Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice.

Background & Aims: Oxidative stress is recognized as a major driver of non-alcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and thus appear to be attractive targets for the treatment of NASH. Methods: Molecular modeling and X-ray crystallography were used to design S217879 - a small molecule that could disrupt the KEAP1-NRF2 interaction. S217879 was highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, namely the methionine and choline-deficient diet (MCDD) and diet-induced obesity NASH (DIO NASH) models. Results: Molecular and cell-based assays confirmed that S217879 is a highly potent and selective NRF2 activator with marked anti-inflammatory properties, as shown in primary human peripheral blood mononuclear cells. In MCDD mice, S217879 treatment for 2 weeks led to a dose-dependent reduction in NAFLD activity score while significantly increasing liver Nqo1 mRNA levels, a specific NRF2 target engagement biomarker. In DIO NASH mice, S217879 treatment resulted in a significant improvement of established liver injury, with a clear reduction in both NAS and liver fibrosis. αSMA and Col1A1 staining, as well as quantification of liver hydroxyproline levels, confirmed the reduction in liver fibrosis in response to S217879. RNA-sequencing analyses revealed major alterations in the liver transcriptome in response to S217879, with activation of NRF2-dependent gene transcription and marked inhibition of key signaling pathways that drive disease progression. Conclusions: These results highlight the potential of selective disruption of the NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis. Impact and implications: We report the discovery of S217879 - a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved in NASH disease progression, leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

Preputial urethrostomy with preservation of the local anatomy in 4 dogs.

OBJECTIVE: To describe a novel modified preputial urethrostomy with preservation of the local anatomy in 4 dogs. STUDY DESIGN: Short case series. ANIMALS: Four client-owned male dogs. METHODS: Dogs presented for dysuria and urethral obstruction and underwent a modified preputial urethrostomy as a salvage procedure after the failure of previous treatments. Urethral stenosis or tear was confirmed by cysto-urethrography in all dogs. The preputial urethrostomy involved anastomosis of the pelvic urethra with the preputial mucosa after caudal laparotomy, without dissection of the prepuce or amputation of the penis. Owner follow up was obtained by telephone interview. RESULTS: Urinary obstruction was due to urethral stenosis in 3 dogs and severe complications following perineal urethrostomy in 1 dog. The urinary obstruction was resolved in all dogs by the modified preputial urethrostomy. None of the dogs had signs of dysuria, urinary tract infection, or dermatitis immediately postoperatively. Two dogs showed signs of urinary incontinence from 15 days to 1 month postoperatively, which persisted throughout the follow-up period. CONCLUSION: A functional urethral stoma was obtained in all dogs. This technique may be an alternative to prepubic urethrostomy in male dogs.

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs.

Background: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Göttingen minipig. Methods: First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results: Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions: These results, taken together, indicate that the diet-induced NASH in the Göttingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development.

Diagnosis and treatment of gastro-oesophageal junction abnormalities in dogs with brachycephalic syndrome.

OBJECTIVES: To determine whether there is a benefit of using pre- and postoperative antacid treatment in dogs undergoing surgery for brachycephalic syndrome. To assess the use of an obstruction manoeuvre during endoscopy for the detection of dynamic gastro-oesophageal junction abnormalities. MATERIALS AND METHODS: Thirty-six client-owned brachycephalic dogs were prospectively included in a randomised trial. Antacid treatment was randomly prescribed in 18 dogs before and after surgery while the other 18 dogs did not receive any gastrointestinal medical treatment. At presentation, at the time of surgery and at recheck, digestive clinical signs and gastro-oesophageal junction abnormalities were assessed using specific scores. Gastro-oesophageal junction abnormalities were assessed during endoscopy in standard conditions as well as during endotracheal tube obstruction. This manoeuvre was also applied in an unrelated control group. RESULTS: The results suggest a beneficial effect of antacid treatment on the improvement of digestive clinical signs and lesions in dogs with brachycephalic syndrome undergoing surgery. At postsurgical control 83% of dogs had a digestive clinical score ≤1 in the treated group in contrast to 44% in the non-treated group and 39% of dogs had a gastro-oesophageal abnormalities score (during obstruction manoeuvre) ≤1 in the treated group in contrast to 16.7% in the non-treated group. The use of the obstruction manoeuvre during endoscopic assessment in a control group revealed that gastro-oesophageal junction movements are negligible in healthy animals. CLINICAL SIGNIFICANCE: The addition of antacid treatment during the pre- and postoperative period for brachycephalic dogs undergoing surgery may result in a faster and greater improvement in treated dogs. The obstruction manoeuvre is an interesting technique to improve detection of gastro-oesophageal junction abnormalities.

Urethral intussusception following traumatic catheterization in a male cat.

An 8-year-old, European male shorthair cat was presented with lower urinary tract obstruction. He was catheterized and referred. Retrograde cysto-urethrography suggested a urethral mass. Intussusception of the urethra with a partial rupture of the urethra was visualized. A perineal urethrostomy was performed. The cat was clinically normal at 15 months' follow-up.

Invagination urétrale secondaire à un cathétérisme traumatique chez un chat mâle. Un chat européen male de 8 ans présentant des signes d'obstruction du bas appareil urinaire a été cathéterisé et référé. L'urétrographie rétrograde suggérait une masse urétrale. Une intussusception de l'urètre avec une rupture partielle de l'urètre a été visualisée. Une urétrostomie périnéale a été réalisée. Le chat était cliniquement normal 15 mois après l'intervention.(Traduit par les auteurs).

Risk factors for tibial damage associated with the modified Maquet technique in 174 stifles.

OBJECTIVE: To identify risk factors for tibial damage associated with the modified Maquet technique (MMT) in dogs with cranial cruciate ligament (CCL) disease. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: One hundred and seventy-four stifles from 147 client-owned dogs. METHODS: Medical records of dogs diagnosed with CCL disease and treated with the current version of MMT were reviewed. Dogs were included if immediate postoperative radiographs were available. Cortical hinge fracture or fissure, tibial tuberosity fracture, and diaphyseal fractures of the tibia were recorded. Age, body weight (BW), thickness of the tibial cortical hinge, and angle of opening of the osteotomy were tested as potential risk factors for tibial damage by univariate logistic regression analysis. RESULTS: Tibial damage included intraoperative tibial fissures in 37% of MMTs, intraoperative fractures of the cortical hinge in 3.4% of MMTs, postoperative tibial fractures in 14% of MMTs. Risk factors for intraoperative fissure included BW (P = .0153) and thickness of cortical hinge (P = .0006). The angle of opening of the osteotomy was identified as a risk factor for intraoperative cortical hinge fracture (P = .0034), angles below 11° being preventive. No risk factor was identified for postoperative fracture. CONCLUSION: Based on these results, preventive measures against tibial damage associated with MMT should include: a thickness of cortical hinge based on the equation related to the BW; a length of osteotomy adjusted to the amount of TTA with an osteotomy angle below 10°; and slow advancement of the tibial tuberosity.

Effects of manipulations to detect sliding hiatal hernia in dogs with brachycephalic airway obstructive syndrome.

OBJECTIVE: To determine the influence of manipulations aimed at increasing the transdiaphragmatic pressure gradient on the gastro-esophageal junction (GEJ) of dogs with brachycephalic airway obstructive syndrome (BAOS), and to identify the manipulation that most improves the detection of GEJ abnormalities and sliding hiatal hernia (SHH) in dogs with BAOS. STUDY DESIGN: In vivo experimental pilot study and prospective clinical study. ANIMALS: Five purpose-bred Beagles and 20 dogs diagnosed with BAOS. METHODS: Respiratory and digestive clinical signs as well as respiratory and GEJ abnormalities were scored. The presence of SHH was investigated using radiography and endoscopy in standard conditions. Endoscopic investigation was repeated after manipulations including manual pressure on the cranial abdomen (MP), Trendelenburg position (30°), or temporary complete endotracheal tube obstruction (ETO). RESULTS: No SHH was detected in any normal dog under any condition. Sixty-five percent of dogs with BAOS presented with digestive clinical signs, including vomiting and/or regurgitation. SHH was observed in only one dog via radiography and was not detected via endoscopy. Manipulations during endoscopy influenced GEJ abnormalities and allowed the detection of SHH in 2 (30°), 4 (ETO), and 5 (MP) dogs, respectively. Digestive clinical signs correlated with GEJ abnormalities observed only in dogs with ETO (P = .02). CONCLUSION: Manipulations aimed at increasing the transdiaphragmatic pressure gradient during endoscopy in BAOS dogs allowed the detection of GEJ abnormalities and SHH that were not detected under standard conditions. Although MP allowed detection of SHH in more dogs than ETO, scores under MP did not correlate with digestive clinical signs. Therefore, ETO may be more accurate manipulation for the detection of GEJ abnormalities in BAOS dogs.

Small molecule glucokinase activators disturb lipid homeostasis and induce fatty liver in rodents: a warning for therapeutic applications in humans.

BACKGROUND AND PURPOSE: Small-molecule glucokinase activators (GKAs) are currently being investigated as therapeutic options for the treatment of type 2 diabetes (T2D). Because liver overexpression of glucokinase is thought to be associated with altered lipid profiles, this study aimed at assessing the potential lipogenic risks linked to oral GKA administration. EXPERIMENTAL APPROACH: Nine GKA candidates were qualified for their ability to activate recombinant glucokinase and to stimulate glycogen synthesis in rat hepatocytes and insulin secretion in rat INS-1E cells. In vivo activity was monitored by plasma glucose and HbA1c measurements after oral administration in rodents. Risk-associated effects were assessed by measuring hepatic and plasma triglycerides and free fatty acids, as well as plasma aminotransferases, and alkaline phosphatase. KEY RESULTS: GKAs, while efficiently decreasing glycaemia in acute conditions and HbA1c levels after chronic administration in hyperglycemic db/db mice, were potent inducers of hepatic steatosis. This adverse outcome appeared as soon as 4 days after daily oral administration at pharmacological doses and was not transient. GKA treatment similarly increased hepatic triglycerides in diabetic and normoglycaemic rats, together with a pattern of metabolic phenotypes including different combinations of increased plasma triglycerides, free fatty acids, alanine and aspartyl aminotransferases, and alkaline phosphatase. GKAs belonging to three distinct structural families induced hepatic steatosis in db/db mice, arguing in favour of a target-mediated, rather than a chemical class-mediated, effect. CONCLUSION AND IMPLICATIONS: Given the risks associated with fatty liver disease in the general population and furthermore in patients with T2D, these findings represent a serious warning for the use of GKAs in humans. LINKED ARTICLE: This article is commented on by Rees and Gloyn, pp. 335-338 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02201.x.

Fructose diet and VEGF-induced plasma extravasation in hamster cheek pouch.

AIM: To determine in the hamster cheek pouch whether or not the changes in plasma extravasation induced by vascular endothelial growth factor (VEGF) could be affected by fructose diet. METHODS: Hamsters were subjected to control drinking water or to water containing fructose (10 %) for 18 weeks. RESULTS: The fructose diet induced a small but significant increase in glycemia (0.80+/-0.11 and 1.09+/-0.15, n=8 and 9 for control and fructose- treated animals, respectively, P<0.05). Bradykinin-induced plasma extravasation was not affected by the fructose diet while the effects of VEGF were markedly increased (maximal number of leakage sites: 76+/-20 and 126+/-55, n = 8 and 9 for control and fructose-treated animals, respectively, P<0.01). CONCLUSION: Even moderate changes in glycemic levels can produce profound alteration in the VEGF response.