Protein profiling of urine from dogs with renal disease using ProteinChip analysis.

Measurement of total urinary proteins in individuals that tested positive by urinary dipstick is a typical method for assessing the presence of potentially serious renal disorders. In the absence of such overt proteinuria, however, measurement of specific urinary proteins may be useful in the diagnosis of nephropathies and may provide greater insight into the pathogenesis. The urine of 28 dogs (16 with renal disease and 12 healthy) was evaluated to determine whether specific low-molecular-weight proteins or the pattern of protein excretion could also be used as a marker of tubular dysfunction in dogs. Specific proteins were assessed by immunological methods, whereas protein profiles were determined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (MS). In particular, changes in the excretion of retinol-binding protein (RBP) and Tamm-Horsfall protein (THP) appear to be of clinical relevance in the diagnosis of canine kidney diseases. The pattern of urinary protein and peptides revealed specific changes in abundance in dogs with renal disease at molecular masses (kD) of 11.58, 12.41, 12.60, 14.58, 20.95 (RBP), 27.85, and 65.69 (albumin). In conclusion, comparable proteins as in humans might be used as urinary markers for proximal (RBP) and distal (THP) tubular dysfunction in dogs. Surface-enhanced laser desorption/ionization time-of-flight MS is a promising tool for the study of kidney physiology and pathophysiology and might aid in the discovery of new biomarkers of renal disease.     

Effects of chronic renal disease on the transport of vitamin A in plasma and urine of dogs

OBJECTIVE: To determine plasma and urine concentrations of retinol, retinyl esters, retinol-binding protein (RBP), and Tamm-Horsfall protein (THP) in dogs with chronic renal disease (CRD). ANIMALS: 17 dogs with naturally developing CRD and 21 healthy control dogs. PROCEDURE: A diagnosis of CRD was established on the basis of clinical signs, plasma concentrations of creatinine and urea, and results of urinalysis. Concentrations of retinol and retinyl esters were measured by use of reverse-phase high-performance liquid chromatography. Concentrations of RBP and THP were measured by use of sensitive ELISA systems. RESULTS: Dogs with CRD had higher plasma concentrations of retinol, which were not paralleled by differences in plasma concentrations of RBP. Calculated ratio of urinary total vitamin A (sum of concentrations of retinol and retinyl esters to creatinine concentration) and ratio of the concentration of urinary retinyl esters to creatinine concentration did not differ between groups. However, we detected a significantly higher retinol-to-creatinine ratio in the urine of dogs with CRD, which was paralleled by a higher urinary RBP-to-creatinine ratio. Thus, in dogs with CRD, the estimated fractional clearance of total vitamin A, retinol, and RBP was increased. Furthermore, dogs with CRD had a reduced urinary THP-to-creatinine ratio. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study documented that CRD affects the concentrations of retinol in plasma and urine of dogs. Analysis of the data indicates that measurement of urinary RBP and urinary THP concentrations provides valuable information that can be helpful in follow-up monitoring of dogs with CRD.     

Immunochemical Localization of Megalin,Retinol-binding protein and Tamm–Horsfall Glycoprotein in the Kidneys of Dogs

Megalin, retinol-binding protein (RBP) and Tamm–Horsfall glycoprotein (THP) are involved in the renal metabolism of vitamin A in canine species. The presence of megalin, RBP and THP in the kidneys of dogs was investigated using immunohistochemical methods. Megalin was highly expressed in the apical membrane of the proximal convoluted and straight tubule cells. Immunoreactive RBP was detected below the apical plasma membrane, as well as in basolateral granules of the proximal convoluted tubule cells. THP immunoreactivity was seen in the epithelial cells lining the thick ascending limb of the loop of Henle. Furthermore, THP was displayed in a scattered pattern within the distal convoluted tubules. The co-localization of megalin and RBP coincides with biochemical studies that have shown megalin to be responsible for renal RBP absorption in the proximal convoluted tubules after filtration through the renal glomerulus. The presence of THP, the carrier for vitamin A in canine urine, showed that vitamin A excretion in the urine of dogs is not merely a filtration process but also seems to be a pathway located in the distal part of the nephron.     

Levels of retinol and retinyl esters in plasma and urine of dogs with urolithiasis

Vitamin A (VA) deficiency and Tamm-Horsfall glycoprotein (THP), a protein that binds retinol and retinyl esters in canine urine, might be involved in the pathogenesis of urolithiasis in dogs. In the present study, we assessed levels of retinol, retinyl esters, retinol-binding protein (RBP) and THP in plasma and urine of dogs with a history of urolithiasis (n = 25) compared with clinically healthy controls (n = 18). Plasma retinol concentrations were higher in dogs with uroliths of struvit (P < 0.01), calcium oxalate (P < 0.05), urate (P < 0.01) and cysteine, but there were no differences in the concentrations of plasma RBP and retinyl esters. Excretion of urinary retinol and retinyl esters were tentatively, but not significantly higher in the stone-forming groups, which was accompanied by increased levels of urinary RBP (P < 0.01) and lower excretions in THP (P < 0.01). The results show that VA deficiency may be excluded as a potential cause for canine urolithiasis. However, the occurrence of RBP and a concomitant reduction of THP in urine indicates a disturbed kidney function as cause or consequence of stone formation in dogs.     

Obstructive protein cast nephropathy in cynomolgus monkeys treated with small organic molecules

We have observed a renal toxicity consistent with an obstructive protein cast nephropathy in cynomolgus macaques but not in other species treated with different therapeutic candidates having a common carboxylic acid moiety, suggesting a species-specific sensitivity. Here, we present renal toxicity findings consistent with a protein cast nephropathy in a 2-week safety study in cynomolgus monkeys. Light microscopic changes consisted of intratubular cast formation, tubular dilatation, interstitial inflammation, and expansion of the medullary interstitium. Tubular cast material was identified as Tamm-Horsfall protein (THP) and, on ultrastructure, crystalloid material was present in vacuoles of tubular epithelium. It is hypothesized that microcrystal formation in the urinary tubular spaces induces aggregation of THP protein and cast formation in monkeys. Drug-induced obstructive nephropathy is not identified as a major problem in humans; thus, the clinical relevance of the above findings in monkeys is not clear.     

Retinol-Binding Protein in Serum and Urine of Hyperthyroid Cats before and after Treatment with Radioiodine

Background: Retinol-binding protein (RBP) is suggested as a clinically useful marker of renal function in cats.
Hypothesis: Serum and urinary RBP concentrations in hyperthyroid (HT) cats differ from those in healthy (H) cats; radioiodine (¹³¹I) treatment influences serum and urinary RBP concentrations in HT cats.
Animals: Ten HT and 8 H cats.
Methods: RBP concentration was evaluated in feline serum and urine samples from a prospective study.
Results: There was a significant ( P = .003) difference in the urinary RBP/creatinine (uRBP/c) ratios of H (−) and untreated HT (1.4 ± 1.5 × 10⁻² μg/mg) cats. Serum total thyroxine concentration (1.8 ± 1.9 μg/dL, 24 weeks) and uRBP/c (0.6 ± 1.0 × 10⁻² μg/mg, 24 weeks) decreased significantly ( P < .001) in HT cats at all time points after treatment with ¹³¹I, and these variables were significantly correlated with one another ( r = 0.42, P = .007). Serum RBP concentrations from HT cats (199 ± 86 μg/L) did not differ significantly ( P = .98) from those of H cats (174 ± 60) and did not change after treatment with ¹³¹I (182 ± 124 μg/L, P = .80).
Conclusion and Clinical Importance: The presence of urinary RBP in HT cats is a potential marker of tubular dysfunction that is correlated to thyroid status, although it is independent of circulating RBP concentrations. The decreased uRBP/c combined with the absence of changes in serum RBP after treatment suggests that the suspected tubular dysfunction was partly reversible with treatment of ¹³¹I.     

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.     

Obstructive struvite ureterolithiasis in 4-month-old intact male Bernese mountain dog

A 4-month-old, 7 kg, intact male, Bernese mountain dog was presented for obstructive struvite ureterolithiasis. Multiple urethroliths, ureteroliths, and urocystoliths were present. Based on an abdominal ultrasound, there was severe left hydronephrosis and hydroureter from distal ureterolith obstruction, just proximal to the vesicoureteral junction. The dog was not azotemic. Successful treatment was accomplished via ventral cystotomy. Bladder wall culture revealed a methicillin-resistant Staphylococcus spp. No predisposing cause was identified. There are no known genetic predispositions in Bernese mountain dogs for struvite urolithiasis. The urinary tract infection resolved with surgical retrieval of the uroliths and antibiotic treatment. The dog remained clinically normal after the cystotomy but developed a subclinical urinary tract infection 4 mo post-operatively.Key clinical message:Urolithiasis is rare in pediatric veterinary patients. To the authors’ knowledge, this is the first report of obstructive ureterolithiasis in a puppy. There is no known genetic predisposition for urolithiasis in Bernese mountain dogs.     

Physiologic and pathophysiologic fundamentals of proteinuria--a review

The term proteinuria is taken to mean abnormally high protein excretion in the urine. Proteinuria is the consequence of glomerular filtration of plasma proteins, their subsequent reabsorption by the proximal tubular cells and secretion of protein by the tubular cells and distal urinary tract. In physiological conditions, the structural integry of the glomerular filtration barrier prevents the abnormal passage of albumin (molecular mass 66 kDa) and high-molecular-weight proteins (> 66 kDa), whereas the passage of low-molecular-weight proteins (< 66 kDa) is almost completely unrestricted. Proteins that arrive the tubular lumen are reabsorbed by endocytosis after binding to the megalin-cubilin complex. An increased load of proteins in the tubular lumen leads to the saturation of the reabsorptive mechanism and higher urinary protein excretion. Proteinuria can originate from prerenal, renal and postrenal causes. Elevated tubular protein concentrations have been recognized to be toxic to tubular cells and associated with the progression of chronic renal disease. Therefore, the quantitative and qualitative evaluation of proteinuria is important for the diagnosis of renal disease.     

Proteinuria in the dog: a clinicopathological study in 51 proteinuric dogs

In 51 dogs with predominantly massive urinary protein loss, the daily loss was quantified and glomerular and tubulointerstitial lesions from renal biopsies were characterised and graded using histology, immune fluorescence and electron microscopy. The highest median daily urinary protein loss occurred in dogs with membranous glomerulonephritis (median 380 mg kg-1 d-1) and renal amyloidosis (median 257 mg kg-1 d-1). Although in nine febrile dogs the urinary protein loss was transient, both glomerular and tubular lesions were diagnosed in five and seven of these dogs, respectively. The pattern of urinary proteins was determined using sodium dodecyl-sulphate polyacrylamide gel electrophoresis. The albumin fractional clearance (FC) was raised in 46 dogs, whereas the FCS of the low molecular weight (MW) protein fraction (MW less than 66,000) and high molecular weight protein fraction (MW more than 66,000) were raised in 42 and 28 dogs, respectively. Both the high molecular weight protein FC and albumin FC significantly correlated to the grade of glomerular lesions, whereas the low molecular weight protein FC only moderately significantly correlated to the grade of tubular lesions. The selectivity index, calculated as (formula; see text) did not differentiate between the various forms of glomerulopathies. The urinary lysozyme concentration was significantly correlated to the grade of tubular lesions. It is concluded that although quantitative and qualitative measurements of urinary proteins can provide additional clinical information, they do not have a reliable predictive value and histopathological examination of renal tissue is still necessary for the final diagnosis.     

Cervical vertebral malformation-malarticulation syndrome in the Bernese mountain dog: clinical and magnetic resonance imaging features

O bjectives : To describe the clinical and magnetic resonance imaging features of cervical vertebral malformation-malarticulation in Bernese mountain dogs.
M ethods : Seven Bernese mountain dogs (four males and three females) were diagnosed with cervical vertebral malformation-malarticulation by magnetic resonance imaging. The following data were evaluated retrospectively: (1) abnormalities of the cervical vertebral column and spinal cord, (2) spinal cord compression, (3) intervertebral disc degeneration and herniation, (4) severity of clinical signs pretreatment and after treatment, (5) type of treatment and (6) outcome.
R esults : Spin echo T1-weighted and T2-weighted images disclosed multi-level, extradural compressive spinal cord lesions (ventral, dorsolateral or both) spanning from intervertebral disc spaces C3-4 to C6-7. In all seven dogs, T2-weighted images disclosed one or more intramedullary hyperintensities associated with extradural spinal cord compression. Surgery was performed in five dogs. Two dogs were managed medically. The prognosis for surgical or conservative management in Bernese mountain dogs was similar to cervical vertebral malformation-malarticulation in other breeds.
C linical S ignificance : Cervical vertebral malformation-malarticulation is an important differential diagnosis for young to middle-aged Bernese mountain dogs with a C1-5 or C6-T2 neuroanatomic localisation. Dorsolateral spinal cord compression associated with articular process hypertrophy was the most common feature of cervical vertebral malformation-malarticulation in the seven Bernese mountain dogs evaluated.     

Multiorgan dysfunction syndrome secondary to joint supplement overdosage in a dog

A 5-year-old spayed female Bernese mountain dog, with a chief complaint of vomiting and melena ingested approximately 200 nutritional joint supplement tablets. Despite aggressive therapy, the patient developed a coagulopathy, pancreatitis, peritonitis, acute kidney injury, and was euthanized. Postmortem examination revealed myocardial necrosis, pneumonia, centrilobular hemorrhage and necrosis of the liver, vasculitis, and acute tubular necrosis.     

Renal biomarkers in domestic species

Current conventional tests of kidney damage and function in blood (serum creatinine and urea nitrogen) and urine (urine protein creatinine ratio and urine specific gravity) are widely used for diagnosis and monitoring of kidney disease. However, they all have important limitations, and additional markers of glomerular filtration rate and glomerular and tubular damage are desirable, particularly for earlier detection of renal disease when therapy is most effective. Additionally, urinary markers of kidney damage and function may help localize damage to the affected portion of the kidney. In general, the presence of high‐ and intermediate‐molecular weight proteins in the urine are indicative of glomerular damage, while low‐molecular weight proteins and enzymes in the urine suggest tubular damage due to decreased reabsorption of proteins, direct tubular damage, or both. This review aims to discuss many of these new blood and urinary biomarkers in domestic veterinary species, focusing primarily on dogs and cats, how they may be used for diagnosis of renal disease, and their limitations. Additionally, a brief discussion of serum creatinine is presented, highlighting its limitations and important considerations for its improved interpretation in domestic species based on past literature and recent studies.     

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis of Canine Urinary Proteins for the Analysis and Differentiation of Tubular and Glomerular Diseases

A preliminary investigation was performed to evaluate the use of a new, noninvasive technique for the localization of canine renal lesions by electrophoresis of urinary proteins. Urine specimens from six clinically healthy, nonproteinuric dogs and 12 dogs with persistent proteinuria were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Urine electrophoretic patterns of proteinuric dogs were classified as glomerular (n=4), tubular (n=2), or mixed (glomerular and tubular) (n=6), based on the number and molecular weight of the silver-stained protein bands. Renal tissues from biopsies or necropsies were obtained from eight of the dogs with proteinuric disease. Interpretation of seven of eight electrophoretograms agreed with the histologic interpretation of renal lesions. We concluded SDS PAGE is a potentially valuable technique for detection and localization of renal lesions in dogs with proteinuric disease.     

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of canine urinary proteins for the analysis and differentiation of tubular and glomerular diseases

A preliminary investigation was performed to evaluate the use of a new, noninvasive technique for the localization of canine renal lesions by electrophoresis of urinary proteins. Urine specimens from six clinically healthy, nonproteinuric dogs and 12 dogs with persistent proteinuria were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Urine electrophoretic patterns of proteinuric dogs were classified as glomerular (n = 4), tubular (n = 2), or mixed (glomerular and tubular) (n = 6), based on the number and molecular weight of the silver-stained protein bands. Renal tissues from biopsies or necropsies were obtained from eight of the dogs with proteinuric disease. Interpretation of seven of eight electrophoretograms agreed with the histologic interpretation of renal lesions. We concluded SDS PAGE is a potentially valuable technique for detection and localization of renal lesions in dogs with proteinuric disease.     

Proteinuria in dogs and cats

Proteinuria is defined as the presence of protein in the urine. Normally, circulating serum proteins are blocked by the glomerulus due to size and/or charge. Any small proteins that pass through a healthy glomerulus are reabsorbed by the renal tubules or broken down by renal tubular epithelial cells. Persistent proteinuria, in the absence of lower urinary tract disease or reproductive tract disease, is usually an indication of renal damage or dysfunction. Less commonly persistent proteinuria can be caused by increased circulating levels of low molecular weight proteins. This article reviews mechanisms of proteinuria in dogs and cats and discusses the importance of screening for and ultimately treating proteinuria.     

Diagnostic relevance of qualitative proteinuria evaluated by use of sodium dodecyl sulfate-agarose gel electrophoresis and comparison with renal histologic findings in dogs

OBJECTIVE: To evaluate results of SDS-agarose gel electrophoresis (AGE) of urinary proteins for use in defining glomerular and tubulointerstitial derangements, investigate patterns of high-molecular-weight (HMW) proteins for differentiating among glomerular disorders, and assess low-molecular-weight (LMW) proteins as markers of severity of tubulointerstitial disease in dogs. ANIMALS: 49 dogs with increased serum creatinine concentrations or abnormal renal protein loss. PROCEDURE: Urinary proteins were examined by use of SDS-AGE and differentiated on the basis of molecular weight. The HMW proteins (> or = 69 kd) were considered indicative of glomerular origin, whereas LMW proteins (< 69 kd) were of tubular origin. Renal specimens were examined by use of light microscopy. Glomerular and tubulointerstitial lesions were differentiated by use of the classification for the World Health Organization and semiquantitative grading, respectively. RESULTS: Sensitivity of SDS-AGE was 100% for detection of glomerular lesions and 92.6% for tubulointerstitial lesions; specificity was 40% and 62.5%, respectively. Although HMW urinary proteins were not significantly associated with the type of glomerular lesion, LMW urinary proteins were significantly associated with the grade of tubulointerstitial damage. Detection of 12- or 15-kd proteins or both was highly indicative of a severe tubulointerstitial lesion. CONCLUSIONS AND CLINICAL RELEVANCE: SDS-AGE of urinary proteins in dogs represents a noninvasive test with high sensitivity for identifying glomerular and tubulointerstitial damage, but low specificity limits its validity as a stand-alone test to differentiate between glomerular and tubulointerstitial lesions. The test is particularly useful for identifying dogs with advanced tubulointerstitial disease but cannot be used to characterize glomerular disorders.     

Urinary gamma-glutamyl transferase and the degree of renal dysfunction in 75 bitches with pyometra

In 75 bitches with pyometra single urine samples were examined for gamma-glutamyl transferase (gamma-GT), protein, glucose, specific gravity, bacteria, red blood cells and white blood cells. Serum samples were examined for urea, creatinine, inorganic phosphate and gamma-GT. Biochemical findings were compared with the degree of illness (clinical signs). Twenty one bitches had no signs of renal disease. Seventeen showed only glomerular damage indicated by proteinuria without signs of proximal tubular damage. Thirty seven bitches had increased urinary gamma-GT levels, indicating proximal tubular lesions, which were associated with proteinuria in 35 and renal failure in 16 of them, and worse clinical findings. In all bitches with pyometra serum levels of gamma-GT were comparable to values in control bitches. Glomerular dysfunction seemed to precede proximal tubular lesions, so that gamma-GT-uria in bitches with pyometra was not an early but rather a late indication of a more profound degree of renal dysfunction, that is, proximal tubular renal damage developed after glomerular dysfunction and preceding renal failure.     

Proteinuria as an indicator of early renal disease in bull terriers with hereditary nephritis

A group of non-azotaemic bull terriers from families with hereditary nephritis had significant subclinical renal disease. Of the renal function tests carried out, proteinuria, almost exclusively albumin, was a reliable and early indicator of glomerular abnormality. While 24-hour urinary protein excretion (24 UPE) in the bull terriers was significantly higher than in a group of normal dogs of other breeds, urinary protein excretion (UPE) was also significantly higher when measured by simple single sample tests such as the urinary protein to urinary creatinine ratio (UP/C). UPE in bull terriers was correlated with increasing age, in contrast to lack of correlation in the normal dogs. The degree of proteinuria in affected bull terriers from which renal biopsies were taken correlated with the severity of histopathological changes which mainly affected glomeruli. Light microscopic examination revealed segmentally thickened glomerular and tubular basement membranes, thickened Bowman's capsules and adhesions between glomerular capillaries and Bowman's capsules. Other renal function tests were performed but failed to detect subclinical disease. It is suggested that hereditary nephritis (HN) in bull terriers is similar to that seen in samoyeds, dober-mann pinschers and humans.     

Comparison of two classification protocols in the evaluation of elbow dysplasia in the dog

The incidence of elbow disease based solely on arthrosis (ARTH) score was compared with a protocol using a combination of ARTH score plus a score for primary lesions (ED score). The population of dogs studied included 425 Bernese mountain dogs and 22 dogs of other breeds. The overall agreement between the two systems was high. However, 12 per cent of ARTH-score negative cases were positive using the ED score. This difference was statistically significant (P<0·005). The female:male ratio of the dogs missed using the ARTH score was 2:1. The proportion of dogs affected with arthrosis increased with age, male dogs being affected more frequently. The development of arthrosis depends not only on age and breed, but probably also on gender. Thus, screening for elbow dysplasia should be based on at least two radiographic projections, including arthrosis and primary lesions. As the vast majority of dogs in this study were Bernese mountain dogs, conclusions are valid only for this breed.