- Email: [email protected]
Ageing changes in the bovine kidney
J. Corn.
PATH.
1986
VOL.
96
AGEING
M.
L.M.
CHANGES
MONAGHAN, Faculty
oj Veterinary
IN
B.J. Medicine,
THE
BOVINE
J. HANNAN
SHEAHAN,
Utniaersity
College
Dublin,
B&bridge,
KIDNEY
and K. MCGILL Dublin
4, Ireland
INTRODUCTION
Histological, ultrastructural and immuno-fluorescence techniques are utilized routinely in the study of kidney disease in man and small domestic animals. Differentiation of chronic glomerulonephritis from chronic interstitial nephritis in the dog, for example, is greatly facilitated by the use of the immuno-fluorescence technique, since the morphological changes visible on light microscopy can be equivocal (Wright, Fisher, Morrison, Thomson and Nash, 1976). Although the glomeruli are normally free from immune reactants in cases of interstitial nephritis, linear deposition of complement on tubular basement membranes has been found occasionally in such cases in the dog (Spencer and Wright, 1981a). Most investigations of renal disease in animals have been conducted into renal disease of the dog and cat, with only occasional reports of such studies in cattle. The immuno-fluorescence technique was used to confirm an individual case of glomerulonephritis in a heifer (Wiseman, Spencer and Petrie, 1980) and to confirm the presence of Bovine Virus Diarrhoea antigen in the glomeruli of four cattle with glomerular lesions (Cutlip, McClurkin and Coria, 1980). Lerner, Dixon and Lee (1968) d escribed a proliferative glomerulonephritis associated with mild proteinuria in 23 of 25 steers which were clinically normal. The diagnosis was based on the proliferation of fixed glomerular cells and linear staining of the glomerular basement membrane with anti-bovine IgG and complement. These findings suggest that most cattle have glomerulonephritis and that the use of techniques for the examination of kidney material from cattle, widely applied in other species, may present difficulties of interpretation. However, the results of an abattoir survey of renal disease in cattle suggest that glomerulonephritis is a relatively unusual condition in this species (Monaghan and Hannan, 1983). The work of Lerner et al. (1968) does not appear to have been repeated and in view of the importance of their findings for the interpretation of bovine renal pathology, it was considered that examination of macroscopically normal kidney material from a number of groups of cattle was an essential prerequisite to a more detailed examination of renal lesions in this species. Macroscopically normal kidneys were therefore collected from three groups of cattle; neonatal calves, young adults and cows. The kidneys were examined using light microscopic, immuno-fluorescence and ultrastructural techniques with a view 002 l-9975/86/060699
+ 12 $03.00/O
0
1986
Academic
Press
Inc.
(London)
Limited
700 to establishing compared.
M.
baseline
L.
values
MATERIALS
M.
t?td.
MONAGHAN
against
AND
which
diseased
kidneys
could
be
METHODS
Kidney tissue was collected from 38 animals (seven neonatal calves, twenty-two 2.5-to-3-year old bullocks and 9 cull cows). The calves were Jersey bull calves, purchased and collected from one farm as soon as possible after birth. The animals were killed within seven days of birth by an intravenous injection of pentobarbitone (200 mg per ml). Urine samples were collected post-mortem by aspiration from the bladder with a needle and syringe. Kidney tissue was collected from the adult cattle in an abattoir. There was an interval of approximately 40 min between exsanguination and removal of the kidneys from the carcase. Before collection of kidneys, a urine sample was aspirated from the bladder of each animal immediately after evisceration and the carcase number was noted. Only macroscopically normal kidneys were selected and the approximate age of the animal was noted from the criteria of Gracey (1981).
Light ib’ficroscopy Histological sections, 2 to 4 pm thick, of formo-fixed, paraffin-embedded tissue were stained with haematoxylin and eosin (HE), Martius scarlet blue and by the periodic acid-Schiff (PAS) method. A semi-quantitative examination of sections from each animal was carried out according to the protocol of Spencer and Wright (1981 b). Total glomerular damage was estimated by counting the glomeruli from two sections so that at least 100 glomeruli were examined and each glomerulus was classified as (a) normal; (b) less than 50 per cent of the tuft obsolescent; (c) 50 per cent or more of the tuft obsolescent or (d) completely obsolescent. Obsolescent glomeruli were sub-classified into the contracted type where tuft scarring predominated and the cystic type where tuft atrophy predominated. A second count all Yved estimation of the basement membrane thickening, incidence of glomerular lesions (glomerular hypercellularity, capsular adhesions and capsular thickening) in non-obsolescent glomeruli. In addition, tubular lesions (atrophy, cystic dilatation and the presence of casts) were recorded and scored for severity: - no lesions recorded + a small number of isolated lesions + + a moderate number of lesions + + + severe and diffuse lesions. A similar scoring system was used for the presence of mononuclear interstitial infiltrates and fibrosis. The chi-squared test (Bradford-Hill, 1977) was used to compare the frequency of lesions between the groups. Immune-Juorescence Slices of kidney, 5 mm in thickness, were snap frozen in liquid nitrogen. Sections were cut 3 pm thick on the cryostat, washed for 30 min in phosphate buffered saline (PBS) pH 7.2 and fixed in acetone for 10 mins. Following 10 mins washing in PBS they were stained with fluorescein isothiocyanate labelled antibovine IgG (Miles Laboratories) in a moist chamber for 30 min and washed for 30 min in three changes of PBS. The sections were mounted and then examined using a microscope equipped for incident light fluorescence. A section of bovine mediastinal lymph node was included in each batch as a positive control.
AGEING
BOVINE
KIDNEYS
701
Electron-microscopy Small pieces (1 mm*) of renal cortex from 11 animals (7 calves and 4 bullocks) were fixed in 4 per cent glutaraldehyde for 3 h, washed in phosphate buffer, post-fixed in osmium tetroxide and, after dehydration in a series of alcohols, embedded in araldite. Ultra-thin sections were cut on an LKB ultramicrotome, stained with uranyl acetate and lead citrate and examined with a Philips 201 electron-microscope. At least three glomeruli which had a normal appearance by light-microscopy were examined from each of the 11 animals.
Urinalysis Analysis of urine was carried out by conventional methods (Benjamin, 1978) and the urine protein content was measured by the Coomassie brilliant blue dye-binding method (Bradford, 1976). The mean urine protein content of the calves and of cows and bullocks combined was compared by the t-test (Snedecor and Cochran, 1980).
RESULTS
Urinalysis No abnormalities were detected on routine urine analysis, the results for all examinations being within the normal range. The urine protein content in calves (Table 1) ranged from 0.025 to 3.81 g per litre. In adults (Tables 2 and 3) the range was from 0.025 to 0.545 g per litre. The mean urine protein content in calves was higher than that in adults (P-c O-01). Immuno-Juorescence Examination of kidney sections from all 38 animals revealed no abnormality. The positive controls showed clear and specific fluorescence of plasma cells with minimal background staining while the kidney sections showed no evidence of fluorescence. Light Microscopy The presence of autolytic change was evident in all sections. In HE sections glomerular tufts appeared as a dense cellular mass contained within Bowman’s capsule, with little or no urinary space. The tubules were invariably collapsed. Glomerular Iesions, in&ding obsolescence, were more obvious in sections stained by PAS than in those stained by HE. The great majority of giomeruli and nephrons were normal in all age groups and no evidence of diffuse lesions was detected. The mesangium was extensive in all casesand clusters of six or more mesangial cells were frequently present. The mesangium was also more easily seen in PAS stained sections. Glomerular scarring was rare, with over 94 per cent of glomeruli showing no evidence of scarring in all age groups. The mean percentage of glomeruli showing no scarring was 98.6 per cent in neonatal calves (Table l), 96.6 per cent in three-year-old-bullocks (Table 2) and 94.5 per cent in cows (Table 3).
GBM
MeatI S.D.
sm.
Mean
= glomerular
membrane.
389 5100
No. glomerulz counted ~___
~--___ Ld
-
_ .___.
OLOMERULAR
Hy~erccllulari~
98.6 f34.0
m&g
406 ilO
No
OF
(per cent)
EXAMINATION
counted
glomeruli
basement
1.518 i 1.62
Urtne protein (g per litre) ~__-.
No.
SEMI-QUANTITATIVE
-
Global
#er
cent)
0.3 f0.3
0.2 f0.2
Total ______
____~
TABLE AND
< 50 per cent scarring
SCARRING
I GLOMERULI
0.2 f0.3
LOW1
CBM
0.4 f0.4
0.3 f0.4
Global
thickening
50 pe7 cm1 D7 more scarring
NON-OBSOLESCENT
-
OF
0.5 ho.6
Total
0.4 f0.5
Contracted
KIDNEYS
(per cent)
IN
NEONATAL
0.5 f0.3
CALVES
0.2 f0.3
Cystic
plomeruli
Adhesions @x77 cent)
Obsolescent
7
0.6 fO.5
Total
1.4 50.6
Capsular thickening (per cent)
(per cent)
Mean
GBM
SD.
Mean
S.D.
= glomerular
basement
0.183 f0.140
Urine protein (g per litre)
membrane.
198 559
No. glomeruli counted
207 -+64
No. gloneruli counted
SEMI-QUANTITATIVE
Global
-
-
Hypercellularity (per cent)
OF
22 2,5-
TABLE SCARRING
Total
1.1 51.2
scarring
i 50 per cent
OF GLOMERULAR NORMAL KIDNEYS
Local
96.6 It2.7
No scarring @er cent)
EXAMINATION
-____-
0.6 f0.6
Local
GEM
0.8 Ltl.1
scarring
0.3 ho.8
Global
thickening (per centj
50 per cent or more
2 AND NON-OBSOLESCENT GLOMERULI T O 3-YEAR-OLD B”JJ..OCKS
0.9 f0.8
Total
0.9 f0.8
Contracted ~.___
0.3 f0.4
Cystic
glomeruli
0.7 f0.8
Adhesions (per cent)
Obsolment
IN MACROSCOPICALLY
cent)
1.2 k-o.95
Total
3.2 f2.5
Capsular thickening (per cent)
(jxr
M 4 v)
;
2
h
5 z
2 G”
Mean S.D.
GBM
MeatI S.D.
Cystic
Total
Global
3.7 f 1.02
Total
1.4 f 1.7
Adhesions (per cent)
glomeruli
Local
1.9 fl.1
Obsolescent
9.0 Ik4.4
Capsular thickening (per cent)
(per cent)
NORMAL KIDNEYS OF 9 CULL cows
Contracted
IN MACROSCOPICALLY
TABLE 3 SCARRING AND NON-OBSOLESCENT
50 per cent or more scarring
Total
1.7 f1.0
thickening (per cent)
-
GBM
GLOMERIJLI
OF GLOMER”LAR
< 50 per cent scarring
0.3 rt 0.4
2.1 f 1.02
EXAMINATION No scarring (per cent)
1.7 fO+il
SEMI-Q”UANTITATI”E No. gloneruli counted
1.3 zkO.6
-
Global
Hypercellularity (per cent)
2.0 f 1.5
-
LOCd
94.5 f 2.5
membrane.
174 f50
No. glomeruli counted
207 l 52
lirine protein (g per litre)
basement
0.131 f 0.039 = glomerular
z
r
z
?
;
z
2
F 2
a
AGEING
BOVINE
KIDNEYS
705
The difference in proportion of scarred glomeruli between the groups was significant (P
706 Transmission
M.
L.
M.
MONAGHAN
et
al.
Electron Microscopy
Post-mortem changes were evident in all cases, the features of which have been described (Monaghan, 1984). No evidence of widespread fusion of epithelial cell foot processes was detected in glomeruli of the calves examined, some of which were excreting considerable amounts of protein (Table 1). Electron-dense areas were not detected in the glomerular basement membrane or in the mesangium of any of the material examined.
DISCUSSION
The smaller plasma proteins that pass through normal glomeruli and the proteins added to urine in the tubules contribute to the small quantity of protein which is present in normal urine (Barsanti and Finco, 1979). However, the concentration of such protein is too low for detection by the routine methods used for urinalysis. The more sensitive Coomassie brilliant blue technique has been used for this purpose in man (McIntosh, 1977) and dog (Barsanti and Finco, 1979) and the results have correlated well with other methods. The protein concentrations found in the calves in this study were in some cases considerably higher than in the other groups of animals. However, a proteinuria in neonatal calves concomitant with the period of permeability of the intestinal mucosa to macromolecules is normal (Pierce, 1961). These proteins consist primarily of non-immune /3 lactoglobulins and lactalbumin (Pierce, 1961) although the finding of IgG in the urine of neonatal calves has also been recorded (Kickhofen, Hammer and Westphal, 1971; Kruse, 1973). The proteinuria reaches a maximum at 25 to 30 h of life and falls off thereafter (Kickhofen et al., 1971). The calves used in the present study varied in age from 2 to 7 days and the varying urine concentrations (Table 1) reflect this gradual reduction in protein loss. Mean urine protein concentration in the bullocks (183 mg per litre) and cows ( 13 1 mg per litre) are slightly higher than those found in a similar group of animals by Lerner et al. ( 1968) and are lower than the average figure of 523 f 115 mg per litre ‘found by Osbaldiston and Moore (197 1). Such amounts of protein are unlikely to be of any clinical significance. Degenerative change is one of the principal characteristics of ageing (Davies, 1983) and the kidney in common with other body systems is subject to morphological and functional alterations with increasing age. Renal morphological changes associated with the ageing process in man are clearly shown in the arterial tree and in glomerular and tubular basement membranes (Darmady and McIver, 1980). Arataki (1926) and Moore (1931) demonstrated the loss of nephrons with increasing age in the albino rat and man, respectively, while a marked reduction in glomerular number has been recorded in man with increasing age (Dunhill and Halley, 1973; McLachlan, Guthrie, Anderson and Fulker, 1977). Darmady, Offer and Woodhouse. (1973) found sclerosed glomeruli in every section from subjects over 30 years old. The range of lesions found in cattle in the present study is similar to those
AGEING
BOVINE
KIDNEYS
707
found in man and the presence of scarred glomeruli, albeit in small numbers, reflects the process of involution of nephrons which continues throughout life. scarring (although not necessarily glomerular In the present study, obsolescence) was present in sections from all animals examined. The proportion increased with age as has been recorded in man and the proportion of scarred glomeruli compares closely with the 4 per cent recorded in a group of normal human subjects aged 50 to 90 years by McGregor ( 1930). The presence of such lesions in neonatal calves is not surprising since sclerosis of a few glomeruli is frequently observed in infants (Heptinstall, 1974) and Craver0 and Culla (1974) found sclerosed glomeruli in all kidneys from 13 aborted bovine foetuses between 6 and 9 months of gestation and from two normal foetuses taken from cows slaughtered at nine months of pregnancy. The proportion of nephrons showing morphological change was very small in the present study. Although there was a slight increase in the proportion affected with increasing age, nephrons showing no evidence of involution comprised well over 90 per cent of the total in all age groups. The age of the cows examined in this study ranged from 5 to 9 years while the maximum life-span of cattle is 15 to 16 years (Miller and Robertson, 1937). It is rare for cattle to reach even half of this maximum under Irish conditions as the culling rate in Irish dairy herds averages 20 per cent (Gleeson, 1979). It seems likely, therefore, that a much greater proportion of nephrons showing involutional change would be found if cattle of advanced age were available for study. Focal accumulations of mononuclear cells were found in most animals and such foci were often associated with small areas of fibrosis and tubular atrophy. These lesions also increased in prevalence with age and this is in keeping with findings in man where increased amounts of interstitial fibrosis and focal accumulations of lymphocytes have also been recorded (Dunhill and Halley, 1973; Darmady et al., 1973). The normal bovine kidney seems, therefore, to undergo a similar range of morphological changes to that of man and the rat, although in the macroscopically normal kidney the proportion of nephrons showing degenerative change is small in all age groups. No evidence of a diffuse proliferative glomerulonephritis as described by Lerner et al. (1968) was found in this study and their finding of an increased number of “fixed glomerular cells” was not corroborated. While IgG is the most common antibody found in glomerulonephritis in other species, the use of antibody against only IgG in the present immuno-fluorescent study could be criticized as being too narrow an investigation. Further work is necessary to evaluate the occurrence of IgA, IgM, IgE and complement in such material. The ultrastructural findings supported the immuno-fluorescence results in that no evidence of electron-dense deposits was found in the glomerular basement membrane or in the mesangium of any of the glomeruli examined. It is interesting to note that, despite the presence of proteinuria to the extent of 3.5 g per litre in some of the calves, no evidence was found of fusion of epithelial cell foot processes, a prominent feature of proteinuria in man and animals. The fusion of foot processes has been shown by scanning electron microscopy to consist of retraction of the foot processes rather than genuine
708
M.
L.
M.
et
MONAGHAN
d.
fusion (Arakawa and Tokunaga, 1972). However, despite the association of foot process fusion with proteinuria in man, this does not invariably occur and a study of the quantitative rela,tionship of foot process width and proteinuria in glomerulonephritis in man (Seefeldt, Bohman, Jorgen, Gunderson, Maunsbach, Petersen and Olsen, 1981) has shown that a large proportion of patients with gross proteinuria had normal foot process width compared with control subjects. It is also likely that the relatively short duration of proteinuria in the neonatal calf is insufficient for the development of this lesion. This study has shown that, in cattle with macroscopically normal kidneys and no detectable abnormality on urinalysis, well in excess of 90 per cent of nephrons were morphologically normal and the process of involution of nephrons became more obvious with increasing age. In contrast to the findings of earlier workers, no evidence of diffuse proliferative glomerulonephritis was seen by histological, immuno-fluorescence or ultrastructural techniques. It is concluded that these techniques should therefore be applied to the investigation of renal disorders in the bovine, as has already been done in man and small domestic animals. SUMMARY
A study of macroscopically normal bovine kidneys from three age groups (neonatal calves, 2.5- to 3-year-old bullocks and cull cows), with no abnormalities on urine analysis, was carried out by light microscopy, immunofluorescence and electron microscopy. There was a slight increase in the proportion of involuted nephrons with increasing age but the proportion of nephrons affected was not greater than 10 per cent in any age group. In contrast to the findings of earlier workers, no evidence of diffuse proliferative glomerulonephritis was found in the material examined. It was concluded that the above techniques should be applied to the investigation of renal disease in cattle,
as has already
been
done
in man
and
small
domestic
animals.
REFERENCES
Arakawa, M. and Tokunaga, J. (1972). A scanning electron microscope study of the glomerulus. Laboratory Investigation, 27, 366-37 1. Arataki, M. (1926). On the post-natal growth of the kidney with special reference to the number 399-436.
and size of the glomeruli
(albino
rat). American Journal
of Anatomy, 36,
Barsanti, J. A. and Finco, D. R. (1979). Protein concentration in urine of normal dogs. American Journal of Veterinary Research, 40, 1583-1587. Benjamin, M. M. (1978). U rinalysis. In: Outline of Veterinary Clinical Pathology. 3rd Edit. Iowa State University Press, Ames, Iowa pp. 180-212. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248-254. Bradford-Hill, A. (1977). A Short Textbook of Medical Statistics. 11 th Edit. Hodder and Stoughton, London pp. 137-144. Glomerulosclerosis in normal Cravero, G. C. and Culla, M. L. (1974). I nvolutional and aborted bovine foetuses. Schzereizer Archiv fir Tierheilkunde, 116, 79-86. Cutlip, R. C., McClurkin, A. W. and Coria, M. F. (1980). Lesions in clinically healthy
AGEING
BOVINE
KIDNEYS
709
viral with the virus of bovine cattle, persistently infected diarrhoea-Glomerulonephritis and Encephalitis. American Journal of Veterinary Research, 41, 1938-1941. London Darmady, E. M. and McIver, A. G. ( 1980). R enal Pathology. Butterworths, pp. 49-57. Darmady, E. M., Offer, J. and Woodhouse, M. A. (1973). The parameters of the ageing kidney. Journal of Pathology, 109, 195-207. Davies, I. (1983). Ageing. Studies in Biology, No. 151. Institute of Biology, Arnold, London pp. l-6. on the quantitative Dunhill, M. S. and Halley, W. J. (1973). S ome observations anatomy of the kidney. Journal of Pathology, 110, 113-12 1. Gleeson, P. A. (1979). Targets in dairy herd replacement rearing. Paper 7. Proceedings of Milk Production Seminar. An Foras Taluntais, Moorepark. Gracy, J. F. (1981). Thornton’s Meat Hygiene. Bailliere Tindall, London p. 51 Heptinstall, R. H. (1974). Pathology of the Kidney. Volume I. 2nd E:dit. Kissane, J. M., Ed., Little, Brown and Company, Boston pp. 65-66. Kickhofen, B., Hammer, D. K. and Westphal, M. (1971). Occurrence of IgG fragments in the urine of the newborn calf. European Journal of Immunology, 1,
49-.54. Kruse, V. (1973). Proteolysis, absorption and excretion of immunoglobulin IgGl in newborn calves. Kongelige Veterinaer-og Landbohojsk Aarsskrift, 173-205. Lerner, R. A., Dixon, F. J. and Lee, S. ( 1968). Sp on t aneous glomerulonephritis in sheep. II. Studies on natural history, occurrence in other species and pathogenesis. American Journal of Pathology, 53, 501-5 12. McGregor, L. (1930). Histological changes in the renal glomerulus in essential rimary) hypertension. A study of fifty-one cases. American Journal of Pathology, 6, k7-369. McIntosh, J. C. (1977). Application of a dye-binding method to the determination of protein in urine and CSF. Clinical Chemistry, 23, 1939-1940. Mclachlan, R. S. F., Guthrie, J. C., Anderson, C. K. and Fulker, M. J. (1977). Vascular and glomerular changes in the ageing kidney. Jourrzal of Pathology, 121, 65-78. Miller, W. C. and Robertson, E. D. S. (1937). Practical Animal Husbandry. 2nd Edit. Oliver and Boyd, London p. 327. Monaghan, M. L. M. (1984). A study of bovine renal disease. PhD. Thesis, University College, Dublin. Monaghan, M. L. M. and Hannan, J. (1983). Abattoir survey of bovine kidney disease. Veterinary Record, 113, 55-57. Moore, R. A. (1931). The total number of glomeruli in the normal human kidney. Anatomical Record, 48, 153-168. Osbaldiston, G. W. and Moore, W. E. (1971). R enal function tests in cattle. Journal of the American Veterinary Medical Association, 159, 292-301. Pierce, A. E. (1961). Proteinuria in the newly-born. Proceedings of the Royal Society of Medicine, 54, 996-999. Seefeldt, T., Bohman, S. O., Jorgen, H., Gunderson, H. J., Maunsbach, A. B., Petersen, V. P. and Olsen, S. (1981). Quantitative relationship between glomerular foot process width and proteinuria in glomerulonephritis. Laboratory Investigation, 44, 541-546. Snedecor, G. W. and Cochran, W. G. (1980). Statistical Methods. 7th Edit. Iowa State University Press, Ames, Iowa pp. 39-63. Spencer, A. J. and Wright, N. G. (1981a). Glomerular lesions in chronic interstitial nephritis in the dog: histological and ultrastructural features. Journal of Comparative Pathology, 91, 393-407. Spencer, A. J. and Wright, N. G. (1981b). Chronic interstitial nephritis in the dog: an immunofluorescence and elution study. Research in Veterinar_ Science, 30, 226-232. Wiseman, A., Spencer, A. J. and Petrie, L. (1980). The nephrotic syndrome in a heifer due to glomerulonephritis. Research in Veterinary Science, 28, 325-329.
710
iv.
L.
M.
MONAGHAN
etal.
Wright, N. G., Fisher, E. W., Morrison, W. I., Thomson, W. B. and Nash, A. S. (1976). Chronic renal failure in dogs: A comparative clinical and morphological study of chronic glomerulonephritis and chronic interstitial nephritis. Veterinary Record, 98, 288-293. [Received for publication,
August 13th, 19851
PATH.
1986
VOL.
96
AGEING
M.
L.M.
CHANGES
MONAGHAN, Faculty
oj Veterinary
IN
B.J. Medicine,
THE
BOVINE
J. HANNAN
SHEAHAN,
Utniaersity
College
Dublin,
B&bridge,
KIDNEY
and K. MCGILL Dublin
4, Ireland
INTRODUCTION
Histological, ultrastructural and immuno-fluorescence techniques are utilized routinely in the study of kidney disease in man and small domestic animals. Differentiation of chronic glomerulonephritis from chronic interstitial nephritis in the dog, for example, is greatly facilitated by the use of the immuno-fluorescence technique, since the morphological changes visible on light microscopy can be equivocal (Wright, Fisher, Morrison, Thomson and Nash, 1976). Although the glomeruli are normally free from immune reactants in cases of interstitial nephritis, linear deposition of complement on tubular basement membranes has been found occasionally in such cases in the dog (Spencer and Wright, 1981a). Most investigations of renal disease in animals have been conducted into renal disease of the dog and cat, with only occasional reports of such studies in cattle. The immuno-fluorescence technique was used to confirm an individual case of glomerulonephritis in a heifer (Wiseman, Spencer and Petrie, 1980) and to confirm the presence of Bovine Virus Diarrhoea antigen in the glomeruli of four cattle with glomerular lesions (Cutlip, McClurkin and Coria, 1980). Lerner, Dixon and Lee (1968) d escribed a proliferative glomerulonephritis associated with mild proteinuria in 23 of 25 steers which were clinically normal. The diagnosis was based on the proliferation of fixed glomerular cells and linear staining of the glomerular basement membrane with anti-bovine IgG and complement. These findings suggest that most cattle have glomerulonephritis and that the use of techniques for the examination of kidney material from cattle, widely applied in other species, may present difficulties of interpretation. However, the results of an abattoir survey of renal disease in cattle suggest that glomerulonephritis is a relatively unusual condition in this species (Monaghan and Hannan, 1983). The work of Lerner et al. (1968) does not appear to have been repeated and in view of the importance of their findings for the interpretation of bovine renal pathology, it was considered that examination of macroscopically normal kidney material from a number of groups of cattle was an essential prerequisite to a more detailed examination of renal lesions in this species. Macroscopically normal kidneys were therefore collected from three groups of cattle; neonatal calves, young adults and cows. The kidneys were examined using light microscopic, immuno-fluorescence and ultrastructural techniques with a view 002 l-9975/86/060699
+ 12 $03.00/O
0
1986
Academic
Press
Inc.
(London)
Limited
700 to establishing compared.
M.
baseline
L.
values
MATERIALS
M.
t?td.
MONAGHAN
against
AND
which
diseased
kidneys
could
be
METHODS
Kidney tissue was collected from 38 animals (seven neonatal calves, twenty-two 2.5-to-3-year old bullocks and 9 cull cows). The calves were Jersey bull calves, purchased and collected from one farm as soon as possible after birth. The animals were killed within seven days of birth by an intravenous injection of pentobarbitone (200 mg per ml). Urine samples were collected post-mortem by aspiration from the bladder with a needle and syringe. Kidney tissue was collected from the adult cattle in an abattoir. There was an interval of approximately 40 min between exsanguination and removal of the kidneys from the carcase. Before collection of kidneys, a urine sample was aspirated from the bladder of each animal immediately after evisceration and the carcase number was noted. Only macroscopically normal kidneys were selected and the approximate age of the animal was noted from the criteria of Gracey (1981).
Light ib’ficroscopy Histological sections, 2 to 4 pm thick, of formo-fixed, paraffin-embedded tissue were stained with haematoxylin and eosin (HE), Martius scarlet blue and by the periodic acid-Schiff (PAS) method. A semi-quantitative examination of sections from each animal was carried out according to the protocol of Spencer and Wright (1981 b). Total glomerular damage was estimated by counting the glomeruli from two sections so that at least 100 glomeruli were examined and each glomerulus was classified as (a) normal; (b) less than 50 per cent of the tuft obsolescent; (c) 50 per cent or more of the tuft obsolescent or (d) completely obsolescent. Obsolescent glomeruli were sub-classified into the contracted type where tuft scarring predominated and the cystic type where tuft atrophy predominated. A second count all Yved estimation of the basement membrane thickening, incidence of glomerular lesions (glomerular hypercellularity, capsular adhesions and capsular thickening) in non-obsolescent glomeruli. In addition, tubular lesions (atrophy, cystic dilatation and the presence of casts) were recorded and scored for severity: - no lesions recorded + a small number of isolated lesions + + a moderate number of lesions + + + severe and diffuse lesions. A similar scoring system was used for the presence of mononuclear interstitial infiltrates and fibrosis. The chi-squared test (Bradford-Hill, 1977) was used to compare the frequency of lesions between the groups. Immune-Juorescence Slices of kidney, 5 mm in thickness, were snap frozen in liquid nitrogen. Sections were cut 3 pm thick on the cryostat, washed for 30 min in phosphate buffered saline (PBS) pH 7.2 and fixed in acetone for 10 mins. Following 10 mins washing in PBS they were stained with fluorescein isothiocyanate labelled antibovine IgG (Miles Laboratories) in a moist chamber for 30 min and washed for 30 min in three changes of PBS. The sections were mounted and then examined using a microscope equipped for incident light fluorescence. A section of bovine mediastinal lymph node was included in each batch as a positive control.
AGEING
BOVINE
KIDNEYS
701
Electron-microscopy Small pieces (1 mm*) of renal cortex from 11 animals (7 calves and 4 bullocks) were fixed in 4 per cent glutaraldehyde for 3 h, washed in phosphate buffer, post-fixed in osmium tetroxide and, after dehydration in a series of alcohols, embedded in araldite. Ultra-thin sections were cut on an LKB ultramicrotome, stained with uranyl acetate and lead citrate and examined with a Philips 201 electron-microscope. At least three glomeruli which had a normal appearance by light-microscopy were examined from each of the 11 animals.
Urinalysis Analysis of urine was carried out by conventional methods (Benjamin, 1978) and the urine protein content was measured by the Coomassie brilliant blue dye-binding method (Bradford, 1976). The mean urine protein content of the calves and of cows and bullocks combined was compared by the t-test (Snedecor and Cochran, 1980).
RESULTS
Urinalysis No abnormalities were detected on routine urine analysis, the results for all examinations being within the normal range. The urine protein content in calves (Table 1) ranged from 0.025 to 3.81 g per litre. In adults (Tables 2 and 3) the range was from 0.025 to 0.545 g per litre. The mean urine protein content in calves was higher than that in adults (P-c O-01). Immuno-Juorescence Examination of kidney sections from all 38 animals revealed no abnormality. The positive controls showed clear and specific fluorescence of plasma cells with minimal background staining while the kidney sections showed no evidence of fluorescence. Light Microscopy The presence of autolytic change was evident in all sections. In HE sections glomerular tufts appeared as a dense cellular mass contained within Bowman’s capsule, with little or no urinary space. The tubules were invariably collapsed. Glomerular Iesions, in&ding obsolescence, were more obvious in sections stained by PAS than in those stained by HE. The great majority of giomeruli and nephrons were normal in all age groups and no evidence of diffuse lesions was detected. The mesangium was extensive in all casesand clusters of six or more mesangial cells were frequently present. The mesangium was also more easily seen in PAS stained sections. Glomerular scarring was rare, with over 94 per cent of glomeruli showing no evidence of scarring in all age groups. The mean percentage of glomeruli showing no scarring was 98.6 per cent in neonatal calves (Table l), 96.6 per cent in three-year-old-bullocks (Table 2) and 94.5 per cent in cows (Table 3).
GBM
MeatI S.D.
sm.
Mean
= glomerular
membrane.
389 5100
No. glomerulz counted ~___
~--___ Ld
-
_ .___.
OLOMERULAR
Hy~erccllulari~
98.6 f34.0
m&g
406 ilO
No
OF
(per cent)
EXAMINATION
counted
glomeruli
basement
1.518 i 1.62
Urtne protein (g per litre) ~__-.
No.
SEMI-QUANTITATIVE
-
Global
#er
cent)
0.3 f0.3
0.2 f0.2
Total ______
____~
TABLE AND
< 50 per cent scarring
SCARRING
I GLOMERULI
0.2 f0.3
LOW1
CBM
0.4 f0.4
0.3 f0.4
Global
thickening
50 pe7 cm1 D7 more scarring
NON-OBSOLESCENT
-
OF
0.5 ho.6
Total
0.4 f0.5
Contracted
KIDNEYS
(per cent)
IN
NEONATAL
0.5 f0.3
CALVES
0.2 f0.3
Cystic
plomeruli
Adhesions @x77 cent)
Obsolescent
7
0.6 fO.5
Total
1.4 50.6
Capsular thickening (per cent)
(per cent)
Mean
GBM
SD.
Mean
S.D.
= glomerular
basement
0.183 f0.140
Urine protein (g per litre)
membrane.
198 559
No. glomeruli counted
207 -+64
No. gloneruli counted
SEMI-QUANTITATIVE
Global
-
-
Hypercellularity (per cent)
OF
22 2,5-
TABLE SCARRING
Total
1.1 51.2
scarring
i 50 per cent
OF GLOMERULAR NORMAL KIDNEYS
Local
96.6 It2.7
No scarring @er cent)
EXAMINATION
-____-
0.6 f0.6
Local
GEM
0.8 Ltl.1
scarring
0.3 ho.8
Global
thickening (per centj
50 per cent or more
2 AND NON-OBSOLESCENT GLOMERULI T O 3-YEAR-OLD B”JJ..OCKS
0.9 f0.8
Total
0.9 f0.8
Contracted ~.___
0.3 f0.4
Cystic
glomeruli
0.7 f0.8
Adhesions (per cent)
Obsolment
IN MACROSCOPICALLY
cent)
1.2 k-o.95
Total
3.2 f2.5
Capsular thickening (per cent)
(jxr
M 4 v)
;
2
h
5 z
2 G”
Mean S.D.
GBM
MeatI S.D.
Cystic
Total
Global
3.7 f 1.02
Total
1.4 f 1.7
Adhesions (per cent)
glomeruli
Local
1.9 fl.1
Obsolescent
9.0 Ik4.4
Capsular thickening (per cent)
(per cent)
NORMAL KIDNEYS OF 9 CULL cows
Contracted
IN MACROSCOPICALLY
TABLE 3 SCARRING AND NON-OBSOLESCENT
50 per cent or more scarring
Total
1.7 f1.0
thickening (per cent)
-
GBM
GLOMERIJLI
OF GLOMER”LAR
< 50 per cent scarring
0.3 rt 0.4
2.1 f 1.02
EXAMINATION No scarring (per cent)
1.7 fO+il
SEMI-Q”UANTITATI”E No. gloneruli counted
1.3 zkO.6
-
Global
Hypercellularity (per cent)
2.0 f 1.5
-
LOCd
94.5 f 2.5
membrane.
174 f50
No. glomeruli counted
207 l 52
lirine protein (g per litre)
basement
0.131 f 0.039 = glomerular
z
r
z
?
;
z
2
F 2
a
AGEING
BOVINE
KIDNEYS
705
The difference in proportion of scarred glomeruli between the groups was significant (P
706 Transmission
M.
L.
M.
MONAGHAN
et
al.
Electron Microscopy
Post-mortem changes were evident in all cases, the features of which have been described (Monaghan, 1984). No evidence of widespread fusion of epithelial cell foot processes was detected in glomeruli of the calves examined, some of which were excreting considerable amounts of protein (Table 1). Electron-dense areas were not detected in the glomerular basement membrane or in the mesangium of any of the material examined.
DISCUSSION
The smaller plasma proteins that pass through normal glomeruli and the proteins added to urine in the tubules contribute to the small quantity of protein which is present in normal urine (Barsanti and Finco, 1979). However, the concentration of such protein is too low for detection by the routine methods used for urinalysis. The more sensitive Coomassie brilliant blue technique has been used for this purpose in man (McIntosh, 1977) and dog (Barsanti and Finco, 1979) and the results have correlated well with other methods. The protein concentrations found in the calves in this study were in some cases considerably higher than in the other groups of animals. However, a proteinuria in neonatal calves concomitant with the period of permeability of the intestinal mucosa to macromolecules is normal (Pierce, 1961). These proteins consist primarily of non-immune /3 lactoglobulins and lactalbumin (Pierce, 1961) although the finding of IgG in the urine of neonatal calves has also been recorded (Kickhofen, Hammer and Westphal, 1971; Kruse, 1973). The proteinuria reaches a maximum at 25 to 30 h of life and falls off thereafter (Kickhofen et al., 1971). The calves used in the present study varied in age from 2 to 7 days and the varying urine concentrations (Table 1) reflect this gradual reduction in protein loss. Mean urine protein concentration in the bullocks (183 mg per litre) and cows ( 13 1 mg per litre) are slightly higher than those found in a similar group of animals by Lerner et al. ( 1968) and are lower than the average figure of 523 f 115 mg per litre ‘found by Osbaldiston and Moore (197 1). Such amounts of protein are unlikely to be of any clinical significance. Degenerative change is one of the principal characteristics of ageing (Davies, 1983) and the kidney in common with other body systems is subject to morphological and functional alterations with increasing age. Renal morphological changes associated with the ageing process in man are clearly shown in the arterial tree and in glomerular and tubular basement membranes (Darmady and McIver, 1980). Arataki (1926) and Moore (1931) demonstrated the loss of nephrons with increasing age in the albino rat and man, respectively, while a marked reduction in glomerular number has been recorded in man with increasing age (Dunhill and Halley, 1973; McLachlan, Guthrie, Anderson and Fulker, 1977). Darmady, Offer and Woodhouse. (1973) found sclerosed glomeruli in every section from subjects over 30 years old. The range of lesions found in cattle in the present study is similar to those
AGEING
BOVINE
KIDNEYS
707
found in man and the presence of scarred glomeruli, albeit in small numbers, reflects the process of involution of nephrons which continues throughout life. scarring (although not necessarily glomerular In the present study, obsolescence) was present in sections from all animals examined. The proportion increased with age as has been recorded in man and the proportion of scarred glomeruli compares closely with the 4 per cent recorded in a group of normal human subjects aged 50 to 90 years by McGregor ( 1930). The presence of such lesions in neonatal calves is not surprising since sclerosis of a few glomeruli is frequently observed in infants (Heptinstall, 1974) and Craver0 and Culla (1974) found sclerosed glomeruli in all kidneys from 13 aborted bovine foetuses between 6 and 9 months of gestation and from two normal foetuses taken from cows slaughtered at nine months of pregnancy. The proportion of nephrons showing morphological change was very small in the present study. Although there was a slight increase in the proportion affected with increasing age, nephrons showing no evidence of involution comprised well over 90 per cent of the total in all age groups. The age of the cows examined in this study ranged from 5 to 9 years while the maximum life-span of cattle is 15 to 16 years (Miller and Robertson, 1937). It is rare for cattle to reach even half of this maximum under Irish conditions as the culling rate in Irish dairy herds averages 20 per cent (Gleeson, 1979). It seems likely, therefore, that a much greater proportion of nephrons showing involutional change would be found if cattle of advanced age were available for study. Focal accumulations of mononuclear cells were found in most animals and such foci were often associated with small areas of fibrosis and tubular atrophy. These lesions also increased in prevalence with age and this is in keeping with findings in man where increased amounts of interstitial fibrosis and focal accumulations of lymphocytes have also been recorded (Dunhill and Halley, 1973; Darmady et al., 1973). The normal bovine kidney seems, therefore, to undergo a similar range of morphological changes to that of man and the rat, although in the macroscopically normal kidney the proportion of nephrons showing degenerative change is small in all age groups. No evidence of a diffuse proliferative glomerulonephritis as described by Lerner et al. (1968) was found in this study and their finding of an increased number of “fixed glomerular cells” was not corroborated. While IgG is the most common antibody found in glomerulonephritis in other species, the use of antibody against only IgG in the present immuno-fluorescent study could be criticized as being too narrow an investigation. Further work is necessary to evaluate the occurrence of IgA, IgM, IgE and complement in such material. The ultrastructural findings supported the immuno-fluorescence results in that no evidence of electron-dense deposits was found in the glomerular basement membrane or in the mesangium of any of the glomeruli examined. It is interesting to note that, despite the presence of proteinuria to the extent of 3.5 g per litre in some of the calves, no evidence was found of fusion of epithelial cell foot processes, a prominent feature of proteinuria in man and animals. The fusion of foot processes has been shown by scanning electron microscopy to consist of retraction of the foot processes rather than genuine
708
M.
L.
M.
et
MONAGHAN
d.
fusion (Arakawa and Tokunaga, 1972). However, despite the association of foot process fusion with proteinuria in man, this does not invariably occur and a study of the quantitative rela,tionship of foot process width and proteinuria in glomerulonephritis in man (Seefeldt, Bohman, Jorgen, Gunderson, Maunsbach, Petersen and Olsen, 1981) has shown that a large proportion of patients with gross proteinuria had normal foot process width compared with control subjects. It is also likely that the relatively short duration of proteinuria in the neonatal calf is insufficient for the development of this lesion. This study has shown that, in cattle with macroscopically normal kidneys and no detectable abnormality on urinalysis, well in excess of 90 per cent of nephrons were morphologically normal and the process of involution of nephrons became more obvious with increasing age. In contrast to the findings of earlier workers, no evidence of diffuse proliferative glomerulonephritis was seen by histological, immuno-fluorescence or ultrastructural techniques. It is concluded that these techniques should therefore be applied to the investigation of renal disorders in the bovine, as has already been done in man and small domestic animals. SUMMARY
A study of macroscopically normal bovine kidneys from three age groups (neonatal calves, 2.5- to 3-year-old bullocks and cull cows), with no abnormalities on urine analysis, was carried out by light microscopy, immunofluorescence and electron microscopy. There was a slight increase in the proportion of involuted nephrons with increasing age but the proportion of nephrons affected was not greater than 10 per cent in any age group. In contrast to the findings of earlier workers, no evidence of diffuse proliferative glomerulonephritis was found in the material examined. It was concluded that the above techniques should be applied to the investigation of renal disease in cattle,
as has already
been
done
in man
and
small
domestic
animals.
REFERENCES
Arakawa, M. and Tokunaga, J. (1972). A scanning electron microscope study of the glomerulus. Laboratory Investigation, 27, 366-37 1. Arataki, M. (1926). On the post-natal growth of the kidney with special reference to the number 399-436.
and size of the glomeruli
(albino
rat). American Journal
of Anatomy, 36,
Barsanti, J. A. and Finco, D. R. (1979). Protein concentration in urine of normal dogs. American Journal of Veterinary Research, 40, 1583-1587. Benjamin, M. M. (1978). U rinalysis. In: Outline of Veterinary Clinical Pathology. 3rd Edit. Iowa State University Press, Ames, Iowa pp. 180-212. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248-254. Bradford-Hill, A. (1977). A Short Textbook of Medical Statistics. 11 th Edit. Hodder and Stoughton, London pp. 137-144. Glomerulosclerosis in normal Cravero, G. C. and Culla, M. L. (1974). I nvolutional and aborted bovine foetuses. Schzereizer Archiv fir Tierheilkunde, 116, 79-86. Cutlip, R. C., McClurkin, A. W. and Coria, M. F. (1980). Lesions in clinically healthy
AGEING
BOVINE
KIDNEYS
709
viral with the virus of bovine cattle, persistently infected diarrhoea-Glomerulonephritis and Encephalitis. American Journal of Veterinary Research, 41, 1938-1941. London Darmady, E. M. and McIver, A. G. ( 1980). R enal Pathology. Butterworths, pp. 49-57. Darmady, E. M., Offer, J. and Woodhouse, M. A. (1973). The parameters of the ageing kidney. Journal of Pathology, 109, 195-207. Davies, I. (1983). Ageing. Studies in Biology, No. 151. Institute of Biology, Arnold, London pp. l-6. on the quantitative Dunhill, M. S. and Halley, W. J. (1973). S ome observations anatomy of the kidney. Journal of Pathology, 110, 113-12 1. Gleeson, P. A. (1979). Targets in dairy herd replacement rearing. Paper 7. Proceedings of Milk Production Seminar. An Foras Taluntais, Moorepark. Gracy, J. F. (1981). Thornton’s Meat Hygiene. Bailliere Tindall, London p. 51 Heptinstall, R. H. (1974). Pathology of the Kidney. Volume I. 2nd E:dit. Kissane, J. M., Ed., Little, Brown and Company, Boston pp. 65-66. Kickhofen, B., Hammer, D. K. and Westphal, M. (1971). Occurrence of IgG fragments in the urine of the newborn calf. European Journal of Immunology, 1,
49-.54. Kruse, V. (1973). Proteolysis, absorption and excretion of immunoglobulin IgGl in newborn calves. Kongelige Veterinaer-og Landbohojsk Aarsskrift, 173-205. Lerner, R. A., Dixon, F. J. and Lee, S. ( 1968). Sp on t aneous glomerulonephritis in sheep. II. Studies on natural history, occurrence in other species and pathogenesis. American Journal of Pathology, 53, 501-5 12. McGregor, L. (1930). Histological changes in the renal glomerulus in essential rimary) hypertension. A study of fifty-one cases. American Journal of Pathology, 6, k7-369. McIntosh, J. C. (1977). Application of a dye-binding method to the determination of protein in urine and CSF. Clinical Chemistry, 23, 1939-1940. Mclachlan, R. S. F., Guthrie, J. C., Anderson, C. K. and Fulker, M. J. (1977). Vascular and glomerular changes in the ageing kidney. Jourrzal of Pathology, 121, 65-78. Miller, W. C. and Robertson, E. D. S. (1937). Practical Animal Husbandry. 2nd Edit. Oliver and Boyd, London p. 327. Monaghan, M. L. M. (1984). A study of bovine renal disease. PhD. Thesis, University College, Dublin. Monaghan, M. L. M. and Hannan, J. (1983). Abattoir survey of bovine kidney disease. Veterinary Record, 113, 55-57. Moore, R. A. (1931). The total number of glomeruli in the normal human kidney. Anatomical Record, 48, 153-168. Osbaldiston, G. W. and Moore, W. E. (1971). R enal function tests in cattle. Journal of the American Veterinary Medical Association, 159, 292-301. Pierce, A. E. (1961). Proteinuria in the newly-born. Proceedings of the Royal Society of Medicine, 54, 996-999. Seefeldt, T., Bohman, S. O., Jorgen, H., Gunderson, H. J., Maunsbach, A. B., Petersen, V. P. and Olsen, S. (1981). Quantitative relationship between glomerular foot process width and proteinuria in glomerulonephritis. Laboratory Investigation, 44, 541-546. Snedecor, G. W. and Cochran, W. G. (1980). Statistical Methods. 7th Edit. Iowa State University Press, Ames, Iowa pp. 39-63. Spencer, A. J. and Wright, N. G. (1981a). Glomerular lesions in chronic interstitial nephritis in the dog: histological and ultrastructural features. Journal of Comparative Pathology, 91, 393-407. Spencer, A. J. and Wright, N. G. (1981b). Chronic interstitial nephritis in the dog: an immunofluorescence and elution study. Research in Veterinar_ Science, 30, 226-232. Wiseman, A., Spencer, A. J. and Petrie, L. (1980). The nephrotic syndrome in a heifer due to glomerulonephritis. Research in Veterinary Science, 28, 325-329.
710
iv.
L.
M.
MONAGHAN
etal.
Wright, N. G., Fisher, E. W., Morrison, W. I., Thomson, W. B. and Nash, A. S. (1976). Chronic renal failure in dogs: A comparative clinical and morphological study of chronic glomerulonephritis and chronic interstitial nephritis. Veterinary Record, 98, 288-293. [Received for publication,
August 13th, 19851