Significance: Necroptosis is a programmed form of necrotic cell death which is implicated in a wide range of human pathological conditions. It is controlled by receptor-interacting protein kinase 3 (RIPK3) and its substrate mixed-lineage kinase domain-like protein (MLKL). Phosphorylated MLKL forms tetramers and translocates to membrane fractions to induce cell death. Here we report that MLKL tetramers further polymerize to form disulfide bond-dependent amyloid-like fibers, which are required for necroptosis. Furthermore, induced polymerization of the MLKL N-terminal domain is sufficient to activate necroptosis without RIPK3. This work reveals a mechanism for MLKL activation and generates exciting directions for necroptosis regulation.

Portal hypertension (PH) is the result of increased vascular resistance in the portal circulation, increased portal venous blood flow, or both. In veterinary medicine, where portal pressure is seldom measured directly, the diagnosis of PH often is inferred from identification of associated complications including multiple acquired portosystemic shunts, ascites, and hepatic encephalopathy. Likewise, treatment of PH primarily is aimed at controlling these complications. The goal of this review is to provide an update on the pathophysiology, diagnosis, and treatment of PH. The review draws from information in the veterinary hepatology literature, reviews, and consensus statements in human hepatology and the literature on experimental models of PH.

Background: Portal vein thrombosis (PVT) has been reported infrequently in dogs. Objectives: To characterize the presentation, associated disease conditions, and outcome in dogs with PVT. Animals: Client-owned dogs with a diagnosis of PVT and a complete medical record. Methods: Records were retrospectively analyzed for presentation, history, physical examination, clinicopathologic data, diagnostic imaging, treatment, and outcome. Results: Thirty-three dogs were included. Common clinical signs were vomiting, diarrhea, abdominal pain, ascites, and signs of hypovolemic shock. Associated disease conditions included hepatic (14/33), neoplastic (7/33), immune (5/33), and infectious (4/33) diseases, protein-losing nephropathy (3/33), hyperadrenocorticism (2/33), protein-losing enteropathy (1/33), and pancreatitis (1/33). Fourteen dogs were receiving glucocorticoids at the time of diagnosis. Twenty-nine dogs had at least 1 predisposing condition for venous thrombosis, and 11 had 2 or more. Thrombocytopenia (24/33), increased liver enzyme activity (23/33), and hypoalbuminemia (20/33) were common laboratory abnormalities. Clinical syndromes at the time of PVT diagnosis included shock (16/33), systemic inflammatory response syndrome (SIRS), (13/33) and disseminated intravascular coagulation (3/33). Twenty-four dogs had acute and 9 had chronic PVT. Multiple thrombi were found in 17/33 dogs. Nineteen dogs survived to discharge. Dogs treated with anticoagulant therapy were more likely, whereas those with acute PVT, multiple thromboses or SIRS were less likely to survive. Conclusions and Clinical Importance: Hepatic disease is a common pre-existing condition in dogs with PVT. PVT should be considered in dogs with risk factors for venous thrombosis presenting with abdominal pain, ascites, and thrombocytopenia. Studies evaluating anticoagulant therapy in the management of PVT are warranted.

Objective—To evaluate differences in hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis. Design—Retrospective case-control study. Sample—Liver tissue specimens from 36 Labrador Retrievers with chronic hepatitis and 36 age- and sex-matched Labrador Retrievers without chronic hepatitis (control dogs). Procedures—Liver tissue specimens were obtained during 2 study periods (1980 to 1997 and 1998 to 2010). For each tissue specimen, a histologic score was assigned independently by each of 2 interpreters, and the hepatic copper concentration was qualitatively determined via rhodanine staining and quantitatively determined via atomic absorption spectroscopy. Results—Mean hepatic copper concentration was significantly higher in dogs with chronic hepatitis (614 μg/g of dry weight [range, 104 to 4,234 μg/g of dry weight]), compared with that in control dogs (299 μg/g of dry weight [range, 93 to 3,810 μg/g of dry weight]), and increased significantly over time. A higher proportion of liver tissue specimens collected during the 1998–2010 study period had hepatic copper concentrations > 400 μg/g of dry weight (the upper limit of the reference range), compared with the proportion of liver tissue specimens collected during the 1980–1997 study period. The qualitative copper score did not accurately predict quantitative hepatic copper concentration in 33% of study dogs. Conclusions and Clinical Relevance—Results suggested that the increase in hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis over time may be the result of increased exposure of dogs to environmental copper, most likely via the diet.

Objectives: The objective of this retrospective study was to describe the clinical signs and diagnostic findings in cats with histopathologically confirmed adrenal neoplasms, and to assess correlations with survival data. Methods: Study data were acquired by reviewing medical records for all cats diagnosed with adrenal neoplasms at seven referral institutions between 2002 and 2013. Inclusion criteria required a histopathologic diagnosis of an adrenal neoplasm (ante-mortem or on necropsy). Results: Thirty-three cats met the inclusion criteria for the study. The most common presenting complaints included weakness (n = 12), respiratory signs (n = 4), blindness (n = 4) or gastrointestinal signs (n = 3). Laboratory abnormalities included hypokalemia (n = 18), alkalemia (n = 12), elevated creatine kinase (>3000, n = 5) and azotemia (n = 4). In addition, hypertension was noted in 13 cats. Thirty cats were diagnosed with cortical tumors (17 carcinomas, 13 adenomas) and three cats were diagnosed with pheochromocytomas. Twenty-five cats underwent tests to evaluate the function of the adrenal tumors; 19/25 cats had functional tumors (hyperaldosteronism [n = 16], hypercortisolemia [n = 1], high estradiol [n = 1], and hypersecretion of aldosterone, estradiol and progesterone [n = 1]). Twenty-six cats underwent adrenalectomy, one cat was medically managed and six were euthanized without treatment. Long-term survival postoperatively ranged from 4–540 weeks, with 20 (77%) cats surviving the perioperative period of 2 weeks. The only variable that was found to be negatively associated with survival was female sex. The most common complications noted during the perioperative period were hemorrhage and progressive lethargy and anorexia. Conclusions and relevance: Surgical treatment for feline adrenal tumors (regardless of tumor type) resulted in good long-term survival. Given that pre- and postoperative hypocortisolemia was identified in this study, and, in addition, hypersecretion of more than one adrenal hormone occurred in one cat, adrenal panels prior to surgery may be beneficial as part of the preoperative work-up.

Necroptosis is a regulated necrotic cell death pathway involved in development and disease. Its signaling cascade results in the formation of disulfide bond-dependent amyloid-like polymers of mixed lineage kinase domain-like protein (MLKL), which mediate proinflammatory cell membrane disruption. We screened compound libraries provided by the National Cancer Institute and identified a small-molecule inhibitor of necroptosis named necroptosis-blocking compound 1 (NBC1). Biotin-labeled NBC1 specifically conjugates to heat shock protein Hsp70. NBC1 and PES-Cl, a known Hsp70 substrate-binding inhibitor, block the formation of MLKL polymers, but not MLKL tetramers in necroptosis-induced cells. In vitro, recombinant Hsp70 interacts with the N-terminal domain (NTD) of MLKL and promotes NTD polymerization, which has been shown to mediate the cell killing activity. Furthermore, the substrate-binding domain (SBD) of Hsp70 is sufficient to promote MLKL polymerization. NBC1 covalently conjugates cysteine 574 and cysteine 603 of the SBD to block its function. In addition, an SBD mutant with both cysteines mutated to serines loses its ability to promote MLKL polymerization. Interestingly, knockdown of Hsp70 in cells leads to MLKL destabilization, suggesting that MLKL might also be a client protein of Hsp70. In summary, using NBC1, an inhibitor of necroptosis, we identified Hsp70 as a molecular chaperone performing dual functions in necroptosis. It stabilizes MLKL protein under normal condition and promotes MLKL polymerization through its substrate-binding domain during necroptosis. Significance Necroptosis is a regulated form of necrotic cell death implicated in many human diseases, including infection, inflammation, neurodegeneration, and cancer. TNF-induced necroptosis results in the formation of MLKL tetramers, which further polymerize to form disulfide bond-dependent amyloid-like fibers to promote cell death. Here we report the identification of a necroptosis-blocking compound, NBC1, which covalently conjugates two cysteines of chaperone Hsp70. Importantly, Hsp70 requires these two cysteines to promote polymerization of MLKL tetramers in an ATP-independent manner. NBC1 blocks MLKL polymerization and subsequent cell death. This work reinforces the importance of MLKL polymer formation and identifies chaperone Hsp70 as an obligatory facilitator for MLKL polymerization, providing further insights for intervention of necroptosis-associated diseases.