- Email: [email protected]
Studies on the nephrotoxicity of p-nitrophenylarsonic acid: Changes in rat kidney and urinary enzyme activities following the administration of p-nitrophenylarsonic acid
Chem.-3ioL Intemctione, 24 (1979) 241-266 8 Elsevier/North-Holland Scientific Publishers Ltd.
241
SPIES ON THR N~HR~XICI~ OF ~~TROP~~A~ON~C ACID: CHANGES IN RAT KIDNEY AND URINARY ENZYME ACTIVITIES FOLLOWING THE ADMINISTRATION OF pNITROPHENYLARSONIC ACID
R.G. PRICE Deportment of Biochemistry, Queen Elizabeth College, University of London, Campden Iiili, London W8 7AH (United Kingdom)
(Received November 2nd. 1977) (Accepted January 14th, 1978)
SUMMARY
Histological studies showed that the administration of p-nitrophenylarsonic acid to rats resulted in renal tubular necrosis. The nephrotoxin was adminis~red ~t~pe~tone~ly and doses greater than 30 m&kg were found to be fatal. The severity of the renal lesion depended on the amount of the nephrotoxin used. Elevated serum urea levels, urinary protein and volume were recorded over an Sday period following the injection of the nephrotoxin. These changerswere paralleled by an increase in the activity of lactate dehydrogenase, acid and alkaline phosphatase, N-acetyl+glucosaminidase and ~-glucosida~ in the urine. &Glycosidase activities increased in kidney homogenates, ~med~tely after the injection of the nephrotoxin, but this eventually fell to well below the normal range. Subcellular fractions were prepared from sucrose homogenates by differential centrifugation and #-glycosidases and cytochrome oxidase were used as enzyme markers. Only minor changes in the activity of cytochrome oxidase activity resulted from the admonition of p-nitrophe~yl~onic acid. One of the earliest indications of renal damage was a decrease in lysosomal latency. The a@vities of the lysosomal and soluble enzymes were elevated above normal during the first two days after the injection of p-nitrophenylarsonic acid, but they fell to values, significantly lower than normal, on the third day. The isoenzymic forms of p-galactosidase, flglucosidase and N-acetylQglucosaminidase in normal and damaged kidneys were studied, using starch gel electrophoresis. The activities of both the lysosomal and the soluble forms of these enzymes decreased following the injection of the nephrotoxin, confirming the results obtained with whole homogenates. The relationship between the changes in renal enzyme acitivity and urinary enzyme excretion during the nephrotoxic process is discussed.
242 INTRODUCI’ION
p-Nitrophenylarsonic acid is a strong acid and is a member of the arsenical group of compounds several of which are used in the treatment of human and animal dliseases. This compound (trade name, Hi&o&at) is an effective drug for the treatment of blackhead in turkeys [I] and possesses growthpromoting activity in poultry [2]. Since p-nitrophenylarsonic acid has been used as a growth promotor and a coccidiostat :in cfiickens, Moody and Williams [3]* carried out a detailed study of the metabolism of this compound. These authors demonstrated that p-nitrophenylarsonic acid is excreted partly unchanged and to a lesser extent as its reduction product, arsanilic acid (4-aminophenylarsonic acid). Armilic acids may be reduced in the gut to more toxic arsenoxides which would then exert a parasitostatic effect accounting for their growth-promoting properties. p-Nitrophenylarsonic acid is several times more toxic than arsanilic acid in chickens [ 41. ‘The report of the elevation of several p-glycosidases enzymes in rat urine [5] was the ffirst indication that p-nitrophenylarsonic acid was nephrotoxic, The change in urinary enzyme profile resulting from the administration of p-nitrophenylarsonic acid was similar to that found after the administration of well established tubular nephrotoxins such as ursnyl nitrate [ 51. The present study forms part of a continuing investigation carried out in our laboratory into urinary enzyme excretion in rene;l damage [5-U). In this paper the site of the lesion induced by p-nitrsphenylarsonic acid in the kidney has been determined using histological methods. The results of an initial experiment when lactate dehydrogenase, acid and alkaline phosphatase were assayed in the urine together with solme P-glycosidase enzymes is reported. The activity of these enzymes in serum and urine were measured at convenient time intervals following the administration of the nephrotoxin. Serum and urine enzyme activities were compared with the activities recovered in Ikidney homogenates and isolated subcellular fractions. &Galactosidase and IV-acetyl+glucosaminidase were selected as lysosomal marker enzymes, while Pglucosidase and lactate dehydrogenase were used as marker enzymes for the cytosol. N-acetyl-&lucosaminid,ase has a bimodal distribution in the rat kidney [12] and in addition to the lysosomal form of the enzyme a second non-latent form is present in the microsomal fraction. This latter isoenzyme of IV-acetylj.?-glucosaminidasewas therefore used as a marker for the microsomal fraction. Cytochrome oxidase was selected as a suitable marker enzyme for mitochondria. Starch gel electrophoresis was used to compare the isoenzyme patterns of the flglycosidases in normal and damaged rat kidney and urine. MATERIALS AND METHODS
Preparation of kidney homogenates. Male Sprague-Dawley rats (200250 g) were used. Rats were killed by cctical dislocation, the kidneys removed, weighed, sliced and suspended in 9 vols. of cold distilled water or
243 0.45 M sucrose (containing 0.68 mM EDTA, adjusted to pH 7.0 with 0.25 M NaOH). The suspension was homogenised in a smooth glass PotterElvehjem homogeniser by two passes of a manually rotated Teflon pestle (diameter clearance 0.48 mm). After filtration though two layers of muslin, the homogenate was made up to 25 ml with the appropriate homogenising medium. Comparison of urine and kidney enzyme activities. A group of 37 rats was injected with p-nitrophenylarsonic acid (30 mg/kg body wt.) Seven rats were placed individually in metabolism cages and the remainder were sacrificed daily in groups of 6 rats for 4 consecutive days and kidney homogenates were prepared. Tissue fractionation studies. Nuclear, lysosomal, mitochondrial, microsomal and soluble fractions were prepared from sucrose homogenates as previously described by Price and Dance [ 121. Latent activities were estimated in the presence and absence of Triton X-100 [ 121. Collection of blood and urine samples. Rats were kept individually or in pairs in metabolism cages (Jencons Ltd., Hemel Hempstead, Herts, U.K.): water and food were provided ad libitum. Twenty-four hour urine samples were collected, filtered through coarse filter paper and used immediately for enzyme analysis. Blood was removed by cardiac puncture an allowed to clot at room temperature for two horus. Serum was separated by low speed centrifugation and stored at -20°C until required. Enzyme assays. The activities of /3-glucosidase (O-D-glucoside-glucohydrolase, EC 3.2.1.21.), @-galactosidase (p-Dgalactoside-galactohydrolase, EC,3.2.1.23) and IV-acetyl-&glucosaminidase (fl-2-acetamido-2-deoxy-Dglucoside acetamidodeoxy glucohydrolase, EC 3.2.1.30) were determined in urine, kidney homogenates and subcellular fractions using the appropriate 4-methylumbelliferyl substrate [ 51. Cytochrcsme oxidase (cytochrome c:02 oxidoreductase, EC 1.11.1.6) activity was assayed as described by Cooperstein and Lazarow [ 131. Lactate dehydrogenase (L-1actate:NAD oxidoreductase, EC 1.1.1.27.) was determined by the procedure of Wacker and Dorfman [14]. Alkaime (orthorp-osphoric monester phosphohydrolase, EC 3.1.3.1) and acid (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2.) phosphatase activities were determined by the methods of Fernley and Walker [15] and Robinson and Wilcox [16] respectively. Gel electrophoresis. Enzyme components were separated in starch gel at 4°C in 0.04 M sodium phosphate buffer [17] and enzyme activities were detected using the appropriate 4-methylumbelliferyl substrate as described previously [17]. The developed gels were either photographed or scanned using an Aminco-Bowman microspectrophotofluorimeter 1181.
Morphology. :Rats were killed at various stages after the administration of p-nitrophenylarsonic acid and the kidneys removed. Thin slices of kidney were fixed in a large volume of formal saline. Paraffin sections of the tissue were prepared and stained with haemotoxylin and eosin. Nephro toxin. p-Nitrophenylarsonic acid (Histostat-50) was a gift from Salisbury’s Laboratories, Charles City, IA, U.S.A. The nephrotoxin was pre:pared in sterile saline (adjusted to pH 7.5 with NaHCOj ) and was administered intraperitoneally as a single does in the range 2-40 mg/kg. RESULTS
Dose range of p-nitrophenylarsonic acid. Groups of rats were injected with varying amounts of p-nitrophenylarsonic acid (2-30 mg/kg). Injection of the highest dose (30 mg/kg) resulted in a 2-3-fold increase in urine volume which reached a maximum (45 ml/day) 2 days after the injection (Fig. lb) and then fell to near normal values 5 days later. During the first 4 days after IbO-
‘ii 320
%
.k i 240 x > '; I60 zL
120; F 80 u : ; 40 E > z w-l
a 80 E" I
o
50
I
2
4
3 Days
P-a II
40-/
4
5
30
-9
\
I c--_Irx
20
8
W
/ .E x 1 E
7
6
\ \ b--
o---A,
-0
xl
i IO
t
L
I--_L-lI IO
I2
3 Days
4
5
._-L-_l___L
15
7
8
Fig. 1. Comparison of daily excretion of urine and protein with serum urea levels in rats injected with p-ntirophenylarsonic acid (30 mg/kg body weight). The arrow indicates the day of the injection. The results are the mean of ten samples, two rats were maintained in each metaboiism cage. Further experimental details are given in the text. (a) Protein in urine and serum urea; (b) daily volume of urine excreted.
245
the injection of p-nitrophenylarsonic acid, the urine had a cloudy white appearance and there was an immediate rise in protein excretion. The rate of protein excretion fell grad.ually to normal levels 5 days after the injection (Fig, la). Some experiments were continued for 28 days and the rate of protein excretion and urine volume remained within the normal range during this period. Abnormal elevation of urea in the serum (Fig. la) followed the injection of p-nitrophenylarsonic acid. The highest value was found 3 daya after the injection, serum urea values then returned gradually to normal. Morphological changes in rat kidney following the administration of p-Nitrophenylarsonic acid. Kidneys removed from rats 48 h after the injection of the nephrotoxin were enlarged, had a grey white appearance and their weight ranged up to twice that of kidneys from normal rats. Kidneys were still abnormally enlarged 28 days after the injection. Injection of p-nitrophenylarsonic acid (30 mg/kg body wt.) produced focal necrosis of the proximal and distal tubules in the outer cortex (Fig. 2,A and B). This was most marked in the second and third days after the administration of the nephrotoxin and the lesion was less severe on the fifth day. Lower doses of p-nitrophenylarsonic acid produced less severe necrosis. No glomerular damage was observed. Focal necrosis waslstill pronounced after three days but thereafter the kidneys showed a gradual recovery. Serum enzyme actiuity. No deviation from the previously published normal ranges [9] of serum enzymes was found following the injection of p-nitrophenylarsonic acid. Urinary enzyme excretion. The pattern of excretion of a number of enzymes was measured following the injection of p-nitrophenylarsonic acid and the activities of acid and alkaline phophatase and lactate dehydrogenase are shown as examples in Fig. 3. Alkaline phosphatase activity rose more rapidly than acid phosphatase during the first 24 h following the injection but the excretion of all the enzymes studied reached a maximum within 2 days of the administration of p-nitrophenylarsonic acid. All the enzyme activities had fallen into the normal range within 5 days of the injection but a secondary and unexplained rise in acid ohosphatase activity occurred on days 6, 7 and 8 of the experiment (Fig. 3). The activity of the lysosomal or brush border enzymes increased to approximately the same extent (Tbble I). Much greater increases in the two enzymes present in the soluble fraction of the kidney were recorded two days after the administration of the nephrotoxin.
Fig. 2. Morphology of proximal and distal convoluted tubules of rat kidney cortex before and 48 h after the administration of p-nitrophenylarsonic acid (30 mg/kg). (a) Control kidney; (b) kidney sbowing necrosis of proximal and distal convoluted tubules. Kidneys were stained with haematoxylin and eosin. Meg. X 660.
247
co---(, 4-
/
IQ
I
f’
\
\ \
:
A
\
I -I
\ \
I o
* I 8
I 7
’ \ \ \
/
Lo4
5
1 6
’
I
I
4
3 Days
I2
w’
I I2
A-_+_
I
-A-
-A-
_A
I
3 Days
4
5
b
7
8
Fig. 3. Mean daily excretion of (a) acid phosphatase activity, o- - -0; alkaline phosphatase activity, O-W; (h) lactate dehydrogenase activity, A- - - 4 The arrow indicates the day of the injection of p-nitrophenylarsonic acid. Further details are given in Fig. 1 and in the text. TABLE I MAXIMUM RECORDED INCREASES IN URINARY ENZYME ACTIVITY FOLLOWING ADMINISTRATION OF p_NITROPHENYLARSONIC ACID Enzyme
Principal subcellular localisation in the kidney
Maxhnum recorded increase in the urine
7.1a Lysosomes ,$-Galactosidase 13.5 Lysosomes N-Acetyl-gglucosam inidase 7.9 Lysosomes Acid phosphatase 13.7 Plasmamembrane Alkaline phosphatase 24.1 Cyt4Xol 1%Glucosidase 50.5 CytosOl Lactate dehydrogenase .-, “Increase is expressed as the number of times the enzyme activity is elevated above the :mean normal value. The increases are calculated from the highest urinary activity recorded for each enzyme. The peak of enzyme activity normally .)ccurred within two days of the injection.
248
v
pot*/ 70 50
-
:“.-%a I
0
-e--
1
Fig. 4. Comparison of the activity of some 8-glycosidase enzymes in kidney and urine of rats following the injection of p-nitrophenylarsonic acid (a) kidney homogenates (b) urine and (c) kidney homogenate activities expressed in terms of recovered protein. The 1%galactosidase, l and pglucosidase, activities of N-acetyl-@-glucosaminidase, o---i) are shown. Each point represents the .nean * S.E.M. of 6 determinations for kidney and 7 for urine. Details of the preparation of kidney homogenates and urine collection are given in the text. The arrow indicates the time of injection of p-nitrophenyalrsonic acid.
Comparison or urine and kidney flglycosidase activitiee following the injection of p-nitrophenybrsonic acid. A detailed study of the changes in the enzymes associated with the renal brush border has been published previously [ll] and for this reason alkaline phosphatase was not further investigated. The total activities of 3 p-glycosidase enzymes measured in urine and in kidney homogenates for three days after the injection are shown in Fig. 4. IN-acetyl+glucosaminidase was the most active of the P-glycosidases in rat kidney and the total activity of this enzyme in kidney homogenates increased by 30% within a day of the injection (Fig. 4a). Renal activities of IV-acetyl- fl-glucosaminidase were still elevated two days after the injection but h,ad dropped to 30% of the normal activity after 3 days (Fig. 4b). P-Galactosidase and @-glucosidase activities in the kidney were elevated 24 b after the injection but both fell to below normal values by the second day and had decreased to less than 50% of the normal activity by day 3. When the activity of the enzymes present in the kidney homogenates were expressed in terms of recovered protein (Fig. 4c) a different pattern emerged. The specific activnties of all three enzymes fell slightly on day 1 and were much lower on days 2 and 3. Comparison of the standard error of the mean values of the recovered enzyme activities (Fig. 4) shows that there was a much greater range in urinary enzyme activities than was found in kidney homogenates. An immediate 5-fold rise in urinary N-acetyl-fl-glucosaminidase in the urine occurred 1 day after the injection (Fig. 3b). Similar elevated activities of this enzyme were found on day 3 and the activities of this enzyme then fell daily towards the normal range. Elevated levels of fl-galactosidase were excreted within 1 day of the injection and abnormal activities were present up to 3 days after the injection. The peak of &glucosidase activity occurred 1 day later, on day 2. When the expeiment was continued for longer periods the activity of all 3 enzymes fell gradually until they reached normal values (day 6).
249
Cc)
+
Fig. 5. Starch gel electrophoresis of rat pglycosidases. p-Nitrophenylarsonic acid was injected at the beginning of day 1. Normal kidney homogenate (H, ) and a kidney homogenate prepared on day 2 (H, ) are compared with a normal urine sample (day 0) and samples obtained in the 3 days immediately following the injection. Samples were prepared for electrophoresis and the separation carried out as described in the text. (a) P-Galactosidase, (h) P-glucosidase and (c) N-acetyl-P-glucosaminidase.
Gel electrophoresis studies. Normal rat kidney contains two forms of pgalactosidase (Fig. Sa), both forms move towards the anode and the more rapidly migrating form is also a fl-glucosidase (Fig. 5b and ref. 5). Two forms of IV-acetyl-fl-glucosaminidase were visible in kidney homogenates. Only the slowly migrating forms of /3-galactosidase and IV-acetyl_P-glucosaminidase were detected in urine but no @-glucosidase acitivity was detectable. Within 2 days of the injection of the nephrotoxin the urine pattern of all 3 &glycosidases changed and closely resembled that seen in rat kidney homogenates on days 2 and 3 of the experiment. No P-glucosidase activity was detected in urine when the experiment was continued past day 3. A kidney homogenate prepared 2 days after the injection of p-nitrophenylarsenic acid is included in Fig. 5 for comparison. The fl-glycosidase pattern was similar to that of normal kidney, but the intensity of the enzymes bands was markedly reduced. The isoenzyme patterns of normal and damaged rat kidney homogenates are compared in Fig. 6. Integration of the peak areas demonstrated that both forms of @-galactosidase decreased to 46% of the activity recovered in normal homogenates, while the @-glucosidase activity also decreased to about 50% of the original activity. The &glucosidase peak in a damaged kidney homogenate had a pronounced shoulder which may indicate the presence of multiple forms of fl-glucosidase. The N-acetyl-p-glucosamidinase activity of
+
v
0
origin
6
I.4 1.2 I-O 0.6 .= 0-b q 0.4
i Q-2 E= .Z
I.0 0.8 0.b 0.4 0.2
Fig. 6. Fluorimetric scaus of starch gel electrophoretograms of rat kidney &glycoaidaeer Starch gels were run and, enzyme activities were visualised and scanned as described i the text. (a) N-Acetyl-fl-glucosaminidase, (b) &galactosidase and (c) Pglucoeidase. Cor tinuous line represents the enzyme activity of normal rat kidney, while the broken lin represents the activity recovered in rat kidney 3 days after the injection of p-nitropheny arsenic acid.
Fig. 7. Recovery of g-glycosidase and cytochrome oxidam activities in subcellular fractior prepared from normal and damaged kidneys. (a) Lysosomal fraction, (b) mitochondrir fraction (c) microsomal fraction and (d) soluble fraction. N-acetyl-~glucosaminidao 0~; @g.Qlact.osidase, ~-5 p-glucosidase, o--o ; cytochrome oxidaQ;e activitie 0~ were assayed as described in the text. Subcellular fractions were prepared ou fou collRecutive days as described in the text and enzyme activities are expressed &Brmoll recovered in each fraction except for cytochrome oxidase activity in whick c88e th activity is expressed in arbitrary units. The arrow indicatee the time of injection c p-nitrophenylarsonic acid.
normal kidney was clearly resolved into three peaks - a slowly rn~t~g form I, an intermediate form 11 and a more rapidly migrating form III. All three forms of N-acetyl+glucosaminidase decreased by approximately equal amounts (59-64%) of their activity in normal kidney homogenates. Recovery of Bglycosidases and cytochrome oxidase activities in subcellukrr fractionsprepared from rats injected with p-nitrophenylarsonic acid. The latency of the two lysosomal marker enzymes &galactosidase and N-acetyl-& glucosaminidase fell from 70% to 35% within 24 h of the administration of ~-ni~ophenyl~onie acid. The enzyme activities recovered in the heavy particulate, microsomal and soluble fractions prepared at daily intervals following the injection of p-ni~ophenyl~oni~ acid are shown in Fig. 7. The nuclear fraction has been omitted from Fig. 7 since this fraction is known to be contaminated by unbroken cells and membranes 1221 and its heterogeneity makes the interpretation of the enzyme activities difficult. The activity of N-acetyl-fl-glucosaminidase recovered in the lysosomal fraction during the two days following the administration of the nephrotoxin was significantly increased above normal (Fig. 7a) but there was a steep fall in activity on the third day. Similar increases were recorded for P-Galactosidase and cytochrome oxidase 1 day after the injection. &Galactosidase activity fell below the normal range for this enzyme 1 day earlier than the In contrast the cyto~~ome oxidase activity ~~~etyl~-glucos~~id~. lay within the normal range on day 2 and 3. A similar increase in N-acetyl-pglucosaminidase activity was also found in the mitochondrial fraction (Fig, 7b). Elevated N-acetyl+glucosaminidase activities were recored on days 1 and 2 and the activities then fell steeply on day 3. A similar profile was found for /3-galactosidase but in this case the maximum recovered activitity was obtained on day 1. A similar fall in activity was found for cytochrome oxidase on days 2 and 3. No significant changes in @-galactosidase activity was found in the microsomal fraction (Fig. 7~). In contrast to the other fractions the N-acety? 3glucos~inida~ activity of this fraction fell slightly on days 1 and 2 and precipitously on day 3. Both &galactosidase and @-gluxosidase activities were slightly elevated in soluble fraction (Fig. 7d) within a day of the injection. The P-glucosidase activity fell steeply to abnormally low levels on days 2 and 3. The 8-glucosidase activity fell more slowly and abnormal values were recorded on day 3. In contrast N-acetyl-fi-glucosaminidase activity increased above normal on day 1 and the highest activities were recorded on day 2. The N-acetyl-Pglucosaminidase activity then fell sharply. DISCUSSION
The possible nephro~oxi~ity of the members of the arsenical group of compounds has not previously been studied, and the discovery that p-nitrophenylarsonic acid is nepbrotoxic is therefore of particular interest. This
252 CO~~OUZI~proved to be lethal at high doses and produced a well defined necrosis at lower doses. Functional changes in the kidney f&lowing the administration of this nephrotoxin were reflected by a sharp rise in the concentration of serum urea which was accompauiell by pr~teinuria and PO&I,&, The rapid rise of three fl-glycosidase enzymes together with acid and alkaline phosphatase and lactate dehydrogenase agreed with our earlier reponcs [ 5,6] and those from other laboratories [23,24,26] that these enzymes are elevated in urine immediately following the administration of tubular nepbrotoxins. Indeed apart from electron microscopy which is not a routine technique, urinary enzymes assay provides the earliest indication of the presence of renal disease [6,11]. Earlier studies in our labo~to~ had demonstrated that tubular damage in rats resulting from the admin~~~on of p-nitrophenylarsonic acid could be distinguished from glomerular damage on the basis of urinary enzyme excretion [ 26f. The absence of significant glomerular damage following the administration of this agent was confirmed in the present study with histological techniques. Moody and Williams [3] suggested that when p-nitrophenylarsonic acid is fed to chickens it is reduced in the gut to arsanilic acid and eventually to the more toxic arsenoxides. In the present study p-nitrophenylarsonic acid was injected by the intraperitoneal route and direct chemical modification by the gut flora was avoided. Little is known of the metabolic fate of ~-nitrophenyla~oni~ acid in the rat and in p~ic~ar whether it is sign~ican~y reduced to ~~n~ic acid or arsenoxides. Further studies will be required to de~rmine whether p-nitrophenylarsonic acid or its reduction products are the major cause of nephrotoxicity. Few studies have monitored renal enzyme activities during the course of renal damage, although because of the high activities of many enzymes in the kidney, it has long been considered to be the principal source of enzymes excreted in the urine. The initial rise in fl-glycosidase activity in kidney homogenates (Fig. 2a) which followed the administration of p-nitrophenylarsenic acid was unexpected, since other studies [l&28] with the tubular nephrotox~ mercuric chloride, demons~~d that the injection of this agent resulted in an immediate fall in renal enzyme activities. This increase in activity is not seen when the enzyme activities are related to protein concentration (Fig. 4~). The expression of results in this way is common practice [ 271, but since protein is lost from the kidney during tubular damage and changes in weight of the kidney are also common, enzyme activities should be expressed as the total activity recovered per kidney and not related to protein or to kidney weight. Tbe increased enzyme activities in the kidney may result from the stimulation of protein synthesis or be caused by the invasion of cells rich in hydrolase activity into the renal tissue. At the dose levels used in the present study, the kidney was unable to fully compensa~ for the initial damage caused by the agent and the development of focal necrosis was eventually accompanied by loss of ki&iey enzymes into the urine. The assay of serum enzymes are of little value in assessing nephrotoxicity since no
253 si~~ic~t changes in the activities of these enzymes were found. The major source of urinary enzymes in renal damage would therefore appear to be the tubular cells. In the present investigation marker enzymes were used to study changes in the principal subcellular organelles in the kidney during the early stages of renal necrosis. A decrease in latency within the first twenty four hours was the earliest detectable change in the properties of subcellular organelles isolated after the administration of p-nitrophenylarsonic acid. An initial increase in lysosomal enzyme activity accompanied the fall in latency which may be a prelude to the loss of hydrolytic enzymes into the cytosol and eventu~y into the urine. This ~~b~ity is suppose by the fading that ~~cetyl~~luco~in~~ activity in the soluble fraction reaches a maximum value on day 2, whereas the lysosomal enzyme activity is greatest on day 1 (Fig. 2a and d). Calder et al. [ 28 ] demonstrated that the administration of paminophenol, o-aminophenol, quinols, catechols and their derivatives to rats, result in a similar type of necrosis in proximal convoluted tubules. These compounds can all participate in redox systems and these authors suggest that there is a relationship between their nephrotoxicity and oxidation-reduction potential. Although ~-nitrophenyla~o~c acid itself would not participate in oxidation-reduction systems the possibility that it is me~bol~ed to an in~~edia~ which could exert its nephroto~c effect in this way emnot be excluded. Indeed phenyla~enoxide is known to inhibit keto acid oxidation in rat liver mitochondria f 291. In a more recent study Crowe et al. [30] demonstrated that the nephrotoxicity of p-aminophenol administered to rats was triggered by mitochondrial injury and Verity and Brown [ 311 have reported that the administration of mercuric chloride results in a significant depression of the renal cytochrome oxidase activity, In contrast, in th present study recovery of cytochrome exidase was normal except for a small increase in the 24 h following the injection (Fig. ?a and b). The effect of ~-nitrophenyl~sonic acid on mitochond~~ function is certainly different from that of ~-~inoph~nol [303 and mercuric chloride f31), but requires more detailed study. The administration of p-nitrophenylarsonic acid results in focal necrosis which is characterised by an early fall in lysosomal latency. Hydrolytic enzymes are then, presumably, lost into the cytosol because of the increased fragility of tht! lysosomes and are eventually recovered in the urine. The elevated enzyme activities in the kidney recovered immediately following the injection may be the result of increased protein synthesis in the unaffected cells. These cells may attempt to compensate for the loss of functional cells in the damaged areas but as the necrosis spreads the expected fail in enzyme activities recovered in kidney homogenates occurred. The rat is a convenient animal for the study of nep~otoxicity and the excretion of urinary enzymes in addition to providing a sensitive screening procedure may also provide information en the localisation of the renal lesion. For example, a rise in the excretion of N-acetyl_P-glucosaminidase
254 in the absence of a corresponding increase in alkaline phosphatase would indicate that the lesion is local&d in the papilla [9]. The elevation of IVacetyl+glucosaminidase and alkaline phosphatase in the urine in the presence of normal &glucosidase activity would indicate that the principal lesion was glomerular since there is no detectable &glucosidase activity in the serum [26]. The simultaneous elevation of fl-glucosidase, alkaline phosphatase and IV-acetyl_P-glucosaminidase would indicate the presence of a lesion of the cortical tubules and these changes were demonstrated in the present study. The assay of a small group of enzymes therefore provides the most convenient and sensitive method of assessing renal damage without recourse to killing the animal. Additional information can be obtained by characterising the isoenzymes present in urine since &glucosidase separates as a rapidly migrating band found in kidney but normally absent from urine. Alternatively a change in the relative proportion of the isoenzymes OPlactate dehydrogenase [ 321 in urine is indicative of renal tubular damage. The results of the present study provide additional evidence that the enzymes excreted in urine as the result of the administration of a tubular nephrotoxin originate primarily from the kidney. They further emphasize the value of studies involving subcellular organelles in elucidation of the biochemical sequence of events occurring in the nephroxtoxic process. ACKNOWLEDGEMENT
This work was supported by a grant from the National Kidney Research Fund, U.K. REFERENCES W.C. McGuire and N.F. Morehouse, Chemotherapy studies in histomoniasis, Poultry Science, 31(1952) 603. N.F. Morehouse and W.C. McGuire, p-Nitrobenzene arsonic acid for blackhead control, U.S. Patent (1953) 2,638,432. 3.P. Moody and R.T. Williams, The fate of 4nitrophenylarsonic acid in hens, Fd. Co&met. Toxicol., 2 (1964) 695. D.$. Frost and H.C. Spruth, Synposium on medicated feeds, in H. Welch and F. Marti-Ibanzed (Eds.), Medical Encylopaedia Inc., New York, p. 136. C. Robinson, R.G. Price and N. Dance, Rat urine glycosidases and kidney damage, Biochem. J., 102 (1967) 533. B.G. Ellis, R.G. Price and J.C. Topham, The effect of tubular damage by mercuric cf;loride on kidney function and some urinary enzymes in the dog, Chem-Biol. Interact., 7 (1973) 101. 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, Chem.-Biol. Interact., 7 (1973) 131. J.M. Wellwood, B.G. Ellis, R.G. Price, K. Hammond, A.E. Thompson and N.F Jones, Urinary N-acetyl-p-glucosaminidase activities in patients with renal disease, Brit. Med. J., 139 (1975) 408. B.G. Ellis and R.G. Price, Urinary enzyme excretion during renal papillary necrosis induced in rats with ethyleneimine, Chem.-Bicl. Interact., 11 (1976) 473.
255 10 11
12 13 14
15 16 17
18 19 20 21 22 23
24 25 26 27 28 29
30 31 32
R.G. Price and 3.0. Ellis, Urinary Enzyme excretion in aminonucleoside nepbrosis in rats, Chem.-Biol. Interact., 13 (1976) 353. S.A. Kempson, B.G. Bllii and R.G. Rice, Changes in rat renal cortex, isolated plasma membranes and urinary enzymes following the injection of mercuric chloride, Chem.Biol. Interact., 18 (1977) 217. R.G. Price and N. Dance, The cellular distribution of some rat kidney glycosidases, Biochem. J., 106 (1967) 877. S.J. Cooperstein and A. Larzarow, A microspectrophotometric method for the deovation of cyhcha:me oxidase, J. Bid. C&m., 189 (1951) 666. W.E.C. Wacker and L.E. Dorfman, Urinary lactate dehydrogenase activity, 1. Screening method for the detection of cancers of the kidney and bladder, J. Am. Med. Ass. 181(1962) 972. H.N. Femley and P.G. Walker, Kinetic behavlour of calf in~sti~ akdine phosphata& with 4-methyl-umbelliferyl phosphate, Biochem. J., 97 (1965) 95. D. Robinson and P. Wilcox, 4-Methylumbelliferyl phosphate as a substrate for lysosomal acid phosphatase, Biochem. Biophys. Acta., 191(1969) 183. D. Robinson, R.G. Price and N. Dance, Separation and properties of &galactosidase, p-glucosidase, ~~lu~u~nid~ and N-a~tyI~~luco~minid~ from rat kidney, Biochem. J., 102 (1967) 525. H.E. Abrahams and D. Robinson, &glucosidsses and related enzymes activities in pig kidney, Biochem. J., 111(1969) 749. 0.11. Lowry, N.J. Rosebrough, AL. Faw and RJ. Randall, Protein me~urements with the Folin phenol reagent, J. Biol. Cbem., 193 (1951) 265. H. Schuel and R. Schuel, Automated determination of protein in the presence of sucrose, Anal. Biochem., 20 (1967) 86. A.C. Gromall, C.S. ~da~l and M.M. David, Destination of serum proteins by means of the Biuret reaction, J. Biol. Chem., 177 (1949) 751. S. Shibko and A.L. Tappel, Rat kidney lysosomes: isolation and properties, Biochem, J., 95 (1966) 731. P.J. Wright and D.T. Plummer, The use of urinary enzyme measurements to detect reteet rend d8Wtgt! caused by nephrotoxic compounds, Biochem. Pharmacol., 23 (1974) 65. W.P. Raab, Diagnostic value of urinary enzyme determinations, Clin. Chem., 18 (1972) 6. J.M. Wellwood, P.M. Simpson, JR. Tighe and A.E. Thompson, Effect of gentamicin nephrotoxicity in patients with renal allograft, Brit. Med. J., 3 (1975) 278. R.G. Price, N. Dance and D. Robinson, A Comparison of the p-glycosidase excretion during damage induced by p-nitrophenylarsonic acid and by rabbit anti-rat kidney antibodies, Eur. J. Clin. Invest., 2 (X971) 47. C. De Duve, Principles of tissue fractionation, J. Theoret. Biol., 6 (1964) 33. I.C. Calder, P.J. Williams, R.A. Woods, C.C. Funder, C.R. Green, K.N. Hamm and J.D. Tange, Nephrotoxicity and molecular structure, Xenobiotica., 5 (1975) 303. M. ~rnst~ng and M. Webb, The reversal of phenyl~noxide inhibition of keto acid oxidation in mitochondria and bacterial suspensions by lipoic acid and other disulphides, Biochem. J., 103 (1967) 913. C.A. Crowe, I.C. Calder, N.P. Madsen, C.C. Funder, C.R. Green, K.N. Hamm and J.D. Tange, An experimental model of analgesic-induced renal damage-some effects of p-aminophenol on rat kidney mitoehondria, Xenobiotica, 7 (1977) 345. M.A. Verity and W.J. Brown, Hg”-induced kidney necrosis, Am. J. Pathol., 61 (1970) 57. P.D. Leathwood, M.K. Gilford and D.T. Plummer, Enzymes in rat urine: lactate dehydrogenase, En~ymolo~, 42 (1971) 286.
241
SPIES ON THR N~HR~XICI~ OF ~~TROP~~A~ON~C ACID: CHANGES IN RAT KIDNEY AND URINARY ENZYME ACTIVITIES FOLLOWING THE ADMINISTRATION OF pNITROPHENYLARSONIC ACID
R.G. PRICE Deportment of Biochemistry, Queen Elizabeth College, University of London, Campden Iiili, London W8 7AH (United Kingdom)
(Received November 2nd. 1977) (Accepted January 14th, 1978)
SUMMARY
Histological studies showed that the administration of p-nitrophenylarsonic acid to rats resulted in renal tubular necrosis. The nephrotoxin was adminis~red ~t~pe~tone~ly and doses greater than 30 m&kg were found to be fatal. The severity of the renal lesion depended on the amount of the nephrotoxin used. Elevated serum urea levels, urinary protein and volume were recorded over an Sday period following the injection of the nephrotoxin. These changerswere paralleled by an increase in the activity of lactate dehydrogenase, acid and alkaline phosphatase, N-acetyl+glucosaminidase and ~-glucosida~ in the urine. &Glycosidase activities increased in kidney homogenates, ~med~tely after the injection of the nephrotoxin, but this eventually fell to well below the normal range. Subcellular fractions were prepared from sucrose homogenates by differential centrifugation and #-glycosidases and cytochrome oxidase were used as enzyme markers. Only minor changes in the activity of cytochrome oxidase activity resulted from the admonition of p-nitrophe~yl~onic acid. One of the earliest indications of renal damage was a decrease in lysosomal latency. The a@vities of the lysosomal and soluble enzymes were elevated above normal during the first two days after the injection of p-nitrophenylarsonic acid, but they fell to values, significantly lower than normal, on the third day. The isoenzymic forms of p-galactosidase, flglucosidase and N-acetylQglucosaminidase in normal and damaged kidneys were studied, using starch gel electrophoresis. The activities of both the lysosomal and the soluble forms of these enzymes decreased following the injection of the nephrotoxin, confirming the results obtained with whole homogenates. The relationship between the changes in renal enzyme acitivity and urinary enzyme excretion during the nephrotoxic process is discussed.
242 INTRODUCI’ION
p-Nitrophenylarsonic acid is a strong acid and is a member of the arsenical group of compounds several of which are used in the treatment of human and animal dliseases. This compound (trade name, Hi&o&at) is an effective drug for the treatment of blackhead in turkeys [I] and possesses growthpromoting activity in poultry [2]. Since p-nitrophenylarsonic acid has been used as a growth promotor and a coccidiostat :in cfiickens, Moody and Williams [3]* carried out a detailed study of the metabolism of this compound. These authors demonstrated that p-nitrophenylarsonic acid is excreted partly unchanged and to a lesser extent as its reduction product, arsanilic acid (4-aminophenylarsonic acid). Armilic acids may be reduced in the gut to more toxic arsenoxides which would then exert a parasitostatic effect accounting for their growth-promoting properties. p-Nitrophenylarsonic acid is several times more toxic than arsanilic acid in chickens [ 41. ‘The report of the elevation of several p-glycosidases enzymes in rat urine [5] was the ffirst indication that p-nitrophenylarsonic acid was nephrotoxic, The change in urinary enzyme profile resulting from the administration of p-nitrophenylarsonic acid was similar to that found after the administration of well established tubular nephrotoxins such as ursnyl nitrate [ 51. The present study forms part of a continuing investigation carried out in our laboratory into urinary enzyme excretion in rene;l damage [5-U). In this paper the site of the lesion induced by p-nitrsphenylarsonic acid in the kidney has been determined using histological methods. The results of an initial experiment when lactate dehydrogenase, acid and alkaline phosphatase were assayed in the urine together with solme P-glycosidase enzymes is reported. The activity of these enzymes in serum and urine were measured at convenient time intervals following the administration of the nephrotoxin. Serum and urine enzyme activities were compared with the activities recovered in Ikidney homogenates and isolated subcellular fractions. &Galactosidase and IV-acetyl+glucosaminidase were selected as lysosomal marker enzymes, while Pglucosidase and lactate dehydrogenase were used as marker enzymes for the cytosol. N-acetyl-&lucosaminid,ase has a bimodal distribution in the rat kidney [12] and in addition to the lysosomal form of the enzyme a second non-latent form is present in the microsomal fraction. This latter isoenzyme of IV-acetylj.?-glucosaminidasewas therefore used as a marker for the microsomal fraction. Cytochrome oxidase was selected as a suitable marker enzyme for mitochondria. Starch gel electrophoresis was used to compare the isoenzyme patterns of the flglycosidases in normal and damaged rat kidney and urine. MATERIALS AND METHODS
Preparation of kidney homogenates. Male Sprague-Dawley rats (200250 g) were used. Rats were killed by cctical dislocation, the kidneys removed, weighed, sliced and suspended in 9 vols. of cold distilled water or
243 0.45 M sucrose (containing 0.68 mM EDTA, adjusted to pH 7.0 with 0.25 M NaOH). The suspension was homogenised in a smooth glass PotterElvehjem homogeniser by two passes of a manually rotated Teflon pestle (diameter clearance 0.48 mm). After filtration though two layers of muslin, the homogenate was made up to 25 ml with the appropriate homogenising medium. Comparison of urine and kidney enzyme activities. A group of 37 rats was injected with p-nitrophenylarsonic acid (30 mg/kg body wt.) Seven rats were placed individually in metabolism cages and the remainder were sacrificed daily in groups of 6 rats for 4 consecutive days and kidney homogenates were prepared. Tissue fractionation studies. Nuclear, lysosomal, mitochondrial, microsomal and soluble fractions were prepared from sucrose homogenates as previously described by Price and Dance [ 121. Latent activities were estimated in the presence and absence of Triton X-100 [ 121. Collection of blood and urine samples. Rats were kept individually or in pairs in metabolism cages (Jencons Ltd., Hemel Hempstead, Herts, U.K.): water and food were provided ad libitum. Twenty-four hour urine samples were collected, filtered through coarse filter paper and used immediately for enzyme analysis. Blood was removed by cardiac puncture an allowed to clot at room temperature for two horus. Serum was separated by low speed centrifugation and stored at -20°C until required. Enzyme assays. The activities of /3-glucosidase (O-D-glucoside-glucohydrolase, EC 3.2.1.21.), @-galactosidase (p-Dgalactoside-galactohydrolase, EC,3.2.1.23) and IV-acetyl-&glucosaminidase (fl-2-acetamido-2-deoxy-Dglucoside acetamidodeoxy glucohydrolase, EC 3.2.1.30) were determined in urine, kidney homogenates and subcellular fractions using the appropriate 4-methylumbelliferyl substrate [ 51. Cytochrcsme oxidase (cytochrome c:02 oxidoreductase, EC 1.11.1.6) activity was assayed as described by Cooperstein and Lazarow [ 131. Lactate dehydrogenase (L-1actate:NAD oxidoreductase, EC 1.1.1.27.) was determined by the procedure of Wacker and Dorfman [14]. Alkaime (orthorp-osphoric monester phosphohydrolase, EC 3.1.3.1) and acid (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2.) phosphatase activities were determined by the methods of Fernley and Walker [15] and Robinson and Wilcox [16] respectively. Gel electrophoresis. Enzyme components were separated in starch gel at 4°C in 0.04 M sodium phosphate buffer [17] and enzyme activities were detected using the appropriate 4-methylumbelliferyl substrate as described previously [17]. The developed gels were either photographed or scanned using an Aminco-Bowman microspectrophotofluorimeter 1181.
Morphology. :Rats were killed at various stages after the administration of p-nitrophenylarsonic acid and the kidneys removed. Thin slices of kidney were fixed in a large volume of formal saline. Paraffin sections of the tissue were prepared and stained with haemotoxylin and eosin. Nephro toxin. p-Nitrophenylarsonic acid (Histostat-50) was a gift from Salisbury’s Laboratories, Charles City, IA, U.S.A. The nephrotoxin was pre:pared in sterile saline (adjusted to pH 7.5 with NaHCOj ) and was administered intraperitoneally as a single does in the range 2-40 mg/kg. RESULTS
Dose range of p-nitrophenylarsonic acid. Groups of rats were injected with varying amounts of p-nitrophenylarsonic acid (2-30 mg/kg). Injection of the highest dose (30 mg/kg) resulted in a 2-3-fold increase in urine volume which reached a maximum (45 ml/day) 2 days after the injection (Fig. lb) and then fell to near normal values 5 days later. During the first 4 days after IbO-
‘ii 320
%
.k i 240 x > '; I60 zL
120; F 80 u : ; 40 E > z w-l
a 80 E" I
o
50
I
2
4
3 Days
P-a II
40-/
4
5
30
-9
\
I c--_Irx
20
8
W
/ .E x 1 E
7
6
\ \ b--
o---A,
-0
xl
i IO
t
L
I--_L-lI IO
I2
3 Days
4
5
._-L-_l___L
15
7
8
Fig. 1. Comparison of daily excretion of urine and protein with serum urea levels in rats injected with p-ntirophenylarsonic acid (30 mg/kg body weight). The arrow indicates the day of the injection. The results are the mean of ten samples, two rats were maintained in each metaboiism cage. Further experimental details are given in the text. (a) Protein in urine and serum urea; (b) daily volume of urine excreted.
245
the injection of p-nitrophenylarsonic acid, the urine had a cloudy white appearance and there was an immediate rise in protein excretion. The rate of protein excretion fell grad.ually to normal levels 5 days after the injection (Fig, la). Some experiments were continued for 28 days and the rate of protein excretion and urine volume remained within the normal range during this period. Abnormal elevation of urea in the serum (Fig. la) followed the injection of p-nitrophenylarsonic acid. The highest value was found 3 daya after the injection, serum urea values then returned gradually to normal. Morphological changes in rat kidney following the administration of p-Nitrophenylarsonic acid. Kidneys removed from rats 48 h after the injection of the nephrotoxin were enlarged, had a grey white appearance and their weight ranged up to twice that of kidneys from normal rats. Kidneys were still abnormally enlarged 28 days after the injection. Injection of p-nitrophenylarsonic acid (30 mg/kg body wt.) produced focal necrosis of the proximal and distal tubules in the outer cortex (Fig. 2,A and B). This was most marked in the second and third days after the administration of the nephrotoxin and the lesion was less severe on the fifth day. Lower doses of p-nitrophenylarsonic acid produced less severe necrosis. No glomerular damage was observed. Focal necrosis waslstill pronounced after three days but thereafter the kidneys showed a gradual recovery. Serum enzyme actiuity. No deviation from the previously published normal ranges [9] of serum enzymes was found following the injection of p-nitrophenylarsonic acid. Urinary enzyme excretion. The pattern of excretion of a number of enzymes was measured following the injection of p-nitrophenylarsonic acid and the activities of acid and alkaline phophatase and lactate dehydrogenase are shown as examples in Fig. 3. Alkaline phosphatase activity rose more rapidly than acid phosphatase during the first 24 h following the injection but the excretion of all the enzymes studied reached a maximum within 2 days of the administration of p-nitrophenylarsonic acid. All the enzyme activities had fallen into the normal range within 5 days of the injection but a secondary and unexplained rise in acid ohosphatase activity occurred on days 6, 7 and 8 of the experiment (Fig. 3). The activity of the lysosomal or brush border enzymes increased to approximately the same extent (Tbble I). Much greater increases in the two enzymes present in the soluble fraction of the kidney were recorded two days after the administration of the nephrotoxin.
Fig. 2. Morphology of proximal and distal convoluted tubules of rat kidney cortex before and 48 h after the administration of p-nitrophenylarsonic acid (30 mg/kg). (a) Control kidney; (b) kidney sbowing necrosis of proximal and distal convoluted tubules. Kidneys were stained with haematoxylin and eosin. Meg. X 660.
247
co---(, 4-
/
IQ
I
f’
\
\ \
:
A
\
I -I
\ \
I o
* I 8
I 7
’ \ \ \
/
Lo4
5
1 6
’
I
I
4
3 Days
I2
w’
I I2
A-_+_
I
-A-
-A-
_A
I
3 Days
4
5
b
7
8
Fig. 3. Mean daily excretion of (a) acid phosphatase activity, o- - -0; alkaline phosphatase activity, O-W; (h) lactate dehydrogenase activity, A- - - 4 The arrow indicates the day of the injection of p-nitrophenylarsonic acid. Further details are given in Fig. 1 and in the text. TABLE I MAXIMUM RECORDED INCREASES IN URINARY ENZYME ACTIVITY FOLLOWING ADMINISTRATION OF p_NITROPHENYLARSONIC ACID Enzyme
Principal subcellular localisation in the kidney
Maxhnum recorded increase in the urine
7.1a Lysosomes ,$-Galactosidase 13.5 Lysosomes N-Acetyl-gglucosam inidase 7.9 Lysosomes Acid phosphatase 13.7 Plasmamembrane Alkaline phosphatase 24.1 Cyt4Xol 1%Glucosidase 50.5 CytosOl Lactate dehydrogenase .-, “Increase is expressed as the number of times the enzyme activity is elevated above the :mean normal value. The increases are calculated from the highest urinary activity recorded for each enzyme. The peak of enzyme activity normally .)ccurred within two days of the injection.
248
v
pot*/ 70 50
-
:“.-%a I
0
-e--
1
Fig. 4. Comparison of the activity of some 8-glycosidase enzymes in kidney and urine of rats following the injection of p-nitrophenylarsonic acid (a) kidney homogenates (b) urine and (c) kidney homogenate activities expressed in terms of recovered protein. The 1%galactosidase, l and pglucosidase, activities of N-acetyl-@-glucosaminidase, o---i) are shown. Each point represents the .nean * S.E.M. of 6 determinations for kidney and 7 for urine. Details of the preparation of kidney homogenates and urine collection are given in the text. The arrow indicates the time of injection of p-nitrophenyalrsonic acid.
Comparison or urine and kidney flglycosidase activitiee following the injection of p-nitrophenybrsonic acid. A detailed study of the changes in the enzymes associated with the renal brush border has been published previously [ll] and for this reason alkaline phosphatase was not further investigated. The total activities of 3 p-glycosidase enzymes measured in urine and in kidney homogenates for three days after the injection are shown in Fig. 4. IN-acetyl+glucosaminidase was the most active of the P-glycosidases in rat kidney and the total activity of this enzyme in kidney homogenates increased by 30% within a day of the injection (Fig. 4a). Renal activities of IV-acetyl- fl-glucosaminidase were still elevated two days after the injection but h,ad dropped to 30% of the normal activity after 3 days (Fig. 4b). P-Galactosidase and @-glucosidase activities in the kidney were elevated 24 b after the injection but both fell to below normal values by the second day and had decreased to less than 50% of the normal activity by day 3. When the activity of the enzymes present in the kidney homogenates were expressed in terms of recovered protein (Fig. 4c) a different pattern emerged. The specific activnties of all three enzymes fell slightly on day 1 and were much lower on days 2 and 3. Comparison of the standard error of the mean values of the recovered enzyme activities (Fig. 4) shows that there was a much greater range in urinary enzyme activities than was found in kidney homogenates. An immediate 5-fold rise in urinary N-acetyl-fl-glucosaminidase in the urine occurred 1 day after the injection (Fig. 3b). Similar elevated activities of this enzyme were found on day 3 and the activities of this enzyme then fell daily towards the normal range. Elevated levels of fl-galactosidase were excreted within 1 day of the injection and abnormal activities were present up to 3 days after the injection. The peak of &glucosidase activity occurred 1 day later, on day 2. When the expeiment was continued for longer periods the activity of all 3 enzymes fell gradually until they reached normal values (day 6).
249
Cc)
+
Fig. 5. Starch gel electrophoresis of rat pglycosidases. p-Nitrophenylarsonic acid was injected at the beginning of day 1. Normal kidney homogenate (H, ) and a kidney homogenate prepared on day 2 (H, ) are compared with a normal urine sample (day 0) and samples obtained in the 3 days immediately following the injection. Samples were prepared for electrophoresis and the separation carried out as described in the text. (a) P-Galactosidase, (h) P-glucosidase and (c) N-acetyl-P-glucosaminidase.
Gel electrophoresis studies. Normal rat kidney contains two forms of pgalactosidase (Fig. Sa), both forms move towards the anode and the more rapidly migrating form is also a fl-glucosidase (Fig. 5b and ref. 5). Two forms of IV-acetyl-fl-glucosaminidase were visible in kidney homogenates. Only the slowly migrating forms of /3-galactosidase and IV-acetyl_P-glucosaminidase were detected in urine but no @-glucosidase acitivity was detectable. Within 2 days of the injection of the nephrotoxin the urine pattern of all 3 &glycosidases changed and closely resembled that seen in rat kidney homogenates on days 2 and 3 of the experiment. No P-glucosidase activity was detected in urine when the experiment was continued past day 3. A kidney homogenate prepared 2 days after the injection of p-nitrophenylarsenic acid is included in Fig. 5 for comparison. The fl-glycosidase pattern was similar to that of normal kidney, but the intensity of the enzymes bands was markedly reduced. The isoenzyme patterns of normal and damaged rat kidney homogenates are compared in Fig. 6. Integration of the peak areas demonstrated that both forms of @-galactosidase decreased to 46% of the activity recovered in normal homogenates, while the @-glucosidase activity also decreased to about 50% of the original activity. The &glucosidase peak in a damaged kidney homogenate had a pronounced shoulder which may indicate the presence of multiple forms of fl-glucosidase. The N-acetyl-p-glucosamidinase activity of
+
v
0
origin
6
I.4 1.2 I-O 0.6 .= 0-b q 0.4
i Q-2 E= .Z
I.0 0.8 0.b 0.4 0.2
Fig. 6. Fluorimetric scaus of starch gel electrophoretograms of rat kidney &glycoaidaeer Starch gels were run and, enzyme activities were visualised and scanned as described i the text. (a) N-Acetyl-fl-glucosaminidase, (b) &galactosidase and (c) Pglucoeidase. Cor tinuous line represents the enzyme activity of normal rat kidney, while the broken lin represents the activity recovered in rat kidney 3 days after the injection of p-nitropheny arsenic acid.
Fig. 7. Recovery of g-glycosidase and cytochrome oxidam activities in subcellular fractior prepared from normal and damaged kidneys. (a) Lysosomal fraction, (b) mitochondrir fraction (c) microsomal fraction and (d) soluble fraction. N-acetyl-~glucosaminidao 0~; @g.Qlact.osidase, ~-5 p-glucosidase, o--o ; cytochrome oxidaQ;e activitie 0~ were assayed as described in the text. Subcellular fractions were prepared ou fou collRecutive days as described in the text and enzyme activities are expressed &Brmoll recovered in each fraction except for cytochrome oxidase activity in whick c88e th activity is expressed in arbitrary units. The arrow indicatee the time of injection c p-nitrophenylarsonic acid.
normal kidney was clearly resolved into three peaks - a slowly rn~t~g form I, an intermediate form 11 and a more rapidly migrating form III. All three forms of N-acetyl+glucosaminidase decreased by approximately equal amounts (59-64%) of their activity in normal kidney homogenates. Recovery of Bglycosidases and cytochrome oxidase activities in subcellukrr fractionsprepared from rats injected with p-nitrophenylarsonic acid. The latency of the two lysosomal marker enzymes &galactosidase and N-acetyl-& glucosaminidase fell from 70% to 35% within 24 h of the administration of ~-ni~ophenyl~onie acid. The enzyme activities recovered in the heavy particulate, microsomal and soluble fractions prepared at daily intervals following the injection of p-ni~ophenyl~oni~ acid are shown in Fig. 7. The nuclear fraction has been omitted from Fig. 7 since this fraction is known to be contaminated by unbroken cells and membranes 1221 and its heterogeneity makes the interpretation of the enzyme activities difficult. The activity of N-acetyl-fl-glucosaminidase recovered in the lysosomal fraction during the two days following the administration of the nephrotoxin was significantly increased above normal (Fig. 7a) but there was a steep fall in activity on the third day. Similar increases were recorded for P-Galactosidase and cytochrome oxidase 1 day after the injection. &Galactosidase activity fell below the normal range for this enzyme 1 day earlier than the In contrast the cyto~~ome oxidase activity ~~~etyl~-glucos~~id~. lay within the normal range on day 2 and 3. A similar increase in N-acetyl-pglucosaminidase activity was also found in the mitochondrial fraction (Fig, 7b). Elevated N-acetyl+glucosaminidase activities were recored on days 1 and 2 and the activities then fell steeply on day 3. A similar profile was found for /3-galactosidase but in this case the maximum recovered activitity was obtained on day 1. A similar fall in activity was found for cytochrome oxidase on days 2 and 3. No significant changes in @-galactosidase activity was found in the microsomal fraction (Fig. 7~). In contrast to the other fractions the N-acety? 3glucos~inida~ activity of this fraction fell slightly on days 1 and 2 and precipitously on day 3. Both &galactosidase and @-gluxosidase activities were slightly elevated in soluble fraction (Fig. 7d) within a day of the injection. The P-glucosidase activity fell steeply to abnormally low levels on days 2 and 3. The 8-glucosidase activity fell more slowly and abnormal values were recorded on day 3. In contrast N-acetyl-fi-glucosaminidase activity increased above normal on day 1 and the highest activities were recorded on day 2. The N-acetyl-Pglucosaminidase activity then fell sharply. DISCUSSION
The possible nephro~oxi~ity of the members of the arsenical group of compounds has not previously been studied, and the discovery that p-nitrophenylarsonic acid is nepbrotoxic is therefore of particular interest. This
252 CO~~OUZI~proved to be lethal at high doses and produced a well defined necrosis at lower doses. Functional changes in the kidney f&lowing the administration of this nephrotoxin were reflected by a sharp rise in the concentration of serum urea which was accompauiell by pr~teinuria and PO&I,&, The rapid rise of three fl-glycosidase enzymes together with acid and alkaline phosphatase and lactate dehydrogenase agreed with our earlier reponcs [ 5,6] and those from other laboratories [23,24,26] that these enzymes are elevated in urine immediately following the administration of tubular nepbrotoxins. Indeed apart from electron microscopy which is not a routine technique, urinary enzymes assay provides the earliest indication of the presence of renal disease [6,11]. Earlier studies in our labo~to~ had demonstrated that tubular damage in rats resulting from the admin~~~on of p-nitrophenylarsonic acid could be distinguished from glomerular damage on the basis of urinary enzyme excretion [ 26f. The absence of significant glomerular damage following the administration of this agent was confirmed in the present study with histological techniques. Moody and Williams [3] suggested that when p-nitrophenylarsonic acid is fed to chickens it is reduced in the gut to arsanilic acid and eventually to the more toxic arsenoxides. In the present study p-nitrophenylarsonic acid was injected by the intraperitoneal route and direct chemical modification by the gut flora was avoided. Little is known of the metabolic fate of ~-nitrophenyla~oni~ acid in the rat and in p~ic~ar whether it is sign~ican~y reduced to ~~n~ic acid or arsenoxides. Further studies will be required to de~rmine whether p-nitrophenylarsonic acid or its reduction products are the major cause of nephrotoxicity. Few studies have monitored renal enzyme activities during the course of renal damage, although because of the high activities of many enzymes in the kidney, it has long been considered to be the principal source of enzymes excreted in the urine. The initial rise in fl-glycosidase activity in kidney homogenates (Fig. 2a) which followed the administration of p-nitrophenylarsenic acid was unexpected, since other studies [l&28] with the tubular nephrotox~ mercuric chloride, demons~~d that the injection of this agent resulted in an immediate fall in renal enzyme activities. This increase in activity is not seen when the enzyme activities are related to protein concentration (Fig. 4~). The expression of results in this way is common practice [ 271, but since protein is lost from the kidney during tubular damage and changes in weight of the kidney are also common, enzyme activities should be expressed as the total activity recovered per kidney and not related to protein or to kidney weight. Tbe increased enzyme activities in the kidney may result from the stimulation of protein synthesis or be caused by the invasion of cells rich in hydrolase activity into the renal tissue. At the dose levels used in the present study, the kidney was unable to fully compensa~ for the initial damage caused by the agent and the development of focal necrosis was eventually accompanied by loss of ki&iey enzymes into the urine. The assay of serum enzymes are of little value in assessing nephrotoxicity since no
253 si~~ic~t changes in the activities of these enzymes were found. The major source of urinary enzymes in renal damage would therefore appear to be the tubular cells. In the present investigation marker enzymes were used to study changes in the principal subcellular organelles in the kidney during the early stages of renal necrosis. A decrease in latency within the first twenty four hours was the earliest detectable change in the properties of subcellular organelles isolated after the administration of p-nitrophenylarsonic acid. An initial increase in lysosomal enzyme activity accompanied the fall in latency which may be a prelude to the loss of hydrolytic enzymes into the cytosol and eventu~y into the urine. This ~~b~ity is suppose by the fading that ~~cetyl~~luco~in~~ activity in the soluble fraction reaches a maximum value on day 2, whereas the lysosomal enzyme activity is greatest on day 1 (Fig. 2a and d). Calder et al. [ 28 ] demonstrated that the administration of paminophenol, o-aminophenol, quinols, catechols and their derivatives to rats, result in a similar type of necrosis in proximal convoluted tubules. These compounds can all participate in redox systems and these authors suggest that there is a relationship between their nephrotoxicity and oxidation-reduction potential. Although ~-nitrophenyla~o~c acid itself would not participate in oxidation-reduction systems the possibility that it is me~bol~ed to an in~~edia~ which could exert its nephroto~c effect in this way emnot be excluded. Indeed phenyla~enoxide is known to inhibit keto acid oxidation in rat liver mitochondria f 291. In a more recent study Crowe et al. [30] demonstrated that the nephrotoxicity of p-aminophenol administered to rats was triggered by mitochondrial injury and Verity and Brown [ 311 have reported that the administration of mercuric chloride results in a significant depression of the renal cytochrome oxidase activity, In contrast, in th present study recovery of cytochrome exidase was normal except for a small increase in the 24 h following the injection (Fig. ?a and b). The effect of ~-nitrophenyl~sonic acid on mitochond~~ function is certainly different from that of ~-~inoph~nol [303 and mercuric chloride f31), but requires more detailed study. The administration of p-nitrophenylarsonic acid results in focal necrosis which is characterised by an early fall in lysosomal latency. Hydrolytic enzymes are then, presumably, lost into the cytosol because of the increased fragility of tht! lysosomes and are eventually recovered in the urine. The elevated enzyme activities in the kidney recovered immediately following the injection may be the result of increased protein synthesis in the unaffected cells. These cells may attempt to compensate for the loss of functional cells in the damaged areas but as the necrosis spreads the expected fail in enzyme activities recovered in kidney homogenates occurred. The rat is a convenient animal for the study of nep~otoxicity and the excretion of urinary enzymes in addition to providing a sensitive screening procedure may also provide information en the localisation of the renal lesion. For example, a rise in the excretion of N-acetyl_P-glucosaminidase
254 in the absence of a corresponding increase in alkaline phosphatase would indicate that the lesion is local&d in the papilla [9]. The elevation of IVacetyl+glucosaminidase and alkaline phosphatase in the urine in the presence of normal &glucosidase activity would indicate that the principal lesion was glomerular since there is no detectable &glucosidase activity in the serum [26]. The simultaneous elevation of fl-glucosidase, alkaline phosphatase and IV-acetyl_P-glucosaminidase would indicate the presence of a lesion of the cortical tubules and these changes were demonstrated in the present study. The assay of a small group of enzymes therefore provides the most convenient and sensitive method of assessing renal damage without recourse to killing the animal. Additional information can be obtained by characterising the isoenzymes present in urine since &glucosidase separates as a rapidly migrating band found in kidney but normally absent from urine. Alternatively a change in the relative proportion of the isoenzymes OPlactate dehydrogenase [ 321 in urine is indicative of renal tubular damage. The results of the present study provide additional evidence that the enzymes excreted in urine as the result of the administration of a tubular nephrotoxin originate primarily from the kidney. They further emphasize the value of studies involving subcellular organelles in elucidation of the biochemical sequence of events occurring in the nephroxtoxic process. ACKNOWLEDGEMENT
This work was supported by a grant from the National Kidney Research Fund, U.K. REFERENCES W.C. McGuire and N.F. Morehouse, Chemotherapy studies in histomoniasis, Poultry Science, 31(1952) 603. N.F. Morehouse and W.C. McGuire, p-Nitrobenzene arsonic acid for blackhead control, U.S. Patent (1953) 2,638,432. 3.P. Moody and R.T. Williams, The fate of 4nitrophenylarsonic acid in hens, Fd. Co&met. Toxicol., 2 (1964) 695. D.$. Frost and H.C. Spruth, Synposium on medicated feeds, in H. Welch and F. Marti-Ibanzed (Eds.), Medical Encylopaedia Inc., New York, p. 136. C. Robinson, R.G. Price and N. Dance, Rat urine glycosidases and kidney damage, Biochem. J., 102 (1967) 533. B.G. Ellis, R.G. Price and J.C. Topham, The effect of tubular damage by mercuric cf;loride on kidney function and some urinary enzymes in the dog, Chem-Biol. Interact., 7 (1973) 101. 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, Chem.-Biol. Interact., 7 (1973) 131. J.M. Wellwood, B.G. Ellis, R.G. Price, K. Hammond, A.E. Thompson and N.F Jones, Urinary N-acetyl-p-glucosaminidase activities in patients with renal disease, Brit. Med. J., 139 (1975) 408. B.G. Ellis and R.G. Price, Urinary enzyme excretion during renal papillary necrosis induced in rats with ethyleneimine, Chem.-Bicl. Interact., 11 (1976) 473.
255 10 11
12 13 14
15 16 17
18 19 20 21 22 23
24 25 26 27 28 29
30 31 32
R.G. Price and 3.0. Ellis, Urinary Enzyme excretion in aminonucleoside nepbrosis in rats, Chem.-Biol. Interact., 13 (1976) 353. S.A. Kempson, B.G. Bllii and R.G. Rice, Changes in rat renal cortex, isolated plasma membranes and urinary enzymes following the injection of mercuric chloride, Chem.Biol. Interact., 18 (1977) 217. R.G. Price and N. Dance, The cellular distribution of some rat kidney glycosidases, Biochem. J., 106 (1967) 877. S.J. Cooperstein and A. Larzarow, A microspectrophotometric method for the deovation of cyhcha:me oxidase, J. Bid. C&m., 189 (1951) 666. W.E.C. Wacker and L.E. Dorfman, Urinary lactate dehydrogenase activity, 1. Screening method for the detection of cancers of the kidney and bladder, J. Am. Med. Ass. 181(1962) 972. H.N. Femley and P.G. Walker, Kinetic behavlour of calf in~sti~ akdine phosphata& with 4-methyl-umbelliferyl phosphate, Biochem. J., 97 (1965) 95. D. Robinson and P. Wilcox, 4-Methylumbelliferyl phosphate as a substrate for lysosomal acid phosphatase, Biochem. Biophys. Acta., 191(1969) 183. D. Robinson, R.G. Price and N. Dance, Separation and properties of &galactosidase, p-glucosidase, ~~lu~u~nid~ and N-a~tyI~~luco~minid~ from rat kidney, Biochem. J., 102 (1967) 525. H.E. Abrahams and D. Robinson, &glucosidsses and related enzymes activities in pig kidney, Biochem. J., 111(1969) 749. 0.11. Lowry, N.J. Rosebrough, AL. Faw and RJ. Randall, Protein me~urements with the Folin phenol reagent, J. Biol. Cbem., 193 (1951) 265. H. Schuel and R. Schuel, Automated determination of protein in the presence of sucrose, Anal. Biochem., 20 (1967) 86. A.C. Gromall, C.S. ~da~l and M.M. David, Destination of serum proteins by means of the Biuret reaction, J. Biol. Chem., 177 (1949) 751. S. Shibko and A.L. Tappel, Rat kidney lysosomes: isolation and properties, Biochem, J., 95 (1966) 731. P.J. Wright and D.T. Plummer, The use of urinary enzyme measurements to detect reteet rend d8Wtgt! caused by nephrotoxic compounds, Biochem. Pharmacol., 23 (1974) 65. W.P. Raab, Diagnostic value of urinary enzyme determinations, Clin. Chem., 18 (1972) 6. J.M. Wellwood, P.M. Simpson, JR. Tighe and A.E. Thompson, Effect of gentamicin nephrotoxicity in patients with renal allograft, Brit. Med. J., 3 (1975) 278. R.G. Price, N. Dance and D. Robinson, A Comparison of the p-glycosidase excretion during damage induced by p-nitrophenylarsonic acid and by rabbit anti-rat kidney antibodies, Eur. J. Clin. Invest., 2 (X971) 47. C. De Duve, Principles of tissue fractionation, J. Theoret. Biol., 6 (1964) 33. I.C. Calder, P.J. Williams, R.A. Woods, C.C. Funder, C.R. Green, K.N. Hamm and J.D. Tange, Nephrotoxicity and molecular structure, Xenobiotica., 5 (1975) 303. M. ~rnst~ng and M. Webb, The reversal of phenyl~noxide inhibition of keto acid oxidation in mitochondria and bacterial suspensions by lipoic acid and other disulphides, Biochem. J., 103 (1967) 913. C.A. Crowe, I.C. Calder, N.P. Madsen, C.C. Funder, C.R. Green, K.N. Hamm and J.D. Tange, An experimental model of analgesic-induced renal damage-some effects of p-aminophenol on rat kidney mitoehondria, Xenobiotica, 7 (1977) 345. M.A. Verity and W.J. Brown, Hg”-induced kidney necrosis, Am. J. Pathol., 61 (1970) 57. P.D. Leathwood, M.K. Gilford and D.T. Plummer, Enzymes in rat urine: lactate dehydrogenase, En~ymolo~, 42 (1971) 286.