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Urinary lysosomal enzymes for detection of acute renal allograft rejection
Urinary Lysosomal Enzymes for Detection of Acute Renal Allograft Rejection Christian Smeesters, MD,” Los Angeles, California Richard M. Ehrllch, MD, Los Angeles, California Eric W. Fonkalsrud, MD, Los Angeles, California
Since the development of fluorometric assays for the measurement of acid hydrolases by Leaback [1] and Leaback and Walker [2] in 1961, there has been considerable interest in the quantitation of enzymes released from injured organs as a method of determining extent of tissue damage. Such measurements
have been helpful in evaluating renal injury after periods of ischemia [3,4]. Urinary levels of N-acetyl-P-glucosaminidase and P-galactosidase [3] and P-glucuronidase [5] were increased in patients with active renal disease, particularly when tubular injury was present. Urine assays for these enzymes have also been performed as a method of detecting episodes of rejection in patients who have received renal allografts [5,6]. To be helpful in identifying episodes of allograft rejection, a new test must be simple, able to be performed quickly, and sufficiently sensitive to detect rejection episodes before conventional clinical and laboratory parameters. The present study was undertaken to determine the practicality and reliability of identifying episodes of renal allograft rejection by fluorometric assay of the urinary activities of two lysosomal enzymes, /3galactosidase and N-acetyl-P-glucosaminidase, in undialyzed urine specimens. These enzymes were selected on the basis of their higher sensitivity as an index of cellular injury, as demonstrated in previous studies on liver preservation and transplantation [7] and on renal rejection [6]. Clinical Methods Release of renal tubular lysosomal enzymes into the urine was evaluated in six patients for one to two months From the Department of Surgery, UCLA School of Medicine, Los Angeles, California. This work was supported by USPHS Grant HL 14333. Reprint requests should be addressed to Eric W. Fonkalsrud. MD, Department of Surgery, UCLA School of Medicine, Los Angeles, California 90024. * Assisted by a Fellowship from the Medical Research Council of Canada.
560
after renal allotransplantation. Three patients had received renal grafts from living related donors (LD), and three patients had received cadaveric renal allografts (CD). Rejection episodes, as diagnosed by conventional parameters of low 24 hour urine volumes, increasing serum creatinine levels, and decreasing urinary sodium concentration, were treated with high-dose intravenous methylprednisolone. Daily measurements of the enzymes P-galactosidase and N-acetyl$-glucosaminidase were obtained from 24 hour urine samples refrigerated at 4’C until testing which was performed within 48 hours of collection. The enzyme levels after refrigeration were stable for three days for @-galactosidase and at least one week for N-acetyl-&glucosaminidase. Fluorometric methods with I-methylumbelliferyl substrates (Koch-Light Labs, Ltd, Buckinghamshire, England) were employed to determine the activity of both enzymes. Assays were performed according to the methods described by Leaback and Walker [2] for N-acetyl-fl-glucosaminidase and by Robinson, Price, and Dance [B] for fl-galactosidase, as modified by Van Hoof [9]. Incubation periods extended up to 30 minutes, and total time to accomplish the test was approximately 60 minutes. Activity w&s expressed in milliunits (mu)/100 ml of urine, 1 mU referring to the amount of enzyme that hydrolyzes 1 nanomole of substrate per minute under the conditions of the assay. The enzyme excretion in the urine was corrected for creatinine content to relate enzyme release to the amount of functioning tubular mass. Results and Comments Increased urine enzyme excretion occurred in the early postoperative period and presumably represented transient renal ischemic injury. Values returned to normal levels by the fourth to sixth day after grafting. In two patients who had an uncomplicated postoperative course, urine enzyme activity remained normal after the seventh day, correlating well with the stabilization of urine volume and serum creatinine level. (Figure 1.)
The American Journal of Surgery
Enzyme Measurements in Altograft Rejection
ENZYMEACTI VU-Y
ENZYME ACTMTY lmlJ/mg CREATINfNE/
CREATlNfNEI
fmWmg
720
24.1
SERUM CREAT/NlNE
3
-c 8 ;
2
t
T;
ID.
SERUM CREAThWVE -i
2
8
;
g
4000
r
URINE VOLU.E
2ooot 5
10
3000-
;
20001000
n
1
5
15
20
DAYS
-
1
5
10
15
I
I
20
25
1 30
35
DAYS
Figure 2. Results of urine vohsne, serum creatkke, and urinary lysosomel enzyme actMy after fvnai transplant8tiOftfram8CMtdW&CdOlUUik)8pdtkntw#tthno~
1. Resufts of urine volume, serum creatkke, and urinary &sosomal enzyme 8ctMty after renal transpkntatkn f&m a llvkg &nor ilkstrate a patient with an uncomplkated postoperative course.
episodes. Treatment of reJectkn episodes lo Indkat~ by high dose adminktratkn of methy@ednkokne. the last determkatkn was made one week after the patient was discharged and two days before she was readm&ted for treatment of severe acute rejectkn. Renal functkn was considered normal at that t/me.
The remaining four patients underwent ten clinical rejection episodes, most of which correlated well with corresponding peaks in urinary activity of both enzymes. (Figures 2 and 3.) Eight of the ten rejection episodes were characterized by simultaneous two- to sixfold increases in enzymatic activity. Although increased activity usually was not demonstrated before manifestation of rejection by clinical and other laboratory parameters, measurements of enzyme excretion were helpful in confirming conventional data compatible with rejection. This is in agreement with the observations of Gonick, Kramer, and Schapiro [5] but does not substantiate those of Wellwood et al [6] who found that urinary activity of N-acetyl-&glucosaminidase was increased up to three weeks before changes in other tests of renal function. However, increased release of acid hydrolases was evident two and four days before clinical confirmation of two rejection episodes. (Figures 2 and 3.) Those rejection episodes were characterized by only minimal changes in conventional criteria of re-
jection and suggest the value of enzymatic determinations in the early detection of rejection. Successful treatment of rejection episodes by high dose corticosteroid therapy was mirrored by a corresponding decrease in enzyme activity, which correlated well with normalization of serum creatinine levels and urine volumes. Although urine acid phosphatase levels were also measured, they were unreliable as an indicator of rejection, especially in adult males, because of the variable contribution of prostatic acid phosphatase to urinary enzymatic activity. This observation agrees with the observations of Kramer and Gonick [IO], who studied urinary acid phosphatase levels in patients with renal disease. An auxiliary study was conducted in eight dogs subjected to renal transplantation after bilateral nephrectomy. In four dogs treated with immunosuppressants, urinary activities of P-galactosidase and N-acetyl-P-glucosaminidase remained normal as long as treatment was effective in preventing re-
Figure
vduru131,Yay
1976
561
Smeesters, Ehrlich, and Fonkalsrud
136 - 32 12-
ENZYME ACTI VI TY fmU/mg CREATNVINEI
- 26 ki - 24 a 0 -20
z-
-
2
-16
g- I
_
-I
-12
:
-
6
-4
2
2
3000 -
1
2000-
levels of fl-galactosidase and N-acetyl-fl-glucosaminidase with conventional clinical and laboratory parameters in the detection of impending rejection. A rapid (60 minute), simple, accurate fluorometric assay was used to measure activities of both enzymes. Eighty per cent of ten rejection episodes were accompanied by a two- to sixfold increase in enzyme release. Parallel changes in serum creatinine levels and urinary volume occurred in six rejection episodes, but in two episodes, elevated urinary enzyme levels were observed two and four days prior to clinical evidence of rejection. It is concluded that urinary lysosomal enzyme measurements by fluorometric assay are valuable indicators of acute renal rejection, particularly when the diagnosis is not clearly established by conventional criteria that show only minimal changes. Continuing studies in a large group of renal transplant recipients are under way to evaluate the validity of this conclusion and to determine whether enzyme measurements will, indeed, be indicative of early rejection. Acknowledgment: We are grateful to Manuel Sanchez and Moon Lee for their excellent technical assistance.
E IOOO-
I 1
5
IO
15
20
25
30
35
References
DAYS
Figure 3. Results of urine volume (ml/24 hr), serum creatinine (mg/lOO mi), and urinary iysosomal enzyme activity (mU/mg of creatintne) after renal transplantation from a cadaveric donor in a patient with multiple rejection eptsodes. Treatment of rejectton episodes is indicated by high dose administration of methylpredntsolone. The third rejection episode, as evidenced by an increase in serum creattntne, was suspected four days earlier because of the increase in enzyme release.
In four untreated dogs, when rejection occurred, urinary activity of the two enzymes started to increase 24 hours prior to any significant change in serum creatinine levels or urinary output. Thereafter, enzymatic activity continued to increase and paralleled changes in conventional data until the death of the animals.
jection.
Summary
Six patients who received renal transplants were closely monitored to compare the sensitivity of urine
562
1. Leaback DH: Properties of some derivatives of 4-methylumbelliferone. Bioc/tem J 78: 22, 1961. 2. Leaback DH, Walker PG: Studies on glucosaminidase. 4. The fluorometric assay on N-acetyl-&glucosaminidase. Biochem J78: 151, 1961. 3. Dance N, Price RG, Cattell WR, et al: The excretion of N-acetyl-&galactosidase by patients with renal disease. C/in Chim Acta 27: 87, 1970. 4. Price RG, Dance N, Richards B, et al: The excretion of N-ace-
tyl-&lucosaminidase and fi-galactosidase following surgery to the kidney. C/in Chim Acta 27: 65, 1970. 5. Gonick HC, Kramer HJ, Schapiro AE: Urinary @-glucuronidase activity in renal disease. Arch intern Med 132: 63, 1973. 6. Wellwood JM. Ellis BG, Hall JH. et al: Early warning of rejection? BrMedJ2:
261, 1973.
7. Smeesters C, Daloze P: Dosage serique de tois. Med 3: 29, 1974.
ChirDig
8. Robinson D, Price RG. Dance N: Separation and properties of &galactosidase, P-glucosidase, P-glucuronidase and Nacetyl-fl-glucosaminidase from rat kidney. Biochem J 102: 525, 1967. 9. Van Hoof F: Les mucopolysaccharidoses en tant que thesaurismoses lysosomiales (Vauder, ed). Belgium, Louvain, 1972, p 145. 10. Kramer HJ, Gonick HD: Studies on human kidney and urine acid phosphatase. II. Measurements of urinary enzyme activity in renal disease. Enzyme 12: 257, 1971.
The American Journal of Surgery
Since the development of fluorometric assays for the measurement of acid hydrolases by Leaback [1] and Leaback and Walker [2] in 1961, there has been considerable interest in the quantitation of enzymes released from injured organs as a method of determining extent of tissue damage. Such measurements
have been helpful in evaluating renal injury after periods of ischemia [3,4]. Urinary levels of N-acetyl-P-glucosaminidase and P-galactosidase [3] and P-glucuronidase [5] were increased in patients with active renal disease, particularly when tubular injury was present. Urine assays for these enzymes have also been performed as a method of detecting episodes of rejection in patients who have received renal allografts [5,6]. To be helpful in identifying episodes of allograft rejection, a new test must be simple, able to be performed quickly, and sufficiently sensitive to detect rejection episodes before conventional clinical and laboratory parameters. The present study was undertaken to determine the practicality and reliability of identifying episodes of renal allograft rejection by fluorometric assay of the urinary activities of two lysosomal enzymes, /3galactosidase and N-acetyl-P-glucosaminidase, in undialyzed urine specimens. These enzymes were selected on the basis of their higher sensitivity as an index of cellular injury, as demonstrated in previous studies on liver preservation and transplantation [7] and on renal rejection [6]. Clinical Methods Release of renal tubular lysosomal enzymes into the urine was evaluated in six patients for one to two months From the Department of Surgery, UCLA School of Medicine, Los Angeles, California. This work was supported by USPHS Grant HL 14333. Reprint requests should be addressed to Eric W. Fonkalsrud. MD, Department of Surgery, UCLA School of Medicine, Los Angeles, California 90024. * Assisted by a Fellowship from the Medical Research Council of Canada.
560
after renal allotransplantation. Three patients had received renal grafts from living related donors (LD), and three patients had received cadaveric renal allografts (CD). Rejection episodes, as diagnosed by conventional parameters of low 24 hour urine volumes, increasing serum creatinine levels, and decreasing urinary sodium concentration, were treated with high-dose intravenous methylprednisolone. Daily measurements of the enzymes P-galactosidase and N-acetyl$-glucosaminidase were obtained from 24 hour urine samples refrigerated at 4’C until testing which was performed within 48 hours of collection. The enzyme levels after refrigeration were stable for three days for @-galactosidase and at least one week for N-acetyl-&glucosaminidase. Fluorometric methods with I-methylumbelliferyl substrates (Koch-Light Labs, Ltd, Buckinghamshire, England) were employed to determine the activity of both enzymes. Assays were performed according to the methods described by Leaback and Walker [2] for N-acetyl-fl-glucosaminidase and by Robinson, Price, and Dance [B] for fl-galactosidase, as modified by Van Hoof [9]. Incubation periods extended up to 30 minutes, and total time to accomplish the test was approximately 60 minutes. Activity w&s expressed in milliunits (mu)/100 ml of urine, 1 mU referring to the amount of enzyme that hydrolyzes 1 nanomole of substrate per minute under the conditions of the assay. The enzyme excretion in the urine was corrected for creatinine content to relate enzyme release to the amount of functioning tubular mass. Results and Comments Increased urine enzyme excretion occurred in the early postoperative period and presumably represented transient renal ischemic injury. Values returned to normal levels by the fourth to sixth day after grafting. In two patients who had an uncomplicated postoperative course, urine enzyme activity remained normal after the seventh day, correlating well with the stabilization of urine volume and serum creatinine level. (Figure 1.)
The American Journal of Surgery
Enzyme Measurements in Altograft Rejection
ENZYMEACTI VU-Y
ENZYME ACTMTY lmlJ/mg CREATINfNE/
CREATlNfNEI
fmWmg
720
24.1
SERUM CREAT/NlNE
3
-c 8 ;
2
t
T;
ID.
SERUM CREAThWVE -i
2
8
;
g
4000
r
URINE VOLU.E
2ooot 5
10
3000-
;
20001000
n
1
5
15
20
DAYS
-
1
5
10
15
I
I
20
25
1 30
35
DAYS
Figure 2. Results of urine vohsne, serum creatkke, and urinary lysosomel enzyme actMy after fvnai transplant8tiOftfram8CMtdW&CdOlUUik)8pdtkntw#tthno~
1. Resufts of urine volume, serum creatkke, and urinary &sosomal enzyme 8ctMty after renal transpkntatkn f&m a llvkg &nor ilkstrate a patient with an uncomplkated postoperative course.
episodes. Treatment of reJectkn episodes lo Indkat~ by high dose adminktratkn of methy@ednkokne. the last determkatkn was made one week after the patient was discharged and two days before she was readm&ted for treatment of severe acute rejectkn. Renal functkn was considered normal at that t/me.
The remaining four patients underwent ten clinical rejection episodes, most of which correlated well with corresponding peaks in urinary activity of both enzymes. (Figures 2 and 3.) Eight of the ten rejection episodes were characterized by simultaneous two- to sixfold increases in enzymatic activity. Although increased activity usually was not demonstrated before manifestation of rejection by clinical and other laboratory parameters, measurements of enzyme excretion were helpful in confirming conventional data compatible with rejection. This is in agreement with the observations of Gonick, Kramer, and Schapiro [5] but does not substantiate those of Wellwood et al [6] who found that urinary activity of N-acetyl-&glucosaminidase was increased up to three weeks before changes in other tests of renal function. However, increased release of acid hydrolases was evident two and four days before clinical confirmation of two rejection episodes. (Figures 2 and 3.) Those rejection episodes were characterized by only minimal changes in conventional criteria of re-
jection and suggest the value of enzymatic determinations in the early detection of rejection. Successful treatment of rejection episodes by high dose corticosteroid therapy was mirrored by a corresponding decrease in enzyme activity, which correlated well with normalization of serum creatinine levels and urine volumes. Although urine acid phosphatase levels were also measured, they were unreliable as an indicator of rejection, especially in adult males, because of the variable contribution of prostatic acid phosphatase to urinary enzymatic activity. This observation agrees with the observations of Kramer and Gonick [IO], who studied urinary acid phosphatase levels in patients with renal disease. An auxiliary study was conducted in eight dogs subjected to renal transplantation after bilateral nephrectomy. In four dogs treated with immunosuppressants, urinary activities of P-galactosidase and N-acetyl-P-glucosaminidase remained normal as long as treatment was effective in preventing re-
Figure
vduru131,Yay
1976
561
Smeesters, Ehrlich, and Fonkalsrud
136 - 32 12-
ENZYME ACTI VI TY fmU/mg CREATNVINEI
- 26 ki - 24 a 0 -20
z-
-
2
-16
g- I
_
-I
-12
:
-
6
-4
2
2
3000 -
1
2000-
levels of fl-galactosidase and N-acetyl-fl-glucosaminidase with conventional clinical and laboratory parameters in the detection of impending rejection. A rapid (60 minute), simple, accurate fluorometric assay was used to measure activities of both enzymes. Eighty per cent of ten rejection episodes were accompanied by a two- to sixfold increase in enzyme release. Parallel changes in serum creatinine levels and urinary volume occurred in six rejection episodes, but in two episodes, elevated urinary enzyme levels were observed two and four days prior to clinical evidence of rejection. It is concluded that urinary lysosomal enzyme measurements by fluorometric assay are valuable indicators of acute renal rejection, particularly when the diagnosis is not clearly established by conventional criteria that show only minimal changes. Continuing studies in a large group of renal transplant recipients are under way to evaluate the validity of this conclusion and to determine whether enzyme measurements will, indeed, be indicative of early rejection. Acknowledgment: We are grateful to Manuel Sanchez and Moon Lee for their excellent technical assistance.
E IOOO-
I 1
5
IO
15
20
25
30
35
References
DAYS
Figure 3. Results of urine volume (ml/24 hr), serum creatinine (mg/lOO mi), and urinary iysosomal enzyme activity (mU/mg of creatintne) after renal transplantation from a cadaveric donor in a patient with multiple rejection eptsodes. Treatment of rejectton episodes is indicated by high dose administration of methylpredntsolone. The third rejection episode, as evidenced by an increase in serum creattntne, was suspected four days earlier because of the increase in enzyme release.
In four untreated dogs, when rejection occurred, urinary activity of the two enzymes started to increase 24 hours prior to any significant change in serum creatinine levels or urinary output. Thereafter, enzymatic activity continued to increase and paralleled changes in conventional data until the death of the animals.
jection.
Summary
Six patients who received renal transplants were closely monitored to compare the sensitivity of urine
562
1. Leaback DH: Properties of some derivatives of 4-methylumbelliferone. Bioc/tem J 78: 22, 1961. 2. Leaback DH, Walker PG: Studies on glucosaminidase. 4. The fluorometric assay on N-acetyl-&glucosaminidase. Biochem J78: 151, 1961. 3. Dance N, Price RG, Cattell WR, et al: The excretion of N-acetyl-&galactosidase by patients with renal disease. C/in Chim Acta 27: 87, 1970. 4. Price RG, Dance N, Richards B, et al: The excretion of N-ace-
tyl-&lucosaminidase and fi-galactosidase following surgery to the kidney. C/in Chim Acta 27: 65, 1970. 5. Gonick HC, Kramer HJ, Schapiro AE: Urinary @-glucuronidase activity in renal disease. Arch intern Med 132: 63, 1973. 6. Wellwood JM. Ellis BG, Hall JH. et al: Early warning of rejection? BrMedJ2:
261, 1973.
7. Smeesters C, Daloze P: Dosage serique de tois. Med 3: 29, 1974.
ChirDig
8. Robinson D, Price RG. Dance N: Separation and properties of &galactosidase, P-glucosidase, P-glucuronidase and Nacetyl-fl-glucosaminidase from rat kidney. Biochem J 102: 525, 1967. 9. Van Hoof F: Les mucopolysaccharidoses en tant que thesaurismoses lysosomiales (Vauder, ed). Belgium, Louvain, 1972, p 145. 10. Kramer HJ, Gonick HD: Studies on human kidney and urine acid phosphatase. II. Measurements of urinary enzyme activity in renal disease. Enzyme 12: 257, 1971.
The American Journal of Surgery