A cautionary tale of low-pass sequencing and imputation with respect to haplotype accuracy.

BACKGROUND: Low-pass whole-genome sequencing and imputation offer significant cost savings, enabling substantial increases in sample size and statistical power. This approach is particularly promising in livestock breeding, providing an affordable means of screening individuals for deleterious alleles or calculating genomic breeding values. Consequently, it may also be of value in companion animal genomics to support pedigree breeding. We sought to evaluate in dogs the impact of low coverage sequencing and reference-guided imputation on genotype concordance and association analyses. RESULTS: DNA isolated from saliva of 30 Labrador retrievers was sequenced at low (0.9X and 3.8X) and high (43.5X) coverage, and down-sampled from 43.5X to 9.6X and 17.4X. Genotype imputation was performed using a diverse reference panel (1021 dogs), and two subsets of the former panel (256 dogs each) where one had an excess of Labrador retrievers relative to other breeds. We observed little difference in imputed genotype concordance between reference panels. Association analyses for a locus acting as a disease proxy were performed using single-marker (GEMMA) and haplotype-based (XP-EHH) tests. GEMMA results were highly correlated (r ≥ 0.97) between 43.5X and ≥ 3.8X depths of coverage, while for 0.9X the correlation was lower (r ≤ 0.8). XP-EHH results were less well correlated, with r ranging from 0.58 (0.9X) to 0.88 (17.4X). Across a random sample of 10,000 genomic regions averaging 17 kb in size, we observed a median of three haplotypes per dog across the sequencing depths, with 5% of the regions returning more than eight haplotypes. Inspection of one such region revealed genotype and phasing inconsistencies across sequencing depths. CONCLUSIONS: We demonstrate that saliva-derived canine DNA is suitable for whole-genome sequencing, highlighting the feasibility of client-based sampling. Low-pass sequencing and imputation require caution as incorrect allele assignments result when the subject possesses alleles that are absent in the reference panel. Larger panels have the capacity for greater allelic diversity, which should reduce the potential for imputation error. Although low-pass sequencing can accurately impute allele dosage, we highlight issues with phasing accuracy that impact haplotype-based analyses. Consequently, if accurately phased genotypes are required for analyses, we advocate sequencing at high depth (> 20X).

Urinary cystatin B differentiates progressive versus stable IRIS Stage 1 chronic kidney disease in dogs.

BACKGROUND: Early identification of dogs with progressive vs stable chronic kidney disease (CKD) might afford opportunity for interventions that would slow progression. However, currently no surrogate biomarker reliably predicts CKD progression. HYPOTHESIS/OBJECTIVES: Urinary cystatin B (uCysB), a novel kidney injury biomarker, predicts progressive disease in International Renal Interest Society (IRIS) CKD Stage 1. ANIMALS: Seventy-two dogs, including 20 dogs from 4 university centers with IRIS CKD Stage 1, with IDEXX symmetric dimethylarginine (SDMA) concentration up to 17 µg/dL and no systemic comorbidities, and 52 clinically healthy staff-owned dogs from a fifth university center. METHODS: A multicenter prospective longitudinal study was conducted between 2016 and 2021 to assess uCysB concentration in IRIS CKD Stage 1 and control dogs. Dogs were followed to a maximum of 3 years (control) or 25 months (CKD). Stage 1 IRIS CKD was classified as stable or progressive using the slope of 1/SDMA, calculated from 3 timepoints during the initial 90-day period. Dogs with slope above or below -0.0007 week × dL/µg were classified as stable or progressive, respectively. Mixed effects modeling was used to assess the association between uCysB and progression rate. RESULTS: Estimates of first visit uCysB results predictive of active ongoing kidney injury based on the mixed effects models were 17 ng/mL for control, 24 ng/mL for stable CKD, and 212 ng/mL for progressive CKD (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary cystatin B differentiated stable vs progressive IRIS CKD Stage 1. Identification of dogs with progressive CKD may provide an opportunity for clinicians to intervene early and slow progression rate.

Differentiation of stable kidney function versus progressive dysfunction in dogs.

BACKGROUND: Circulating creatinine and symmetric dimethylarginine (SDMA) are biomarkers of kidney function that have been used variously to define stable vs progressive chronic kidney disease (CKD). Slope monitoring of inverse biomarker values (creatinine-1 or SDMA-1 ) has shown promise, but quantitative criteria to distinguish stable vs progressive CKD using this approach are lacking. OBJECTIVE: Assessment of creatinine-1 and SDMA-1 slope cutoffs to distinguish stable vs progressive CKD. ANIMALS: One hundred ten clinically healthy university staff-owned dogs and 29 male colony dogs with progressive X-linked hereditary nephropathy (XLHN). METHODS: Retrospective analysis combining 2 prospective observational studies, 1 tracking kidney function biomarkers in healthy dogs (HDs) to a maximum of 3 years, and 1 tracking kidney function biomarkers in male colony dogs with progressive XLHN to a maximum of 1 year. The minimum slope of creatinine-1 or SDMA-1 as measured using the IDEXX SDMA test from HD was assigned as the slope cutoff for stable kidney function. RESULTS: The stable vs progressive slope cutoff was -0.0119 week × dL/mg for creatinine-1 and -0.0007 week × dL/µg for SDMA-1 . CONCLUSIONS AND CLINICAL IMPORTANCE: In the studied CKD population, progressive dysfunction can be distinguished from stable kidney function by using the slope of creatinine-1 or SDMA-1 . These criteria may serve to characterize CKD in other cohorts of dogs and to establish guidelines for degrees of progression rate in dogs with naturally occurring CKD.

Prospective evaluation of novel biomarkers of acute kidney injury in dogs following cardiac surgery under cardiopulmonary bypass.

OBJECTIVE: To assess the occurrence of acute kidney injury (AKI) in dogs undergoing cardiac surgery under cardiopulmonary bypass (CPB) and explore associations between traditional and novel serum and urinary biomarkers. DESIGN: Prospective cohort study conducted between July 2018 and April 2019. SETTING: University teaching hospital. ANIMALS: Nineteen dogs undergoing cardiac surgery under CPB with preoperative serum creatinine <140 µmol/L (<1.6 mg/dl). INTERVENTIONS: Blood and urine samples were obtained at 4 time points: preoperatively following general anesthesia induction, immediately postoperatively, and 2 and 4 days postoperatively (T1 , T2 , T3 , and T4 ). AKI was defined as an increase in serum creatinine ≥26.4 µmol/L (≥0.3 mg/dl) above baseline within 48 hours. Serum creatinine, C-reactive protein (CRP), symmetric dimethylarginine (SDMA), inosine, beta-aminoisobutyric acid (BAIB), urinary clusterin (uClus), and urinary cystatin B (uCysB) were measured. Data were log-transformed (log10 ) when appropriate and assessed using linear mixed-effects models. MEASUREMENTS AND MAIN RESULTS: AKI occurred in 3 of 19 dogs (15.8%, 95% confidence interval: 0.047-0.384). Inosine increased at T2 (adjusted mean ± standard error: 53 ± 5.6) in all dogs, and then gradually decreased. Log10 uCysB increased at T2 (2.3 ± 0.1) in all dogs and remained high. Log10 CRP and log10 uClus increased significantly at T3 (1.9 ± 0.1 and 3.6 ± 0.1, respectively) in all dogs and remained increased. There was a significant positive association between serum creatinine and SDMA (P < 0.001, estimate ± standard error: 0.06 ± 0.00), between log10 CRP and log10 uClus (P < 0.001, 0.35 ± 0.08), between SDMA and creatinine as well as between SDMA and BAIB (P < 0.001, 11.1 ± 0.83 and P < 0.001, 1.06 ± 0.22, respectively) for all dogs at all time points. CONCLUSIONS: Inosine and uCysB concentrations changed in all dogs immediately following a surgery under CPB and may indicate tubular injury. Further studies are required to ascertain the usefulness of those biomarkers in early detection of AKI.

Comparison of IDEXX SediVue Dx® urine sediment analyzer to manual microscopy for detection of casts in canine urine.

BACKGROUND: Detection of urinary casts is difficult due to their intermittent presence and deterioration in urine samples. OBJECTIVE: To compare the performance of the IDEXX SediVue Dx® Urine Sediment Analyzer (SediVue) with manual microscopy for the detection of urinary casts. We hypothesized that the SediVue analyzer would perform similarly to manual microscopy in cast detection. ANIMALS: Four hundred forty-three samples from 420 dogs from a hospital population. METHODS: This is a prospective, cross-sectional study. For SediVue analysis (software version [SW] 1.0.1.3), uncentrifuged urine was pipetted into a disposable cartridge. Seventy images were captured and processed by an onboard algorithm. For manual microscopy, urine was centrifuged to obtain sediment. Any cast identified by either method was considered a positive result (>0/low-power field [LPF]). SediVue images were evaluated if casts were detected by either methodology. A revised sensitivity and specificity were calculated after image review and when using a threshold of >1 cast/LPF. RESULTS: The sensitivity of the SediVue analysis for the detection of urinary casts was 53.7% (43.85%-63.35%), and specificity was 86.0% (81.78%-89.51%). After image review, the revised sensitivity/specificity was 52.0% (42.89%-61.02%) and 90.6% (86.81%-93.54%), respectively. When using a more clinically relevant threshold of >1/LPF, the sensitivity was 52.6% (35.82%-69.02%) and specificity was 99.3% (97.85%-99.85%). CONCLUSIONS AND CLINICAL IMPORTANCE: The SediVue provides moderate agreement to manual methodology for detection of casts in urine.

The impact of periodontal disease and dental cleaning procedures on serum and urine kidney biomarkers in dogs and cats.

The objective of this study was to evaluate the benefits and inherent risks of dental cleaning procedures, based on serum and urine biomarkers for kidney function and tissue damage, in dogs and cats. Thirty-one asymptomatic, mostly older dogs (14 neutered male and 17 ovariohysterectomized female dogs of various breeds between 3 and 14 years old) and cats (19 neutered male and 12 ovariohysterectomized female domestic short hair cats between 2 and 16 years old) diagnosed with periodontal disease on physical exam, and recommended by their veterinarian to have dental cleaning under general anesthesia were evaluated in a prospective study. Serum and urine samples were collected from dogs and cats 1 week before, 6 hours after, and again 1 week after the dental cleaning procedure. Samples were analyzed for biomarkers of kidney function [serum creatinine (Cr), symmetric dimethylarginine (SDMA), and blood urea nitrogen (BUN), and urine for specific gravity (USG) and protein:creatinine (UPC) ratio]. A panel of biomarkers for renal tissue damage was also assessed [serum ß-aminoisobutyric acid (BAIB), and urine cystatin B and clusterin]. Samples collected one week before dental cleaning procedures showed that increased age and severity of dental disease were linked to abnormal kidney function biomarker values (age: elevated SDMA and Cr concentrations and isosthenuric USG values; disease severity: elevated UPC ratios) as well as elevated urine cystatin B and clusterin concentrations. Directly after the dental cleaning procedure, an increased number of cats with elevated SDMA concentrations was observed (specifically in cats with longer duration of dental procedures). Extended duration of dental procedures (≥60 min) was linked to increased urine cystatin B and clusterin concentrations, whereas shorter duration procedures was linked to decreased urine cystatin B and clusterin. Higher SDMA concentrations persisted in cats one week after the dental cleaning procedures and were linked to elevated UPC ratios one week before cleaning procedures. In conclusion, the results of this study indicate a link between severity of dental disease, renal tissue injury, and impaired renal function. Longer duration dental procedures in cats may carry inherent risks of kidney injury and impaired renal function.

Effects of low-dose meloxicam in cats with chronic kidney disease.

OBJECTIVES: Meloxicam therapy may benefit cats with degenerative joint disease, and retrospective studies suggest it could slow kidney disease progression and increase survival. This study aimed to prospectively evaluate the renal effects of low-dose meloxicam treatment (0.02 mg/kg/day) over 6 months in cats with chronic kidney disease (CKD). METHODS: Twenty-one cats with stable International Renal Interest Society stage 2 or 3 CKD were recruited and randomized to placebo or meloxicam groups. Cats were evaluated at baseline and at 1, 3 and 6 months, including blood pressure, chemistry, symmetric dimethylarginine (SDMA), glomerular filtration rate (GFR), urinalysis, urine protein:creatinine ratio (UPC), urine transforming growth factor-beta (ß):creatinine ratio, urine clusterin, urine cystatin B and serum inosine. RESULTS: No statistical difference was observed in systolic blood pressure, blood urea nitrogen, creatinine, SDMA, GFR, urine transforming growth factor-ß:creatinine ratio, urine clusterin, urine cystatin B or serum inosine in cats receiving meloxicam vs placebo. Mean UPC was greater in the meloxicam group (0.33) than the placebo group (0.1) at 6 months (P = 0.006). Four cats had meloxicam discontinued owing to potential (mainly gastrointestinal) adverse effects. CONCLUSIONS AND RELEVANCE: No decline in renal excretory function was observed when meloxicam was administered to cats with CKD. However, gastrointestinal adverse effects were observed, and cats that received meloxicam had greater proteinuria at 6 months than cats that received placebo. As proteinuria is associated with negative outcomes (progression of azotemia and hypertension) in cats with CKD, this finding suggests that meloxicam should be used with caution in cats with CKD and UPC monitored. Until further research is available, clinicians should weigh the risk of potential increased proteinuria against quality of life benefits when considering meloxicam for analgesia in cats with renal disease.

Serum concentrations of symmetric dimethylarginine and creatinine in cats with kidney stones.

Serum concentrations of symmetric dimethylarginine (SDMA) correlate with renal function in cats and SDMA has been shown to be a more reliable and earlier marker for chronic kidney disease (CKD) compared with serum creatinine (Cr). Calcium oxalate uroliths tend to develop in mid-to-older aged cats and kidney stones may cause a reduction in renal function with increased SDMA, but normal serum Cr. The purpose of this retrospective study was to determine if cats with kidney stones had increased serum SDMA concentrations, and whether SDMA increased earlier than serum creatinine concentrations. Cats in the colony with kidney stones diagnosed between August 2010 and December 2015 (n = 43) were compared with healthy geriatric cats (n = 21) without kidney stones. Serum SDMA concentrations were determined by liquid chromatography-mass spectrometry and serum Cr concentrations were determined by enzymatic colorimetry. Cats with kidney stones were diagnosed antemortem by radiographic imaging (n = 12) or by postmortem necropsy (n = 31). Retrospectively, serum SDMA was found to be increased above the upper reference limit in 39 of 43 cats with kidney stones. Serum Cr was increased above the upper reference limit in 18 of 43 cats; 6 of these 18 cats had terminal azotemia only. The mean time that serum SDMA was increased before serum Cr was increased was 26.9 months (range 0 to 60 months). Kidney stones were composed of calcium oxalate in 30 of 34 cats. The lifespan for cats with kidney stones (mean, 12.5 years; range, 6.1 to 18.1 years) was shorter (P < 0.001) than for control cats (mean, 15.2 years; range, 13.0 to 17.2 years), suggesting that non-obstructive kidney stones have an effect on mortality rate or rate of CKD progression. In conclusion, if SDMA concentrations are elevated in mid-to-older aged cats, further imaging studies are warranted to check for the presence of kidney stones.

Kidney Disease and the Nexus of Chronic Kidney Disease and Acute Kidney Injury: The Role of Novel Biomarkers as Early and Accurate Diagnostics.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are interconnected and the presence of one is a risk for the other. CKD is an important predictor of AKI after exposure to nephrotoxic drugs or major surgery, whereas persistent or repetitive injury could result in the progression of CKD. This brings new perspectives to the diagnosis and monitoring of kidney diseases highlighting the need for a panel of kidney-specific biomarkers that reflect functional as well as structural damage and recovery, predict potential risk and provide prognosis. This article discusses the kidney-specific biomarkers, symmetric dimethylarginine (SDMA), clusterin, cystatin B, and inosine.

Positive Impact of Nutritional Interventions on Serum Symmetric Dimethylarginine and Creatinine Concentrations in Client-Owned Geriatric Cats.

A prospective study was conducted in client-owned geriatric cats to evaluate the short- term effects of a test food on serum symmetric dimethylarginine (SDMA) and creatinine (Cr) concentrations. Test food contained functional lipids (fish oil), antioxidants (vitamins C and E), L-carnitine, botanicals (vegetables), highly bioavailable protein, and amino acid supplements. Cats (n = 80) were fed either test food or owner's-choice foods (non-nutritionally controlled cohort). Cats were included based on age (≥ 9 years), indoor only, neutered, and free of chronic disease. At baseline, all cats had serum Cr concentrations within the reference interval. Renal function biomarkers and urinalysis results at baseline and after consuming test food or owner's-choice foods for 3 and 6 months were evaluated. Cats consuming test food showed significant decreases in serum Cr and BUN concentrations across time. Overall, cats consuming owner's-choice foods showed significant increases in serum SDMA concentrations at 3 and 6 months compared with baseline (P ≤ 0.05), whereas in cats consuming test food serum SDMA concentrations did not change. At baseline or during the 6-month feeding trial, 23 (28.8%) cats had increased serum SDMA, but normal serum Cr consistent with IRIS Stage 1 chronic kidney disease. This included 6 cats fed test food and 17 cats fed owner's-choice foods. In the 6 cats fed test food, serum SDMA decreased in 3 cats and remained stable in 1 cat, whereas in the 17 cats fed owner's-choice foods, serum SDMA increased in 13 cats and decreased or remained stable in 4 cats. The increase in serum SDMA concentration was significant (P = 0.02) only for cats fed owner's-choice foods. These results suggest that nonazotemic cats with elevated serum SDMA (early renal insufficiency) when fed a food designed to promote healthy aging are more likely to demonstrate stable renal function compared with cats fed owner's-choice foods. Cats fed owner's-choice foods were more likely to demonstrate progressive renal insufficiency.

Positive Impact of Nutritional Interventions on Serum Symmetric Dimethylarginine and Creatinine Concentrations in Client-Owned Geriatric Dogs.

A prospective study was conducted in client-owned geriatric dogs to evaluate the short-term effects of a test food on serum symmetric dimethylarginine (SDMA) and creatinine (Cr) concentrations. Test food contained functional lipids (fish oil), antioxidants (lipoic acid, vitamins C and E), L-carnitine, botanicals (fruits and vegetables), controlled sodium concentration, and high quality protein sources (high bioavailability and an ideal amino acid composition). Dogs (n = 210) were fed either test food or owner's-choice foods (non-nutritionally controlled cohort). Dogs were included based on age and body weight: small (6.8 to 11.4 kg) and medium dogs (11.5 to 22.7 kg) were ≥ 9 years, whereas dogs >22.7 kg were ≥ 7 years at baseline. At baseline, all dogs had to have serum Cr concentrations within the reference interval and be free of chronic disease. Renal function biomarkers and urinalysis results at baseline, and after consuming test food or owner's-choice foods for 3 and 6 months, were evaluated. Only dogs consuming test food showed significant decreases in serum SDMA and Cr concentrations (both P ≤ 0.05) across time. At baseline or during the 6-month feeding trial, 18 dogs (8.6%) had increased serum SDMA, but normal serum Cr, consistent with IRIS Stage 1 chronic kidney disease. This included 9 dogs fed test food and 9 dogs fed owner's-choice foods. Compared with baseline, after feeding 9 dogs test food for 6 months, serum SDMA decreased in 8 dogs and increased in 1 dog. After feeding 9 dogs owner's-choice foods for 6 months, serum SDMA decreased in 4 dogs and increased in 4 dogs (remained stable in 1 dog). The decreases in serum SDMA and Cr concentrations were significant (both P = 0.03) only for dogs fed test food. These results suggest that nonazotemic dogs with elevated serum SDMA (early renal insufficiency) when fed a test food designed to promote healthy aging are more likely to demonstrate improved renal function compared with dogs fed owner's-choice foods.

Relationship between lean body mass and serum renal biomarkers in healthy dogs.

BACKGROUND: Symmetric dimethylarginine (SDMA) is an accurate and precise biomarker for estimating glomerular filtration rate (GFR) in humans and cats. Serum creatinine (sCr) also correlates with GFR, but has limitations as a biomarker of renal function because nonrenal factors can influence its concentration. HYPOTHESIS: Differences in lean body mass (LBM) influence sCr, but not serum SDMA concentrations. ANIMALS: Forty-one healthy Beagles, mean age 9.9 years (range: 3.1-14.8 years), were studied over a 6 month period. METHODS: Serum biomarkers of renal function were measured prospectively at baseline, and 1, 3, and 6 months. SDMA concentrations were measured by liquid chromatography-mass spectroscopy and sCr concentrations by enzymatic colorimetry. Body composition was determined by dual energy x-ray absorptiometry. RESULTS: LBM (P < .001) and age (P = .006) were significant explanatory variables for sCr concentration (R(2) = 0.38), but not SDMA concentration. Time on food was the only significant explanatory variable for SDMA concentration (R(2) = 0.49). SDMA concentrations decreased across time (P < .001). LBM was affected by sex (males > females; P = .02). Mature adult dogs (<8 years) had greater LBM compared with geriatric dogs (≥8 years; P < .001). CONCLUSION AND CLINICAL IMPORTANCE: sCr concentrations, but not SDMA concentrations, are influenced by LBM, which limits sCr utility as a biomarker for monitoring renal function in dogs with decreased LBM. Reductions in LBM can lower sCr concentration and overestimate GFR. SDMA concentrations, but not sCr concentrations were influenced by time on food. SDMA could have clinical advantages over sCr in monitoring response to nutritional interventions.