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Juvenile renal disease in the Doberman Pinscher: Ultrastructural changes of the glomerular basement membrane
J. COMP.
PATH.
1987
VOL.
97
JUVENILE RENAL DISEASE IN PINSCHER: ULTRASTRUCTURAL GLOMERULAR BASEMENT CATHERINE Department
ff
Pathology,
NW,
THE DOBERMAN CHANGES OF THE MEMBRANE
A. PICUT and R. M. LEWIS
Ybrk State College
of Veterinq 1:s.d.
Medicine.
Cornell
Uniuersi@.
Ithaca,
.2’ew
York.
INTRODUCTION
Juvenile Renal Disease progressive renal disease
ORD) resulting
in the Doberman Pinscher is an hereditary in azotaemia, marked proteinuria, and renal
failure in young male and female dogs (Wilcock
and Patterson,
1979; Chew,
1983). Histologically, the lesion is DiBartola, Boyce, Hayes and Brace, characterized as an idiopathic membrano-proliferative glomerulonephritis (MPGN, Chew et al., 1983). Although the primary lesion is believed to exist in the glomerulus, no underlying structural or immunological abnormality has been reported. This paper describes ultrastructural changes of the glomerular in Doberman Pinschers with JRD. The nature of basement membrane (GBM) the changes suggests a primary structural or metabolic defect of the GBM. MATERIALS
AND
METHODS
Ten cases of idiopathic membrano-proliferative glomerulonephritis in juvenile and adolescent Doberman Pinschers were selected from material submitted to the diagnostic pathology services at the Drew York State College of Veterinary Medicine between January 1977 and December 1985. Renal tissue obtained at necropsy was fixed in 10 per cent neutral buffered formalin and embedded in paraffin wax for histopathology. Sections (2 to 4 pm thick) were stained with haematoxylin and eosin (HE), alcian blue, Masson’s trichrome, Jones’ methenamine silver (JMS) and periodic-acid Schiff (PAS) stains. Electron microscopy of renal tissue was performed on eight cases. Tissues fixed as above were post-fixed in 1 per cent osmium tetroxide, embedded in epon-araldite, stained with uranyl acetate and lead citrate and observed under a Phillips EM-301 electron miscroscope. Indirect immunofluorescence of paraffin-embedded renal tissue was performed on 6 urn thick tissue sections by a standard protocol established in our laboratory. Deparaffinized tissue was trypsinized for 6 h in 0.5 per cent trypsin in 0.1 per cent CaCl, (pH 7.8 at 37 “C) and stored in phosphate buffered saline (PBS, pH 7.2) overnight at 4 “C. Each section was incubated with rabbit anti-dog IgA, IgM, complement and IgG (100 mg antibody protein per ml) in a humid chamber for 2 h at 37 “C (Reagents: Rabbit anti-dog IgA, Pel Freeze Biologicals; Rabbit anti-dog IgG and IgM, Miles Scientific; Rabbit anti-dog Complement, U.S. Biochemical Corporation). Fluorescein-labelled goat anti-rabbit immunoglobulin (100 mg antibody protein per ml) was applied to washed tissue and incubated for 30 min at room temperature. Following two washes in PBS, the tissue was mounted for 002lL9975/87/050587+
10 $03.00/O
0
1987
Academic
Press
Limited
588
CATHERINE
A.
PICUT
AND
R.
M.
LEWIS
fluorescent microscopy. Positive fluorescence of plasma cells served as an internal positive control while positive fluorescence of a documented case of immune-mediated glomerulonephritis in the dog served as an external positive control. RESULTS
The age and sex of the ten Doberman Pinschers included in this study are listed in Table 1. There were seven males and three females, with the age at death or euthanasia ranging from 4 to 42 months. All animals showed clinical signs of advanced renal failure, including azotaemia, polyuria, polydipsia, vomiting, anorexia and/or elevated blood urea nitrogen and creatinine. Urine analysis in 7 dogs revealed marked proteinuria (3+ to 4+ on an arbitrary scale) and one dog had microscopic haematuria.
JUVENILE
RENAL
DISEASE
TABLE 1 IN DOBERMAN
PINSCHERS
(IO
Lesion
Case No.
Age* (months)
1
4 9
2 3 4 5
24 42
6 7 8
5 8 36
9 10
36
10
9
Lanellation of Lamina densa (Lesion No. I)
sex
M M M F M F M M M F
IgM -
CASES)
of GBM
Electron-dense deposits
+ + + + + -
+
-
-
NOT NOT
Collagtn jbre (Lesion No. 2)
+ + +
EXAMINED EXAMINED
*Age at death or time of euthanasia. GBM = Glomerular basement membrane
Gross Pathology Examination of the gross lesion was possible in four cases. The kidneys were of normal size and had a coarsely granular to finely pitted subcapsular surface. The cortex was finely mottled dark brown to yellow in two of these cases (Fig. 1). Histopathology All lesions were classified as a membrano-proliferative glomerulonephritis (MPGN), with varying degrees of tubulo-interstitial disease [Figs 2(a) and (b)] . Lobular accentuation of glomerular tufts, increased mesangial matrix,
589
GLOMERULARBASEMENTMEMBRANES
Fig.
1. Kidney surface.
from
Doberman
Pinscher
with
Juvenile
Renal
Disease.
Note
fine
mottling
of subcapsular
hypercellularity, intraglomerular adhesions, exudation of fibrin and fibroepithelial crescents characterized the glomerular lesion. In three cases, the glomerular lesion was diffuse, with minimal interstitial fibrosis or tubular atrophy. In the remaining seven cases, the glomerular lesion was multifocal and accompanied by severe tubulo-interstitial disease. Patchy areas of tubular atrophy, interstitial fibrosis, periglomerular fibrosis, cystic distention of Bowman’s capsule (cystic glomerular atrophy) and a, mild to moderate lymphoplasmacytic interstitial nephritis characterized the tubulo-interstitial changes. In one case, cystic glomerular atrophy dominated the morphological lesion.
Nine cases were negative for the immunoreactants, complement. In one case, scattered deposits of IgM glomerular basement membrane. Electron
IgG, IgM, IgA were present within
and the
Microscopy
Among the eight cases examined, two distinct structural lesions of the glomerular basement membrane (GBM) were apparent. The first, observed in five cases, was characterized by multifocal irregular thickening of the GBM with lamellation of the lamina densa [Fig. 3(a)]. Focal areas of lucency
590
CATHERINE
A.
PICUT
AND
R.
M.
LEWIS
GLOMERULARBASEMENTMEMBRANES
591
containing electron-dense particles were observed. In one of these cases, intramembranous electron-dense deposits were present. The second lesion, observed in three cases, was characterized by diffuse thickening of the GBM zone [Fig. 3(b)]. There was marked attenuation of the lamina densa with intramembranous and/or subendothelial deposition of matrix composed of cross-banded fibres [Figs 4(a) and (b)]. These prominent, randomly dispersed cross-banded fibres had the periodicity of collagen [Fig. 4(b)]. Focal fusion of overlying foot-processes was a general finding in all cases examined. DISCUSSION
The histopathological features of these ten cases of JRD in Doberman Pinschers were similar to those lesions described previously for this condition (Chew et al., 1983; Wilcock and Patterson, 1979). The morphological picture ranged from a multifocal MPGN with severe tubulo-interstitial inflammation to a severe diffuse glomerulonephritis with minimal tubulo-interstitial disease. Cystic glomerular atrophy commonly accompanied severe interstitial nephritis. Two distinct ultrastructural lesions of the GBM were observed. The first was characterized by irregular thickening of the GBM with lamellation of the lamina densa, focal areas of lucency and intramembranous aggregates of electron dense particles. The second lesion was a diffuse change manifested by attenuation of the lamina densa with thickening of the GBM which contained randomly dispersed intramembranous collagen fibres. There was no apparent relationship between the two distinct histopathological patterns and the two ultrastructural lesions. These ultrastructural changes may be due to a primary structural defect of the GBM or they may represent metabolic or biochemical abnormalities. Similar lesions have been observed in other hereditary nephropathies in man and dog. The basic structural lesion associated with hereditary nephritis (Alport’s syndrome) in man and familial glomerulopathy in Samoyeds resembles one of the alterations seen in Doberman Pinschers (i.e. irregular thickening of the GBM, lamellation of the lamina densa and focal areas of lucency containing electron dense granules) (Churg and Sherman, 1973; Gubler, Levy, Broyer, Naizot, Gonzales, Perrin and Habib, 1981; Heptinstall, 1974; Jansen, Thorner, Singh, Patterson, Lumsden, Valli, Baumal and Basrur, 1984; Krickstein, Gloor and Balogh, 1966; Perkoff, 1983; Spear, 1984; Suki and Caskey, 1979; Yoshikawa, Cameron and White, 1981; Bernstein, 1979). Limited biochemical analyses of the GBM in hereditary nephritis reveals abnormal amounts of hydroxyproline, cysteine and sialic acid (DiBona, 1983), Fig.
2. Light microscopy of Juvenile Renal Disease in Doberman Pincshers. (a) Membrano-proliferative glomerulonephritis with pronounced tubule-interstitial disease and severe cystic glomerular atrophy. HE x 40. (b) Membrane-proliferative glomerular lesion with fibro-epithelial crescent, lobular accentuation of tuft, and exudation of fibrin into Bowman’s space (arrows). The limit of Bowman’s capsule is difficult to discern due to surrounding interstitial fibrosis. HE x 320.
CATHERINE
A.
PICUT
AND
R.
M.
LEWIS
Fig. 3. Ultrastructural changes of the glomerular basement membrane (GBM) in Juvenile Renal Dise Doberman Pincshers. (a) Irregularly thickened GBM with lamellation oflamina densa (arrowhead). Also present al -e two dense large electron dense deposits (arrows) within focal lucent zones surrounded by fine electron particles. Note fusion of overlying foot processes. (EP= epithelial cell). x 9100. (b) Diffusely thickened GBM zone composed of subendothelial fibrillar matrix (arrows) al nd an attenuated lamina densa (arrowhead). (EN=endothelial cell, EP=epithelial cell). X 7500.
GLOMERULARBASEMENTMEMBRANES
of GBM lesion [Fig. 3(b)]: 4. Higher m agnification basement membrane zone characterized by attenuated lamina de nsa (arrow; (a) Glom xular fibrillar matrix (arrowhead). (EN = endothelial cell). x 25 000. abundant (b) Glomc zrular basement membrane with overlying epithelial foot processes. Notes< :vere attenua densa in subepithelial zone (arrow) and numerous cross-banded fib1 yes (arrowhe of lamina x 45 000.
593
and tion ad).
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CATHERINE
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thereby implying an underlying defect in collagen synthesis or maturation. Such a maturational defect is further substantiated by the absence of Goodpasture’s antigen in some patients with HN (DiBona, 1983; Jeraj, Kim, Vernier, Fish and Michael, 1983; McCoy, Johnson, Stone and Wilson, 1982; Olson, Anand, Landing, Heuser, Grushkin and Lieberman, 1980; Spear, 1984), thus suggesting a failure of maturation of the GBM with retention of fetal glomerular antigens (Grunfeld, 1985; Martinez-Hernandez and Amenta, 1983; Olson et al., 1980). Recently the absence of the Goodpasture’s antigen has also been reported in Samoyeds with familial glomerulopathy (Thorner, Jansen, Baumal and Valli, 1986). Attenuation of the lamina densa with intramembranous and/or subendothelial collagen fibres is a unique finding and has not been previously reported in the dog. Intramembranous collagen fibres have been associated only with the human disorder, hereditary osteo-onychodysplasia (nail-patella syndrome). This is a rare syndrome consisting of multiple skeletal anomalies and renal disease in 30 to 40 per cent of patients (Leaky, 1966; Silverman, Goodman and Cuppage, 1967; Similia, Vesa and Wasz-Hockert, 1970; Bernstein, 1979). Histologically, the renal lesion is characterized by segmental glomerulosclerosis and chronic interstitial nephritis (Bernstein, 1979; Silverman et al., 1967; Hoyer, Michael and Vernier, 1972). Ultrastructurally, diffuse irregular thickening of the GBM containing lucent areas and randomly arranged collagen fibres characterizes the lesion (Ben-Bassat, Cohen and Rosenfeld, 1971; Bennet, Musgrave, Campbell, Elliot, Cox, Brooks, Lovrien, Beals and Porter, 1973). The presence of these collagen fibres is considered to be a manifestation of a basic, yet undefined structural membrane abnormality (MartinezzHernandez and Amenta, 1983). Type IV collagen in all basement membranes differs from other types of collagen in that it is incorporated into basement membrane with its pro-peptide extensions intact (Minor, Clark, Strause, Koszalka, Brent and Kefalides, 1976), thereby preventing the assembly of cross-banded fibres (MartinezzHernandez and Amenta, 1983). The observed presence of cross-banded collagen fibres in the GBM of affected Doberman Pinschers suggests either a switch in collagen phenotype to other than type IV collagen or extracellular cleavage of type IV collagen pro-peptides resulting in cross-banding formation (Martinez-Hernandez and Amenta, 1983). The significance of intramembranous electron-dense deposits in one dog may represent resolving immune complexes trapped in an abnormal GBM by non-immunological mechanisms. Equally important is the possibility that abnormalities, as have been underlying biochemical or immunological described in HN and familial glomerulopathy in Samoyeds, might lead to increased capillary affinity for circulating antigens with subsequent in situ immune complex formation (Bertani, Appel, D’Agati, Nash and Pirani, 1983). explain the occasional reports of weakly positive This would immunofluorescent tests for intraglomerular IgG, IgM or complement in hereditary nephritis, osteo-onychodysplasia and JRD in Doberman Pinschers (Gubler et al., 1981; Bernstein, 1979; Bennett et al., 1973; Hoyer et al., 1972; Chew et al., 1983).
GLOMERULAR
BASEMENT
595
MEMBRANES
To date, JRD in Doberman Pinschers has been considered to be an acquired entity occurring with a familial distribution. The ultrastructural lesions described in this report suggest the alternative possibility of a primary metabolic or developmental defect involving the extracellular matrices of the glomerulus. To define the pathogenesis of the glomerulopathy in Doberman Pinschers, prospective morphological, biochemical, and immunological studies of early lesions will be necessary in which the primary structural alteration or metabolic abnormality may be identified. Clinicopathological correlations will help to determine to what extent these structural alterations relate to functional impairment of the GBM. Pedigree analyses are needed, not only to determine the precise mode of inheritance of this disorder, but to help to clarify the genetic bases of possible structural or metabolic defects associated with JRD in the Doberman Pinscher. SUMMARY
Ten cases ofjuvenile renal disease in Doberman Pinschers were examined by light microscopy and 8 of them additionally by electron microscopy. Two distinct basic ultrastructural lesions of the glomerular basement membrane (GBM) were observed. One is characterized by lamellation of the lamina densa with intramembranous focal areas of lucency containing electron-dense particles, the second by diffuse attenuation of the lamina densa with intramembranous and/or subendothelial deposition of matrix entrapping cross-banded fibres (collagen). Based on similar ultrastructural changes in other hereditary nephropathies in man and dogs, a metabolic or biochemical basis for the structural lesions is suspected. ACKNOWLEDGMENT
This study was 088T22EF07 105.
supported
by
the
National
Institute
of Health
Grant
No.
REFERENCES
Ben-Bassat, M., Cohen, L. and Rosenfeld, J. (1971). The glomerular basement membrane in the nail-patella syndrome. Archives of Pathology, 92, 350-355. Bennett, W. M., Musgrave, J. E., Campbell, R. A., Elliot, D., Cox, R., Brooks, R. E., Lovrien, E. W., Beals, R. K. and Porter, G. A. (1973). The nephropathy of the nail-patella syndrome. Clinicopathological analysis of 11 kindred. American Journal of Medicine, 54, 304-3 19. Bernstein, J. (1979). Hereditary renal disease. In Kidny Disease: Present Status. J. Schurg, B. H. Spargo, F. K. Mostofi, M. R. Abell, Eds, Williams & Wilkins Co., Baltimore. Bertani, T., Appel, G. B., D’Agati, V., Nash, M. A. and Pirani, C. L. (1983). Focal segmental membranous glomerulonephropathy associated with other glomerular diseases. American Journal of Kidny Disease, 11, 439-448. Chew, D. J., DiBartola, S. P., Boyce, J. T., Hayes, H. M. and Brace, J. J. (1983). Juvenile renal disease in Doberman Pinscher Dogs. Journal of the American Veterinary Medical Association, 182, 481-485. Churg, J. and Sherman, R. L. (1973). Pathologic characteristics of hereditary nephritis. Archives of Pathology, 95, 374-379.
596
CATHERINE
A.
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AND
R.
M.
LEWIS
DiBona, G. (1983). Alport’s syndrome: a genetic defect in biochemical composition of basement membrane of glomerulus, lens, and inner ear. Journal of Laboratory and Clincial Medicine, 101, 817-820. Grunfeld, J. P. (1985). The clinical spectrum of hereditary nephritis. Kidnq International, 27, 83-92. Gubler, M., Levy, M., Broyer, M., Naizot, C., Gonzales, G., Perrin, D. and Habib, R. (1981). Alport’s syndrome: a report of 58 cases and a review of the literature. American Journal of Medicine, 70, 493-505. Heptinstall, R. H. (1974). Miscellaneous conditions affecting the kidney. In Pathology of the Kidney, R. H. Heptinstall, Ed., Little, Brown and Co., Boston, pp. 1135-l 145. Hoyer, J. R., Michael, A. F. and Vernier, K. L. (1972). Kenal disease in nail-patella syndrome: clinical and morphologic studies. Kidney International, 2, 23 l-238. Jansen, B., Thorner, P. S., Singh, A., Patterson, J. M., Lumsden, J. H., Valli, V. E., Baumal, R. and Basrur, P. K. (1984). Hereditary nephritis in Samoyed dogs. American Journal of Pathology, 116, 175-178. Jeraj, K., Kim, Y., Vernier, R. L., Fish, A. J. and Michael, ,4. F. ( 1983). Absence of Goodpasture’s antigen in male patients with familial nephritis. American Journal of Kidng Disease, 2, 626. Krickstein, H. I., Gloor, F. J. and Balogh, K. ( 1966). R enal pathology in hereditary nephritis with nerve deafness. Archiues of Pathology, 82, 506-5 17. Leaky, M. S. (1966). The hereditary nephropathy of osteo-onychodysplasia, (nail-patella Syndrome). American Journal of Diseases of Children, 112, 237-241. Martinez-Hernandez, A. and Amenta, P. S. (1983). The basement membrane in pathology. Laboratov Investigation, 48, 656-677. McCoy, R. C., Johnson, H. K., Stone, W. J. and Wilson, C. B. (1982). Absence of nephritogenic GBM antigen(s) in some patients with hereditary nephritis. Kidney International, 21, 642-652. Minor, R. R., Clark, C. C., Strause, E. I,., Koszalka, T. R., Brent, R. L. and Kefalides, N. A. (1976). Basement membrane procollagen is not converted to collagen in organ cultures of parietal yolk sac endoderm. Journal of Biological Chemistry, 251, 1789-l 794. Olson, D. L., Anand, S. K., Landing, B. H., Heuser, E., Grushkin, C. M. and Lieberman, E. ( 1980). Diagnosis of hereditary nephritis by failure of glomeruli to bind anti-glomerular basement membrane antibodies. Journal of Pediatrics, 96, 697-699. Perkoff, G. T. (1983). The hereditary renal diseases. .New England journal of Medicine, 277, 79-85, 129-138. Silverman, ,M. E., Goodman, R. M. and Cuppage, F. E. (1967). The nail-pateila syndrome. Clinical findings and ultrastructural observations in the kidney. Archives of Internal Medicine, 120, 68-74. Similia, S., Vesa, L. and Wasz-Hockert, 0. (1970). H ereditary osteo-onychodyspfasia (nail-pateha syndrome) with nephrosis-like renal disease in a newborn boy. Pedia tries, 46, 6 1-65. Spear, G. S. (1984). Hereditary Nephritis (Alport’s syndrome). Clinical Nephrology, 21, 3-6. In Strauss and Suki, W. N. and Caskey, C. T. (1979). H ereditary chronic nephropathies. Welt’s Diseases of the Kidney, Earley and Gottschalk, Eds, 3rd Edit., Little, Brown and Co., Boston. Thorner, P., Jansen, B., Baumal, R. and Valli, V. (1986). Hereditary glomerulopathy (HG) in Samoyed dogs: a model for Alport’s syndrome in man (abst). Laboratory Inuestigation, 54, 63A. in Doberman Wilcock, B. P. and Patterson, J. M. (1979). F amilial glomerulonephritis Pinscher dogs. Canadian Veterinary Journal, 20, 244-245. Yoshikawa, N., Cameron, A. H. and White, R. H. R. (1981). The glomerular basal lamina in hereditary nephritis. Journal of Pathology, 135, 199-205. [Received for publication,
June 1Oth, 19861
PATH.
1987
VOL.
97
JUVENILE RENAL DISEASE IN PINSCHER: ULTRASTRUCTURAL GLOMERULAR BASEMENT CATHERINE Department
ff
Pathology,
NW,
THE DOBERMAN CHANGES OF THE MEMBRANE
A. PICUT and R. M. LEWIS
Ybrk State College
of Veterinq 1:s.d.
Medicine.
Cornell
Uniuersi@.
Ithaca,
.2’ew
York.
INTRODUCTION
Juvenile Renal Disease progressive renal disease
ORD) resulting
in the Doberman Pinscher is an hereditary in azotaemia, marked proteinuria, and renal
failure in young male and female dogs (Wilcock
and Patterson,
1979; Chew,
1983). Histologically, the lesion is DiBartola, Boyce, Hayes and Brace, characterized as an idiopathic membrano-proliferative glomerulonephritis (MPGN, Chew et al., 1983). Although the primary lesion is believed to exist in the glomerulus, no underlying structural or immunological abnormality has been reported. This paper describes ultrastructural changes of the glomerular in Doberman Pinschers with JRD. The nature of basement membrane (GBM) the changes suggests a primary structural or metabolic defect of the GBM. MATERIALS
AND
METHODS
Ten cases of idiopathic membrano-proliferative glomerulonephritis in juvenile and adolescent Doberman Pinschers were selected from material submitted to the diagnostic pathology services at the Drew York State College of Veterinary Medicine between January 1977 and December 1985. Renal tissue obtained at necropsy was fixed in 10 per cent neutral buffered formalin and embedded in paraffin wax for histopathology. Sections (2 to 4 pm thick) were stained with haematoxylin and eosin (HE), alcian blue, Masson’s trichrome, Jones’ methenamine silver (JMS) and periodic-acid Schiff (PAS) stains. Electron microscopy of renal tissue was performed on eight cases. Tissues fixed as above were post-fixed in 1 per cent osmium tetroxide, embedded in epon-araldite, stained with uranyl acetate and lead citrate and observed under a Phillips EM-301 electron miscroscope. Indirect immunofluorescence of paraffin-embedded renal tissue was performed on 6 urn thick tissue sections by a standard protocol established in our laboratory. Deparaffinized tissue was trypsinized for 6 h in 0.5 per cent trypsin in 0.1 per cent CaCl, (pH 7.8 at 37 “C) and stored in phosphate buffered saline (PBS, pH 7.2) overnight at 4 “C. Each section was incubated with rabbit anti-dog IgA, IgM, complement and IgG (100 mg antibody protein per ml) in a humid chamber for 2 h at 37 “C (Reagents: Rabbit anti-dog IgA, Pel Freeze Biologicals; Rabbit anti-dog IgG and IgM, Miles Scientific; Rabbit anti-dog Complement, U.S. Biochemical Corporation). Fluorescein-labelled goat anti-rabbit immunoglobulin (100 mg antibody protein per ml) was applied to washed tissue and incubated for 30 min at room temperature. Following two washes in PBS, the tissue was mounted for 002lL9975/87/050587+
10 $03.00/O
0
1987
Academic
Press
Limited
588
CATHERINE
A.
PICUT
AND
R.
M.
LEWIS
fluorescent microscopy. Positive fluorescence of plasma cells served as an internal positive control while positive fluorescence of a documented case of immune-mediated glomerulonephritis in the dog served as an external positive control. RESULTS
The age and sex of the ten Doberman Pinschers included in this study are listed in Table 1. There were seven males and three females, with the age at death or euthanasia ranging from 4 to 42 months. All animals showed clinical signs of advanced renal failure, including azotaemia, polyuria, polydipsia, vomiting, anorexia and/or elevated blood urea nitrogen and creatinine. Urine analysis in 7 dogs revealed marked proteinuria (3+ to 4+ on an arbitrary scale) and one dog had microscopic haematuria.
JUVENILE
RENAL
DISEASE
TABLE 1 IN DOBERMAN
PINSCHERS
(IO
Lesion
Case No.
Age* (months)
1
4 9
2 3 4 5
24 42
6 7 8
5 8 36
9 10
36
10
9
Lanellation of Lamina densa (Lesion No. I)
sex
M M M F M F M M M F
IgM -
CASES)
of GBM
Electron-dense deposits
+ + + + + -
+
-
-
NOT NOT
Collagtn jbre (Lesion No. 2)
+ + +
EXAMINED EXAMINED
*Age at death or time of euthanasia. GBM = Glomerular basement membrane
Gross Pathology Examination of the gross lesion was possible in four cases. The kidneys were of normal size and had a coarsely granular to finely pitted subcapsular surface. The cortex was finely mottled dark brown to yellow in two of these cases (Fig. 1). Histopathology All lesions were classified as a membrano-proliferative glomerulonephritis (MPGN), with varying degrees of tubulo-interstitial disease [Figs 2(a) and (b)] . Lobular accentuation of glomerular tufts, increased mesangial matrix,
589
GLOMERULARBASEMENTMEMBRANES
Fig.
1. Kidney surface.
from
Doberman
Pinscher
with
Juvenile
Renal
Disease.
Note
fine
mottling
of subcapsular
hypercellularity, intraglomerular adhesions, exudation of fibrin and fibroepithelial crescents characterized the glomerular lesion. In three cases, the glomerular lesion was diffuse, with minimal interstitial fibrosis or tubular atrophy. In the remaining seven cases, the glomerular lesion was multifocal and accompanied by severe tubulo-interstitial disease. Patchy areas of tubular atrophy, interstitial fibrosis, periglomerular fibrosis, cystic distention of Bowman’s capsule (cystic glomerular atrophy) and a, mild to moderate lymphoplasmacytic interstitial nephritis characterized the tubulo-interstitial changes. In one case, cystic glomerular atrophy dominated the morphological lesion.
Nine cases were negative for the immunoreactants, complement. In one case, scattered deposits of IgM glomerular basement membrane. Electron
IgG, IgM, IgA were present within
and the
Microscopy
Among the eight cases examined, two distinct structural lesions of the glomerular basement membrane (GBM) were apparent. The first, observed in five cases, was characterized by multifocal irregular thickening of the GBM with lamellation of the lamina densa [Fig. 3(a)]. Focal areas of lucency
590
CATHERINE
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M.
LEWIS
GLOMERULARBASEMENTMEMBRANES
591
containing electron-dense particles were observed. In one of these cases, intramembranous electron-dense deposits were present. The second lesion, observed in three cases, was characterized by diffuse thickening of the GBM zone [Fig. 3(b)]. There was marked attenuation of the lamina densa with intramembranous and/or subendothelial deposition of matrix composed of cross-banded fibres [Figs 4(a) and (b)]. These prominent, randomly dispersed cross-banded fibres had the periodicity of collagen [Fig. 4(b)]. Focal fusion of overlying foot-processes was a general finding in all cases examined. DISCUSSION
The histopathological features of these ten cases of JRD in Doberman Pinschers were similar to those lesions described previously for this condition (Chew et al., 1983; Wilcock and Patterson, 1979). The morphological picture ranged from a multifocal MPGN with severe tubulo-interstitial inflammation to a severe diffuse glomerulonephritis with minimal tubulo-interstitial disease. Cystic glomerular atrophy commonly accompanied severe interstitial nephritis. Two distinct ultrastructural lesions of the GBM were observed. The first was characterized by irregular thickening of the GBM with lamellation of the lamina densa, focal areas of lucency and intramembranous aggregates of electron dense particles. The second lesion was a diffuse change manifested by attenuation of the lamina densa with thickening of the GBM which contained randomly dispersed intramembranous collagen fibres. There was no apparent relationship between the two distinct histopathological patterns and the two ultrastructural lesions. These ultrastructural changes may be due to a primary structural defect of the GBM or they may represent metabolic or biochemical abnormalities. Similar lesions have been observed in other hereditary nephropathies in man and dog. The basic structural lesion associated with hereditary nephritis (Alport’s syndrome) in man and familial glomerulopathy in Samoyeds resembles one of the alterations seen in Doberman Pinschers (i.e. irregular thickening of the GBM, lamellation of the lamina densa and focal areas of lucency containing electron dense granules) (Churg and Sherman, 1973; Gubler, Levy, Broyer, Naizot, Gonzales, Perrin and Habib, 1981; Heptinstall, 1974; Jansen, Thorner, Singh, Patterson, Lumsden, Valli, Baumal and Basrur, 1984; Krickstein, Gloor and Balogh, 1966; Perkoff, 1983; Spear, 1984; Suki and Caskey, 1979; Yoshikawa, Cameron and White, 1981; Bernstein, 1979). Limited biochemical analyses of the GBM in hereditary nephritis reveals abnormal amounts of hydroxyproline, cysteine and sialic acid (DiBona, 1983), Fig.
2. Light microscopy of Juvenile Renal Disease in Doberman Pincshers. (a) Membrano-proliferative glomerulonephritis with pronounced tubule-interstitial disease and severe cystic glomerular atrophy. HE x 40. (b) Membrane-proliferative glomerular lesion with fibro-epithelial crescent, lobular accentuation of tuft, and exudation of fibrin into Bowman’s space (arrows). The limit of Bowman’s capsule is difficult to discern due to surrounding interstitial fibrosis. HE x 320.
CATHERINE
A.
PICUT
AND
R.
M.
LEWIS
Fig. 3. Ultrastructural changes of the glomerular basement membrane (GBM) in Juvenile Renal Dise Doberman Pincshers. (a) Irregularly thickened GBM with lamellation oflamina densa (arrowhead). Also present al -e two dense large electron dense deposits (arrows) within focal lucent zones surrounded by fine electron particles. Note fusion of overlying foot processes. (EP= epithelial cell). x 9100. (b) Diffusely thickened GBM zone composed of subendothelial fibrillar matrix (arrows) al nd an attenuated lamina densa (arrowhead). (EN=endothelial cell, EP=epithelial cell). X 7500.
GLOMERULARBASEMENTMEMBRANES
of GBM lesion [Fig. 3(b)]: 4. Higher m agnification basement membrane zone characterized by attenuated lamina de nsa (arrow; (a) Glom xular fibrillar matrix (arrowhead). (EN = endothelial cell). x 25 000. abundant (b) Glomc zrular basement membrane with overlying epithelial foot processes. Notes< :vere attenua densa in subepithelial zone (arrow) and numerous cross-banded fib1 yes (arrowhe of lamina x 45 000.
593
and tion ad).
594
CATHERINE
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PICUT
AND
R.
M.
LEWIS
thereby implying an underlying defect in collagen synthesis or maturation. Such a maturational defect is further substantiated by the absence of Goodpasture’s antigen in some patients with HN (DiBona, 1983; Jeraj, Kim, Vernier, Fish and Michael, 1983; McCoy, Johnson, Stone and Wilson, 1982; Olson, Anand, Landing, Heuser, Grushkin and Lieberman, 1980; Spear, 1984), thus suggesting a failure of maturation of the GBM with retention of fetal glomerular antigens (Grunfeld, 1985; Martinez-Hernandez and Amenta, 1983; Olson et al., 1980). Recently the absence of the Goodpasture’s antigen has also been reported in Samoyeds with familial glomerulopathy (Thorner, Jansen, Baumal and Valli, 1986). Attenuation of the lamina densa with intramembranous and/or subendothelial collagen fibres is a unique finding and has not been previously reported in the dog. Intramembranous collagen fibres have been associated only with the human disorder, hereditary osteo-onychodysplasia (nail-patella syndrome). This is a rare syndrome consisting of multiple skeletal anomalies and renal disease in 30 to 40 per cent of patients (Leaky, 1966; Silverman, Goodman and Cuppage, 1967; Similia, Vesa and Wasz-Hockert, 1970; Bernstein, 1979). Histologically, the renal lesion is characterized by segmental glomerulosclerosis and chronic interstitial nephritis (Bernstein, 1979; Silverman et al., 1967; Hoyer, Michael and Vernier, 1972). Ultrastructurally, diffuse irregular thickening of the GBM containing lucent areas and randomly arranged collagen fibres characterizes the lesion (Ben-Bassat, Cohen and Rosenfeld, 1971; Bennet, Musgrave, Campbell, Elliot, Cox, Brooks, Lovrien, Beals and Porter, 1973). The presence of these collagen fibres is considered to be a manifestation of a basic, yet undefined structural membrane abnormality (MartinezzHernandez and Amenta, 1983). Type IV collagen in all basement membranes differs from other types of collagen in that it is incorporated into basement membrane with its pro-peptide extensions intact (Minor, Clark, Strause, Koszalka, Brent and Kefalides, 1976), thereby preventing the assembly of cross-banded fibres (MartinezzHernandez and Amenta, 1983). The observed presence of cross-banded collagen fibres in the GBM of affected Doberman Pinschers suggests either a switch in collagen phenotype to other than type IV collagen or extracellular cleavage of type IV collagen pro-peptides resulting in cross-banding formation (Martinez-Hernandez and Amenta, 1983). The significance of intramembranous electron-dense deposits in one dog may represent resolving immune complexes trapped in an abnormal GBM by non-immunological mechanisms. Equally important is the possibility that abnormalities, as have been underlying biochemical or immunological described in HN and familial glomerulopathy in Samoyeds, might lead to increased capillary affinity for circulating antigens with subsequent in situ immune complex formation (Bertani, Appel, D’Agati, Nash and Pirani, 1983). explain the occasional reports of weakly positive This would immunofluorescent tests for intraglomerular IgG, IgM or complement in hereditary nephritis, osteo-onychodysplasia and JRD in Doberman Pinschers (Gubler et al., 1981; Bernstein, 1979; Bennett et al., 1973; Hoyer et al., 1972; Chew et al., 1983).
GLOMERULAR
BASEMENT
595
MEMBRANES
To date, JRD in Doberman Pinschers has been considered to be an acquired entity occurring with a familial distribution. The ultrastructural lesions described in this report suggest the alternative possibility of a primary metabolic or developmental defect involving the extracellular matrices of the glomerulus. To define the pathogenesis of the glomerulopathy in Doberman Pinschers, prospective morphological, biochemical, and immunological studies of early lesions will be necessary in which the primary structural alteration or metabolic abnormality may be identified. Clinicopathological correlations will help to determine to what extent these structural alterations relate to functional impairment of the GBM. Pedigree analyses are needed, not only to determine the precise mode of inheritance of this disorder, but to help to clarify the genetic bases of possible structural or metabolic defects associated with JRD in the Doberman Pinscher. SUMMARY
Ten cases ofjuvenile renal disease in Doberman Pinschers were examined by light microscopy and 8 of them additionally by electron microscopy. Two distinct basic ultrastructural lesions of the glomerular basement membrane (GBM) were observed. One is characterized by lamellation of the lamina densa with intramembranous focal areas of lucency containing electron-dense particles, the second by diffuse attenuation of the lamina densa with intramembranous and/or subendothelial deposition of matrix entrapping cross-banded fibres (collagen). Based on similar ultrastructural changes in other hereditary nephropathies in man and dogs, a metabolic or biochemical basis for the structural lesions is suspected. ACKNOWLEDGMENT
This study was 088T22EF07 105.
supported
by
the
National
Institute
of Health
Grant
No.
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