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Increased levels of urinary adenosine deaminase binding protein in children treated with cisplatin or methotrexate
Clinrca Chimica Elsevier
157
Acta, 160 (1986) 157-161
CCA 03543
Increased levels of urinary adenosine deaminase binding protein in children treated with cisplatin or methotrexate Marshall Departments
P. Goren
‘, Reba
K. Wright
’ and Marc
E. Horowitz
of’ Pathology and Laboratory Medicine and ’ Hematology-Oncology, Research Hospital, P.O. Box 318, Memphis, TN 38101 (USA)
(Received
20 February
1986; revision received 15 May 1986; accepted
Kqv words: Adenosine deaminase binding
protein;
Nephrotoxicity;
2
St. Jude Children >
15 May 1986)
Cisplatin; Methotrexate;
Renal tubules
Summary Levels of adenosine deaminase binding protein (ABP), a renal tubular cell antigen, were determined by enzyme immunoassay in urine specimens from seven children with solid tumors who were receiving the recognized nephrotoxins cisplatin or methotrexate. ABP excretion was uniformly increased within the first 3 days after administration of either drug. Elevated ABP levels were usually accompanied by increased excretion of the urinary enzymes N-acetyl-P-D-glucosaminidase and alanine aminopeptidase. In alkaline urine specimens associated with methotrexate therapy, ABP levels were increased whereas enzyme activities appeared to be unstable. Hence, immunochemical measurement of urinary ABP levels may be adjunctively useful for clinical studies of renal tubular damage.
Introduction Renal damage in children receiving antineoplastic drugs is usually monitored by determination of serum creatinine; however, this is a relatively insensitive measure of change in renal function, as well as being age dependent. More sensitive assays of renal tubular damage might be useful for identifying subclinical renal damage induced by antineoplastic drugs. Urinary levels of adenosine deaminase binding protein (ABP), an antigen localized to proximal renal tubular cells [1,2], are reportedly increased lo-fold in patients with acute tubular necrosis in comparison to marginal changes in patients with glomerular disease [3]. We demonstrate increased excretion of urinary ABP in children receiving two nephrotoxic antineoplastic drugs, and show that ABP can be 0009-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
158
measured immunochemically be unstable.
in instances
when the activity
of urinary
enzymes
may
Materials and methods Daily urine specimens were obtained for up to 1 week after doses of either cisplatin or high-dose methotrexate (MTX) administered to children with osteosarcoma. After intravenous pre-hydration consisting of 10 g/m* mannitol in 500 ml/m2 of 5% glucose and 0.22% saline, cisplatin was administered over 6 hours with 10 g/m2 mannitol in 1000 ml/m2 of 0.22% saline. MTX was infused over 4 hours after the initiation of alkaline diuresis; this therapy was followed by ten oral or i.v. doses of calcium leucovorin, 15 mg every 6 hours; hydration, sufficient to induce a urine output of at least 3 liters/m2 per day was continued for 2 days after the infusion. Before analysis, the specimens were stored at 4°C and centrifuged (1500 X g, 10 min) to remove insoluble salts and debris. ABP was assayed with reagents supplied by Cambridge Research Laboratory, Cambridge, MA 02139 131. Briefly, duplicate 100 ~1 samples of urine or assay standards were transferred to the wells of microtiter plates precoated with URO-4, an ABP-specific monoclonal antibody. After a 1 hour incubation at 25°C. the plates were washed three times with Tween-containing phosphate-buffered saline to remove nonbound antigen, and then 100 ~11of the second ABP-specific monoclonal antibody, URO-4a, conjugated to horseradish peroxidase was added to each well. After 1 hour incubation, the wells were rinsed three times prior to the addition of the chromogen solution, 18.5 mmol/l of O-phenylenediamine and 4.4 mmol/l H,O, in citrate (19 mmol/l)-Na2HP0, (62 mmol/l) buffer, pH 5.0. After 1 hour, the reaction was stopped with 100 ~1 of 4.5 mol/l sulfuric acid, and the relative peroxidase activity in each well was determined by comparing the absorbance at 490 nm with that for a standard curve. Activity was expressed in arbitrary units {AU), with 1 AU defined as the amount of ABP iq 100 ~1 of sample that generates an increase of 1 absorbance unit. Urinary N-acetyl-/?-D-glucosaminidase (NAG, EC 3.2.1.30) and alanine aminopeptidase (AAP, EC 3.4.11.2) activity were assayed by modifications of calorimetric methods [4,5] for automated determinations on a Micro-KDA analyzer (American Monitor Carp, Indianapolis, IN); the procedure included a I : 20 dilution of the sample. Total urinary protein was assayed with Coomassie brilliant blue (Bio-Rad, Anaheim, CA). ABP, NAG, AAP and total protein levels were divided by the concentration of urinary creatinine to adjust for variations in urine flow in untimed specimens. Results We verified that the ABP assay was linear to 1.5 AU by diluting two clinical specimens with high activity. A precision study of control materials measured on 11 days yielded means (AU) and coefficients of variation of 0.065 (33%), 0.44 (10.4%) and 0.93 (9.9%). The highest value for 30 healthy children without renai disease was 30 AU/mol creatinine.
159
50
1
-0
I
2
3
4
5
Time (days) Fig. 1. Mean urinary levels of adenosine deaminase binding protein (ABP) (AU/m01 children receiving cisplatin. Vertical bars indicate standard error of the mean.
creatinine)
in
In five patients who received seven courses of cisplatin, ABP levels increased within 3 days after therapy to a mean (k2 SD) maximum value of 90 If: 51 AU/mol creatinine, ranging from 63 to 136 AU/mol creatinine (Fig. 1). ABP did not return to pretreatment levels by 3 weeks after two of four evaluable cisplatin courses. The
Methotrexote ( 12q/m2)
I
0
1
I
I
I
I
I
1
I
2
3
4
5
6
7
0
Time (days) Fig. 2. Mean urinary
ABP levels in children
receiving
methotrexate.
Bars indicate
SEM.
160
80 70 1
60 -
E \
50-
z
40-
k v 301 2 $j zo-
LB,
i
.9-----e
AAP
IO 0 -. 0
I2
3 Time
4
5
z
(Days)
Fig. 3. Urinary ABP, NAG and AAP levels in a patient who received high-dose methotrexate therapy on day 1. Urine specimens obtained on days 2 and 3 were dilute, alkaline (pH 2 8) and intensely yellow with methotrexate concentrations b 0.5 mmol/l.
serum creatinine concentrations in these two patients were also persistently increased by 26-61 pmol/l. After 11 doses of high-dose methotrexate administered to six patients (Fig. 2), ABP excretion increased to a mean (k 2 SD) maximum value of 111 f 70 AU/mol creatinine (range, 53-152; n = 10); there was an exceptional value of 737 AU/mol creatinine achieved in one other instance on the day of methotrexate therapy. ABP returned to pretreatment levels within 1 week after methotrexate therapy. The results of the assays for ABP were compared by regression analysis with those for NAG, r = 0.52 (n = 94, p < O.OOl), AAP, r = 0.51 (n = 94, p < 0.001) and total protein r = 0.04, (n = 86, N.S.). Exclusion from analysis of results for specimens obtained on days 1 and 2 of methotrexate therapy improved the degree of correlation with NAG, r = 0.63; enzyme activity in many of these specimens was undetectable because the urine was dilute and alkaline, pH 2 8, or intensely colored by concentrations of methotrexate, resulting in high background absorbance at the wavelength used in both enzyme assays. ABP levels were increased in these specimens despite the low enzyme levels. Fig. 3 shows a representative example of a patient with depressed levels of NAG and AAP following methotrexate therapy, but increased ABP levels. Discussion The transiently increased urinary ABP levels after chemotherapy are consistent with reports of cisplatin [6] and methotrexate [7] renal tubular pathology. The
161
persistent elevation of urinary ABP in two patients after cisplatin therapy may reflect irreversible drug-induced alterations of renal tubules [8]. Our correlations between measurements of ABP, NAG and AAP might be improved by pre-analysis filtration [9] or dialysis of specimens to remove reversible inhibitors of enzyme activity. The selective depression of enzymatic activity in alkaline urine indicates irreversible inactivation of enzymes; the increased level of ABP in these specimens suggests that immunochemical assays can be adjunctively useful with conventional urinary enzyme measurements. The ABP procedure is as sensitive as enzymatic methods, and includes a step to remove most interfering substances. Determinations of urinary ABP may be useful for screening investigational drugs for acute tubular nephrotoxicity. Persistently increased ABP levels could indicate alterations of the renal tubules that affect processing of drugs by the tubules or increase the patient’s risk of subsequent drug-induced nephrotoxicity. As additional monoclonal antibodies are characterized, it should be possible to localize drug-induced damage to specific portions of the nephron. Acknowledgements Supported in part by Biomedical Research Support Grant RR05584 (M.P.G.), Cancer Center Support (Core) Grant CA-21765, Childhood Solid Tumor Program Project Grant CA-23099, American Lebanese Syrian Associated Charities and Cambridge Research Laboratories. References Cordon-Cardo C, Bander NH, Fradet Y, Finstad CL, Whitmore WF, Lloyd KO, Oettgen HF. Melamed MR, Old LJ. Immunoanatomic dissection of the human urinary tract by monoclonal antibodies. J Histochem Cytochem 1984; 32: 1035-1040. Andy RJ, Finstad CL, Old LJ, Lloyd KO, Kornfeld R. The antigen identified by a mouse monoclonal antibody raised against human renal cancer cells is the adenosine deaminase binding protein. J Biol Chem 1984; 259: 12844-12849. Thompson RE, Piper DJ, Galberg C, Chan TH, Tolkoff-Rubin NE, Rubin RH. Adenosine deaminase binding protein, a new diagnostic marker for kidney disease. Clin Chem 1985; 31: 679-683. Horak E, Hopfer SM, Sunderman FW Jr. Spectrophotometric assay for urinary N-acetyI+-n-glucosaminidase. Clin Chem 1981; 27: 1180-1185. Jung K, Scholl D. An optimized assay of alanine aminopeptidase activity in urine. Chn Chem 1980; 26: 1251-1254. Gonzalez-Vitale JC, Hayes DM, Cvitkovic E, Stemberg SS. The renal pathology in clinical trials of &-platinum (II) diamminedichloride. Cancer 1977; 39: 1362-1371. Condit PT, Chanes RE, Joel W. Renal toxicity of methotrexate. Cancer 1969; 23: 126-131. Goren MP, Wright RK, Horowitz ME. Cumulative renal tubular damage associated with cisplatin nephrotoxicity. Cancer Chemother Pharmacol, in press. Werner M, Gabrielson D. Ultrafiltration for improved assay of urinary enzymes. Clin Chem 1977; 23: 700-704.
157
Acta, 160 (1986) 157-161
CCA 03543
Increased levels of urinary adenosine deaminase binding protein in children treated with cisplatin or methotrexate Marshall Departments
P. Goren
‘, Reba
K. Wright
’ and Marc
E. Horowitz
of’ Pathology and Laboratory Medicine and ’ Hematology-Oncology, Research Hospital, P.O. Box 318, Memphis, TN 38101 (USA)
(Received
20 February
1986; revision received 15 May 1986; accepted
Kqv words: Adenosine deaminase binding
protein;
Nephrotoxicity;
2
St. Jude Children >
15 May 1986)
Cisplatin; Methotrexate;
Renal tubules
Summary Levels of adenosine deaminase binding protein (ABP), a renal tubular cell antigen, were determined by enzyme immunoassay in urine specimens from seven children with solid tumors who were receiving the recognized nephrotoxins cisplatin or methotrexate. ABP excretion was uniformly increased within the first 3 days after administration of either drug. Elevated ABP levels were usually accompanied by increased excretion of the urinary enzymes N-acetyl-P-D-glucosaminidase and alanine aminopeptidase. In alkaline urine specimens associated with methotrexate therapy, ABP levels were increased whereas enzyme activities appeared to be unstable. Hence, immunochemical measurement of urinary ABP levels may be adjunctively useful for clinical studies of renal tubular damage.
Introduction Renal damage in children receiving antineoplastic drugs is usually monitored by determination of serum creatinine; however, this is a relatively insensitive measure of change in renal function, as well as being age dependent. More sensitive assays of renal tubular damage might be useful for identifying subclinical renal damage induced by antineoplastic drugs. Urinary levels of adenosine deaminase binding protein (ABP), an antigen localized to proximal renal tubular cells [1,2], are reportedly increased lo-fold in patients with acute tubular necrosis in comparison to marginal changes in patients with glomerular disease [3]. We demonstrate increased excretion of urinary ABP in children receiving two nephrotoxic antineoplastic drugs, and show that ABP can be 0009-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
158
measured immunochemically be unstable.
in instances
when the activity
of urinary
enzymes
may
Materials and methods Daily urine specimens were obtained for up to 1 week after doses of either cisplatin or high-dose methotrexate (MTX) administered to children with osteosarcoma. After intravenous pre-hydration consisting of 10 g/m* mannitol in 500 ml/m2 of 5% glucose and 0.22% saline, cisplatin was administered over 6 hours with 10 g/m2 mannitol in 1000 ml/m2 of 0.22% saline. MTX was infused over 4 hours after the initiation of alkaline diuresis; this therapy was followed by ten oral or i.v. doses of calcium leucovorin, 15 mg every 6 hours; hydration, sufficient to induce a urine output of at least 3 liters/m2 per day was continued for 2 days after the infusion. Before analysis, the specimens were stored at 4°C and centrifuged (1500 X g, 10 min) to remove insoluble salts and debris. ABP was assayed with reagents supplied by Cambridge Research Laboratory, Cambridge, MA 02139 131. Briefly, duplicate 100 ~1 samples of urine or assay standards were transferred to the wells of microtiter plates precoated with URO-4, an ABP-specific monoclonal antibody. After a 1 hour incubation at 25°C. the plates were washed three times with Tween-containing phosphate-buffered saline to remove nonbound antigen, and then 100 ~11of the second ABP-specific monoclonal antibody, URO-4a, conjugated to horseradish peroxidase was added to each well. After 1 hour incubation, the wells were rinsed three times prior to the addition of the chromogen solution, 18.5 mmol/l of O-phenylenediamine and 4.4 mmol/l H,O, in citrate (19 mmol/l)-Na2HP0, (62 mmol/l) buffer, pH 5.0. After 1 hour, the reaction was stopped with 100 ~1 of 4.5 mol/l sulfuric acid, and the relative peroxidase activity in each well was determined by comparing the absorbance at 490 nm with that for a standard curve. Activity was expressed in arbitrary units {AU), with 1 AU defined as the amount of ABP iq 100 ~1 of sample that generates an increase of 1 absorbance unit. Urinary N-acetyl-/?-D-glucosaminidase (NAG, EC 3.2.1.30) and alanine aminopeptidase (AAP, EC 3.4.11.2) activity were assayed by modifications of calorimetric methods [4,5] for automated determinations on a Micro-KDA analyzer (American Monitor Carp, Indianapolis, IN); the procedure included a I : 20 dilution of the sample. Total urinary protein was assayed with Coomassie brilliant blue (Bio-Rad, Anaheim, CA). ABP, NAG, AAP and total protein levels were divided by the concentration of urinary creatinine to adjust for variations in urine flow in untimed specimens. Results We verified that the ABP assay was linear to 1.5 AU by diluting two clinical specimens with high activity. A precision study of control materials measured on 11 days yielded means (AU) and coefficients of variation of 0.065 (33%), 0.44 (10.4%) and 0.93 (9.9%). The highest value for 30 healthy children without renai disease was 30 AU/mol creatinine.
159
50
1
-0
I
2
3
4
5
Time (days) Fig. 1. Mean urinary levels of adenosine deaminase binding protein (ABP) (AU/m01 children receiving cisplatin. Vertical bars indicate standard error of the mean.
creatinine)
in
In five patients who received seven courses of cisplatin, ABP levels increased within 3 days after therapy to a mean (k2 SD) maximum value of 90 If: 51 AU/mol creatinine, ranging from 63 to 136 AU/mol creatinine (Fig. 1). ABP did not return to pretreatment levels by 3 weeks after two of four evaluable cisplatin courses. The
Methotrexote ( 12q/m2)
I
0
1
I
I
I
I
I
1
I
2
3
4
5
6
7
0
Time (days) Fig. 2. Mean urinary
ABP levels in children
receiving
methotrexate.
Bars indicate
SEM.
160
80 70 1
60 -
E \
50-
z
40-
k v 301 2 $j zo-
LB,
i
.9-----e
AAP
IO 0 -. 0
I2
3 Time
4
5
z
(Days)
Fig. 3. Urinary ABP, NAG and AAP levels in a patient who received high-dose methotrexate therapy on day 1. Urine specimens obtained on days 2 and 3 were dilute, alkaline (pH 2 8) and intensely yellow with methotrexate concentrations b 0.5 mmol/l.
serum creatinine concentrations in these two patients were also persistently increased by 26-61 pmol/l. After 11 doses of high-dose methotrexate administered to six patients (Fig. 2), ABP excretion increased to a mean (k 2 SD) maximum value of 111 f 70 AU/mol creatinine (range, 53-152; n = 10); there was an exceptional value of 737 AU/mol creatinine achieved in one other instance on the day of methotrexate therapy. ABP returned to pretreatment levels within 1 week after methotrexate therapy. The results of the assays for ABP were compared by regression analysis with those for NAG, r = 0.52 (n = 94, p < O.OOl), AAP, r = 0.51 (n = 94, p < 0.001) and total protein r = 0.04, (n = 86, N.S.). Exclusion from analysis of results for specimens obtained on days 1 and 2 of methotrexate therapy improved the degree of correlation with NAG, r = 0.63; enzyme activity in many of these specimens was undetectable because the urine was dilute and alkaline, pH 2 8, or intensely colored by concentrations of methotrexate, resulting in high background absorbance at the wavelength used in both enzyme assays. ABP levels were increased in these specimens despite the low enzyme levels. Fig. 3 shows a representative example of a patient with depressed levels of NAG and AAP following methotrexate therapy, but increased ABP levels. Discussion The transiently increased urinary ABP levels after chemotherapy are consistent with reports of cisplatin [6] and methotrexate [7] renal tubular pathology. The
161
persistent elevation of urinary ABP in two patients after cisplatin therapy may reflect irreversible drug-induced alterations of renal tubules [8]. Our correlations between measurements of ABP, NAG and AAP might be improved by pre-analysis filtration [9] or dialysis of specimens to remove reversible inhibitors of enzyme activity. The selective depression of enzymatic activity in alkaline urine indicates irreversible inactivation of enzymes; the increased level of ABP in these specimens suggests that immunochemical assays can be adjunctively useful with conventional urinary enzyme measurements. The ABP procedure is as sensitive as enzymatic methods, and includes a step to remove most interfering substances. Determinations of urinary ABP may be useful for screening investigational drugs for acute tubular nephrotoxicity. Persistently increased ABP levels could indicate alterations of the renal tubules that affect processing of drugs by the tubules or increase the patient’s risk of subsequent drug-induced nephrotoxicity. As additional monoclonal antibodies are characterized, it should be possible to localize drug-induced damage to specific portions of the nephron. Acknowledgements Supported in part by Biomedical Research Support Grant RR05584 (M.P.G.), Cancer Center Support (Core) Grant CA-21765, Childhood Solid Tumor Program Project Grant CA-23099, American Lebanese Syrian Associated Charities and Cambridge Research Laboratories. References Cordon-Cardo C, Bander NH, Fradet Y, Finstad CL, Whitmore WF, Lloyd KO, Oettgen HF. Melamed MR, Old LJ. Immunoanatomic dissection of the human urinary tract by monoclonal antibodies. J Histochem Cytochem 1984; 32: 1035-1040. Andy RJ, Finstad CL, Old LJ, Lloyd KO, Kornfeld R. The antigen identified by a mouse monoclonal antibody raised against human renal cancer cells is the adenosine deaminase binding protein. J Biol Chem 1984; 259: 12844-12849. Thompson RE, Piper DJ, Galberg C, Chan TH, Tolkoff-Rubin NE, Rubin RH. Adenosine deaminase binding protein, a new diagnostic marker for kidney disease. Clin Chem 1985; 31: 679-683. Horak E, Hopfer SM, Sunderman FW Jr. Spectrophotometric assay for urinary N-acetyI+-n-glucosaminidase. Clin Chem 1981; 27: 1180-1185. Jung K, Scholl D. An optimized assay of alanine aminopeptidase activity in urine. Chn Chem 1980; 26: 1251-1254. Gonzalez-Vitale JC, Hayes DM, Cvitkovic E, Stemberg SS. The renal pathology in clinical trials of &-platinum (II) diamminedichloride. Cancer 1977; 39: 1362-1371. Condit PT, Chanes RE, Joel W. Renal toxicity of methotrexate. Cancer 1969; 23: 126-131. Goren MP, Wright RK, Horowitz ME. Cumulative renal tubular damage associated with cisplatin nephrotoxicity. Cancer Chemother Pharmacol, in press. Werner M, Gabrielson D. Ultrafiltration for improved assay of urinary enzymes. Clin Chem 1977; 23: 700-704.