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A model of autosomal recessive Alport syndrome in English ... · PDF fileA model of autosomal recessive Alport syndrome in English cocker spaniel dogs GEORGE E. LEES,R.GAYMAN HELMAN,CLIFFORD

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  • A model of autosomal recessive Alport syndrome in Englishcocker spaniel dogs


    Texas Veterinary Medical Center, Texas A&M University, College Station, Texas, Oklahoma Animal Disease Diagnostic Laboratory,Oklahoma State University, Stillwater, Oklahoma, and Department of Pediatrics, University of Minnesota Medical School, Minneapolis,Minnesota, USA; Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama, and Divisions ofImmunology and Ultrastructural Biology, Shigei Medical Research Institute, Okayama, Japan

    A model of autosomal recessive Alport syndrome in Englishcocker spaniel dogs.

    Background. Dogs with naturally occurring genetic disorders ofbasement membrane (type IV) collagen may serve as animalmodels of Alport syndrome.

    Methods. An autosomal recessive form of progressive heredi-tary nephritis (HN) was studied in 10 affected, 3 obligate carrier,and 4 unaffected English cocker spaniel (ECS) dogs. Clinical,pathological, and ultrastructural features of the disease werecharacterized. Expression of basement membrane (BM) proteinswas examined with an immunohistochemical technique usingmonospecific antibodies.

    Results. Affected dogs had proteinuria and juvenile-onsetchronic renal failure. Glomerular basement membrane (GBM)thickening and multilamellation typical of HN were observed inall renal specimens obtained from proteinuric dogs, and severityof GBM ultrastructural abnormalities varied with the clinicalstage of disease. Expression of a3(IV) and a4(IV) chains wastotally absent in the kidney of affected dogs. Expression of a5(IV)and a6(IV) chains was normal in Bowmans capsule, collectingtubular BM and epidermal BM of affected dogs. The a5(IV)chain was not expressed in distal tubular BM of affected dogs.Expression of a5(IV) chains was markedly reduced but notabsent, and expression of a6(IV) chains was present in GBM ofaffected dogs. Expression of a1-a2(IV) chains in GBM of affecteddogs was increased. Features of obligate carriers were similar tothose of unaffected dogs.

    Conclusions. We conclude that HN in ECS dogs is a naturallyoccurring animal model of autosomal recessive Alport syndrome.However, it differs from human disease in the persistence ofa5(IV) chains in GBM and in the appearance of a6(IV) chains inGBM.

    Hereditary nephritis (HN) refers to a group of geneticdisorders of basement membrane (type IV) collagen that

    cause progressive glomerular disease [13]. In humans withAlport syndrome (AS), the nephropathy is frequently asso-ciated with sensorineural hearing loss and ocular abnormal-ities. Distinctive ultrastructural changes in glomerular base-ment membranes (GBM) of affected individuals is aprominent characteristic of these disorders [13].

    In humans AS usually is X-linked, resulting from muta-tions in the COL4A5 gene, which encodes the a5 chain oftype IV collagen [1, 4]. Some families have an autosomalrecessive form of AS, due to mutations in the COL4A3 orCOL4A4 gene on chromosome 2, that affect the a3(IV) ora4(IV) chain [58]. Rare families have AS with an auto-somal dominant pattern of inheritance [9]. Jefferson et alhave mapped autosomal dominant AS to the region ofCOL4A3 and COL4A4 in a single family, but have yet toidentify a specific mutation [10].

    Naturally occurring diseases that are animal models forAS have been identified in dogs. An X-linked form of HNin a family of Samoyed dogs has been thoroughly charac-terized [1116]. The causative mutation, located in exon 35of the COL4A5 gene, is a single nucleotide substitutionthat produces a premature stop codon [17]. Additionally,autosomal dominant HN has been described in bull terrierdogs, but the causative mutation is unknown [18].

    We have identified an autosomal recessive form of HN inEnglish cocker spaniel (ECS) dogs. Initially, ultrastructuralGBM lesions similar to those of HN were identified inseveral juvenile ECS dogs that had advanced renal failure[19]. Subsequently, repeated matings of the parents ofaffected dogs produced additional affected dogs [20]. Oc-currence of an inherited nephropathy in ECS dogs has beenrecognized for many years; however, previous investigatorshad not clearly established that the disease was a form ofHN [2125]. Nonetheless, studies had shown that thedisease was a rapidly progressive glomerular disorder thatwas inherited in an autosomal recessive fashion [26, 27].Pedigrees of affected ECS dogs that we studied also were

    Key words: genetic disorder, chronic renal failure, hereditary nephritis,basement membrane, type IV collagen, progressive glomerular disease,multilamination.

    Received for publication August 11, 1997and in revised form April 6, 1998Accepted for publication April 14, 1998

    1998 by the International Society of Nephrology

    Kidney International, Vol. 54 (1998), pp. 706719


  • compatible with autosomal recessive transmission of thedisease [20].

    Immunohistochemical techniques have been used tostudy distribution of collagen a(IV) chains in basementmembranes of humans with AS, dogs with HN, and trans-genic mice with COL4A3 mutations. Expression of a3(IV),a4(IV), and a5(IV) usually is absent in the GBM of menwith X-linked AS, while women who are heterozygous forX-linked AS mutations frequently lack these chains inportions of their GBM [2831]. In people with autosomalrecessive AS, a3(IV), a4(IV), and a5(IV) chains usuallyare lacking in GBM; however, the a5(IV) chain is presentin other basement membranes, for example, Bowmanscapsule, collecting tubular basement membrane, and epi-dermal basement membrane [32]. Transgenic COL4A3mutant mice lack a3(IV), a4(IV) and a5(IV) chains intheir GBM and tubular basement membranes [33, 34].Human sera from patients with Goodpasture disease havebeen used to study X-linked HN in Samoyed dogs. Thesesera, which contain antibodies reacting mainly with a3(IV)chains, bind to normal dog GBM but not to the GBM ofaffected males [14]. Segmental GBM staining is seen inyoung heterozygous females, but staining becomes global asthe dogs grow older [16]. In bull terrier dogs with autoso-mal dominant HN, renal expression of a3(IV) and a5(IV)chains appeared normal using monospecific antibodies[18].

    This article describes the clinical, ultrastructural andimmunohistochemical features of autosomal recessive HNin ECS dogs. Monospecific antibodies were used to deter-

    mine the distribution of a(IV) chains in renal and epider-mal basement membranes of affected dogs.



    Subjects were 17 ECS dogs that were selected in severalways (Table 1). Seven dogs (numbers 1 to 7) were identifiedwhen they developed juvenile-onset chronic renal failure.As described in a previous clinical report, which includedfour of these animals, such dogs were found be HN-affected by postmortem examinations that revealed char-acteristic ultrastructural GBM changes [19]. Four dogs(numbers 8 to 10, and 14) were offspring from repeatmatings of dogs that had produced an affected dog in aprevious litter. Three of these four dogs also were affected[20]. Three dogs (numbers 11 to 13) were obligate HNcarriers, identified by having at least one offspring in whichHN had been diagnosed. Three dogs (numbers 15 to 17)were healthy animals that served as controls.

    Each affected dog met two criteria for diagnosis of HN.First, the kidney disease exhibited clinicopathologic fea-tures identical to those described for the familial nephrop-athy that has been recognized in ECS dogs for many years.Second, examination with transmission electron micros-copy (TEM) demonstrated characteristic abnormalities ofGBM ultrastructure. Every dog with the clinicopathologicfeatures of familial nephropathy that was examined withTEM had similar GBM changes.

    Table 1. English cocker spaniel dogs studied

    Dog no. FamilyHN

    phenotype Gender

    Age atinitialexam

    Age atlast





    1 A Affected M 10 11 Pos Pos Pos2 B Affected M 27 Pos Pos Pos3 C Affected M 15 22 Pos Pos Pos4 D Affected F 13 Pos Pos Pos5 E Affected M 9 9 Pos Pos Pos6 F Affected M 10 Pos Pos Pos7 G Affected F 15 Pos Pos Pos8 A Affected M 3 8 Pos Pos Pos9 H Affected F 3 13 Pos Pos Pos

    10 H Affected F 3 17 Pos Pos Pos11 A Carrier M 88 Neg Neg ND12 A Carrier F 59 73 Neg Neg Neg13 I Carrier F 102 104 Neg Neg Neg14 H Unaffected M 3 50 Neg Neg Neg15 J Normal M 19 Neg Neg Neg16 K Normal F 76 Neg Neg Neg17 L Normal F 9 Neg Neg Neg

    Dogs 1 and 8 were siblings but not littermates; dogs 11 and 12 were parents of dogs 1 and 8. Dogs 9, 10, and 14 were littermate siblings. Abbreviationsare: HN, hereditary nephritis; TEM, transmission electron microscopy; Affected, HN diagnosis based on clinicopathologic features and TEM findings;Carrier, parent of an HN-affected dog; Unaffected, healthy dog from HN-carrier parents; could be a carrier or a normal dog; Normal, kindred containsno HN-affected dogs, and little chance of being a HN-carrier; ND, not determined.

    Lees et al: Autosomal recessive Alport syndrome in dogs 707

  • Clinical evaluations

    Eight dogs were evaluated once, and nine dogs wereevaluated on 2 to 26 occasions. Five dogs with multipleevaluations were examined two to six times at irregularintervals. The other four dogs were evaluated monthlybeginning when they were three months old and continuinguntil they developed renal failure and were euthanatized orbecame two years old and were still healthy [20].

    Clinical evaluations were performed by conventionalmethods. Histories were reviewed, and complete physicalexaminations were performed. Renal ultrasonography wasperformed on 29 occasions in 15 d

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