Mary B. Nabity

ASVCP guidelines: allowable total error guidelines for biochemistry

As all laboratory equipment ages and contains components that may degrade with time, initial and periodically scheduled performance assessment is required to verify accurate and precise results over the life of the instrument. As veterinary patients may present to general practitioners and then to referral hospitals (both of which may each perform in-clinic laboratory analyses using different instruments), and given that general practitioners may send samples to reference laboratories, there is a need for comparability of results across instruments and methods. Allowable total error (TEa ) is a simple comparative quality concept used to define acceptable analytical performance. These guidelines are recommendations for determination and interpretation of TEa for commonly measured biochemical analytes in cats, dogs, and horses for equipment commonly used in veterinary diagnostic medicine. TEa values recommended herein are aimed at all veterinary settings, both private in-clinic laboratories using point-of-care analyzers and larger reference laboratories using more complex equipment. They represent the largest TEa possible without generating laboratory variation that would impact clinical decision making. TEa can be used for (1) assessment of an individual instruments analytical performance, which is of benefit if one uses this information during instrument selection or assessment of in-clinic instrument performance, (2) Quality Control validation, and (3) as a measure of agreement or comparability of results from different laboratories (eg, between the in-clinic analyzer and the reference laboratory). These guidelines define a straightforward approach to assessment of instrument analytical performance.

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.

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.

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.

Safety of Masitinib Mesylate in Healthy Cats

BACKGROUND Masitinib mesylate is a PO-administered tyrosine kinase inhibitor developed both for human and animal diseases with activity against both mutated and wild type forms of the c-kit receptor and platelet-derived growth factor receptors α and β, and is currently registered in Europe for the treatment of mast cell tumors in dogs. HYPOTHESIS/OBJECTIVES The objective of this study was to determine if healthy cats can tolerate administration of masitinib without clinically relevant adverse effects. ANIMALS Twenty healthy research colony-specific pathogen-free cats. METHODS This study was a prospective, randomized phase 1 clinical trial. Masitinib was administered PO to 20 healthy cats. Ten cats received 50 mg masitinib every other day for 4 weeks, and 10 cats received 50 mg masitinib daily for 4 weeks. RESULTS Clinically relevant proteinuria was noted in 2/20 (10%) cats (both treated daily), and neutropenia was noted in 3/20 (15%) (seen in both treatment groups). An increase in serum creatinine concentration and adverse gastrointestinal effects were noted in some cats. CONCLUSIONS AND CLINICAL IMPORTANCE Masitinib mesylate was tolerated in the majority of cats. Long-term administration and pharmacokinetic studies are needed to further assess the use of masitinib in cats.

Prevalence of Immune-Complex Glomerulonephritides in Dogs Biopsied for Suspected Glomerular Disease: 501 Cases (2007-2012)

BACKGROUND Glomerulonephropathies are common causes of kidney disease in dogs. OBJECTIVE To determine the prevalence of immune-complex glomerulonephritis (ICGN) in North American dogs biopsied for suspected glomerular disease. ANIMALS Renal biopsies (n = 733) submitted to the Texas Veterinary Renal Pathology Service between January 1, 2007 and December 31, 2012 were reviewed. Dogs were included if the biopsy was performed for suspected glomerular disease. METHODS Specimens were evaluated by light microscopy (LM), immunofluorescence (IF), and transmission electron microscopy (TEM). Findings were retrospectively evaluated to categorize the diagnosis for each case. For the diagnosis of ICGN, TEM findings were considered conclusive when LM and IF were equivocal. RESULTS Of the 501 dogs included in the study, 241 (48.1%) had ICGN; 103 (20.6%) had primary glomerulosclerosis; 76 (15.2%) had amyloidosis; 45 (9.0%) had nonimmune complex (IC) glomerulopathy; 24 (4.8%) had non-IC nephropathy; and, 12 (2.4%) had primary tubulointerstitial disease. Many (66/241; 27.4%) ICGN cases required TEM for definitive diagnosis, including 14 cases (5.8%) that were not suspected on LM. Of cases not diagnosed as ICGN, a substantial proportion (60/260; 23.1%) required TEM to rule out immune complex deposits, including 14 of 189 cases (7.4%) presumptively diagnosed as ICGN on LM. CONCLUSIONS AND CLINICAL IMPORTANCE Approximately half of all dogs biopsied for suspected glomerular disease had conditions other than ICGN. Renal biopsy is needed to accurately categorize the underlying disease and direct appropriate treatment. Additionally, TEM and IF evaluations by experienced nephropathologists are necessary to obtain an accurate diagnosis in many cases.

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.

Effects of dietary protein content on renal parameters in normal cats

This study evaluates the effect of dietary protein content on renal parameters in 23 healthy spayed female cats. The objective was to determine if cats eating diets high in protein will have higher serum urea nitrogen (UN) and creatinine values without a detectable change in kidney function, as assessed by urinalysis. A single random cross-over design was used. Cats were fed a standard maintenance diet for at least 1 month prior to the dietary trial. They were fed in two phases. For the first phase, cats were randomly assigned to receive either a high protein [HP = 46% metabolizable energy (ME)] or low protein (LP = 26% ME) diet. For the second phase, cats were fed whichever diet they were not fed during the phase I period. Blood and urine samples were collected at 2-week intervals for the duration of the study (10 weeks). UN, albumin, alanine aminotransferase and urine specific gravity were significantly higher, and creatinine and phosphorus were significantly lower (P < 0.05) when cats were fed the HP diet as compared to when they were fed the LP diet, although none of the mean values were found to be outside of the corresponding reference interval. Dietary intake can result in clinically significant changes in UN and statistically significantly changes in several other biochemical analytes, although all analytes are likely to remain within normal reference intervals. Therefore, an accurate dietary history is necessary to help determine if renal parameters are being influenced by diet in a particular patient.

Correlation of Urine and Serum Biomarkers with Renal Damage and Survival in Dogs with Naturally Occurring Proteinuric Chronic Kidney Disease

Background Urine protein loss is common in dogs with chronic kidney disease (CKD). Hypothesis/Objectives To evaluate new biomarkers of glomerular and tubulointerstitial (TI) damage compared with histology and as survival indicators in dogs with naturally occurring, proteinuric CKD. Animals One hunderd and eighty dogs with naturally occurring kidney disease. Methods Retrospective study using urine, serum, and renal biopsies from dogs with kidney disease, 91% of which had proteinuric CKD. Biomarkers were evaluated and correlated with pathologic renal damage, and significant associations, sensitivities, and specificities of biomarkers for renal disease type were determined. Results Fractional excretions of immunogloblin M (IgM_FE) and immunoglobulin G (IgG_FE) correlated most strongly with glomerular damage based on light microscopy (r = 0.58 and 0.56, respectively; P < .01). Serum creatinine (SCr) correlated most strongly with TI damage (r = 0.70, P < .01). Urine IgM/creatinine and urine NAG/creatinine had the highest sensitivity (75%) and specificity (78%) for detection of immune complex‐mediated glomerulonephritis. Although individually most biomarkers were significantly associated with decreased survival time (P < .05), in a multivariate analysis, SCr, IgM_FE, and glomerular damage based on transmission electron microscopy (TEM) were the only biomarkers significantly associated with survival time (SCr: P = .001; IgM_FE: P = .008; TEM: P = .017). Conclusions and Clinical Importance Novel urine biomarkers and FEs are useful for detection of glomerular and TI damage in dogs with proteinuric CKD and might predict specific disease types and survival.