A novel model of autosomal dominant Alport syndrome in Dalmatian dogs
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Nephrol Dial Transplant (2002) 17: 2094–2098
Original Article
A novel model of autosomal dominant Alport syndrome in
Dalmatian dogs
Jennifer C. Hood1, Clive Huxtable1, Ichiro Naito2, Carole Smith3, Roger Sinclair3 and Judy Savige4
1
Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Australia, 2Shigei Medical Research
Institute, Okayama, Japan and 3Division of Laboratory Medicine and 4University of Melbourne Department of Medicine,
Austin and Repatriation Medical Centre, Heidelberg, Australia
Abstract intramembranous deposits. All a1(IV)–a5(IV) type IV
Background. Autosomal dominant Alport syndrome is collagen chains were present in the affected GBM and
a rare inherited disease characterized clinically by haem- Bowman’s capsule.
aturia, renal failure and deafness, and ultrastructurally Conclusions. Autosomal dominant Alport syndrome
by a lamellated glomerular basement membrane (GBM). in Dalmatians resembles the disease in Bull terriers but
It is usually caused by mutations in the COL4A3 or has arisen independently. These models will enable
COL4A4 genes which code for the a3 and a4 chains of us to determine how genetic mutations affect the cor-
type IV collagen. We describe here a novel spontaneous responding proteins and overall membrane structure
model of autosomal dominant Alport syndrome in in autosomal dominant Alport syndrome.
Dalmatian dogs.
Methods. Affected dogs were identified by a urinary Keywords: autosomal dominant Alport syndrome;
protein creatinine P0.3. A total of 10 affected adult Dalmatian dogs; glomerular basement membrane;
Dalmatians and eight unaffected age- and sex-matched hereditary nephritis
dogs from breeds other than Dalmatians were exam-
ined. In addition, kidneys from five Dalmatian fetuses
from affected mothers were examined histologically
and ultrastructurally.
Introduction
Results. All affected dogs were purebred Dalmatians
and had a common progenitor. Successive generations Most individuals with Alport syndrome have X-linked
were affected, and males and females were affected or autosomal recessive disease which is characterized
equally often and equally severely, consistent with clinically by haematuria, progressive renal failure,
autosomal dominant inheritance. The median age at hearing loss, lenticonus and dot-and-fleck retinopathy
onset of renal failure was 18 months (range 8 months [1,2]. The glomerular basement membrane (GBM) is
to 7 years). Affected dogs were not clinically deaf, and typically lamellated with vacuolation and subepithelial
did not have the ocular abnormalities seen in human frilling [3]. In X-linked Alport syndrome, mutations
X-linked or autosomal recessive Alport syndrome. In affect the COL4A5 gene [4] and result in the loss of
addition, they did not have the leucocyte inclusions, the a5(IV) chain together with the a3(IV) and a4(IV)
low platelet counts or large platelets seen in autosomal collagen chains from the glomerular and other base-
dominant hereditary nephritis due to MYH9 muta- ment membranes [5]. Mutations in the less common
tions. The renal histology and ultrastructural appear- autosomal recessive disease affect the COL4A3 and
ance of the GBM appeared to be normal in utero. COL4A4 genes [6] which again usually cause the loss
However, affected adult kidneys demonstrated seg- of the a3(IV)–a5(IV) collagen chains from affected
mental glomerular hyalinosis and sclerosis with glomerular membranes [7].
tubulo-interstitial inflammation and fibrosis, and on Autosomal dominant Alport syndrome is rare, and
ultrastructural examination the GBM was lamellated the clinical phenotype differs from that seen with
with subepithelial frilling, vacuolation and occasional X-linked and autosomal recessive inheritance. Haemat-
uria is associated with both normal renal function and
progressive renal impairment, hearing loss is common,
but ocular abnormalities do not occur [8,9]. Again, the
Correspondence and offprint requests to: Professor Judy Savige,
University of Melbourne Department of Medicine, Austin and GBM is lamellated but the a3(IV)–a5(IV) collagen
Repatriation Medical Centre, Heidelberg, VIC 3084, Australia. chains are present in the affected GBM [10]. Mutations
Email: jsavige@austin.unimelb.edu.au affect the COL4A3 or COL4A4 genes [8,9] which are
# 2002 European Renal Association–European Dialysis and Transplant AssociationAutosomal dominant Alport syndrome 2095
also abnormal in autosomal recessive inheritance. (Hoffman–LaRoche, Basel, Switzerland) and the results
Autosomal dominant Alport syndrome has to be confirmed on a second specimen taken at least 1 month later.
distinguished from autosomal dominant hereditary Hearing was tested clinically at 4 weeks by a veterinarian.
nephritis with progressive renal failure, deafness, and Eyes were dilated with topical 1.0% tropicamide and exam-
ined by direct ophthalmoscopy by an experienced observer
leucocyte inclusions, low platelet counts and large
for the ‘oil droplet’ sign of anterior lenticonus and the dot-
platelets [11,12]. This is caused by mutations in the and-fleck retinopathy seen in human X-linked and autosomal
MYH9 gene which encodes a non-muscle myosin recessive Alport syndrome. Blood films were made within 3 h
heavy chain [13,14]. of collection, stained with the May Grunwald Giemsa stain,
The diagnosis of all forms of Alport syndrome and examined for neutrophil inclusions and platelet number
depends on the presence of the typical clinical features and size.
together with a family history of the disease, or on the
ultrastructural demonstration of a lamellated GBM. It
is always caused by a mutation in one of the type IV Histological examination
collagen genes. Autosomal dominant inheritance is Renal tissue obtained from biopsies, collected under general
characterized by disease in successive generations, and anaesthesia or after euthanasia for medical indications, was
males and females being affected equally often and fixed immediately in neutral-buffered formalin and processed
equally severely. It is the only form of inheritance in for light microscopy by standard methods. Three micron
which disease is transmitted from father to son. paraffin-embedded sections were stained with haematoxylin
Canine models of X-linked, autosomal recessive and and eosin, periodic acid-Schiff and silver methenamine.
dominant Alport syndrome in Samoyed and Navasota
dogs, Cocker spaniels and Bull terriers, respectively
Ultrastructural examination
[15–19], have contributed enormously to our under-
standing of the pathogenesis and pathology of Alport Renal tissue was fixed in 1.5% chilled glutaraldehyde in 0.1 M
syndrome. We describe here a novel canine model of phosphate-buffered saline, post-fixed in 1% Dalton’s osmium
autosomal dominant Alport syndrome in Dalmatians tetroxide and embedded in Epon 812 (TAAB Laboratories,
and compare its clinical, histological and ultrastruc- TAAB Lab Equipment, Berkshire, UK). Thin sections were
tural features with those seen in the Bull terrier model cut on a Reichert Ultra Cut E microtome, supported on 200
and the human disease. mesh copper grids, and stained with saturated uranyl acetate
and lead citrate. After carbon coating, the grids were
examined in a Philips 301 transmission electron microscope.
Animals and methods
Basement membrane composition
Dogs
Fixed, embedded blocks of kidney tissue were incubated
Affected adult Dalmatians (ns10) were identified by a in 0.2 M HCl at 110–1278C for 6 min depending on the
urinary protein creatinine ratio (UPC) P0.3, which has requirements of individual antibodies. Sections were then
previously corresponded with histological evidence of renal incubated with rat monoclonal antibodies against the a1(IV)–
disease in Bull terrier hereditary nephritis [19,20]. Some a6(IV) collagen chains and the colour developed with the
animals had, in addition, laboratory evidence of renal fail- LSAB2 kit (DAKO, Glostrup, Denmark). These antibodies
ure or the typical light microscopic abnormalities. Normal had been prepared by immunizing rats with synthetic pep-
adult dogs (ns8) were either cross-breeds or from pedigrees tides from the C-termini of the human a(IV) non-collagenous
where there was no known history of renal disease, and were domains. Their use has been described previously [21].
approximately the same age and size as adult Dalmatians. This project had the approval of the Animal Ethics’
Normal animals had a UPC -0.3 on two occasions Committee of Murdoch University.
at least 1 month apart, no laboratory evidence of renal
failure, and renal biopsies with a normal light microscopic and
ultrastructural appearance. Results
Dalmatian fetuses (ns5) were from litters with an affected
mother and were aged ;35 days (canine gestation is 61–63
days). Normal fetuses (ns5) were also at about 35 days of Clinical features
gestation, and were from dog breeds where there was no
Ten affected adult dogs comprising five males and five
known history of renal disease. Fetuses were obtained either
at the time of routine hysterectomy or after euthanasia for females were examined over a 10 year period. All
medical reasons. affected dogs were purebred Dalmatians, and their
pedigrees demonstrated a common progenitor and
affected dogs in successive generations.
Clinical features Haematuria was present in four of the five affected
dogs (80%) tested, and none of the normals. Four
Random voided urine specimens were tested for haematuria
and UPC. Haematuria was present if the urine was positive males and four females had renal failure. Their median
for blood on testing with Multistix (Ames Co., Miles age at onset of renal failure was 18 months (range
Laboratories, Elkhart, IN, USA) or if any red cells were 8 months to 7 years). None of the affected dogs was
observed in a field at 3 600 magnification by light micro- clinically deaf at 4 weeks of age (ns10) or subsequently
scopy. UPC was estimated using a Cobas Mira autoanalyser demonstrated any features suggesting deafness to their2096 J. C. Hood et al.
A B
C D
Fig. 1. Ultrastructural appearance of glomerular basement membrane from affected Dalmatians with autosomal dominant Alport syndrome:
(A) normal membrane in 35-day-old embryo (3 5700); (B) lamellated membrane with fusion of overlying foot processes in adult dog (3 1850);
(C) irregular thickened lamellated membrane with subepithelial frilling and fusion of foot processes in adult dog (3 1850); and (D) membrane
showing irregular basket weave appearance in adult dog (3 5700).
owner-breeders, and none had the ocular abnormalities Ultrastructural examination of the GBM from
seen in human X-linked or autosomal recessive Alport biopsies from affected dogs early in the disease and
syndrome (ns6). In addition, none had the leucocyte later at post-mortem showed lamellation, subepithelial
inclusions, thrombocytopenia or large platelets pre- frilling and vacuolation (Figure 1). These abnorm-
sent in autosomal dominant hereditary nephritis with alities all increased the width of the affected GBM
haematological abnormalities due to MYH9 mutations compared with normal. The frilling affected both the
(ns6). paramesangium and the peripheral capillary loops
in individual glomeruli. Vacuolation and occasional
intramembranous deposits were present. Foot pro-
Histological and ultrastructural appearance
cess effacement overlying the abnormal GBM, and
Adult kidneys. The light microscopic appearance was mesangial matrix expansion were noted. The ultra-
nearly normal in biopsies from dogs with early dis- structural abnormalities were, however, less prominent
ease (ns3). Later, at post-mortem (ns4), the kidneys than in affected Bull terriers since some glomerular
were macroscopically small with moderate pallor and loops from each dog were only mildly abnormal,
firmness. Histologically there was segmental hyalinosis and GBM changes often affected only part of the
and glomerular sclerosis, tuft adhesions without an circumference of the glomerular loop. The tubular
increase in tuft cellularity, mild through to marked basement membranes and Bowman’s capsule were
tubulo-interstitial inflammation and fibrosis, and gross of normal thickness and appearance in the affected
capillary wall thickening. kidneys.Autosomal dominant Alport syndrome 2097
The normal dogs had no GBM abnormalities, but models of autosomal dominant Alport syndrome. The
one had a minor rare subepithelial irregularity and only distinguishing features are the possible absence
another had a minor TBM lamellation. Bowman’s of ultrastructural changes in utero in Dalmatians and
capsule appeared split in some places in all dogs. their less prominent GBM abnormalities. All affected
Dalmatians and Bull terriers have arisen from two
Fetal kidneys. The light microscopic appearance was independent progenitors in each breed within the past
normal in all five fetuses from litters where one 50 years, and are likely to have two different muta-
parent was affected, and ultrastructural examination tions. The mutation in Bull terriers is also likely to
showed that the GBM had none of the subepithelial affect the COL4A3 or COL4A4 genes and the minor
frilling, lamellation or vacuolation seen in affected differences in phenotype could be due to the small
neonatal Bull terriers. The absence of ultrastructural number of Dalmatians examined, differences in the
GBM changes in these embryos did not, however, underlying mutations or to environmental effects.
preclude in utero abnormalities in this model since it The abundance of canine models of Alport syn-
was not possible to confirm that any fetus was drome may reflect an increased mutation rate in the
affected. The histological and ultrastructural appear- causative genes, the care with which inherited disease
ances of the normal fetal kidneys (ns5) showed no is monitored and investigated in pedigree dogs, or
significant abnormalities. the sophistication of diagnostic techniques available
to veterinary medicine. The Bull terrier model of auto-
Basement membrane composition somal dominant Alport syndrome in particular has
contributed enormously to our understanding of a
Each of the a1(IV)–a6(IV) collagen chains was condition that is rare or infrequently recognized. The
demonstrated in the renal basement membranes from Bull terrier model has suggested that the abnormal-
affected dogs. The a1(IV)–a2(IV) chains were present ities in autosomal dominant Alport syndrome begin
in all the basement membranes, the a3(IV)–a5(IV) in utero, that impaired renal function is principally
chains in the GBM, Bowman’s capsule and the distal due to tubulo-interstitial disease, and that extrarenal
TBM, and a tiny amount of the a6(IV) chain was basement membranes are not affected [18–21,23].
demonstrated in Bowman’s capsule. This distribution The Dalmatian and Bull terrier models of autosomal
was identical to that seen in Bull terriers with dominant Alport syndrome can be used to explore the
hereditary nephritis and in normal dogs. relationship between genetic mutations, the corres-
ponding biochemical defects, and the clinical and
ultrastructural phenotypes.
Discussion
Acknowledgements. This work was supported by the National
In affected Dalmatians, the diagnosis of Alport syn- Health and Medical Research Council of Australia.
drome and its autosomal dominant nature were
confirmed by the demonstration of GBM lamellation,
and by the presence of disease affecting males and References
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Received for publication: 21.1.02
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