• Alport syndrome;
  • Canine;
  • Hereditary disease;
  • Type IV collagen


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References


Autosomal recessive hereditary nephropathy (ARHN) was diagnosed in 2 English Springer Spaniels (ESS), a breed not previously reported to be affected by hereditary nephropathy (HN).


To identify and characterize the genetic cause of ARHN in ESS.


Sixty-three ESS (2 with ARHN, 2 obligate carriers, and 59 others), 2 mixed-breed dogs with X-linked HN, and 2 English Cocker Spaniels (ECS) with ARHN were included.


ARHN was diagnosed based on transmission electron microscopy and immunostaining of kidney. DNA from affected dogs was screened for the mutation known to cause ARHN in ECS. Quantities of COL4A3,COL4A4, and COL4A5 mRNA transcripts in renal cortex were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for ARHN-affected dogs and 7 other dogs. The coding regions of COL4A3 and COL4A4 were sequenced for the 2 ARHN-affected ESS and an unaffected dog. Exon 30 of COL4A4 was sequenced for all 63 ESS.


qRT-PCR indicated a significant reduction in transcript levels of both COL4A3 and COL4A4 mRNA in the kidney of ARHN-affected ESS. Sequencing identified a single nucleotide substitution in COL4A4 at base 2806 resulting in a premature stop codon. Thirteen of 25 related dogs were identified as carriers.

Conclusions and Clinical Importance

A mutation highly likely to cause ARHN in ESS has been identified.


autosomal recessive hereditary nephropathy


blood urea nitrogen


complementary DNA


English Cocker Spaniel


English Springer Spaniel


glomerular basement membrane


hereditary nephropathy


polymerase chain reaction


quantitative reverse transcription-polymerase chain reaction


serum albumin


serum creatinine


urine protein : creatinine ratio


urine-specific gravity


X-linked hereditary nephropathy

Hereditary nephropathy (HN) is a progressive fatal renal disease that has been identified in several domestic dog breeds or kindreds. The disease was first described as renal cortical hypoplasia[1] and also has been called familial nephropathy[2-4] and hereditary nephritis[5-7] in various published accounts. HN is the result of glomerular basement membrane (GBM) defects that alter GBM structure and function in the mature kidney. The GBM is a sheet-like layer of extracellular matrix that lies between and supports the endothelial and visceral epithelial cell components of the glomerular capillary walls. This structure is primarily composed of heterotrimers of laminins and type IV collagens (α1–α5 chains). During glomerular development, the GBM is initially composed of α1α1α2(IV) heterotrimers, but as the glomeruli mature, α3α4α5(IV) heterotrimers gradually replace the α1α1α2(IV) network. Moreover, normal synthesis and assembly of α3α4α5(IV) heterotrimers is crucial for maintaining the structure and function of the GBM in adult kidney. Defects in any of the genes encoding α3(IV), α4(IV), or α5(IV) collagen chains can cause the GBM to develop distinctive abnormalities during adolescence that initiate a progressive renal disease culminating in juvenile-onset chronic renal failure.[8] In humans, the nephropathy caused by type IV collagen defects is usually called Alport syndrome and is associated with certain hearing and ocular abnormalities in many affected individuals. In both humans and dogs, type IV collagen-related renal disorders have both X-linked and autosomal patterns of inheritance. This is because the COL4A5 gene is on the X-chromosome and the COL4A3 and COL4A4 genes are on an autosome (chromosome 2 in humans and chromosome 25 in dogs) in both species. For these reasons, COL4A3, COL4A4, and COL4A5 are candidate genes for identifying the causative mutations in subjects with HN.

An autosomal recessive form of HN (ARHN) has occurred in the English Cocker Spaniel (ECS) for more than 50 years.[1-4, 6] Affected dogs typically develop severe (end-stage) renal disease by the time they are 6–24 months of age. In dogs with ARHN, clinical signs often are not observed until late in the course of disease,[4] although proteinuria invariably develops a few months before other clinicopathologic changes can be detected. There is no cure for HN; all currently available treatments are symptomatic or supportive in nature. Previous work identified the mutation in COL4A4 that causes ARHN in ECS.[9] Efforts to eliminate the disease from the ECS breed using genetic testing coupled with careful breeding practices (ie, preventing the breeding of 2 carriers) are ongoing and have been highly successful.

The English Springer Spaniel (ESS) is closely related to the ECS and, according to the American Kennel Club, originally came from the same litters.[10] Despite common ancestry, ARHN has not been described previously in the ESS breed. This report describes the discovery of ARHN in a family of ESS and subsequent identification of a novel COL4A4 mutation in this kindred.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clinical and Pathologic Evaluations

Two female ESS littermates were evaluated at the Texas A&M University College of Veterinary Medicine and Biomedical Sciences (TAMU-CVM) for renal disease that was first detected when the dogs were 7 months old. Each dog's renal disease was observed to progress to a near-terminal stage over the next 2–3 months, whereupon each dog was euthanized.

Clinical examinations, laboratory testing, and diagnostic imaging of the 2 dogs were performed by conventional methods. Pathologic evaluations of kidney obtained immediately after euthanasia included histologic, ultrastructural, and immunostaining examinations utilizing methods similar to those previously described.[6] Formalin-fixed tissue was routinely embedded in paraffin and sections cut 3 μm thick were prepared with H&E, PAS, Jones' methenamine silver, and Masson's trichrome stains for light microscopic examination. Cortical tissue for transmission electron microscopic examination was fixed in chilled 3% glutaraldehyde, further processed, sectioned, and stained by conventional methods,[6] and examined in a JEOL TEM-1230 transmission electron microscope. Immunolabeling of renal basement membranes for their content of certain collagen IV proteins was performed with indirect immunofluorescence staining methods using monoclonal antibodies specific for α1/2 chains (MAB M3F7), α3 chains (MAB A2), or α5 chains (MAB A7), as previously described.[6]

Samples for Molecular Genetic Evaluation

Whole blood and kidney tissue were collected from both affected ESS. DNA was extracted from whole blood using a commercially available kit,1 and renal tissue was stored in an RNA stabilization solution.2 Additional blood samples were collected from 25 related ESS (including both parents) and 1 unrelated ESS owned by the breeder. Subsequently, DNA was isolated from buccal cell samples from 35 ESS, unrelated to the kindred, obtained from owners.

RNA was extracted from the kidney tissues of the 2 affected ESS, as well as from archived frozen kidney tissue from 2 ARHN-affected ECS, 2 X-linked hereditary nephropathy (XLHN)-affected mixed-breed (NAV) dogs[11] and 3 mixed-breed dogs unaffected by any form of HN. Reverse transcription was performed to synthesize complementary DNA (cDNA) from the RNA. The ESS were client-owned; breed-matched tissue samples from unaffected dogs were not available. DNA and RNA quantification was determined using a spectrophotometer.3

Genomic Screening of COL4A4 Exon 3

Exon 3 of COL4A4 was amplified by polymerase chain reaction (PCR) to screen for the previously identified ARHN mutation[9] using genomic DNA from the 2 affected ESS and 3 ECS with known clinical status: 1 ARHN-affected dog, 1 ARHN-carrier, and 1 unaffected dog. Each reaction contained 12.5 μL 2× all-inclusive PCR premix,4 0.25 μM of each primer and 50 ng of DNA, brought to a final volume of 25 μl with sterilized water. Primer sequences and cycling conditions have been published previously.[9] Product sizes were verified using agarose gel electrophoresis. The PCR amplicons were treated with 0.5 units of Exonuclease I5 and 0.25 units of shrimp alkaline phosphatase.6 Purified amplicons then were sequenced7 and resolved.8

Quantitative Reverse Transcription-PCR (qRT-PCR)

The mRNA transcript numbers of COL4A3, COL4A4, and COL4A5 were quantified for 9 dogs: 2 ESS and 2 ECS with ARHN, 2 NAV dogs with XLHN, and 3 dogs unaffected by any form of HN. Primer/probe assays9 specific to Canis familiaris and spanning 2 exons were selected for qRT-PCR. Probes were labeled at the 5′ end with 6-carboxyfluorescein (6-FAM) and at the 3′ end with a nonfluorescent quencher. Each 25 μl reaction contained 12.5 μl 2× QuantiTect Probe RT-PCR Master Mix,10 0.25 μl QuantiTect RT Mix,10 0.72 nM of each primer, 0.2 nM of probe, and 7 ng of RNA. Reverse transcription, amplification, and detection were performed11 under the following cycling conditions: reverse transcription at 50°C for 30 minutes and inactivation at 95°C for 13 minutes 30 seconds, followed by 45 cycles of 94°C for 15 seconds, 60°C for 60 seconds, and 70°C for 30 seconds. All reactions were performed in triplicate. For each sample, quantification of collagen mRNA transcripts was determined by normalization against β-actin mRNA transcript numbers, and the magnitude of change between groups was analyzed using the Pfaffl method.[12] Any changes in transcript numbers greater than 2-fold were considered significant.

COL4A3 and COL4A4 cDNA Sequencing

Amplification of the coding regions of COL4A3 and COL4A4 was performed for both affected ESS and a normal control dog. RNA was reverse transcribed,12 and the resulting cDNA was used as a template for amplification. Amplicons were 500-base pairs (bp) long and designed to overlap by approximately 75 bp at each end, spanning the coding regions for both genes (see Table 1 for primer sequences). Each 25 μL reaction contained 3–5 μL of cDNA, 2.5 μL of 10× Taq DNA polymerase buffer B,13 2.0 units of Taq DNA polymerase,13 0.2 mM of each dexoynucleotide triphosphate, 1.5 mM magnesium chloride, 0.4 μM of each primer, 1 M betaine,14 and 5% dimethyl sulfoxide. PCR amplification was carried out under the following conditions: a single cycle at 94°C for 5 minutes, 35 cycles of 94°C for 1 minute, 50–54°C for 1 minute, and 72°C for 1 minute, and a single cycle of 72°C for 10 minutes. Purified amplicons were sequenced and analyzed as described above.

Table 1. Primers and melting temperatures (Tm) (°C) used for polymerase chain reaction amplification of canine COL4A3 and COL4A4.
Primer SetTmSequencePrimer SetTmSequence

Genomic Screening of COL4A4 Exon 30

PCR amplification of exon 30 of COL4A4 was performed as above for exon 3 using genomic DNA from the 2 affected ESS and 61 ESS of unknown status. Cycling conditions were as follows: 94°C for 5 minutes, 35 cycles of 94°C for 1 minute, 54°C for 1 minute, and 72°C for 1 minute, and 1 cycle of 72°C for 10 minutes. PCR products were treated with 0.5 units of Exonuclease I and 0.25 units of shrimp alkaline phosphatase, and then sequenced and analyzed as described for exon 3 above.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clinical Findings

Two female ESS littermates (Dog 14 and Dog 1515) were owned by 1 individual who did not observe any signs of ill health before taking them to a veterinarian to be spayed at 7 months of age. Laboratory tests performed then showed that both dogs had marked proteinuria (Dog 14: urine-specific gravity [USG] 1.036; urine protein : creatinine ratio [UPC] 7.9 and Dog 15: USG 1.042; UPC 7.0). Both dogs were mildly hypoalbuminemic (Dog 14: serum albumin [SA] 2.2 g/dL and Dog 15: SA 2.5 g/dL; reference range 2.7–4.4 g/dL). Dog 14 also was mildly azotemic (blood urea nitrogen [BUN] 48 mg/dL; reference range 6–25 mg/dL; and serum creatinine [SCR] 1.6 mg/dL; reference range 0.5–1.6 mg/dL), and values for Dog 15 (BUN 38 mg/dL; SCR 1.3 mg/dL) were borderline high.

Additional testing performed 2 months later showed persistent proteinuria and progression to renal failure in both dogs, albeit more rapidly in Dog 14 (UPC 9.8; BUN 119 mg/dL; SCR 6.2 mg/dL; and SA 2.2 g/dL) than in Dog 15 (UPC 8.1; BUN 65 mg/dL; SCR 3.0 mg/dL; SA 2.2 g/dL). A renal ultrasound examination of Dog 14 showed hyperechoic cortices with normal corticomedullary definition in both kidneys. Renal ultrasound findings for Dog 15 were similar except that the left kidney also exhibited a large wedge-shaped cortical defect that was thought likely to be the site of a previous infarct, and the right kidney was appreciably larger (about 1.5 cm longer) than the left kidney.

One week later, Dog 14 began exhibiting uremic signs and was brought to TAMU-CVM for euthanasia and postmortem examination of the kidneys. When examined, the dog was weak and moderately dehydrated. Laboratory tests performed on this occasion showed severe azotemia (BUN 203 mg/dL; SCR 11.3 mg/dL) and persistent proteinuria (USG 1.021; UPC 9.8).

Dog 15 remained clinically well for 1 additional month despite increasing azotemia (SCR concentration increased from 3.0 to 4.4 mg/dL over a 12-day interval) before the dog became lethargic and began vomiting. The dog then was brought to TAMU-CVM for euthanasia and postmortem examinations of the kidneys. The dog had a subdued demeanor and was marginally dehydrated when examined, and laboratory tests showed azotemia (BUN 82 mg/dL; SCR 6.2 mg/dL) and persistent proteinuria (USG 1.016; UPC 11.9).

Pathologic Findings

The gross appearance of both kidneys of Dog 14 was similar. They were symmetrically shaped and had smooth capsular contours. However, the kidneys of Dog 15 were not symmetrical; the left kidney was smaller than the right, and its middle third was smaller than its poles.

Histologic examination disclosed similar severe changes in the glomeruli and tubulointerstitium of both dogs. Glomeruli had thickened and often laminated Bowman's capsules. Glomerular tufts were lobulated and markedly hypercellular with widespread mesangial expansion and effacement of peripheral capillary loops. Hypercellularity within glomeruli consisted of increased numbers of mesangial cells coupled with hypertrophy of endothelia as well as visceral and parietal epithelial cells. Where present, patent glomerular capillary loops frequently were circumscribed by mildly to moderately thickened walls. Glomerular adhesions to Bowman's capsule (synechia) were common, and occasionally glomerular adhesions were accompanied by crescents of increased cells at the interface of the adherent glomerular tuft and the thickened capsule. Hyaline eosinophilic amorphous coagula and eosinophilic globular material were common within the urinary space. The cortical interstitium was diffusely and mildly to moderately expanded by dense to regionally loose (edematous) collagenous connective tissue containing widely scattered infiltrates of predominantly mixed mononuclear inflammatory cells. Cortical tubules often were mildly dilated and lined by mildly attenuated epithelia. Tubular dilatation also was regionally accentuated and often most prominent in distal segments. Eosinophilic proteinaceous fluid and protein casts were commonly present within tubular lumina and extended into the medulla. The medulla was diffusely and homogenously expanded by dense collagenous connective tissue containing widely scattered small foci of mixed mononuclear inflammatory cells.

Ultrastructural evaluation of the glomeruli identified diffuse effacement of visceral epithelial cell foot processes. Glomerular capillary walls exhibited multifocal wrinkling and mesangial cell interpositioning. Basement membranes within the glomerular capillary walls were globally thickened because of the distortion of the lamina densa, which was characterized by longitudinal splitting and fragmentation that created the appearance of a “net-like” or “woven” pattern (Fig 1A). Mesangium had increased cells and matrix.


Figure 1. Transmission electron photomicrograph of a glomerular capillary loop from an ARHN-affected ESS (Dog 15; panel A) that shows marked thickening of the glomerular basement membrane with longitudinal splitting and fragmentation of its lamina densa, as compared with a capillary loop from a normal dog (panel B).

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Immunostaining of kidneys from both Dog 14 and Dog 15 produced similar results that demonstrated an abnormal pattern of expression of type IV collagens in their glomeruli (Fig 2). Labeling of GBM for α3 chains was completely absent (Fig 2C–D), whereas labeling for α5 chains was present but of decreased fluorescent intensity compared with normal dog kidney (Fig 2E–F) and labeling for α1/2 chains was increased (Fig 2A–B). This pattern of abnormal expression of type IV collagens in GBM was identical to that previously observed in ECS with ARHN.[6, 9]


Figure 2. Fluorescence photomicrographs showing immunolabeling of renal sections from an ARHN-affected ESS (Dog 14; panels A, C, and E) compared with that for a normal dog (panels B, D, and F) for α1/2 chains (panels A and B), α3 chains (panels C and D), or α5 chains (panels E and F) of collagen IV. Panel A shows increased labeling of α1/2 chains, especially in the capillary walls, compared with normal dog kidney (panel B). In panel C, which shows completely negative labeling for α3 chains in the ARHN-affected dog, half of the microscopic field is shown with phase-contrast illumination to verify presence of a glomerulus at that location. The negative labeling for α3 chains contrasts with the uniformly positive labeling of the normal dog's glomerular capillary walls for α3 chains (panel D). Panel E shows labeling of the affected dog's capillary walls for α5 chains that is substantially reduced in its intensity and extent compared with the labeling of the normal dog's glomerular capillary walls for α5 chains (panel F).

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Taken together, the pathologic findings were sufficient for an unequivocal diagnosis of ARHN in both affected ESS (Dog 14 and Dog 15) and identified COL4A3 and COL4A4 as candidate genes in which the underlying causative mutation was expected to exist in the dogs' kindred.

Genomic Screening of COL4A4 Exon 3

Exon 3 was sequenced and analyzed from the ARHN-affected ESS and compared to the sequences obtained from ECS of varying ARHN status to determine if this disease in ESS was caused by the same mutation identified in ECS. All sequences were aligned with the published genomic sequence (CanFam 2.0[13]) for reference. Neither ARHN-affected ESS possessed the nonsense mutation found in affected and carrier ECS (data not shown).

Quantitative Reverse Transcription-PCR

Transcript quantities of COL4A3, COL4A4, and COL4A5 mRNA were evaluated with qRT-PCR. Relative to unaffected dogs, quantities of COL4A3 and COL4A4 mRNA transcripts were decreased 14.33-fold and 10.60-fold, respectively, in the kidneys of ARHN-affected ESS. COL4A5 mRNA numbers in kidneys of affected ESS were not significantly different from those seen in unaffected dogs (Fig 3).


Figure 3. Quantitative reverse transcription-PCR analysis of COL4A3,COL4A4, and COL4A5 mRNA transcript levels revealed a 14.33- and 10.6-fold decrease of COL4A3 and COL4A4, respectively, in ARHN-affected ESS compared with mRNA levels in normal dogs. Transcript levels of COL4A5 were not significantly different between the 2 groups.

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COL4A3 and COL4A4 cDNA Sequencing

To identify the causative mutation of ARHN in ESS, overlapping 500-bp segments spanning the coding regions of both COL4A3 and COL4A4 were sequenced. For comparison, cDNA from an unaffected, mixed-breed dog was sequenced with the same primer sets. All sequences were aligned with the published mRNA sequences for COL4A3 (GenBank accession number AY263362.1) and COL4A4 (GenBank accession number AY263363.1). A single nucleotide substitution was found at base 2806 in COL4A4, changing a cytosine to a thymine in a region corresponding to exon 30. This substitution creates a nonsense mutation and is predicted to change the glutamine at position 904 to a stop codon, resulting in a prematurely truncated transcript (Fig 4). No other deleterious mutations were found in the coding sequences of either gene.


Figure 4. Diagram representing COL4A4 including the mutation causing ARHN in the ESS. (A) Model of the gene including the 47 exons and their introns. (B) Magnification of the region encompassing exons 28, 29, and 30; the identified mutation is located in exon 30. (C) The predicted change to the protein sequence of the α4 (IV) collagen chain corresponding to the exon boundary between exons 29 and 30 (Modified from Davidson et al[9]).

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Genomic Screening of COL4A4 Exon 30

To confirm the mutation found in COL4A4 cDNA, exon 30 was sequenced from the genomic DNA of 63 ESS. Both ARHN-affected ESS were homozygous for the nonsense mutation (thymine). Thirteen of the 25 related ESS and 1 of the unrelated ESS were identified as carriers of the mutation, having both a cytosine and a thymine at base 2806. All other dogs screened were homozygous at this locus for cytosine (data not shown).


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This report documents the occurrence of ARHN in a family of ESS, a breed not previously reported to be afflicted with this disease. Affected ESS did not harbor the particular COL4A4 mutation that is known to cause ARHN in ECS; therefore a different mutation in COL4A4 or a mutation in COL4A3 was anticipated. qRT-PCR was used to evaluate mRNA transcript quantities of the 2 candidate genes (COL4A3 and COL4A4) as well as those of COL4A5 in the kidney tissue of affected dogs with the intention of focusing the search for the mutation preferentially on 1 of the 2 genes. Because transcript numbers were similarly decreased for both COL4A3 and COL4A4, sequencing of the coding regions proceeded for both genes. Sequencing of COL4A3 cDNA did not reveal any mutations, but a nonsense C[RIGHTWARDS ARROW]T transition was found at base 2806, located in exon 30, of COL4A4. This point mutation changes a codon for a glutamine to a premature stop codon, and thus is predicted to produce a protein approximately half the normal length (904 of 1688 amino acids) and lacking the NC1 domain that is required for normal assembly of α3α4α5(IV) heterotrimers. The data strongly suggest this nucleotide change is the causative mutation that results in the disease state.

Mutation screening for C2806T in related and unrelated ESS identified 13 carriers out of 25 related dogs (Fig 5) and 1 carrier among 35 unrelated dogs. Detailed pedigree analysis identified a common ancestor for the unrelated carrier (Dog 27) and the 2 founders of the family (dogs 1 and 2) 4 generations back. The common ancestor indicates that the mutation is not de novo within the nuclear family in Figure 5, and that additional carriers within the breed are likely. Given the small number of unrelated dogs tested in this study, it is not possible to predict the frequency of the mutant allele in the ESS population. Therefore, a population study to estimate the prevalence of carriers within the breed is warranted.


Figure 5. Pedigree of the related ESS. Squares represent male dogs; circles represent female dogs. Filled shapes indicate affected animals, dark circles within shapes indicate carrier status, and gray circles within shapes indicate presumed carrier status. Dogs 14 and 15 are the probands. Dog 27 is unrelated and shares a common ancestor (*) with Dogs 1 and 2 four or more generations back. The genotyping results from 1 related dog (†) were inconclusive; however, some of its progeny in multiple litters were carriers, and the dog thus is presumed to be a carrier. Only numbered dogs were included in the analysis.

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A genetic test for the mutant allele would be useful in confirming a diagnosis of suspected ARHN in an affected ESS. Such a test, however, would be most beneficial to ESS breeders in identifying carriers, thus aiding in breeding decisions to prevent the propagation of the disease. Because the prevalence of the mutant allele in the ESS population is unknown, all dogs in the breeding population might be tested to prevent the mating of 2 carriers and thus prevent further production of any affected dogs. Offspring from matings in which 1 parent is a carrier can be tested to select for dogs not carrying the mutation to be used as future breeding stock. In this way, the mutation can be removed from the ESS breeding lines in as few as 2 generations.

Finally, the findings in this report, together with the findings in previous reports, support several general statements about HN. To date, there are 4 different, fully characterized genetic forms of HN that have been identified. Two forms are X-linked, affecting Navasota mixed-breed dogs[11] and Samoyeds,[14] and each breed possesses a unique mutation in COL4A5. Two forms are autosomal recessive and are caused by distinct mutations in COL4A4, affecting ECS[9] and ESS (described here). However, all 4 mutations cause disruptions of gene translation leading to the synthesis of truncated collagen IV alpha-chains, thus preventing the assembly of the α3α4α5(IV) network that is crucial for maintenance of GBM structure and function in the adult kidney. Therefore, the clinical disease produced by all 4 conditions is similar. Affected dogs appear completely healthy as puppies, developing persistent proteinuria of glomerular origin as their first manifestation of disease.[4, 15, 16] The onset of proteinuria most often is at 4–6 months of age, but can be as early as 2–3 months or as late as 6–8 months of age. Progression of the disease thereafter is characterized by a period of increasing magnitude of proteinuria, followed by a period of gradually decreasing glomerular filtration rate and increasing azotemia, leading to end-stage renal disease, which most often occurs at about 12 months of age, but can occur as early as 6 months or as late as 24 months of age.[4, 6, 15, 17] When dogs with juvenile-onset nephropathies are carefully evaluated, recognition of the clinical features of HN and exclusion of the clinical features of other conditions generally are sufficient to raise a high index of suspicion for the diagnosis of HN, but definitive diagnosis of the condition requires appropriate pathologic evaluations. Nevertheless, light microscopic examinations alone are not sufficient. The histologic lesions in the kidneys of HN-affected dogs are nonspecific changes that are indicative of glomerular disease but are not unique to HN. The distinctive morphologic feature of HN, which is the unique ultrastructural appearance of the GBM, can only be demonstrated by transmission electron microscopy. Notably, the distinctive ultrastructural GBM change is common to all of the fully characterized forms of HN that have been described, regardless of their mode of inheritance.[4, 15, 17] The type of pathologic evaluation that can discriminate between the 2 genetic forms of HN is immunostaining that demonstrates the presence or absence of particular type IV collagen alpha-chains in the renal or epidermal basement membranes of affected dogs. The pattern of collagen IV chain expression observed in the ESS in this report is the same as that observed in ECS and is characteristic of ARHN.[6] There is no GBM labeling for α3(IV) chains, and GBM labeling for α5(IV) is decreased but not completely absent because α5(IV) chains combine with α6(IV) chains to form a network composed of α5α5α6(IV) heterotrimers in the GBM of ARHN-affected dogs. This contrasts with the complete absence of labeling for α3(IV), α4(IV), α5(IV), or α6(IV) chains in XLHN-affected male dogs,[15, 18] which occurs because they cannot synthesize α5(IV) chains and therefore cannot assemble either α3α4α5(IV) or α5α5α6(IV) heterotrimers for expression in any basement membrane. Of note, despite the clinical and pathologic similarities that exist among these inherited nephropathies, each of the breeds or kindreds of dogs with fully characterized HN that have been identified to date have had a different (ie, unique to that breed or kindred) underlying causative mutation.[9, 11, 14] The practical consequence of breed-specific mutations is the requirement of a different DNA-based genetic test in order to identify the mutant allele in each separate dog breed or kindred. Such an outcome is not surprising because several hundred different causative mutations have been identified in people with Alport syndrome, and nearly every affected family worldwide has its own unique causative mutation in COL4A3, COL4A4, or COL4A5.[19]


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors gratefully acknowledge Mary Sanders and Kayla Alling for their technical assistance and the owners and breeders of the English Springer Spaniels who provided samples from the dogs that were used in this study.

This study was supported in part by funding from Clemson University.

  1. 1

    Gentra Puregene Blood Kit, Qiagen, Valencia, CA

  2. 2

    RNA later, Applied Biosystems(ABI)/Ambion, Austin, TX

  3. 3

    NanoDrop, Fisher Scientific, Pittsburgh, PA

  4. 4

    ReddyMix Master Mix, AbGene, Rochester, NY

  5. 5

    New England Biolabs, Ipswich, MA

  6. 6

    Promega, Madison, WI

  7. 7

    Big Dye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems, Foster City, CA

  8. 8

    ABI 3730 Genetic Analyzer, Applied Biosystems

  9. 9

    TaqMan Gene Expression Assays, Applied Biosystems

  10. 10

    Qiagen Inc

  11. 11

    BioRad iQ5 Real-Time PCR Detection system, BioRad Inc, Hercules, CA

  12. 12

    High Capacity Reverse Transcription kit, Applied Biosystems

  13. 13

    Fisher Scientific

  14. 14

    Sigma-Aldrich, St. Louis, MO

  15. 15

    In keeping constant with the numbering of the dogs in Figure 5, the affected ESS are referred to as dogs 14 and 15


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References