Familial nephropathy in Bracchi Italiani: 8 cases (2012-2019).

OBJECTIVE: To characterize the signalment, clinical signs, clinical pathological and histologic findings, and outcome in 8 related Bracchi Italiani with proteinuric kidney disease. ANIMALS: 8 client-owned Bracchi Italiani. PROCEDURES: Health records submitted to the Bracco Italiano Health Foundation and the Bracco Italiano Club of America between 2012 and 2019 were reviewed for dogs with evidence of nephropathy for which histologic diagnoses were obtained. Pedigree, signalment, clinical signs, diagnostic test results (including microscopic examination of kidney tissue samples collected ante- or postmortem), and outcome were acquired. Results were presented as descriptive statistics. RESULTS: The most common clinical sign in affected dogs was inappetence. All dogs were proteinuric, and 4 dogs were azotemic. Seven dogs developed clinical signs of kidney disease and were euthanized a median of 75 days postdiagnosis. Six dogs had glomerular amyloidosis, and 1 dog each had nephrosclerosis and nonamyloidotic fibrillar glomerulopathy. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the clinical presentation may vary in affected dogs, and proteinuria in young or middle-aged Bracchi Italiani should raise the concern for hereditary nephropathy. Prognosis is likely poor once clinical signs are noted.

Abnormal Expression of miR-21 in Kidney Tissue of Dogs With X-Linked Hereditary Nephropathy: A Canine Model of Chronic Kidney Disease.

MicroRNAs (miRNAs) are a group of small noncoding RNAs that act as regulators of posttranslational gene/protein expression and are known to play a key role in physiological and pathological processes. The objective of our study was to compare expression of miR-21 in renal tissue from dogs affected with chronic kidney disease (CKD) caused by X-linked hereditary nephropathy (XLHN), a disease equivalent to human Alport syndrome, to that from unaffected dogs. Additionally, we sought to characterize changes in relative mRNA expression of various genes associated with miR-21 function. miRNA was isolated from kidney tissue collected from both affected dogs and unaffected, age-matched littermates at defined milestones of disease progression, including end-stage renal disease (ESRD). Additionally, autopsy samples from affected dogs at ESRD and corresponding unaffected dogs were evaluated. Samples were scored based on histological changes, and relative expression of miR-21 and kidney disease-related genes was determined using quantitative real-time polymerase chain reaction. In affected dogs, significant upregulation of kidney miR-21 was first detected at the milestone corresponding with increased serum creatinine. Furthermore, miR-21 expression correlated significantly with urine protein: urine creatinine ratio, serum creatinine concentration, glomerular filtration rate, and histologic lesions (glomerular damage, tubular damage, chronic inflammation, and fibrosis). At end-stage disease, COL1A1, TGFB1 and its receptor, TGFB2, and Serpine1 were upregulated, while PPARA, PPARGC1A, ACADM, SOD1, and EGF were downregulated. In conclusion, miR-21 is abnormally upregulated in the kidneys of dogs with CKD caused by XLHN, which may play an important pathologic role in the progression of disease by dysregulating multiple pathways.

RNA-seq of serial kidney biopsies obtained during progression of chronic kidney disease from dogs with X-linked hereditary nephropathy.

Dogs with X-linked hereditary nephropathy (XLHN) have a glomerular basement membrane defect that leads to progressive juvenile-onset renal failure. Their disease is analogous to Alport syndrome in humans, and they also serve as a good model of progressive chronic kidney disease (CKD). However, the gene expression profile that affects progression in this disease has only been partially characterized. To help fill this gap, we used RNA sequencing to identify differentially expressed genes (DEGs), over-represented pathways, and upstream regulators that contribute to kidney disease progression. Total RNA from kidney biopsies was isolated at 3 clinical time points from 3 males with rapidly-progressing CKD, 3 males with slowly-progressing CKD, and 2 age-matched controls. We identified 70 DEGs by comparing rapid and slow groups at specific time points. Based on time course analysis, 1,947 DEGs were identified over the 3 time points revealing upregulation of inflammatory pathways: integrin signaling, T cell activation, and chemokine and cytokine signaling pathways. T cell infiltration was verified by immunohistochemistry. TGF-ß1 was identified as the primary upstream regulator. These results provide new insights into the underlying molecular mechanisms of disease progression in XLHN, and the identified DEGs can be potential biomarkers and therapeutic targets translatable to all CKDs.

X-Linked Hereditary Nephropathy in Navasota Dogs: Clinical Pathology, Morphology, and Gene Expression During Disease Progression.

X-linked hereditary nephropathy (XLHN) in Navasota dogs is a spontaneously occurring disease caused by a mutation resulting in defective production of type IV collagen and juvenile-onset renal failure. The study was aimed at examining the evolution of renal damage and the expression of selected molecules potentially involved in the pathogenesis of XLHN. Clinical data and renal samples were obtained in 10 XLHN male dogs and 5 controls at 4 (T0), 6 (T1), and 9 (T2) months of age. Glomerular and tubulointerstitial lesions were scored by light microscopy, and the expression of 21 molecules was investigated by quantitative real-time polymerase chain reaction with selected proteins evaluated by immunohistochemistry. No significant histologic lesions or clinicopathologic abnormalities were identified in controls at any time-point. XLHN dogs had progressive proteinuria starting at T0. At T1, XLHN dogs had a mesangioproliferative glomerulopathy with glomerular loss, tubular necrosis, and interstitial fibrosis. At T2, glomerular and tubulointerstitial lesions were more severe, particularly glomerular loss, interstitial fibrosis, and inflammation. At T0, transforming growth factor ß, connective tissue growth factor, and platelet-derived growth factor α mRNA were overexpressed in XLHN dogs compared with controls. Clusterin and TIMP1 transcripts were upregulated in later stages of the disease. Transforming growth factor ß, connective tissue growth factor, and platelet-derived growth factor α should be considered as key players in the initial events of XHLN. Clusterin and TIMP1 appear to be more associated with the progression rather than initiation of tubulointerstitial damage in chronic renal disease.

Pathological features of proteinuric nephropathy resembling Alport syndrome in a young Pyrenean Mountain dog.

The renal biopsy tissue from a 9-month-old, male Pyrenean Mountain dog with renal disorder and severe proteinuria was examined. Ultrastructural examination revealed multilaminar splitting and fragmentation of the glomerular basement membrane (GBM) and diffuse podocyte foot process effacement. Immunofluorescent staining for α(IV) chains revealed presence of α5(IV) and complete absence of α3(IV) and α4(IV) chains in the GBM. Immunohistochemistry also revealed decreased and altered expression of nephrin and podocin in the glomeruli compared with normal canine glomeruli. These results suggested that the glomerular disease of the present case might be consistent with canine hereditary nephropathy resembling human Alport syndrome caused by genetic defect of type IV collagen, and indicated possible contribution of podocyte injury to severe proteinuria in this case.

A clinical study of canine collagen type III glomerulopathy.

BACKGROUND: Collagen type III glomerulopathy (Col3GP), also known as collagenofibrotic glomerulonephropathy, is a rare renal disease with unknown pathogenesis that occurs in animals and humans. We recently described a naturally occurring canine autosomal recessive model of Col3GP, and the aim of the present work was to study the clinical features of canine Col3GP and compare with the human phenotype. In humans two different clinical syndromes with different age at onset (child- or adulthood) have been observed. In children a more aggressive course with familial occurrence is described, characterized by progressively increasing proteinuria, nephrotic syndrome, hypertension and chronic renal failure. A markedly increased serum level of the aminoterminal propeptide of type III procollagen (PIIINP) is considered a useful marker for the disease. Since Col3GP and concurrent hypocomplementemia have been observed in humans, we also aimed to investigate if hypocomplementemia was present in Col3GP affected dogs. A litter consisting of seven puppies, four Col3GP affected and three healthy unaffected, was observed from the day of birth until the affected puppies developed a mild or moderate renal azotemia. RESULTS: During the period of observation growth retardation, increasing blood pressure, progressive proteinuria, azotemia, hypoalbuminemia, hypercholesterolemia and increased serum PIIINP were observed in all the affected dogs. Hypocomplementemia was not detected. Affected dogs were euthanized between 109 and 144 days of age, and pathological examinations revealed ascites and massive glomerular accumulations of collagen type III, consistent with Col3GP. CONCLUSIONS: Dogs with Col3GP develop juvenile chronic renal failure, preceded by nephrotic syndrome, elevated serum PIIINP and hypertension, thus have similar clinical features as the juvenile Col3GP in humans. Further studies of this naturally occurring canine phenotype may provide more information on the pathogenesis and genetics of Col3GP in both animals and humans.

Characterization of the genetic basis for autosomal recessive hereditary nephropathy in the English Springer Spaniel.

BACKGROUND: 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). OBJECTIVE: To identify and characterize the genetic cause of ARHN in ESS. ANIMALS: 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. METHODS: 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. RESULTS: 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.

Urinary biomarkers of renal disease in dogs with X-linked hereditary nephropathy.

BACKGROUND: Sensitive and specific biomarkers for early tubulointerstitial injury are lacking. HYPOTHESIS: The excretion of certain urinary proteins will correlate with the state of renal injury in dogs with chronic kidney disease. ANIMALS: Twenty-five male colony dogs affected with X-linked hereditary nephropathy (XLHN) and 19 unaffected male littermates were evaluated. METHODS: Retrospective analysis of urine samples collected every 2-4 weeks was performed. Urine proteins evaluated were retinol binding protein (uRBP/c), ß2-microglobulin (uB2M), N-acetyl-ß-D-glucosaminidase (uNAG/c), neutrophil gelatinase-associated lipocalin (uNGAL/c), and immunoglobulin G (uIgG/c). Results were correlated with serum creatinine concentration (sCr), glomerular filtration rate (GFR), urine protein : creatinine ratio, and histopathologic analysis of serial renal biopsies. Analytical validation was performed for all assays; uNAG stability was evaluated. RESULTS: All urinary biomarkers distinguished affected dogs from unaffected dogs early in their disease process, increasing during early and midstages of disease. uRBP/c correlated most strongly with conventional measures of disease severity, including increasing sCr (r = 0.89), decreasing GFR (r = -0.77), and interstitial fibrosis (r = 0.80), P < .001. However, multivariate analysis revealed age, sCr, uIgG/c, and uB2M, but not uRBP/c, as significant independent predictors of GFR (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: All urinary biomarkers were elevated before sCr increased, but typically after proteinuria developed in dogs with progressive glomerular disease because of XLHN. uRBP/c measurement might be promising as a noninvasive tool for diagnosis and monitoring of tubular injury and dysfunction in dogs.

Dysregulation of renal MMP-3 and MMP-7 in canine X-linked Alport syndrome.

Matrix metalloproteinases (MMPs) play an important regulatory role in many biological and pathological processes and their specific role in Alport syndrome (AS) is not yet clearly defined. In this study, the naturally occurring canine X-linked AS was used to demonstrate a potential role for MMP-3 and MMP-7 in Alport renal pathogenesis. Recently, we demonstrated that the expression of MMP-2, MMP-9 and MMP-14 was upregulated in the renal cortex of dogs with a spontaneous form of XLAS. In the present study, we examined necropsy samples of renal cortex from normal and XLAS dogs for MMP-3 and MMP-7 as they have the potential to activate MMP-2 and MMP-9. Immunohistochemical analysis showed strong immunostaining for both MMP-3 and MMP-7 in the interstitial space of XLAS kidneys, while virtually no immunostaining was observed in similar fields from normal dogs. RT-PCR and casein zymography confirmed that both mRNA transcripts and activities of MMP-3 and MMP-7 are elevated in XLAS kidneys. The induction of these MMPs likely contributes to tissue destruction associated with the fibrogenic process, while augmenting the activation of MMP-2 and MMP-9 by MMP-3 and MMP-7 in XLAS. Thus, these data further implicate a role for the MMPs in progressive renal pathogenesis associated with AS.

Genetic cause of X-linked Alport syndrome in a family of domestic dogs.

Alport syndrome is a hereditary disease of type IV (basement membrane) collagens that occurs spontaneously in humans and dogs. In the human, X-linked Alport syndrome (XLAS) is caused by mutations in COL4A5, resulting in absence of type IV collagen alpha5 chains from the glomerular basement membrane (GBM) of affected individuals. The consequence of this defect is progressive renal failure, for which the only available treatments are dialysis and transplantation. Recent studies support the prospect of gene transfer therapy for Alport syndrome, but further development of required technologies and demonstration of safety and efficacy must be accomplished in a suitable animal model. We previously identified and have propagated a family of mixed-breed dogs with an inherited nephropathy that exhibits the clinical, immunohistochemical, pathological, and ultrastructural features of human XLAS. To identify the causative mutation, COL4A5 cDNAs from normal and affected dogs were sequenced in their entirety. Sequence analyses revealed a 10-bp deletion in exon 9 of affected dogs. This deletion causes a frame-shift that results in a premature stop codon in exon 10. Characterization of the causative mutation was followed by development of an allele-specific test for identification of dogs in this kindred that are destined to develop XLAS.

Renal pathology of polycystic kidney disease and concurrent hereditary nephritis in Bull Terriers.

OBJECTIVE: To describe the renal lesions in Bull Terrier polycystic kidney disease (BTPKD), to confirm that the renal cysts in BTPKD arise from the nephron or collecting tubule, and to identify lesions consistent with concurrent BTPKD and Bull Terrier hereditary nephritis (BTHN). DESIGN: Renal tissue from five Bull Terriers with BTPKD and eight control dogs was examined by light and transmission electron microscopy. Clinical data were collected from all dogs, and family history of BTPKD and BTHN for all Bull Terriers. RESULTS: In BTPKD the renal cysts were lined by epithelial cells of nephron or collecting duct origin that were usually squamous or cuboidal, with few organelles. They had normal junctional complexes, and basal laminae of varying thicknesses. Glomeruli with small, atrophic tufts and dilated Bowman's capsules, tubular loss and dilation, and interstitial inflammation and fibrosis were common. Whereas the lesions seen in BTHN by light microscope were nonspecific, the presence of characteristic ultrastructural glomerular basement membrane (GMB) lesions and a family history of this disease indicated concurrent BTHN was likely in three of five cases of BTPKD. CONCLUSION: This paper provides evidence that renal cysts in BTPKD are of nephron or collecting duct origin. In addition, GBM lesions are described that strongly suggest that BTPKD and BTHN may occur simultaneously.

New form of X-linked dominant hereditary nephritis in dogs.

OBJECTIVE: To determine features of a new form of hereditary nephritis (HN) in dogs. ANIMALS: Parents and 16 first-generation offspring (8 males, 8 females). PROCEDURE: Adolescent dogs that developed renal failure were euthanatized and necropsied. Unaffected dogs were monitored until they were at least 2 years old. Studies included light and electron microscopy of kidneys obtained from affected and unaffected dogs and immunolabeling for collagen-IV chains in renal and epidermal basement membranes (BM). The nucleotide sequence of a portion of exon 35 of the COL4A5 gene was determined in genomic DNA isolated from affected and unaffected males. RESULTS: 7 of 8 male and 2 of 8 female offspring had proteinuria and juvenile-onset chronic renal failure, which progressed more rapidly in the males. Labeling for alpha3-alpha6(IV) chains was completely absent in renal BM of affected males and segmentally absent in affected females. Expression of alpha1-alpha2(IV) chains in glomerular BM (GBM) of affected dogs was increased. Labeling for alpha5-alpha6(IV) chains in epidermal BM was absent in affected males and segmental in affected females. Ultrastructural changes characteristic of HN were observed in GBM of affected dogs. The sequence of exon 35 of COL4A5 was normal in affected dogs. CONCLUSIONS: This renal disease is an example of X-linked dominant HN, with typical abnormalities of GBM ultrastructure and alpha(IV) chain expression. CLINICAL RELEVANCE AND IMPLICATIONS FOR HUMAN MEDICINE: Dogs with this naturally acquired progressive renal disease can be used to investigate the pathogenesis and treatment of similar disorders in human beings and dogs.

Glomerular ultrastructural findings similar to hereditary nephritis in 4 English cocker spaniels.

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 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.

Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the alpha 5 chain of collagen type IV.

Many families with X-chromosome linked hereditary nephritis (HN) have mutations in the gene on the X chromosome that codes for the alpha 5 chain of collagen type IV. Canine X-linked HN is an animal model for human X-linked HN. To study the alpha 5(IV) gene in this model, we used the nucleotide sequence published for the human alpha 5(IV) cDNA to construct sets of primers covering approximately 95% of the complete cDNA. cDNA from both affected and normal dog kidneys was amplified by PCR in nine overlapping regions. The nucleotide sequence encoding the noncollagenous domain NC1 hybridized to the human X chromosome and was 93% identical at the DNA level and 97% identical at the protein level to the human alpha 5(IV) NC1 domain, confirming that the canine alpha 5(IV) cDNA had been amplified. Sequence analysis of the alpha 5(IV) cDNA detected a single nucleotide substitution, G-->T, in affected dogs, changing a codon for a conserved glycine residue (GGA) to a stop codon (TGA). When genomic DNA was amplified, the same abnormality was found in exon 35. Using the canine NC1 domain cDNA as a probe for Northern analysis, two transcripts of approximately 8.6 kb and approximately 6.7 kb were identified in both normal and affected male dog kidney RNA. However, the abundance of both transcripts was decreased by a factor of approximately 10 in the affected dog. These results establish at the molecular level that canine X-linked HN is a model for human X-linked HN. This model provides an opportunity to determine the efficacy of new therapies and to investigate the role of the alpha 5(IV) chain in type IV collagen assembly.

Hereditary nephritis in the bull terrier: evidence for inheritance by an autosomal dominant gene.

A high prevalence of renal failure has been reported in bull terriers in Australia. The pattern of inheritance was analysed in a family of 33 bull terriers in which 10 dogs had renal disease manifested by proteinuria, ultrastructural abnormalities in the glomerular basement membrane, renal failure, or 'end stage' kidneys. The presence of at least one affected parent for each affected offspring, the approximately equal male/female ratio and the apparent absence of 'generation-skipping', strongly supported an autosomal dominant mode of inheritance, assuming a fully penetrant single major gene locus. Further evidence was not compatible with either an autosomal recessive or X-linked inheritance pattern. This contrasts with the X-linked inheritance shown in Alport's-type human hereditary nephritis and hereditary glomerulopathy in the samoyeds. Hereditary nephritis in the bull terrier should be a useful model for non-Alport's-type human hereditary nephritis, which is also reported to have an autosomal dominant inheritance pattern.

Abnormalities in the NC1 domain of collagen type IV in GBM in canine hereditary nephritis.

Samoyed hereditary glomerulopathy (SHG) in dogs serves as a model for human X-linked hereditary nephritis (HN). We previously showed that glomerular capillaries of affected males did not stain by immunofluorescence (IF) using serum from a patient with Goodpasture's syndrome. Our goal in the present study was to determine whether the NC1 domain of the collagen type IV molecule, which contains Goodpasture antigen (GPA), could be demonstrated in these dogs, and to assess its immunological reactivity. By SDS-PAGE, NC1 in collagenase digests of glomerular basement membranes (GBM) of unaffected and carrier female dogs in the family with SHG showed 24 kilodalton (kD), 26 kD and 28 kD monomer, and 46 kD and 47 kD dimer components, but the 24 kD monomer was diminished in the affected males. By IF, a rabbit antibody to NCl stained glomerular capillaries of unaffected, affected male, and carrier female dogs. In contrast, a human anti-GBM plasmapheresis fluid (PPF) stained glomerular capillaries of only the unaffected and carrier female dogs. By RIA, both antibodies reacted strongly with NCl in collagenase digests of GBM of the unaffected and carrier female dogs, but showed reduced reactivity with NCl of affected males. By Western blotting, both antibodies bound to dimers and 24 kD and 26 kD monomers of the NCl domain in collagenase digests of GBM of unaffected and carrier female dogs. However, in affected males, the rabbit anti-NCl antibody did not bind to the 24 kD monomer, while the human anti-GBM PPF showed weak binding to the 24 kD and 26 kD monomers.(ABSTRACT TRUNCATED AT 250 WORDS)