Pulmonary hypertension is associated with hypocoagulability in dogs: a retrospective analysis of 66 cases (2013-2021).

OBJECTIVE: To describe coagulation profiles in dogs with echocardiographic evidence of pulmonary hypertension (PH), to compare them to coagulation profiles in dogs without echocardiographic evidence of PH, and to determine the relationship between coagulation profiles and echocardiographic probability of PH. ANIMALS: 66 dogs with PH (cases) and 86 dogs without PH (controls). METHODS: Retrospective evaluation of records between 2013 and 2021 of dogs that had both an echocardiogram and a coagulation panel performed within 7 days. Dogs that received antithrombotics within 7 days of evaluation and dogs diagnosed with congenital or acquired coagulopathy or other severe systemic disease that could lead to coagulopathy were excluded. Dogs with a low echocardiographic probability of PH were also excluded. The dogs were divided into a PH group and non-PH group based on echocardiographic results. Demographic, clinicopathologic, and traditional coagulation parameters and VCM Vet (Entegrion) parameters were compared between the 2 groups. RESULTS: Dogs with PH were significantly older (median, 11 years vs 9.5 years, P = .02) and had a significantly lower body weight (median, 7.3 kg vs 19.3 kg, P < .001) than controls. Dogs with PH also had a significantly greater percent increase in prothrombin time (PT; P = .02), partial thromboplastin time (PTT; P < .0001), and fibrinogen (P = .045); however, their antithrombin concentration was lower (P = .005) compared to controls. Eight of 65 dogs (12.3%) in the PH group and 1/86 (1.2%) dogs in the non-PH group had an elevation of PT and/or PTT greater than 50% above the reference interval (P = .005). Dogs with PH had 11.9 times (95% CI, 1.5 to 97.9; P = .02) greater odds of being hypocoagulable than dogs without PH based on PT and PTT. CLINICAL RELEVANCE: This study demonstrated an association between a moderate to high echocardiographic probability of PH and a hypocoagulable state in dogs as determined by traditional coagulation assays. It underscores the importance of monitoring the coagulation status in canine patients with PH, particularly before initiating antithrombotic medications.

MicroRNA-126 in dogs with immune complex-mediated glomerulonephritis.

BACKGROUND: Most proteinuric dogs with naturally occurring chronic kidney disease have amyloidosis (AMYL), glomerulosclerosis (GS), or immune complex-mediated glomerulonephritis (ICGN), each with different treatment and prognosis. A noninvasive and disease-specific biomarker is lacking. HYPOTHESIS: We hypothesized that the expression pattern of biofluid microRNA (miRNAs and miRs) would correlate with disease progression and categorization. ANIMALS: Archived serum and urine samples from 18 dogs with glomerular disease and 6 clinically healthy dogs; archived urine samples from 49 dogs with glomerular disease and 13 clinically healthy dogs. METHODS: Retrospective study. Archived biofluid samples from adult dogs with biopsy-confirmed glomerular disease submitted to the International Veterinary Renal Pathology Service between 2008 and 2016 were selected. Serum and urinary miRNAs were isolated and profiled using RNA sequencing. Urinary miR-126, miR-21, miR-182, and miR-486 were quantified using quantitative reverse transcription PCR. RESULTS: When comparing more advanced disease with earlier disease, no serum miRNAs were differentially expressed, but urinary miR-21 and miR-182 were 1.63 (95% CI: .86-3.1) and 1.45 (95% CI: .82-2.6) times higher in azotemic dogs, respectively (adjusted P < .05) and weakly correlated with tubulointerstitial fibrosis (miR-21: r = .32, P = .03; miR-182: r = .28, P = .05). Expression of urinary miR-126 was 10.5 (95% CI: 4.1-26.7), 28.9 (95% CI: 10.5-79.8), and 126.2 (95% CI: 44.7-356.3) times higher in dogs with ICGN compared with dogs with GS, AMYL, and healthy controls, respectively (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: The miR-126 could help identify dogs that might benefit from immunosuppressive therapy in the absence of a biopsy. MiR-21 and miR-182 are potential markers of disease severity and fibrosis.

Effect of withholding food versus feeding on creatinine, symmetric dimethylarginine, cholesterol, triglycerides, and other biochemical analytes in 100 healthy dogs.

BACKGROUND: Withholding food is often recommended before collection of blood for routine biochemical analysis in dogs despite a paucity of evidence to support this requirement. OBJECTIVES: To compare measurements of selected biochemical analytes collected before and after feeding in clinically healthy dogs. ANIMALS: One hundred clinically healthy staff- and student-owned dogs weighing ≥15 kg. METHODS: Prospective observational study. Food was withheld from the dogs for 10-26 hours. Preprandial serum was collected, and then dogs were fed their usual food at an amount equivalent to at least 2/3 resting energy requirement (RER). Selected serum analytes were measured at 2-, 4-, 6-, and 8-hours postprandially. The proportion of postprandial values that exceeded either the reported allowable total error (TEa), or for symmetric dimethylarginine (SDMA), the reference change value (RCV), was determined. As neither TEa nor RCV is available for lipase, comparison was made to the high end of the reference interval (RI). RESULTS: The proportion of dogs with at least 1 postprandial measurement that exceeded the TEa or RCV was 92/100 for triglycerides, 66/100 for blood urea nitrogen (BUN), 46/100 for phosphorus, 17/100 for glucose, 9/100 for bilirubin, 5/100 for SDMA, 2/100 for creatinine, and 0/100 for cholesterol and albumin. Postprandial lipase never exceeded the RI in dogs with normal fasted lipase. CONCLUSIONS AND CLINICAL IMPORTANCE: Withholding food is generally not necessary before performing routine biochemical analysis in clinically healthy dogs. Withholding food might be helpful to limit variability in analytes impacted by feeding, such as triglycerides and phosphorus.

Accuracy of the IDEXX SediVue Dx analyzer for quantifying RBC and WBC indices in the urine sediments of cats and dogs compared with manual microscopic evaluations.

BACKGROUND: The IDEXX SediVue Dx (SediVue) is an automated, in-clinic urine sediment analyzer for veterinary patients. The bias between the results from manual microscopy and the SediVue is currently unknown. OBJECTIVES: To assess the diagnostic accuracy of the SediVue, we aimed to determine the bias between the SediVue (index test) and manual microscopy (reference standard) for the quantification of RBCs and WBCs in urine. METHODS: Urine remnant samples were collected from cats and dogs that contained RBCs (n = 462) and WBCs (n = 510). Retrospective analysis of results from urine sediment examinations using both manual microscopy (using a KOVA and DeciSlide system) and the SediVue (1.0.1.3) was performed. Bias was determined with Bland-Altman plots. SediVue-captured images from high-bias samples were reviewed, and biases were compared. RESULTS: The median bias for semi-quantitative RBC and WBC counts was determined for RBC and WBC counts. The cutoffs were RBC ≤ 5/HPF, 0.3; RBC 5.1-10/HPF, 10.1; RBC 10.1-20/HPF, 10.6; and RBC > 20/HPF, 28.93; WBC ≤ 5/HPF, 0.1; WBC 5.1-10/HPF, 2.2; WBC 10.1-20/HPF, 9.4; and WBC > 20/HPF, 26.6. High bias between the methods was identified in 98 samples (21.0%) with RBCs and 77 samples (15.7%) with WBCs. Reviewer-based enumeration of the SediVue-captured images decreased the percentage of samples with high bias to 17.3% for RBCs and to 11.4% for WBCs. CONCLUSIONS: Bias in the RBC and WBC counts between manual microscopy and the SediVue was unlikely to impact clinical interpretations in a majority of cases. Although reviewer enumeration of SediVue-captured images reduced observed bias, inherent differences between methodologies appeared to have a larger impact on the bias.

Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy.

Dogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

Mucocutaneous nasal histoplasmosis in an immunocompetent dog.

BACKGROUND: Histoplasma (H.) capsulatum is a dimorphic fungus, and infection is typically via inhalation of microconidia. After conversion to the yeast phase within the lung, the organism is subsequently disseminated to other tissues by macrophages. Nasal histoplasmosis appears to be a rare condition in dogs. CASE PRESENTATION: We report the clinical case of a 4.5-year-old male neutered Cocker spaniel/Poodle mix, 7.7 kg, body condition score 6/9, that presented with a 3-month history of sneezing and left-sided mucoid nasal discharge. The history also included a mild swelling (transient) of the right carpus with a lameness (grade II-III/IV), coinciding with the onset of sneezing and nasal discharge. The dog lived primarily indoors in the Texas Gulf Coast area. On physical examination, the dog was febrile, and the left nostril was swollen, ulcerative, deformed, and hypopigmented. Mandibular lymph nodes were firm and mildly enlarged bilaterally. Mild lymphopenia, thrombocytopenia, and hyperglobulinemia were noted. Thoracic radiographs were unremarkable. Computed tomography and rhinoscopy revealed swelling of the rostral portion of the left and right nasal passages. Cytology and histology of biopsies of the affected nasal tissue showed pyogranulomatous inflammation and yeast organisms consistent with H. capsulatum. Weak antigenuria was detected on the MVista H. capsulatum antigen test. Treatment with oral itraconazole led to a resolution of the nasal signs and normalization of the appearance of the nostril over 13 weeks, and neither antigenuria nor antigenemia was detected on several recheck examinations. The dog remained in good general and physical condition and showed no signs of disease recurrence more than 6 years after the last examination. CONCLUSION: We report a rare case of nasal mucocutaneous histoplasmosis in an immunocompetent dog, with an excellent clinical response to oral itraconazole. This case documents that histoplasmosis in dogs can affect primarily the nasal cavity, which responds rapidly to triazole antifungal therapy and has a good prognosis. A similar case has only been reported in human medicine in a young adult.

A Kidney-Targeted Nanoparticle to Augment Renal Lymphatic Density Decreases Blood Pressure in Hypertensive Mice.

Chronic interstitial inflammation and renal infiltration of activated immune cells play an integral role in hypertension. Lymphatics regulate inflammation through clearance of immune cells and excess interstitial fluid. Previously, we demonstrated increasing renal lymphangiogenesis prevents hypertension in mice. We hypothesized that targeted nanoparticle delivery of vascular endothelial growth factor-C (VEGF-C) to the kidney would induce renal lymphangiogenesis, lowering blood pressure in hypertensive mice. A kidney-targeting nanoparticle was loaded with a VEGF receptor-3-specific form of VEGF-C and injected into mice with angiotensin II-induced hypertension or LNAME-induced hypertension every 3 days. Nanoparticle-treated mice exhibited increased renal lymphatic vessel density and width compared to hypertensive mice injected with VEGF-C alone. Nanoparticle-treated mice exhibited decreased systolic blood pressure, decreased pro-inflammatory renal immune cells, and increased urinary fractional excretion of sodium. Our findings demonstrate that pharmacologically expanding renal lymphatics decreases blood pressure and is associated with favorable alterations in renal immune cells and increased sodium excretion.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characteristics associated with bacterial growth in urine in 451 proteinuric dogs (2008-2018).

BACKGROUND: Urine cultures are frequently recommended to rule out infection as a postrenal cause of proteinuria. OBJECTIVE: Identify characteristics associated with bacterial growth in urine in proteinuric dogs. ANIMALS: Four hundred and fifty-one dogs admitted to a teaching hospital between January 2008 and January 2018 with urine protein-to-creatinine ratios (UPCs) >0.5. METHODS: Retrospective study included dogs with a UPC, urinalysis, and quantitative urine culture (QUC) performed within a 72-hour period by searching electronic records. Dogs with recent antimicrobial therapy, urine collected by methods other than cystocentesis, or UPC ≤0.5 were excluded. Signalment, comorbidities, serum BUN and creatinine concentrations, urinalysis findings, and QUC results were recorded. The association between these characteristics and presence of bacterial growth in urine was assessed by univariable and multivariable analysis. RESULTS: Thirty of four hundred fifty-one dogs (6.7%) had bacterial growth in urine. Of these, 18 (60.0%) had active urine sediment. Bacterial growth in urine was associated with pyuria (odd ratio [OR] 25.1, 95% confidence interval [CI] 7.9-79.6, P < .001), bacteriuria (OR 11.1, 95% CI 3.2-39.1, P < .001), and lower urinary tract disease (OR 6.7, 95% CI 1.9-23.0; P = .0028). If QUC was prompted based on these criteria, 8/451 (1.8%) of proteinuric dogs would have had undetected bacterial growth. CONCLUSIONS AND CLINICAL IMPORTANCE: The proportion of proteinuric dogs with both inactive urine sediment and bacterial growth in urine was low, suggesting that QUC might not be necessary in the evaluation of all proteinuric dogs. An active urine sediment or lower urinary tract disease should prompt QUC for proteinuric dogs.

Oral delivery of nanoparticle urolithin A normalizes cellular stress and improves survival in mouse model of cisplatin-induced AKI.

The popular anticancer drug cisplatin causes many adverse side effects, the most serious of which is acute kidney injury (AKI). Emerging evidence from laboratory and clinical studies suggests that the AKI pathogenesis involves oxidative stress pathways; therefore, regulating such pathways may offer protection. Urolithin A (UA), a gut metabolite of the dietary tannin ellagic acid, possesses antioxidant properties and has shown promise in mouse models of AKI. However, therapeutic potential of UA is constrained by poor bioavailability. We aimed to improve oral bioavailability of UA by formulating it into biodegradable nanoparticles that use a surface-conjugated ligand targeting the gut-expressed transferrin receptor. Nanoparticle encapsulation of UA led to a sevenfold enhancement in oral bioavailability compared with native UA. Treatment with nanoparticle UA also significantly attenuated the histopathological hallmarks of cisplatin-induced AKI and reduced mortality by 63% in the mouse model. Expression analyses indicated that nanoparticle UA therapy coincided with oxidative stress mitigation and downregulation of nuclear factor erythroid 2-related factor 2- and P53-inducible genes. Additionally, normalization of miRNA (miR-192-5p and miR-140-5p) implicated in AKI, poly(ADP-ribose) polymerase 1 levels, antiapoptotic signaling, intracellular NAD+, and mitochondrial oxidative phosphorylation were observed in the treatment group. Our findings suggest that nanoparticles greatly increase the oral bioavailability of UA, leading to improved survival rates in AKI mice, in part by reducing renal oxidative and apoptotic stress.

Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids.

BACKGROUND: Small RNA sequencing (RNA-seq) of biofluids is challenging due to the relative scarcity of microRNAs (miRNAs), limited sample volumes, and the lack of a gold standard isolation method. Additionally, few comparisons exist for the RNA isolation and sequencing methods of biofluids. OBJECTIVES: We aimed to compare the performance of six commercial RNA isolation kits and two library preparation methods for small RNA-seq using canine serum and urine. METHODS: Serum and urine were collected from seven dogs with protein-losing nephropathy, and the samples were pooled. Total RNA from serum (2 mL) and urine (10 mL) was isolated in triplicate using three methods each for serum (Zymo Direct-zol, mirVana PARIS, miRCURY Biofluids) and urine (Qiagen exoRNeasy, Norgen Urine Exosome, miRCURY Exosome). For each sample type, the two kits yielding the highest RNA concentration were selected, and small RNA-seq was performed using TruSeq and NEXTflex library preparations. Data were analyzed by CPSS 2.0 and DESeq2. RESULTS: For serum, Zymo Direct-zol combined with NEXTflex was the only combination that enabled successful library preparation, while for urine, Qiagen exoRNeasy combined with NEXTflex outperformed other combinations for detecting miRNAs. The total number of miRNAs detected in serum and urine was 198 and up to 115, respectively. miRNA expression in serum was distinct from urine. Furthermore, the library preparation method introduced a higher variation of urine results than the RNA isolation method. CONCLUSIONS: Different isolation and library preparation methods show significant differences in miRNA results that could affect biomarker discovery. Small RNA-seq provides an unbiased, global assessment to compare these methods in canine biofluids.

Comparison of the performance of the IDEXX SediVue Dx® with manual microscopy for the detection of cells and 2 crystal types in canine and feline urine.

BACKGROUND: Microscopic evaluation of urine is inconsistently performed in veterinary clinics. The IDEXX SediVue Dx® Urine Sediment Analyzer (SediVue) recently was introduced for automated analysis of canine and feline urine and may facilitate performance of urinalyses in practice. OBJECTIVE: Compare the performance of the SediVue with manual microscopy for detecting clinically relevant numbers of cells and 2 crystal types. SAMPLES: Five-hundred thirty urine samples (82% canine, 18% feline). METHODS: For SediVue analysis (software versions [SW] 1.0.0.0 and 1.0.1.3), uncentrifuged urine was pipetted into a cartridge. Images were captured and processed using a convolutional neural network algorithm. For manual microscopy, urine was centrifuged to obtain sediment. To determine sensitivity and specificity of the SediVue compared with manual microscopy, thresholds were set at ≥5/high power field (hpf) for red blood cells (RBC) and white blood cells (WBC) and ≥1/hpf for squamous epithelial cells (sqEPI), non-squamous epithelial cells (nsEPI), struvite crystals (STR), and calcium oxalate dihydrate crystals (CaOx Di). RESULTS: The sensitivity of the SediVue (SW1.0.1.3) was 85%-90% for the detection of RBC, WBC, and STR; 75% for CaOx Di; 71% for nsEPI; and 33% for sqEPI. Specificity was 99% for sqEPI and CaOx Di; 87%-90% for RBC, WBC, and nsEPI; and 84% for STR. Compared to SW1.0.0.0, SW1.0.1.3 had increased sensitivity but decreased specificity. Performance was similar for canine versus feline and fresh versus stored urine samples. CONCLUSIONS AND CLINICAL IMPORTANCE: The SediVue exhibits good agreement with manual microscopy for the detection of most formed elements evaluated, but improvement is needed for epithelial cells.

Traditional Renal Biomarkers and New Approaches to Diagnostics.

Traditional biomarkers of renal disease have a number of limitations, whether evaluating veterinary patients or performing preclinical toxicity studies. Serum creatinine and urea nitrogen are affected by nonrenal influences that limit their usefulness for detecting small but significant decreases in glomerular filtration rate (GFR) in veterinary patients. These nonrenal influences can be more controlled in preclinical studies than in clinical patients; however, because of its high functional reserve, these estimates of GFR are insensitive for detecting kidney injury prior to loss of a substantial proportion of functioning nephrons. Urine biomarkers can be highly sensitive for tubular or glomerular injury that might lead to irreversible damage to the nephron. Several proteins are qualified by the Food and Drug Administration for nonclinical application as urinary biomarkers of drug-induced nephrotoxicity, and many of these also have preliminary data supporting their usefulness for kidney injury in dogs and cats. In addition to these relatively recently identified biomarkers, efforts are underway to discover new renal biomarkers using a variety of techniques including liquid chromatography-mass spectrometry and small RNA sequencing. Ultimately, the interplay between preclinical studies and clinical patients in discovery and validation of renal biomarkers is critical to their successful implementation.

New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity.

A scientific session entitled "New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity" focused on novel biomarkers to monitor kidney injury both preclinically and clinically, as well as providing mechanistic insight of the induced injury. Further, the role and impact of kidney membrane transporters in drug-induced kidney toxicity provided additional considerations when understanding kidney injury and the complex role of drug transporters in either sensitivity or resistance to drug-induced injury. The onset of nephropathy in diabetic patients was also presented, focusing on the quest to discover novel biomarkers that would differentiate diabetic populations more susceptible to nephropathy and renal failure. The session highlighted exciting new research areas and novel biomarkers that will enhance our understanding of kidney injury and provide tools for ensuring patient safety clinically.

Correlation of electrophoretic urine protein banding patterns with severity of renal damage in dogs with proteinuric chronic kidney disease.

BACKGROUND: Urine protein loss is common in dogs with chronic kidney disease (CKD). Currently available noninvasive means of evaluating CKD in dogs cannot accurately predict the severity of glomerular and tubulointerstitial damage. Electrophoretic analysis of urine proteins can indicate the compromised renal compartment (glomerular vs tubular), but extensive evaluation of protein banding pattern associations with histologic damage severity has not been performed in dogs. OBJECTIVES: We aimed to evaluate electrophoretic banding patterns as indicators of the presence and severity of glomerular and tubulointerstitial damage in dogs with naturally occurring, predominantly proteinuric CKD. METHODS: We performed a retrospective study using urine and renal tissue from 207 dogs with CKD. Urine protein banding patterns were correlated with histologic severity of renal damage. Sensitivity and specificity of banding patterns for the detection of glomerular and tubulointerstitial damage were determined. RESULTS: Banding patterns were 97% sensitive and 100% specific for the detection of glomerular damage and 90% sensitive and 100% specific for the detection of tubulointerstitial damage. Correlations between composite banding patterns and the severity of renal damage were strong, while glomerular banding patterns correlated moderately with glomerular damage severity, and tubular gel scores correlated weakly to moderately with the severity of tubulointerstitial damage. CONCLUSIONS AND CLINICAL IMPORTANCE: Urine protein banding patterns are useful for the detection of glomerular and tubulointerstitial damage in dogs with proteinuric CKD.

ASVCP guidelines: Allowable total error hematology.

The purpose of this document is to provide total allowable error (TEa ) recommendations for commonly analyzed hematology measurands for veterinary personnel. These guidelines define relevant terminology and highlight considerations specific to hematology measurands. They also provide reasons and guidelines for using TEa in instrument performance evaluation, including recommendations for when the total observed error exceeds the recommended TEa . Biological variation-based quality specifications are briefly discussed. The appendix describes the derivation of the hematology TEa recommendations and provides resources for external quality assurance/proficiency testing programs and a worksheet for implementation of the guidelines.

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.