Dog Kidney Research Articles

In Vitro Nephrotoxicity and Permeation of Vancomycin Hydrochloride Loaded Liposomes.

Drugs can be toxic to the fetus depending on the amount that permeates across the maternal–fetal barrier. One way to limit the amount which penetrates this barrier is to increase the molecular size of the drug. In this study, we have achieved this by encapsulating our model antibiotic (vancomycin hydrochloride, a known nephrotoxic agent) in liposomes. PEGylated and non-PEGylated liposomes encapsulating vancomycin hydrochloride were prepared using two different methods: thin-film hydration followed by the freeze–thaw method and the reverse-phase evaporation method. These liposomes were characterized by their hydrodynamic size and zeta potential measurements, CryoTEM microscopy, loading and encapsulation efficiency studies, in vitro release measurements and in vitro cytotoxicity assays using NRK-52 E rat kidney cells. We also determined the in vitro permeability of these liposomes across the human placental cell and dog kidney cell barriers. Vancomycin hydrochloride-loaded PEGylated liposomes (VHCL-lipo) of a size less than 200 nm were prepared. The VHCL-lipo were found to have the faster release of vancomycin hydrochloride and resulted in greater viability of NRK-52E cells. In vitro, the VHCL-lipo permeated the human placental cell and dog kidney cell barriers to a lesser extent than the free vancomycin hydrochloride. The data suggest a reduction in nephrotoxicity and permeability of vancomycin hydrochloride after encapsulation in PEGylated liposomes.

ZO‐1 Knockdown in MDCK Type II Cells Decreases Leak Pathway Opening Size and Increases Opening Number

Paracellular solute movement across epithelial cell sheets is divided into at least two pathways, the Pore Pathway (high capacity, small ions and solutes, charge selective) and the Leak Pathway (low capacity, large molecules, charge insensitive). Improved understanding of the Leak Pathway is essential to evaluate its role in epithelial physiology and pathophysiology, as well as, its potential use as a pathway for the delivery of macromolecule drugs. Depletion of the tight junction‐associated cytoplasmic protein Zonula Occludens‐1 (ZO‐1) in the dog kidney epithelial cell line, MDCK Type II, increases Leak Pathway apparent permeability (Papp). There are multiple parameters that could affect Leak Pathway Papp, including the density of the pores/openings (number/unit membrane area), their length, their tortuosity, and their size (radius). A theoretical computational analysis revealed marked differences in the effect of pore size versus pore density on Papp­ as a function of solute size. We, therefore, compared the dependence of Papp on solute size in populations of wild type MDCK Type II cells, ZO‐1 knockdown MDCK Type II (ZO‐1 KD) cells, and ZO‐2 knockdown MDCK Type II (ZO‐2 KD) cells. The flux of fluorescein‐dextrans of different sizes across monolayers of wild type MDCK Type II, ZO‐1 KD, and ZO‐2 KD cell populations grown on permeable supports was measured as a function of time. Flux was linear as a function of time for all solutes across all cell lines. As expected, flux rate decreased with increasing fluorescein‐dextran Stokes radius (3 kDa ‐ ~12 Å to 70 kDa ‐ ~60 Å) in all three cell lines. For the smaller fluorescein‐dextrans, ZO‐1 KD cell populations exhibited several‐fold higher Papp values than wild type MDCK Type II cells. As solute size increased, however, the extent of the increase over the wild type MDCK Type II cells diminished progressively. In contrast, the Papp values for the wild type MDCK Type II cell populations and the ZO‐2 KD cell populations were similar throughout the fluorescein‐dextran size range. These results revealed that Papp values decreased more rapidly as a function of solute size in ZO‐1 KD cell populations than in wild type MDCK Type II cell populations or ZO‐2 KD cell populations. While we cannot determine Leak Pathway opening radius using this approach, our results argue that the Leak Pathway openings in ZO‐1 KD cell populations are smaller than those in either wild type MDCK Type II cell populations or ZO‐2 KD cell populations. Since the ZO‐1 KD cell populations exhibit an increased flux rate for smaller fluorescein‐dextrans, this suggests that the ZO‐1 KD cell populations have an increased number of Leak Pathway openings compared to wild type MDCK Type II cell populations and ZO‐2 KD cell populations. Differences in opening length and/or tortuosity may also play a role in the observed differences in solute flux rates.

A Small-Plaque Isolate of the Zika Virus with Envelope Domain III Mutations Affect Viral Entry and Replication in Mammalian but Not Mosquito Cells.

An Asian Zika virus (ZIKV) isolated from a Thai patient that was serially passaged in Primary Dog Kidney (PDK) cells for attenuation displayed both big and small plaque-forming viruses by the 7th passage. Two small-plaque isolates were selected and purified for characterization as attenuated ZIKV candidates. In vitro growth kinetics showed significantly reduced titers for small-plaque isolates in Vero cells early post-infection compared to the parental ZIKV and a big-plaque isolate, but no significant difference was observed in C6/36 cells. Viral entry experiments elucidate that titer reduction likely occurred due to the diminished entry capabilities of a small-plaque isolate. Additionally, a small-plaque isolate displayed lowered neurovirulence in newborn mice compared to 100% lethality from infection with the parental ZIKV. Genomic analysis revealed the same three unique non-synonymous mutations for both small-plaque isolates: two on the envelope (E) protein at residues 310, alanine to glutamic acid (A310E), and 393, glutamic acid to lysine (E393K), and one on residue 355 of NS3, histidine to tyrosine (H355Y). Three-dimensional (3D) mapping suggests that the E protein mutations located on the receptor-binding and fusion domain III likely affect cell entry, tropism, and virulence. These ZIKV isolates and genotypic markers will be beneficial for vaccine development.

Preclinical safety profile of a liver-localized mitochondrial uncoupler: OPC-163493.

Mitochondrial uncouplers (mUncouplers) are known to exhibit a variety of toxic effects in animals. Here we report a safety profile of an mUncoupler, OPC-163493, recently synthesized at Otsuka Pharmaceutical Co, Ltd, and its development as a therapeutic agent for treating diabetes. To understand the acute and subchronic toxicity of OPC-163493, single and repeated oral dose studies in rats, dogs, and monkeys were performed. In the rat studies, rigor mortis and increased body temperatures were observed in the high dose group. Focal necrosis, fatty change, and granular eosinophilic cytoplasm of the hepatocytes were also observed in the high dose group. In the dog studies, gastrointestinal manifestations were observed with decreased body weight and decreased food consumption in the high dose group. Necrotizing arteritis was observed in multiple organs as well as meningitis with hemorrhage in the brain. In the monkey studies, vomiting, decreased food consumption, and decreased locomotor activity were observed in the high dose group. Degeneration of the proximal convoluted tubules and the straight tubular epithelium, regeneration of the proximal tubular epithelium, and degeneration of the collecting tubular epithelium were observed. The target organs of OPC-163493 were liver, blood vessels, and kidney in rats, dogs, and monkeys, respectively. In rats, dogs, and monkeys, safety ratios were 100:1, 13:1, and 20:1, respectively, in terms of total exposure (AUC24h). These safety ratios showed clear separation between exposure to OPC-163493 in animals at NOAEL and the exposure at the effective dose in ZDF rats. This information should contribute to the drug development of new and effective mUncoupler candidates.

Comparative quantitation of aquaporin-2 and arginine vasopressin receptor-2 localizations among chronic kidney disease and healthy kidney in dogs.

Background and Aim:Aquaporin-2 (AQP2) and arginine vasopressin receptor-2 (AVPR2) are proteins that control water homeostasis in principal cells. Chronic kidney disease (CKD) is defined as the impairment and irreversible loss of kidney function and/or structure, which causes water imbalances and polyuria. The study aimed to know the expression of AQPs and AVPR2 in the kidneys of a canine with CKD.Materials and Methods:The kidneys were collected from two dog carcasses from Small Animal Teaching Hospital, Faculty of Veterinary Medicine, Chiang Mai University. The kidney tissue was prepared for immunohistochemistry and investigated the expression and localization of tissue’s AQP2 and AVPR2. For statistical analysis, the Mann–Whitney U-test was applied to the data.Results:By immunohistochemistry, AQP2 was expressed strongly in the basolateral and apical membranes of the principal cells, whereas AVPR2 was localized in the principal cell’s basolateral membrane in both renal cortex and renal medulla. In the normal kidney, the semi-quantitative immunohistochemistry for the percentage of protein expression of AQP2 and AVPR2 was 5.062±0.4587 and 4.306±0.7695, respectively. In contrast, protein expression of AQP2 and AVPR2 in CKD was found to be 1.218±0.1719 and 0.8536±0.1396, respectively. The data shows that the percentage of AQP2 and AVPR2 expression was decreased, corresponding to a 4-fold and 5-fold in CKD (p<0.001).Conclusion:Our findings revealed that CKD was a marked decrease in AQP2 and AVPR2 expression. The central role of specific AQP2 and AVPR2 in regulating water homeostasis will provide correlations in case of CKD with polyuria.

Renal Diffusion-Weighted Imaging in Healthy Dogs: Reproducibility, Test-Retest Repeatability, and Selection of the Optimal b-value Combination.

Diffusion-weighted imaging (DWI) magnetic resonance imaging can evaluate alterations in the microstructure of the kidney. The purpose of this study was to assess the apparent diffusion coefficient (ADC) and the intravoxel incoherent motion model (IVIM) parameters of a normal kidney in healthy dogs, to evaluate the effect of b-value combinations on the ADC value, and the reproducibility and test-retest repeatability in monoexponential and IVIM analysis. In this experimental study, the ADC, pure diffusion coefficient (D), pseudodiffusion coefficient (D*), and perfusion fraction (f p) were measured from both kidneys in nine healthy beagles using nine b-values (b = 0, 50, 70, 100, 150, 200, 500, 800, and 1,000 s/mm2) twice with a 1-week interval between measurements. Interobserver and intraobserver reproducibility, and test-retest repeatability of the measurements were calculated. ADC values were measured using 10 different b-value combinations consisting of three b-values each, and were compared to the ADC obtained from nine b-values. All the ADC, D, D*, and f p values measured from the renal cortex, medulla, and the entire kidney had excellent interobserver and intraobserver reproducibility, and test-retest repeatability. The ADC obtained from a b-value combination of 0, 100, and 800 s/mm2 had the highest intraclass correlation coefficient with the ADC from nine b-values. The results of this study indicated that DWI MRI using multiple b-values is feasible for the measurement of ADC and IVIM parameters with high reproducibility and repeatability in the kidneys of healthy dogs. A combination of b = 0, 100, and 800 s/mm2 can be used for ADC measurements when multiple b-values are not available in dogs.

The evolutionary development of the renin angiotensin aldosterone system and its importance for the survival of the human species

Kidneys produce a number of substances that affect intrarenal blood circulation; however, the key system that regulates blood flow in both general and local circulation (including the renal circulation) is the renin-angiotensinaldosterone system (RAAS). Individual elements of the RAA system are synthesized in separate tissues of the body under the influence of specific local factors. The system functions as a whole due to mutual relations based on feedback and it consists of three basic elements: renin, angiotensin and aldosterone. The history of research on the RAA system dates back to the late 19th century. One of the important stages of exploring the mechanisms related to RAA system functioning was the publication (in 1898) of the results of research on the hypertensive effect on blood pressure of rabbit kidney extracts (containing renin). The observations from 1934 were of similar significance: the correlation between dog kidney ischaemia and the occurrence of hypertension was found. In the following years, the enzymatic properties and structure of renin and angiotensin peptides, resulting from the action of renin and the enzyme converting angiotensin I (Ang I) to its active form — angiotensin II (Ang II), were clarified. The latter belongs to the most important regulators of aldosterone secretion. In 1939, it was proved that under the influence of renin blood pressure-rising peptides are formed. Consequently, it was documented that angiotensin was the cause of hypertension in animals with ischaemic kidney, and in 1954 the sequence of angiotensin I and II was described. In 1960–1961 systemic RAA occurrences were identified. However, to provide the insight of evolutionary significance of the RAA system for humans, the phylogenetic development of this enzyme-endocrine system in vertebrates should be investigated. The largest database of information regarding this system in the aforementioned group of animals is the research of Hirofumi Sokabe and Hiroko Nishimura, which, among others, is the basis for this manuscript.

МОРФОЛОГИЯ ВЕНОЗНОЙ СИСТЕМЫ ПОЧЕК СОБАК В ПОСТНАТАЛЬНОМ ПЕРИОДЕ ОНТОГЕНЕЗА

The article presents data on the dynamics of morphometric parameters of the diameter of the caudal vena cava and renal veins of dogs in the postnatal period of ontogenesis, the peculiarities of the course and branching of extra- and intraorganic venous vessels of the kidneys of dogs, as well as the formation of the right and left renal veins in the gates of the organ. The increase in the diameter of the caudal vena cava and renal veins of dogs in the postnatal period of ontogenesis is uneven. An intense increase in the diameter of the caudal vena cava in dogs was detected at 3 months. and seven years. The diameter of the renal veins increases rapidly by 3 months. the age of the animals. In most cases, the intraorgan renal veins of the dogs on the right and left form one trunk of the efferent renal vein. The formation of the right renal vein in the gates of the organ occurs in three ways: two, three and four trunks, and the left renal vein by the fusion of two interlobar veins. The double trunk of the right renal vein is rarely recorded. The data obtained in the course of morphological study on the venous system of the kidneys of dogs can be used for op-erative access to organs.

Hydrodynamic particle focusing enhanced by femtosecond laser deep grooving at low Reynolds numbers

Microfluidic focusing of particles (both synthetic and biological), which enables precise control over the positions of particles in a tightly focused stream, is a prerequisite step for the downstream processing, such as detection, trapping and separation. In this study, we propose a novel hydrodynamic focusing method by taking advantage of open v-shaped microstructures on a glass substrate engraved by femtosecond pulse (fs) laser. The fs laser engraved microstructures were capable of focusing polystyrene particles and live cells in rectangular microchannels at relatively low Reynolds numbers (Re). Numerical simulations were performed to explain the mechanisms of particle focusing and experiments were carried out to investigate the effects of groove depth, groove number and flow rate on the performance of the groove-embedded microchannel for particle focusing. We found out that 10-µm polystyrene particles are directed toward the channel center under the effects of the groove-induced secondary flows in low-Re flows, e.g. Re < 1. Moreover, we achieved continuous focusing of live cells with different sizes ranging from 10 to 15 µm, i.e. human T-cell lymphoma Jurkat cells, rat adrenal pheochromocytoma PC12 cells and dog kidney MDCK cells. The glass grooves fabricated by fs laser are expected to be integrated with on-chip detection components, such as contact imaging and fluorescence lifetime-resolved imaging, for various biological and biomedical applications, where particle focusing at a relatively low flow rate is desirable.

Expression of neuronal nitric oxide synthase and renin in dysplastic kidneys of young dogs.

Renin and neuronal nitric oxide synthase in the kidney control the renin-angiotensin and tubuloglomerular feedback systems. The present study investigated the expression of renin and neuronal nitric oxide synthase in the dysplastic kidneys of three young dogs. Renin-immunoreactivity, which occurs in the juxtaglomerular and tubular cells of dysplastic kidneys, did not differ from that in the normal kidneys of young dogs. Macula densa cells in the normal kidneys showed neuronal nitric oxide synthase -immunoreactivity, but those in the dysplastic kidneys showed no apparent signals. This observation may be correlated with the pathological mechanisms of renal failure in young dogs.

Assessment of neutrophil gelatinase-associated lipocalin as an early biomarker for canine renal ischemia-reperfusion injury.

BackgroundThe pathological mechanism of ischemia/reperfusion acute kidney injury (I/R-AKI) differs from other forms of AKI. Neutrophil gelatinase-associated lipocalin (NGAL) is a sensitive biomarker for early diagnosis of AKI, but its utility for diagnosis of canine I/R-AKI remains to be evaluated. The aims of this study were to establish an I/R-AKI model in dogs and to evaluate the diagnostic value of NGAL for canine I/R-AKI.MethodsWe randomly divided 12 beagle dogs into a sham and an I/R group. Artery and vein of the left kidneys of I/R group were cross-clamped for 60 min followed by reperfusion. The kidney samples were analyzed for histopathological lesions. Serum and urinary samples were analyzed for blood urea nitrogen (BUN), serum creatinine (sCr), serum NGAL (sNGAL), urinary creatinine (uCr), and urinary NGAL (uNGAL). Their detection sensitivities and specificities were compared using a receiver operating characteristics (ROC) method. The expression of NGAL in the renal tissues was analyzed by quantitative RT-PCR (qRT-PCR) and immunohistochemical (IHC) analysis.ResultsAfter I/R, histopathological analysis showed typical AKI lesions in the dog kidneys of the I/R group, but not in the sham group. Compared to that of the sham group, BUN and sCr of the I/R group rose to significant high levels from 24 h after I/R. Both uNGAL and sNGAL rose rapidly from 2 h, reached to the peak levels at 12 h, and then receded to the pre-operation levels by 72 h after I/R. The uNGAL/uCr ratio (uNCR) rose rapidly from 2 h and remained at variably high levels from 6 to 60 h after I/R. The ROC analysis showed that detection sensitivities of uNCR, uNGAL, and sNGAL were significantly (P<0.0001) higher than that of sCr, without significant difference in specificity. The cut-off values of sNGAL, uNGAL and uNCR were 14,642 pg/mL, 6,773 pg/mL, and 6,701 pg/mg, respectively. Both qRT-PCR and IHC analyses confirmed the dynamic expression of NGAL in the dog kidneys with ischemic acute kidney injury (I-AKI).ConclusionsThere is potential for NGAL to be used as a sensitive biomarker for early diagnosis of canine I-AKI.

Expression of Phosphatonin-Related Genes in Sheep, Dog and Horse Kidneys Using Quantitative Reverse Transcriptase PCR.

Simple SummaryTraditionally, it has been thought that control of body phosphorus was secondary to the tighter control of calcium. However, over the last 20 years, an extensive system for control of body phosphorus by proteins called phosphatonins has been shown to exist. Most research on phosphatonins has been done in rat or mouse models. This paper looks at whether important proteins and phosphorus channels in the phosphatonin pathways are present in the kidneys of dogs, horses and sheep. The results showed that all of the components of the phosphatonin system are present in these species, but that there are species differences in which protein or channel is most common, and in the relationships between the proteins and channels. This research is important because the phosphatonin system is involved in the progression of chronic kidney disease in humans and animals, and differences in the systems between animal species may affect treatment of chronic kidney disease.The aim of this preliminary study was to determine the relative expression of phosphatonin pathway-related genes in normal dog, sheep and horse kidneys and to explore the relationships between the different genes. Kidneys were collected post-mortem from 10 sheep, 10 horses and 8 dogs. RNA was extracted, followed by reverse transcriptase quantitative polymerase chain reaction for fibroblast growth factor receptor 1 IIIc (FGFR1IIIC), sodium-phosphate co-transporter (NPT) 1 (SLC17A1), NPT2a (SLC34A1), NPT2c (SLC34A3), parathyroid hormone 1 receptor (PTH1R), klotho (KL), vitamin D receptor (VDR), 1a-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1). NPT2a was highly expressed in the dog kidneys, compared with those of the horses and sheep. NPT1 had greatest expression in horses and sheep, although the three different NPTs all had relatively similar expression in sheep. There was little variability in FGFR1IIIc expression, particularly in the dogs and horses. FGFR1IIIc expression was negatively correlated with NPT genes (except NPT2a in sheep), while NPT genes were all positively correlated with each other. Unexpectedly, klotho was positively correlated with NPT genes in all three species. These results provide the basis for further research into this important regulatory system. In particular, species differences in phosphatonin gene expression should be considered when considering the pathogenesis of chronic kidney disease.

Chronic Inflammatory Disease in the Pancreas, Kidney and Salivary Glands of English Cocker Spaniels and Dogs of Other Breeds Shows Similar Histological Features to Human IgG4-related Disease

Chronic pancreatitis (CP) is a common disease in the English cocker spaniel (ECS) and is characterized histologically by duct destruction, interlobular fibrosis and dense periductular and perivenous lymphocytic aggregates. These features are also found in human autoimmune pancreatitis type 1, part of a glucocorticoid-responsive, multiorgan syndrome, newly recognized as IgG4-related disease (IgG4-RD). Human IgG4-RD affects one or several organs, often showing a predominance of IgG4+ plasma cells histologically, with an IgG4+:total IgG+ plasma cell ratio of >40%. This study investigated whether ECSs with CP and/or inflammatory disease in several organs show an increase in IgG4+ plasma cells within affected tissues. Histological sections of pancreas, liver, kidney, salivary gland and conjunctiva were obtained from ECSs with idiopathic chronic inflammatory disease affecting those tissues. Tissue samples from age-matched dogs of other breeds with similar diseases were also sampled. Control diseased tissue samples, from dogs without a suspected immune-mediated disease, were included. A subset of ECSs and dogs of other breeds presented with disease in more than one organ. Immunohistochemistry was performed with primary reagents detecting total IgG and three of the four canine IgG subclasses (IgG2, IgG3 and IgG4). Normal sections of pancreas and liver showed an absence of labelled plasma cells of any subclass. Normal kidney and salivary gland sections showed the presence of a few labelled plasma cells (<10 plasma cells/high-power field). Fourteen tissue sections from 12 ECSs and seven sections from six dogs of other breeds showed elevated numbers of IgG4+ plasma cells and IgG4+:IgG+ ratios >40%. Individual dogs (ECSs and other breeds) showed marked increases in IgG4+ cells. There were no significant differences in the number of IgG4+ plasma cells between ECSs and dogs of other breeds for affected pancreas, liver, salivary glands and conjunctiva. Kidney sections had more IgG4+ cells, for both ECSs and dogs of other breeds, than did sections from other organs. Dogs of other breeds had significantly more IgG4+ plasma cells in affected kidneys than ECSs. In conclusion, several ECSs and dogs of other breeds fulfilled the histological criteria for the diagnosis of IgG4-RD, supporting the existence of a multiorgan immune-mediated disease in ECSs and some dogs of other breeds.

Novel Effect of Chitooligosaccharide on Cyst Growth Retardation in an in vitro Cyst Model of Polycystic Kidney Disease

Polycystic kidney disease (PKD) is a common inherited disorder characterized by multiple fluid‐filled cysts that replace normal renal parenchyma along the renal tubules leading to impaired renal function and end‐stage renal disease (ESRD). It has recently been reported that the activation of AMP‐activated protein kinase (AMPK) by metformin inhibits mammalian target of rapamycin (mTOR)‐mediated cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated chloride secretion in Madin‐Darby canine kidney (MDCK) cell, a cell line derived from distal nephron of dog kidneys. Therefore, AMPK activators are proposed to be a promising agent for PKD treatment. Recently, our research group has shown that chitooligosaccharide (COS) derived from chitin/chitosan activates AMPK in intestinal epithelial cells. This study was conducted based on the hypothesis that AMPK activation by COS may inhibit cyst growth in the in vitro cyst model of PKD. We found that COS at the concentrations of 10, 50, and 100 μg/mL decreased cyst growth in a concentration‐dependent manner without toxicity measured by MTT assays. Immunoblotting analysis showed that COS at 100 μg/mL activated AMPK and this effect was abolished by STO‐609, a calcium/calmodulin‐dependent kinase kinase beta (CaMKKβ) inhibitor. Moreover, COS could elevate the level of intracellular calcium. These results suggest that COS inhibits cyst progression by activation of AMPK and the increment of intracellular calcium level via CaMKKβ. Therefore, COS has novel potential application for the treatment of polycystic kidney disease.Support or Funding InformationFaculty of Medicine Ramathibodi Hospital, Mahidol University

The positive effect of selective prostaglandin E2 receptor EP2 and EP4 blockade on cystogenesis in vitro is counteracted by increased kidney inflammation in vivo

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major cause of end-stage kidney disease in man. The central role of cyclic adenosine monophosphate (cAMP) in ADPKD pathogenesis has been confirmed by numerous studies including positive clinical trial data. Here, we investigated the potential role of another major regulator of renal cAMP, prostaglandin E2 (PGE2), in modifying disease progression in ADPKD models using selective receptor modulators to all four PGE2 receptor subtypes (EP1-4). In 3D-culture model systems utilizing dog (MDCK) and patient-derived (UCL93, OX161-C1) kidney cell lines, PGE2 strikingly promoted cystogenesis and inhibited tubulogenesis by stimulating proliferation while reducing apoptosis. The effect of PGE2 on tubulogenesis and cystogenesis in 3D-culture was mimicked or abolished by selective EP2 and EP4 agonists or antagonists but not those specific to EP1 or EP3. In a Pkd1 mouse model (Pkd1nl/nl), kidney PGE2 and COX-2 expression were increased by two-fold at the peak of disease (week four). However, Pkd1nl/nl mice treated with selective EP2 (PF-04418948) or EP4 (ONO-AE3-208) antagonists from birth for three weeks had more severe cystic disease and fibrosis associated with increased cell proliferation and macrophage infiltration. A similar effect was observed for the EP4 antagonist ONO-AE3-208 in a second Pkd1 model (Pax8rtTA-TetO-Cre-Pkd1f/f). Thus, despite the positive effects of slowing cyst growth invitro, the more complex effects of inhibiting EP2 or EP4 invivo resulted in a worse outcome, possibly related to unexpected pro-inflammatory effects.

DIAGNOSTICS AND TREATMENT OF DIOCTOPHYMOSIS IN DOGS

This article includes materials on the rare disease in domestic and wild carnivores, dioctophimosis, caused by a helminth from the group of aphasmidia nematodes, Dioctophyme renale, which is parasitic in the kidneys, recently registered in veterinary clinics in Almaty. The disease has important social significance - a person is susceptible to it, the infection of which occurs when raw fish is eaten. In Almaty, according to the statistics of veterinary clinics in recent years (2018-2019), 17 cases of dioctophimosis in dogs have been registered, that is up to 0.3% of the number of dogs examined for helminthiases. The appearance of this disease, which is relatively new for our region, can have a significant impact on the epizootiological and epidemiological situation. The results of in vivo laboratory diagnosis of dioctophimosis in dogs by ovoscopic methods are presented, with the provision of microphotos of the parasite eggs; methods of instrumental diagnostics - ultrasound examination (ultrasound) of the kidneys with the provision of photos proving the presence of a parasite in the renal pelvis. Since the only treatment for dioctophimosis is surgery and extraction of dioctophyma from the affected organ (kidney), our own original materials are presented for a step by step surgical intervention to extract helminth from the kidneys of dogs and treat animals. The condition of the operated animals is satisfactory, the prognosis for surgery is favorable.

Evaluation of the serum biochemistry and histopathology of kidney and bladder of dogs with Leishmania sp. in their urine

The visceral establishment of Leishmania infantum in dogs may result in kidney and bladder tissue injury, with L. infantum ending up in urine. This study therefore aimed at investigating the presence of Leishmania sp. in urinary sediments, and correlating the results with those from renal and bladder serum biochemistry and histopathology. Thirty dogs with negative Nested-Polymerase Chain Reaction (PCR) for E. canis were used in the experiment, and were divided into three groups: control group (10 dogs), neither leishmaniasis nor clinical changes; group I (15 dogs), leishmaniasis but no Leishmania sp. in urine; and group II (5 dogs), leishmaniasis, as well as Leishmania sp. in urine. All animals were submitted to clinical, serological, and parasitological diagnosis for leishmaniasis, biochemical exams, and kidney and bladder histopathology. The parasite was also detected in the bladder imprint of one group II dog. Group II dogs presented with very low albumin concentrations, low albumin/globulin ratios, and kidney and bladder lesions. In the kidneys, hydropic degeneration, thickened Bowman's capsule, and thickening of the tubular capsule were detected in all dogs with positive urinary sediment. However, no significant difference in these renal changes was observed between groups. The intensity and distribution of bladder inflammatory infiltrates were significantly (p-value &lt; 0.05, Kruskal-Wallis’ and Dunn’s tests) higher in group II dogs, compared with those of the other groups. The presence of Leishmania sp. in the urine of infected dogs appeared to be related to low serum albumin concentrations and more severe bladder lesions.

A GM1b/asialo-GM1 oligosaccharide-binding R-type lectin from purplish bifurcate mussels Mytiliseptavirgata and its effect on MAP kinases.

A 15‐kDa lectin, termed SeviL, was isolated from Mytilisepta virgata (purplish bifurcate mussel). SeviL forms a noncovalent dimer that binds strongly to ganglio‐series GM1b oligosaccharide (Neu5Acɑ2‐3Galβ1‐3GalNAcβ1‐4Galβ1‐4Glc) and its precursor, asialo‐GM1 (Galβ1‐3GalNAcβ1‐4Galβ1‐4Glc). SeviL also interacts weakly with the glycan moiety of SSEA‐4 hexaose (Neu5Acα2‐3Galβ1‐3GalNAcβ1‐3Galα1‐4Galβ1‐4Glc). A partial protein sequence of the lectin was determined by mass spectrometry, and the complete sequence was identified from transcriptomic analysis. SeviL, consisting of 129 amino acids, was classified as an R(icin B)‐type lectin, based on the presence of the QxW motif characteristic of this fold. SeviL mRNA is highly expressed in gills and, in particular, mantle rim tissues. Orthologue sequences were identified in other species of the family Mytilidae, including Mytilus galloprovincialis, from which lectin MytiLec‐1 was isolated and characterized in our previous studies. Thus, mytilid species contain lectins belonging to at least two distinct families (R‐type lectins and mytilectins) that have a common β‐trefoil fold structure but differing glycan‐binding specificities. SeviL displayed notable cytotoxic (apoptotic) effects against various cultured cell lines (human breast, ovarian, and colonic cancer; dog kidney) that possess asialo‐GM1 oligosaccharide at the cell surface. This cytotoxic effect was inhibited by the presence of anti‐asialo‐GM1 oligosaccharide antibodies. With HeLa ovarian cancer cells, SeviL showed dose‐ and time‐dependent activation of kinase MKK3/6, p38 MAPK, and caspase‐3/9. The transduction pathways activated by SeviL via the glycosphingolipid oligosaccharide were triggered apoptosis.DatabaseNucleotide sequence data have been deposited in the GenBank database under accession numbers MK434191, MK434192, MK434193, MK434194, MK434195, MK434196, MK434197, MK434198, MK434199, MK434200, and MK434201.

Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells.

Bats are natural reservoirs of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Scotophilus bat CoV-512 demonstrates potential for cross-species transmission because its viral RNA and specific antibodies have been detected in three bat species of Taiwan. Understanding the cell tropism of Scotophilus bat CoV-512 is the first step for studying the mechanism of cross-species transmission. In this study, a lentivirus-based pseudovirus was produced using the spike (S) protein of Scotophilus bat CoV-512 or SARS-CoV as a surface protein to test the interaction between coronaviral S protein and its cell receptor on 11 different cells. Susceptible cells expressed red fluorescence protein (RFP) after the entry of RFP-bound green fluorescence protein (GFP)-fused S protein of Scotophilus bat CoV-512 (RFP-Sco-S-eGFP) or RFP-SARS-S pseudovirus, and firefly luciferase (FLuc) activity expressed by cells infected with FLuc-Sco-S-eGFP or FLuc-SARS-S pseudovirus was quantified. Scotophilus bat CoV-512 pseudovirus had significantly higher entry efficiencies in Madin Darby dog kidney epithelial cells (MDCK), black flying fox brain cells (Pabr), and rat small intestine epithelial cells (IEC-6). SARS-CoV pseudovirus had significantly higher entry efficiencies in human embryonic kidney epithelial cells (HEK-293T), pig kidney epithelial cells (PK15), and MDCK cells. These findings demonstrated that Scotophilus bat CoV-512 had a broad host range for cross-species transmission like SARS-CoV.

Evaluation of Fraction Unbound Across 7 Tissues of 5 Species

Binding to various tissues and species is frequently assessed in drug discovery and development to support safety and efficacy studies. To reduce time, cost, and labor requirements for binding experiments, we conducted a large comparison study to evaluate the correlation of fraction unbound (fu) across 7 tissues of 5 species, including white adipose, brain, heart, kidney, liver, lung, and skeletal muscle of mouse, rat, dog, monkey, and human. The results showed that there were no significant species differences of fu for tissue binding, and a single-species (e.g., rat) tissue fu can be used as a surrogate for binding in other species. Cross-tissue comparison indicated that brain, heart, liver, and muscle had quite similar fu values; rat liver binding can be used as a surrogate for binding of the other 3 tissues without any scaling factors. Binding to adipose, kidney, and lung can also be estimated with rat liver fu with scaling factors. This study suggests that a single tissue of a single species (e.g., rat liver) is a good predictor for fu of other tissues of various species with or without scaling factors. Molecular size, lipophilicity, pKa, and topological polar surface area are important physiochemical properties influencing tissue fu.