Characterization of clinical presentation, histological features, ultrasonographic findings, and survival in 29 dogs with granulomatous hepatitis.

BACKGROUND: Granulomatous hepatitis (GH) is a form of chronic hepatitis (CH) in dogs for which limited information is published. HYPOTHESIS: Describe the clinical presentation, clinical pathology, ultrasound, and hepatic histopathology findings and to report survival times in dogs with GH. ANIMALS: Twenty-nine client-owned dogs with GH. METHODS: Retrospective observational study. Pathology records were searched. Inclusion criteria included a histopathologic diagnosis of GH, absence of an identified etiology or evidence of extrahepatic granulomatous disease, and a medical record available for review. Clinical presentation, clinical pathologic findings, treatment protocols, and survival times were recorded. Available hepatic biopsy material was graded and scored, and ultrasound evaluations reviewed. RESULTS: The median age was 7 years (range, 0.66-12 years). Nineteen breeds were represented. Decreased appetite (19/29), lethargy (16/29), and fever (13/29) were seen most commonly. All dogs had increased serum transaminase activities, whereas 21/29 and 12/24 had hyperbilirubinemia and neutrophilia, respectively. Ultrasonographic findings included hepatomegaly (12/22), nodular parenchymal lesions (9/22), and hyperechoic parenchymal bands (8/22). Histopathologic necroinflammatory scores were moderate to severe in 16/19 dogs, and fibrosis scores were mild in 14/19 dogs. Treatments varied and included antibiotics, immunosuppressive drugs, and hepatoprotectants. Overall median survival was 635 days (range, 1-2482 days). CONCLUSION AND CLINICAL IMPORTANCE: Granulomatous hepatitis in dogs is associated with high histopathologic grade, fever, neutrophilia, and a high incidence of hepatomegaly and focal parenchymal lesions on ultrasound examination. Despite disease severity on presentation, dogs with GH can have a good outcome with prolonged survival.

Pre-operative Hemostatic Status in Dogs Undergoing Splenectomy for Splenic Masses.

Portal system thrombosis is a rare but potentially fatal complication of splenectomy in dogs. The mechanism behind development of post-operative portal system thrombosis is unclear but may include alterations of portal blood flow following surgery, acquired hypercoagulability and endothelial dysfunction. The aim of the study was to evaluate hemostatic biomarkers in hemodynamically stable (heart rate <130 beats/min, blood lactate < 2.5 mMol/L) and non-anemic (hematocrit >35%) dogs prior to splenectomy for splenic masses. Our hypothesis was that this population of stable dogs would have pre-existing laboratory evidence of hypercoagulability unrelated to shock, bleeding, anemia, or other pre-operative comorbidities. Pre-operatively, abdominal ultrasonography was performed and blood was collected for platelet enumeration, prothrombin time (PT), activated partial thromboplastin time (aPTT), kaolin-activated thromboelastography (TEG), fibrinogen, von Willebrand factor activity (vWF:Ag), antithrombin and thrombin-antithrombin complex (TAT). Histopathological diagnosis and 30-day survival were recorded. None of the 15 enrolled dogs had pre-operative sonographic evidence of portal system thrombosis. Three of fifteen dogs were thrombocytopenic, three had thrombocytosis, three were hyperfibrinogenemic, one had low vWF:Ag, three had mild prolongations of PT and none had abnormal aPTT. Based on the TEG G value, 13/15 dogs were hypercoagulable (mean ± SD 13.5 ± 5.4 kd/s). Antithrombin deficiency was identified in 9/15 dogs (mean ± SD 68.7 ± 22.7%) with 5/9 having concurrently elevated TAT suggesting active thrombin generation. No dogs developed portal system thrombosis and all achieved 30-day survival. Pre-operative hypercoagulability was recognized commonly but its association with post-operative thrombosis remains undetermined.

Serum 25-hydroxyvitamin D and C-reactive protein and plasma von Willebrand concentrations in 23 dogs with chronic hepatopathies.

BACKGROUND: Serum concentrations of 25-hydroxyvitamin D (25(OH)VD) and C-reactive protein (CRP) and von Willebrand's factor (vWF) concentration correlate with histopathologic disease grade and stage in chronic inflammatory and fibrotic hepatopathies (CH) in humans. OBJECTIVES: To evaluate serum 25(OH)VD and serum CRP concentrations and plasma vWF concentration and determine if they correlate with histopathologic and biochemical variables in dog with CH. ANIMALS: Twenty-three client-owned dogs with a histopathologic diagnosis of CH were prospectively enrolled. METHODS: Blood samples were collected before liver biopsy. Correlations between biomarkers and clinical pathological and histopathologic variables were evaluated using Pearson's or Spearman's test. RESULTS: Serum 25(OH)VD concentration (median, 213 nmol/L; range, 42-527 nmol/L) was negatively correlated with serum aspartate aminotransferase activity (AST; rho = -0.59, P < .01), polymorphonuclear neutrophil count (PMN; r = -0.46, P < .05), and positively correlated with serum albumin concentration (r = 0.69, P < .001). Serum CRP concentration (median, 7.4 µg/L; range, 1-44.9 µg/L) was positively correlated with overall histopathologic necroinflammatory activity (r = 0.78, P < .001) and fibrosis score (rho = 0.49, P < .05). Plasma vWF concentration (median, 73.3%; range, 15-141%) was positively correlated with fibrosis score (r = 0.53, P < .05) and prothrombin time (rho = 0.67, P < .01), and negatively correlated with serum albumin concentration (r = -0.73, P < .001). CONCLUSION AND CLINICAL IMPORTANCE: In dogs with CH, serum 25(OH)VD concentration was negatively correlated with disease activity, whereas serum CRP concentration and plasma vWF concentration were positively correlated with histopathologic grade and stage. Our results provide preliminary evidence that these biomarkers may be useful to assess grade and stage of CH in dogs in the absence of liver biopsy.

Evaluation of coagulation parameters in dogs with gallbladder mucoceles.

BACKGROUND: Gallbladder mucocele (GBM) is a common biliary disorder in dogs. Limited information is available on the coagulation status of dogs with GBM. HYPOTHESIS/OBJECTIVES: To determine patterns of coagulation alterations in dogs with GBM and correlate them with clinicopathologic abnormalities and ultrasonographic findings of disease severity. ANIMALS: Twenty-three dogs with GBM identified on ultrasound examination were prospectively enrolled. METHODS: At the time of GBM identification, blood and urine were collected for CBC, serum biochemical panel, urinalysis, prothrombin time, activated partial thromboplastin time (aPTT), factor VIII, protein C (PC), von Willebrand's factor (vWF), antithrombin activity, fibrinogen, D-dimers, and thromboelastrography (TEG). Gallbladder mucoceles were classified into ultrasound types 1 to 5. Medical records were reviewed for clinical presentation, underlying conditions and to determine if systemic inflammatory response syndrome (SIRS) was present. RESULTS: Based on TEG parameters, maximal amplitude, and G, 19/23 (83%) of dogs with GBM had evaluations consistent with hypercoagulability. On plasma-based coagulation testing, dogs with GBM had increased total PC activity (20/23, 87%), fibrinogen (9/23, 39%), platelet count (9/23, 39%), and D-dimers (6/15, 40%) as well as prolongations in aPTT (9/22, 41%) and low vWF activity (5/21, 24%). No correlation was found between TEG G value and any coagulation or clinical pathology variables, ultrasound stage of GBM or disease severity as assessed by the presence of SIRS. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with ultrasonographically identified GBM have changes in whole blood kaolin-activated TEG supporting a hypercoagulable state although traditional plasma-based coagulation testing suggests that a complex state of hemostasis exists.

Hepatocyte ploidy in cats with and without hepatocellular carcinoma.

BACKGROUND: Domestic cats rarely develop hepatocellular carcinoma. The reason for the low prevalence is unknown. Reductions in hepatocellular ploidy have been associated with hepatic carcinogenesis. Recent work in mice has shown that livers with more polyploid hepatocytes are protected against the development of hepatocellular carcinoma. Hepatocyte ploidy in the domestic cat has not been evaluated. We hypothesized that ploidy would be reduced in peri-tumoral and neoplastic hepatocytes compared to normal feline hepatocytes. Using integrated fluorescence microscopy, we quantified the spectra of ploidy in hepatocellular carcinoma and healthy control tissue from paraffin embedded tissue sections. RESULTS: Feline hepatocytes are predominantly mononuclear and the number of nuclei per hepatocyte did not differ significantly between groups. Normal cats have a greater number of tetraploid hepatocytes than cats with hepatocellular carcinoma. CONCLUSIONS: Total hepatocellular polyploidy in normal cat liver is consistent with values reported in humans, yet cellular ploidy (nuclei per cell) is greater in humans than in cats. Tetraploid cat hepatocytes are predominantly mononuclear.

Approach to the Diagnosis of Hepatocutaneous Syndrome in Dogs: A Retrospective Study and Literature Review.

Superficial necrolytic dermatitis (SND) is a rare and often fatal disease in dogs that has been associated with pancreatic neuroendocrine neoplasia (SND/EN) and hepatocutaneous syndrome (SND/HCS). Although various combinations of diagnostics have been used to differentiate these two causes of SND, there are currently no data on which combination would enable the most timely and noninvasive way to diagnose HCS. Medical records were reviewed retrospectively (2004-2018) for dogs with SND/HCS (n = 24) and SND/EN (n = 1). These data were compared with cases found by review of the literature of dogs with SND/HCS (n = 105) and SND/EN (n = 13). The most consistent findings with SND were dermatological lesions affecting paw pads or mucocutaneous junctions (143/143, 100%) and marked plasma hypoaminoacidemia (58/58, 100%). On ultrasound, a honeycomb liver was seen in 62/63 (98%) dogs with SND/HCS but none with SND/EN. Six out of 23 (26%) dogs in the retrospective study with SND/HCS had marked keratinocyte apoptosis, a finding that was associated with diabetes mellitus. This study suggests that in dogs with characteristic skin lesions, an amino acid profile permits a noninvasive diagnosis of SND. An abdominal ultrasound can then assist in the differentiation of SND/HCS and SND/EN.

Hemorrhage and complications associated with percutaneous ultrasound guided liver biopsy in dogs.

BACKGROUND: Liver biopsy is often necessary to obtain a diagnosis in dogs with hepatobiliary disease. Hemorrhage after biopsy is a concern. OBJECTIVE: To describe the extent of hemorrhage and incidence of complications after percutaneous ultrasound guided liver biopsy (PUGLB) in dogs and to examine risk factors for hemorrhage or complications. ANIMALS: One hundred two client owned dogs with suspected hepatobiliary disease that underwent PUGLB. METHODS: Medical records were retrospectively reviewed. Using human guidelines, major hemorrhage was defined as an absolute decrease in the PCV (ΔPCV) ≥ 6%. Complications were defined separately as clinically relevant physiologic compromise that necessitated intervention or death. The relationship between ΔPCV and the occurrence of complications and the initial PCV, coagulation variables, serum activity of liver-derived enzymes, serum bilirubin concentration, number of biopsies, biopsy needle gauge, radiologist experience, histological diagnosis, and ultrasound variables were compared. RESULTS: Before PUGLB, most aberrations in coagulation variables were mild. After biopsy a decrease in PCV occurred in 87/102 (85.3%) dogs. The mean ΔPCV was -7.2% ± 4.5%. Major hemorrhage occurred in 43/102 (42.2%) dogs and complications in 2/102 (1.9%). ΔPCV was significantly positively correlated with PCV before biopsy (r = .47, P = .004). There was no correlation between ΔPCV or complications with any of the variables examined. CONCLUSION AND CLINICAL IMPORTANCE: Percutaneous ultrasound guided liver biopsy in the population of dogs in the current study, with normal or mild abnormalities in coagulation, results in a high incidence of clinically silent, major hemorrhage (42.5%), but few complications (1.9%).

ACVIM consensus statement on the diagnosis and treatment of chronic hepatitis in dogs.

This consensus statement on chronic hepatitis (CH) in dogs is based on the expert opinion of 7 specialists with extensive experience in diagnosing, treating, and conducting clinical research in hepatology in dogs. It was generated from expert opinion and information gathered from searching of PubMed for manuscripts on CH, the Veterinary Information Network for abstracts and conference proceeding from annual meetings of the American College of Veterinary Medicine and the European College of Veterinary Medicine, and selected manuscripts from the human literature on CH. The panel recognizes that the diagnosis and treatment of CH in the dog is a complex process that requires integration of clinical presentation with clinical pathology, diagnostic imaging, and hepatic biopsy. Essential to this process is an index of suspicion for CH, knowledge of how to best collect tissue samples, access to a pathologist with experience in assessing hepatic histopathology, knowledge of reasonable medical interventions, and a strategy for monitoring treatment response and complications.

Mechanism of inhibition of taurolithocholate-induced retrieval of plasma membrane MRP2 by cyclic AMP and tauroursodeoxycholate.

Taurolithocholate (TLC) produces cholestasis by inhibiting biliary solute secretion in part by retrieving MRP2 from the plasma membrane (PM). Tauroursodeoxycholate (TUDC) and cAMP reverse TLC-induced cholestasis by inhibiting TLC-induced retrieval of MRP2. However, cellular mechanisms for this reversal are incompletely understood. Recently, we reported that TLC decreases PM-MRP2 by activating PKCε followed by phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS). Thus, cAMP and TUDC may reverse TLC-induced cholestasis by inhibiting the TLC/PKCε/MARCKS phosphorylation pathway. We tested this hypothesis by determining whether TUDC and/or cAMP inhibit TLC-induced activation of PKCε and phosphorylation of MARCKS Studies were conducted in HuH-NTCP cell line and rat hepatocytes. Activation of PKCε was determined from the translocation of PKCε to PM using a biotinylation method. Phosphorylation of MARCKS was determined by immunoblotting with a phospho-MARCKS antibody. TLC, but not cAMP and TUDC, activated PKCε and increased MARCKS phosphorylation in HuH-NTCP as well in rat hepatocytes. Treatment with TUDC or cAMP inhibited TLC-induced activation of PKCε and increases in MARCKS phosphorylation in both cell types. Based on these results, we conclude that the reversal of TLC-induced cholestasis by cAMP and TUDC involves, at least in part, inhibition of TLC-mediated activation of the PKCε/MARCKS phosphorylation pathway.

Retrospective evaluation of acute liver failure in dogs (1995-2012): 49 cases.

OBJECTIVE: To characterize the clinical presentation and outcome of dogs with acute liver failure (ALF). DESIGN: Retrospective case series from January 1995 to December 2012. SETTING: University teaching hospital. ANIMALS: Forty-nine dogs were diagnosed with ALF defined as the acute onset of clinical signs accompanied by serum hyperbilirubinemia and coagulopathy (prothrombin time >1.5 times the upper limit of the reference interval) with or without signs of hepatic encephalopathy. METHODS: Medical records were retrospectively analyzed for clinical presentation, history, physical examination findings, clinicopathologic data, diagnostic imaging findings, hepatic histopathology, treatment, and outcome. MAIN RESULTS: Presenting signs included anorexia (28/49, 57%), vomiting (25/49, 51%), neurologic abnormalities (17/49, 35%), and polydipsia/polyuria (10/49, 20%). Neurologic impairment compatible with hepatic encephalopathy occurred at some point during hospitalization in 28/49 (57%) of dogs. Common clinicopathologic abnormalities on presentation other than hyperbilirubinemia and increased serum liver enzyme activity included thrombocytopenia (25/49, 51%), hypoalbuminemia (23/49, 46%), leukocytosis (17/49, 34%), anemia (14/49, 29%), hypokalemia (13/49, 27%), and hypoglycemia (10/49, 20%). The causes of ALF included neoplasia (13/49, 27%), presumptive leptosporosis (4/49, 8%), and ischemia (1/49, 2%). The remaining cases were idiopathic although 15 of these dogs had exposure to possible hepatotoxins. Common lesions in the 35/49 (71%) dogs that had hepatic histopathology were necrosis (19/39, 48%), lipidosis (16/39, 41%), vacuolar change (7/49, 14%), and inflammation (4/49, 8%). Complications included ascites (20/49, 41%), bleeding tendencies (14/49, 29%), pancreatitis (12/49, 24%), and acute tubular necrosis (11/49, 22%). Seven (14%) dogs survived to discharge. Survivors had higher alanine aminotransferase activity, and were more likely to maintain normal albumin concentrations and not develop clinical bleeding or ascites during hospitalization. CONCLUSIONS: Canine ALF is associated with multiple etiologies and a high mortality rate. Strategies to increase survival are urgently required.

Hydrophobic bile acid apoptosis is regulated by sphingosine-1-phosphate receptor 2 in rat hepatocytes and human hepatocellular carcinoma cells.

The hepatotoxic bile acid glycochenodeoxycholate (GCDC) modulates hepatocyte cell death through activation of JNK, Akt, and Erk. The nonhepatotoxic bile acid taurocholate activates Akt and Erk through the sphingosine-1-phosphate receptor 2 (S1PR2). The role of the S1PR2 in GCDC-mediated apoptosis and kinase activation is unknown. Studies were done in rat hepatocytes, HUH7 cells, and HUH7 cells stably transfected with rat Ntcp (HUH7-Ntcp). Cells were treated with GCDC and apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for the active cleaved fragment of caspase 3. Kinase activation was determined by immunoblotting with phospho-specific antibodies. JTE-013, an inhibitor of S1PR2, significantly attenuated morphological evidence of GCDC-induced apoptosis and prevented caspase 3 cleavage in rat hepatocytes and HUH7-Ntcp cells. In hepatocytes, JTE-013 mildly suppressed, augmented, and had no effect on GCDC-induced JNK, Akt, and Erk phosphorylation, respectively. Similar results were seen in HUH7-Ntcp cells except for mild suppression of JNK and Erk phosphorylation. Knockdown of S1PR2 in HUH7-Ntcp augmented Akt, inhibited JNK, and had no effect on Erk phosphorylation. GCDC failed to induce apoptosis or kinase activation in HUH7 cells. In conclusion, SIPR2 inhibition attenuates GCDC-induced apoptosis and inhibits and augments GCDC-induced JNK and Akt phosphorylation, respectively. In addition, GCDC must enter hepatocytes to mediate cell death or activate kinases. These results suggest that SIPR2 activation is proapoptotic in GCDC-induced cell death but that this effect is not due to direct ligation of the S1PR2 by the bile acid.

p38 MAPK α and ß isoforms differentially regulate plasma membrane localization of MRP2.

In hepatocytes, cAMP both activates p38 mitogen-activated protein kinase (MAPK) and increases the amount of multidrug resistance-associated protein-2 (MRP2) in the plasma membrane (PM-MRP2). Paradoxically, taurolithocholate (TLC) activates p38 MAPK but decreases PM-MRP2 in hepatocytes. These opposing effects of cAMP and TLC could be mediated via different p38 MAPK isoforms (α and ß) that are activated differentially by upstream kinases (MKK3, MKK4, and MKK6). Thus we tested the hypothesis that p38α MAPK and p38ß MAPK mediate increases and decreases in PM-MRP2 by cAMP and TLC, respectively. Studies were conducted in hepatocytes isolated from C57BL/6 wild-type (WT) and MKK3-knockout (MKK3(-/-)) mice and in a hepatoma cell line (HuH7) that overexpresses sodium-taurocholate cotransporting polypeptide (NTCP) (HuH-NTCP). Cyclic AMP activated MKK3, p38 MAPK, and p38α MAPK and increased PM-MRP2 in WT hepatocytes, but failed to activate p38α MAPK or increase PM-MRP2 in MKK3(-/-) hepatocytes. In contrast to cAMP, TLC activated total p38 MAPK but decreased PM-MRP2, and did not activate MKK3 or p38α MAPK in WT hepatocytes. In MKK3(-/-) hepatocytes, TLC still decreased PM-MRP2 and activated p38 MAPK, indicating that these effects are not MKK3-dependent. Additionally, TLC activated MKK6 in MKK3(-/-) hepatocytes, and small interfering RNA knockdown of p38ß MAPK abrogated TLC-mediated decreases in PM-MRP2 in HuH-NTCP cells. Taken together, these results suggest that p38α MAPK facilitates plasma membrane insertion of MRP2 by cAMP, whereas p38ß MAPK mediates retrieval of PM-MRP2 by TLC.

Protein kinase Cδ protects against bile acid apoptosis by suppressing proapoptotic JNK and BIM pathways in human and rat hepatocytes.

Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.

Rab11, but not Rab4, facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation to the plasma membrane.

Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking. Rab4 and Rab11 are involved in vesicular trafficking to the plasma membrane from early endosomes and recycling endosomes, respectively. Tauroursodeoxycholate (TUDC) and cAMP increase bile formation, in part, by increasing plasma membrane localization of multidrug resistance-associated protein 2 (MRP2). The goal of the present study was to determine the role of these Rab proteins in the trafficking of MRP2 by testing the hypothesis that Rab11 and/or Rab4 facilitate cAMP- and TUDC-induced MRP2 translocation to the plasma membrane. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which constitutively express MRP2. HuH-NTCP cells were transfected with Rab11-WT and GDP-locked dominant inactive Rab11-GDP or with Rab4-GDP to study the role of Rab11 and Rab4. A biotinylation method and a GTP overlay assay were used to determine plasma membrane MRP2 and activation of Rab proteins (Rab11 and Rab4), respectively. Cyclic AMP and TUDC increased plasma membrane MRP2 and stimulated Rab11 activity. Plasma membrane translocation of MRP2 by cAMP and TUDC was increased and inhibited in cells transfected with Rab11-WT and Rab11-GDP, respectively. Cyclic AMP (previous study) and TUDC increased Rab4 activity. However, cAMP- and TUDC-induced increases in MRP2 were not inhibited by Rab4-GDP. Taken together, these results suggest that Rab11 is involved in cAMP- and TUDC-induced MRP2 translocation to the plasma membrane.

Clinicopathological and ultrasonographic features of cats with eosinophilic enteritis.

Eosinophilic enteritis (EE) in cats is poorly characterized. The aim of the current study was to retrospectively evaluate the clinical and ultrasonographic findings in cats with histologic evidence of eosinophilic inflammation on gastrointestinal biopsy. Twenty-five cats with tissue eosinophilia on surgical (10) or endoscopic (15) biopsy of the gastrointestinal tract, having an abdominal ultrasound performed within 48 h of biopsy acquisition, were enrolled. History, clinical presentation, clinical pathology and abdominal ultrasound findings were reviewed. Intestinal biopsies were evaluated by a single pathologist and separated into two groups based on the degree of eosinophilic infiltrate: mild (<10 eosinophils/high-power field [HPF], 11/25 cats), or moderate/marked (>10 eosinophils/HPF, 14/25 cats). The former were considered primary lymphoplasmacytic or lymphocytic inflammatory bowel disease (LPE) with subtle eosinophilic infiltrates, and the latter to have EE. Signalment, history and clinical signs were similar in all cats. Only cats with EE (6/14) had palpably thickened intestines. The only distinguishing clinicopathological feature of cats with EE was the presence of peripheral eosinophilia (6/14). On ultrasound, when compared with cats with LPE, cats with EE had a greater mean jejunal wall thickness (3.34 mm ± 0.72 mm vs 4.07 mm ± 0.58 mm, respectively) and an increased incidence of thickening of the muscularis layer (1/11 and 11/14, respectively). In conclusion, ultrasonographic evidence of a prominent intestinal muscularis layer, palpably thickened intestines and peripheral eosinophilia can serve as biomarkers for the presence of EE in cats with chronic intestinal signs.

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells.

UNLABELLED: Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. CONCLUSION: Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane.

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane.

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

Phosphatidylinositol-3-kinase p110γ contributes to bile salt-induced apoptosis in primary rat hepatocytes and human hepatoma cells.

BACKGROUND & AIMS: Glycochenodeoxycholate (GCDC) and taurolithocholate (TLC) are hepatotoxic and cholestatic bile salts, whereas tauroursodeoxycholate (TUDC) is cytoprotective and anticholestatic. Yet they all act, in part, through phosphatidylinositol-3-kinase(PI3K)-dependent mechanisms ("PI3K-paradox"). Hepatocytes express three catalytic PI3K Class I isoforms (p110α/ß/γ), specific functions of which, in liver, are unclear. In other cell types, p110γ is associated with detrimental effects. To shed light on the PI3K enigma, we determined whether hydrophobic and hydrophilic bile salts differentially activate distinct p110 isoforms in hepatocytes, and whether p110γ mediates bile salt-induced hepatocyte cell death. METHODS: Isoform-specific PI3K activity assays were established to determine isoform activation by bile salts in rat hepatocytes. Activation of Akt and JNK was determined by immunoblotting. Following stimulation with hydrophobic bile salts, hepatocellular apoptosis was determined morphologically after Hoechst staining and by analysis of caspase-3/-7 activity or caspase-3 cleavage. Activity or expression of PI3K p110γ was inhibited pharmacologically (AS604850) or by knock-down using specific siRNA. RESULTS: All bile salts tested activated p110ß, while p110α was activated by TUDC and GCDC. Intriguingly, only hydrophobic bile salts activated p110γ. Inhibition of p110γ attenuated GCDC-induced Akt- and JNK-activation, but did not alter TUDC- or cAMP-induced Akt-signaling in rat hepatocytes. Inhibition or knock-down of p110γ markedly attenuated hydrophobic bile salt-induced apoptosis in rat hepatocytes and human hepatoma cell lines but did not alter Fas-, tumor necrosis factor α- and etoposide-induced apoptosis. Depletion of Ca(++) prevented GCDC-induced toxicity in rat hepatocytes but did not affect GCDC-induced Akt- and JNK-activation. CONCLUSIONS: PI3K p110γ is activated by hydrophobic, but not hydrophilic bile salts. Bile salt-induced hepatocyte apoptosis is partly mediated via a PI3K p110γ dependent signaling pathway, potentially involving JNK.

Cyclic AMP stimulates Mrp2 translocation by activating p38{alpha} MAPK in hepatic cells.

Cyclic AMP (cAMP) induces translocation of multidrug resistant protein 2 (Mrp2) to the canalicular membrane and activates p38 MAPK in rat hepatocytes. In this study, we tested the hypothesis that cAMP-induced Mrp2 translocation may be mediated via p38 MAPK. Studies were conducted in rat hepatocytes and in a human hepatoma cell line, HuH-7. In rat hepatocytes, cAMP increased Mrp2 translocation and p38 MAPK activity. These effects of cAMP were inhibited by SB203580, an inhibitor of p38 MAPK. Wortmannin, a specific inhibitor of phosphoinositide-3-kinase (PI3K), did not inhibit cAMP induced activation of p38 MAPK, indicating PI3K-independent activation of p38 MAPK by cAMP. To further define the role of p38 MAPK, molecular approaches were used to up- or downregulate p38 MAPK activity in HuH-7 cells using constitutively active (CA) and dominant-negative (DN) MAPK kinase 3 and 6 (MKK3/6). MKK3/6 are upstream kinases responsible for the activation of p38 MAPK. Cells transfected with CAMKK6 showed increased p38 MAPK activity and MRP2 translocation compared with empty vector. cAMP-induced activation of p38 MAPK was inhibited in cells transfected with DNMKK3/6 and DNMKK3, but not with DNMKK6. DNMKK3/6 and DNMKK3 also inhibited cAMP-induced MRP2 translocation. cAMP selectively activated p38alpha MAPK in HuH-7 cells. Knockdown of p38alpha MAPK by short heterodimer RNA resulted in decreased level of p38 MAPK and failure of cAMP to stimulate MRP2 translocation. Taken together, these results suggest that cAMP-induced MRP2 translocation in hepatic cells is mediated via PI3K-independent and MKK3-mediated activation of p38alpha MAPK.

PKC{epsilon}-dependent and -independent effects of taurolithocholate on PI3K/PKB pathway and taurocholate uptake in HuH-NTCP cell line.

The cholestatic bile acid taurolithocholate (TLC) inhibits biliary secretion of organic anions and hepatic uptake of taurocholate (TC). TLC has been suggested to induce retrieval of Mrp2 from the canalicular membrane via the phosphoinositide-3-kinase (PI3K)/PKB-dependent activation of novel protein kinase Cepsilon (nPKCepsilon) in rat hepatocytes. The aim of the present study was to determine whether TLC-induced inhibition of TC uptake may also involve PI3K-dependent activation of PKCepsilon in HuH7 cells stably transfected with human Na(+)-dependent TC-cotransporting polypeptide (NTCP) (HuH-NTCP cells). To avoid direct competition for uptake, cells were pretreated with TLC, washed, and then incubated with (3)H-TC to determine TC uptake. TLC produced time- and dose-dependent inhibition of TC uptake. TLC inhibited TC uptake competitively without affecting NTCP membrane translocation. A PI3K inhibitor failed to reverse TLC-induced TC uptake inhibition and TLC-inhibited PKB phosphorylation. TLC did activate nPKCepsilon as evidenced by increased membrane translocation and nPKCepsilon-Ser(729) phosphorylation. Overexpression of dominant negative-nPKCepsilon reversed TLC-induced inhibition of PKB phosphorylation but not of TC uptake. Finally, cAMP prevented TLC-induced inhibition of TC uptake via the PI3K pathway, and the prevention is due to the sum of cAMP-induced stimulation and TLC-induced inhibition of TC uptake. Taken together, these results suggest that TLC-induced inhibition of PKB, but not of TC uptake, is mediated via nPKCepsilon. Activation of nPKCepsilon and inhibition of TC uptake by TLC are not mediated via the PI3K/PKB pathway.