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Membranous nephropathy and the nephrotic syndrome in the cat
J. COMP.
PATH.
1971. VOL.
MEMBRANOUS
81.
463
NEPHROPATHY SYNDROME
B. R. H. Departments
AND IN
THE
and C. R. R.
FARROW
of Vettrinary Uniwrsi~
Medicine of Sydq,
and Vehhny Australia
THE
NEPHROTIC
CAT
HUXTABLE Pathology,
INTRODUCTION
The nephrotic syndrome has been well documented in the dog (Lewis, 1968 ; Slauson, Gribble and Russell, 1970), but it is considered to be rare in other domestic animals (Blood and Henderson, 1968). This paper records the clinical and laboratory data, and light and electron-microscopic findings on four cats with the nephrotic syndrome due to membranous nephropathy. This type of glomerular lesion was originally described in man by Bell (1938) as a diffuse membranous glomerulonephritis. Subsequently Ehrenreich and Churg (1968) introduced the term membranous nephropathy to describe these changes and this term seems to us a more suitable one than that previously used because of the absence of glomerular inflammation.
MATERIALS
AND
METHODS
The 4 cases described were submitted to the University of Sydney Veterinary Clinic. The cats were between 14 months and 3 years old. Two were castrated males and 2 were ovariectomised females. The first case in this series has already been reported separately (Farrow, Huxtable and McGovern, 1969). Electrophoretic analyses of serum and urinary protein were performed using cellulose acetate as the support medium (Gelman Instrument Company). The protein bands were stained with Ponceau red. A Joyce Loebl recording and integrating densitometer (Chromoscan) was used to quantitate the various protein fractions. Samples of kidney taken at autopsy were fixed in 12.5 per cent. buffered formalin, Zenker’s solution and corrosive formalin. Paraffin sections were cut at 3 to 4 p and stained with haematoxylin and eosin, PAS-Wilder and chromatrope-silver methenamine. For electronmicroscopy, small pieces of kidney cortex were removed under pentobarbitone sodium anaesthesia prior to euthanasia, and diced into 1 mm. cubes in a solution of 1 per cent. osmium tetroxide buffered with 0.1 M cacodylate and containing 0.01 per cent. calcium chloride. Fixation was continued in this solution for 3 to 5 hours at 4OC., following which the cubes were transferred to a 1 per cent. solution of aqueous uranyl acetate for one hour. They were then dehydrated in graded ethanol solutions, cleared in propylene oxide and soaked overnight in a mixture of equal parts of propylene oxide and araldite. Final infiltration and embedding were done in araldite. Thin sections were cut and stained on the grids with lead citrate and many1 acetate and were examined in a Philips E.M. 200 electron microscope.
464
FELINE
Clinical
Findings
MEMBRANOUS
NEPHROPATHY
AND
NEPHROTIC
SYNDROME
RESULTS
All 4 animals had the nephrotic syndrome. The feature of each case was oedema which varied in extent and disposition from animal to animal and also from day to day in each animal. Subcutaneous oedema of the limbs, head, abdomen and thorax, as well as ascites were observed. A physical examination of each cat revealed no other abnormality. They were afebrile, alert and eating well. TABLE SERUM
AND URINARY LFXELS IN
4
1
PROTEIN FRACTIONS AND CATS WITH TEE NEPHROTIC
2 Male 2 years
3 Female 3 years
4 Female I4 months
3.7
4-4
4.6
3;:: 25.9 31.3 O-08 323
3i.i 20.4 31.6 0.11 240
3Y.i 19.5 39.4 0.09 176
4E * 14.2 30.8 0.07 208
-
4:.dj 23.6 13-3 15.8
2i.i 28.8 13.4 28.5
2% * 29.6 12.4 31.0
1
Male 2 years Serum Total Protein Albumin t( Globulin @ Globulin y Globulin Albumin: globulin ratio mg. per cent. Cholesterol Urine
protein Albumin (L Globulin p Globulin y Globulin Total
- = Not performed * All figures except
cholesterol,
SERUM CHOLESTEROL SYNDROME
4+%*
in g./per
cent.
In each case urinalysis revealed heavy proteinuria. Serum protein levels were below 5 g./ 100 ml. Electrophoretic analysis of serum and urine gave the figures indicated in Table 1. The protein in the urine was principally albumin with smaller amounts of the larger molecular weight plasma proteins. Serum protein analysis revealed a severe hypoalbuminaemia. Fig. 1 shows a typical densitometric tracing of serum and urinary protein from an affected animal. In addition to oedema, proteinuria and hypoproteinaemia, the cats showed hypercholesterolaemia, thus satisfying the well-established criteria for the nephrotic syndrome (Johnson and Reader, 1959).
Treatment In 2 cats, corticosteroid therapy (Prednisolone 1 mg./Kg./day) was followed by a remission of clinical signs. The proteinuria became less pronounced, but did not stop entirely, the total serum protein level returned to within normal limits and the oedema disappeared. Therapy was continued until clinical signs disappeared and then the dose rate was gradually reduced. The nephrotic syndrome recurred 4 months after cessation of therapy in 1 case and 2 weeks after cessation
B. R. H.
I Fig.
1.
FARROW
y 0
a
AND
Alb.
Densitometric tracing of serum (left) syndrome. Note the reduction in serum urine.
of therapy in the other. Treatment animals were ultimately destroyed. Post-mortem
C. R. R. HUXTABLE
IJ
y
B
a
465
Albi
and urinary protein from a cat with the nephrotic albumin and the high concentration of albumin in the
of the other 2 cats was not attempted.
The
Findings
Oedema of the limbs and ascites were the most constant finding although subcutaneous oedema of the head, abdomen and thorax was also observed. In 2 of the cases subcutaneous oedema of the head was prominent. The a&tic fluid in each instance was characteristic of a transudate. It was clear and colourless, had a low specific gravity and contained only a trace of protein. The kidneys were macroscopically normal and there were no changes in other tissues. Histological
Examination
The changes seen in the kidneys on microscopic examination were similar in each case. The glomeruli showed a diffuse thickening of the capillary walls without cellular proliferation (Fig. 2). Protein was present in many glomerular spaces and in the tubules. Proximal and distal convoluted tubules showed extensive vacuolar degeneration. Sections stained by the PAS-Wilder method revealed bristle or club-like projections from the subepithelial aspect of the glomerular basement membrane which are characteristic of membranous nephropathy (Fig. 3). of glomerular capillary walls revealed Electronmicroscopic examination electron-dense deposits on the epithelial side of the basement membrane between which were irregularly shaped projections of basement membrane. In some areas these projections spread laterally to surround the electron-dense deposits within the thickened basement membrane. Immediately above the areas of altered basement membrane the foot processes of the epithelial cells were fused. These changes produced a marked increase in thickness of the glomerular capillary wall (Fig. 4).
466
FELINE
MEMBRANOUS
NEPHROPATHY
AND
NEPHROTIC
Fig. 5 is an electronmicrograph at the same magnification cat for comparison.
SYNDROME
of a glomerular
capillary
wall from a normal
DISCUSSION
In the normal animal the filtrate produced by the passage of blood through the glomerular capillaries is essentially protein-free. Small quantities of albumin (molecular weight approx. 70,000) may pass the glomerular filter to be later removed from the filtrate by active tubular resorption giving rise to protein-free urine. Various diseases involving the kidneys may affect the glomeruli in such a way that their permeability to plasma proteins is increased. Albumin, having the lowest molecular weight of the normal plasma proteins, passes through the glomerular membrane most readily. When serum proteins are lost at a greater rate than they can be replaced, hypoproteinaemia results. As the albumin concentration in serum is critical for the maintenance of intravascular osmotic pressure, hypoalbuminaemia resulting from excessive loss to the urine causes lowering of intravascular osmotic pressure and consequent oedema. The kidney changes which resulted in the nephrotic syndrome in the 4 cases described were similar, in every respect, to membranous nephropathy in man as described by Ehrenreich and Churg (1968). The aetiology of membranous nephropathy is not known, but there is considerable speculation on the possible pathogenesis. Immune, autoimmune, metabolic and toxic factors are all suspected. The common pathway in all cases is believed to be the deposition of protein complexes from the blood stream in the wall of the glomerular capillaries (Ehrenreich and Churg, 1968). Several workers, using immunofluorescent techniques, have demonstrated the presence of gamma globulin and complement in the glomerular deposits in this disease in man (Lange, Tresser, Sage& Ty and Waserman, 1966; McCluskey, Vassalli, Gallo and Baldwin, 1966). Slauson ( 1970), using fluorescence microscopy, suggested that such deposits in a cat were due to glomerular localisation of circulating antigen-antibody complexes. Ehrenreich and Churg (1968) consider that in man these deposits stimulate a reaction of the basement membrane resulting in the formation of subepithelial projections, fusion of foot-processes of the epithelial cells and eventual diffuse thickening of the basement membrane. In addition to the cases described in this paper another cat with the nephrotic syndrome has been seen by the authors. While membranous nephropathy was not confirmed by renal biopsy a good response to corticosteroid therapy was observed, but daily dosing with prednisolone was necessary to prevent recurrence of the nephrotic syndrome and at the time of writing the clinical signs have been controlled for nine months. Therapy was reduced on 2 occasions and the nephrotic syndrome reappeared. A response to steroid therapy has also been observed in some cases of membranous nephropathy in man (Ehrenreich and Churg, 1968). As with the 3 cats treated so far, the human cases showed a remission of clinical and biochemical symptoms although a mild degree of proteinuria persisted. Subcutaneous oedema in the cat is an uncommon clinical sign and hypoproteinaemia due to loss of serum proteins to the urine should be considered in the differential diagnosis of oedema in this species. Membranous nephropathy in the cat is similar in its clinical and biochemical
B. R. H. FARROW AND C. R. R. HUXTABLE
Fig. 2. Fig. 3.
Renal cortex. Diffuse thickening of the glomerular capillary walls, leakage of protein into Bowman’s space and hyaline castsin some tubules. The tubular epithelial cells are swollen and vacuolated. H & E x 400. Glomerular capillary loops showing characteristic projections from the basement membrane (arrows). PAS-Wilder x 1000. TOfac~paga466
FELINE MEMBRANOUS NEPHROPATHY
Fig. 4.
Fig. 5.
AND NEPHROTIC SYNDROME
Glomerular capillary wall. Electron-dense deposits are visible in the basement membrane, which has lost its trilaminar structure. A spike-like projection of the basement membrane can be seen partially enclosing a dense deposit (arrow). There is complete fusion of foot processes. P = podocyte, U = urinary space, F = foot processes(fused), M = basement membrane. L = capillary lumen, N = neutrophil. x 14,000. Glomerular capillary wall from a six-month old normal cat. Symbols as Fig. 4. x 14,000.
B.
R.
H.
FARROW
AND
C. R.
R.
HUXTABLE
467
manifestations to the disease in man. In addition, limited observations indicate that steroids have a place in the management of this disorder in the cat. SUMMARY
Four cases of membranous nephropathy in cats are described. The condition in cats is similar in its clinical and biochemical manifestations to the disease in man. Each cat had the nephrotic syndrome characterised by oedema, proteinuria, hypoproteinaemia and hypercholesterolaemia. The kidneys were macroscopically normal, but microscopic examination of kidney sections and electronmicroscopic examination of glomerular capillary walls revealed changes similar to those described in human cases of membranous nephropathy. Corticosteroid therapy produced a remission of clinical signs in the 2 cats treated although a mild degree of proteinuria persisted. REFERENCES
Bell, E. T. (1938). Amer. f. Path., 14, 691. Blood, D. C., and Henderson, J. A. (1968). Veterinary Medicine, 3rd Ed., p. 153, Baillihre, Tindall and Cassell; London. Ehrenreich, T., and Churg, J. (1968). Pathology Annual, p. 145, Appleton-CenturyCrofts; New York. Farrow, B. R. H., Huxtable, C. R., and McGovern, V. J. (1969). Pathology, 1, 67. Johnson, J. R., and Reader, R. (1959). Amt. Ann. Med., 8, 200. Lange, K., Tresser, G., Sagel, I., Ty, A., and Wasserman, E. (1966). Ann. intern. Med.,
64, 25.
Lewis, R. M. (1968). In Current Veterinary Therapy, p. 654, Ed. by Kirk, R. W., W. B. Saunders Company; Philadelphia, London and Toronto. McCluskey, R. T., Vassalli, P., Gallo, G., and Baldwin, D. S. (1966). New Eng. 1. Med.,
274, 695.
Slauson, D. 0. (1970). J. Path., in press. [Received
for publication,
October
21st, 19701
PATH.
1971. VOL.
MEMBRANOUS
81.
463
NEPHROPATHY SYNDROME
B. R. H. Departments
AND IN
THE
and C. R. R.
FARROW
of Vettrinary Uniwrsi~
Medicine of Sydq,
and Vehhny Australia
THE
NEPHROTIC
CAT
HUXTABLE Pathology,
INTRODUCTION
The nephrotic syndrome has been well documented in the dog (Lewis, 1968 ; Slauson, Gribble and Russell, 1970), but it is considered to be rare in other domestic animals (Blood and Henderson, 1968). This paper records the clinical and laboratory data, and light and electron-microscopic findings on four cats with the nephrotic syndrome due to membranous nephropathy. This type of glomerular lesion was originally described in man by Bell (1938) as a diffuse membranous glomerulonephritis. Subsequently Ehrenreich and Churg (1968) introduced the term membranous nephropathy to describe these changes and this term seems to us a more suitable one than that previously used because of the absence of glomerular inflammation.
MATERIALS
AND
METHODS
The 4 cases described were submitted to the University of Sydney Veterinary Clinic. The cats were between 14 months and 3 years old. Two were castrated males and 2 were ovariectomised females. The first case in this series has already been reported separately (Farrow, Huxtable and McGovern, 1969). Electrophoretic analyses of serum and urinary protein were performed using cellulose acetate as the support medium (Gelman Instrument Company). The protein bands were stained with Ponceau red. A Joyce Loebl recording and integrating densitometer (Chromoscan) was used to quantitate the various protein fractions. Samples of kidney taken at autopsy were fixed in 12.5 per cent. buffered formalin, Zenker’s solution and corrosive formalin. Paraffin sections were cut at 3 to 4 p and stained with haematoxylin and eosin, PAS-Wilder and chromatrope-silver methenamine. For electronmicroscopy, small pieces of kidney cortex were removed under pentobarbitone sodium anaesthesia prior to euthanasia, and diced into 1 mm. cubes in a solution of 1 per cent. osmium tetroxide buffered with 0.1 M cacodylate and containing 0.01 per cent. calcium chloride. Fixation was continued in this solution for 3 to 5 hours at 4OC., following which the cubes were transferred to a 1 per cent. solution of aqueous uranyl acetate for one hour. They were then dehydrated in graded ethanol solutions, cleared in propylene oxide and soaked overnight in a mixture of equal parts of propylene oxide and araldite. Final infiltration and embedding were done in araldite. Thin sections were cut and stained on the grids with lead citrate and many1 acetate and were examined in a Philips E.M. 200 electron microscope.
464
FELINE
Clinical
Findings
MEMBRANOUS
NEPHROPATHY
AND
NEPHROTIC
SYNDROME
RESULTS
All 4 animals had the nephrotic syndrome. The feature of each case was oedema which varied in extent and disposition from animal to animal and also from day to day in each animal. Subcutaneous oedema of the limbs, head, abdomen and thorax, as well as ascites were observed. A physical examination of each cat revealed no other abnormality. They were afebrile, alert and eating well. TABLE SERUM
AND URINARY LFXELS IN
4
1
PROTEIN FRACTIONS AND CATS WITH TEE NEPHROTIC
2 Male 2 years
3 Female 3 years
4 Female I4 months
3.7
4-4
4.6
3;:: 25.9 31.3 O-08 323
3i.i 20.4 31.6 0.11 240
3Y.i 19.5 39.4 0.09 176
4E * 14.2 30.8 0.07 208
-
4:.dj 23.6 13-3 15.8
2i.i 28.8 13.4 28.5
2% * 29.6 12.4 31.0
1
Male 2 years Serum Total Protein Albumin t( Globulin @ Globulin y Globulin Albumin: globulin ratio mg. per cent. Cholesterol Urine
protein Albumin (L Globulin p Globulin y Globulin Total
- = Not performed * All figures except
cholesterol,
SERUM CHOLESTEROL SYNDROME
4+%*
in g./per
cent.
In each case urinalysis revealed heavy proteinuria. Serum protein levels were below 5 g./ 100 ml. Electrophoretic analysis of serum and urine gave the figures indicated in Table 1. The protein in the urine was principally albumin with smaller amounts of the larger molecular weight plasma proteins. Serum protein analysis revealed a severe hypoalbuminaemia. Fig. 1 shows a typical densitometric tracing of serum and urinary protein from an affected animal. In addition to oedema, proteinuria and hypoproteinaemia, the cats showed hypercholesterolaemia, thus satisfying the well-established criteria for the nephrotic syndrome (Johnson and Reader, 1959).
Treatment In 2 cats, corticosteroid therapy (Prednisolone 1 mg./Kg./day) was followed by a remission of clinical signs. The proteinuria became less pronounced, but did not stop entirely, the total serum protein level returned to within normal limits and the oedema disappeared. Therapy was continued until clinical signs disappeared and then the dose rate was gradually reduced. The nephrotic syndrome recurred 4 months after cessation of therapy in 1 case and 2 weeks after cessation
B. R. H.
I Fig.
1.
FARROW
y 0
a
AND
Alb.
Densitometric tracing of serum (left) syndrome. Note the reduction in serum urine.
of therapy in the other. Treatment animals were ultimately destroyed. Post-mortem
C. R. R. HUXTABLE
IJ
y
B
a
465
Albi
and urinary protein from a cat with the nephrotic albumin and the high concentration of albumin in the
of the other 2 cats was not attempted.
The
Findings
Oedema of the limbs and ascites were the most constant finding although subcutaneous oedema of the head, abdomen and thorax was also observed. In 2 of the cases subcutaneous oedema of the head was prominent. The a&tic fluid in each instance was characteristic of a transudate. It was clear and colourless, had a low specific gravity and contained only a trace of protein. The kidneys were macroscopically normal and there were no changes in other tissues. Histological
Examination
The changes seen in the kidneys on microscopic examination were similar in each case. The glomeruli showed a diffuse thickening of the capillary walls without cellular proliferation (Fig. 2). Protein was present in many glomerular spaces and in the tubules. Proximal and distal convoluted tubules showed extensive vacuolar degeneration. Sections stained by the PAS-Wilder method revealed bristle or club-like projections from the subepithelial aspect of the glomerular basement membrane which are characteristic of membranous nephropathy (Fig. 3). of glomerular capillary walls revealed Electronmicroscopic examination electron-dense deposits on the epithelial side of the basement membrane between which were irregularly shaped projections of basement membrane. In some areas these projections spread laterally to surround the electron-dense deposits within the thickened basement membrane. Immediately above the areas of altered basement membrane the foot processes of the epithelial cells were fused. These changes produced a marked increase in thickness of the glomerular capillary wall (Fig. 4).
466
FELINE
MEMBRANOUS
NEPHROPATHY
AND
NEPHROTIC
Fig. 5 is an electronmicrograph at the same magnification cat for comparison.
SYNDROME
of a glomerular
capillary
wall from a normal
DISCUSSION
In the normal animal the filtrate produced by the passage of blood through the glomerular capillaries is essentially protein-free. Small quantities of albumin (molecular weight approx. 70,000) may pass the glomerular filter to be later removed from the filtrate by active tubular resorption giving rise to protein-free urine. Various diseases involving the kidneys may affect the glomeruli in such a way that their permeability to plasma proteins is increased. Albumin, having the lowest molecular weight of the normal plasma proteins, passes through the glomerular membrane most readily. When serum proteins are lost at a greater rate than they can be replaced, hypoproteinaemia results. As the albumin concentration in serum is critical for the maintenance of intravascular osmotic pressure, hypoalbuminaemia resulting from excessive loss to the urine causes lowering of intravascular osmotic pressure and consequent oedema. The kidney changes which resulted in the nephrotic syndrome in the 4 cases described were similar, in every respect, to membranous nephropathy in man as described by Ehrenreich and Churg (1968). The aetiology of membranous nephropathy is not known, but there is considerable speculation on the possible pathogenesis. Immune, autoimmune, metabolic and toxic factors are all suspected. The common pathway in all cases is believed to be the deposition of protein complexes from the blood stream in the wall of the glomerular capillaries (Ehrenreich and Churg, 1968). Several workers, using immunofluorescent techniques, have demonstrated the presence of gamma globulin and complement in the glomerular deposits in this disease in man (Lange, Tresser, Sage& Ty and Waserman, 1966; McCluskey, Vassalli, Gallo and Baldwin, 1966). Slauson ( 1970), using fluorescence microscopy, suggested that such deposits in a cat were due to glomerular localisation of circulating antigen-antibody complexes. Ehrenreich and Churg (1968) consider that in man these deposits stimulate a reaction of the basement membrane resulting in the formation of subepithelial projections, fusion of foot-processes of the epithelial cells and eventual diffuse thickening of the basement membrane. In addition to the cases described in this paper another cat with the nephrotic syndrome has been seen by the authors. While membranous nephropathy was not confirmed by renal biopsy a good response to corticosteroid therapy was observed, but daily dosing with prednisolone was necessary to prevent recurrence of the nephrotic syndrome and at the time of writing the clinical signs have been controlled for nine months. Therapy was reduced on 2 occasions and the nephrotic syndrome reappeared. A response to steroid therapy has also been observed in some cases of membranous nephropathy in man (Ehrenreich and Churg, 1968). As with the 3 cats treated so far, the human cases showed a remission of clinical and biochemical symptoms although a mild degree of proteinuria persisted. Subcutaneous oedema in the cat is an uncommon clinical sign and hypoproteinaemia due to loss of serum proteins to the urine should be considered in the differential diagnosis of oedema in this species. Membranous nephropathy in the cat is similar in its clinical and biochemical
B. R. H. FARROW AND C. R. R. HUXTABLE
Fig. 2. Fig. 3.
Renal cortex. Diffuse thickening of the glomerular capillary walls, leakage of protein into Bowman’s space and hyaline castsin some tubules. The tubular epithelial cells are swollen and vacuolated. H & E x 400. Glomerular capillary loops showing characteristic projections from the basement membrane (arrows). PAS-Wilder x 1000. TOfac~paga466
FELINE MEMBRANOUS NEPHROPATHY
Fig. 4.
Fig. 5.
AND NEPHROTIC SYNDROME
Glomerular capillary wall. Electron-dense deposits are visible in the basement membrane, which has lost its trilaminar structure. A spike-like projection of the basement membrane can be seen partially enclosing a dense deposit (arrow). There is complete fusion of foot processes. P = podocyte, U = urinary space, F = foot processes(fused), M = basement membrane. L = capillary lumen, N = neutrophil. x 14,000. Glomerular capillary wall from a six-month old normal cat. Symbols as Fig. 4. x 14,000.
B.
R.
H.
FARROW
AND
C. R.
R.
HUXTABLE
467
manifestations to the disease in man. In addition, limited observations indicate that steroids have a place in the management of this disorder in the cat. SUMMARY
Four cases of membranous nephropathy in cats are described. The condition in cats is similar in its clinical and biochemical manifestations to the disease in man. Each cat had the nephrotic syndrome characterised by oedema, proteinuria, hypoproteinaemia and hypercholesterolaemia. The kidneys were macroscopically normal, but microscopic examination of kidney sections and electronmicroscopic examination of glomerular capillary walls revealed changes similar to those described in human cases of membranous nephropathy. Corticosteroid therapy produced a remission of clinical signs in the 2 cats treated although a mild degree of proteinuria persisted. REFERENCES
Bell, E. T. (1938). Amer. f. Path., 14, 691. Blood, D. C., and Henderson, J. A. (1968). Veterinary Medicine, 3rd Ed., p. 153, Baillihre, Tindall and Cassell; London. Ehrenreich, T., and Churg, J. (1968). Pathology Annual, p. 145, Appleton-CenturyCrofts; New York. Farrow, B. R. H., Huxtable, C. R., and McGovern, V. J. (1969). Pathology, 1, 67. Johnson, J. R., and Reader, R. (1959). Amt. Ann. Med., 8, 200. Lange, K., Tresser, G., Sagel, I., Ty, A., and Wasserman, E. (1966). Ann. intern. Med.,
64, 25.
Lewis, R. M. (1968). In Current Veterinary Therapy, p. 654, Ed. by Kirk, R. W., W. B. Saunders Company; Philadelphia, London and Toronto. McCluskey, R. T., Vassalli, P., Gallo, G., and Baldwin, D. S. (1966). New Eng. 1. Med.,
274, 695.
Slauson, D. 0. (1970). J. Path., in press. [Received
for publication,
October
21st, 19701