Canine Liver Research Articles

Fecal Bile Acids in Canine Chronic Liver Disease: Results from 46 Dogs.

The concentrations of fecal and serum bile acids (BAs) are known to be altered in human patients with chronic liver diseases (CLDs), especially those with biliary tract involvement (BTD). Scarce literature is available regarding fecal BA modifications during canine CLDs. This study aimed to evaluate fecal BAs in canine CLDs according to different clinical and clinicopathological variables. Forty-six dogs were enrolled. Canine feces were analyzed by HPLC. Cholic Acid (CA), Chenodeoxycholic Acid (CDCA), Ursodeoxycholic Acid (UDCA), Deoxycholic Acid (DCA), and Lithocholic Acid (LCA) were measured, and primary BAs (CA + CDCA), secondary BAs (UDCA + DCA + LCA), and the primary/secondary (P/S) ratio were calculated. Primary BAs (p < 0.0001), CA (p = 0.0003), CDCA (p = 0.003), the P/S ratio (p = 0.002), and total BAs (p = 0.005) were significatively higher in BTD dogs (n = 18) compared to in non-BTD dogs (n = 28). Fecal secondary BAs did not statistically differ between BTD and non-BTD dogs. Gastrointestinal clinical signs (p = 0.028) and diarrhea (p = 0.03) were significantly more prevalent in BTD dogs compared to in non-BTD dogs, supporting the hypothesis of some pathological mechanisms assimilable to bile acid diarrhea (BAD). Our results could reflect imbalances of the fecal BA metabolism in dogs with CLDs. Further studies involving gut microbiome and metabolomic assessment are needed to better understand the possible clinical implications of BA metabolism disruption and their potential role in canine CLDs.

Exploring the Relationship between Canine Paraoxonase-1 (PON-1) Serum Activity and Liver Disease Classified by Clinico-Pathological Evaluation.

Paraoxonase-1 (PON-1), a liver-synthesized enzyme, acts as a negative acute-phase reactant during systemic inflammation in dogs. Given the hepatic synthesis of this enzyme, the presence of liver diseases may influence PON-1, thus affecting its reliability as a biomarker for inflammatory/oxidative systemic diseases. The aim of this study is to investigate PON-1 activity variations among dogs suspected of liver injury or failure, evaluating the influence of hepatic diseases on PON-1 activity. A total of one-hundred-sixty dogs were retrospectively enrolled and categorized into three groups based on clinical presentation and laboratory results: control (C = 20), suspected liver injury (INJ = 114), and suspected liver failure (FAIL = 26). The INJ group was further divided into subgroups based on the severity of the alanine aminotransferase (ALT) increase. Both the INJ and FAIL groups were further divided based on serum macroscopic appearance. The PON-1 activity was quantified using a paraoxon-based method, which is already validated in dogs. No significant difference in PON-1 activity was observed between the C and INJ groups, despite a significant increase in the subgroups with moderate and severe elevations of ALT. The dogs with icteric serum exhibited decreased PON-1 activity, while lipemic serum was associated with an increased PON-1 activity. A significant reduction in PON-1 activity was noted in the FAIL group, compared to both C and INJ groups (p < 0.0001), regardless of serum appearance. Given the retrospective nature of this study, additional evaluations (e.g., histopathology, imaging) were not performed. The results obtained here suggest the importance of interpreting PON-1 activity cautiously in dogs with suspected liver disease.

A randomized comparison of an adhesive gelatin sponge and a plain collagen sponge for hemostatic control during canine liver surgery.

To compare the effectiveness of a modified surface gelatin sponge to a plain collagen sponge for hemostasis of parenchymal hepatic bleeding. Prospective, randomized trial of two hemostatic agents. A total of 45 dogs undergoing elective liver surgery were randomly allocated into two groups: 22 in the adhesive gelatin (AG) group and 23 in the plain collagen (PC) group. A total of 20 patients per group underwent liver biopsy to create a uniformly sized bleeding surface, with the remaining patients (AG = 2, PC = 3) undergoing liver lobectomy. Evaluation of hemostatic effectiveness and tissue adhesion of each sponge type was performed by the operating surgeon using structured scoring systems. Hemostatic parameters were primarily evaluated at the liver biopsy site to maintain homogeneity of bleeding surface size. For the liver biopsy group (n = 40), 5 min after hemostatic sponge application, 10/20 dogs were bleeding in the PC group, compared to 2/20 in AG group (p = .0138). The PC bleeding was significantly higher than AG across the 3 to 6 min evaluation period (p < .001). When surgeons tested the adhesion of the sponge across the whole cohort (n = 45), AG scored 2 (of 3) against 1 for PC (p < .001). In group PC, 5/23 sponges dislodged during abdominal lavage and preparations for closure and had to be replaced due to recurrence of bleeding, compared with no AG sponges dislodging (p = .042). There were no further complications related to the use of either sponge. In the dogs with hepatic parenchymal incision, use of an adhesive gelatin sponge improved intraoperative attachment and haemostatic effectiveness, compared to a collagen sponge. Based on our clinical experience in these cases, adhesive gelatin sponges could be considered an effective option when selecting a hemostatic agent for liver surgery in dogs.

Etiological Tracing Test and Drug Efficacy Test in a Case of Clinical Canine Toxic Liver Disease

This experiment is divided into three parts. A typical case report of canine toxic liver disease was recorded at the Huazhong Agricultural University Animal Hospital. The etiology tracing test focused on the daily life risk factors that induced the disease. The pathological model of toxic liver disease in mice was successfully established by the administration of household formaldehyde, floor cleaner and chlorine-containing disinfectant tablets. The drug efficacy test was carried out by the administration of self-made traditional Chinese medicine to the model mice, and to observe the therapeutic effect of traditional Chinese medicine prescription on the mice pathological model. It can be concluded that the daily life risk factors should be paid enough attention to in the dog's toxic liver disease. In comparison, within households that own dogs, the toxicity of chlorine-containing disinfectant is low in the normal dilution range, and its liver damage to mice is very light during the test. However, caution should be exercised when using floor cleaners and formaldehyde. Self-made traditional Chinese medicine has a good effect on this type of liver disease, but it should also pay attention to the dose, concentration and usage cycle.

Metabolic reprogramming of corn oligopeptide in regulating sodium nitrite-induced canine hepatocyte injury via TGF/NF-κB signaling pathways and aminoacyl-tRNA biosynthesis

Sodium nitrite (SN), a prevalent food preservative, is known to precipitate hepatotoxicity upon exposure. This study elucidates the hepatoprotective effects of corn oligopeptide (COP) and vitamin E (VE) against SN-induced hepatic injury in canine hepatocytes. Canine liver cells were subjected to SN to induce hepatotoxicity, followed by treatment with COP and VE. Evaluations included assays for cell viability, oxidative stress markers, apoptosis, and inflammatory cytokines. Additionally, transcriptomic and metabolomic analyses were performed to delineate the underlying molecular mechanisms. The findings demonstrated that COP and VE significantly ameliorated SN-induced cytotoxicity, oxidative stress, and apoptosis. It was evidenced by restored cell viability, enhanced antioxidant enzyme activity, reduced cytoplasmic enzyme leakage, and decreased levels of malondialdehyde and inflammatory cytokines, with COP showing superior efficacy. The RNA sequencing revealed that COP treatment suppressed the SN-activated aminoacyl-tRNA biosynthesis pathway and TGF-β/NF-κB signaling pathways, thereby mitigating amino acid depletion, apoptosis, and inflammation. Moreover, COP treatment upregulated genes associated with protein folding, bile acid synthesis, and DNA repair. Metabolomic analysis corroborated these results, showing that COP restored amino acid levels and enhanced bile acid metabolism, alleviating SN-induced metabolic disruptions. These findings offered significant insights into the protective mechanisms of COP underscoring its prospective application in treating liver injuries.

Overactivation of Signal Transducer and Activator of Transcription 3 in Canine Hepatocellular Carcinoma and Its Prognostic Significance.

Phosphorylated signal transducer and activator of transcription 3 (pSTAT3), which is related to anti-apoptosis, cellular proliferation, invasion and migration of tumours, has prognostic significance in malignant tumours in humans as well as in canine melanoma. However, the significance of pSTAT3 in canine liver tissues has not yet been evaluated. This study's objective was to compare its expression in canine normal, non-neoplastic hepatic disease and hepatocellular carcinoma (HCC) tissues by immunohistochemical analysis. Furthermore, the association between pSTAT3 immunostaining and clinicopathological factors was investigated. Overall, 68 canine liver tissues, including 10 normal liver tissues, 30 non-neoplastic hepatic disease tissues and 28 HCC tissues were examined, revealing distinct differences in pSTAT3 immunostaining among the groups. (p < 0.001). Additionally, high pSTAT3 immunostaining was significantly associated with increased tumour size (5 > cm) (p = 0.041), and metastasis (p = 0.046). Furthermore, Kaplan-Meier survival curve analysis revealed a correlation between high pSTAT3 immunostaining and poor disease-free survival (p = 0.013) and overall survival (p = 0.011). These findings suggest that overactivation of STAT3 is associated with poor prognosis in canine HCC. Therefore, pSTAT3 is considered a potential prognostic marker and therapeutic target for canine HCC.

Relationship between the sonographic foetal lung-to-liver ratio of mean grey level and progesterone concentration in canine late gestation

A study was conducted to perform the quantitative analysis of canine foetal lung and liver sonographic images in the late term of gestation (58-67 days) and to evaluate its association with serum progesterone concentration. Thirty-two pregnant bitches were selected for the study. Lung development of foetus occurs in three different stages viz the pseudo glandular (40-48), canalicular (49-56 d) and saccular phase (57-63 d) of pregnancy. Lungs of the foetus at the time of parturition are in saccular phase of development and the alveolar phase occurs in the neonatal period. In the present study, sonographic changes occurring in the lung during the saccular phase of foetal lung development was evaluated. Mean grey level of the foetal lung and liver sonographic images were estimated using dedicated image analysis software Image J. The lung-to-liver ratio (LLR) of mean grey level (MGL) was recorded. Corresponding progesterone concentrations were also estimated for each animal. Correlation statistical analysis revealed a significant correlation between LLR of MGL and progesterone concentration in the animals selected for the study. LLR of MGL is a prognostic indicator of foetal survival and since LLR of MGL and progesterone are positively correlated, it can be used as an indicator of foetal maturity in place of progesterone concentration. Keywords: Lung-to-liver ratio of mean grey level, ultrasonography, progesterone

Evaluating the Effectiveness of Ursodeoxycholic Acid in Various Treatment Approaches for Canine Liver Dysfunction: A Comparative Study

Evaluating the Effectiveness of Ursodeoxycholic Acid in Various Treatment Approaches for Canine Liver Dysfunction: A Comparative Study

Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model

Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions.Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant.Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.

RNAscope: a novel method for the detection of Heterobilharzia americana ova in canine liver.

Canine schistosomiasis caused by Heterobilharzia americana can lead to severe morbidity and eventual mortality, in part due to the deposition of fluke ova in the liver and gastrointestinal tract, which promotes an influx of peri-ova inflammatory cells. Although fluke eggs can be identified in H&E-stained histologic sections, cases exist in which only fragments of the ova persist, or the egg is obscured by inflammatory infiltrates, which can confound definitive histologic diagnosis. Unfortunately, antibodies specific to Heterobilharzia are not commercially available for immunohistochemical labeling. Therefore, we aimed to use an RNA in situ hybridization strategy to fluorescently label Heterobilharzia ova. Using the H. americana 18S rRNA sequence, we developed an RNA probe and validated its performance on archival formalin-fixed, paraffin-embedded canine tissue. A positive signal was observed for all identifiable ova, fragmented and whole. Use of this methodology could aid understanding of the pathogenesis of H. americana infection in dogs. This technique augments standard diagnostic methodology, enabling spatial colocalization of fluke ova and inflammatory infiltrates when using fluorescent techniques.

The choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function.

Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments.

Canine primary liver tumors treated with stereotactic body radiation therapy: A case series.

Stereotactic body radiation therapy (SBRT) is an increasingly used alternative treatment option for nonresectable hepatocellular carcinoma (HCC) in people. Comparatively, the publication of SBRT of dogs with HCC is limited. The objective of this retrospective, descriptive case series was to evaluate the clinical outcomes and toxicity data of SBRT in dogs with HCC and imaging-documented primary liver tumors using volumetric-modulated arc therapy delivery at two private institutions. Medical records of 14 dogs treated between 2018 and 2023 were reviewed. All dogs had macroscopic tumors, and 9 of 14 dogs had HCC diagnoses confirmed on cytology or histopathology. The median longest tumor diameter was 5.5cm. The median percentage of planning target volume relative to liver volume was 27.1%. Most dogs were treated with three daily fractions of 7-7.5Gy. All dogs completed their radiotherapy protocols. Three of nine HCC dogs experienced partial responses and clinical improvement. Five of nine HCC dogs had stable disease. Overall median survival time was 164 days for nine HCC dogs (range: 93-706 days). One late grade 5 liver and two late grade 3 kidney side effects were reported. One dog received repeated SBRT to the same HCC treatment field, and one dog had two courses of SBRT to bifocal HCC treatment fields, both with no more than grade 2 acute and chronic toxicities.

Harmonic Motion Imaging-Guided Focused Ultrasound Ablation: Comparison of Three Focused Ultrasound Interference Filtering Methods

Harmonic motion imaging (HMI) is an acoustic radiation force-based elasticity imaging technique, which can be used to monitor changes in tissue mechanical properties caused by focused ultrasound (FUS)-induced thermal ablation. In conventional HMI, the amplitude-modulated FUS sequence and imaging pulse are transmitted simultaneously. With this method, the high-amplitude FUS signal must be separated from the imaging data for tissue displacement estimation. Frequency domain notch filtering and bandpass filtering were previously used to filter FUS interference from imaging data. However, FUS interference becomes more pronounced at the increased intensities and durations necessary for thermal ablation, rendering frequency domain filtering challenging. In this study, three methods for FUS interference filtering during HMI-guided FUS ablation (HMIgFUS) were compared. The methods were notch filtering; interleaved HMIgFUS, with interleaved FUS and imaging pulses; and FUS-net, a convolutional neural network-based U-Net autoencoder developed by our group for FUS interference filtering in radiofrequency data. FUS-net was applied here for the first time for the purpose of ablation displacement monitoring. The three filtering methods were tested during 20 HMIgFUS acquisitions in an ex vivo canine liver using a range of peak positive pressures from 11 to 18 MPa and durations ranging from 60 to 180 seconds. The B-mode mean squared errors (MSEs) and displacement amplitude contrast-to-noise ratios (CNRs) of the three methods were calculated and compared. The interleaved method for HMIgFUS was found to be significantly robust in avoiding FUS interference in all tested cases for FUS ablation monitoring, especially cases with high FUS pressure and long durations, as opposed to traditional notch filtering and FUS-net filtering. CNRs obtained from displacement amplitude maps in the interleaved data set were significantly higher in all cases than those obtained from the notch filtered and FUS-net data sets. There was not a significant trend in displacement CNR between the FUS-net and notch filtered data sets. However, B-mode MSE was found to be significantly higher when comparing the FUS-net and interleaved data sets as opposed to the notch filtered and interleaved data sets, suggesting further potential of FUS-net as an FUS interference filtering method. These findings indicate the robustness of interleaved HMIgFUS in avoiding FUS interference during HMIgFUS monitoring and the advantages, limitations and future potential of FUS-net and notch filtering.

Diagnostic quality of ultrasound-guided fine-needle aspirates samples from the canine liver and spleen is not significantly affected by using 22-, 23-, and 25-gauge needles.

Ultrasound-guided fine-needle aspirates (FNA) of the liver and spleen for cytological analysis are a commonly performed procedure in canine veterinary practice. Based on our review of the literature, this is the first published study investigating whether needle size affects the diagnostic quality of hepatic and splenic samples. The aim of this prospective analytical study was to compare the diagnostic quality of ultrasound-guided FNA cytological samples of canine liver and spleen based on cellularity, blood contamination, and overall cell preservation between three different needle sizes (22-, 23-, and 25-gauge). A total of 282 splenic aspirates from 94 dogs and 348 hepatic aspirates from 116 dogs were enrolled in the study and examined by two board-certified veterinary clinical pathologists. In this study, no significant differences in diagnostic quality were identified between different needle gauge sizes when sampling canine liver and spleen. Blood contamination was higher using 22-gauge needles compared with 25-gauge needles (P=0.024) when sampling the liver.

Canine intrahepatic portosystemic shunts: Interlobar and intralobar classifications.

This two-part study design showed that a canine congenital intrahepatic portosystemic shunt (IPSS) may be classified by its location within a liver fissure (interlobar) or lobe (intralobar). A prospective anatomic study reviewed normal canine liver morphology and showed the CT angiography (CTA) appearance of the normal canine ductus venosus (DV), which was confirmed via dissection and literature review to be between the papillary process and left-lateral liver lobe (in the fissure for ligamentum venosum). A retrospective multi-institutional case series documented the frequency of imaging findings in 56 dogs with a single IPSS that underwent portal CTA at Cornell University or the Schwarzman Animal Medical Center between June 2008 and August 2022. An interlobar IPSS was seen in 24 of 56 (43%) dogs, all arose from the left portal branch except one. These shunts were typically near the median plane, remained interlobar throughout the course, and were nearly always (96%) craniodorsal to the porta hepatis. Four types were distinguished: patent DV (11 dogs), left interlobar (11 dogs), right interlobar (1 dog), and ventral interlobar (1 dog). Only about half (46%) were in the fissure for ligamentum venosum and therefore classified as a patent DV. An intralobar IPSS was seen in 32 of 56 (57%) dogs, most (88%) originated from the right portal branch and were in the right-lateral liver lobe (21 dogs) or caudate process (7 dogs). During canine portal CTA, documenting the interlobar or intralobar location of an IPSS might increase the consistency and validity of IPSS description.

The comparative performance of a custom Canine NanoString® panel on FFPE and snap frozen liver biopsies

Formalin-Fixed Paraffin Embedded (FFPE) biopsies would provide a critical mass of cases to allow investigation of canine liver disease, however their use is often limited by challenges typically associated with transcriptomic analysis. This study evaluates the capability of NanoString® to measure the expression of a broad panel of genes in FFPE liver samples. RNA was isolated from matched histopathologically normal liver samples using FFPE (n = 6) and snap frozen in liquid nitrogen (n = 6) and measured using a custom NanoString® panel. Out of the 40 targets on the panel, 27 and 23 targets were above threshold for non-diseased snap frozen and FFPE tissue respectively. The binding density and total counts were significantly reduced in the FFPE samples relative to the snap frozen samples (p = 0.005, p = 0.01, respectively), confirming a reduction in sensitivity. The concordance between the snap frozen and FFPE samples was high, with correlations (R) ranging between 0.88 and 0.99 between the paired samples. An additional 14 immune-related targets, undetectable the non-diseased FFPE liver, were above threshold when the technique was applied to a series of diseased samples, further supporting their inclusion on this panel. This use of NanoString® based analysis opens up huge opportunity for retrospective evaluation of gene signatures in larger caseloads through harnessing the capacity of archived FFPE samples This information used alongside clinical and histological data will not only afford a way to explore disease etiopathogenesis, it may also offer insight into sub-types of liver disease in dogs, which cannot be discerned using more traditional diagnostic methods.

Lyciumbarbarum polysaccharides ameliorate canine acute liver injury by reducing oxidative stress, protecting mitochondrial function, and regulating metabolic pathways.

The development of acute liver injury can result in liver cirrhosis, liver failure, and even liver cancer, yet there is currently no effective therapy for it. The purpose of this study was to investigate the protective effect and therapeutic mechanism of Lyciumbarbarum polysaccharides (LBPs) on acute liver injury induced by carbon tetrachloride (CCl4). To create a model of acute liver injury, experimental canines received an intraperitoneal injection of 1 mL/kg of CCl4 solution. The experimental canines in the therapy group were then fed LBPs (20 mg/kg). CCl4-induced liver structural damage, excessive fibrosis, and reduced mitochondrial density were all improved by LBPs, according to microstructure data. By suppressing Kelch-like epichlorohydrin (ECH)-associated protein 1 (Keap1), promoting the production of sequestosome 1 (SQSTM1)/p62, nuclear factor erythroid 2-related factor 2 (Nrf2), and phase II detoxification genes and proteins downstream of Nrf2, and restoring the activity of anti-oxidant enzymes like catalase (CAT), LBPs can restore and increase the antioxidant capacity of liver. To lessen mitochondrial damage, LBPs can also enhance mitochondrial respiration, raise tissue adenosine triphosphate (ATP) levels, and reactivate the respiratory chain complexes I‒V. According to serum metabolomics, the therapeutic impact of LBPs on acute liver damage is accomplished mostly by controlling the pathways to lipid metabolism. 9-Hydroxyoctadecadienoic acid (9-HODE), lysophosphatidylcholine (LysoPC/LPC), and phosphatidylethanolamine (PE) may be potential indicators of acute liver injury. This study confirmed that LBPs, an effective hepatoprotective drug, may cure acute liver injury by lowering oxidative stress, repairing mitochondrial damage, and regulating metabolic pathways.

Association of cytochrome P450 2D15 (CYP2D15) nonsynonymous polymorphisms and exon 3 deleted RNA splice variant with CYP2D15 protein content and enzyme function in dog liver microsomes.

CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3-delta) was associated with differences in CYP2D15-selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15-selective activities measured included dextromethorphan o-demethylation and tramadol o-demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3-delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3-delta was not correlated with CYP2D15 activities or protein content. Compared with wild-type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p< .05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O-demethylation activities versus wild-type livers (p< .05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver.

Ultrasonographic appearance and possible clinical relevance of hyperechoic foci of mineralization in the canine intrahepatic biliary tree

Background: The evidence of mineralizations in the canine liver is usually considered an incidental finding of unclear clinical significance, frequently observed in small size old dogs. Aim: To describe the ultrasound features of intrahepatic biliary tree foci of mineralization, to assess their clinical relevance and their possible relationship with other gastrointestinal pathological disorders. Methods: A retrospective analysis evaluating the database of canine patients admitted to two referral veterinary centers we carried out. All dogs under study underwent an abdominal ultrasound examination in which intrahepatic biliary tree mineralization was found. Clinical and anamnestic data of the included dogs were reviewed. Results: Approximatively 90% of the patients showed ultrasonographic abnormalities regarding the biliary system, and over 85% presented ultrasonographic abnormalities of the hepatic parenchyma. In 81.2% of dogs, ultrasonographic anomalies in the digestive tract were observed. In approximately the half of our patients, we evidenced increased liver enzymes (ALP, ALT and GGT). At clinical evaluation, 84.4% (23 out of 32 dogs) of patients showed signs of gastrointestinal disease that persisted for over three months. Conclusion: The presence of intrahepatic biliary tree mineralizations is an unusual and frequently incidental finding that could be related to a bile stasis condition, a chronic inflammatory disease involving the biliary system and the hepatic parenchyma, and it could be associated to a liver-gut axis alteration.

Reduced Klotho expression and its prognostic significance in canine hepatocellular carcinoma.

Klotho is an anti-ageing gene and is known to act as a tumour suppressor in human hepatocellular carcinoma (HCC). According to a previous study, Klotho is present in normal canine mammary glands, and down-expression in tumours is positively associated with negative prognosis. However, the presence and significance of Klotho in canine HCC has not yet been reported. This study aimed to confirm Klotho expression in normal canine liver tissues using western blotting and immunohistochemistry, and whether the expression differed in non-neoplastic liver disease and HCC. Furthermore, correlation between clinicopathologic features and expression of Klotho was evaluated. All of the normal liver tissues showed the presence of Klotho, and Klotho expression was significantly decreased in the HCC tissue as compared to the non-neoplastic hepatic tissue. Additionally, Klotho expression was significantly associated with tumour size (P=.045), liver enzyme (alanine aminotransferase (ALT)) (P=.018), and metastasis (P=.024). Analysis of the survival curve revealed that reduced Klotho expression was significantly associated with poor disease-free survival (P=.041) in HCC. These results show that Klotho expression is present in normal canine liver tissue and that reduced Klotho expression is associated with poor prognosis in canine HCC. Thus, Klotho was presumed to be a potential clinical prognostic marker for canine HCC.