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Use of urinary enzymes as markers of nephrotoxicity
Toxicology Letters,
193
46 (1988) 193-196
TXL 02130
Use of urinary enzymes as markers of nephrotoxicity U.C. Dubach, M. Le Hir and R. Gandhi Medizinische
Universitiits-Poliklinik, Enzym-Biologie-Labor,
fiir Innere Medizin, Kantonsspital,
Departement Forschung und Departement
CH-4031 Base1 (Switzerland)
SUMMARY Urinary laboratory
excretion
of enzymes
equipment;
conditions,
the time-course
be followed
by measuring
a large number the kidney injury quence
by urinary
application analysis
of the nephrotoxicity
enzymes.
even under of urinary
may be possible
continuously
for the animal
and dose-dependence urinary
of parameters,
renders
can be monitored
there is little discomfort
investigated.
of some pathophysiological
The activities physiological
enzymes
at low cost, with commonly or person
of enzymes conditions.
for diagnostic
Furthermore,
only if the pathophysiological
controlled
events in the kidney
in urine are, however,
purposes
available
Under
difficult.
affected
the heterogeneity Assessment
mechanisms
can by of
of renal
and the time se-
are known.
INTRODUCTION
The symposium on ‘Enzymes in Urine and Kidney’ [l] included papers on increased urinary enzyme activity in patients with kidney disease [2]. Approximately 100 urinary enzymes have since been introduced for the diagnosis of renal damage. The enzymes that have been used most often are N-acetyl-/3-D-glucosaminidase (NAG), y-glutamyltransferase, lysozyme and alkaline phosphatase (Table I). La Due and Wroblewski [3] considered that serum enzymes would provide a ‘biochemical biopsy’ of organs affected by disease. While the routine use of certain serum enzymes has been useful in the diagnosis of diseases such as myocardial infarction, pancreatitis and chronic liver disease, this has not been the case for the kidney. The reasons for this failure are due largely to the fact that measurement of a single enzyme may give misleading information about the site, type and extent of injury. Enzymuria is associated with the acute and not the chronic effects of toxins, because the urine represents an open system.
194 HETEROGENEITY
OF THE KIDNEY
The metabolic organization of the nephron has been assessed [I] and shown to be highly heterogeneous morphologicalIy. About 12 segments can be distinguished according to their enzymatic activities [3,4], and this has been used as the rational basis for the use of urinary enzymes as a diagnostic tool for assessing injury (Tables I and II). Many of the factors that regulate enzyme excretion in healthy persons are known. The epithelium of the nephron sheds cells continuously; it is under hormone influence: excretion of lysosomal hydrolases into the urine from the proximal tubufe is a physiological phenomenon which can be stimulated by hormones without induction of cell necrosis [.5]. Furthermore, simple variables, such as urine concentration, age and sex, may influence the results [6]. The wide variation in normal enzyme activity and a number of methodological probIems have resulted in a reIuctance to accept normal urinary enzyme values, and therefore in a reduced use of these determinations as a clinical tool. TABLE I SELECTED URINARY ENZYMES USED TO DIAGNOSE RENAL DISEASES Site of injury ~__--._____ Glomerular
~-
Disease/Toxin
Enzymes .______ ~_____.~. NAG, j%galactosidase NAG
.__-
Aminonucleoside Glomerulonephritis
Proximal tubular
Mercuric chloride Chromate [VI] Cadmium
Ligandin Aianine aminopeptidase Alkaline phosphatase, r-glutamyltransferase, NAG, glucosidase
Distal tubular
Folate
Lactic dehydrogenase
Papillary
Ethylene imine _
Alkaline phosphatase, NAG, glucosidase -_
TABLE II ORIGINS OF URINARY ENZYMES ____________ ~__._~___~ -_.__ Enzymes Origin _.__ -Brush border Alkaline phosphatase, aminopeptidases
______~. _ _.__________~~
Lysosomes
iv-Acetyl-@-D-glucosaminidase,
p-galactosidase
Cytosol
/%Glucosidase, ligandin, lactic dehydrogenase
Proximal tubule
Ligandin, alkaline phosphatase,
Distal tubule
Lactic dehydrogenase (also present in other parts of the nephron)
aminopeptidases
___
19s
Injury to the nephron has been associated with an increase in the presence of enzymes in the urine (Table I), but this may reflect neither functional nor morphological changes. Thus, despite its location in the proximal tubule, NAG may increase as a result of both proximal tubular and glomerular injuries; levels of alkaline phosphatase are elevated after injuries to all parts of the nephron. The diagnostic use of urinary enzymes as opposed to functional and histological data on kidney damage is of limited value. There are many parameters that are likely to modify the activities of enzymes in urine, including inhibitors, activators, pH value and osmolality. These vary widely according to the physiological status of the subject, and it is still not clear whether reproducible diagnostic results can be obtained in either healthy persons or patients [7]. In addition, diagnostic procedures and drugs may affect urinary enzyme determinations in patients with kidney disease. USE OF URINARY
ENZYMES
FOR MONITORING
NEPHROTOXICITY
A rise in serum creatinine or a drop in creatinine clearance after administration of antibiotics, analgesics and other substances signals the presence of extensive damage. More sensitive methods, such as elevated levels of urinary enzymes, are used, but these may reflect only an innocuous functional change, reversible damage or cell necrosis. This central problem is very difficult to resolve [8,9]. The proposal to establish a battery of tests of urinary enzymes in order to define renal damage has not been materialized, since in both experimental animals and in humans all conditions must be carefully standardized. Factors such as sex, age, volume depletion and diuresis influence the amount of damage and the time of appearance of enzymes in urine. Therefore, although urinary enzymes may constitute a sensitive indicator of parenchymal damage, their diagnostic reliability remains questionable. However, a number of situations in which urinary enzymes have been used are described below. Hypertonic solutions Burchardt and Peters [lo] examined the influence of intravenous administration of hypertonic solutions (10% mannitol, 10% dextran or the X-ray contrast medium ‘Visostrat 370’) to patients with pre-existing nephropathy. They observed a reproducible increase in the activity of urinary alanine aminopeptidase and suggested that it was due to osmotic nephropathy. They did not find such changes in persons without renal disease. Antibiotics There are dramatic changes in the excretion of several urinary enzymes after the administration of gentamicin [l 11. The potential nephrotoxicity of many other antimicrobial agents, mainly aminoglycosides and cephalosporins, represents an important clinical problem [ 121.
196
Occupational exposure to nephrotoxic chemicals Meyer et al. [13] reported on the renal effects of exposure to lead, mercury and organic solvents. None of the persons in their study had a history of clinically evident renal disease or hypertension, but in comparison with an appropriate control population, workers exposed to lead, mercury and two of three groups of workers exposed to organic solvents had significant increases in urinary NAG activity. Laboratory workers exposed to low levels of organic solvents showed no increase in urinary NAG activity. The authors concluded that exposure to environmental nephrotoxic agents (at levels currently considered safe) can produce renal effects as manifested by elevations of urinary NAG excretion. However, the question of the relevance of these changes in groups with well-defined exposure remains uncertain, especially with respect to their health significance. ACKNOWLEDGEMENTS
Supported
by Schweiz Nationalfonds,
Bern.
REFERENCES 1 Dubach, 2
U.C. (Ed.) (1968) Enzymes
2). Hans
Huber,
Rosalki,
S.B. and Wilkinson,
in Urine and Kidney (Current
Problems
in Clinical
Biochemistry
Bern. J.H.
(1959) Urinary
lactic dehydrogenase
in renal disease.
Lancet
ii,
327. 3
Guder,
W.G. and Heidland,
man Society
for Clinical
1985. J. Clin. Chem. 4
6 7
Biochem.
Ross, B.D. and Ciuder, W.G. (Ed.),
5
A. (1986) Urine analysis:
Chemistry
Metabolic
Koenig,
A., Goldstone, A manifestation
on the workshop
conference
in Wiirzburg,
(1981) Heterogeneity Academic
A. and Hughes,
and compartmentation Press,
C. (1978) Lysosomal
of cell defecation
in the kidney.
of residual
enzymuria
bodies.
Lab.
39, 339.
Dubach,
and Schmidt,
U.C.
8
Price,
R.G.
9
Berlyne,
U. (Eds.) (1979) Diagnostic
in Clinical
(1982) Urinary
G.M.
of the kidney. Effects
Problems
enzymes,
Biochemistry
In: M.A.
of Petroleum
Mehlmann,
Hydrocarbons,
G.P.
Significance
9). Hans
nephrotoxicity
(1984) Toxic nephropathies
Huber,
Hemstreet,
methods J.J.
Vol. 7. Princeton
and N-acetyl-P-D-
of Enzymes
and Proteins
Toxicology
23, 99-134.
for early detection
Thorpe Scientific
in
Bern.
and renal disease.
and current
In: H. Sies
in the testosterone-treated
Invest.
(1986) The effects of age and urine concentration on lysozyme (NAG) content in urine. Ann. Clin. Biochem. 23, 297.
(Current
25th-26th,
New York.
Houser, M.T. glucosaminidase Urine
of the Ger-
October
24, 611-620.
Compartmentation.
mouse.
report
and the Society of Nephrology
and N.K. Weaver Publishers,
of the toxicity (Eds.),
Princeton,
Renal
NJ, pp.
173-184. 10
Burchardt,
U. and Peters,
J.E. (1975) Enzyme
im Harn
nach Applikation
hypertoner
Losungen.
Z.
Ges. Inn. Med. 30, 248-251. 11
Hautmann,
R.,
Kisters,
glutamyltranspeptidase. 12 Bianchi, C., Bertelli, Karger,
R. and
Lutzeyer,
Urology 7, 12-16. A. and Duarte, C.G.
(Eds.)
(1976)
Diagnosis
of renal
(1984) Contributions
tumor
to Nephrology,
by gammaVol. 42.
Basel.
Rosenman, 13 Meyer, B.R., Fischbein, A.L.F., (1984) Increased urinary enzyme excretion Med.
W.
76. 989-999.
K., Lerman, Y., Drayer, D.E. and Reidenberg, M.M. in workers exposed to nephrotoxic chemicals. Am. J.
193
46 (1988) 193-196
TXL 02130
Use of urinary enzymes as markers of nephrotoxicity U.C. Dubach, M. Le Hir and R. Gandhi Medizinische
Universitiits-Poliklinik, Enzym-Biologie-Labor,
fiir Innere Medizin, Kantonsspital,
Departement Forschung und Departement
CH-4031 Base1 (Switzerland)
SUMMARY Urinary laboratory
excretion
of enzymes
equipment;
conditions,
the time-course
be followed
by measuring
a large number the kidney injury quence
by urinary
application analysis
of the nephrotoxicity
enzymes.
even under of urinary
may be possible
continuously
for the animal
and dose-dependence urinary
of parameters,
renders
can be monitored
there is little discomfort
investigated.
of some pathophysiological
The activities physiological
enzymes
at low cost, with commonly or person
of enzymes conditions.
for diagnostic
Furthermore,
only if the pathophysiological
controlled
events in the kidney
in urine are, however,
purposes
available
Under
difficult.
affected
the heterogeneity Assessment
mechanisms
can by of
of renal
and the time se-
are known.
INTRODUCTION
The symposium on ‘Enzymes in Urine and Kidney’ [l] included papers on increased urinary enzyme activity in patients with kidney disease [2]. Approximately 100 urinary enzymes have since been introduced for the diagnosis of renal damage. The enzymes that have been used most often are N-acetyl-/3-D-glucosaminidase (NAG), y-glutamyltransferase, lysozyme and alkaline phosphatase (Table I). La Due and Wroblewski [3] considered that serum enzymes would provide a ‘biochemical biopsy’ of organs affected by disease. While the routine use of certain serum enzymes has been useful in the diagnosis of diseases such as myocardial infarction, pancreatitis and chronic liver disease, this has not been the case for the kidney. The reasons for this failure are due largely to the fact that measurement of a single enzyme may give misleading information about the site, type and extent of injury. Enzymuria is associated with the acute and not the chronic effects of toxins, because the urine represents an open system.
194 HETEROGENEITY
OF THE KIDNEY
The metabolic organization of the nephron has been assessed [I] and shown to be highly heterogeneous morphologicalIy. About 12 segments can be distinguished according to their enzymatic activities [3,4], and this has been used as the rational basis for the use of urinary enzymes as a diagnostic tool for assessing injury (Tables I and II). Many of the factors that regulate enzyme excretion in healthy persons are known. The epithelium of the nephron sheds cells continuously; it is under hormone influence: excretion of lysosomal hydrolases into the urine from the proximal tubufe is a physiological phenomenon which can be stimulated by hormones without induction of cell necrosis [.5]. Furthermore, simple variables, such as urine concentration, age and sex, may influence the results [6]. The wide variation in normal enzyme activity and a number of methodological probIems have resulted in a reIuctance to accept normal urinary enzyme values, and therefore in a reduced use of these determinations as a clinical tool. TABLE I SELECTED URINARY ENZYMES USED TO DIAGNOSE RENAL DISEASES Site of injury ~__--._____ Glomerular
~-
Disease/Toxin
Enzymes .______ ~_____.~. NAG, j%galactosidase NAG
.__-
Aminonucleoside Glomerulonephritis
Proximal tubular
Mercuric chloride Chromate [VI] Cadmium
Ligandin Aianine aminopeptidase Alkaline phosphatase, r-glutamyltransferase, NAG, glucosidase
Distal tubular
Folate
Lactic dehydrogenase
Papillary
Ethylene imine _
Alkaline phosphatase, NAG, glucosidase -_
TABLE II ORIGINS OF URINARY ENZYMES ____________ ~__._~___~ -_.__ Enzymes Origin _.__ -Brush border Alkaline phosphatase, aminopeptidases
______~. _ _.__________~~
Lysosomes
iv-Acetyl-@-D-glucosaminidase,
p-galactosidase
Cytosol
/%Glucosidase, ligandin, lactic dehydrogenase
Proximal tubule
Ligandin, alkaline phosphatase,
Distal tubule
Lactic dehydrogenase (also present in other parts of the nephron)
aminopeptidases
___
19s
Injury to the nephron has been associated with an increase in the presence of enzymes in the urine (Table I), but this may reflect neither functional nor morphological changes. Thus, despite its location in the proximal tubule, NAG may increase as a result of both proximal tubular and glomerular injuries; levels of alkaline phosphatase are elevated after injuries to all parts of the nephron. The diagnostic use of urinary enzymes as opposed to functional and histological data on kidney damage is of limited value. There are many parameters that are likely to modify the activities of enzymes in urine, including inhibitors, activators, pH value and osmolality. These vary widely according to the physiological status of the subject, and it is still not clear whether reproducible diagnostic results can be obtained in either healthy persons or patients [7]. In addition, diagnostic procedures and drugs may affect urinary enzyme determinations in patients with kidney disease. USE OF URINARY
ENZYMES
FOR MONITORING
NEPHROTOXICITY
A rise in serum creatinine or a drop in creatinine clearance after administration of antibiotics, analgesics and other substances signals the presence of extensive damage. More sensitive methods, such as elevated levels of urinary enzymes, are used, but these may reflect only an innocuous functional change, reversible damage or cell necrosis. This central problem is very difficult to resolve [8,9]. The proposal to establish a battery of tests of urinary enzymes in order to define renal damage has not been materialized, since in both experimental animals and in humans all conditions must be carefully standardized. Factors such as sex, age, volume depletion and diuresis influence the amount of damage and the time of appearance of enzymes in urine. Therefore, although urinary enzymes may constitute a sensitive indicator of parenchymal damage, their diagnostic reliability remains questionable. However, a number of situations in which urinary enzymes have been used are described below. Hypertonic solutions Burchardt and Peters [lo] examined the influence of intravenous administration of hypertonic solutions (10% mannitol, 10% dextran or the X-ray contrast medium ‘Visostrat 370’) to patients with pre-existing nephropathy. They observed a reproducible increase in the activity of urinary alanine aminopeptidase and suggested that it was due to osmotic nephropathy. They did not find such changes in persons without renal disease. Antibiotics There are dramatic changes in the excretion of several urinary enzymes after the administration of gentamicin [l 11. The potential nephrotoxicity of many other antimicrobial agents, mainly aminoglycosides and cephalosporins, represents an important clinical problem [ 121.
196
Occupational exposure to nephrotoxic chemicals Meyer et al. [13] reported on the renal effects of exposure to lead, mercury and organic solvents. None of the persons in their study had a history of clinically evident renal disease or hypertension, but in comparison with an appropriate control population, workers exposed to lead, mercury and two of three groups of workers exposed to organic solvents had significant increases in urinary NAG activity. Laboratory workers exposed to low levels of organic solvents showed no increase in urinary NAG activity. The authors concluded that exposure to environmental nephrotoxic agents (at levels currently considered safe) can produce renal effects as manifested by elevations of urinary NAG excretion. However, the question of the relevance of these changes in groups with well-defined exposure remains uncertain, especially with respect to their health significance. ACKNOWLEDGEMENTS
Supported
by Schweiz Nationalfonds,
Bern.
REFERENCES 1 Dubach, 2
U.C. (Ed.) (1968) Enzymes
2). Hans
Huber,
Rosalki,
S.B. and Wilkinson,
in Urine and Kidney (Current
Problems
in Clinical
Biochemistry
Bern. J.H.
(1959) Urinary
lactic dehydrogenase
in renal disease.
Lancet
ii,
327. 3
Guder,
W.G. and Heidland,
man Society
for Clinical
1985. J. Clin. Chem. 4
6 7
Biochem.
Ross, B.D. and Ciuder, W.G. (Ed.),
5
A. (1986) Urine analysis:
Chemistry
Metabolic
Koenig,
A., Goldstone, A manifestation
on the workshop
conference
in Wiirzburg,
(1981) Heterogeneity Academic
A. and Hughes,
and compartmentation Press,
C. (1978) Lysosomal
of cell defecation
in the kidney.
of residual
enzymuria
bodies.
Lab.
39, 339.
Dubach,
and Schmidt,
U.C.
8
Price,
R.G.
9
Berlyne,
U. (Eds.) (1979) Diagnostic
in Clinical
(1982) Urinary
G.M.
of the kidney. Effects
Problems
enzymes,
Biochemistry
In: M.A.
of Petroleum
Mehlmann,
Hydrocarbons,
G.P.
Significance
9). Hans
nephrotoxicity
(1984) Toxic nephropathies
Huber,
Hemstreet,
methods J.J.
Vol. 7. Princeton
and N-acetyl-P-D-
of Enzymes
and Proteins
Toxicology
23, 99-134.
for early detection
Thorpe Scientific
in
Bern.
and renal disease.
and current
In: H. Sies
in the testosterone-treated
Invest.
(1986) The effects of age and urine concentration on lysozyme (NAG) content in urine. Ann. Clin. Biochem. 23, 297.
(Current
25th-26th,
New York.
Houser, M.T. glucosaminidase Urine
of the Ger-
October
24, 611-620.
Compartmentation.
mouse.
report
and the Society of Nephrology
and N.K. Weaver Publishers,
of the toxicity (Eds.),
Princeton,
Renal
NJ, pp.
173-184. 10
Burchardt,
U. and Peters,
J.E. (1975) Enzyme
im Harn
nach Applikation
hypertoner
Losungen.
Z.
Ges. Inn. Med. 30, 248-251. 11
Hautmann,
R.,
Kisters,
glutamyltranspeptidase. 12 Bianchi, C., Bertelli, Karger,
R. and
Lutzeyer,
Urology 7, 12-16. A. and Duarte, C.G.
(Eds.)
(1976)
Diagnosis
of renal
(1984) Contributions
tumor
to Nephrology,
by gammaVol. 42.
Basel.
Rosenman, 13 Meyer, B.R., Fischbein, A.L.F., (1984) Increased urinary enzyme excretion Med.
W.
76. 989-999.
K., Lerman, Y., Drayer, D.E. and Reidenberg, M.M. in workers exposed to nephrotoxic chemicals. Am. J.