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Urinary enzyme excretion during renal papillary necrosis induced in rats with ethyleneimine
433
ta,&
474 Zollingerl and analgesic abuse is now thought to account for approximately 6 % of the patients suffering from renal insufficiency 2. Burrys has suggested on the basis of clinical data that the pathogenesis of analgesic nephropathy may begin as papillary necrosis with secondary cortical features developing later. Due to the complexities of interpreting clinical data increasing use has been made of animal models. Davies” induced papillary necrosis in rabbits with ethyleneimine, an agent known to cause selective necrosis of the renal pyramid 58s.Ham and Tange7 injected ethyleneimine into rats and reported that the initial lesions occurred in the renal papilla. The same groups compared the nephrotoxicity of aspirin and phenacetin derivatives in the rat and reported that the lesions were confined to the proximal convoluted tubules. Papillary lesions have also been described following the administration of bromoethylamine hydrobromides. Rarely have studies on papillary necrosis included the assay of urinary enzymes as well as histology. It has been suggested by Schonfieldl” that the output of urinary enzymes reflects the anatomic integrity of the kidney. Studies in the dog*’ demonstmted that the assay of urinary enzymes provides an early index of deteriorating renal integrity and an indication of the localisation of the lesions. In the present study the excretion of urinary enzymes was followed in rats injected with ethyleneimine. Histology was carried out to determine the sequence of regional damage and in addition urinary volume, protein and serum urea were monitored as indicators of renal function. MATERIALS AND METHODS
Preparation
of tissues
Pairs of male Wistar-derived rats (200 to 220 g) were kept in glass Met&owl Metabolism cages (Jencons Ltd., Hemel Hempstead, U.K.) which facilitate complete separation of urine and faeces and limit contamination with either food or water. Rats were allowed food and water ad libitum and urine samples were toll 24-h period in flasks maintained at 4”. Urine samples were filtered thr filter paper and stored at 4” until required. Blood was obtained by cardiac puncture and allowed to coagulate for I h at 3 before serum was collected and stored at 4”. 6 of rats were killed by cervical dislocation and their kidneys removed and h nised in cola distilled water. Homogenates (IOU/,) were prepared with a smooth glass Potter geniser with a Teflon pestle (clearance 0.48 mm) rotuted manually. were filtered through two layers of muslin and stored at 4”. Adn~kistratiort
of the twptvotositr
Two groups of rats were used in these studies, the first a single intraperitoneal injection of ethyleneimine (OS @/ had been adjusted to pH 7.2 with 5 N hydrochloric acid. A seco was given a single intraperitoneal injection of ethyleneimine
Light mkroscopy
ed with sa!ine and rats which had been injected tilted at various time intervals. Prior to sacrifice s removed and placed in a large w were prepared and stain-
p~vio~~i~l3. Serum urea eh”“. Protein was estimated by
in the
kidney which resulted
ion but necrosis was
nt
tubules also inmembrane was ob-
Plate I. Morphology of rat kidney papilla bcbrc and 24 h after the nilministmticln of cthylcnviminc (5.0 [d/kg). (a) control kidney ; (1))kidncy showing nwrosis ofthc loops of Hcnlc and collecting ducts iIt the tip of the papilla. Kidneys wwc st;rillcd with hnumntoxylin and cosin. Originnl m;rgnitiw;ation. x 66.
477 e
inactionof the
dose of crease in the rate of values on day 8 but ~~~~~1for the rest of of the lower higher
I
0
I
I
1
I
I
1
I
2
4
6
8
10
12
14
Days
Fig. 2. Mean urinary excretion of (a) protein (0 - - - 0) and urine (@ - - -0); (b) N-acetyl-/Iglucosaminidase (m - - - n ) and alkaline phosphatase (Cl - - - Cl); (c)acid phosphatase (A - - -aI and /?-galactosidase (A - - - A). The arrow indicates the day of injection of ethyleneimine (0.5 /#l/kg). Details of the number of rats used are given in the legend to Fig. 1. TABLE I ENZYME ACTIVlT1E.S IN SOME NORMAL RAT TISSUES
Kidney homogenates were prepared and urine and serum obtained from control groups of rats as described in the text. The activities of the five hydrolytic enzymes nre expressed as the number of ,rtmoles of the appropriate 4-methylumbelliferyl substrate hydrolysed/h. All results are the mean rt standard deviation. Values in parentheses indicate the number of salnples used. Enzyme
Activities Kidttev
Uritte prttoles/h/24-h
(2s)
snwtple Itntoll~slhlki~tte~~ (20)
P-
__ss__
PI__
N-acetyl-t%glucosami~tidctse
Acid phosphatase Alkaline phosphatase P-Galactosidase &Glucosidase
1.1 2.4 I.1 2.7 0.21
kO.4
f & f f
2.7 0.62 0.9 0.05
128
f
214 f 58 % 75 f 19.3 :t
37.0
22 5 8.0 3
Sertont pntoles/h/rttl
(22) ----.-0.3 rt: 0.02 1.02 rl: 0.29 0.24 :b: 0.03 0.13 f 0.01 0.0
on day 7 of the experiment (Fig. 2b and c). Maximum recorded increases of all the enzymes were between 2- and 3-fold.
A IO-fold increase in the dose of ethyleneimine
administered
resulted in a
479
b into rats. Mean
Fig. 3. Wrinary ewnx excrer’ daily excrctisn of (831hcetyllactosidaseIO---O)an iajection. Details of the nwn
pattern of urinary enzyme excretion which resembled but was not identical with that observed after the administmtion
of the lower dose. The higher dose, however, resuft-
cd in a more pronounced increase (Mold) The injection
of ethylcneimine
N-acetyl-&glucosnminidase
in the activity of all the urinary enzymes.
was follow
to a second peak on days IQ and I I. Ur%ay then gradually Urinary
by an ~mmcdiate rise in urinary
(Fig. 3a). The activity fell slightly on day 5 and then rose N-acetyl-/3-
i~co~aminida~
activity
returned to normal. alkaline phosphatase also showed a biphasic pattern of excretion with
peak values being recorded on da> 4 (24 h after the injection) and
tweeen days 7 and
I 1 (Fig. 3a). The excretion profile of acid phosphatase was similar to that of alkaline phosphatase with two peaks of enzyme excretion occurrin on days 4 and IQ. Urinary &galactosidase activity was elevated with the maxi urn excretion of this enzyme occurring on day 10, There was no significant rise in hmcosidase activity at this dose level.
Studies on the mobility of urinary ensrymeswith starch trop~or~~i~ failed nzyme patterns of any of the en~ymc~ studied strate any chungc in the dose of cthyleneimine. the administration of tit
Only alkaline phosphatase activity changed in the serum as a result of t administration of ethyleneimine (5 id/kg). The activity of th:s enzyme 1-0 aflcr the
TABLE II SOME ENZYME ACTIVITIES IN RAT KIDNEY CORTEX, MEDULLA AND PAPILLA
Results are expressed as pmoles 4-methylumbelliferone f standard deviation. The number of determinations
liberated/h/g wet weight. Results are the mean are shown in parentheses.
Enzyme
Cortex (10)
Medulla (8)
Papilla (10)
N-acetyl-/Q$cosaminidase Acid phosphatase Alkaline phosphatase fi-Galactosidase fi-Glucosidase
136 f 27 214 + 22 116f37 49 & 15 19 & 0.6
67 56 39 17 6.0
11.0 f 6.0 & 0.6 f 3.0 & 0.2f
&IS f 8 i13 &- 2 j, 0.6
2.0 1.0 0.5 1.0 1
injection to a maximum (5- to &fold increase) on day 12, the activity then fell gradually to normal (Table I). Enzyme activities in kidney homogenates The enzyme activity in kidney homogenates prepared at various time intervals after the administration of ethyleneimine (5 $/kg) were compared with normal renal enzyme activies (Table I). A decrease in renal activity occurred immediately after the injection and the lowest activities (50 “/, of normal values given in Table I) were recorded on day 5. The activities of all the enzymes studied then increased until 90 % of the normal activity was recovered by day 24 of the experiment. DlSCUSSION
The administration of ethyleneimine to rats resulted in a two-phase response. After an initial selective necrosis to the tip of the papilla a secondary low-grade chronic inflammatory reaction occurred in the cortex. These findings agree closely with those described following the administration of ethyleneimine to rabbits4 and rats’. It has also been proposed that damage to the tip of the papilla is the primary event in analgesic nephropathys although there is evidence that the initial lesion may be cortical. It can be seen from Figs. 1 and 2 that there are two peaks of proteinuria and polyuria which coincided with the observed progression from a papillary to a cortical phase of’necrosis. The second peak of proteinuria and polyuria extended over a larger number of days than the first peak reflecting the presence of chronic rather than acute damage to the cortical tubules. No change in serum urea was recorded and histological studies failed to find any glomerular lesions indicating that damage was restricted to the renal tubules. Two dose levels of ethyleneimine were used in these studies. The administration of a low dose of ethyleneimine (0.5 PI/kg) was followed by the elevation of urinary N-acetyl-P-glucosaminidase in the first 3 days after the injection (Fig. 2). Minor papillary necrosis induced by a low dose of ethyleneimine in dogs was also characterised by an immediate rise in urinary N-acetyl-@-glucosaminidase activitylt. It can be seen in Table II that this enzyme is the most active of the enzymes assayed in the
481 papilla. Once the damage becomes more generalised, an elevation of urinary alkaline phosphatase, acid phosphatase, and p-galactosidase (Table II) would be expected (Fig. 2). No significant elevation of urinary fl-glucosidase occurred during the second phase of enzyme excretion and chronic cortical damage resulting from the administration of ethyleneimine therefore differs in this respect from the acute damage resulting from the administration of mercuric chloride 17. The occurrence of this enzyme in the urine provides an excellent indicator of acute tubular necrosis and the absence of a sharp rise of the activity of this enzyme in the urine distinguishes the cortical lesions in tubular necrosis from the cortical involvement which follows papillary necrosis. pGlucosidase is the least active of the enzymes in the renal cortex (Table II) but it can be readily detected if it is present in the urine by its characteristic migration on starch gel electrophoresis 13. This isoenzyme was not found in the urine excreted by rats injected with cthyleneimine. The profile of urinary enzyme excretion closely resembled the pattern of protein excretion (Figs. 1 and 2) which followed the administration of the higher dose of ethyleneimine (5.0 PI/kg). The early rise in N-acetyl+glucosaminidase found following the administration of the higher dose is probably masked because of the more generalised necrosis. The elevation of enzyme activities and protein fell on the third and fourth days after the injection but once cortical involvement occurred they again rose to high levels and remained elevated for a further 11 days. Serum and kidney enzyme activities were also assayed at this dose level and only serum alkaline phosphatase was elevated. This is in contrast to the finding that iV-acetyl-@-glucosaminidase was the only serum enzyme elevated in the dog following ethpleneimine administrationtl. The activities of the enzymes studied fell in the kidney and the decrease coincided with the two phases of urinary enzyme excretion providing evidence that the increased urinary enzyme activity probably originates from the kidney. It seems clear from these and earlier studies with dog+ that the assay of urinary enzymes provides a useful adjunct to histological and functional procedures previously used in the study of papillary nephropathy. Because of the regional distribution of enzyme activities along the nephron, urinary enzyme profiles reflect the site of damage in the kidney. When the disease is progressive and involves a second area of the nephron then the urinary enzyme pattern will change. This was clearly demonstrated in a previous study in rats which glomerular damage could be distinguished from tubular damage by the excretion of /%glucosidase which only occurred when tubular damage was present”. When necrosis is limited to the tip of the papilla, IV-acetyl-@-glucosaminidase is the only urinary enzyme to be significantly elevated. The assay of urinary glycosidases in rats provides a convenient method for the early detection of renal damage. The differential excretion of these enzymes can be used to determine the region of the kidney which is the site of the initial lesion. Changes in the urinary enzyme profile reflect an extension of necrosis from one region to another. ACKNOWLEDGEMENTS
This work was supported by the National Kidney Research Fund (U.K.). The
4212 authors are indebted to Miss Rowena Barlow for technical assistance and Dr. J. C. Topham for advice during the course of this study.
REFERENCES 1 0. Spuhler and H. U. Zollinger, Die chronische interstitielle Nephritis, He/v. Med. Acta, 17, (1950) 564-567. 2 W. F. Clark and A. L. Linton, The problem of analgesic nephropathy, C&t. Toxicol., 6 (1973) 39-43. 3 A. F. Bursy, The evolution of analgesic nephropathy, Nephron, 5 (1968) 185. 4 D. J. Davies. Changes in the renal cortex following experimental medullary necrosis, Arc/t. Patho/., 86 (1968) 377-382. 5 C. Levaditi, Experimentelle Untersuchungen tiber die Nekrose der Nierenpapilie, Arch. I#~fer,r. Pharnlucodyn., 8 (1901) 45-63. 6 E. E. Mandel and H. Popper, Experimental medullary necrosis of kidney: morphological and functional study, Arch. futhol., 52 (1951) I-17. 7 K. N. Ham and J. D. Tange, Some experimental observations relating to the pathogenesis of renal papillary necrosis, Amt. Ann. Med., I8 (1969) 199-208. 8 I. C. Calder, C. C. Funder, C. R. Green, K. N. Ham and J. D. Tange, Comparative nephrotoxicity of aspirin and phenacetin derivatives, Brir. Med. J., 4 (1971) 518-521. 9 G. S. Hill, R. G. Wyllie, M. Miller and R. H. Heptinstall, Experimental papillary necrosis of the kidney, II. Electron microscopy and histochemical studies, Aster. J. Puthol., 68 (1972) 213-234. 10 M. R. Schonfield, Acid phosphatase activity in ureteral urine, J. Amer. Med. Ass., 193 (1965) 618-621. 11 B. G. Ellis, R. G. Price and J. C. Topham, The effect of papillary damage by ethyleneimine on kidney function and some urinary enzymes in the dog, Chettt.-Biol. fttternct., 7 (1973) 131-142. 12 B. G. Ellis, R. G. Price and J. C. Topham, The effect of tubular damage by mercuric chloride on kidney function and some ‘:, irlary enzymes in the dog, Chcttt.-Biol. lttreract., 7 (1973) 101-l 13. 13 D. Robinson, R. G. Price i, : ‘i. Dance, Separation and properties of /J-galactosidase, &glucosidase, /%glucuronidase, and ‘\-acetyl-&glucosaminidase from rat kidney, Biochem. J., 102 (1967) 525-532. I4 A. L. Chaney and E. P. M:rrbach, Modified reagents for the determination of urea and ammonia, Chit. Chetn., 8 (1962) 130-132. 15 A. C. Gromall, C. S. Bardawill and M. M. David, Determination of serum proteins by means of the Biurct reaction, J. Biol. Chettl., 177 (1949) 751-766. 16 D. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Cktn., 193 (195 1) 265-275. I7 R. G. Price, N. Dance and D. Robinson, A comparison of the /I-glycosidase excretion during kidney damage induced by 4-nitrophenylarsonic acid and by rabbit anti-rat kidney antibodies, Europ. J. Chit. Invest., 2 (1971) 47-5 I.
ta,&
474 Zollingerl and analgesic abuse is now thought to account for approximately 6 % of the patients suffering from renal insufficiency 2. Burrys has suggested on the basis of clinical data that the pathogenesis of analgesic nephropathy may begin as papillary necrosis with secondary cortical features developing later. Due to the complexities of interpreting clinical data increasing use has been made of animal models. Davies” induced papillary necrosis in rabbits with ethyleneimine, an agent known to cause selective necrosis of the renal pyramid 58s.Ham and Tange7 injected ethyleneimine into rats and reported that the initial lesions occurred in the renal papilla. The same groups compared the nephrotoxicity of aspirin and phenacetin derivatives in the rat and reported that the lesions were confined to the proximal convoluted tubules. Papillary lesions have also been described following the administration of bromoethylamine hydrobromides. Rarely have studies on papillary necrosis included the assay of urinary enzymes as well as histology. It has been suggested by Schonfieldl” that the output of urinary enzymes reflects the anatomic integrity of the kidney. Studies in the dog*’ demonstmted that the assay of urinary enzymes provides an early index of deteriorating renal integrity and an indication of the localisation of the lesions. In the present study the excretion of urinary enzymes was followed in rats injected with ethyleneimine. Histology was carried out to determine the sequence of regional damage and in addition urinary volume, protein and serum urea were monitored as indicators of renal function. MATERIALS AND METHODS
Preparation
of tissues
Pairs of male Wistar-derived rats (200 to 220 g) were kept in glass Met&owl Metabolism cages (Jencons Ltd., Hemel Hempstead, U.K.) which facilitate complete separation of urine and faeces and limit contamination with either food or water. Rats were allowed food and water ad libitum and urine samples were toll 24-h period in flasks maintained at 4”. Urine samples were filtered thr filter paper and stored at 4” until required. Blood was obtained by cardiac puncture and allowed to coagulate for I h at 3 before serum was collected and stored at 4”. 6 of rats were killed by cervical dislocation and their kidneys removed and h nised in cola distilled water. Homogenates (IOU/,) were prepared with a smooth glass Potter geniser with a Teflon pestle (clearance 0.48 mm) rotuted manually. were filtered through two layers of muslin and stored at 4”. Adn~kistratiort
of the twptvotositr
Two groups of rats were used in these studies, the first a single intraperitoneal injection of ethyleneimine (OS @/ had been adjusted to pH 7.2 with 5 N hydrochloric acid. A seco was given a single intraperitoneal injection of ethyleneimine
Light mkroscopy
ed with sa!ine and rats which had been injected tilted at various time intervals. Prior to sacrifice s removed and placed in a large w were prepared and stain-
p~vio~~i~l3. Serum urea eh”“. Protein was estimated by
in the
kidney which resulted
ion but necrosis was
nt
tubules also inmembrane was ob-
Plate I. Morphology of rat kidney papilla bcbrc and 24 h after the nilministmticln of cthylcnviminc (5.0 [d/kg). (a) control kidney ; (1))kidncy showing nwrosis ofthc loops of Hcnlc and collecting ducts iIt the tip of the papilla. Kidneys wwc st;rillcd with hnumntoxylin and cosin. Originnl m;rgnitiw;ation. x 66.
477 e
inactionof the
dose of crease in the rate of values on day 8 but ~~~~~1for the rest of of the lower higher
I
0
I
I
1
I
I
1
I
2
4
6
8
10
12
14
Days
Fig. 2. Mean urinary excretion of (a) protein (0 - - - 0) and urine (@ - - -0); (b) N-acetyl-/Iglucosaminidase (m - - - n ) and alkaline phosphatase (Cl - - - Cl); (c)acid phosphatase (A - - -aI and /?-galactosidase (A - - - A). The arrow indicates the day of injection of ethyleneimine (0.5 /#l/kg). Details of the number of rats used are given in the legend to Fig. 1. TABLE I ENZYME ACTIVlT1E.S IN SOME NORMAL RAT TISSUES
Kidney homogenates were prepared and urine and serum obtained from control groups of rats as described in the text. The activities of the five hydrolytic enzymes nre expressed as the number of ,rtmoles of the appropriate 4-methylumbelliferyl substrate hydrolysed/h. All results are the mean rt standard deviation. Values in parentheses indicate the number of salnples used. Enzyme
Activities Kidttev
Uritte prttoles/h/24-h
(2s)
snwtple Itntoll~slhlki~tte~~ (20)
P-
__ss__
PI__
N-acetyl-t%glucosami~tidctse
Acid phosphatase Alkaline phosphatase P-Galactosidase &Glucosidase
1.1 2.4 I.1 2.7 0.21
kO.4
f & f f
2.7 0.62 0.9 0.05
128
f
214 f 58 % 75 f 19.3 :t
37.0
22 5 8.0 3
Sertont pntoles/h/rttl
(22) ----.-0.3 rt: 0.02 1.02 rl: 0.29 0.24 :b: 0.03 0.13 f 0.01 0.0
on day 7 of the experiment (Fig. 2b and c). Maximum recorded increases of all the enzymes were between 2- and 3-fold.
A IO-fold increase in the dose of ethyleneimine
administered
resulted in a
479
b into rats. Mean
Fig. 3. Wrinary ewnx excrer’ daily excrctisn of (831hcetyllactosidaseIO---O)an iajection. Details of the nwn
pattern of urinary enzyme excretion which resembled but was not identical with that observed after the administmtion
of the lower dose. The higher dose, however, resuft-
cd in a more pronounced increase (Mold) The injection
of ethylcneimine
N-acetyl-&glucosnminidase
in the activity of all the urinary enzymes.
was follow
to a second peak on days IQ and I I. Ur%ay then gradually Urinary
by an ~mmcdiate rise in urinary
(Fig. 3a). The activity fell slightly on day 5 and then rose N-acetyl-/3-
i~co~aminida~
activity
returned to normal. alkaline phosphatase also showed a biphasic pattern of excretion with
peak values being recorded on da> 4 (24 h after the injection) and
tweeen days 7 and
I 1 (Fig. 3a). The excretion profile of acid phosphatase was similar to that of alkaline phosphatase with two peaks of enzyme excretion occurrin on days 4 and IQ. Urinary &galactosidase activity was elevated with the maxi urn excretion of this enzyme occurring on day 10, There was no significant rise in hmcosidase activity at this dose level.
Studies on the mobility of urinary ensrymeswith starch trop~or~~i~ failed nzyme patterns of any of the en~ymc~ studied strate any chungc in the dose of cthyleneimine. the administration of tit
Only alkaline phosphatase activity changed in the serum as a result of t administration of ethyleneimine (5 id/kg). The activity of th:s enzyme 1-0 aflcr the
TABLE II SOME ENZYME ACTIVITIES IN RAT KIDNEY CORTEX, MEDULLA AND PAPILLA
Results are expressed as pmoles 4-methylumbelliferone f standard deviation. The number of determinations
liberated/h/g wet weight. Results are the mean are shown in parentheses.
Enzyme
Cortex (10)
Medulla (8)
Papilla (10)
N-acetyl-/Q$cosaminidase Acid phosphatase Alkaline phosphatase fi-Galactosidase fi-Glucosidase
136 f 27 214 + 22 116f37 49 & 15 19 & 0.6
67 56 39 17 6.0
11.0 f 6.0 & 0.6 f 3.0 & 0.2f
&IS f 8 i13 &- 2 j, 0.6
2.0 1.0 0.5 1.0 1
injection to a maximum (5- to &fold increase) on day 12, the activity then fell gradually to normal (Table I). Enzyme activities in kidney homogenates The enzyme activity in kidney homogenates prepared at various time intervals after the administration of ethyleneimine (5 $/kg) were compared with normal renal enzyme activies (Table I). A decrease in renal activity occurred immediately after the injection and the lowest activities (50 “/, of normal values given in Table I) were recorded on day 5. The activities of all the enzymes studied then increased until 90 % of the normal activity was recovered by day 24 of the experiment. DlSCUSSION
The administration of ethyleneimine to rats resulted in a two-phase response. After an initial selective necrosis to the tip of the papilla a secondary low-grade chronic inflammatory reaction occurred in the cortex. These findings agree closely with those described following the administration of ethyleneimine to rabbits4 and rats’. It has also been proposed that damage to the tip of the papilla is the primary event in analgesic nephropathys although there is evidence that the initial lesion may be cortical. It can be seen from Figs. 1 and 2 that there are two peaks of proteinuria and polyuria which coincided with the observed progression from a papillary to a cortical phase of’necrosis. The second peak of proteinuria and polyuria extended over a larger number of days than the first peak reflecting the presence of chronic rather than acute damage to the cortical tubules. No change in serum urea was recorded and histological studies failed to find any glomerular lesions indicating that damage was restricted to the renal tubules. Two dose levels of ethyleneimine were used in these studies. The administration of a low dose of ethyleneimine (0.5 PI/kg) was followed by the elevation of urinary N-acetyl-P-glucosaminidase in the first 3 days after the injection (Fig. 2). Minor papillary necrosis induced by a low dose of ethyleneimine in dogs was also characterised by an immediate rise in urinary N-acetyl-@-glucosaminidase activitylt. It can be seen in Table II that this enzyme is the most active of the enzymes assayed in the
481 papilla. Once the damage becomes more generalised, an elevation of urinary alkaline phosphatase, acid phosphatase, and p-galactosidase (Table II) would be expected (Fig. 2). No significant elevation of urinary fl-glucosidase occurred during the second phase of enzyme excretion and chronic cortical damage resulting from the administration of ethyleneimine therefore differs in this respect from the acute damage resulting from the administration of mercuric chloride 17. The occurrence of this enzyme in the urine provides an excellent indicator of acute tubular necrosis and the absence of a sharp rise of the activity of this enzyme in the urine distinguishes the cortical lesions in tubular necrosis from the cortical involvement which follows papillary necrosis. pGlucosidase is the least active of the enzymes in the renal cortex (Table II) but it can be readily detected if it is present in the urine by its characteristic migration on starch gel electrophoresis 13. This isoenzyme was not found in the urine excreted by rats injected with cthyleneimine. The profile of urinary enzyme excretion closely resembled the pattern of protein excretion (Figs. 1 and 2) which followed the administration of the higher dose of ethyleneimine (5.0 PI/kg). The early rise in N-acetyl+glucosaminidase found following the administration of the higher dose is probably masked because of the more generalised necrosis. The elevation of enzyme activities and protein fell on the third and fourth days after the injection but once cortical involvement occurred they again rose to high levels and remained elevated for a further 11 days. Serum and kidney enzyme activities were also assayed at this dose level and only serum alkaline phosphatase was elevated. This is in contrast to the finding that iV-acetyl-@-glucosaminidase was the only serum enzyme elevated in the dog following ethpleneimine administrationtl. The activities of the enzymes studied fell in the kidney and the decrease coincided with the two phases of urinary enzyme excretion providing evidence that the increased urinary enzyme activity probably originates from the kidney. It seems clear from these and earlier studies with dog+ that the assay of urinary enzymes provides a useful adjunct to histological and functional procedures previously used in the study of papillary nephropathy. Because of the regional distribution of enzyme activities along the nephron, urinary enzyme profiles reflect the site of damage in the kidney. When the disease is progressive and involves a second area of the nephron then the urinary enzyme pattern will change. This was clearly demonstrated in a previous study in rats which glomerular damage could be distinguished from tubular damage by the excretion of /%glucosidase which only occurred when tubular damage was present”. When necrosis is limited to the tip of the papilla, IV-acetyl-@-glucosaminidase is the only urinary enzyme to be significantly elevated. The assay of urinary glycosidases in rats provides a convenient method for the early detection of renal damage. The differential excretion of these enzymes can be used to determine the region of the kidney which is the site of the initial lesion. Changes in the urinary enzyme profile reflect an extension of necrosis from one region to another. ACKNOWLEDGEMENTS
This work was supported by the National Kidney Research Fund (U.K.). The
4212 authors are indebted to Miss Rowena Barlow for technical assistance and Dr. J. C. Topham for advice during the course of this study.
REFERENCES 1 0. Spuhler and H. U. Zollinger, Die chronische interstitielle Nephritis, He/v. Med. Acta, 17, (1950) 564-567. 2 W. F. Clark and A. L. Linton, The problem of analgesic nephropathy, C&t. Toxicol., 6 (1973) 39-43. 3 A. F. Bursy, The evolution of analgesic nephropathy, Nephron, 5 (1968) 185. 4 D. J. Davies. Changes in the renal cortex following experimental medullary necrosis, Arc/t. Patho/., 86 (1968) 377-382. 5 C. Levaditi, Experimentelle Untersuchungen tiber die Nekrose der Nierenpapilie, Arch. I#~fer,r. Pharnlucodyn., 8 (1901) 45-63. 6 E. E. Mandel and H. Popper, Experimental medullary necrosis of kidney: morphological and functional study, Arch. futhol., 52 (1951) I-17. 7 K. N. Ham and J. D. Tange, Some experimental observations relating to the pathogenesis of renal papillary necrosis, Amt. Ann. Med., I8 (1969) 199-208. 8 I. C. Calder, C. C. Funder, C. R. Green, K. N. Ham and J. D. Tange, Comparative nephrotoxicity of aspirin and phenacetin derivatives, Brir. Med. J., 4 (1971) 518-521. 9 G. S. Hill, R. G. Wyllie, M. Miller and R. H. Heptinstall, Experimental papillary necrosis of the kidney, II. Electron microscopy and histochemical studies, Aster. J. Puthol., 68 (1972) 213-234. 10 M. R. Schonfield, Acid phosphatase activity in ureteral urine, J. Amer. Med. Ass., 193 (1965) 618-621. 11 B. G. Ellis, R. G. Price and J. C. Topham, The effect of papillary damage by ethyleneimine on kidney function and some urinary enzymes in the dog, Chettt.-Biol. fttternct., 7 (1973) 131-142. 12 B. G. Ellis, R. G. Price and J. C. Topham, The effect of tubular damage by mercuric chloride on kidney function and some ‘:, irlary enzymes in the dog, Chcttt.-Biol. lttreract., 7 (1973) 101-l 13. 13 D. Robinson, R. G. Price i, : ‘i. Dance, Separation and properties of /J-galactosidase, &glucosidase, /%glucuronidase, and ‘\-acetyl-&glucosaminidase from rat kidney, Biochem. J., 102 (1967) 525-532. I4 A. L. Chaney and E. P. M:rrbach, Modified reagents for the determination of urea and ammonia, Chit. Chetn., 8 (1962) 130-132. 15 A. C. Gromall, C. S. Bardawill and M. M. David, Determination of serum proteins by means of the Biurct reaction, J. Biol. Chettl., 177 (1949) 751-766. 16 D. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Cktn., 193 (195 1) 265-275. I7 R. G. Price, N. Dance and D. Robinson, A comparison of the /I-glycosidase excretion during kidney damage induced by 4-nitrophenylarsonic acid and by rabbit anti-rat kidney antibodies, Europ. J. Chit. Invest., 2 (1971) 47-5 I.