Hepatocyte expression and prognostic importance of senescence marker p21 in liver histopathology samples from dogs with chronic hepatitis

Abstract

BackgroundChronic hepatitis (CH) occurs commonly in dogs but is associated with a variable and largely unpredictable prognosis. p21, a cell‐cycle inhibitor and marker of cellular senescence, is upregulated in human liver disease and is a better prognostic marker than histological or clinical scoring systems.ObjectiveTo quantify hepatocyte p21 immunopositivity in histopathology samples from dogs with CH and determine its association with outcome.AnimalsTwenty‐six client‐owned dogs with histologically confirmed CH, and 15 dogs with normal liver histology.MethodsMedical records and liver histopathology samples were retrospectively reviewed to identify cases of CH. Immunohistochemistry for p21 was performed on all samples and hepatocyte immunopositivity was visually quantified. Relationships between p21 and dog age and dog survival time were statistically evaluated.ResultsHepatocyte p21 immunopositivity in dogs with CH was high (median percentage of positive hepatocytes: 90%, range: 20%‐98%) and exceeded 70% in 23/26 cases with no association with age. In control dogs, p21 immunopositivity was low (≤15% positive hepatocytes in 12/15 cases) and was positively correlated with age (r s = 0.63; P = .011). Dogs with p21 immunopositivity exceeding 91.8% (upper tercile) had significantly shorter survival compared to dogs with less than 88.9% immunopositivity (lowest tercile; 218 versus 874 days, P = .006). Increasing hepatocyte p21 immunopositivity was significantly negatively associated with survival time (HR 4.12; 95% CI 1.34‐12.63; P = .013).Conclusions and Clinical ImportanceMarked p21 immunopositivity in dogs with CH might be indicative of widespread hepatocellular senescence. A significant association with survival time also suggests a potential value for p21 quantification in determining prognosis.

Breed-related expression patterns of Ki67, γH2AX, and p21 during ageing in the canine liver

“…Senescence, as defined by γH2AX expression, is reported to increase with age in mouse hepatocytes (Wang et al 2009 ), and accumulation of these cells has prognostic significance in human liver diseases, with molecular mechanisms now being elucidated (Aravinthan and Alexander 2016 ). A previous study by our group reveals a similar increase in hepatocyte p21 expression in dogs with idiopathic chronic hepatitis (Kortum et al 2018 ), and while breed-related differences in prevalence and prognosis are recognised (Bexfield et al 2012 ), the aetiology, pathogenesis, and progression of this disease remains poorly understood.…”

“…Dogs were recruited in seven different breed categories with the aim of spanning the widest possible size and age range. Three dogs had been included as controls in the previous hepatitis study (Kortum et al 2018 ). Haematoxylin and eosin (H&E) stained sections of liver were assessed by LH under the supervision of FCC to ensure that only microscopically normal tissues were included.…”

“…An HRP-conjugated secondary antibody was applied for 30 minutes at room temperature (Dako EnVision FLEX/HRP, Agilent Dako), followed by incubation for five minutes with 3,3’-diaminobenzidine chromogen (Agilent Dako) and counterstain with Mayer’s haematoxylin for five minutes. Sections of canine skin were used as a positive control for each antibody (for example, as in (Kortum et al 2018 )), and negative control slides were prepared using commercial species-matched immunoglobulins (Agilent Dako).…”

“…Hepatocytes with brown-stained nuclei were considered positive, irrespective of staining intensity (Fig. 1 ) and as conducted in (Kortum et al 2018 ), but these were also subjectively assigned to either strong or weak staining intensity categories with full dataset provided in the electronic supplementary material . Using total numbers of positive hepatocytes from eight hpf, a mean number per hpf was calculated for each marker in each dog.…”

A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues

“…Current traditional medical treatments have a poor effect in curing numerous dog diseases and injuries, including nerve damage, kidney damage, hepatitis, and diabetes. Therefore, the scientists are constantly exploring new medical tools [14,20,21,22,23]. The emergence of stem cell therapy brings new hope for the treatment of these diseases [24,25].…”