111
Clinica Chimica Acta, 91 ( 1 9 7 9 ) 1 1 1 - - 1 1 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
CCA 9919
URINARY LYSOSOMAL ENZYME EXCRETION AFTER RENAL ALLOTRANSPLANTATION
ROLAND
F. D Y C K
*, C A R L J. C A R D E L L A
and M A R I K A
A. S A C K S
Division o f Nephrology, Toron to Western Hospital and The Gage Research Itzslil u re, University o f Toronto, Toronto (Canada) (Received July 22nd, 1 9 7 8 )
Summary Three urinary lysosomal enzymes, ~-glucuronidase (~-Gluc), fl-galactosidase (~-Gal) and N-acetyl-fl-D-glucosaminidase (NAG), were measured in twenty-one renal allograft recipients to evaluate their role in the diagnosis and prediction of rejection episodes, and in the prediction of eventual graft outcome. A fluorometric assay using methylumbelliferone substrates was used to measure the three enzymes in morning urine samples and enzyme activity was defined in terms of urine creatinine concentration. Urinary NAG levels increased significantly in 13/16 first rejection episodes and 4/4 instances of acute tubular necrosis and graft infarction. In 5 of the 16 first rejection episodes the NAG was predictive of the rejection. NAG was not useful ill diagnosing second or subsequent rejections and/3-Gluc and ~-Gal were of little value in assessing any component of renal transplant pathology. As a prognostic index of eventual graft outcome, the peak urinary NAG was particularly encouraging. It correlated strongly with deterioration in graft function as time passed such that only 2/10 patients with peak NAG >1400 Units had normal serum creatinines at 6 months post transplantation. Conversely 4/4 patients with peak NAG levels < 7 0 0 Units had normal serum creatinine at that time. In our series the measurement of ,Lrinary NAG was a useful adjunct to the diagnosis of first rejections but appears to be more valuable in predicting graft outcome.
Introduction Urinary enzymes have been used with variable success to diagnose and predict renal allograft rejection [1--8]. Diagnostic limitations have arisen * Correspondence should b e addressed to: Dr. R.F. Dyck, The Gage Research Institute, Division of Nephrology, 223 College Street, Toronto M5T 1R4, Canada.
because enzyme elevations usually occur concurrently with increases in the re, an initial claim that enzymes could rise ection diagnosis [2] has not been substantiated 5,7 ] This study was undertaken in order to clarify the value of urinary lysosr::.~al enzymes in the diagnosis and prediction of acute renal allograft rejection episodes; in addition we explored a new function for this test, the prediction of eventual graft outcome. Patients and methods
Twenty-one renal transplant recipients were studied at the Toronto Western Hospital between January 1, 1977 and November 30, 1977. Morning urine specimens were collected from each patient from the day that urine production started post transplantation until the time of hospital discharge. Urine was kept at 4°C until centrifuged at 3000 r.p.m, for 20 rain. The supernatant was then stored at--20°C until tested Each specimen underwent the following analysis: 1. Lysosomal enzymes Three urinary lysosomal enzymes were measured in each sample. These were ~-glucuronidase (fl-Gluc), ~-galactosidase (fl-Gal), and N-acetyl-~-D-glucosaminidase (NAG}. They were measured fluorometrica~ly using appropriate 4-methylumbelliferone (MU) substrates according to the method described by Sandman et al. [ 1]. This technique was modified by using 0.5 ml of the buffer-substrate solutions instead of 0.4 ml and by adding 2.5 ml of stopping buffer instead of 2.0 ml. Pre-incubation steps as well as termination of hydrolysis were carried out in an ice water bath. 2. Creatinine Urine creatinine concentration was measured using the Jaff~ reaction as described by Husdan and Rapoport [9]. Instead of filtering precipitated protein, however, samples were centrifuged at 5500 r.p.m, for 20 rain before removing the supernatant for further analysis. 3. Enzyme activity One unit of urinary lysosomal enzyme activity [ 10] is defined as 1 nmole of MU released per hour of incubation per milligram of urinary creatinine. All renal allograft recipients received the same immunosuppressive regimen consisting of azathioprine and corticosteroids. A rejection episode was defined as a significant rise of the serum creatinine above baseline values, and most rejection episodes were confirmed by renal biopsy. The diagnosis and treatment of each rejection episode was undertaken without knowledge of urinary enzyme values. Acute tubular necrosis (ATN) and graft infarction were diagnosed clinically and the diagnoses were confirmed by renal biopsy, renal scans and renal arteriograms.
113
The first part of this study correlated elevations in uri,mry lysosomal enzyme levels with significant increases in the serum creatinine. For each patient a baseline excretion of urinary lysosomal enzymes was established. Subsequent elevations above this established baseline were calculated and these increases were then grouped into two categories: those occurring when the serum creatinine was stable and those occurring in relationship to an event causing tissue damage such as rejection, ATN or graft infarction. The mean increase in each category was then calculated and the total population data was analyzed using a Student's t-test. In the second part of the study peak urinary lysosomal enzyme levels in the immediate post-transplantation period were correlated with graft function at 1, 2, 3 and 6 months post transplantaticJn.
Results Sixteen of the 21 transplant recipients had rejection episodes and, in addition, ATN occurred in two patients as did graft infarction. Both ATN and infarction each occurred in 1 patient with a rejection and 1 patient without a rejection. Only three recipients had an uneventful post-operative course. Of the three urinary enzymes measured, only NAG showed consistent corTABLE I MEAN ELEVATION Patient No.
OF URINE NAG FROM BASELINE Graft pathology
Mean elevation of NAG(U) With pathology
1 2 3 4 5 6 7 8 8 9 10 11 12 13 13 14 15 16 17 18 19 20 21
None Graft infarction Rejection Rejection Rejection Rejection Rejection Rejection Infarction None None Rejection Acute tubular necrosis Acute tubular necrosis Rejection Rejection ~ Rejection Rejection Rejection Rejection Rejection Rejection Rejection Mean S.E.
382 582 399 437 853 904 205 421 162 574 985 557 577 * 63
|
* P '~ 0 . 0 0 1 .
1069 528 1004 588 350 749 112 676
N o pathology 236 113 206 125 159 249 89 89 78 113 162
186 97 170 108 42 139 15
relations :with :alteration in transplant function, fl-Gal and /3-Glue frequently remained unClmnged, increased marginally or actually decreased during rejection episodes. Table I lists the mean elevation of urine NAG from baseline in each instance
p l ~ t d ~ a g e was I39 U (S!E. 15). The difference between these values was statistically si~ificant with a p < 0.001. Elevations in urinary NAG did not differentiate rejection episodes from ATN or infarction although values of urinary NAG > 250 U were always associated with transplant pathology. This arbitrarily chosen level resulted in 3/20 (15%} false negative diagnoses. In 5 of the 16 first rejection episodes the urinary NAG was predictive of the rejection. Since urinary NAG levels tended to stay elevated after a first rejection, NAG was of little value in diagnosing or predicting second and subsequent rejection episodes. Table II lists the peak urinary NAG values in each patient in the immediate post surgical period along with the serum creatinines at 1, 2, 3 and 6 months post transplantation. The correlation between peak NAG and deteriorating graft function was increasingly significant as time passed with a final "r" value of 0.6685 at 6 months (p < 0.001). All patients in Group 1 (peak NAG < T A B L E 1I Patient No.
Peak NAG (U)
Comments
Serum creatinines (mg/dl)
1 Month
2 Month
3 ~'lonth
6 Months
645 674 590 580
1.38 1.23 0.81 1.18
1.10 1.46 0.80 1.20
1.28 1.25 0.85 1.03
1.03 1.19 0.85 1.20
1243 736 1026 862 950 1103 1079
2.04 3.50 1.50 1.60 3.00 2.08 2.00
1.61 3.07 2.55 1.32 1.32 1.92 2.11
1.57 • 2.01 2.30 1.30 1.50 2.23 2.49
1.50 1.93 4.00 1.22 2.79 1.95 2.10
1768 1574 1812 1422 1406 1691 2362 1529 1463 1906
10+ 1.10 1.00 2.65 10+ 2.40 8.70 6,20 3.40 1.01
Group 1
1 3 9 10 Group 2
7 11 12 13 16 18 21 , Group 3
2 4 5 6 8 14 15 17 19 20
. . 1.03 1.08 5.80 -3.46 10+ 3.80 2.80 1.00
.
. 1.37 -10+ -4.29 -5.28 2.53 1.20
10 days, graft loss
1.35 ---7.86 -4.70 2.65 1.15
Peak NAG VS.
graft function
r = 0.,187
(p < 0 . 0 5 )
r = 0.5410 (p < 0 . 0 5 )
r = 0.6567 (p < 0 . 0 5 )
r = 0.6685 (p < 0 . 0 0 1 )
died 2 months 4 months, graft loss 24 days, graft loss
6 weeks, graft loss
115
700 U) had nermal serum creatinines up to 6 months post transplantation, while only 1/7 patients in Group 2 (peak NAG = 700--1400 U) and 2/10 patients in Group 3 (peak NAG > 1400 U) had normal serum creatinines at that time. Conversely, 6/7 patients in Group 2 had mild to moderate graft functional impairment at 6 months, while in Group 3, 4/10 patients had lost their grafts by this time, and 3/10 had moderate to severe impairment of their renal function. An additional patient in Group 3 had died of infectious complications resulting from prolonged rejection therapy.
Discussion The first part of this study confirmed previous investigators' findings by demonstrating that elevation of urinary NAG is not specific for transplant rejection episodes [1,4] and that its predictive value in such events is inconstant [5--7] (5 of 16 first rejections). Moreover, there were 3/16 false negatives in our series due to significant overlap between elevations of NAG associated with transplant pathology and those elevations occurring for no apparent reason. In our series, therefore, urinary NAG was useful in the early diagnosis of some first rejections but was not useful either in the mlalysis of subsequent rejection episodes or in distinguishing the type of graft pathology. ~-Gal and /~-Gluc were neither diagnostic nor predictive of renal allograft rejections at any time. The second part of this study provides evidence that the measurement of urinary NAG may be useful in the promising new role of predicting renal allograft outcome. The excretion of urinary NAG is directly proportional to the amount of renal tissue damaged [11] and may reflect the graft's ability to recover. This is in contrast to the creatinine clearance which is a functional measurement and is influenced by many factors apart from renal tissue damage. To our knowledge urinary lysosomal enzymes have not previously been used to predict eventual graft outcome although recently the serum lactate dehydrogenase has been used for this purpose [ 12]. The significant correlation between peak urinary NAG and eventual graft outcome indicates that' NAG could be a ~aluable tool in early post surgical graft assessment. Rejection remains the leading cause of g~'aft loss in the first year post transplantation and since rejection therapy is associated with significant morbidity and mortality, prognostic indices of eventual graft outcome could be valuable in tailoring rejection therapy to the individual.
Acknowledgements This work was supported in part by the Medical Research Council of Canada and the Kidney Foundation of Canada. We wish to thank Mary Harding for her assistance in obtaining statistical data for this study.
References 1 Sandman, R., Margules, K.M. and Kountz, S.L. (1973) Clin. Chim. Acta 45, 349 2 WeUwood, J.M., Ellis. B.G., Hall, J.H., Robinson, D.R. and Thompson, A.E. (1973) Br. Med. J. 2 , 2 6 1
A.E. (1973) Arch. Intern. Med. 132, 63 5 Keyser, J,W., Watkins, G,L. and Salaman, J.R. (1976) Clin. Chem. 2 2 , 9 2 5 6 Smeesters, C., Ehrlich, R.M. a n d Fonkalsrud, E.W. (1976) Am. J. Surg. 1 3 1 , 5 6 0 ~bel, M. (1977) Clin. Chem. 2 3 . 7 7 0 14, 222 ~pson, A.E. (1976) Clin. Chim. Acta 69, 85 and Richards, B. (1970) Clin. Chim. Acta 27, 87 12 Roses, J,, Woods, J,E. and Zincke, If. (1977) Am. J. Surg. 1 3 3 , 7 2 6
Clinica Chimica Acta, 91 ( 1 9 7 9 ) 1 1 1 - - 1 1 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
CCA 9919
URINARY LYSOSOMAL ENZYME EXCRETION AFTER RENAL ALLOTRANSPLANTATION
ROLAND
F. D Y C K
*, C A R L J. C A R D E L L A
and M A R I K A
A. S A C K S
Division o f Nephrology, Toron to Western Hospital and The Gage Research Itzslil u re, University o f Toronto, Toronto (Canada) (Received July 22nd, 1 9 7 8 )
Summary Three urinary lysosomal enzymes, ~-glucuronidase (~-Gluc), fl-galactosidase (~-Gal) and N-acetyl-fl-D-glucosaminidase (NAG), were measured in twenty-one renal allograft recipients to evaluate their role in the diagnosis and prediction of rejection episodes, and in the prediction of eventual graft outcome. A fluorometric assay using methylumbelliferone substrates was used to measure the three enzymes in morning urine samples and enzyme activity was defined in terms of urine creatinine concentration. Urinary NAG levels increased significantly in 13/16 first rejection episodes and 4/4 instances of acute tubular necrosis and graft infarction. In 5 of the 16 first rejection episodes the NAG was predictive of the rejection. NAG was not useful ill diagnosing second or subsequent rejections and/3-Gluc and ~-Gal were of little value in assessing any component of renal transplant pathology. As a prognostic index of eventual graft outcome, the peak urinary NAG was particularly encouraging. It correlated strongly with deterioration in graft function as time passed such that only 2/10 patients with peak NAG >1400 Units had normal serum creatinines at 6 months post transplantation. Conversely 4/4 patients with peak NAG levels < 7 0 0 Units had normal serum creatinine at that time. In our series the measurement of ,Lrinary NAG was a useful adjunct to the diagnosis of first rejections but appears to be more valuable in predicting graft outcome.
Introduction Urinary enzymes have been used with variable success to diagnose and predict renal allograft rejection [1--8]. Diagnostic limitations have arisen * Correspondence should b e addressed to: Dr. R.F. Dyck, The Gage Research Institute, Division of Nephrology, 223 College Street, Toronto M5T 1R4, Canada.
because enzyme elevations usually occur concurrently with increases in the re, an initial claim that enzymes could rise ection diagnosis [2] has not been substantiated 5,7 ] This study was undertaken in order to clarify the value of urinary lysosr::.~al enzymes in the diagnosis and prediction of acute renal allograft rejection episodes; in addition we explored a new function for this test, the prediction of eventual graft outcome. Patients and methods
Twenty-one renal transplant recipients were studied at the Toronto Western Hospital between January 1, 1977 and November 30, 1977. Morning urine specimens were collected from each patient from the day that urine production started post transplantation until the time of hospital discharge. Urine was kept at 4°C until centrifuged at 3000 r.p.m, for 20 rain. The supernatant was then stored at--20°C until tested Each specimen underwent the following analysis: 1. Lysosomal enzymes Three urinary lysosomal enzymes were measured in each sample. These were ~-glucuronidase (fl-Gluc), ~-galactosidase (fl-Gal), and N-acetyl-~-D-glucosaminidase (NAG}. They were measured fluorometrica~ly using appropriate 4-methylumbelliferone (MU) substrates according to the method described by Sandman et al. [ 1]. This technique was modified by using 0.5 ml of the buffer-substrate solutions instead of 0.4 ml and by adding 2.5 ml of stopping buffer instead of 2.0 ml. Pre-incubation steps as well as termination of hydrolysis were carried out in an ice water bath. 2. Creatinine Urine creatinine concentration was measured using the Jaff~ reaction as described by Husdan and Rapoport [9]. Instead of filtering precipitated protein, however, samples were centrifuged at 5500 r.p.m, for 20 rain before removing the supernatant for further analysis. 3. Enzyme activity One unit of urinary lysosomal enzyme activity [ 10] is defined as 1 nmole of MU released per hour of incubation per milligram of urinary creatinine. All renal allograft recipients received the same immunosuppressive regimen consisting of azathioprine and corticosteroids. A rejection episode was defined as a significant rise of the serum creatinine above baseline values, and most rejection episodes were confirmed by renal biopsy. The diagnosis and treatment of each rejection episode was undertaken without knowledge of urinary enzyme values. Acute tubular necrosis (ATN) and graft infarction were diagnosed clinically and the diagnoses were confirmed by renal biopsy, renal scans and renal arteriograms.
113
The first part of this study correlated elevations in uri,mry lysosomal enzyme levels with significant increases in the serum creatinine. For each patient a baseline excretion of urinary lysosomal enzymes was established. Subsequent elevations above this established baseline were calculated and these increases were then grouped into two categories: those occurring when the serum creatinine was stable and those occurring in relationship to an event causing tissue damage such as rejection, ATN or graft infarction. The mean increase in each category was then calculated and the total population data was analyzed using a Student's t-test. In the second part of the study peak urinary lysosomal enzyme levels in the immediate post-transplantation period were correlated with graft function at 1, 2, 3 and 6 months post transplantaticJn.
Results Sixteen of the 21 transplant recipients had rejection episodes and, in addition, ATN occurred in two patients as did graft infarction. Both ATN and infarction each occurred in 1 patient with a rejection and 1 patient without a rejection. Only three recipients had an uneventful post-operative course. Of the three urinary enzymes measured, only NAG showed consistent corTABLE I MEAN ELEVATION Patient No.
OF URINE NAG FROM BASELINE Graft pathology
Mean elevation of NAG(U) With pathology
1 2 3 4 5 6 7 8 8 9 10 11 12 13 13 14 15 16 17 18 19 20 21
None Graft infarction Rejection Rejection Rejection Rejection Rejection Rejection Infarction None None Rejection Acute tubular necrosis Acute tubular necrosis Rejection Rejection ~ Rejection Rejection Rejection Rejection Rejection Rejection Rejection Mean S.E.
382 582 399 437 853 904 205 421 162 574 985 557 577 * 63
|
* P '~ 0 . 0 0 1 .
1069 528 1004 588 350 749 112 676
N o pathology 236 113 206 125 159 249 89 89 78 113 162
186 97 170 108 42 139 15
relations :with :alteration in transplant function, fl-Gal and /3-Glue frequently remained unClmnged, increased marginally or actually decreased during rejection episodes. Table I lists the mean elevation of urine NAG from baseline in each instance
p l ~ t d ~ a g e was I39 U (S!E. 15). The difference between these values was statistically si~ificant with a p < 0.001. Elevations in urinary NAG did not differentiate rejection episodes from ATN or infarction although values of urinary NAG > 250 U were always associated with transplant pathology. This arbitrarily chosen level resulted in 3/20 (15%} false negative diagnoses. In 5 of the 16 first rejection episodes the urinary NAG was predictive of the rejection. Since urinary NAG levels tended to stay elevated after a first rejection, NAG was of little value in diagnosing or predicting second and subsequent rejection episodes. Table II lists the peak urinary NAG values in each patient in the immediate post surgical period along with the serum creatinines at 1, 2, 3 and 6 months post transplantation. The correlation between peak NAG and deteriorating graft function was increasingly significant as time passed with a final "r" value of 0.6685 at 6 months (p < 0.001). All patients in Group 1 (peak NAG < T A B L E 1I Patient No.
Peak NAG (U)
Comments
Serum creatinines (mg/dl)
1 Month
2 Month
3 ~'lonth
6 Months
645 674 590 580
1.38 1.23 0.81 1.18
1.10 1.46 0.80 1.20
1.28 1.25 0.85 1.03
1.03 1.19 0.85 1.20
1243 736 1026 862 950 1103 1079
2.04 3.50 1.50 1.60 3.00 2.08 2.00
1.61 3.07 2.55 1.32 1.32 1.92 2.11
1.57 • 2.01 2.30 1.30 1.50 2.23 2.49
1.50 1.93 4.00 1.22 2.79 1.95 2.10
1768 1574 1812 1422 1406 1691 2362 1529 1463 1906
10+ 1.10 1.00 2.65 10+ 2.40 8.70 6,20 3.40 1.01
Group 1
1 3 9 10 Group 2
7 11 12 13 16 18 21 , Group 3
2 4 5 6 8 14 15 17 19 20
. . 1.03 1.08 5.80 -3.46 10+ 3.80 2.80 1.00
.
. 1.37 -10+ -4.29 -5.28 2.53 1.20
10 days, graft loss
1.35 ---7.86 -4.70 2.65 1.15
Peak NAG VS.
graft function
r = 0.,187
(p < 0 . 0 5 )
r = 0.5410 (p < 0 . 0 5 )
r = 0.6567 (p < 0 . 0 5 )
r = 0.6685 (p < 0 . 0 0 1 )
died 2 months 4 months, graft loss 24 days, graft loss
6 weeks, graft loss
115
700 U) had nermal serum creatinines up to 6 months post transplantation, while only 1/7 patients in Group 2 (peak NAG = 700--1400 U) and 2/10 patients in Group 3 (peak NAG > 1400 U) had normal serum creatinines at that time. Conversely, 6/7 patients in Group 2 had mild to moderate graft functional impairment at 6 months, while in Group 3, 4/10 patients had lost their grafts by this time, and 3/10 had moderate to severe impairment of their renal function. An additional patient in Group 3 had died of infectious complications resulting from prolonged rejection therapy.
Discussion The first part of this study confirmed previous investigators' findings by demonstrating that elevation of urinary NAG is not specific for transplant rejection episodes [1,4] and that its predictive value in such events is inconstant [5--7] (5 of 16 first rejections). Moreover, there were 3/16 false negatives in our series due to significant overlap between elevations of NAG associated with transplant pathology and those elevations occurring for no apparent reason. In our series, therefore, urinary NAG was useful in the early diagnosis of some first rejections but was not useful either in the mlalysis of subsequent rejection episodes or in distinguishing the type of graft pathology. ~-Gal and /~-Gluc were neither diagnostic nor predictive of renal allograft rejections at any time. The second part of this study provides evidence that the measurement of urinary NAG may be useful in the promising new role of predicting renal allograft outcome. The excretion of urinary NAG is directly proportional to the amount of renal tissue damaged [11] and may reflect the graft's ability to recover. This is in contrast to the creatinine clearance which is a functional measurement and is influenced by many factors apart from renal tissue damage. To our knowledge urinary lysosomal enzymes have not previously been used to predict eventual graft outcome although recently the serum lactate dehydrogenase has been used for this purpose [ 12]. The significant correlation between peak urinary NAG and eventual graft outcome indicates that' NAG could be a ~aluable tool in early post surgical graft assessment. Rejection remains the leading cause of g~'aft loss in the first year post transplantation and since rejection therapy is associated with significant morbidity and mortality, prognostic indices of eventual graft outcome could be valuable in tailoring rejection therapy to the individual.
Acknowledgements This work was supported in part by the Medical Research Council of Canada and the Kidney Foundation of Canada. We wish to thank Mary Harding for her assistance in obtaining statistical data for this study.
References 1 Sandman, R., Margules, K.M. and Kountz, S.L. (1973) Clin. Chim. Acta 45, 349 2 WeUwood, J.M., Ellis. B.G., Hall, J.H., Robinson, D.R. and Thompson, A.E. (1973) Br. Med. J. 2 , 2 6 1
A.E. (1973) Arch. Intern. Med. 132, 63 5 Keyser, J,W., Watkins, G,L. and Salaman, J.R. (1976) Clin. Chem. 2 2 , 9 2 5 6 Smeesters, C., Ehrlich, R.M. a n d Fonkalsrud, E.W. (1976) Am. J. Surg. 1 3 1 , 5 6 0 ~bel, M. (1977) Clin. Chem. 2 3 . 7 7 0 14, 222 ~pson, A.E. (1976) Clin. Chim. Acta 69, 85 and Richards, B. (1970) Clin. Chim. Acta 27, 87 12 Roses, J,, Woods, J,E. and Zincke, If. (1977) Am. J. Surg. 1 3 3 , 7 2 6
