George E. Lees

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), β2-microglobulin (uB2M), N-acetyl-β-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.

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 α5 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.

The effect of progressive glomerular disease on megalin-mediated endocytosis in the kidney

BACKGROUND A well-characterized dog model of the X-linked collagen disease Alport syndrome (XLAS) was used to study the effect of progressive glomerular disease on megalin-mediated endocytosis. In XLAS, altered structure and function of the glomerular basement membrane induces a progressive proteinuric nephropathy. METHODS The investigation was performed in male XLAS dogs and age-matched normal male littermates. The urine profile and megalin-mediated endocytosis in the proximal tubule of six healthy and six XLAS dogs were examined at 2, 4, 6, 8 and 10 months of age using SDS-PAGE, immunoblotting and immunohistochemistry. RESULTS Gradually increasing urinary excretion of proteins over time and a reduced content of the same proteins in proximal tubule cells were found. Besides the glomerular component of the proteinuria, a significant tubular component was seen, which is due to a progressive change in the uptake of low-molecular-weight (LMW) ligands by megalin. Furthermore, the protein overload present in the lumen of the proximal tubule exceeds the reabsorption capacity of megalin and the co-receptor cubilin and results in a combined low- and high-molecular-weight (HMW) proteinuria. Also, a shift in the distribution of lysosomes was seen in the XLAS dogs suggesting changes in the lysosomal degradation pattern in response to the altered endocytosis. CONCLUSIONS The present study shows that the increased glomerular permeability and the subsequently altered megalin-mediated and megalin-dependent cubilin-mediated endocytosis lead to a partial LMW proteinuria and partial HMW proteinuria.

Symmetric Dimethylarginine Assay Validation, Stability, and Evaluation as a Marker for the Early Detection of Chronic Kidney Disease in Dogs

Background Symmetric dimethylarginine (SDMA) is a small molecule formed by methylation of arginine, and released into blood during protein degradation. SDMA is primarily eliminated by renal excretion and is a promising endogenous marker of glomerular filtration rate (GFR). Objectives To validate an assay for SDMA measurement, determine stability of SDMA in blood, and compare SDMA with serum creatinine concentration (sCr) and GFR for early detection of decreasing kidney function in dogs with chronic kidney disease (CKD). Animals Eight male dogs affected with X‐linked hereditary nephropathy and 4 unaffected male littermates. Methods Prospective study validating SDMA measurement using liquid chromatography‐mass spectrometry, assessing stability of SDMA in serum and plasma, and serially determining sCr, SDMA, and GFR (using iohexol clearance) in dogs during progression from preclinical disease to end‐stage renal failure. Correlations were determined using linear regression. Timepoints at which sCr, SDMA, and GFR identified decreased renal function were compared using defined cutoffs, trending in an individual dog, and comparison with unaffected littermates. Results Symmetric dimethylarginine was highly stable in serum and plasma, and the assay demonstrated excellent analytical performance. In unaffected dogs, SDMA remained unchanged whereas in affected dogs, SDMA increased during disease progression, correlating strongly with an increase in sCr (r = 0.95) and decrease in GFR (r = −0.95). Although trending improved sCrs sensitivity, SDMA identified, on average, <20% decrease in GFR, which was earlier than sCr using any comparison method. Conclusions and Clinical Importance Symmetric dimethylarginine is useful for both early identification and monitoring of decreased renal function in dogs with CKD.

Stem cell therapy for Alport syndrome: the hope beyond the hype

Alport syndrome is a hereditary glomerulopathy leading to end-stage renal disease (ESRD), frequently during adolescence. It is caused by the absence or abnormal composition of the type IV collagen α3/4/5 network normally present in the glomerular basement membrane (GBM) [1]. In the September 2008 issue of the Journal of the American Society of Nephrology, Katayama and colleagues reported that bone marrow transplantation (BMT) following lethal irradiation with either wild-type or Col4a3−/− BM prolonged the lifespan of Alport mice with similar efficiencies. Sublethal irradiation alone also provided significant benefits [2].

Day‐to‐Day Variation of the Urine Protein: Creatinine Ratio in Female Dogs with Stable Glomerular Proteinuria Caused by X‐Linked Hereditary Nephropathy

BACKGROUND Interpretation of serial urine protein:creatinine (UPC) values is confounded by a lack of data regarding random biologic variation of UPC values in dogs with stable glomerular proteinuria. HYPOTHESIS That there is minimal day-to-day variability in the UPC of dogs with unchanging proteinuria and the number of measurements needed to reliably estimate UPC varies with the magnitude of proteinuria. ANIMALS Forty-eight heterozygous (carrier) female dogs with X-linked hereditary nephropathy (XLHN) causing stable proteinuria. METHODS Urine samples were obtained daily by cystocentesis for 3 consecutive days on 183 occasions (549 samples). The UPC was measured for each sample with a single dry-film chemistry auto-analyzer. Data were analyzed retrospectively by a power of the mean model because the variance of UPC values within the 3-day evaluation periods increased as the magnitude of proteinuria increased. RESULTS To demonstrate a significant difference (P < .05) between serial values in these proteinuric dogs, the UPC must change by at least 35% at high UPC values (near 12) and 80% at low UPC values (near 0.5). One measurement is adequate to reliably estimate the UPC when UPC <4, but 2-5 determinations are necessary at higher UPC values. CONCLUSIONS AND CLINICAL IMPORTANCE These guidelines for interpretation of serial UPC values in female dogs with XLHN may also be helpful for interpretation of UPC values in dogs with other glomerulopathies.

Proteomic analysis of urine from male dogs during early stages of tubulointerstitial injury in a canine model of progressive glomerular disease

BACKGROUND Sensitive and specific noninvasive biomarkers for tubulointerstitial injury are lacking, and proteomic techniques provide a powerful tool for biomarker discovery. OBJECTIVE The aim of this study was to identify novel urinary biomarkers of early tubulointerstitial injury in canine progressive renal disease using both 2-dimensional differential in-gel electrophoresis (2-D DIGE), which identifies individual proteins, and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF), which generates protein peak profiles. METHODS Urine was collected from 6 male dogs with X-linked hereditary nephropathy (XLHN) at 2 time points (TP): 1) the onset of overt proteinuria (urine protein:creatinine ratio>2) and 2) the onset of azotemia (creatinine ≥ 1.2 mg/dL); corresponding renal biopsies were analyzed from 3 of the dogs. Urine samples from the 6 dogs were subjected to analysis by 2-D DIGE and SELDI-TOF. Urinary retinol-binding protein (RBP) was evaluated in 25 male dogs with XLHN and normal control dogs by Western blot analysis. RESULTS Clinical data and histologic evaluation revealed reduced renal function and increased tubulointerstitial fibrosis at TP 2. A number of urine proteins and protein peaks were differentially present at the 2 time points, with several known biomarkers of renal disease identified in addition to several promising new biomarkers. RBP was first detected in urine approximately 2 months before onset of azotemia (TP 2), but after onset of overt proteinuria, and amounts increased with progression of disease. CONCLUSIONS Proteomic techniques were successfully used to identify urinary biomarkers of renal disease in dogs with XLHN. Urinary RBP is a promising biomarker for early detection of tubulointerstitial damage and progression to end-stage renal disease.

Early diagnosis of familial nephropathy in English cocker spaniels

Two litters of English cocker spaniels (ECSs) produced by familial nephropathy (FN) carriers were evaluated to characterize the early features of this disease. Three puppies developed FN. Proteinuria, which began when these puppies were five-to-eight months old, was the first abnormality detected. Proteinuria persisted while each puppys growth rate slowed, and renal function gradually deteriorated. The interval from onset of proteinuria to development of azotemia was two-to-nine months. Characteristic glomerular capillary basement membrane (GCBM) lesions were seen with transmission electron microscopy (TEM) of renal biopsy specimens obtained during this interval. Ultrastructural GCBM lesions progressed substantially during the interval from biopsy to necropsy. However, routine light microscopic findings did not allow definitive diagnosis of FN in either biopsy or necropsy specimens. Detection of FN can be accomplished by screening at-risk ECSs for proteinuria. Renal biopsies are required to confirm the diagnosis in dogs for which proteinuria cannot be explained otherwise. Percutaneous needle biopsy specimens sufficient for TEM must be used to examine the GCBM to make a definitive diagnosis.

X-inactivation modifies disease severity in female carriers of murine X-linked Alport syndrome

BACKGROUND Female carriers of X-linked Alport syndrome (XLAS) demonstrate variability in clinical phenotype that, unlike males, cannot be correlated with genotype. X-inactivation, the method by which females (XX) silence transcription from one X chromosome in order to achieve gene dosage parity with males (XY), likely modifies the carrier phenotype, but this hypothesis has not been tested directly. METHODS Using a genetically defined mouse model of XLAS, we generated two groups of Alport female (Col4a5(+/-)) carriers that differed only in the X-controlling element (Xce) allele regulating X-inactivation. We followed the groups as far as 6 months of age comparing survival and surrogate outcome measures of urine protein and plasma urea nitrogen. RESULTS Preferential inactivation of the mutant Col4a5 gene improved survival and surrogate outcome measures of urine protein and plasma urea nitrogen. In studies of surviving mice, we found that X-inactivation in kidney, measured by allele-specific mRNA expression assays, correlated with surrogate outcomes. CONCLUSIONS Our findings establish X-inactivation as a major modifier of the carrier phenotype in X-linked Alport syndrome. Thus, X-inactivation patterns may offer prognostic information and point to possible treatment strategies for symptomatic carriers.

Genetic cause of autosomal recessive hereditary nephropathy in the english cocker spaniel

BACKGROUND Autosomal recessive hereditary nephropathy (ARHN) in the English Cocker Spaniel is caused by a type IV collagen defect, but the underlying mutation is unknown. ANIMALS One hundred thirty-four English Cocker Spaniels (12 with ARHN, 8 obligate carriers, and 114 others), 3 mixed breed dogs with X-linked hereditary nephropathy (XLHN), and 7 other dogs without hereditary nephropathy were included. METHODS Diagnosis of ARHN was based on transmission electron microscopy and immunostaining of kidney. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to compare COL4A3, COL4A4, and COL4A5 mRNA concentrations in the renal cortex from ARHN-affected English Cocker Spaniels, XLHN-affected dogs, and dogs without hereditary nephropathy. The entire coding region of COL4A4 was sequenced in 2 ARHN-affected dogs, 2 obligate carriers, 2 English Cocker Spaniels of unknown status, and 2 healthy mixed breed dogs. The exon containing the mutation was sequenced for all 134 English Cocker Spaniels. RESULTS Quantitative real time RT-PCR implicated COL4A4 as the gene harboring the mutation, and sequencing identified a single nucleotide substitution at base 115 as the cause of ARHN in English Cocker Spaniels. This mutation, which causes a premature stop codon in exon 3 of COL4A4, was segregated with clinical status in all affected dogs and obligate carriers. The mutation also was identified in 39 of 114 other English Cocker Spaniels with previously unknown status. CONCLUSIONS AND CLINICAL IMPORTANCE The cause of this disease has been identified, and use of a test for the mutation will permit eradication of ARHN in the English Cocker Spaniel.