BIOCHEMICAL
MEDICINt
21, 220-225
1197%
Alanine Aminopeptidase Excretion After Mercuric Chloride Renal Failure BARRY B. KIRSCHBAUM Medical
College of Virginia. Box 197. MCV
Division Szation,
Received
of Nephrology, Richmond,
November
Department Virginia 23298
of Medicine.
15, 1978
The search for a sensitive and specific indicator of renal parenchymal damage has included the measurement of several urinary enzymes (1). Ideally, the enzyme to be studied should be stable, easily assayed without elaborate or time-consuming urine purification procedures, present with high activity in a compartment of the renal cell readily accessible to the tubular lumen, and of sufficient molecular size to minimize the contribution of glomerular filtration of circulating enzyme protein derived from nonrenal sources to the determined enzymuria. Alanine aminopeptidase (AAP), a brush-border glycoprotein enzyme of molecular weight greater than 200,000 (2), satisfies these requirements. AAP activity may be readily measured in unconcentrated human urine. In our studies, no inhibition of enzyme activity was observed by high concentrations of albumin (10 g/liter) or low-molecular-weight urine solutes assessed by ultrafiltration of urine across Amicon XM-1OOA membranes. However, a large percentage of urines obtained randomly from critically ill patients in intensive care units and from people with chronic renal disease contained a higher AAP activity per gram creatinine than did urines from healthy nonhospitalized controls. Therefore, to evaluate AAP excretion as a potentially useful indicator of acute renal injury, we elected to study enzymuria in the homogeneous setting of mercuric chloride-induced renal failure in the rat (3,4). MATERIALS
AND METHODS
Female Sprague-Dawley rats were housed two per cage, fed standard rat chow, and maintained on 0.2% saline orally in order to increase urine volumes. After 48 hr, three baseline 24hr urines were collected (days -3, 220 Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in any form reserved.
AAP
EXCRETION
IN RENAL
FAILURE
221
-2, - 1). Each animal received one subcutaneous injection of HgCl,, 4.7 mg/kg body weight, and urine collections were continued. Each 24hr urine was filtered through glass wool to remove particulate matter, centrifuged for 60 min at lO,OOOg, and concentrated in an Amicon filtration cell using an XM-1OOA membrane. The retentate was washed with 0.1 M NaPO, buffer, pH 7.0, and diluted with the same buffer. AAP activity was determined on a Zeiss PMdK spectrophotometer at 37°C by adding to each cuvette 1.0 ml of 0.1 M NaPO, buffer and an equal volume of concentrated urine plus buffer to give 1.0 ml. The reaction was initiated by pipetting 1.0 ml of 3 mM L-alanine /9-naphthylamide except for the blank which received buffer (5). The change in absorption at 340 nm was followed with continuous zeroing of the enzyme blank. The data were converted to pmole substrate hydrolyzed per minute by reference to a standard curve for naphthylamide. Urinary creatinine was measured manually by the alkaline picrate method. Statistical analysis was conducted with the t test for paired data acceptingP G 0.05 as significant. Data are given as mean _’ SD. RESULTS During the 3-day control period, there were no statistically significant differences in the mean AAP excretion rates whether expressed as activity per 24 hr or activity per gram creatinine. The mean 2 SD of urine AAP for each pair of rats for the three baseline collections is recorded in Tables 1 and 2 as pmole substrate/min/24-hr urine, and pmole/min/g creatinine, respectively. A highly significant (P < 0.001) increase in AAP excretion was measured in the first 24-hr urine after mercuric chloride injection. Compared to the mean value of the control period, AAP activity rose 11.5fold when expressed per 24hr urine volume and l&2-fold when related to urine creatinine content. Significant hyperenzymuria was also documented on Days 2 and 3 after HgCl, injection, but the mean AAP values on these days were only 22 to 40% of that present during the first collection. By Day 4, no significant increase in AAP activity could be demonstrated. Between 8 and 11 days after HgCl*, a modest secondary rise in AAP excretion was seen which varied considerably among the animals both in magnitude and timing. As a result of this variability, only the values recorded on Day 8 were significant (P < 0.05). In Fig. 1, the mean AAP activities per gram urine creatinine for all five pairs of rats are plotted against time. Although serum urea nitrogen and creatinine levels were not measured on these animals, companion studies indicate that the peak of renal failure occurred about 72 hr after HgC12 and, by Day 5, function was rapidly recovering. In general, expressing urine AAP activity as pmole/min/g Cr or as ~molelminl2Chr urine gave equivalent results suggesting that random urines corrected for creatinine content could replace timed urine collections.
s
0.051 *0.006 0.858 0.463 0.347 0.171 0.113 0.231 0.2% 0.112
1
URINE
3 0.065 20.005 0.642 0.193 0.212 0.055 0.047 0.089 0.1% 0.107
2
0.058 20.012 0.680 0.159 0.126 0.035 0.011 0.139 0.112 0.075
1 AAP
4
EXCRETIONS
0.074 r!IO.029 0.961 0.439 0.229 0.145 0.194 0.209 0.210 0.314
urine)
TABLE Animal set substrate/min/24-hr
2~HOUR
0.086 AIO.000 0.704 0.336 0.1% 0.059 0.030 0.128 0.154 0.150
5
0.40 >0.90 co.05 >o. 10 =o. 10
P value
D Each 24hr AAP excretion was compared to the control value for that set of rats. P value calculated from t test for paired data, column) refers to AAP activity for all pairs of animals on each day.
Control Mean 2 SD 1 2 3 4 5 8 10 11
Day
&mole
TOTAL
Mean
(last
0.121 0.112 0.072 0.054 0.038 0.053 0.145 0.084
F SD
2 SD
0.067 k-O.019 0.769 i 0.306 t 0.221 2 0.093 rt 0.065 k 0.159 t 0.216 + 0.152 +
Mean
M w
2.71 20.43 58.3 22.5 19.6 7.24 14.2 33.2 8.51
I
3.50 r0.29 74.0 10.0 9.69 0.95 9.14 5.88 4.78
2 4.03 20.73 63.3 17.9 17.0 4.09 6.31 4.67 5.10
3
2
4.65 21.66 101 29.1 15.2 3.51 10.6 11.5 18.0
4
CREATININE”
4.42 20.77 53.7 21.8 15.7 3.51 5.92 6.76 6.57
5
0.90
MEDICINt
21, 220-225
1197%
Alanine Aminopeptidase Excretion After Mercuric Chloride Renal Failure BARRY B. KIRSCHBAUM Medical
College of Virginia. Box 197. MCV
Division Szation,
Received
of Nephrology, Richmond,
November
Department Virginia 23298
of Medicine.
15, 1978
The search for a sensitive and specific indicator of renal parenchymal damage has included the measurement of several urinary enzymes (1). Ideally, the enzyme to be studied should be stable, easily assayed without elaborate or time-consuming urine purification procedures, present with high activity in a compartment of the renal cell readily accessible to the tubular lumen, and of sufficient molecular size to minimize the contribution of glomerular filtration of circulating enzyme protein derived from nonrenal sources to the determined enzymuria. Alanine aminopeptidase (AAP), a brush-border glycoprotein enzyme of molecular weight greater than 200,000 (2), satisfies these requirements. AAP activity may be readily measured in unconcentrated human urine. In our studies, no inhibition of enzyme activity was observed by high concentrations of albumin (10 g/liter) or low-molecular-weight urine solutes assessed by ultrafiltration of urine across Amicon XM-1OOA membranes. However, a large percentage of urines obtained randomly from critically ill patients in intensive care units and from people with chronic renal disease contained a higher AAP activity per gram creatinine than did urines from healthy nonhospitalized controls. Therefore, to evaluate AAP excretion as a potentially useful indicator of acute renal injury, we elected to study enzymuria in the homogeneous setting of mercuric chloride-induced renal failure in the rat (3,4). MATERIALS
AND METHODS
Female Sprague-Dawley rats were housed two per cage, fed standard rat chow, and maintained on 0.2% saline orally in order to increase urine volumes. After 48 hr, three baseline 24hr urines were collected (days -3, 220 Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in any form reserved.
AAP
EXCRETION
IN RENAL
FAILURE
221
-2, - 1). Each animal received one subcutaneous injection of HgCl,, 4.7 mg/kg body weight, and urine collections were continued. Each 24hr urine was filtered through glass wool to remove particulate matter, centrifuged for 60 min at lO,OOOg, and concentrated in an Amicon filtration cell using an XM-1OOA membrane. The retentate was washed with 0.1 M NaPO, buffer, pH 7.0, and diluted with the same buffer. AAP activity was determined on a Zeiss PMdK spectrophotometer at 37°C by adding to each cuvette 1.0 ml of 0.1 M NaPO, buffer and an equal volume of concentrated urine plus buffer to give 1.0 ml. The reaction was initiated by pipetting 1.0 ml of 3 mM L-alanine /9-naphthylamide except for the blank which received buffer (5). The change in absorption at 340 nm was followed with continuous zeroing of the enzyme blank. The data were converted to pmole substrate hydrolyzed per minute by reference to a standard curve for naphthylamide. Urinary creatinine was measured manually by the alkaline picrate method. Statistical analysis was conducted with the t test for paired data acceptingP G 0.05 as significant. Data are given as mean _’ SD. RESULTS During the 3-day control period, there were no statistically significant differences in the mean AAP excretion rates whether expressed as activity per 24 hr or activity per gram creatinine. The mean 2 SD of urine AAP for each pair of rats for the three baseline collections is recorded in Tables 1 and 2 as pmole substrate/min/24-hr urine, and pmole/min/g creatinine, respectively. A highly significant (P < 0.001) increase in AAP excretion was measured in the first 24-hr urine after mercuric chloride injection. Compared to the mean value of the control period, AAP activity rose 11.5fold when expressed per 24hr urine volume and l&2-fold when related to urine creatinine content. Significant hyperenzymuria was also documented on Days 2 and 3 after HgCl, injection, but the mean AAP values on these days were only 22 to 40% of that present during the first collection. By Day 4, no significant increase in AAP activity could be demonstrated. Between 8 and 11 days after HgCl*, a modest secondary rise in AAP excretion was seen which varied considerably among the animals both in magnitude and timing. As a result of this variability, only the values recorded on Day 8 were significant (P < 0.05). In Fig. 1, the mean AAP activities per gram urine creatinine for all five pairs of rats are plotted against time. Although serum urea nitrogen and creatinine levels were not measured on these animals, companion studies indicate that the peak of renal failure occurred about 72 hr after HgC12 and, by Day 5, function was rapidly recovering. In general, expressing urine AAP activity as pmole/min/g Cr or as ~molelminl2Chr urine gave equivalent results suggesting that random urines corrected for creatinine content could replace timed urine collections.
s
0.051 *0.006 0.858 0.463 0.347 0.171 0.113 0.231 0.2% 0.112
1
URINE
3 0.065 20.005 0.642 0.193 0.212 0.055 0.047 0.089 0.1% 0.107
2
0.058 20.012 0.680 0.159 0.126 0.035 0.011 0.139 0.112 0.075
1 AAP
4
EXCRETIONS
0.074 r!IO.029 0.961 0.439 0.229 0.145 0.194 0.209 0.210 0.314
urine)
TABLE Animal set substrate/min/24-hr
2~HOUR
0.086 AIO.000 0.704 0.336 0.1% 0.059 0.030 0.128 0.154 0.150
5
0.40 >0.90 co.05 >o. 10 =o. 10
P value
D Each 24hr AAP excretion was compared to the control value for that set of rats. P value calculated from t test for paired data, column) refers to AAP activity for all pairs of animals on each day.
Control Mean 2 SD 1 2 3 4 5 8 10 11
Day
&mole
TOTAL
Mean
(last
0.121 0.112 0.072 0.054 0.038 0.053 0.145 0.084
F SD
2 SD
0.067 k-O.019 0.769 i 0.306 t 0.221 2 0.093 rt 0.065 k 0.159 t 0.216 + 0.152 +
Mean
M w
2.71 20.43 58.3 22.5 19.6 7.24 14.2 33.2 8.51
I
3.50 r0.29 74.0 10.0 9.69 0.95 9.14 5.88 4.78
2 4.03 20.73 63.3 17.9 17.0 4.09 6.31 4.67 5.10
3
2
4.65 21.66 101 29.1 15.2 3.51 10.6 11.5 18.0
4
CREATININE”
4.42 20.77 53.7 21.8 15.7 3.51 5.92 6.76 6.57
5
0.90
