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Studies on Experimental Oxaluria in Pigs
By. vet.
J.
(1977), 133.4 18
STUDIES ON EXPERIMENTAL OXALURIA IN PIGS By G. D. A. WILSON* AND D. G. HARVEY Department of Pathology, Royal Veterinary College, London SUMMARY
Oral doses of 20 to 1600 mg sodium oxalate (COONah/kg bodyweight were given to 8 to 12 week-old SPF pigs whose water intake was slightly restricted. Oxalate crystals were detected in the urine only when the oxalate content was high or the dose of oxalate large. Diuresis and proteinuria followed dosing. About 45% of the given dose was recovered in the urine when the sodium salt was given by capsule. Recoveries were less (6·0 and 11·4%) when oxalate was mixed with the food. Impaired kidney function following the highest dose was suggested by histological lesions observed after slaughter. Serum protein, urea and cholesterol concentrations, and arginase activity were increased by the diuresis and subsequent haemoconcentration following the highest dose of oxalate. Serum alkaline phosphatase also was increased shortly after dosing. Low T t values for PSP elimination suggest that clearance of the dye by the kidney was increased after dosing. INTRODUCTION
The occurrence of 'mould nephrosis' in pigs in Denmark and experimental damage in pigs induced by feeding mouldy grain contaminated with Penicillium viridicatum have been described by Krogh & Hasselager (1968, 1970). Feeding toxic barley resulted in chronic kidney degeneration in pigs and rats. Buckley (197 I) described a field outbreak of fungal nephrotoxicity in pigs in Ireland. Pigs experimentally fed the contaminated barley developed polydipsia, polyuria, and renal tubular degeneration. Carlton & Tuite (1970) described the production of kidney damage and a perirenal oedema syndrome in immature pigs following the administration of cultures of P. viridicatum grown on autoclaved corn. The extensive development of oedema especially in the perirenal region was considered by the authors to be a species reaction to severe kidney damage. Friis, Hasselager & Krogh (1969) reported the isolation of citrinin and oxalic acid from a nephrotoxin strain of P. viridicatum, and concluded from feeding experiments that citrinin was mainly responsible for naturally-occurring kidney degeneration in Danish pigs. The isolation of oxalate is interesting, since cases of perirenal oedema in pigs have been described by Buck et al. (1966) and Osweiler, Buck & Bicknell (1969) following poisoning by Amaranthus retroflexus, a weed rich in oxalate.
* Present address: Ruakura Agricultural Research Centre (Private Bag), Hamilton, New Zealand.
OXALURIA IN PIGS
In pig urines examined at slaughter by Wilson, H arvey & Snook (1972) the incidence of oxalate crystals was 32.1 % in nephritic pigs and 27.0 % in pigs without kidney damage. High values of urinary oxalate were associated with proteinuria, and high levels of proteinuria were associated with the presence of oxalate and casts. Experimental studies on oxaluria following oral dosing of sodium oxalate to pigs are reported in this paper. MATERIALS AND METHODS
Animals and experimental design SPF pigs were used throughout. All animals were fed I kg per animal per day commercial pig food containing 3% oil, 16% crude protein, 5"5% crude fibre and 199 parts/10 6 copper. Water intake was slightly restricted to ensure that each animal received an identical measured volume, usually between 2 and 4 litres per day. In experiment I, two of four 8-week-old pigs weighing 20 to 24·5 kg were given 20 mg (COONa)2/kg by capsule on days 7 and 8 and 50 mg/kg on day 9. The control animals were fed empty capsules. All four animals were slaughtered when 19 weeks old. In experiment 2, two of four I3-week-old pigs weighing 33 to 37 kg were given daily doses of 50 to 60 mg (COONa)2/kg per day throughout the experiment, the dose being mixed with the food. All four animals were slaughtered when 18 weeks old. In experiment 3, a single pig, 12 weeks old and weighing 25 kg, was given oxalate in the diet as follows: 400 mg/kg on day 1,800 mg/kg on days 2 to 7 inclusive, and 1600 mg/kg on days 8, 9 and 10. This animal and a control were slaughtered on day I I. Methods Estimations were made essentially as described by Wilson et al. (1972). In addition, the following were added: serum arginase (Cornelius & Freedland, 1962), serum GOT and GPT (Reitman & Frankel, 1957), serum cholesterol (Zurkowski, 1964), serum alkaline phosphatase (Wootton, 1964). Fractional clearance of phenol-sulphonphthalein (PSP) as T i was measured essentially according to the method of Mixner & Anderson (1958), but using reagent volumes identical with those recommended by Prinz & Fiesel (1957) for the measurement of BSP clearance in pigs. Sampling Blood samples were taken each day from each animal just prior to the morning feed. Serum was separated in the usual way. PSP clearance was determined between days 3 and 4 and between days 8 and 9, i.e. before and after oxalate dosing in experiment I, and between days 9 and loin experiment 2. RESULTS
Blood parameters Blood parameter values for all animals in experiments I and 2 are summarized in Table I. Results of experiment 3 are shown in Fig. I. Only the urea levels
to
SERUM VALUES (MEAN
±
Total protein (gj roo ml)
TABLE I 1.S,D, ) ON OXALATE DOSING EXPERIMENTS (VALUES IN PARENTHESIS ARE PRE-DOSING LE VELS)
0:1 :;tI
....
....>-l
en
:r:
Urea (mg j roo ml)
Arginase (U jml)
Clwlesterol (mgjroo mi)
GOT (mU/ml)
GPT (mU/ml)
AP (KAU)
N
<: t'fj >-l t'fj
Experiment I Controls
Dosed Experiment 2 Controls
Dosed
5'18 (5'01 5'07 (5'53
± 0 '32 ± 0'38) ± 0'33 ± 0'43)
5,85 ± 0' 70 (6'10) 5'69 ± 0,66 (6' 13)
35 (28 27 (26
± ± ± ±
3'4* 10'0) 3'4 10'1 )
0' 76 (0,85 0,80 (0'93
± 0'44 ± 0,63) ± 0'37 ± 0'70 )
91 (105 83 103
± ± ± ±
15' 1 40'6) 11'3 22 '0
26 (28 25 (3 0
± 9'9 ± 5'5) ± 6'5 ± 6' 7)
12'3 (6,6 13'3 (7'4
± ± ± ±
7'0 1'3) 7'0 1'5)
22 (39 19 (33
± ± ± ±
....
:;tI
10'5 12'9) 7,8 4' 1)
12 8 12 8
Z
;l> :;tI
><:
~
0
29 ± (35) 31 ± (39)
8'4 5,8
0'34 ± 0'20 (0,66) 0'47 ± 0'3 2 (0' 38)
* Significantly different from dosed group at P = < 0'001.
84 ± 19'4 (119) 77 ± 15'0 (84)
3 1 ± 9'9 (3 1) 28 ± 8'5 (23)
12'5 ± 3,0 (12'5) 13'5 ± 1,6 (12'0)
28 ± 10'9 (4 1) 17 ± 7'7 (23)
L! 13 4 13 4
:;tI
z
;l> t""
~
~
"'S"" ~
OXALURIA IN PIGS
~
150
~!_ .... _
2
100
~-.-~
E
421
....--.-.
50
E
80
z8
:5, 4 0
'"
E
cE ~8
0: , '"
6
4
2
0....
/\
l ___
.~ ~=~~----~~--------~
Fig.
I.
Serum values in experiment 3. • - . Dosed pig. 0 - 0 Control pig.
of the dosed and control animals show any statistical difference. In experiment 2 a marked fall in the alkaline phosphatase activity occurred in both dosed and control groups. However, the fall was less in the dosed group. In experiment 3, the most marked changes were increases in the concentrations of total protein, urea, cholesterol and arginase activity on days 8 and 9. Increases in S-GOT and S-GPT were less obvious, as were their differences from the control values. The pattern of S-AP variation is that of an early increase followed by a steady decline in activity.
Urine The results of urine analysis are given in Fig. 2 and are not particularly spectacular. There appear to be three main findings. First, that towards the end of all three experiments there are increases in urinary protein, and this is particularly marked in experiment 3 where the dose levels of oxalate were high. However, there is a lack of consistency in the increases, and in the differences between the dosed and control values. Secondly, in experiment I the dosed pigs excreted large urine volumes on day 7 and smaller ones on day 8, and in experiment 3 the volumes excreted by the dosed pig were generally greater than those excreted by the control. pH and specific gravities were also higher in the dosed pig urine. Thirdly, oxalate crystals were observed in the urine of one dosed pig in experiment 2 on days 9 and 10, and in the urine of the pig in experiment 3
BRITISH VETERINARY JOURNAL, 133, 4
422
E.. pe riment I
Elperimenf 2
Con lrol
Contro l
Dosed
Dosed
Experiment 3 Control
Ur inory
prote in mg /l OO ml Urine volume L pH Okalole
g/ lOO ml 500
Urinar y pro tein
~
mg /lOO m l
Urine
vol ume
l pH
Oxolo le g /iOO ml Olto late dose mg / kg
1500 1000
500
50
50
I 2345678910
I 23 4 567 8 9 10
Day s
Days
Fig.
2.
Days
Urine data following oxalate dosing.
at the end of days 4, 5, 6, 8 and g. Some diuresis occurred in dosed pigs in all three experiments following initial dosing, usually within 24 to 48 h.
Oxalate exretion Mean oxalate excretion, expressed as (COONah for all animals prior to dosing was 0'052 g/24 h in experiment I . In the four-day period following dosing the dosed pigs excreted 0'343 g and 0'246 g, or 46. I % and 45'8% of the dose. In experiment 2 mean urinary oxalate excretion for the control pigs in the experimental period was o' I 20 g/24 h. For the dosed pigs the mean oxalate excretion was 0'348 g and 0'240 g/24 h, or I 1'4% and 6'0% of the dose. Urinary oxalate determinations were not carried out on the pigs in experiment 3. PSP elimination These are summarized in Table II. Histopathology No lesions were demonstrated in the kidneys of dosed pigs in experiments I and 2 . In experiment 3, the kidneys of the dosed pig were firm, rounded, pale brown, and large. On incision, they were pale, and the cortical area apparently enlarged, the ratio of the width of the cortex to the width of the medulla being 4: I for the dosed pig and approximately I: I for the control. Microscopically, the kidneys of the dosed pig showed dilated glomerular spaces and tubules in the cortex. Much more eosinophilic material was seen in the Bowman's capsules of the dosed pig than in the control pig. Eosinophilic material in the kidney tubules, as casts, was seen only in the kidneys of the dosed pig.
OXALURIA IN
PIG~
423
TABLE II HALF-TIME OF ELIMINATION OF PSP FOLLOWING OXALATE DOSING
Tt (minutes)
Pig Experiment OR OR WH WH
I
Experiment BN BK
2
Experiment BNA
3
17. 8 18·6 24'4 22'0
Day
9
Pre-dosing 2 d X 20 mg/kg Pre-dosing 2 d X 20 mg/kg 8
4 10 3
Hours since last dose
Dosing regime (COONah
+I +I
d
X 50
mg/kg
26
d
X 50
mg/kg
2
d X 50 mg/kg d X 50 mg/kg
22'2
8
23 ' 2
10
10
18·8
10
I
d
X 400 3
d
mg/kg X
1600
24 24
+6
d mg/kg
X 800
mg/kg
+
2
Crystalline material was visible microscopically in the kidney tubules, but this material was only von Kossa positive in the tubules of the animal receiving oxalate. At slaughter both animals showed some evidence of chronic nephritis in the form of focal and diffuse areas of mononuclear infiltration. The livers of both animals did not appear to be macroscopically abnormal. Histological sections of the liver of the dosed pig showed that the cells surrounding the central vein had vacuolation typical of fatty infiltration. There was no sign of oedema in either carcass, but there was some interstitial oedema in the kidney of the dosed pig. DISCUSSION
Experiment
I
The lower levels of blood urea in the experimental animals following oxalate administration may be due to a diuretic effect of the dietary oxalate at the dose level employed. However, diuresis following oxalate administration on day 7 seems to be a real effect, since low blood urea levels were found in the experimental pigs in the second week. About 45% of the initial dose of sodium oxalate was excreted in the urine, which was larger than expected, since Archer et ai. (1957) found only about 5% increase in urinary oxalate in human patients. This difference could be attributed to the different methods of administration, since Archer et ai. gave their oxalate in four equal portions after food, whereas in the experiment described in this paper, the animals were not allowed access to food until go min after dosing. In these conditions rapid uptake of oxalate may have occurred, and this would have precluded much of the bacterial decomposition of oxalate reported by Brune & Bredehorn (1961) after they had fed calcium oxalate to pigs. It is possible that the stomach contents of the fasted pig may present less opportunity for the d ecomposition of sodium oxalate or the precipitation of oxalate as the calcium salt. Archer et ai. (1957) have shown that calcium oxalate does not cause an
BRITISH VETERINARY JOURNAL, 133, 4
increase in urinary oxalate in human patients, and Dodson (1959) has shown that in sheep it is excreted mainly in the faeces if the rumen is by-passed. Urinary excretion of protein was transiently increased after dosing with oxalate. This finding has some significance in view of the observations on slaughterhouse material (Wilson et ai., 1972), where certain pigs showed high levels of urinary protein associated with high urinary oxalate. Any functional impairment of the kidneys which may have resulted from oxalate administration in this experiment could not be detected by histological examination post mortem.
Experiment 2 Although most changes in the concentrations of serum parameters in this experiment must be regarded as insignificant, dietary oxalate does seem to have some effect on S-AP activity. Although reduced levels of activity were found in both groups of animals, the reduction was less in the dosed group. This observation has some similarity to that of Tuba et ai. (1952) who noted that dietary sodium oxalate appeared to cause an increase in S-AP activity in the young growing rat. The recovery of urinary oxalate in this experiment was about the same order as that reported by Archer et ai. (1957) in their experiments on human subjects. In their experiments and in those reported in this particular experiment, the methods of administration were similar in that food was present. Similar results were reported by Hardy & Baumann (1959). Absorption of oxalate through the gut (Radeleff, 1964) is probably followed by storage in the tissues, since Curtin & King (1955) demonstrated that injection of l4C labelled oxalate into rats was followed by a wide distribution of oxalic acid in the tissues with specially high concentrations in bone. Accordingly, the increased urinary excretion of oxalate of the dosed pigs in the latter part of this experiment may have reflected an effect of the administered oxalate in exceeding the binding capacity of the pigs' tissues for this substance. The rise in urinary protein on days 3 and 4 which occurred in the pigs dosed with oxalate probably represents a real effect due to sodium oxalate since no similar change was noted in the controls. This effect was similar to, but more pronounced than, the changes observed in the first oxalate experiment. Dietary oxalate, however, seems to be only one of a series of factors involved in determining the level of proteinuria, since the controls also showed a degree of proteinuria, and this was broadly inversely proportional to the daily volume of urine. In this experiment high protein levels in the urine were generally associated with low urinary volumes. Therefore, it seems reasonable to assume that urinary protein excretion in the dosed pigs is affected by oxalate and its effect in reducing urine volume. This hypothesis is substantiated, in part, by the work of other authors. For example, reduction in urine volume in cattle, associated with water deprivation causes a marked proteinuria (Weeth et ai., 1970), and reduced urinary output has been reported by Dodson (1959) in sheep following intravenous administration of sodium oxalate. Further, Derivaux & Liegeois (1962) have reported oliguria and anuria as symptoms in oxalate poisoning in livestock.
OXALURIA IN PIGS
Experiment 3 The increases in the levels of the serum parameters in this experiment appear to be due to d ehydration since the volumes of urine particularly on days 7 and 8, were large. Similar results were noted by Baur & Filer (1959). However, the dehydration effects are probably reversible since McCance (1960) noted the return of blood urea levels to normal once dehydrated pigs became rehydrated. The diuretic effects of sodium oxalate seen in these studies are similar to those noted by James et al. (I 968a) in their experiments on la mbs. The diuretic effect is not solely attributable to oxalate; sodium chloride has been shown to have similar activity. Thus, Ek (1965) has shown that in cases of salt poisoning of pigs in Norway, there were considerable increases in serum electrolytes, total protein and urea. Similarly, Kwatra & Khera (1965a) have reported similar effects when hill cattle in India were fed with potassium oxalate. The observations regarding the increase in S-AP activity in the early stages of the experiment are interesting, and it would appear that this phenomenon is due to the oxalate per se, and not due to any haemoconcentration effect since none was observed at this early stage in the experiment. The precise mechanism of this is difficult to explain. However, the secondary fall in S-AP activity in the dosed pig could be explained by the fact that, as the experiment progressed, less of the administered dose combined with the calcium of the bone, thus reducing the contribution of AP derived from that tissue to the total S-AP. The high specific gravities observed in the urines of the dosed pig were probably a function of dehydration and increased oxalate excretion, and this assumption receives some support from the observations of Bianca (1970) and Baur & Filer (1959) who demonstrated that dehydration is associated with increased specific gravity of the urines of cattle and pigs respectively. Similarly the effect of oxalate in increasing pH values has been noted by James et al. ( 1 968b) in their feeding experiments on lambs. The more pronounced excretion of urinary protein by the dosed pig is in general agreement with the findings of the first two experiments. However, the presence of kidney lesions in both dosed and control animals at slaughter suggests that the high proteinuria may not be attributable solely to oxalate. Nevertheless, the possibility that oxalate exacerbated the nephritis cannot be discounted since K watra & Khera (I 965b) noted this phenomenon in cattle in which Corynebacterium renale infection was superimposed on an oxalate nephritis. How far oxalate can be deemed responsible for kidney damage in 'natural' forms of oxalate poisoning cannot accurately be measured. Friis et al. (1969) in their studies on P. viridicatum poisoning in pigs demonstrated that citrinin was the prime cause of nephrotoxicity, but that other compounds, such as oxalate, might have a powerful synergistic effect. However, they found that feeding pigs with 1'0 g sodium oxalate per kg per day for 42 days resulted only in a slight interstitial renal fibrosis. On these results, they concluded that sodium oxalate toxicity for pigs was low. The work presented in this paper partly substantiates the results of Friis and his colleagues, but suggests that changes in kidney function and pathology occur somewhat earlier when pigs do not have continuous access to drinking
BRITISH VETERINARY JOURNAL, 133, 4
water. Feeding toxic food, not unexpectedly, increases the water reqt'Iirement of pigs (Krogh & Hasselager, 1968; Krogh, 1969) so that synergistic effect of oxalate with citrinin may be powerfully enhanced when water consumption is restricted. AC KN o-WLE D GEMENTS
Our thanks are due to Mr C. R. Snook, F.I.S.T., for valuable technical assistance and the Agricultural Research Council for financial aid. REFERENCES
ARCHER, H. E., DORMER, A. E., SCOWEN, E. F. & WATI'S, R. W. E. (1957). Clin. Sci. 16,4°5. BAUR, L. S. & FILER, L. j. (1959).]. Nutr. fi9, 128. BIANCA, W. (1970). Br. vet.]. 126, 121. BRUNE, N. & BREDEHORN, H. (1961). Z. Tierphysiol. Tiererniihr. Futtermittelk. 16,214. BUCK, W. B., PRESTON, K . S., ABEL, M. & MARSHALL, V. L. (1966).]. Am. vet. med. Ass. 148, 15 25. BUCKLEY, H . G. (1971). Ir. vet.]. 115, 194. CARLTON, W. W. & TUITE, J. (1970). Path. vet. 7,68. CORNELIUS, C. E. & FREEDLAND, R. A. (1962). Cornell vet. 511,344. CURTIN, C. 0. & KING, C. G. (1955).]. biol. Chem. 1116,539. DERIVAUX,j. & LIEGEOIS, F. (1962). In Toxicologie Veterinaire. Paris: Vigot Freres. DODSON, M. E. (1959). Aust. vet.]. 35, 225. EK, N. (1965). Nord. VetMed. 17,6°4. FRIIS, P., HASSE LAGER, E. & KROGH, P. (1969). Acta path. microbiol. scand. 77,559. HARDY, R. W. F. & BAUMANN, C. A. (1959).]. Nutr. 6g, 429. JAMES, L. F., STREET,j. C., BUTCHER,J. E. & BINNS, W. (1968a).]. Anim. Sci. 27, 718. JAMES, L. F., STREET,j. C., BUTCHER,J. E. & BINNS, W. (1968b).]. Anim. Sci. 27,724. KROGH, P. (1969).]. stored Prod. Res. 5, 259. KROGH, P. & HASSELAGER, E. (1968). Arsskr. K. Vet.-Landbohojsk. 198. KROGH, P. & HASSELAGER, E . (1970). Nord. VetMed.22, 141. KWATRA, M. S. & KHERA, S. S. (1965a). Indian]. vet. Sci. 35,157. KWATRA, M. S. & KHERA, S. S. (1965b). Indian]. vet. Sci. 35, 165. MCCANCE, R. A. (1960) . Br.]. Nutr. 140 59. MIXNER, j. P. & ANDERSON, R. R. (1958).]. Dairy Sci. 41, 306. OSWEILER, G. D., BUCK, W. D. & BICKNELL, E. j. (1969). Am.]. vet. Res. 30, 557. PRINZ, W. & FIESEL, A. (1957). Z. Tierphysiol. TieremMr. Futtermittelk. III, 170. RADELEFF, R. D. (1964). In Veterinary Toxicology. Philadelphia: Lea & Febiger. REITMAN, R. S. & FRANKEL, S. (1957). Am.]. clin. Path. 28,56. TUBA, j., SILUCH, K. A., ROBINSON, M. I. & MADSEN, N. B. (1952). Can.]. med. Sci. 30,515. WEETH, H.j., WITTON, R., SPETH, C. F. & BLINCOE, C. R . (1970).]. Anim. Sci. 30, 219. WILSON, G. D. A., HARVEY, D. G. & SNOOK, C. R. (1972). Br. vet. ]. 128, 512. WOOTTON, I. D. P. (1964). In Microanalysis in Medical Biochemistry. London: Churchill. ZURKOWSKl, P. (1964). Clin. Chem. 10,451. (Accepted for publication 3 December 1974)
J.
(1977), 133.4 18
STUDIES ON EXPERIMENTAL OXALURIA IN PIGS By G. D. A. WILSON* AND D. G. HARVEY Department of Pathology, Royal Veterinary College, London SUMMARY
Oral doses of 20 to 1600 mg sodium oxalate (COONah/kg bodyweight were given to 8 to 12 week-old SPF pigs whose water intake was slightly restricted. Oxalate crystals were detected in the urine only when the oxalate content was high or the dose of oxalate large. Diuresis and proteinuria followed dosing. About 45% of the given dose was recovered in the urine when the sodium salt was given by capsule. Recoveries were less (6·0 and 11·4%) when oxalate was mixed with the food. Impaired kidney function following the highest dose was suggested by histological lesions observed after slaughter. Serum protein, urea and cholesterol concentrations, and arginase activity were increased by the diuresis and subsequent haemoconcentration following the highest dose of oxalate. Serum alkaline phosphatase also was increased shortly after dosing. Low T t values for PSP elimination suggest that clearance of the dye by the kidney was increased after dosing. INTRODUCTION
The occurrence of 'mould nephrosis' in pigs in Denmark and experimental damage in pigs induced by feeding mouldy grain contaminated with Penicillium viridicatum have been described by Krogh & Hasselager (1968, 1970). Feeding toxic barley resulted in chronic kidney degeneration in pigs and rats. Buckley (197 I) described a field outbreak of fungal nephrotoxicity in pigs in Ireland. Pigs experimentally fed the contaminated barley developed polydipsia, polyuria, and renal tubular degeneration. Carlton & Tuite (1970) described the production of kidney damage and a perirenal oedema syndrome in immature pigs following the administration of cultures of P. viridicatum grown on autoclaved corn. The extensive development of oedema especially in the perirenal region was considered by the authors to be a species reaction to severe kidney damage. Friis, Hasselager & Krogh (1969) reported the isolation of citrinin and oxalic acid from a nephrotoxin strain of P. viridicatum, and concluded from feeding experiments that citrinin was mainly responsible for naturally-occurring kidney degeneration in Danish pigs. The isolation of oxalate is interesting, since cases of perirenal oedema in pigs have been described by Buck et al. (1966) and Osweiler, Buck & Bicknell (1969) following poisoning by Amaranthus retroflexus, a weed rich in oxalate.
* Present address: Ruakura Agricultural Research Centre (Private Bag), Hamilton, New Zealand.
OXALURIA IN PIGS
In pig urines examined at slaughter by Wilson, H arvey & Snook (1972) the incidence of oxalate crystals was 32.1 % in nephritic pigs and 27.0 % in pigs without kidney damage. High values of urinary oxalate were associated with proteinuria, and high levels of proteinuria were associated with the presence of oxalate and casts. Experimental studies on oxaluria following oral dosing of sodium oxalate to pigs are reported in this paper. MATERIALS AND METHODS
Animals and experimental design SPF pigs were used throughout. All animals were fed I kg per animal per day commercial pig food containing 3% oil, 16% crude protein, 5"5% crude fibre and 199 parts/10 6 copper. Water intake was slightly restricted to ensure that each animal received an identical measured volume, usually between 2 and 4 litres per day. In experiment I, two of four 8-week-old pigs weighing 20 to 24·5 kg were given 20 mg (COONa)2/kg by capsule on days 7 and 8 and 50 mg/kg on day 9. The control animals were fed empty capsules. All four animals were slaughtered when 19 weeks old. In experiment 2, two of four I3-week-old pigs weighing 33 to 37 kg were given daily doses of 50 to 60 mg (COONa)2/kg per day throughout the experiment, the dose being mixed with the food. All four animals were slaughtered when 18 weeks old. In experiment 3, a single pig, 12 weeks old and weighing 25 kg, was given oxalate in the diet as follows: 400 mg/kg on day 1,800 mg/kg on days 2 to 7 inclusive, and 1600 mg/kg on days 8, 9 and 10. This animal and a control were slaughtered on day I I. Methods Estimations were made essentially as described by Wilson et al. (1972). In addition, the following were added: serum arginase (Cornelius & Freedland, 1962), serum GOT and GPT (Reitman & Frankel, 1957), serum cholesterol (Zurkowski, 1964), serum alkaline phosphatase (Wootton, 1964). Fractional clearance of phenol-sulphonphthalein (PSP) as T i was measured essentially according to the method of Mixner & Anderson (1958), but using reagent volumes identical with those recommended by Prinz & Fiesel (1957) for the measurement of BSP clearance in pigs. Sampling Blood samples were taken each day from each animal just prior to the morning feed. Serum was separated in the usual way. PSP clearance was determined between days 3 and 4 and between days 8 and 9, i.e. before and after oxalate dosing in experiment I, and between days 9 and loin experiment 2. RESULTS
Blood parameters Blood parameter values for all animals in experiments I and 2 are summarized in Table I. Results of experiment 3 are shown in Fig. I. Only the urea levels
to
SERUM VALUES (MEAN
±
Total protein (gj roo ml)
TABLE I 1.S,D, ) ON OXALATE DOSING EXPERIMENTS (VALUES IN PARENTHESIS ARE PRE-DOSING LE VELS)
0:1 :;tI
....
....>-l
en
:r:
Urea (mg j roo ml)
Arginase (U jml)
Clwlesterol (mgjroo mi)
GOT (mU/ml)
GPT (mU/ml)
AP (KAU)
N
<: t'fj >-l t'fj
Experiment I Controls
Dosed Experiment 2 Controls
Dosed
5'18 (5'01 5'07 (5'53
± 0 '32 ± 0'38) ± 0'33 ± 0'43)
5,85 ± 0' 70 (6'10) 5'69 ± 0,66 (6' 13)
35 (28 27 (26
± ± ± ±
3'4* 10'0) 3'4 10'1 )
0' 76 (0,85 0,80 (0'93
± 0'44 ± 0,63) ± 0'37 ± 0'70 )
91 (105 83 103
± ± ± ±
15' 1 40'6) 11'3 22 '0
26 (28 25 (3 0
± 9'9 ± 5'5) ± 6'5 ± 6' 7)
12'3 (6,6 13'3 (7'4
± ± ± ±
7'0 1'3) 7'0 1'5)
22 (39 19 (33
± ± ± ±
....
:;tI
10'5 12'9) 7,8 4' 1)
12 8 12 8
Z
;l> :;tI
><:
~
0
29 ± (35) 31 ± (39)
8'4 5,8
0'34 ± 0'20 (0,66) 0'47 ± 0'3 2 (0' 38)
* Significantly different from dosed group at P = < 0'001.
84 ± 19'4 (119) 77 ± 15'0 (84)
3 1 ± 9'9 (3 1) 28 ± 8'5 (23)
12'5 ± 3,0 (12'5) 13'5 ± 1,6 (12'0)
28 ± 10'9 (4 1) 17 ± 7'7 (23)
L! 13 4 13 4
:;tI
z
;l> t""
~
~
"'S"" ~
OXALURIA IN PIGS
~
150
~!_ .... _
2
100
~-.-~
E
421
....--.-.
50
E
80
z8
:5, 4 0
'"
E
cE ~8
0: , '"
6
4
2
0....
/\
l ___
.~ ~=~~----~~--------~
Fig.
I.
Serum values in experiment 3. • - . Dosed pig. 0 - 0 Control pig.
of the dosed and control animals show any statistical difference. In experiment 2 a marked fall in the alkaline phosphatase activity occurred in both dosed and control groups. However, the fall was less in the dosed group. In experiment 3, the most marked changes were increases in the concentrations of total protein, urea, cholesterol and arginase activity on days 8 and 9. Increases in S-GOT and S-GPT were less obvious, as were their differences from the control values. The pattern of S-AP variation is that of an early increase followed by a steady decline in activity.
Urine The results of urine analysis are given in Fig. 2 and are not particularly spectacular. There appear to be three main findings. First, that towards the end of all three experiments there are increases in urinary protein, and this is particularly marked in experiment 3 where the dose levels of oxalate were high. However, there is a lack of consistency in the increases, and in the differences between the dosed and control values. Secondly, in experiment I the dosed pigs excreted large urine volumes on day 7 and smaller ones on day 8, and in experiment 3 the volumes excreted by the dosed pig were generally greater than those excreted by the control. pH and specific gravities were also higher in the dosed pig urine. Thirdly, oxalate crystals were observed in the urine of one dosed pig in experiment 2 on days 9 and 10, and in the urine of the pig in experiment 3
BRITISH VETERINARY JOURNAL, 133, 4
422
E.. pe riment I
Elperimenf 2
Con lrol
Contro l
Dosed
Dosed
Experiment 3 Control
Ur inory
prote in mg /l OO ml Urine volume L pH Okalole
g/ lOO ml 500
Urinar y pro tein
~
mg /lOO m l
Urine
vol ume
l pH
Oxolo le g /iOO ml Olto late dose mg / kg
1500 1000
500
50
50
I 2345678910
I 23 4 567 8 9 10
Day s
Days
Fig.
2.
Days
Urine data following oxalate dosing.
at the end of days 4, 5, 6, 8 and g. Some diuresis occurred in dosed pigs in all three experiments following initial dosing, usually within 24 to 48 h.
Oxalate exretion Mean oxalate excretion, expressed as (COONah for all animals prior to dosing was 0'052 g/24 h in experiment I . In the four-day period following dosing the dosed pigs excreted 0'343 g and 0'246 g, or 46. I % and 45'8% of the dose. In experiment 2 mean urinary oxalate excretion for the control pigs in the experimental period was o' I 20 g/24 h. For the dosed pigs the mean oxalate excretion was 0'348 g and 0'240 g/24 h, or I 1'4% and 6'0% of the dose. Urinary oxalate determinations were not carried out on the pigs in experiment 3. PSP elimination These are summarized in Table II. Histopathology No lesions were demonstrated in the kidneys of dosed pigs in experiments I and 2 . In experiment 3, the kidneys of the dosed pig were firm, rounded, pale brown, and large. On incision, they were pale, and the cortical area apparently enlarged, the ratio of the width of the cortex to the width of the medulla being 4: I for the dosed pig and approximately I: I for the control. Microscopically, the kidneys of the dosed pig showed dilated glomerular spaces and tubules in the cortex. Much more eosinophilic material was seen in the Bowman's capsules of the dosed pig than in the control pig. Eosinophilic material in the kidney tubules, as casts, was seen only in the kidneys of the dosed pig.
OXALURIA IN
PIG~
423
TABLE II HALF-TIME OF ELIMINATION OF PSP FOLLOWING OXALATE DOSING
Tt (minutes)
Pig Experiment OR OR WH WH
I
Experiment BN BK
2
Experiment BNA
3
17. 8 18·6 24'4 22'0
Day
9
Pre-dosing 2 d X 20 mg/kg Pre-dosing 2 d X 20 mg/kg 8
4 10 3
Hours since last dose
Dosing regime (COONah
+I +I
d
X 50
mg/kg
26
d
X 50
mg/kg
2
d X 50 mg/kg d X 50 mg/kg
22'2
8
23 ' 2
10
10
18·8
10
I
d
X 400 3
d
mg/kg X
1600
24 24
+6
d mg/kg
X 800
mg/kg
+
2
Crystalline material was visible microscopically in the kidney tubules, but this material was only von Kossa positive in the tubules of the animal receiving oxalate. At slaughter both animals showed some evidence of chronic nephritis in the form of focal and diffuse areas of mononuclear infiltration. The livers of both animals did not appear to be macroscopically abnormal. Histological sections of the liver of the dosed pig showed that the cells surrounding the central vein had vacuolation typical of fatty infiltration. There was no sign of oedema in either carcass, but there was some interstitial oedema in the kidney of the dosed pig. DISCUSSION
Experiment
I
The lower levels of blood urea in the experimental animals following oxalate administration may be due to a diuretic effect of the dietary oxalate at the dose level employed. However, diuresis following oxalate administration on day 7 seems to be a real effect, since low blood urea levels were found in the experimental pigs in the second week. About 45% of the initial dose of sodium oxalate was excreted in the urine, which was larger than expected, since Archer et ai. (1957) found only about 5% increase in urinary oxalate in human patients. This difference could be attributed to the different methods of administration, since Archer et ai. gave their oxalate in four equal portions after food, whereas in the experiment described in this paper, the animals were not allowed access to food until go min after dosing. In these conditions rapid uptake of oxalate may have occurred, and this would have precluded much of the bacterial decomposition of oxalate reported by Brune & Bredehorn (1961) after they had fed calcium oxalate to pigs. It is possible that the stomach contents of the fasted pig may present less opportunity for the d ecomposition of sodium oxalate or the precipitation of oxalate as the calcium salt. Archer et ai. (1957) have shown that calcium oxalate does not cause an
BRITISH VETERINARY JOURNAL, 133, 4
increase in urinary oxalate in human patients, and Dodson (1959) has shown that in sheep it is excreted mainly in the faeces if the rumen is by-passed. Urinary excretion of protein was transiently increased after dosing with oxalate. This finding has some significance in view of the observations on slaughterhouse material (Wilson et ai., 1972), where certain pigs showed high levels of urinary protein associated with high urinary oxalate. Any functional impairment of the kidneys which may have resulted from oxalate administration in this experiment could not be detected by histological examination post mortem.
Experiment 2 Although most changes in the concentrations of serum parameters in this experiment must be regarded as insignificant, dietary oxalate does seem to have some effect on S-AP activity. Although reduced levels of activity were found in both groups of animals, the reduction was less in the dosed group. This observation has some similarity to that of Tuba et ai. (1952) who noted that dietary sodium oxalate appeared to cause an increase in S-AP activity in the young growing rat. The recovery of urinary oxalate in this experiment was about the same order as that reported by Archer et ai. (1957) in their experiments on human subjects. In their experiments and in those reported in this particular experiment, the methods of administration were similar in that food was present. Similar results were reported by Hardy & Baumann (1959). Absorption of oxalate through the gut (Radeleff, 1964) is probably followed by storage in the tissues, since Curtin & King (1955) demonstrated that injection of l4C labelled oxalate into rats was followed by a wide distribution of oxalic acid in the tissues with specially high concentrations in bone. Accordingly, the increased urinary excretion of oxalate of the dosed pigs in the latter part of this experiment may have reflected an effect of the administered oxalate in exceeding the binding capacity of the pigs' tissues for this substance. The rise in urinary protein on days 3 and 4 which occurred in the pigs dosed with oxalate probably represents a real effect due to sodium oxalate since no similar change was noted in the controls. This effect was similar to, but more pronounced than, the changes observed in the first oxalate experiment. Dietary oxalate, however, seems to be only one of a series of factors involved in determining the level of proteinuria, since the controls also showed a degree of proteinuria, and this was broadly inversely proportional to the daily volume of urine. In this experiment high protein levels in the urine were generally associated with low urinary volumes. Therefore, it seems reasonable to assume that urinary protein excretion in the dosed pigs is affected by oxalate and its effect in reducing urine volume. This hypothesis is substantiated, in part, by the work of other authors. For example, reduction in urine volume in cattle, associated with water deprivation causes a marked proteinuria (Weeth et ai., 1970), and reduced urinary output has been reported by Dodson (1959) in sheep following intravenous administration of sodium oxalate. Further, Derivaux & Liegeois (1962) have reported oliguria and anuria as symptoms in oxalate poisoning in livestock.
OXALURIA IN PIGS
Experiment 3 The increases in the levels of the serum parameters in this experiment appear to be due to d ehydration since the volumes of urine particularly on days 7 and 8, were large. Similar results were noted by Baur & Filer (1959). However, the dehydration effects are probably reversible since McCance (1960) noted the return of blood urea levels to normal once dehydrated pigs became rehydrated. The diuretic effects of sodium oxalate seen in these studies are similar to those noted by James et al. (I 968a) in their experiments on la mbs. The diuretic effect is not solely attributable to oxalate; sodium chloride has been shown to have similar activity. Thus, Ek (1965) has shown that in cases of salt poisoning of pigs in Norway, there were considerable increases in serum electrolytes, total protein and urea. Similarly, Kwatra & Khera (1965a) have reported similar effects when hill cattle in India were fed with potassium oxalate. The observations regarding the increase in S-AP activity in the early stages of the experiment are interesting, and it would appear that this phenomenon is due to the oxalate per se, and not due to any haemoconcentration effect since none was observed at this early stage in the experiment. The precise mechanism of this is difficult to explain. However, the secondary fall in S-AP activity in the dosed pig could be explained by the fact that, as the experiment progressed, less of the administered dose combined with the calcium of the bone, thus reducing the contribution of AP derived from that tissue to the total S-AP. The high specific gravities observed in the urines of the dosed pig were probably a function of dehydration and increased oxalate excretion, and this assumption receives some support from the observations of Bianca (1970) and Baur & Filer (1959) who demonstrated that dehydration is associated with increased specific gravity of the urines of cattle and pigs respectively. Similarly the effect of oxalate in increasing pH values has been noted by James et al. ( 1 968b) in their feeding experiments on lambs. The more pronounced excretion of urinary protein by the dosed pig is in general agreement with the findings of the first two experiments. However, the presence of kidney lesions in both dosed and control animals at slaughter suggests that the high proteinuria may not be attributable solely to oxalate. Nevertheless, the possibility that oxalate exacerbated the nephritis cannot be discounted since K watra & Khera (I 965b) noted this phenomenon in cattle in which Corynebacterium renale infection was superimposed on an oxalate nephritis. How far oxalate can be deemed responsible for kidney damage in 'natural' forms of oxalate poisoning cannot accurately be measured. Friis et al. (1969) in their studies on P. viridicatum poisoning in pigs demonstrated that citrinin was the prime cause of nephrotoxicity, but that other compounds, such as oxalate, might have a powerful synergistic effect. However, they found that feeding pigs with 1'0 g sodium oxalate per kg per day for 42 days resulted only in a slight interstitial renal fibrosis. On these results, they concluded that sodium oxalate toxicity for pigs was low. The work presented in this paper partly substantiates the results of Friis and his colleagues, but suggests that changes in kidney function and pathology occur somewhat earlier when pigs do not have continuous access to drinking
BRITISH VETERINARY JOURNAL, 133, 4
water. Feeding toxic food, not unexpectedly, increases the water reqt'Iirement of pigs (Krogh & Hasselager, 1968; Krogh, 1969) so that synergistic effect of oxalate with citrinin may be powerfully enhanced when water consumption is restricted. AC KN o-WLE D GEMENTS
Our thanks are due to Mr C. R. Snook, F.I.S.T., for valuable technical assistance and the Agricultural Research Council for financial aid. REFERENCES
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