D have appeared intermittently for more than 35 years.

Transcription

1 Glomerular Ultrastructural Findings Similar to Hereditary Nephritis in 4 English Cocker Spaniels George E. Lees, Patrick D. Wilson, R. Gayman Helman, Linda D. Homco, and Miles S. Frey Renal disease affecting 3 male and 1 female English Cocker Spaniels was studied. Clinical features of the disease included proteinuria and progressive deterioration of renal function. Dogs were 11 to 27 months old when euthanized because of severe chronic renal failure. Grossly, the renal cortices were thin. Light microscopic evaluation revealed diffuse glomerular disease characterized by mesangial thickening, glomerular fibrosis, periglomerular fibrosis, and glomerular obsolescence. Based on these clinical and pathologic features, familial nephropathy of English Cocker Spaniels was suspected despite the fact that the individual dogs were not closely related. On transmission electron microscopy, a distinctive ultrastructural lesion was observed escriptions of renal disease in young Cocker Spaniels D have appeared intermittently for more than 35 years. Reduced thickness of the renal cortex of both kidneys was observed in young dogs with severe renal failure, and the condition was first called renal cortical hypoplasia in reports from Sweden,'.' Switzerland,' and Australia.4 Early investigators believed that the disease was inherited,'.' and pedigree analysis indicated that the condition indeed was familial.'~" More recently, investigators recognized that the primary lesions affected glomeruli, and that renal cortical hypoplasia probably was not the cause. Consequently, familial nephropathy (FN) has become the preferred name for the disease? Morphometric studies of kidney specimens from dogs with FN subsequently provided persuasive evidence that the disease was a primary glomerulopathy and not renal hypoplasia." Although FN in Cocker Spaniels was the first hereditary renal disease of dogs to be characterized, its pathogenesis remains unknown. The ultrastructural lesions in Cocker Spaniels with FN have been reported to be similar to those observed in human beings with Alport ~yndrome,~,'~' but transmission electron microscopic (TEM) findings have been described from only 2 young affected Cocker spaniel^.^," The purpose of the present report is to describe the clinical and pathologic findings in 4 juvenile English Cocker Spaniels with advanced renal failure due to suspected FN. Materials and Methods The subjects of this report are 4 English Cocker Spaniels that were referred to the Texas Veterinary Medical Center (TVMC) for in the glomerular basement membranes (GBM) of all dogs. The GBM exhibited extensive thickening, multilaminar splitting, and fragmentation. Electron dense deposits, suggestive of immunocomplex glomerular disease, were notably absent. A similar ultrastructural GBM lesion is found in human beings and Samoyeds with hereditary nephritis, diseases caused by mutations in the type IV collagen genes. Familial nephropathy in English Cocker Spaniels may be a form of hereditary nephritis caused by a mutation in one of the collagen IV genes. J Vet Intern Med 1997;11: Copyright by the American College of Veterinary Internal Medicine. evaluation of renal disease between 1992 and After clinical evaluation, the affected dogs were euthanized and necropsied. For comparative purposes, a renal biopsy specimen was obtained from a young English Cocker Spaniel with normal renal function at the time of routine ovariohysterectoniy. Clinical evaluations were performed by conventional methods. Histories were reviewed, and coniplete physical examinations were performed. CBCs, serum biochemistry, and urinalyses were performed in our hospital using standard methods. For urine protein-creatinine ratios, urine protein concentrations were determined by a turbidimetric method (DuPont Discrete Clinical Analyzer, Medical Products Division, DuPont Co., Wilmington, DE). and urine creatinine concentrations were measured by an enzymatic method (Kodak Ektacheni Crcatinine [single-slide method], two-point rate test, Clinical Chemistry Products Division, Eastinan Kodnk Co., Rochester, NY). Urine specimens for bacteriologic culture were inoculated on cystine lactose electrolyte-deticient and blood agar plates using volume-calibrated loops. Renal ultrasonography was performed with a 7.5 MHz mechanical sector transducer (ATL Ultramark 4, Advanced Technology Laboratories, Bothell, WA). Nccropsies were performed immediately after euthanasia, and renal tissue was placed in fixatives within IS minutes after euthanasia. For light microscopy, tissues were tixed in 10% neutral burlered formalin and embedded in paraffin. Sections wei-c cut at 3 p i and stained with hematoxylin and eosin (H&E), periodicacid Schiff (PAS), and Masson's trichrome. For electron microscopy, minced I-enal corlex was tixed overnight at 4 C in 4576 paraforinaldchyde and 6.25% glutaraldehyde in 0. I M sodium cacodylate buffer with 0.05% CaCI, (ph, 7.4)." Ti washed 3 times with 0. I M sodium cacodylate bu fixed for 2 hours at 4 C in 1 ';% osmium tetroxide in 0. I M sodium cacodylate buffer. Tissues again were washed 3 times with 0.1 M sodium cacodylate buffer, rinsed 3 times in distilled water, and stained ovei-night at 4 C in a saturated uranyl acetate solution. Tissues then were dehydrated in ethanol and propylene oxide, and embedded in Epon Araldite. Sections cut at 0.5 pm were stained with toluidine blue and examined to identify glomeruli. Blocks then were trimmed accordingly. Thin (60-90 nm) sections then were cut, mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (Zeiss IOC, Carl Zeiss, Inc., New York, NY). Clinical Findings Dog 1 A I0-month-old, 10.7-kg male blue roan English Cocker Spaniel was referred to TVMC for evaluation of suspected 80 Journal of Veterinary Internal Medicine, Vol 7 7, No 2 (March-April), 1997: pp 80-85

2 GLOMERULAR ULTRASTRUCTURAL FINDINGS 81 Table 1. Selected Test Results Obtained* From Juvenile English Cocker Spaniels Suspected to Have Familial Nephropathy Dogs-Number Reference Parameters Range Gender Male Male Male Female Age (months) Urine tests Specific gravity > PH Proteint f 2+ Negative Protein-creatinine ratio Serum chemistry tests Creatinine (mg/dl) Urea nitrogen (mg/dl) Phosphorus (mg/dl) Calcium (rng/dl) Total protein (g/dl) Albumin (g/dl) Sodium (meq/l) Potassium (meq/l) Chloride (meq/l) Total CO, (rneq/l) CBC Hematocrit (YO) Leukocytes (x 103/pL) Platelets ix i03/pl) * Data from each dog's initial evaluation at Texas A&M University. t Semiquantitative turbidometric acid precipitation tests scored negative, trace, 1 +, 2+, 3+, or 4+. renal disease. The owner, who had obtained the dog when it was 3 months old, observed that the dog had been difficult to housebreak and had always produced colorless urine. Polyuria and polydipsia had been present for 3 months prior to evaluation. Mild lethargy, slight weight loss, and occasional vomiting had been noted in the last month. Urinalyses performed on 3 occasions in the preceding week had demonstrated proteinuria (4+) and low urine specific gravity ( ). Azotemia (serum urea nitrogen concentration, 69 mg/dl; reference range, mg/dl; and serum creatinine concentration, 3.7 mg/dl; reference range, 1.O- 2.2 mg/dl) and hyperphosphatemia (12.2 mg/dl; reference range, mg/dl) were detected 3 days prior to referral. The dog was slightly thin, but there were no other abnormalities on physical examination. Clinicopathologic evaluation at TVMC confirmed the presence of proteinuria and renal failure (Table 1). Aerobic urine culture produced no growth. Renal ultrasonographic examination showed that both kidneys were similar in size and echogenicity. The renal medulla appeared normal, but the renal cortex was hyperechoic. A presumptive diagnosis of FN was made. Recommendations for dietary management of chronic renal failure were provided, but the owner was advised to expect rapid progression of the disease. When evaluated 3 weeks later, the dog was anorexic, lethargic, and had been vomiting. On physical examination, the dog's mucous membranes were pale, it was moderately (8%) dehydrated, and it weighed 10 kg. Clinicopathologic evaluation revealed persistent proteinuria (urine protein-creatinine ratio, 1 1. I), dilute urine (specific gravity, 1.019), severe azoteniia (serum urea nitrogen concentration, 267 mg/dl; serum creatinine concentration, 12.8 mgldl), and hyperphosphatemia (25.2 mg/dl). The dog was euthanized, and a limited necropsy performed. Dog 2 A 27-month-old, 8.9-kg male blue roan English Cocker Spaniel was referred to TVMC for evaluation of renal disease. Two days previously, a veterinarian had examined the dog because of anorexia and vomiting. Findings included normal rectal temperature, pale mucous membranes, and abdominal tenderness in the area of the kidneys. On laboratory testing, there were anemia (PCV, 14.7%), leukopenia (3.5 X 10' WBCIpL), proteinuria (4+), and severe renal failure (urine specific gravity, 1.017; serum urea nitrogen concentration, 235 mg/dl, reference range, 6-30 mg/dl; serum creatinine concentration, 1 I.7 mg/dl, reference range, mg/dl; serum phosphorus concentration, 19.3 mg/dl, reference range, mg/dl; and serum total carbon dioxide concentration, 8 mmolb, reference range, mmol/l). Nephritis due to an unknown cause was suspected, and lactated Ringer's solution was administered IV. On examination at TVMC, the dog was thin and lethargic. Its mucous membranes were pale, but the dog was normally hydrated. Palpation near the kidneys did not elicit pain, but the peripheral lymph nodes were slightly enlarged. Clinical pathology studies (Table 1) showed pancytopenia, proteinuria, and renal failure, but the azotemia was less severe

3 82 LEES ET AL than that observed prior to IV fluid administration. On renal ultrasonography, there was hyperechogenicity of the cortical and medullary areas of both kidneys. The dog transiently responded to supportive care, but was euthanized and necropsied on hospital day 6. Dog 3 A 22-month-old, 13.2-kg male blue roan English Cocker Spaniel was referred to TVMC for evaluation of renal disease. The owner previously had owned another English Cocker Spaniel that died of renal failure at a young age, and was concerned that this dog also might be affected. Polyuria and polydipsia also had been observed. Urinalyses had been performed monthly or bimonthly since the dog was 15 months old. These urinalyses had documented persistent proteinuria (urine protein-creatinine ratio, ) and gradually diminishing urine concentration (sequential urine specific gravities of 1.027, 1.018, 1.017, 1.017, and 1.012). The dog was referred when it developed azotemia (serum creatinine concentration, 2.3 mg/dl; reference range mg/dl) and anemia (PCV, ls%). At the time of referral, the dog was thin, and its mucous membranes were pale, but its general appearance was otherwise normal. During the next 2 days, the dog remained active and well hydrated but polydipsia and polyuria were observed. Arterial blood pressure determined by an indirect method was normal. Severe anemia, proteinuria, and renal failure were also found (Table I). An aerobic urine culture was negative. Renal ultrasonography showed slight irregularity in the shape of both kidneys, and the right kidney was slightly larger than the left. Hyperechogenicity of the cortex and medulla of both kidneys was present. The renal pelves of both kidneys were mildly dilated, but ureteral enlargement was not seen. After a presumptive diagnosis of FN, euthanasia and necropsy were performed. Dog 4 A 13-month-old, 6.3-kg female orange roan English Cocker Spaniel was referred to TVMC for evaluation of renal disease. Three days previously, a veterinarian had examined the dog because of reduced appetite. Clinicopathologic evaluation revealed anemia (PCV, 14%, reference range, 37-5S%), azotemia (serum urea nitrogen concentration, 117 mg/dl, reference range, mg/dl; serum creatinine concentration, 4.0; reference range, mg/dl), and hyperphosphatemia (1 9.2 mg/dl, reference range, mg/dl). At TVMC, the dog was thin, had pale mucous membranes, but was otherwise normal. During the next 5 days, the dog remained active and well hydrated, but polydipsia and polyuria were observed. Arterial blood pressure determined using an indirect method was normal. Severe anemia, proteinuria and renal failure were found (Table I). Additionally, glucosuria (2+) was present despite a normal blood glucose concentration (105 mg/dl). An aerobic urine culture was negative. Renal ultrasonography showed hyperechogenicity of both kidneys and reduced thickness of the renal cortices. The right ludney was slightly larger than the left. After a presumptive diagnosis of FN, euthanasia and necropsy were performed. Normal Dog A 9-month-old, female blue roan English Cocker Spaniel was presented to TVMC for ovariohysterectomy. She had normal urine concentrating ability and lack of proteinuria or azotemia. During ovariohysterectomy, a wedge biopsy specimen was taken from the left kidney. Pathologic Findings Both kidneys of each dog were smaller thlui normal, excessively firm, and diffusely pale tan to gray. In dog 2, the kidneys were unequally reduced in size. The renal capsules were adherent to the underlying cortical surfaces, which were diffusely pitted and granular. On their cut surfaces, the renal cortices were diffusely but irregularly thinned, having corticalto-medullary thickness ratios of 0.17 to 0.36 (normal, at the poles and 0.75? 0.06 in the middle of the kidney). In dog 2, the parathyroid glands were enlarged, the ribs were pliable, and punctate gastric ulcers were present. The light microscopic appearance of the kidneys was similar in all 4 dogs. The principal change was diffuse glomerular disease. Mesangial thickening, glomerular fibrosis, periglomerular fibrosis, and glomerular obsolescence were noted. These glomerular lesions were not associated with abnormal amounts of either PAS- or GMS-positive basement membrane material, as expected in dogs with immune-mediated glomerulonephritis. In the interstitium, patchy fibrosis that was sometimes radially oriented was accompanied by moderate to severe lymphoplasmacytic cellular infiltration. Neutrophils were rare in the inflammatory cell infiltrates. Renal tubules in the fibrotic areas were atrophic and often contained luminal protein casts. Medullary fibrosis was mild to moderate in all 4 dogs, and 2 dogs (dogs 1 and 4) had nephrocalcinosis. On TEM, a distinctive ultrastructural lesion was observed in the GBM of all 4 dogs. The GBM was greatly thickened and exhibited multilaminar, longitudinal splitting (Fig 1 A). The GBM thickening and fragmentation were extensive, and all of the GBM examined in each dog were similarly affected. Electron dense GBM deposits, such as those observed in immunocomplex glomerulonephritis, were notably absent. Other ultrastructural changes observed in all dogs included fusion of visceral epithelial cell foot processes, large mesangial collagen deposits, and prominent glomerular macrophages. No lesions were found on light microscopic or TEM examination (Fig I B) of the renal biopsy specimen from the young English Cocker Spaniel with normal renal function. Discussion Familial nephropathy in Cocker Spaniels is a progressive, invariably fatal di~ease., ~ It occurs both in male and female dogs, and the age at onset of illness in most dogs is 6 to 24 months. Early clinical signs include polyuria, polydipsia, weight loss, anorexia, vomiting, and diarrhea, but many affected dogs show few clinical signs before their renal failure becomes severe. The clinicopathologic findings are typical

4 GLOMERULAR ULTRASTRUCTURAL FINDINGS 83 Fig 1. Transmission electron micrographs of glomerular capillaries in young English Cocker Spaniels. (A) Capillary from a 22-month-old male dog with familial nephropathy (dog 3). The capillary lumen, containing a leukocyte (L) and 2 erythrocytes (E), is lined by endothelial cell processes (arrowheads) arrayed on the inner surface of the glomerular basement membrane (GBM). The GBM is greatly thickened and exhibits multilaminar longitudinal splitting (between solid arrows). Foot processes of visceral epithelial cells arrayed on the outer surface of the GBM are fused (open arrows). (B) Capillary from a normal 9-month-old female English Cocker Spaniel. Normal ultrastructural appearance of the GEM (between solid arrows) and visceral epithelial cell foot processes (open arrows). Bars = 1.0 pm. of dogs with chronic renal failure, with severity depending on the stage of disease at presentation. Proteinuria occurs prior to loss of urine concentrating ability or onset of azotemia, persists as the disease progresses, and is indicative of the underlying primary glomerulopathy. The gross and light microscopic lesions of Cocker Spaniels with FN have been described.'.' Both kidneys are pale and smaller than normal, but not always equally reduced in size. The renal capsule is adherent to the underlying parenchyma, and once stripped reveals a finely granular renal cortical surface. Gross scarring is not a feature, but the kidneys are abnormally firm when cut. The thickness of the renal cortex is irregularly reduced, and streaks of calcification are sometimes noted in the medulla. The microscopic renal lesions usually have been interpreted as membranoproliferative or sclerosing glomerulonephritis. Glomerular fibrin deposition and periglomerular fibrosis are prominent and believed to lead to glomerular obsolescence. Secondary tubulointerstitial lesions also are found. Interstitial fibrosis is prominent, but inflammation usually is mild. Calcification of basement membranes often is conspicuous. Previous reports of FN have identified affected dogs only as Cocker Spaniels. In North America, however, Cocker Spaniel breeds are named differently than in the rest of the world. Since 1936, nomenclature used by the American Kennel Club (AKC) has referred to the breed that is called Cocker Spaniel elsewhere in the world as the English Cocker Spaniel. The dog breed called Cocker Spaniel by the AKC is known outside the United States and Canada as the American Cocker Spaniel. The previous report" of renal cortical hypoplasia affecting 2 Cocker Spaniel littermates in the United States identified the breed only as Cocker Spaniel, but an author of that report has confirmed that the affected dogs were American Cocker Spaniels (Graber ER, personal communication, 1994). At that time, however, the term renal corticcd hypoplasia commonly was used as a general morphologic diagnosis, regardless of the breed of dog affected." The clinical and clinicopathologic features, as well as the gross and microscopic lesions that characterized the renal disease in the 4 dogs of this report were consistent with previous descriptions of FN. We were unable to investigate the familial occurrence of renal disease in the kindreds of the dogs in this report. The affected dogs were not closely related (ie, they were not full or half siblings). The pedigrees of the affected dogs show that they have some ancestors in common; but this likely is true of many English Cocker Spaniels in North America, regardless of their health status. The mode of inheritance of FN in English Cocker Spaniels is reported to be autosoinal recessive,x but the data and analysis supporting this conclusion have not been published. Distinctive alterations in GBM ultrastructure were observed in all English Cocker Spaniels with FN examined by TEM. Our findings are similar to those reported previously in a Cocker Spaniel with FN.' The GBM in the dogs in this study and the one previously reported had widespread thickening, multilaminar splitting, and fragmentation. The electron-dense deposits in glomerular capillary walls that characterize immunocomplex glomerulonephritis were not noted. These ultrastructural GBM lesions appear to be characteristic of FN in English Cocker Spaniels, and TEM is recommended for diagnostic confirmation of suspected cases.

5 84 LEES ET AL The ultrastructural GBM lesions found in these English Cocker Spaniels are similar to the lesions noted in hereditary nephritis (HN) in human beings and Samoyeds.21~2' Indeed, when these distinctive ultrastructural changes are extensive, as they were in the dogs we examined, they are accepted as sufficient evidence for a morphologic diagnosis of HN in human patient^.'^," Therefore, we believe that FN in English Cocker Spaniels is a form of HN. Hereditary nephritis refers to a specific group of progressive renal diseases that have been characterized in human beings and Samoyeds. Alport syndrome is the most common form of HN in humans, and also is associated with sensorineural hearing loss and ocular lesions. "-'" Affected persons usually develop renal failure when they are children or young adults. Genetic transmission of HN is X-linked in most kindreds, but autosomal transmission occurs in 10% to 15% of affected families. The light microscopic features in HN are nonspecific and variable, especially late in the disease, and tissue diagnosis of HN rests on TEM and immunohistologic findings. Often, the diagnosis of HN is made by TEM alone. The distinctive feature of HN is extensive thickening with longitudinal splitting or layering and fragmentation of the GBM. Tmmunofluorescence tests using anti-gbm antibodies also have shown that the antigenic composition of GBM from patients with HN is abnorma1.l""~'" An X-linked form of HN in Samoyeds has been well characterized.2'-2' In affected neonatal Samoyeds, the GBM appears normal by light and electron microscopic examination, but antigenic composition of GBM is demonstrably abnormal even at this time. The first structural changes can be observed by TEM as early as 1 month of age, and proteinuria, which is the earliest clinical marker of the condition, usually appears at approximately 3 months of age. In HN in Samoyeds, both affected male and carrier female dogs develop proteinuria associated with characteristic TEM lesions in their GBM. The difference between affected male and carrier female dogs is that changes in the GBM of affected male dogs steadily become more extensive, whereas the changes in carrier female dogs do not progress. Affected male Samoyeds usually develop severe renal failure before they are 1-year old. Studies of HN in human beings and in Samoyeds have shown that these conditions are caused by defective type IV collagen in the GBM.'X-2'.2h Collagen IV is an important protein component of GBM. Each collagen monomer is a triple helix of polypeptides (a chains), and six different a(tv) chains have been identified. The gene that encodes a5(iv) chains is located on the X chromosome. Analyses of the a5(iv) gene in X-linked Alport syndrome kindreds have now identified more than 50 different deletions or mutations in this gene." The mutation that causes X-linked HN in Samoyed Dogs also has been identified in the d(1v) gene." Human genes for a3(iv) and a4(1v) chains are on chromosome 2, and mutations of each of these genes have been demonstrated in families with autosomal recessive Alport ~yndrome.'~ If FN of Cocker Spaniels is indeed a form of HN, as suggested by the ultrastructural features of the GBM in the affected dogs that we have studied, pathogenesis of the condition is likely to be coupled to defective synthesis of type IV collagen. Familial nephropathy in Cocker Spaniels has been reported to be inherited as an autosomal trait, and the underlying genetic defect is likely to be found in the genes that encode a3(iv) or a4(tv) collagen chains of dogs. References I. Krook L. The pathology of renal cortical hypopl dog. Nord Vet-Med 1957;9: Persson F, Persson S, Asheim A. Renal cortical hypoplasia in dogs: A clinical study on uraemia and secondary hyperparathyroidism. Acta Vet Scand 1961;2: Freudiger U. 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Proceedings of the 40th Annual Meeting of the American College of Veterinary Pathologists, Baltimore, MD, 1989; Lucke VM, Kelly DF, Darke PGG, et al. Chronic rcnal failure in young dogs-possible renal dysplasia. J Small Anim Pract 1980; 2 I : 169- I Karnovsky hij. A formaldehyde-gllitaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 196.5; 27: I37A- I38A. 13. Picut CA, Lewis RM. Hereditary kidney disease in dogs. Kal Kan Forum 1989:8: Kelly DF. Renal dysplasia in the dog. In: Grunsell CSG, Hill FWG, eds. Veterinary Annual, 13th ed. Bristol: John Wright; 1972: IS. Johnson ME, Denhart JD, Graher ER. Renal cortical hypoplasia in a litter of Cocker Spaniels. J Am Anim Hosp Assoc 1972;8: Glassock RJ, Cohen AH, Adler SG, et al. Sccondary glomerular diseases. In: Brenner BM, Rector FC, eds. The Kidncy, 4th ed. Philadelphia: WB Saunders; 1991: Striker LJ, Olson JL, Striker GE. The Renal Biopsy, 2nd ed. Philadelphia: WB Saunders: 1990: Kashtan CE, Kleppel MM, Butkowski RJ, et al. Alport syndrome, basement membranes and collagen. Pediatr Nephrol 1990: Hudson BG, Reeders ST, Tryggvason K. Type IV collagen: Structure, gene organization, and role in human diseases. J Biol Chem 1993:268: Kashtan CE, Michael AF. Alport syndrome: From bedside to genome to bedside. Am J Kidney Dis 1993;22: Zheiig K, Thoi-iier PS, Marrano P, et al. Canine X chroinosome-linked hereditary nephritis: A genetic model for human X- linked hereditary nephritis resulting from a single base mutation in

6 GLOMERULAR ULTRASTRUCTURAL FINDINGS 85 the gene encoding the a5 chain of collagen type IV. Proc Natl Acad Sci USA 1994;91: Jansen B, Valli VEO, Thorner P, et al. Samoyed hereditary glomerulopathy: Serial clinical and laboratory (urine, serum biochemistry, and hematology) studies. Can J Vet Res 1987;51: Thorner P, Jansen B, Baumal R, et al. Samoyed hereditary glomerulopathy: Immunohistochemical staining of basement membranes of kidney for laminin, collagen type IV, fibronectin, and Goodpasture antigen, and correlation with electron microscopy of glomerular capillary basement membranes. Lab Invest 1987; 56: Thorner P, Baumal R, Valli VEO, et al. Abnormalities in the NCI domain of collagen type IV in GBM in canine hereditary nephritis. Kidney Int 1989;35: Thorner PS, Baumal R, Valli VEO, et al. Production of anti- NCl antibody by affected male dogs with X-linked hereditary nephritis: A probe for assessing the NC 1 domain of collagen type IV in dogs and humans with hereditary nephritis. Virchows Arch A Pathol Anat Histopathol 1992;421: Reeders ST. Molecular genetics of hereditary nephritis. Kidney Int 1992;42: Mochizuki T, Lemmink HH, Mariyama M, et al. Identification of mutations in the alpha-3(iv) and alpha-4(lv) collagen genes in autosomal recessive Alport syndrome. Nat Genet 1994; 8:77-82.