protein, was initially isolated by Ekstrom et al. [2]. This protein is produced in lymphocytes and probably in hepatocytes [6, 71. It is expected that cyl-m is handled in a similar manner by the kidney as those of other low-M, proteins such as &m, retinol binding protein or lysozyme. They are freely filtered through the renal glomeruli and absorbed by the proximal tubular cells where they are degraded. Only trace amounts of these proteins are excreted into the urine because the reabsorption of the substances is almost complete. As shown in Table I, the cadmium-exposed subjects used in the present study were proven to have had renal damage caused by cadmium. They showed significant excretions of low-M, protein, glucose, amino acid, total protein and cadmium in the urine. It was demonstrated that large amounts of ai-m were also excreted in the urine of the cadmium-exposed subjects. As shown in Fig. 1, the concentrations of urinary al-m were significantly correlated with those of the other urinary parameters. al-m was highly correlated with &-m. A correlation coefficient between al-m and &-m was 0.968. Lyl-m was less correlated with all other parameters, e.g., protein, glucose and cadmium, than with 02-m. Therefore it is reasonable to say that urinary amounts of al-m increase significantly in the case of cadmiuminduced renal damage and the usefulness of al-m as an indicator in determining the extent of renal dysfunction caused by cadmium is almost equal to that of urinary P2-m.
There has been considerable discussion on the biological significance of fi2microglobulinuria. Increased Pa-m in urine in cadmium-polluted areas is considered to be caused by a decreased reabsorption capacity of the renal tubules [8, 91. However, Nomiyama et al., reported that the remarkable increase in urinary &-m
68
is associated with renal dysfunction, while the slight increase is caused by mechanisms other than renal dysfunctions [lo]. Tsuchiya et al., also reported that an increase of 02-m in blood and urine in an early stage of cadmium exposure is caused by the increased level of Pz-m in blood, which may be a result of stimulation due to cadmium, but not necessarily by the clinical dysfunction of the reabsorption of f12-m in the renal tubules [ 111. The present study strongly indicates that renal handling of al-m was quite similar to that of 02-m. A study on the mechanism of urinary excretion of al-m would be of great assistance in explaining the biological significance of increased 02-m in urine in cadmium intoxication. REFERENCES 1 T. Kjellstom and M. Piscator, Quantitative Analysis of &Microglobulin as an Indicator of Renal Tubular Function, Pharmacia Diagnostics, Uppsala, Sweden, 1977. 2 B. Ekstrom, P.A. Peterson and I. Berggard, A urinary and plasama al-glycoprotein of low molecular weight: isolation and some properties, Biochem. Biophys. Res. Commun., 65 (1975) 1427-1433. 3 L. Svensson and U. Ravnskov, err-Microglobulin, a new low molecular weight plasma protein, Clin. Chim. Acta, 73 (1976) 415-422. 4 K. Takagi, K. Kin, K. Itoh, H. Enomoto and T. Kawai, Human or-microglobulin levels in various body fluids, J. Clin. Pathol., 33 (1980) 786-791. 5 K. Nogawa, E. Kobayashi, R. Honda, A. Ishizaki, S. Kawano and H. Matsuda, Renal dysfunctions of inhabitants in a cadmium-polluted area, Environ. Res., 23 (1980) 13-23. 6 K. Takagi, K. Kin, Y. Itoh, Y. Kawano, T. Kasahara, T. Shimada and T. Shikata, Tissue distribution of human cYr-microglobulin, J. Clin. Invest., 63 (1979) 318-325. 7 H. Enomoto, Y. Itoh, K. Takagi, H. Harada, T. Kawai, T. Kasahara and F. Sasaki, Studies on human or-microglobulin, VII. Production of or-microglobulin by hepatoma cell line, Physicochem. Biol., 24 (1981) 327-333. 8 K. Shitomi, H. Saito, A. Nakano, H. Unakami, K. Takada, T. Sato, T. Furuyama, K. Yoshinaga, T. Arikawa and K. Nagai, Urinary &-microglobulin for residents in an environmentally cadmiumpolluted area. Studies of generational and sexual differences, and a comparison with the results of proximal renal tubular function tests, Jap. J. Nephrol., 23 (1981) 45-62. 9 K. Nogawa, Biologic indicators of cadmium nephrotoxicity in persons with low-level cadmium exposure, Environ. Health Perspect., (1984) (in press). 10 K. Nomiyama, M. Yotoriyama and H. Nomiyama, Mechanism of flz-microglobulinuria in experimental chronic cadmium intoxication, Jpn. J. Ind. Health, 25 (1983) 23-27. 11 K. Tsuchiya, S. Iwao, M. Sugita and H. Sakurai, Increased &-microglobulin in cadmium exposure: dose-effect relationship and biological significance of a;?-microglobulin, Environ. Health Perspect., 28 (1979) 147-153.