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The effect of tubular damage by mercuric chloride on kidney function and some urinary enzymes in the dog
ChewBfol. 8
Intet%lctions,
7 (1973)
Scimtidc Publishing
Elrvicr
IOI-
I13
Company,
Amstcr
w--Printed
101
in The Netherlands
THE EFFECT OF TUBULAR DAMAG’E BY MERCURIC CHLORIDE ON KIDNEY FUNCTION AND SOME URINARY ENZYMES IN THE DOG
8. G. ELLIS,
R. G. PRICE
Depwtment of Biockemistry. W8 7AH and SK 10 4TG
AND
lPhurmacerrtlcals
fGrat January
16th. 1973)
(Revision
received
May
May
C. TOPHAM
Division.
College.
Imperial
lUniversity
of London),
Che. nical Industries Limited,
Cumpden Hill, A/de&y
London
Park, Cheshire
Brituin)
(Received (Accepted
lJ.
Queen Elizabeth
7th. 1973)
8th. 1973)
SUMMARY
Alkaline and acid phosphatases, &glucosidase, /?-galactosidase, A’-acetyl-,9glucosaminidase and lactate dehydrogenase Tvere monitored in the urine and serum of dog.. with renal tubular damage induced b) a series of increasing doses of mercuric chloride. Tsocitrate dehydrogenase, glutamatb oxaloacetate transaminase and glutamate pyruvate transaminase were assayed in the serum. The functional capacity of the kidney was determined by creatinine, inulin and p-aminohippurate (P/.H) clearances and tubular maxi na tests. Serum urea levels, total protein in the urine and the specific gravity of the urine were monitored throughout the study. The extent and location of the kidney damage was determined by histological investigation. Evidence is presented that the assay ofurinary alkaline and acid phosphatase is the most sensitive method of detecting renal tubular damage in the dog. The clearance of [‘*Clpropranolol was compared before and after the administration of mercuric chloride. In the presence of tubular damage the blood half-life of propranolol and the rate of excretion of metabolites in the urine were increased.
INTRODUCTION
The kidney nephron is divided into distinct morphological and functional regions, each having a characteristic complement of enzymes’.2. Damage to one of these regions results in the liberation of enzymes into the urine which are characteristic of that region of the nephron. Much interest has been shown recently in the use of the assay of urinary enzymes in the early detection of kidney damage and disease. Investigations on the excretion of lactate dehydrogenase3.4, alkaline* and acid’ phosAbbreviations:
PAH,
p-ominohippurate:
TmPAH.
tubular
maximal
rate of reabsorption
of PAH.
phatases, leucine aminopeptidase*, muramidase’, glutamate oxaloacetate transamina&‘, and several JI-glycosidases10-‘3 demonstrated that these enzymes are elevated in the urine following kidney damage in a variety of animal species and in man. SCHONFIELD~
has suggested that the output of urinary enzymes reflects the anatomic integrity of :he kidney and because the kidney has a large functional reserve capacity, urinary enzyme excretion should vary prior to any change in physiological function. RMB’*
has postulated that the assay of urinary enzymes provides a more sensitive method for the detection of kidney damage than histological studies. The morphological and functional changes which occur in rats following tubular damage induced by mercuric chloride have been studied by MCCREIGHT AND WiTQFSKi”, but these authors did not monitor urinary enzyme excretion. On the basis of previous experiments with n~ts’~*‘~ we believe that the measurement of enzyme excretion in the urine is in fact a more sensitive and an earlier means of detection of kidney damage than the commonly used functional tests such as clearances of specific substances or the blood levels of certain excretory products. In this and the accompanying paper”’ urinary enzyme excretion is monitored in tubular and papillary damage. The change in the excretion of urinary enzymes is compared with kidney function tests in order to determine whicn provides the more sensitive method of detecting kidney damage. Six enzyme activities are assayed in an attempt to establish whether a characteristic enzyme excretion pattern results from damage to a specific region of the kidney. Many drugs are excreted in the urine and impairment of renal function has been shown to alter their excretion distribution and pharmacodynamics”. Propranolol is a drug which is used in the management of hypertension and mcent studiesrs with [‘*C]propranolol have shown that in patients with functional renal impairment the rate of urinary excretion of total radioactivity (propranolol plus metabolites) is greatly reduced. while the half-life in blood of propranolol itself is also reduced. The effect of renai tubular damage induced by mercuric chloride in dogs on the pharmacodynamics of propranolol is now reported and related to the accompanying functional impairment and changes in urinary enzymes. MATERIALS AND METHOIX
Collection of urine and blood samples Aldcrley Park-bred beagle dogs (IO-13 kg) were housed individually in galvanised wire mesh metabolism cages, each supported over a large separating funnel. Water was available ud libitum and food was supplied in a bowl placed temporarily inside the cage. The base of the cage was a wire mesh platform through which faeces and urine could readily pass. A second wire mesh held back faeces but allowed urine to pass into a collecting flask which was maintained at 4”. 24-h urine samples were tittered through coarse filter paper and were used immediately for enzyme analysis or stored at - 10’. Blood was removed from tae cephalic vein and allowed to clot at room temperature for 2 h. Serum was separated. by low-speed centrifugation and stored at -20” until required.
liFFECTOOPMERCURlCCHLORIDEON DOG KIDNEYFUNCTION
103
Ittdttclion of tubrtlar damage
A series of doses of mercuric chloride was admimstered to 4 dogs. Initially a solution of mercuric chloride (0.5 mg/ml in sterile saline, adjusted to pH 7.2 with NaHCOJ) was injected into the cephalic vein in daily doses of 0.1 mg/kg. Because this procedure resulted in fibrosis of the blood vessels the injections were terminated after 7 days. The dogs were then allowed to rest until 6 days later when a capsule containing mercuric chloride (2.5 mpjkg) was administered orally to each dog and the dose repeated daily for the 9 subsequent days. Mercuric chloride (0.5 mg/kg) was injected into the jugular vein on the next 2 consecutive days; after 4 weeks each dog was given a single intravenous injection of mercuric chloride (2.0 mgjkg). Assays
The activities of /r-glucosidascrP(EC 3.2.1.21), /?-galactosidase*g(EC 3.2.1.23), alkaline phosphatasczO (EC 3. I .?.I), acid phosphata@ (EC 3.1.3.2) and N-acetyl/%glucosaminidase22 (EC 3.2.1.3C~ wers determined Huorimetrically in Serum and urine using the appropriate 4-methylum~~lliferyl substrates. All samples were diluted IO-fold except in the cast of alkaline phosphatase. where a IOO-fold dilution was used. The diluted enzyme sample (I ml) was incubated with the appropriate substrate (I ml) in buffer for 30 min at 37”. The reaction was stopped with glycine-NaOH buffer (pH 10.4, 3 ml). The fluorescence of liberated 4-methylumbclliferone waj determined at 440 nm in an Aminco-Bowman fluorimeter using quinine sulphate as standard (4 @ml in 0.1 N H2SOI). Lactate dehydrogenase (EC I _I. I .27) and isocitrate dehydrogenase (EC I. I. I .4l) were assayed on a LKB 860@reaction rate analyser using the methods of WROBLEWSK! AND LA DUE= and WOLFROM AND WILLIA~~+&HMAN’~ respectively. Glutamate oxaloacetate transaminasez’ (EC 2.6.1.1) and glutamate pyruvate transaminase23 (EC 2.6.1.2) activities in serum were determined spectrophotometrically. Total urinary protein was determined by the procedure of GROMALL et01.~~and creatinine in serum and urine by the method of BOSNFS AND TAUSSKY~'. Serum urea was measured according to CHANEY AND MARBACH’~. The specific gravity of urine was measured with a hydrome.er (range 1000-l 100). Estimation
o$ the clearance qf imrlin and pamittohippurate
The infusion methods used in the estimation of the clearance of inulin and PAH were adapted from procedures by LINGARDH'~ and described by VARLEY~". Each dog was given an initial subcutaneous injection of acetopromazine (0.5 ml).
After 30 min the dogs were anaesthetised with a single injection of sodium metheheuitone (5 ml) into the cephalic vein. An endotracheal tube was inserted and connected to a mixture of oxygen containing I-27; halothane maintaioed at a flow rate of 3 I/min. A solution of saline and dextrose (3W ml) was infused into the jugular vein over IO min. An infusion solution (30 ml) containing loo/, inulin, 20% PAH and isotonic saline (pH 7.2) in the ratio 5 : 2 : 30 was passed into a blood deserette inserted into the other jugular vein as a priming solution. More of this solution was then infused as a constant flow rate of 1 ml/mm for the duration of ihe test. The first blood
104
R. 0.
ELLlS ct al.
and urine samplea were obtained SO min a&r the infusion of the priming solution. Urine was colkctcd via n catheter and over a 15.mitt period. After 7.5 mitt 8 sample of Mood (4 ml) was withdrawn from the jugular deaerettc and shaken in a oxalated tube. Heparinised saline (2 ml) was injected into the dcselrtte after each blood sample was withdrawn, Plasma was separated from the oxalated. blood by lowspeed centrifugation. Six determinations were made for each clearance estimation. The dogs were allowed to recover and were given an injection of ampicillin as a prophylactic. lnulin and PAH concentration in urine and plasma were determined as described by vARl.E+‘.
Histoiogy All dogs were killed at th: end of the experiment and the kidneys and livers removed. Thin slices of these tissues were fixed in a large vobtme of formal saline. Paraftin sections of the tissue we, e prepared and stained with haematoxyiin and eosin. Blood levels and urinary excretiojz of [I 4C]propranolol and its metabolites [W]Propranolol labelled at position 1 in the naphthalene ring3’ (specific activity 3.86 ,uCi/mg) was diluted with carrier drug (non-labelled propranolol) such that each dog received 1 $i of labelled drug in a single intravenous injection (0.1 mg/kg). After [‘“C]propranolol administration, blood samples (2 ml) were collected every 30 min for 4 h and assayed for [‘4Clpropranolol as described by HAIFS AND COOPER~~. 24-h urine volumes were collected after the injection of propranolol and the total ‘*C in urine was determined by liquid scintillation countingsz. RESULTS
Enzyme activity in norm01dog urine and serum In order to determine whether low molecular weight inhibitors were present in the urine, &glucosidase, /3galactosidase, h’-acetyl+glucosaminidase, alkaline phosphatase, acid phosphatase and lactate dehydrogenase activities were assayed in urine before and after dialysis against cold distilled water for 4 h. No increase in enzyme activity could be detected at the urine dilutions used and the urine samples were not dialysed prior to assay. Average values for the daily excretion of the 6 urinary enzymes studied are shown in Table I. Alkaline phosphatase was found to be the most active urinary hydrolase and was six times more active than N-acetyl-P-glucosaminidase. Lower activities of acid phosphatase, &glucosidase and @-galactosidase were detected. Lactate dehydrogenase is included for comparison. The 6 enzymes assayed in the urine were also assayed in serum together with 3 additional enzymes-isocitrate dehydrogenase, glutamate pyruvate transaminase and glutamate oxaloacetate transaminase. The activities of these enzymes arc given in Table I. Alkaline phosphatase and N-acetyl-@glucosaminidase were again the most active hydrolytic enzymes.
105
ENZYMB ACTIVIIIUI IN NQRMAL
MCI “RING ANU SERUM
WUfits are expressedUS the mean f S.D. Sample number was !2. Urinary enzyme activities are expressedas @iofes ~methyi~~il~ferone l~~ral~~~~h urine sample,except tactate dehydro~nPM activ&s wkich art expressedas I.U./24-h urine sample, Serum enzyme activities are expressedas aBIrO& 4methylumbellifbrone liberated/h/ml of serum Pxcept for iacfate dehydrogenase,iz&trate dehydro~na~ g~u~m~te pyruvate traosami~asc and Elutamate ox&acetate tran~mioase which are cxprassedas l.U,lmt serum. ” “. ,. _^._^ .._____,._ .._.... ..__, “l.l.. .” I. _. ..__“_ _.___, Urine SWWN .._. “. ...l .__.,.^_l_l “._, ..___... “_.,”_“.. ._.“” _ &Glucosidase 5.0 rfr 0.6 ‘2.1 & 0.3 ~Galactosida~ 4.9 f 0.3 3.8 & 0.5 33.8 ri: 6.8 N-acetyl.~-SIueo~minidase 125.0 f 13.u 201.0 * 31.0 AI+aline phosphatase 133.0 f 14,o 9‘7 f I.8 43.0 f 9.6 Acid phosphatase 59.0 i 7.0 Lactate dehydro~n~e 12.6 f 2.0 bocitrate dehydrogenase n.d.’ 6.4 & 0.5 Glutamate pyruvare transaminase n.d. 14.2 * 1.3 Glutamate oxaloacetate 14.4 * 0.5 n.d. transaminse ._ .-__- ..- _..-.. -- .-- .._. -....-...-. -. ..-.-.-...- ._ ..- -. * n.d., not determined
The rate of ctearance of creatinine, inulin and PAN was monitored in normal dogs together with the Tm PAH. The values obtained are shown in Table II: also included are the normal levels of urea in the serum and protein in the urine. Daily urine volumes were measured and the specific gravity of these samples monitored. Ail the parameters included in Table II are commonly used to detect and diagnose kidney damage and disease.
The changes in the pattern of excretion of a number of enzymes during gradually increasing mercuric chloride intoxication are shown in Tables III and IV. Serum UWd and urinary protein levels monitored over the same period are included for comparison, Serum enzyme activities, urinary volume and specific gravity were measured throughout the serial injection of mercuric chloride and kidney function tests were regularly performed on at1 4 dogs. The serum enzyme activities and kidney function TABLE II RENM..FUNCTION ,N NORMAL DOGS data are presented as the mean f S.D. The units and number of determinations are givea ia parentheses. The
____.____--.--..--
-._.
Clearance creatinine (miiminikg~ Ctearance inulin ~ml~min~kS) CtearancePAH ~ml/min/kg) Tm PAN (mllminlkg) Serum urea (mg %I Urine protein fmg/24-h urine sample> Urine volume (ml/N-h urine sample) Urine specificgrmity ,,____ ~~_.~..~ _,__..^” _...._..-.-
-_I.. .-
0.39 (20) 3.24 & 0.42 (81 3.89 & 1.0 (8) 8.8 ; O.il (8) 0.5 5 3.9 (20) 34.4 117 (20) i 15 * 125 (20) 600 0.003 (20) 1.026 + ___~~ -.
l
I.3
2.0 -.
f 9.6 * 1.4 * 3.1 * 3.3 * 2.2 f 3.5
8.1 t
n.d.. not determined
_.
f
6.4 n.d. 14.7 I 5.0 7.1 12.5 7.1 10.6
ii 23 24 25 26 27 28
29 ___ _.-
& ;t rt f * *
4.7 4.9 8.7 5.4 13.8 11.7
15 16 17 I8 19 20
0.4 1.2 20 0.3 3.5 4.5
9.4 f. 1.6 11.0 f 2.8 3.4 * I.1
0.2 0.9 1.0 0.8 5.5 1.0 1.3 1.0 3.4
IO II 12
fi f It * & f f &
4.3 4.5 5.0 6.2 12.2 8.5 8.1 9.1 12.6
: 3 4 5 6 7 8 9
0.5
0.2 0.2 0.4 0.4 0.1
3.6 2.9 3.3 3.1 3.7
3.7 f 0.1 n.d. 3.4 f 0.3 4.8 =t 0.9 4.1 f 0.6 4.9 IO.6 2.7 z&z0.2 3.1 & 0.3 3.5 rt 0.2
4.8 $ 0.3 4.9 * 0.5 4.6 + 0.5
f f f f f
4.8 f
f 0.3 * 0.3 i 0.3 f 0.2
5.9 4.7 4.5 4.2
0.5 0.6 0.2 0.3 0.2
f f i f +
5.4 4.8 4.6 4.3 3.9 10.0 16.1 8.2 9.6 13. 48 7.1 5.0 6.7 6.7 8.7
54.9 f 52.9 f 50.1 f 55.1 i 4’13_e
53.2 + 57.7 *
36.2 40.1 49.8 64.7
5&l 94.6 57.6 41.4 uI.0
0.4 30
83
35
+ 0.g f 22 i I2 f 10 f 3.6
39.1 f n.d. 45.3 + 87.7 +
47.3 f
45.0 -t 3.1 49.1 f 10.4
f rt f A-
2.0 2.0 3.5 2.7
f I_ f &
40.1 30.4 39.6 53.4
41.0 i 33.0 f IL0 f
1477 rf: 410 1079 f 380 6oOf 2cm
-.
13 I5 8.6
f 8.9 + 16.3 f 12.8 + 11.0
222 n.d. 24.5 45.6 34.1 127.9
______
1.7 10.8 0.7 0.7 2.9 7.6 5.7 14.4 4.8
2.6 23 2.1 2.6 5.5 3.1 2.2 3.6 4.6
It. 3.3
17.2 f 22.6 i 8.0 f 8.0 & 17.0 f 21.3 f 20.6 f 30.6 -t 211.1f
* I .:
10.6 f 9.8 f 8.3 f 8.6 i 15.3 f 19.1 * 13.1 * 14.3 f
1296* I66 ad. 1119* 3xl 14071;; 364 15P9* 500 3428 rt II22
70 187 30 45 177 271 105 635 267
78 ‘dC
5011 -,., :
698 * 587 I 201* 27?J& 643& 814f 541 rt 1382 f lll7&
29 49 50 % 159 I05 72
2llf 156+ 18Ot 2411f 459 f 575 f 406f
2.9
3.6
. __
l 2.0
.- _.-.
8.0
68f2.6 14.4 l 4 8.5 ;1;22 1.7 f t.4
17.5 f n.d. 10.4 f nd.
7
6 6
II 5 4 3 II
102f 82i 75 f 138 f 95f nd. IlO&
qd.
IO
5 I4 18 I5 7
I58 It I2
123f19
139& 13.7 + 3.5
18.9 + 5.1
117*24 72f no* PO& 97% 136 i 139i
f f 5 f f f
f
I I4 +I 22 120 rt 17 103 :i I2 93i IS
ljl
08f 7 1:2j,21 w*l2 91* 7
12 3.3 1.4 26 5.2 2.9
8.3 9.2 12.5 8.0 14.4 14.3
I.1
t 1.0 * 2.0 f 2.6 f 2.0
5.4 11.4 9.0 8.6 9.8 f
f I.6 & 2.1 f 1.4 f I.0 -Ir 13
II.2 6.8 12.7 5.9 8.5
21 31 n.d. _w n.d
29 nd. nd 39
26
29 29 n.d. 23 26 20
I9
n.d.
29 n.d.~ 31 n.d.
39 30 35 29 24
-
2.5 mdtg u mdL8
t5raJb 25e 2.5 -8 2.5 wg
-
0.1 mg/kg 0.1 mgJkg 0.1 q/kg
0.1 mg/kg
0.1 mg/kg 0.1 mgflcg 0.1 mg/kg
-
R
7.8 13.1 32.8 13.4 6.8 8.9 10.0 8.8
i f 5 f. f & & f
4.0 4.9 10.0 3.6 1.3 3.8 2.0 1.8
_--
& 0.4 * 0.9 & 3.5 f 0.7 & 0.9 f I.5 f 1.0 f I.1
---_.
2.5 6.4 8.5 10.1 6.9 5.3 5.6 4.5
21.4 + 56.1 & 145.2 f 221.4 * 97.0 l 55.8 f 57.9 f 66.3 + .-_....
3.5 4.6 19.0 31.0 21.0 1 I.5 8.9 2.7
.-._.
588 f 280 777 f 481 4106 f I712 3319 i 1197 1437 rt 3271 945 f 293 1713 If: 297 977 + 2% __-- - .-. _ ___-.
.-_-
Alkoliru
IS.9 * 5.0 23.0 -r_ 4.5 194.6 f 52.0 63.5 f 28.4 62.0 f 11.3 60.8 rt 9.2 73.8 rt 6.9 57.5 f 11.0 ___-...
7.5 f I.8 14.2 * 0.6 251.0 + 8.7 346.0 * 20.0 112.0 f 56.0 21.5 f 13.0 19.0 + 6.0 14.2 f 2.8 .___.
l7Si 70 253 c 45 1724 f 471 2217 + 931 449 * I.44 363 * IO7 330*100 29srt 40
24.0 24.0 30.0 64.0 130.0 144.0 128.0 39.0 _-
* 4.0 rfr 3.1 5 5.3 * Y-3.0 f 61.0 f 73.0 + 31.0 * 6.3 . --
1.1
IS.1 *
I.!
16.1 i
_._ _.
14.6 Jr 2.4
,-20 i IS *I5
15.3 $- 3.0
I20 15s 44
3.0 i
.--.-_ 0.7
..-.--_
89 5 I4 4.9 5 0.9
i 21 c 13 r”l3
I.1
60 __I-.
--.-.
64 -23 5.5 f I.3
IiS 165 37
2.5 i:
_._-.
59 ..-.--
Lactate dchydrogenase lsocitrate dchydrogcnax Glutamate pyruvate transaminase Glutamate oz.&acetate transaminase _. .____ ._.. -* n.d., not determined
---. -. _ .-.. fl-Galactosidxc N-ZiWyl-,Uglucosaminidase Alkaline phosphatase Acid phosphatase
-._-
33.0 _i 4.3
3.0
k S8 f 9.3
12.3 f
98 87
* 19 h48 k.2
_ ___ 3.5 f I.1
153 165 48
61 0.6
4.0 25.0 * 13.9
13.5 *
64 i 12.5 17.0 * 3.4
+ 18 I27 fl7
4.4 f I59 184 28
62
18.0 It
14.3 *
46 f 6.8 It
. I.0
2.4
3.3
7.8 0.9
* 59 f32 f 30
4.5 * 113 167 45
63
_..__-_
0.4
16.0 *
12.5 f
2.0
2.9
6.0 0.8
f I4 i 20 f 20 4s f 6.0 f
Ii6 180 37
64 __. 3.6 f
0.7
f 18
16.5 f
1.8
13.8 rfr 3.1
70 fl3 S.8
n.d: ISI C.d.
65 __ _.... 3.3 f
-
n.d. _
n.d.
n.d. 2.:
IS3 140 50
64 _.._-._ n.d.
..__
f 19 * 13 f18
_.._
THEAC~VIT,~ OF * NUMBEROF SERUMENZYMEIN DOCISrot,t.owm~ THEFIN*L INTRAVEN~WM~LC~,ONOF WLERCURIC CHLO~DE(2.0 mglks) ADI(MISTRED ON DAY60 Activities ate expressed as in Table I. The number of dogs used was 4. /?-Glucosidase was not assayed.
TABLE V
59 60 61 62 63 64 65 66 -..
-. .- _.----.
__..
IO8 test
8. G. IiLLIS
values
el fJ/.
found after the final injection of mercuric chloride ure given in Tables V
and VI respectively. A comparison of Tables I and 111shows that both acid a Id alkaline phosphatase activities were elevated in the urine 48 h after the first injection of mercuric chloride (0.1 mdkg) and the activities of these enzymes continued to rise with each subsequent injection. The highest levels were recorded after the 6th injection. The activities of both enzymes returned to normal 72 h after the final injection. /?-glucosidase and N-acetyl-r!Lglucosaminidase were also elevated, although the excretion of these enzymes fluctuated. The activity of /I-galactosidase and lactate dehydrogenase remained within the normal range as did the excretion of urinary protein. No change in serum urea or enzyme activities was detected at this dose level. Glomerular filtration rate measured by the rate of cicarance of creatinine and inulin remained normal as did Tm PAH and the rate of clearance of PAH. Urine volumes and specific gravity fall within the normal range (Table If). Oral dosing of mercuric chloride (2.5 mg/kg) was carried out over a IO-day period and urinary alkaline phosphatase activity rose gradually to values which were 7 times greater than normal (Table III). Acid phosphatase, P-glucosidase, N-acetyl/?-glucosaminidaseand lactate dehydrogenase activities were also elevated (2-J-fold) but urinary /?-galactosidaseactivity was within the normal range. Again no significant change was found in kidney function, serum enzyme activities, serum urea, urinary protein or urinary specific gravity. Some enzyme activities were still slightly elevated (Table III) when the next stage of the dosing regimen was started. The intravenous injection of mercuric chloride (0.5 mg/kg) on consecutive days resulted in an immediate and marked elevation of all the urinary enzymes studied except &galactosidase. Alkaline phosphatase (17-fold) and acid phosphatase (IZfold)
TABLE
were constdernbly elevated above
\‘I
RENAL FIJVCTION IN DOGS FOLLOWINO (2.0 mglkg) AC~~TERED ON DAY 60 Results areexprewd
as in Table
THE FINN,
II. The number
INTRAVENoUS
lWECT,ON
cw MERC”R,C
of dogs injected was 4. Where nc S.D.
than 4 do@ were used and fhc values shown represent
CHLORIDE is shown
less
a mean of 2 or 3 dogs.
l-r,1 PAH
n.d.=
n.d.
o.d.
1.026 + 0.003
3.78
9.0
0.58
I.028
IT 0.004
1.32
2.10
0.30
I.016
+. 0.003
0.25 .+ 0.17
1.022 * 0.003 I.018 2. 0.002
61
3.43 i 0.37 _t 0.21 1.60 ,. 0.33
62 63
0.80 0.74
It 0.26 f 0.33
0.51
64
1.39 If: 0.41 1.20 + 0.32
n.d.
1.95 jz 0.52
n.d.
59~60
65 66
3.86
’ n.d.. not determined
0.27 1.52
x 0.22
I.0
& I.1
2.4 ad.
0.44
3.5 n.d.
0.48
n.d. n.d.
I.020
-; 0.001
I .018* I.019 f
0.002 0.002
their aetivity iir normal urine and smaller increases in ~-glucosidase (bfold), N~cetyl~~-gluco~minida~ C&fold) were also found. Lactate dehydrogenase activity was elevated @-fold) in 2 of the 4 dogs used in this study, but again no change jn @-galactosidase activity was found. In contrast to the earlier doses of mercuric chloride, the injection of 0.5 m&kg resulted in changes in some of the other parameters studied as well as the elevation of urinary enzymes. In one dog which excreted the highest activities of urinary enzymes an elevation (Zfold) in serum urea was found and the rate of clearance of creat@ne fell below the normal range (Table 1111to I .3ml/min/kgon the day following maximum excretion of urinary enzymes (day 27) but had returned to normal 24 h Iater. TWQ serum enzymes, acid phosphata~ (3.fold) and ~-a~tyl-~-glucosaminida~ (Z-fold) were slightly elevated in all 4 dogs immediately aker the injection. Following a rest period of 30 days all 4 dogs were given a single injection (on day 60) of an increased dose of mercuric chloride (2.0 mgikg). Urinary alkaline phospbata~ activity was still elevated prior to the injection. Massive increases in urinary alkaline phosph~lta~ (2O_fold), acid phosphatase (20-fobtd) and lactate dehydrogenase (30-fold) activities above the normal followed the administration of this dose (Table IV), Lower increases in @-glucosidase f7-fold) and N-aeetyl-@-glucosaminida~ (S-fold) activities were alsa found, together with a slight elevation of &I-galactosidase activity (2-fold). Proteinuria coincided with the increased excretion of enzymes and reached a maximum of 2.0 g/day 48 h after the injection. The elevation of enzyme activities in the urine preceded a significant rise in urea in the se?um (Table IV). Isocitrate dehydrogenase activity was elevated 14”fold In the serum and smaller rises in lactate defiydro~enese and glutamate oxaloacet%te traa~~minase (Table V) activities were also observed on days 61 and 62. Renal function was impaired in all 4 dogs (Table VI) following the final injection af mercuric chloride. The rate of clearance of creatinine, inulin and PAH fell significantly within 48 h of the injection as did Tm PAH. The specific gravity of the urine also felt within 24 h of the injection. Renal function test values were still abnormal 10 days after the injection but urinary enzyme excretion fell steadily from the peak values which were recorded OR day 61 (Table IV).
Examination of the kidneys of all 4 dogs at the termination of the experiment revealed a marked dilation and some basophilia of the tubules in the medullary rays of the cortex. There was some interstitial inffamm~tion in the medullary ray and small areas of inflammation in both the medulla and papilla. A number GCtnb;>les iu the medulla were dilated and seven Jf these contained hyaline casts but no glomeruiar damage was detected. The livers of all the dogs were also examined but were found to be normal.
E&x? of tubular dunmge on the excretion of ~14C~~ro~r~~o~oj The rate of excretion of ~14C]propranolol from normal dogs was monitored
and the half-life of [*~CJpropranolol in blood and the excretion ofzotai W (propranolol plus metaboiites) into urine is shown in Table WI. The functional status of the kidneys and excretion of alkaline phosphata~ in the urine was atso monitored in ail 4 dogs (Table VII). After the administration of mercuric chloride both the propranolol blood half-life and the rate of urinary excretion of total ‘+C in urine were increased (Table VII). The magnitude of the increase in these parameters appears to be related to the degree of damage to the kidney as measured by kidney function tests and to the level of excretion of alkaline phosphatase in the urine.
The a~nistration of gradually increasing doses of mercuric chloride to dogs resulted in the elevation of urinary enzyme activities prior to any changes in the other parameters measured. The extent of the elevation was directly related to the magnitude of the dase. The most sensitive indication of tubular damage in the dog appears to be the excretion of atkaline and acid phosphatase which are the only enzymes elevated in the urine at the lowest dose used. Increase in dosage resulted in rises of /&lucosidase and N-acetyI+gJucosaminidase activities, but a further increase was necessary before an elevation of Lactatedehydrogenase took place. Abnormal Bgalactosidase activities were only detected following the administration of the highest dose. In contrast to the urinary enzymes, serum enzymes were unaffected by the two lOtVi2i &XX%, bu: ma!! increases of serum lactate dehydrogenase and glutamate oxaloacetate transaminase were found following the administration of the highest dose of mercuric chloride (2 mgjkg), together with a massive elevation of isocitrate dehydrogena~. Although slight increases (2-Fold) in acid phosphate and N-acetyl~-gluco~minida~ activities were occasionally found in individual dogs, the elevation of serum isocitrate dehydrogenase was the only significant change in serum enzyme TABLE VII THE R.%TE09 LOSSOS [‘~c]FROPRA~OLOL FROM TM BLOOD AND THE EXCRETIONOF ‘*c TOTAL IN THE URME OF 4 DOGS IWORE AND AFTER THE INTRAVENOUS~NJEC~CJN OF MERCURIC CHLORIDE (2.0 m&kg)
‘*C-propranolol was administeredas a single intravenous injection (0.1 m&kg). Renal function test zbta arc cxpresscdas in Table I1 and urinary alkaline phosphataseactivities as in Table 1,
Dog
Urittary .-_---_-__.-.
.._
ClearanceClearance iwlin PAH Nmmal
^_.__.._ Ttn
PAH
alkalitte phosphatme activity
40 60 60 60
30 44 59 45
6.7 8.0 8.4 5.9
2.90 3.20 2.80 2.30
0.40 0.30 0.49 0.40
180 252 179 115
24 h after J 53 injection 2 77 of mercuric 3 chloride 4 64 --___ _ _.. _ sn.d., not determined
41 70
I,1 2.0 2.4
0.80 0.90 0.92
0.18 0.11 0.3 I 0.35
ill0 6700 1450 330
1 2 3
4
53
ad.’
Loo
EFFECT
OF MERCURlC CHLORIL)E
ON DOG KIDNEY FUNCTtON
Ill
activity found in this study. The changein serum zsocitrate dehydrogenase activity was only detected when severe kidney damage was present and the other commonly u~l prmedures for the detection of kidney damage and disease were no more sensitive than the assay of serum enzymes. Significant changes in Serum urea and proteinuria were again only detected after the highest dose of mercuric chloride: si~:arly, changes in the functional capacity of the kidney were only recorded when extensive tubular necrosis was present. Sequential studies in the rat of the histological changes produced by mercuric chloride (ELLIS, unpublished results) showed that the initial area damaged was the terminal straight portion of the proximal convotuted tubules, but necrosis spread to the distal convoluted tubules and the medulla in time. These results may provide an explanation as to why, in the studies described in this paper, only certain enzymes were elevated at the lowest dose Ievel, whilst all the urinary enzymes studied were elevated at the higher dose level. Alkaline phosphatase and acid phosphatase are present in the proximal convoluted tubule of the dog in high activities33a34 and mild damage to this region would result in their excretion in abnormal amounts in the urine. More extensive damage would result in their further loss from cells over a greater area of the tubule, together with other enzymes which may have a different subcellular localisation. The data presented in this paper demonstrate that the assay of urinary enzymes provides the most sensitive method of detecting tubular damage. In the dog the elevation of urinary acid and alkaline phosphatase activities gave the earliest indication of kidney damage produced by mercuric chloride intoxication, whereas previously it was shown that the assay of urinary &glucosidase can be used to monitor tubular damage in the rat j3. The fluorimetric assays of these urinary enzymes areeasy to perform and could be readily inco~orated into toxicological trials or used for the differential diagnosis of kidney disease. A recent study investigated the handling of propranolol in patients with renal failure and it was considered of interest to compare the excretion of this drug in dogs with well characterised kidney damage. Propranolof is rapidly meta~lised in dog and man to 4-hydroxypropranolol, naphthoxylactic acid and an acid-labile conjugate of a mixture of propranolol and ~hydrox~r~pranolol which is identical to the major urinary metabolite. Only traces of propranoiol and ~hydroxypropranolol are found in urine. The main urinary components are naphthoxylactic acid and tie partially characterised conjugates in both species3z*J5. The more rapid excretion of metabolites in urine and the increase in proprano101blood half-life in dogs after tubular damage was unexpected as it has been reported that the rate of urinary excretion of metabolites in patients with renal funCtiORa1 impairment was greatly reduced and the blood half-life of propranolol itself was reduced. The reason for this difference may be that the metabolites in the filtrate are not reabsorbed because of the tubular damage caused by mercuric chloride (although glomerular filtration proceeds at a reduced rate), resulting in an increase in the rate of urinary excretion of metabolites. On the other hand filtration of metabolites would be reduced in the presence of giomerular damage, while reabsorption by the tubules
B. a.
112
ELLIS Ct a/.
would be normal, so that the rate of urinary excretion would be towered as observed by THOMPSONet al lb. The changes in the pIalma half-life of propranolol observed may be due to an alteration in the volume of distribution of propranolol. For example,
patients with severe glomerular damage may have a smaller volume of distribution than normal patients, because of a change in the ratio of the distribution rate constants. This change may also lead to a decrease in plasm half-life of the drug”. The pharmacodynamic consequencesof renal tubular damage have not been investigated and the reason for the increase in plasma half-life of propmnolol in dogs under these conditions is not understood. It is concluded that the consequencesof renal functional impairment on the pharmacodynamics of propranolol may be dependent on the site of the damage within the kidney. ACKNOWLEDGEMENTS We acknowledge
a Science Research Council Studentship (CAPS) to B. G. U. M. FOULKES for their kind
ELLIS. We thank Professor D. ROBINSON and Dr.
interest and encouragement and Dr. J. S. L. FOWLER fo:. assistance with the kidney function tests. We are indebted to Miss R. V. H. BARLOW for assistance with the histological studies and Mrs. S. LONGSHAW and Mr. I. M. OLIVER for technical assistam. REF&RENCES H. MATTENHEIHER.in U. C. DUBACH (Ed.). &mymcr in Urine und Kidney, Huber. Bcrnc, 1968. p. 119. H. MAITENHEIWER, V. E. POLLAKE AND R. C. MUEHREKE. Quantitativeenzymepatternsin the nephron of the healthy human kidney, Neplnon.. 7 (1970) 144-154. E. AHADOR, L. E. DOI~FMAN AND W. E. C. WACKER.Urinary alkaline phosphutascand LDH activities in the differenrial diagnosisof renal disease,Ann. Inrem. Med.. 62 (1965) 30-40. L. E. D~IIFMAN, E. AIUWR AND W. E. C. WACYER, Urinary lactic dehydrogenaseactivi(y. 1. Am. Med. Ass.. 188 (1964) 671-676. C. BREEDIS. C. M. FL.ORYANDJ. FURTH, Alkaline phosphatasclevel in urine in relation 10 renal injury, Arch. Pnrhol., 36 (1943) 402-412. M. R. SCHMNFELD.Acid phosphataseactivity in ureteral urine. J. Ant. Med. Ass., 193 (1965) 618-621. L. J. G~EENB~RO.Fluorometric measurement of alkaline phosphatase and aminopeptidase activities in rhe order of lo-‘* mole, Biochcm. Biophys. Res. Commun., 9 (1962) 430-435. J. P. H*JLIZT.P. E. PERILLI~ANDS. C. FINCH. Urinary muramidase and renal disease,Ncm &ag/. J. Med., 279 (1968) 506-512. L. F. PRESCOTTAND S. ANSAFX The effects of rcocated administration of mercuric chloride on exfoliation of renal tubular cells and urinary glutamic-oxaloacetictransaminase activity in the rat, Toxicol. AnpI. Phannacol., 14 (!969) 97-107. D. ROBI)CSON. R.G. PRIC~ANON. DANCZ,Rat-urine glycosidasesandkidneydamsge.Bloche/f~.1.. 102 (1967) 533438. R. G. PRICE,N. DANCE,R. R~cfl~aos AND W. R. CA~~ELL.The excretion of N-acctyl-&@cosaminidasaand fl-gdlactosidasefollowing surgeryto the kidney, C/in. Chin!. Acra,27 (197016 i-72. N. DANCE. R. 0. PRICE.W. R. CATTELL. J. LANSDELL AND B. RICHARDS, The excretion .*f Nacetyl-&glucosuninidaseand fi-galanosidaae by patients with renal disease, C/in. Chim. Attn. 27 (1970) 87-92. R. G. PRICE.N. DANCEAND D. ROBINSON,A comparison of the fl-glycosidaseexcreticn durin(( kidney damage inducedby 4-nitrophenylarsonic acid and by rabbit anti-rat kidney antibodies. EuropeanJ. CIltt. Inwr., 2 (1970) 47-51.
EFFECTOF MERCURICCHLORIDEON DOG KIDNEY PUNCTION
113
14 W. !?. RAAa. DiagnOStiCVahtc of Urinary Cnyae delcrminations. C/in. Chrm.. 5 (1972) 5-25. IS C. I!. bfcC*PtOtlT AND R. I.. W~mnXt. Scquencc of morphological and functional changes in fend epi~hella following henvy metal poisoning, in W. Now~mrr AND R. cou (ws.) Cornpemaror~ Renal Hypwtrophy, Academic Press. London, 1969. pp. 251-269. 8. G. ~LLts. R. G. Patt’t ANT)J. C. TOPHAM,The e&cl of papillary damage by ethyleneimineon kidrcy function and some urinary cnaymorin the dop, Chem.-Biol. Ioteroctlotts, 7(1973) 131. M. GlSALot AND D. PERRI~R.Drug distribulion nnd renal failure, 1. C/in. PbormocoL, 5.6 (1972) 201..204. F. D. THOMPSON. A. M. JO~WUAND D. M. FOULKES.Pharmacodynamicsof propranolol in renal failure, &if. Med. J.. 2 (1972) 434-436. D. ROBINSON.R. G. PRICEAM) N. DANCE. Separation and properties of P-galactosidase,/I&tcosiducR. /I-glucutonidase and N-acelyl-fl-glucosaminidascfrom rat kidney, Bioch~~n.1.. 102 (1967) 525-532. 20 H. N. FERNLEY AND P. G. WALKER.Kinetic behaviour of calf-intestinal alkaline phosphatase with 4_n~thylu~.~belliferyIphosphate, Biochr~tr.J.. 97 (1965) 95-103. 21 D. KJaIYsONAND P. WILCOX,4~Mcthylumbellirerylphosphate as a substrate for lysosomalacid phosphatasc, Biochtm. Eiophys. Acto, 191 (1969) 183-186. 22 D. H. LEA~ACKAND P. G. WALKER.The Ruorimetric assay of N-acetyl-/I-glucosaminidase. Elochrm. J., 78 (1961) 151-156. 23 F. WRO~LEWSWI AND1. S. LA DUE. Serum glutamic pyruvic transaminasein cardiac and hcpatic disease,Proc. Sot. Exptl. Biol. Med.. 91 (1956) 569-571. 24 S. K. WOLFROMAND H. G. Wtt,LtAMs-AsHMAN, lsocitric and 6-phosphogluconicdehydrogenases in human blood serum. Proc. Sot. Exptl. B/o/. M-d.. 96 (19S7) 231-234. 25 A. KAIIMEN,Note on spcctrophotometricassay of glutamic oxaloacetic transaminare in human blood xrum, J. C/lo. Invest., 14 (1955) 131-133. 26 A. C. GROMALL.C. S. BARDAWILLANDM. M. DAVID. Determination of serum proteinsby means of the Biuret reaction. J. Biol. Chem., 177 (1949) 751-766. 27 R. W. &xNEs AND H. H. TAUSSKY.On the colorimelric determination of creatininc by the Jaffe reaction. 1. Biol. Chcm.. I58 (1945) 581-591. 28 A. L. CHANEYAND E. P. UAR~ACH. Modified reagents for the determination of urea and ammonia, C/in. Chem., 8 (1962) 130.132. 29 G. LINOARDHAND L. ANDER~~~N.Urinary enzyme studies following renal ischaemia in dog, SN&. 1. Ural. Nephrol.. 2 ( 1968) 165-l 72. 30 H. VARLEY,Chemical (cs(s in kidney disease,in frocticol Cfinicd Biochemistv. 4th ed.. Heinemann, London, 1967, pp. 168-189. 31 J. BURNS.The preparation and puriticationof labelled &adrcnergic blocking agents. I. Synthesis of carbon-14 and tritium labelled I-isopropylamino-3-(I-naphthyfoxy)-propan-2-01 hydrochloride propranolcl hydrochloride, Inderal. J. Lobe//cd Camp., 6 (1970146-W. 32 A. HAY= AND R. G. COOPER.Sludics on the absorption, distribution and excretion of Propranolol in rat. dog and monkey, J. Phormocol. E:rprl. Thcr.. 176 (1970) 302-31 I. 33 P. J. C. VAN BRED&VRIESHAN,M. LINK AND R. G. J. WtLLtattAGEN.Histochemical studies Of canine renal homotransplanrswith special reference to alkaline phosphataseand its isoenrymcs, Traosplaototion.
34 35
5 (1967)
420434.
P. J. C. VAN BREDAVRIESMANAND R. G. J. WILLICHAG~N.Activity of alkaline phosphataw and isocnzymepattern of normal dog glomerulus. &it. J. Exptl. Pothol.. 49 (1968) 324-330. J. W. PA.I-~EERSON, M. E. CONNELLY.C. T. DOLLERY,A. HAYESAND R. G. COOPER.The Pharmacodynamicsand metabolismof propranolol in man, Phormocol.. C/in.. 2 (1970) 127-134.
Intet%lctions,
7 (1973)
Scimtidc Publishing
Elrvicr
IOI-
I13
Company,
Amstcr
w--Printed
101
in The Netherlands
THE EFFECT OF TUBULAR DAMAG’E BY MERCURIC CHLORIDE ON KIDNEY FUNCTION AND SOME URINARY ENZYMES IN THE DOG
8. G. ELLIS,
R. G. PRICE
Depwtment of Biockemistry. W8 7AH and SK 10 4TG
AND
lPhurmacerrtlcals
fGrat January
16th. 1973)
(Revision
received
May
May
C. TOPHAM
Division.
College.
Imperial
lUniversity
of London),
Che. nical Industries Limited,
Cumpden Hill, A/de&y
London
Park, Cheshire
Brituin)
(Received (Accepted
lJ.
Queen Elizabeth
7th. 1973)
8th. 1973)
SUMMARY
Alkaline and acid phosphatases, &glucosidase, /?-galactosidase, A’-acetyl-,9glucosaminidase and lactate dehydrogenase Tvere monitored in the urine and serum of dog.. with renal tubular damage induced b) a series of increasing doses of mercuric chloride. Tsocitrate dehydrogenase, glutamatb oxaloacetate transaminase and glutamate pyruvate transaminase were assayed in the serum. The functional capacity of the kidney was determined by creatinine, inulin and p-aminohippurate (P/.H) clearances and tubular maxi na tests. Serum urea levels, total protein in the urine and the specific gravity of the urine were monitored throughout the study. The extent and location of the kidney damage was determined by histological investigation. Evidence is presented that the assay ofurinary alkaline and acid phosphatase is the most sensitive method of detecting renal tubular damage in the dog. The clearance of [‘*Clpropranolol was compared before and after the administration of mercuric chloride. In the presence of tubular damage the blood half-life of propranolol and the rate of excretion of metabolites in the urine were increased.
INTRODUCTION
The kidney nephron is divided into distinct morphological and functional regions, each having a characteristic complement of enzymes’.2. Damage to one of these regions results in the liberation of enzymes into the urine which are characteristic of that region of the nephron. Much interest has been shown recently in the use of the assay of urinary enzymes in the early detection of kidney damage and disease. Investigations on the excretion of lactate dehydrogenase3.4, alkaline* and acid’ phosAbbreviations:
PAH,
p-ominohippurate:
TmPAH.
tubular
maximal
rate of reabsorption
of PAH.
phatases, leucine aminopeptidase*, muramidase’, glutamate oxaloacetate transamina&‘, and several JI-glycosidases10-‘3 demonstrated that these enzymes are elevated in the urine following kidney damage in a variety of animal species and in man. SCHONFIELD~
has suggested that the output of urinary enzymes reflects the anatomic integrity of :he kidney and because the kidney has a large functional reserve capacity, urinary enzyme excretion should vary prior to any change in physiological function. RMB’*
has postulated that the assay of urinary enzymes provides a more sensitive method for the detection of kidney damage than histological studies. The morphological and functional changes which occur in rats following tubular damage induced by mercuric chloride have been studied by MCCREIGHT AND WiTQFSKi”, but these authors did not monitor urinary enzyme excretion. On the basis of previous experiments with n~ts’~*‘~ we believe that the measurement of enzyme excretion in the urine is in fact a more sensitive and an earlier means of detection of kidney damage than the commonly used functional tests such as clearances of specific substances or the blood levels of certain excretory products. In this and the accompanying paper”’ urinary enzyme excretion is monitored in tubular and papillary damage. The change in the excretion of urinary enzymes is compared with kidney function tests in order to determine whicn provides the more sensitive method of detecting kidney damage. Six enzyme activities are assayed in an attempt to establish whether a characteristic enzyme excretion pattern results from damage to a specific region of the kidney. Many drugs are excreted in the urine and impairment of renal function has been shown to alter their excretion distribution and pharmacodynamics”. Propranolol is a drug which is used in the management of hypertension and mcent studiesrs with [‘*C]propranolol have shown that in patients with functional renal impairment the rate of urinary excretion of total radioactivity (propranolol plus metabolites) is greatly reduced. while the half-life in blood of propranolol itself is also reduced. The effect of renai tubular damage induced by mercuric chloride in dogs on the pharmacodynamics of propranolol is now reported and related to the accompanying functional impairment and changes in urinary enzymes. MATERIALS AND METHOIX
Collection of urine and blood samples Aldcrley Park-bred beagle dogs (IO-13 kg) were housed individually in galvanised wire mesh metabolism cages, each supported over a large separating funnel. Water was available ud libitum and food was supplied in a bowl placed temporarily inside the cage. The base of the cage was a wire mesh platform through which faeces and urine could readily pass. A second wire mesh held back faeces but allowed urine to pass into a collecting flask which was maintained at 4”. 24-h urine samples were tittered through coarse filter paper and were used immediately for enzyme analysis or stored at - 10’. Blood was removed from tae cephalic vein and allowed to clot at room temperature for 2 h. Serum was separated. by low-speed centrifugation and stored at -20” until required.
liFFECTOOPMERCURlCCHLORIDEON DOG KIDNEYFUNCTION
103
Ittdttclion of tubrtlar damage
A series of doses of mercuric chloride was admimstered to 4 dogs. Initially a solution of mercuric chloride (0.5 mg/ml in sterile saline, adjusted to pH 7.2 with NaHCOJ) was injected into the cephalic vein in daily doses of 0.1 mg/kg. Because this procedure resulted in fibrosis of the blood vessels the injections were terminated after 7 days. The dogs were then allowed to rest until 6 days later when a capsule containing mercuric chloride (2.5 mpjkg) was administered orally to each dog and the dose repeated daily for the 9 subsequent days. Mercuric chloride (0.5 mg/kg) was injected into the jugular vein on the next 2 consecutive days; after 4 weeks each dog was given a single intravenous injection of mercuric chloride (2.0 mgjkg). Assays
The activities of /r-glucosidascrP(EC 3.2.1.21), /?-galactosidase*g(EC 3.2.1.23), alkaline phosphatasczO (EC 3. I .?.I), acid phosphata@ (EC 3.1.3.2) and N-acetyl/%glucosaminidase22 (EC 3.2.1.3C~ wers determined Huorimetrically in Serum and urine using the appropriate 4-methylum~~lliferyl substrates. All samples were diluted IO-fold except in the cast of alkaline phosphatase. where a IOO-fold dilution was used. The diluted enzyme sample (I ml) was incubated with the appropriate substrate (I ml) in buffer for 30 min at 37”. The reaction was stopped with glycine-NaOH buffer (pH 10.4, 3 ml). The fluorescence of liberated 4-methylumbclliferone waj determined at 440 nm in an Aminco-Bowman fluorimeter using quinine sulphate as standard (4 @ml in 0.1 N H2SOI). Lactate dehydrogenase (EC I _I. I .27) and isocitrate dehydrogenase (EC I. I. I .4l) were assayed on a LKB 860@reaction rate analyser using the methods of WROBLEWSK! AND LA DUE= and WOLFROM AND WILLIA~~+&HMAN’~ respectively. Glutamate oxaloacetate transaminasez’ (EC 2.6.1.1) and glutamate pyruvate transaminase23 (EC 2.6.1.2) activities in serum were determined spectrophotometrically. Total urinary protein was determined by the procedure of GROMALL et01.~~and creatinine in serum and urine by the method of BOSNFS AND TAUSSKY~'. Serum urea was measured according to CHANEY AND MARBACH’~. The specific gravity of urine was measured with a hydrome.er (range 1000-l 100). Estimation
o$ the clearance qf imrlin and pamittohippurate
The infusion methods used in the estimation of the clearance of inulin and PAH were adapted from procedures by LINGARDH'~ and described by VARLEY~". Each dog was given an initial subcutaneous injection of acetopromazine (0.5 ml).
After 30 min the dogs were anaesthetised with a single injection of sodium metheheuitone (5 ml) into the cephalic vein. An endotracheal tube was inserted and connected to a mixture of oxygen containing I-27; halothane maintaioed at a flow rate of 3 I/min. A solution of saline and dextrose (3W ml) was infused into the jugular vein over IO min. An infusion solution (30 ml) containing loo/, inulin, 20% PAH and isotonic saline (pH 7.2) in the ratio 5 : 2 : 30 was passed into a blood deserette inserted into the other jugular vein as a priming solution. More of this solution was then infused as a constant flow rate of 1 ml/mm for the duration of ihe test. The first blood
104
R. 0.
ELLlS ct al.
and urine samplea were obtained SO min a&r the infusion of the priming solution. Urine was colkctcd via n catheter and over a 15.mitt period. After 7.5 mitt 8 sample of Mood (4 ml) was withdrawn from the jugular deaerettc and shaken in a oxalated tube. Heparinised saline (2 ml) was injected into the dcselrtte after each blood sample was withdrawn, Plasma was separated from the oxalated. blood by lowspeed centrifugation. Six determinations were made for each clearance estimation. The dogs were allowed to recover and were given an injection of ampicillin as a prophylactic. lnulin and PAH concentration in urine and plasma were determined as described by vARl.E+‘.
Histoiogy All dogs were killed at th: end of the experiment and the kidneys and livers removed. Thin slices of these tissues were fixed in a large vobtme of formal saline. Paraftin sections of the tissue we, e prepared and stained with haematoxyiin and eosin. Blood levels and urinary excretiojz of [I 4C]propranolol and its metabolites [W]Propranolol labelled at position 1 in the naphthalene ring3’ (specific activity 3.86 ,uCi/mg) was diluted with carrier drug (non-labelled propranolol) such that each dog received 1 $i of labelled drug in a single intravenous injection (0.1 mg/kg). After [‘“C]propranolol administration, blood samples (2 ml) were collected every 30 min for 4 h and assayed for [‘4Clpropranolol as described by HAIFS AND COOPER~~. 24-h urine volumes were collected after the injection of propranolol and the total ‘*C in urine was determined by liquid scintillation countingsz. RESULTS
Enzyme activity in norm01dog urine and serum In order to determine whether low molecular weight inhibitors were present in the urine, &glucosidase, /3galactosidase, h’-acetyl+glucosaminidase, alkaline phosphatase, acid phosphatase and lactate dehydrogenase activities were assayed in urine before and after dialysis against cold distilled water for 4 h. No increase in enzyme activity could be detected at the urine dilutions used and the urine samples were not dialysed prior to assay. Average values for the daily excretion of the 6 urinary enzymes studied are shown in Table I. Alkaline phosphatase was found to be the most active urinary hydrolase and was six times more active than N-acetyl-P-glucosaminidase. Lower activities of acid phosphatase, &glucosidase and @-galactosidase were detected. Lactate dehydrogenase is included for comparison. The 6 enzymes assayed in the urine were also assayed in serum together with 3 additional enzymes-isocitrate dehydrogenase, glutamate pyruvate transaminase and glutamate oxaloacetate transaminase. The activities of these enzymes arc given in Table I. Alkaline phosphatase and N-acetyl-@glucosaminidase were again the most active hydrolytic enzymes.
105
ENZYMB ACTIVIIIUI IN NQRMAL
MCI “RING ANU SERUM
WUfits are expressedUS the mean f S.D. Sample number was !2. Urinary enzyme activities are expressedas @iofes ~methyi~~il~ferone l~~ral~~~~h urine sample,except tactate dehydro~nPM activ&s wkich art expressedas I.U./24-h urine sample, Serum enzyme activities are expressedas aBIrO& 4methylumbellifbrone liberated/h/ml of serum Pxcept for iacfate dehydrogenase,iz&trate dehydro~na~ g~u~m~te pyruvate traosami~asc and Elutamate ox&acetate tran~mioase which are cxprassedas l.U,lmt serum. ” “. ,. _^._^ .._____,._ .._.... ..__, “l.l.. .” I. _. ..__“_ _.___, Urine SWWN .._. “. ...l .__.,.^_l_l “._, ..___... “_.,”_“.. ._.“” _ &Glucosidase 5.0 rfr 0.6 ‘2.1 & 0.3 ~Galactosida~ 4.9 f 0.3 3.8 & 0.5 33.8 ri: 6.8 N-acetyl.~-SIueo~minidase 125.0 f 13.u 201.0 * 31.0 AI+aline phosphatase 133.0 f 14,o 9‘7 f I.8 43.0 f 9.6 Acid phosphatase 59.0 i 7.0 Lactate dehydro~n~e 12.6 f 2.0 bocitrate dehydrogenase n.d.’ 6.4 & 0.5 Glutamate pyruvare transaminase n.d. 14.2 * 1.3 Glutamate oxaloacetate 14.4 * 0.5 n.d. transaminse ._ .-__- ..- _..-.. -- .-- .._. -....-...-. -. ..-.-.-...- ._ ..- -. * n.d., not determined
The rate of ctearance of creatinine, inulin and PAN was monitored in normal dogs together with the Tm PAH. The values obtained are shown in Table II: also included are the normal levels of urea in the serum and protein in the urine. Daily urine volumes were measured and the specific gravity of these samples monitored. Ail the parameters included in Table II are commonly used to detect and diagnose kidney damage and disease.
The changes in the pattern of excretion of a number of enzymes during gradually increasing mercuric chloride intoxication are shown in Tables III and IV. Serum UWd and urinary protein levels monitored over the same period are included for comparison, Serum enzyme activities, urinary volume and specific gravity were measured throughout the serial injection of mercuric chloride and kidney function tests were regularly performed on at1 4 dogs. The serum enzyme activities and kidney function TABLE II RENM..FUNCTION ,N NORMAL DOGS data are presented as the mean f S.D. The units and number of determinations are givea ia parentheses. The
____.____--.--..--
-._.
Clearance creatinine (miiminikg~ Ctearance inulin ~ml~min~kS) CtearancePAH ~ml/min/kg) Tm PAN (mllminlkg) Serum urea (mg %I Urine protein fmg/24-h urine sample> Urine volume (ml/N-h urine sample) Urine specificgrmity ,,____ ~~_.~..~ _,__..^” _...._..-.-
-_I.. .-
0.39 (20) 3.24 & 0.42 (81 3.89 & 1.0 (8) 8.8 ; O.il (8) 0.5 5 3.9 (20) 34.4 117 (20) i 15 * 125 (20) 600 0.003 (20) 1.026 + ___~~ -.
l
I.3
2.0 -.
f 9.6 * 1.4 * 3.1 * 3.3 * 2.2 f 3.5
8.1 t
n.d.. not determined
_.
f
6.4 n.d. 14.7 I 5.0 7.1 12.5 7.1 10.6
ii 23 24 25 26 27 28
29 ___ _.-
& ;t rt f * *
4.7 4.9 8.7 5.4 13.8 11.7
15 16 17 I8 19 20
0.4 1.2 20 0.3 3.5 4.5
9.4 f. 1.6 11.0 f 2.8 3.4 * I.1
0.2 0.9 1.0 0.8 5.5 1.0 1.3 1.0 3.4
IO II 12
fi f It * & f f &
4.3 4.5 5.0 6.2 12.2 8.5 8.1 9.1 12.6
: 3 4 5 6 7 8 9
0.5
0.2 0.2 0.4 0.4 0.1
3.6 2.9 3.3 3.1 3.7
3.7 f 0.1 n.d. 3.4 f 0.3 4.8 =t 0.9 4.1 f 0.6 4.9 IO.6 2.7 z&z0.2 3.1 & 0.3 3.5 rt 0.2
4.8 $ 0.3 4.9 * 0.5 4.6 + 0.5
f f f f f
4.8 f
f 0.3 * 0.3 i 0.3 f 0.2
5.9 4.7 4.5 4.2
0.5 0.6 0.2 0.3 0.2
f f i f +
5.4 4.8 4.6 4.3 3.9 10.0 16.1 8.2 9.6 13. 48 7.1 5.0 6.7 6.7 8.7
54.9 f 52.9 f 50.1 f 55.1 i 4’13_e
53.2 + 57.7 *
36.2 40.1 49.8 64.7
5&l 94.6 57.6 41.4 uI.0
0.4 30
83
35
+ 0.g f 22 i I2 f 10 f 3.6
39.1 f n.d. 45.3 + 87.7 +
47.3 f
45.0 -t 3.1 49.1 f 10.4
f rt f A-
2.0 2.0 3.5 2.7
f I_ f &
40.1 30.4 39.6 53.4
41.0 i 33.0 f IL0 f
1477 rf: 410 1079 f 380 6oOf 2cm
-.
13 I5 8.6
f 8.9 + 16.3 f 12.8 + 11.0
222 n.d. 24.5 45.6 34.1 127.9
______
1.7 10.8 0.7 0.7 2.9 7.6 5.7 14.4 4.8
2.6 23 2.1 2.6 5.5 3.1 2.2 3.6 4.6
It. 3.3
17.2 f 22.6 i 8.0 f 8.0 & 17.0 f 21.3 f 20.6 f 30.6 -t 211.1f
* I .:
10.6 f 9.8 f 8.3 f 8.6 i 15.3 f 19.1 * 13.1 * 14.3 f
1296* I66 ad. 1119* 3xl 14071;; 364 15P9* 500 3428 rt II22
70 187 30 45 177 271 105 635 267
78 ‘dC
5011 -,., :
698 * 587 I 201* 27?J& 643& 814f 541 rt 1382 f lll7&
29 49 50 % 159 I05 72
2llf 156+ 18Ot 2411f 459 f 575 f 406f
2.9
3.6
. __
l 2.0
.- _.-.
8.0
68f2.6 14.4 l 4 8.5 ;1;22 1.7 f t.4
17.5 f n.d. 10.4 f nd.
7
6 6
II 5 4 3 II
102f 82i 75 f 138 f 95f nd. IlO&
qd.
IO
5 I4 18 I5 7
I58 It I2
123f19
139& 13.7 + 3.5
18.9 + 5.1
117*24 72f no* PO& 97% 136 i 139i
f f 5 f f f
f
I I4 +I 22 120 rt 17 103 :i I2 93i IS
ljl
08f 7 1:2j,21 w*l2 91* 7
12 3.3 1.4 26 5.2 2.9
8.3 9.2 12.5 8.0 14.4 14.3
I.1
t 1.0 * 2.0 f 2.6 f 2.0
5.4 11.4 9.0 8.6 9.8 f
f I.6 & 2.1 f 1.4 f I.0 -Ir 13
II.2 6.8 12.7 5.9 8.5
21 31 n.d. _w n.d
29 nd. nd 39
26
29 29 n.d. 23 26 20
I9
n.d.
29 n.d.~ 31 n.d.
39 30 35 29 24
-
2.5 mdtg u mdL8
t5raJb 25e 2.5 -8 2.5 wg
-
0.1 mg/kg 0.1 mgJkg 0.1 q/kg
0.1 mg/kg
0.1 mg/kg 0.1 mgflcg 0.1 mg/kg
-
R
7.8 13.1 32.8 13.4 6.8 8.9 10.0 8.8
i f 5 f. f & & f
4.0 4.9 10.0 3.6 1.3 3.8 2.0 1.8
_--
& 0.4 * 0.9 & 3.5 f 0.7 & 0.9 f I.5 f 1.0 f I.1
---_.
2.5 6.4 8.5 10.1 6.9 5.3 5.6 4.5
21.4 + 56.1 & 145.2 f 221.4 * 97.0 l 55.8 f 57.9 f 66.3 + .-_....
3.5 4.6 19.0 31.0 21.0 1 I.5 8.9 2.7
.-._.
588 f 280 777 f 481 4106 f I712 3319 i 1197 1437 rt 3271 945 f 293 1713 If: 297 977 + 2% __-- - .-. _ ___-.
.-_-
Alkoliru
IS.9 * 5.0 23.0 -r_ 4.5 194.6 f 52.0 63.5 f 28.4 62.0 f 11.3 60.8 rt 9.2 73.8 rt 6.9 57.5 f 11.0 ___-...
7.5 f I.8 14.2 * 0.6 251.0 + 8.7 346.0 * 20.0 112.0 f 56.0 21.5 f 13.0 19.0 + 6.0 14.2 f 2.8 .___.
l7Si 70 253 c 45 1724 f 471 2217 + 931 449 * I.44 363 * IO7 330*100 29srt 40
24.0 24.0 30.0 64.0 130.0 144.0 128.0 39.0 _-
* 4.0 rfr 3.1 5 5.3 * Y-3.0 f 61.0 f 73.0 + 31.0 * 6.3 . --
1.1
IS.1 *
I.!
16.1 i
_._ _.
14.6 Jr 2.4
,-20 i IS *I5
15.3 $- 3.0
I20 15s 44
3.0 i
.--.-_ 0.7
..-.--_
89 5 I4 4.9 5 0.9
i 21 c 13 r”l3
I.1
60 __I-.
--.-.
64 -23 5.5 f I.3
IiS 165 37
2.5 i:
_._-.
59 ..-.--
Lactate dchydrogenase lsocitrate dchydrogcnax Glutamate pyruvate transaminase Glutamate oz.&acetate transaminase _. .____ ._.. -* n.d., not determined
---. -. _ .-.. fl-Galactosidxc N-ZiWyl-,Uglucosaminidase Alkaline phosphatase Acid phosphatase
-._-
33.0 _i 4.3
3.0
k S8 f 9.3
12.3 f
98 87
* 19 h48 k.2
_ ___ 3.5 f I.1
153 165 48
61 0.6
4.0 25.0 * 13.9
13.5 *
64 i 12.5 17.0 * 3.4
+ 18 I27 fl7
4.4 f I59 184 28
62
18.0 It
14.3 *
46 f 6.8 It
. I.0
2.4
3.3
7.8 0.9
* 59 f32 f 30
4.5 * 113 167 45
63
_..__-_
0.4
16.0 *
12.5 f
2.0
2.9
6.0 0.8
f I4 i 20 f 20 4s f 6.0 f
Ii6 180 37
64 __. 3.6 f
0.7
f 18
16.5 f
1.8
13.8 rfr 3.1
70 fl3 S.8
n.d: ISI C.d.
65 __ _.... 3.3 f
-
n.d. _
n.d.
n.d. 2.:
IS3 140 50
64 _.._-._ n.d.
..__
f 19 * 13 f18
_.._
THEAC~VIT,~ OF * NUMBEROF SERUMENZYMEIN DOCISrot,t.owm~ THEFIN*L INTRAVEN~WM~LC~,ONOF WLERCURIC CHLO~DE(2.0 mglks) ADI(MISTRED ON DAY60 Activities ate expressed as in Table I. The number of dogs used was 4. /?-Glucosidase was not assayed.
TABLE V
59 60 61 62 63 64 65 66 -..
-. .- _.----.
__..
IO8 test
8. G. IiLLIS
values
el fJ/.
found after the final injection of mercuric chloride ure given in Tables V
and VI respectively. A comparison of Tables I and 111shows that both acid a Id alkaline phosphatase activities were elevated in the urine 48 h after the first injection of mercuric chloride (0.1 mdkg) and the activities of these enzymes continued to rise with each subsequent injection. The highest levels were recorded after the 6th injection. The activities of both enzymes returned to normal 72 h after the final injection. /?-glucosidase and N-acetyl-r!Lglucosaminidase were also elevated, although the excretion of these enzymes fluctuated. The activity of /I-galactosidase and lactate dehydrogenase remained within the normal range as did the excretion of urinary protein. No change in serum urea or enzyme activities was detected at this dose level. Glomerular filtration rate measured by the rate of cicarance of creatinine and inulin remained normal as did Tm PAH and the rate of clearance of PAH. Urine volumes and specific gravity fall within the normal range (Table If). Oral dosing of mercuric chloride (2.5 mg/kg) was carried out over a IO-day period and urinary alkaline phosphatase activity rose gradually to values which were 7 times greater than normal (Table III). Acid phosphatase, P-glucosidase, N-acetyl/?-glucosaminidaseand lactate dehydrogenase activities were also elevated (2-J-fold) but urinary /?-galactosidaseactivity was within the normal range. Again no significant change was found in kidney function, serum enzyme activities, serum urea, urinary protein or urinary specific gravity. Some enzyme activities were still slightly elevated (Table III) when the next stage of the dosing regimen was started. The intravenous injection of mercuric chloride (0.5 mg/kg) on consecutive days resulted in an immediate and marked elevation of all the urinary enzymes studied except &galactosidase. Alkaline phosphatase (17-fold) and acid phosphatase (IZfold)
TABLE
were constdernbly elevated above
\‘I
RENAL FIJVCTION IN DOGS FOLLOWINO (2.0 mglkg) AC~~TERED ON DAY 60 Results areexprewd
as in Table
THE FINN,
II. The number
INTRAVENoUS
lWECT,ON
cw MERC”R,C
of dogs injected was 4. Where nc S.D.
than 4 do@ were used and fhc values shown represent
CHLORIDE is shown
less
a mean of 2 or 3 dogs.
l-r,1 PAH
n.d.=
n.d.
o.d.
1.026 + 0.003
3.78
9.0
0.58
I.028
IT 0.004
1.32
2.10
0.30
I.016
+. 0.003
0.25 .+ 0.17
1.022 * 0.003 I.018 2. 0.002
61
3.43 i 0.37 _t 0.21 1.60 ,. 0.33
62 63
0.80 0.74
It 0.26 f 0.33
0.51
64
1.39 If: 0.41 1.20 + 0.32
n.d.
1.95 jz 0.52
n.d.
59~60
65 66
3.86
’ n.d.. not determined
0.27 1.52
x 0.22
I.0
& I.1
2.4 ad.
0.44
3.5 n.d.
0.48
n.d. n.d.
I.020
-; 0.001
I .018* I.019 f
0.002 0.002
their aetivity iir normal urine and smaller increases in ~-glucosidase (bfold), N~cetyl~~-gluco~minida~ C&fold) were also found. Lactate dehydrogenase activity was elevated @-fold) in 2 of the 4 dogs used in this study, but again no change jn @-galactosidase activity was found. In contrast to the earlier doses of mercuric chloride, the injection of 0.5 m&kg resulted in changes in some of the other parameters studied as well as the elevation of urinary enzymes. In one dog which excreted the highest activities of urinary enzymes an elevation (Zfold) in serum urea was found and the rate of clearance of creat@ne fell below the normal range (Table 1111to I .3ml/min/kgon the day following maximum excretion of urinary enzymes (day 27) but had returned to normal 24 h Iater. TWQ serum enzymes, acid phosphata~ (3.fold) and ~-a~tyl-~-glucosaminida~ (Z-fold) were slightly elevated in all 4 dogs immediately aker the injection. Following a rest period of 30 days all 4 dogs were given a single injection (on day 60) of an increased dose of mercuric chloride (2.0 mgikg). Urinary alkaline phospbata~ activity was still elevated prior to the injection. Massive increases in urinary alkaline phosph~lta~ (2O_fold), acid phosphatase (20-fobtd) and lactate dehydrogenase (30-fold) activities above the normal followed the administration of this dose (Table IV), Lower increases in @-glucosidase f7-fold) and N-aeetyl-@-glucosaminida~ (S-fold) activities were alsa found, together with a slight elevation of &I-galactosidase activity (2-fold). Proteinuria coincided with the increased excretion of enzymes and reached a maximum of 2.0 g/day 48 h after the injection. The elevation of enzyme activities in the urine preceded a significant rise in urea in the se?um (Table IV). Isocitrate dehydrogenase activity was elevated 14”fold In the serum and smaller rises in lactate defiydro~enese and glutamate oxaloacet%te traa~~minase (Table V) activities were also observed on days 61 and 62. Renal function was impaired in all 4 dogs (Table VI) following the final injection af mercuric chloride. The rate of clearance of creatinine, inulin and PAH fell significantly within 48 h of the injection as did Tm PAH. The specific gravity of the urine also felt within 24 h of the injection. Renal function test values were still abnormal 10 days after the injection but urinary enzyme excretion fell steadily from the peak values which were recorded OR day 61 (Table IV).
Examination of the kidneys of all 4 dogs at the termination of the experiment revealed a marked dilation and some basophilia of the tubules in the medullary rays of the cortex. There was some interstitial inffamm~tion in the medullary ray and small areas of inflammation in both the medulla and papilla. A number GCtnb;>les iu the medulla were dilated and seven Jf these contained hyaline casts but no glomeruiar damage was detected. The livers of all the dogs were also examined but were found to be normal.
E&x? of tubular dunmge on the excretion of ~14C~~ro~r~~o~oj The rate of excretion of ~14C]propranolol from normal dogs was monitored
and the half-life of [*~CJpropranolol in blood and the excretion ofzotai W (propranolol plus metaboiites) into urine is shown in Table WI. The functional status of the kidneys and excretion of alkaline phosphata~ in the urine was atso monitored in ail 4 dogs (Table VII). After the administration of mercuric chloride both the propranolol blood half-life and the rate of urinary excretion of total ‘+C in urine were increased (Table VII). The magnitude of the increase in these parameters appears to be related to the degree of damage to the kidney as measured by kidney function tests and to the level of excretion of alkaline phosphatase in the urine.
The a~nistration of gradually increasing doses of mercuric chloride to dogs resulted in the elevation of urinary enzyme activities prior to any changes in the other parameters measured. The extent of the elevation was directly related to the magnitude of the dase. The most sensitive indication of tubular damage in the dog appears to be the excretion of atkaline and acid phosphatase which are the only enzymes elevated in the urine at the lowest dose used. Increase in dosage resulted in rises of /&lucosidase and N-acetyI+gJucosaminidase activities, but a further increase was necessary before an elevation of Lactatedehydrogenase took place. Abnormal Bgalactosidase activities were only detected following the administration of the highest dose. In contrast to the urinary enzymes, serum enzymes were unaffected by the two lOtVi2i &XX%, bu: ma!! increases of serum lactate dehydrogenase and glutamate oxaloacetate transaminase were found following the administration of the highest dose of mercuric chloride (2 mgjkg), together with a massive elevation of isocitrate dehydrogena~. Although slight increases (2-Fold) in acid phosphate and N-acetyl~-gluco~minida~ activities were occasionally found in individual dogs, the elevation of serum isocitrate dehydrogenase was the only significant change in serum enzyme TABLE VII THE R.%TE09 LOSSOS [‘~c]FROPRA~OLOL FROM TM BLOOD AND THE EXCRETIONOF ‘*c TOTAL IN THE URME OF 4 DOGS IWORE AND AFTER THE INTRAVENOUS~NJEC~CJN OF MERCURIC CHLORIDE (2.0 m&kg)
‘*C-propranolol was administeredas a single intravenous injection (0.1 m&kg). Renal function test zbta arc cxpresscdas in Table I1 and urinary alkaline phosphataseactivities as in Table 1,
Dog
Urittary .-_---_-__.-.
.._
ClearanceClearance iwlin PAH Nmmal
^_.__.._ Ttn
PAH
alkalitte phosphatme activity
40 60 60 60
30 44 59 45
6.7 8.0 8.4 5.9
2.90 3.20 2.80 2.30
0.40 0.30 0.49 0.40
180 252 179 115
24 h after J 53 injection 2 77 of mercuric 3 chloride 4 64 --___ _ _.. _ sn.d., not determined
41 70
I,1 2.0 2.4
0.80 0.90 0.92
0.18 0.11 0.3 I 0.35
ill0 6700 1450 330
1 2 3
4
53
ad.’
Loo
EFFECT
OF MERCURlC CHLORIL)E
ON DOG KIDNEY FUNCTtON
Ill
activity found in this study. The changein serum zsocitrate dehydrogenase activity was only detected when severe kidney damage was present and the other commonly u~l prmedures for the detection of kidney damage and disease were no more sensitive than the assay of serum enzymes. Significant changes in Serum urea and proteinuria were again only detected after the highest dose of mercuric chloride: si~:arly, changes in the functional capacity of the kidney were only recorded when extensive tubular necrosis was present. Sequential studies in the rat of the histological changes produced by mercuric chloride (ELLIS, unpublished results) showed that the initial area damaged was the terminal straight portion of the proximal convotuted tubules, but necrosis spread to the distal convoluted tubules and the medulla in time. These results may provide an explanation as to why, in the studies described in this paper, only certain enzymes were elevated at the lowest dose Ievel, whilst all the urinary enzymes studied were elevated at the higher dose level. Alkaline phosphatase and acid phosphatase are present in the proximal convoluted tubule of the dog in high activities33a34 and mild damage to this region would result in their excretion in abnormal amounts in the urine. More extensive damage would result in their further loss from cells over a greater area of the tubule, together with other enzymes which may have a different subcellular localisation. The data presented in this paper demonstrate that the assay of urinary enzymes provides the most sensitive method of detecting tubular damage. In the dog the elevation of urinary acid and alkaline phosphatase activities gave the earliest indication of kidney damage produced by mercuric chloride intoxication, whereas previously it was shown that the assay of urinary &glucosidase can be used to monitor tubular damage in the rat j3. The fluorimetric assays of these urinary enzymes areeasy to perform and could be readily inco~orated into toxicological trials or used for the differential diagnosis of kidney disease. A recent study investigated the handling of propranolol in patients with renal failure and it was considered of interest to compare the excretion of this drug in dogs with well characterised kidney damage. Propranolof is rapidly meta~lised in dog and man to 4-hydroxypropranolol, naphthoxylactic acid and an acid-labile conjugate of a mixture of propranolol and ~hydrox~r~pranolol which is identical to the major urinary metabolite. Only traces of propranoiol and ~hydroxypropranolol are found in urine. The main urinary components are naphthoxylactic acid and tie partially characterised conjugates in both species3z*J5. The more rapid excretion of metabolites in urine and the increase in proprano101blood half-life in dogs after tubular damage was unexpected as it has been reported that the rate of urinary excretion of metabolites in patients with renal funCtiORa1 impairment was greatly reduced and the blood half-life of propranolol itself was reduced. The reason for this difference may be that the metabolites in the filtrate are not reabsorbed because of the tubular damage caused by mercuric chloride (although glomerular filtration proceeds at a reduced rate), resulting in an increase in the rate of urinary excretion of metabolites. On the other hand filtration of metabolites would be reduced in the presence of giomerular damage, while reabsorption by the tubules
B. a.
112
ELLIS Ct a/.
would be normal, so that the rate of urinary excretion would be towered as observed by THOMPSONet al lb. The changes in the pIalma half-life of propranolol observed may be due to an alteration in the volume of distribution of propranolol. For example,
patients with severe glomerular damage may have a smaller volume of distribution than normal patients, because of a change in the ratio of the distribution rate constants. This change may also lead to a decrease in plasm half-life of the drug”. The pharmacodynamic consequencesof renal tubular damage have not been investigated and the reason for the increase in plasma half-life of propmnolol in dogs under these conditions is not understood. It is concluded that the consequencesof renal functional impairment on the pharmacodynamics of propranolol may be dependent on the site of the damage within the kidney. ACKNOWLEDGEMENTS We acknowledge
a Science Research Council Studentship (CAPS) to B. G. U. M. FOULKES for their kind
ELLIS. We thank Professor D. ROBINSON and Dr.
interest and encouragement and Dr. J. S. L. FOWLER fo:. assistance with the kidney function tests. We are indebted to Miss R. V. H. BARLOW for assistance with the histological studies and Mrs. S. LONGSHAW and Mr. I. M. OLIVER for technical assistam. REF&RENCES H. MATTENHEIHER.in U. C. DUBACH (Ed.). &mymcr in Urine und Kidney, Huber. Bcrnc, 1968. p. 119. H. MAITENHEIWER, V. E. POLLAKE AND R. C. MUEHREKE. Quantitativeenzymepatternsin the nephron of the healthy human kidney, Neplnon.. 7 (1970) 144-154. E. AHADOR, L. E. DOI~FMAN AND W. E. C. WACKER.Urinary alkaline phosphutascand LDH activities in the differenrial diagnosisof renal disease,Ann. Inrem. Med.. 62 (1965) 30-40. L. E. D~IIFMAN, E. AIUWR AND W. E. C. WACYER, Urinary lactic dehydrogenaseactivi(y. 1. Am. Med. Ass.. 188 (1964) 671-676. C. BREEDIS. C. M. FL.ORYANDJ. FURTH, Alkaline phosphatasclevel in urine in relation 10 renal injury, Arch. Pnrhol., 36 (1943) 402-412. M. R. SCHMNFELD.Acid phosphataseactivity in ureteral urine. J. Ant. Med. Ass., 193 (1965) 618-621. L. J. G~EENB~RO.Fluorometric measurement of alkaline phosphatase and aminopeptidase activities in rhe order of lo-‘* mole, Biochcm. Biophys. Res. Commun., 9 (1962) 430-435. J. P. H*JLIZT.P. E. PERILLI~ANDS. C. FINCH. Urinary muramidase and renal disease,Ncm &ag/. J. Med., 279 (1968) 506-512. L. F. PRESCOTTAND S. ANSAFX The effects of rcocated administration of mercuric chloride on exfoliation of renal tubular cells and urinary glutamic-oxaloacetictransaminase activity in the rat, Toxicol. AnpI. Phannacol., 14 (!969) 97-107. D. ROBI)CSON. R.G. PRIC~ANON. DANCZ,Rat-urine glycosidasesandkidneydamsge.Bloche/f~.1.. 102 (1967) 533438. R. G. PRICE,N. DANCE,R. R~cfl~aos AND W. R. CA~~ELL.The excretion of N-acctyl-&@cosaminidasaand fl-gdlactosidasefollowing surgeryto the kidney, C/in. Chin!. Acra,27 (197016 i-72. N. DANCE. R. 0. PRICE.W. R. CATTELL. J. LANSDELL AND B. RICHARDS, The excretion .*f Nacetyl-&glucosuninidaseand fi-galanosidaae by patients with renal disease, C/in. Chim. Attn. 27 (1970) 87-92. R. G. PRICE.N. DANCEAND D. ROBINSON,A comparison of the fl-glycosidaseexcreticn durin(( kidney damage inducedby 4-nitrophenylarsonic acid and by rabbit anti-rat kidney antibodies. EuropeanJ. CIltt. Inwr., 2 (1970) 47-51.
EFFECTOF MERCURICCHLORIDEON DOG KIDNEY PUNCTION
113
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34 35
5 (1967)
420434.
P. J. C. VAN BREDAVRIESMANAND R. G. J. WILLICHAG~N.Activity of alkaline phosphataw and isocnzymepattern of normal dog glomerulus. &it. J. Exptl. Pothol.. 49 (1968) 324-330. J. W. PA.I-~EERSON, M. E. CONNELLY.C. T. DOLLERY,A. HAYESAND R. G. COOPER.The Pharmacodynamicsand metabolismof propranolol in man, Phormocol.. C/in.. 2 (1970) 127-134.