Innovative application of an implantable venous access system in the portal vein: technique, results and complications in three dogs.

BACKGROUND: Vascular access port (VAP) systems are widely used in human medicine to provide long-term venous access. However, in veterinary medicine the use of VAP systems is not common practice and publications on their potential applications have been limited. A VAP system was used as part of an experimental study on liver regeneration and implanted in the canine portal vein to create direct access to the portal venous circulation of the liver. The aim of the present study is to describe the surgical technique, its use, and the complications of a VAP system in three research dogs. RESULTS: The VAP system was successfully used for the intraoperative measurement of portal blood pressure, the administration of cell suspensions, and the collection of portal venous blood samples. Long-term complications consisted of dislocation of the VAP system in one dog (2 months after implantation) and thrombus formation at the catheter tip in two dogs (3 months after implantation). Both complications prevented further use of the VAP but had no adverse clinical implications. CONCLUSIONS: This pilot study suggests that the VAP system is an effective and safe technique to obtain long term access to the portal venous system in dogs. However, complications with port detachment and thrombosis may limit long term use of VAPs in the portal system of dogs.

SUCNR1-mediated chemotaxis of macrophages aggravates obesity-induced inflammation and diabetes.

AIMS/HYPOTHESIS: Obesity induces macrophages to drive inflammation in adipose tissue, a crucial step towards the development of type 2 diabetes. The tricarboxylic acid (TCA) cycle intermediate succinate is released from cells under metabolic stress and has recently emerged as a metabolic signal induced by proinflammatory stimuli. We therefore investigated whether succinate receptor 1 (SUCNR1) could play a role in the development of adipose tissue inflammation and type 2 diabetes. METHODS: Succinate levels were determined in human plasma samples from individuals with type 2 diabetes and non-diabetic participants. Succinate release from adipose tissue explants was studied. Sucnr1 -/- and wild-type (WT) littermate mice were fed a high-fat diet (HFD) or low-fat diet (LFD) for 16 weeks. Serum metabolic variables, adipose tissue inflammation, macrophage migration and glucose tolerance were determined. RESULTS: We show that hypoxia and hyperglycaemia independently drive the release of succinate from mouse adipose tissue (17-fold and up to 18-fold, respectively) and that plasma levels of succinate were higher in participants with type 2 diabetes compared with non-diabetic individuals (+53%; p < 0.01). Sucnr1 -/- mice had significantly reduced numbers of macrophages (0.56 ± 0.07 vs 0.92 ± 0.15 F4/80 cells/adipocytes, p < 0.05) and crown-like structures (0.06 ± 0.02 vs 0.14 ± 0.02, CLS/adipocytes p < 0.01) in adipose tissue and significantly improved glucose tolerance (p < 0.001) compared with WT mice fed an HFD, despite similarly increased body weights. Consistently, macrophages from Sucnr1 -/- mice showed reduced chemotaxis towards medium collected from apoptotic and hypoxic adipocytes (-59%; p < 0.05). CONCLUSIONS/INTERPRETATION: Our results reveal that activation of SUCNR1 in macrophages is important for both infiltration and inflammation of adipose tissue in obesity, and suggest that SUCNR1 is a promising therapeutic target in obesity-induced type 2 diabetes. DATA AVAILABILITY: The dataset generated and analysed during the current study is available in GEO with the accession number GSE64104, www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64104 .

Sex specific differences in hepatic and plasma lipid profiles in healthy cats pre and post spaying and neutering: relationship with feline hepatic lipidosis.

BACKGROUND: A link between lipid metabolism and disease has been recognized in cats. Since hepatic lipidosis is a frequent disorder in cats, the aim of the current study was to evaluate liver and plasma lipid dimorphism in healthy cats and the effects of gonadectomy on lipid profiling. From six female and six male cats plasma and liver lipid profiles before and after spaying/neutering were assessed and compared to five cats (three neutered male and two spayed female) diagnosed with hepatic lipidosis. RESULTS: Intact female cats had a significantly lower level of plasma triacylglycerides (TAG) and a higher liver level of the long chain polyunsaturated fatty acid arachidonic acid (AA) compared to their neutered state. Both male and female cats with lipidosis had a higher liver, but not plasma TAG level and an increased level of plasma and liver sphingomyelin compared to the healthy cats. CONCLUSION: Although lipid dimorphism in healthy cats resembles that of other species, intact female cats show differences in metabolic configuration that could predispose them to develop hepatic lipidosis. The increased sphingomyelin levels in cats with lipidosis could suggest a potential role in the pathogenesis of hepatic lipidosis in cats.

Negative pressure therapy versus passive open abdominal drainage for the treatment of septic peritonitis in dogs: A randomized, prospective study.

OBJECTIVE: To compare passive open abdominal drainage (POAD) and negative-pressure abdominal drainage (NPAD) using the ABThera™ system in the treatment of septic peritonitis. STUDY DESIGN: Randomized prospective clinical trial. ANIMALS: Dogs (n = 16) with septic peritonitis. METHODS: Dogs with septic peritonitis were randomly assigned to one of two treatment protocols: NPAD versus POAD. Anesthesia time, operating time, duration of drainage, costs, survival, and complications were compared between techniques. Hematological and biochemical parameters in blood and abdominal fluid, and histopathological findings of omentum and abdominal wall tissue samples were compared between NPAD and POAD at time of initial surgery and at time of closure. RESULTS: Overall survival was 81%. Treatment costs, anesthesia and operating time, drainage time, survival, and postoperative complications were similar between techniques. Loss of total plasma protein and decreased inflammation-related factors in abdominal fluid at time of closure were noted in all patients. Neutrophilic inflammation was greater in abdominal wall samples after NPAD. POAD patients showed discomfort during bandage changes and had frequent leakage of abdominal fluid outside of the bandage. CONCLUSION: NPAD is an effective alternative to POAD for treatment of septic peritonitis, based on costs and survival. NPAD resulted in less abdominal fluid leakage, and evidence of superior healing on histological evaluation of abdominal tissues.

No up-regulation of the phosphatidylethanolamine N-methyltransferase pathway and choline production by sex hormones in cats.

BACKGROUND: Feline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT expression is known to be upregulated by oestrogens, protecting the females in these species from the development of HL when exposed to choline deficient diets. The aim of the present study was to evaluate the influence of sex hormones on choline synthesis via the PEMT pathway in healthy male and female cats before and after spaying/neutering, when fed a diet with recommended dietary choline content. RESULTS: From six female and six male cats PEMT activity was assayed directly in liver biopsies taken before and after spaying/neutering, and assessed indirectly by analyses of PEMT-specific hepatic phosphatidylcholine (PC) species and plasma choline levels. Hepatic PEMT activity did not differ between intact female and male cats and no changes upon spaying/neutering were observed. Likewise, no significant differences in liver PC content and PEMT-specific polyunsaturated PC species were found between the sexes and before or after spaying/neutering. CONCLUSION: These results suggest that choline synthesis in cats differs from what is observed in humans, mice and rats. The lack of evident influence of sex hormones on the PEMT pathway makes it unlikely that spaying/neutering predisposes cats for HL by causing PC deficiency as suggested in other species.

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit ß-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of ß-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Assessment of misdiagnosis in small animal intensive care patients using the Modified Goldman criteria.

The postmortem examination can be used as a means of quality control for clinical diagnoses. A retrospective study on 300 dogs and cats that had been admitted to a small animal intensive care unit was performed comparing the clinical and postmortem findings, using the Modified Goldman criteria. All patient files were reevaluated for clinical diagnoses and all postmortem material was reevaluated for pathological diagnoses. After this, the Modified Goldman criteria were applied to score the discrepancies between them, and factors associated with the occurrence of an undiagnosed major unexpected finding were analyzed. The postmortem examination revealed additional findings in 65% of the cases. Major discrepancies, defined as those affecting treatment and possibly outcome of the patient, were present in 21.3% of the cases. The most frequently missed diagnoses detected at necropsy were pneumonia of various etiologies, meningitis/meningoencephalitis, myocarditis and generalized vasculitis. A shorter ICU stay was associated with increased odds of a major discrepancy. Conditions affecting the urinary or gastrointestinal system were negatively associated with major discrepancy.

Evidence of a sudden increase in α-chloralose poisoning in dogs and cats in the Netherlands between 2018 and 2021.

BACKGROUND: After changes in European Union biocide legislation, the Dutch Poisons Information Center observed a strong increase in information requests concerning dogs and cats exposed to α-chloralose. To investigate whether α-chloralose-based rodenticides are safe for non-professional use, additional information regarding poisoning scenarios and clinical course was collected. METHODS: Veterinarians reporting α-chloralose exposure over a 2.5-year period were contacted by mail for follow-up information concerning exposure scenario, product formulation, clinical course and treatment, and outcome. In total, information was collected for 96 dogs and 41 cats. RESULTS: Fifty-three of 96 dogs and 17 of 19 cats known to have been exposed to α-chloralose-based rodenticides developed signs of central nervous system (CNS) depression or sensory-induced CNS excitation. Mortality in dogs and cats following exposure was 1% and 18%, respectively. An additional 22 cats presented with clinical signs suggestive of α-chloralose poisoning, with a mortality of 5%. LIMITATIONS: Exposure to α-chloralose was not confirmed by biochemical analyses. CONCLUSION: Dogs and especially cats were at risk of poisoning from α-chloralose. If criteria such as acute toxicity and risk of (secondary) poisoning are applied during the approval of α-chloralose-based rodenticides, similar to anticoagulant-based rodenticides, it can be concluded that α-chloralose is also not safe for non-professional use.

Intracellular activation of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus by nonpeptide agonists.

Binding of the peptide hormone vasopressin to its type-2 receptor (V2R) in kidney triggers a cAMP-mediated translocation of Aquaporin-2 water channels to the apical membrane, resulting in water reabsorption and thereby preventing dehydration. Mutations in the V2R gene lead to Nephrogenic Diabetes Insipidus (NDI), a disorder in which this process is disturbed, because the encoded, often intrinsically functional mutant V2 receptors are misfolded and retained in the endoplasmic reticulum (ER). Since plasma membrane expression is thought to be essential for V2R activation, cell permeable V2R antagonists have been used to induce maturation and rescue cell surface expression of V2R mutants, after which they need to be displaced by vasopressin for activation. Here, however, we show that 3 novel nonpeptide V2R agonists, but not vasopressin, activate NDI-causing V2R mutants at their intracellular location, without changing their maturation and at a sufficient level to induce the translocation of aquaporin-2 to the apical membrane. Moreover, in contrast to plasma membrane V2R, degradation of intracellular V2R mutants is not increased by their activation. Our data reveal that G protein-coupled receptors (GPCRs) normally active at the plasma membrane can be activated intracellularly and that intracellular activation does not induce their degradation; the data also indicate that nonpeptide agonists constitute highly promising therapeutics for diseases caused by misfolded GPCRs in general, and NDI in particular.

Succinate receptors in the kidney.

The G protein-coupled succinate and α-ketoglutarate receptors are closely related to the family of P2Y purinoreceptors. Although the α-ketoglutarate receptor is almost exclusively expressed in the kidney, its function is unknown. In contrast, the succinate receptor, SUCRN1, is expressed in a variety of tissues, including blood cells, adipose tissue, liver, retina, and the kidney. Recent evidence suggests SUCRN1 and its succinate ligand are novel detectors of local stress, including ischemia, hypoxia, toxicity, and hyperglycemia. Local levels of succinate in the kidney also activate the renin-angiotensin system and together with SUCRN1 may play a key role in the development of hypertension and the complications of diabetes mellitus, metabolic disease, and liver damage. This makes the succinate receptor a promising drug target to counteract an expanding number of interrelated disorders.

Genomic Investigation of Two Acinetobacter baumannii Outbreaks in a Veterinary Intensive Care Unit in The Netherlands.

Acinetobacter baumannii is a nosocomial pathogen that frequently causes healthcare-acquired infections. The global spread of multidrug-resistant (MDR) strains with its ability to survive in the environment for extended periods imposes a pressing public health threat. Two MDR A. baumannii outbreaks occurred in 2012 and 2014 in a companion animal intensive care unit (caICU) in the Netherlands. Whole-genome sequencing (WGS) was performed on dog clinical isolates (n = 6), environmental isolates (n = 5), and human reference strains (n = 3) to investigate if the isolates of the two outbreaks were related. All clinical isolates shared identical resistance phenotypes displaying multidrug resistance. Multi-locus Sequence Typing (MLST) revealed that all clinical isolates belonged to sequence type ST2. The core genome MLST (cgMLST) results confirmed that the isolates of the two outbreaks were not related. Comparative genome analysis showed that the outbreak isolates contained different gene contents, including mobile genetic elements associated with antimicrobial resistance genes (ARGs). The time-measured phylogenetic reconstruction revealed that the outbreak isolates diverged approximately 30 years before 2014. Our study shows the importance of WGS analyses combined with molecular clock investigations to reduce transmission of MDR A. baumannii infections in companion animal clinics.

Regulation of AQP2 localization by S256 and S261 phosphorylation and ubiquitination.

Vasopressin-induced water reabsorption coincides with phosphorylation of aquaporin-2 (AQP2) at S256 (pS256), dephosphorylation at S261, and its translocation to the apical membrane, whereas treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA) induces AQP2 ubiquitination at K270, its internalization, and lysosomal degradation. In this study we investigated the relationship between S256 and S261 phosphorylation in AQP2 and its ubiquitination and trafficking in MDCK cells. Forskolin stimulation associated with increased pS256 and decreased pS261 AQP2, indicating that MDCK cells are a good model. After forskolin stimulation, TPA-induced ubiquitination of AQP2 preceded phosphorylation of AQP2 at S261, which in the first instance occurred predominantly on ubiquitinated AQP2. Forskolin-induced changes in pS261 were also observed for AQP2-S256A and AQP2-S256D, which constitutively localize in vesicles and the apical membrane, respectively. Although pS261 varies with forskolin as with wild-type AQP2, AQP2-S256A is not increased in its ubiquitination. Our data reveal that pS261 occurred independently of AQP2 localization and suggest that pS261 follows ubiquitination and endocytosis and may stabilize AQP2 ubiquitination and intracellular localization. The absence of increased ubiquitination of AQP2-S256A indicates that its intracellular location is due to the lack of pS256. Furthermore, AQP2-S261A and AQP2-S261D localized to vesicles, which was due to their increased ubiquitination, because changing K270 into Arg in both mutants resulted in their localization in the apical membrane. Although still increased in its ubiquitination, AQP2-S256D-S261D localized in the apical membrane. AQP2-S256D-K270R-Ub, however, localized to intracellular vesicles. Although our localization of AQP2-S261A/D is different from that of others, these data indicate that constitutive S256 phosphorylation counterbalances S261D-induced ubiquitination and internalization or changes its structure to allow distribution to the apical membrane. The vesicular localization of AQP2-S256D-K270R-Ub, however, indicates that the dominant apical sorting of S256D can again be overruled by constitutive ubiquitination. These data indicate that the membrane localization of AQP2 is determined by the balance of the extents of phosphorylation and ubiquitination.

Counteracting vasopressin-mediated water reabsorption by ATP, dopamine, and phorbol esters: mechanisms of action.

Water homeostasis is regulated by a wide variety of hormones. When in need for water conservation, vasopressin, released from the brain, binds renal principal cells and initiates a signaling cascade resulting in the insertion of aquaporin-2 (AQP2) water channels in the apical membrane and water reabsorption. Conversely, hormones, including extracellular purines and dopamine, antagonize AVP-induced water permeability, but their mechanism of action is largely unknown, which was investigated here. Addition of these hormones to mpkCCD cells decreased total and plasma membrane abundance of AVP-induced AQP2, partly by increasing its internalization to vesicles and lysosomal degradation. This internalization was ubiquitin dependent, because the hormones increased AQP2 ubiquitination, and the plasma membrane localization of AQP2-K270R, which cannot be monoubiquitinated, was unaffected by these hormones. Both hormones also increased AQP2 phosphorylation at S261, which followed ubiquitination, but was not essential for hormone-induced AQP2 degradation. A similar process occurs in vivo, as incubation of dDAVP-treated kidney slices with both hormones also resulted in the internalization and S261 phosphorylation of AQP2. Both hormones also reduced cAMP and AQP2 mRNA levels, suggesting an additional effect on AQP2 gene transcription. Interestingly, phorbol esters only reduced AQP2 through the first pathway. Together, our results indicate that ATP and dopamine counteract AVP-induced water permeability by increasing AQP2 degradation in lysosomes, preceded by ubiquitin-dependent internalization, and by decreasing AQP2 gene transcription by reducing the AVP-induced cAMP levels.

SLC29A3 gene is mutated in pigmented hypertrichosis with insulin-dependent diabetes mellitus syndrome and interacts with the insulin signaling pathway.

Pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID) syndrome is a recently described autosomal recessive disorder associated with predominantly antibody negative, insulin-dependent diabetes mellitus. In order to identify the genetic basis of PHID and study its relationship with glucose metabolism, we performed homozygosity mapping in five unrelated families followed by candidate gene sequencing. Five loss-of-function mutations were identified in the SLC29A3 gene which encodes a member of a highly conserved protein family that transports nucleosides, nucleobases and nucleoside analogue drugs, hENT3. We show that PHID is allelic with a related syndrome without diabetes mellitus, H syndrome. The interaction of SLC29A3 with insulin signaling pathways was then studied using an established model in Drosophila melanogaster. Ubiquitous knockdown of the Drosophila ortholog of hENT3, dENT1 is lethal under stringent conditions; whereas milder knockdown induced scutellar bristle phenotypes similar to those previously reported in the knockdown of the Drosophila ortholog of the Islet gene. A cellular growth assay showed a reduction of cell size/number which could be rescued or enhanced by manipulation of the Drosophila insulin receptor and its downstream signaling effectors, dPI3K and dAkt. In summary, inactivating mutations in SLC29A3 cause a syndromic form of insulin-dependent diabetes in humans and in Drosophila profoundly affect cell size/number through interactions with the insulin signaling pathway. These data suggest that further investigation of the role of SLC29A3 in glucose metabolism is a priority for diabetes research.

The Effect of the Preoperative Fasting Regimen on the Incidence of Gastro-Oesophageal Reflux in 90 Dogs.

This study aimed to investigate the effect of three different preoperative fasting regimens on the incidence of gastro-oesophageal reflux (GOR) in dogs under general anaesthesia. Ninety dogs undergoing non-abdominal and non-thoracic elective surgery were included in the study and equally allocated to three groups. Dogs received canned food providing half the daily resting energy requirements (RER) 3 h prior to premedication (group 3H), a quarter of the daily RER 3 h before premedication (group 3Q), and half the daily RER 12 h before premedication (group 12H). The animals were premedicated with acepromazine and pethidine, anaesthesia was induced with propofol and maintained with isoflurane vaporised in oxygen. Oesophageal pH was monitored throughout anaesthesia. Demographic and surgery-related parameters were not different among groups. The incidence of GOR was 11/30 in group 3H (36.7%), 9/30 in group 3Q (30.0%) and 5/30 in group 12H (16.7%), which was not statistically different (p = 0.262). Reduction of the amount of the preoperative meal from half to a quarter of the daily RER did not reduce the incidence of GOR but resulted in a lower oesophageal pH (p = 0.003). The results of this study suggest that the administration of a meal 3 h before anaesthesia does not have any beneficial effect in the reduction of GOR incidence in dogs compared to the administration of a meal 12 h before anaesthesia.

Effect of the cGMP pathway on AQP2 expression and translocation: potential implications for nephrogenic diabetes insipidus.

BACKGROUND: Arginine vasopressin (AVP) binding to the V2 receptor (V2R) in renal collecting duct principal cells induces a cAMP signalling cascade resulting in the activation of protein kinase A (PKA), translocation of aquaporin-2 (AQP2) to the apical membrane and an increase in AQP2 expression. Consequently, concentration of urine is initiated. X-linked nephrogenic diabetes insipidus (NDI), characterized by the inability to concentrate urine in response to AVP, is caused by mutations in the V2R gene. Initiation of AQP2 translocation, while circumventing the V2R-cAMP-PKA pathway has been suggested as a putative therapy for these patients. In this respect, the activation of a cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion by different cyclic guanosine monophosphate (cGMP) pathway activators, such as atrial natriuretic peptide (ANP), l-arginine and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), has been put forward. However, it is unclear whether they can increase AQP2 expression. METHODS: Mouse cortical collecting duct (mpkCCD) cells were incubated with ANP, l-arginine and 8-Br-cGMP for 2 h and subjected to immunocytochemistry and cell surface biotinylation assays to examine their effect on AQP2 translocation. To test the effect of cGMP pathway activators on AQP2 expression, the mpkCCD cells were treated with dDAVP, ANP and l-arginine for 4 days, or with 8-Br-cGMP for the last day. AQP2 protein levels were determined by immunoblotting. RESULTS: ANP, l-arginine and 8-Br-cGMP induced the translocation of AQP2 in the mpkCCD cells. However, in contrast to dDAVP, ANP, l-arginine and 8-Br-cGMP did not increase the expression of AQP2. CONCLUSIONS: Our results suggest that while activators of the cGMP pathway are likely beneficial in the treatment of X-linked NDI, their ability to relieve NDI in the patients may be improved when combined with agents stimulating AQP2 expression.

LIP5 interacts with aquaporin 2 and facilitates its lysosomal degradation.

Vasopressin binding to the V2 receptor in renal principal cells leads to activation of protein kinase A, phosphorylation of aquaporin 2 (AQP2) at Ser256, and the translocation of AQP2 to the apical membrane, resulting in concentration of the urine. In contrast, phorbol ester-induced activation of protein kinase C pathway leads to ubiquitination of AQP2 at Lys270 and its internalization to multivesicular bodies, where it is targeted for lysosomal degradation or stored for recycling. Because little is known about the regulation of AQP2 trafficking, we used the carboxy-terminal tail of constitutively nonphosphorylated AQP2 (S256A) as a bait for interacting proteins in a yeast two-hybrid assay. We isolated lysosomal trafficking regulator-interacting protein 5 (LIP5) and found that LIP5 interacted with the proximal carboxy-terminal tail (L230-D243) of AQP2 in vitro but not with AQP3 or AQP4, which are also expressed in principal cells. Immunohistochemistry revealed that LIP5 co-localized with AQP2 in principal cells. LIP5 binding occurred independent of the state of Ser256 phosphorylation or Lys270 ubiquitination. LIP5 has been shown to facilitate degradation of the EGF receptor; here, LIP5 seemed to bind this receptor. Knockdown of LIP5 in mouse renal cells (mpkCCD) reduced the phorbol ester-induced degradation of AQP2 approximately two-fold. In summary, LIP5 binds cargo proteins and, considering the role of LIP5 in protein sorting to multivesicular bodies, plays a role in the degradation of AQP2, possibly by reducing the formation of late endosomes.

A novel insertion technique for urinary catheters in female dogs with the use of a guidewire.

BACKGROUND: Complicated transurethral urinary bladder catheterization in female dogs is a commonly encountered urologic problem, often causing inadvertent trauma to the lower urinary tract and breach in aseptic technique. KEY CONCEPTS: A novel method for transurethral insertion of urinary catheters in female dogs is described. The main variation from current procedures involves the application of the Seldinger technique with the use of an introduction catheter that is specifically designed to feed into the urethra, and the use of a guidewire to replace the introducer with an indwelling balloon catheter. SIGNIFICANCE: The technique presented offers a step-by-step approach that could improve success in urinary catheter placement. This modification may facilitate catheter insertion, be less traumatic, and offer better control of asepsis, especially with challenging urethral catheterization. The technique also offers an easy method to replace the catheter. Application of the technique may reduce catheter-associated urinary tract infections as it addresses certain infection-related risk factors. Prospective validation studies are needed to support its advantages over current urinary catheter placement techniques.

Genes Involved in Energy Metabolism Are Differentially Expressed During the Day-Night Cycle in Murine Retinal Pigment Epithelium.

Purpose: The functional interaction between photoreceptors and retinal pigment epithelium (RPE) cells is essential for vision. Phagocytosis of photoreceptor outer segments (POSs) by the RPE follows a circadian pattern; however, it remains unknown whether other RPE processes follow a daily rhythm. Therefore, our aim was to identify RPE processes following a daily rhythm. Methods: Murine RPE was isolated at Zeitgeber time (ZT) 0, 2, 4, 9, 14, and 19 (n = 5 per time point), after which RNA was isolated and sequenced. Genes with a significant difference in expression between time points (P < 0.05) were subjected to EnrichR pathway analysis to identify daily rhythmic processes. Results: Pathway enrichment revealed 13 significantly enriched KEGG pathways (P < 0.01), including the metabolic pathway (P = 0.002821). Analysis of the metabolic pathway differentially expressed genes revealed that genes involved in adenosine triphosphate production, glycolysis, glycogenolysis, and glycerophospholipid were low at ZT0 (light onset) and high at ZT19 (night). Genes involved in fatty acid degradation and cholesterol synthesis were high at light onset and low at night. Conclusions: Our transcriptome data suggest that the highest energy demand of RPE cells is at night, whereas POS phagocytosis and degradation take place in the morning. Furthermore, we identified genes involved in fatty acid and glycerophospholipid synthesis that are upregulated at night, possibly playing a role in generating building blocks for membrane synthesis.

Gene expressions of de novo hepatic lipogenesis in feline hepatic lipidosis.

OBJECTIVES: The aim of this study was to evaluate if de novo hepatic lipid synthesis contributes to fatty acid overload in the liver of cats with feline hepatic lipidosis (FHL). METHODS: Lipogenic gene expression of peroxisome proliferator-activated receptor-alpha (PPAR-α), peroxisome proliferator-activated receptor-gamma (PPAR-γ), fatty acid synthase (FASN) and sterol regulatory element-binding factor (SREBF1) were evaluated using quantitative RT-PCR in liver tissue of six cats with FHL and compared with the liver tissue of eight healthy cats. RESULTS: In liver tissue, PPAR-α, PPAR-γ and FASN mRNA expression levels were not significantly different (P >0.12, P >0.89 and P >0.5, respectively) in the FHL group compared with the control group. SREBF1 gene expression was downregulated around 10-fold in the FHL group vs the control group (P = 0.039). CONCLUSIONS AND RELEVANCE: The downregulation of SREBF1 in the liver tissue of cats with FHL does not support the hypothesis that de novo lipogenesis in the liver is an important pathway of fatty acid accumulation in FHL.