Kidney Samples Research Articles

Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC-1α-mediated Transcriptional Coactivation.

Calcium oxalate (CaOx) crystals induce renal tubular epithelial cell injury and subsequent nephropathy. However, the underlying mechanisms remain unclear. In the present study, single-cell transcriptome sequencing is performed on kidney samples from mice with CaOx nephrocalcinosis. Renal proximal tubular cells are identified as the most severely damaged cell population and are accompanied by elevated ferroptosis. Further studies demonstrated that sirtuin1 (Sirt1) effectively reduced ferroptosis and CaOx crystal-induced kidney injury in a glutathione peroxidase 4 (GPX4)-dependent manner. Mechanistically, Sirt1 relies on peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) to promote resistance to ferroptosis in the tubular epithelium, and PGC-1α can recruit nuclear factor erythroid 2-related factor 2 (NRF2) to the promoter region of GPX4 and co-activate GPX4 transcription. This work provides new insight into the mechanism of CaOx crystal-induced kidney injury and identifies Sirt1 and PGC-1α as potential preventative and therapeutic targets for crystal nephropathies.

Toxicological Evaluation of Sargassum polycystum in Mice with Loperamide-Induced Constipation

Constipation gradually increases in people after the age of 50 years leading to difficulty in stool passage. Until now, there have been no effective drugs without side effects. This study investigated the preventive potential and toxicity of Sargassum polycystum extract (SPE) against loperamide-induced constipation in mice. Daily oral administration of SPE (100, 500 and 1000 mg/kg) for 14 days significantly increased defecation frequency in constipation mice. Additionally, no significant differences in blood haematological and biochemical indices between the control, constipation and SPE-treated groups were detected. Histopathological examinations revealed non-toxic effects in liver, kidney and colon tissue samples from the SPE-treated groups compared with the control and constipation groups. Real-time polymerase chain reaction showed that the administration of SPE significantly modulated Saa3, Odc1 and Clu expressions that promote the health of living organisms compared with the control group. Overall, this study showed that daily oral administration of Sargassum polycystumfor 14 days could prevent constipation without any adverse effects. Sargassum polycystumis suitable for further use as a supplement or drug to manage constipation.

Factors affecting the concentration of metals and metalloids in the kidneys of a top predator, the Eurasian Buzzard (Buteo buteo) wintering in farmland in Poland.

During late autumn and winter, raptors in the western Palearctic face challenges due to food scarcity and dropping temperatures. That time they can be exposed to various elements including toxic ones ingested with food. Kidney samples from 22 females and 19 males of a medium-sized raptor, the Common Buzzard Buteo buteo found dead in farmland of Eastern Poland in winter were analyzed for a concentration of 21 elements. Elemental concentrations were analyzed regarding the age and sex of birds. Results revealed that only 4.9% of individuals had kidney lead levels exceeding 8.0mg, while 9.8% showed cadmium levels above 8.0mg/kg, indicating potential poisoning. The study also highlighted the limited entry of arsenic into agricultural ecosystems exploited by Common Buzzards. Sex differences were noted, with females accumulating more lead and vanadium than males which can be associated with foraging niche partitioning between sexes driven by body size dimorphism. Sulfur showed complex interactions with cadmium, mercury, and zinc, with a positive correlation between sulfur and zinc levels in the kidneys, emphasizing dietary needs during food scarcity. A positive correlation was found between zinc and lead concentrations, indicating zinc's role in mitigating lead's impact. The study also revealed positive correlations between selenium and highly toxic elements like mercury (Spearman correlation, rs = 0.41) and cadmium (rs = 0.51), suggesting a mitigating effect of selenium on exposure to heavy metals. This study enhances understanding of year-round environmental contamination exposure for raptors and sheds light on bioaccumulation in a top predator.

Pharmacokinetics and cardioprotective efficacy of intravenous miR-125b* microRNA mimic in a mouse model of acute myocardial infarction.

MicroRNA (miRNA) therapy is a promising approach to induce cardioprotection. We have previously identified cardiac microRNA-125b* (microRNA-125b-2-3p; miR-125b*) as a potential cardioprotective miRNA, termed ProtectomiR. We aimed to characterize the pharmacokinetics and pharmacodynamics, and the effect of miR-125b* mimic on infarct size using an in vivo mouse model. To characterize the pharmacokinetics properties of miR-125b* mimic, a single injection of 10-μg miR-125b* mimic or its scramble miRNA control, or vehicle i.v. was given to C57BL/6 mice. MiR-125b* expression was measured from plasma, heart, kidney and liver samples. Effect of miR-125b* on area at risk and infarct size was assessed after 45-min coronary occlusion, followed by 24-h reperfusion; 10-μg miR-125b* mimic or 10-μg non-targeting miRNA mimic control or vehicle were administered via the right jugular vein at 10th mins of coronary occlusion. To assess molecular mechanism involved in cardioprotection, expression of mRNA targets of miR-125b* were measured from ventricular myocardium at 1, 2, 4, 8 or 24 hpost-treatment using quantitative real time polymerase chain reaction. MiR-125b* expression was markedly increased in plasma and myocardium 1h, and in the liver 2h after treatment. Infarct size was significantly reduced after miR-125b* mimic treatment when compared to the vehicle. The expression of Ccna2, Eef2k and Cacnb2 target mRNAs was significantly reduced 8h after injection of miR-125b* mimic. This is the first demonstration of pharmacokinetic and molecular pharmacodynamic properties as well as the cardioprotective effect of miR-125b* mimic in vivo.

First detection of D181 genotype of infectious bronchitis in poultry flocks of Morocco

BackgroundThis paper reports the first pathological and molecular characterization of the novel variant of infectious bronchitis virus (IBV) D181 in poultry flocks in Morocco and Africa.MethodsThe study includes six poultry farms, involving three flocks of layers aged between 28 and 67 weeks and three broiler flocks aged 27, 39 and 42 days from different regions of Morocco. In all affected layer flocks, a severe drop in egg production with poor eggshell quality was reported. Necropsy of dead birds was carried out, and samples of trachea, lungs, oviduct, ovaries, and kidneys were fixed in 10% neutral buffered formalin for histopathologic examinations, while other portions were stored at -20 °C for molecular analysis. Real time RT-qPCR for IBV gene group was performed, and IBV variants were identified. Partial S1 gene sequences were amplified by conventional RT-PCR, sequenced, and aligned for phylogenetic and amino acid similarity analysis.ResultsNecropsy of dead birds revealed misshapen and hemorrhagic ovarian follicles with an edematous oviduct and severe reaction in the cecal tonsils. A caseous material accumulation in the sinus was noted in few birds. In contrast, the broiler flocks exhibited respiratory clinical signs such as difficulty in breathing, sneezing, tracheal rales, watery eyes and lethargy, associated with a decrease in feed consumption. Mortality in broiler ranged from 2 to 15%. Histopathological analysis of samples showed a lympho-plasmocytic inflammation in the oviduct, trachea, and lungs. Individual necrosis of epithelial cells, with sloughing of the bronchial epithelium and accumulation of desquamated cells with mucus in the airways, was observed in some birds. Partial S1 gene sequencing and phylogenetic analyses showed that the Moroccan strains were very closely related to D181 strains isolated in Dutch layers and breeders in 2018. Nucleotide sequence identities reached 90.9–95% with the Dutch isolates (strain CK/NL/D181/2018).ConclusionOur sequencing results demonstrate for the first time that the D181 IBV genotype is circulating in Moroccan poultry. These findings justify permanent monitoring of circulating strains in order to appropriately adjust vaccination strategies to align with the evolving field situation.

Empagliflozin reduces renal calcium oxalate deposition in hyperoxaluria rats induced with ethylene glycol-ammonium chloride

A retrospective study reported that empagliflozin reduced the risk of urinary stone events in patients with diabetes mellitus. To further investigate empagliflozin's potential, we conducted an animal experiment to determine whether empagliflozin can prevent renal stone formation in hyperoxaluria rats. Hyperoxaluria rat models were constructed by administrating 0.75 % ethylene glycol and 1 % ammonium chloride in water. The empagliflozin-treated rats were gauged with empagliflozin at different concentrations, and their body weight and blood sugar data were recorded. After 30 days of treatment, we obtained 24-h urine, kidney, and blood samples. The urine samples were subjected to component detection. Blood samples were prepared for component detection and cytokines detection. Renal samples were subjected to von Kossa staining, transmission electron microscopy, immunohistochemistry, and transcriptome sequencing analysis. Results showed that in empagliflozin-treated hyperoxaluria rats, renal crystal deposition and mitochondria injury, urinary concentration, and excretion of oxalate were significantly decreased. Additionally, plasma levels of VEGF, IL-2, IL-1β, and MCP-1 were decreased. Immunohistochemistry showed that renal expression of KIM-1, MCP-1 was significantly decreased in empagliflozin-treated hyperoxaluria rats. Transcriptome sequencing of renal tissue represented that 25 genes were down-regulated while 12 were up-regulated in empagliflozin-treated hyperoxaluria rats. These regulated genes were mainly enriched in fatty acid metabolism, insulin resistance, muscle contraction, bile secretion, and parathyroid metabolism. Our animal experiments found that empagliflozin could reduce urinary concentration and excretion of oxalate and inhibit renal inflammation, then abating renal calcium oxalate deposition in hyperoxaluria rats in a non-diabetic state.

Neuropilin-1 as a Key Molecule for Renal Recovery in Lupus Nephritis: Insights from an NZB/W F1 Mouse Model

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs, with lupus nephritis (LN) occurring in 40–50% of SLE patients. Despite advances in diagnosis and treatment, LN remains a major cause of morbidity and mortality, with 10–20% of patients progressing to end-stage renal disease (ESRD). While knowledge of LN’s pathogenesis has improved, mechanisms of renal recovery are still largely unexplored. Neuropilin-1 (NRP-1), a transmembrane receptor expressed in renal tissue, has emerged as a promising biomarker for assessing renal recovery in LN. This study evaluates and correlates longitudinal levels of NRP-1 with kidney histology using an NZB/W F1 mouse model of LN. A total of 30 mice were used, with 15 receiving intravenous cyclophosphamide (CYC) and 15 being untreated. NRP-1 levels were measured in urine and serum, and kidney samples were taken from a subgroup of mice for histological evaluation. The results demonstrated a progressive increase in renal and urinary NRP-1 expression, particularly notable at weeks 26 and 32. Urinary NRP-1 levels above 34.40 ng/mL were strong predictors of favorable renal response, showing 100% sensitivity and 88% specificity. These findings indicate a robust correlation between urinary NRP-1 levels and renal histological recovery, underscoring the potential of NRP-1 as a valuable biomarker for assessing renal response in LN. This study demonstrates that renal production of NRP-1 is linked to histological recovery and establishes a foundation for future research into the role of NRP-1 in lupus kidney recovery.

Rapid identification of antibiotic residues in bovine kidney using coated blade spray-mass spectrometry.

The use of certain antibiotics in food-producing animals is allowed in Europe following Regulation (EU) 2017/625. However, use could result in antibiotic residues in foodstuffs of animal origin. Maximum residue limits (MRLs) are in place to protect consumers. For monitoring purposes, animal matrices are tested to verify their compliance with these MRLs. Initially, matrices of (slaughtered) food animals are screened, often using a microbiological assay. Faster screening tests for antibiotics would be an advantage for control laboratories. Therefore, the present study describes, for the first time, the use of coated blade spray (CBS) followed by direct mass spectrometry (MS) analysis for the screening of tetracyclines, sulfonamides, quinolones, and macrolides residues from the renal area of intact bovine kidneys. An optimized workflow using two different desorption/ionization solutions per blade allowed screening of target compounds within 1min per sample. The proof-of-principle of the CBS-MS method is validated according to (EU) 2021/808, presenting CCβ screening values of 0.1 × MRL for 43 analytes, 0.5 × MRL for 4 analytes, and 2.5µgkg-1 for the prohibited substance dapsone, respectively. The developed method was successfully applied to seven official control samples of bovine kidneys. One of these samples was found to be positive using the CBS-MS method, which was confirmed as a true positive by LC-MSMS analysis. The developed method demonstrates that CBS devices can directly extract and analyze kidney samples for food safety testing.

Simultaneous determination of macrolide and quinolone antibiotics in animal tissues for human consumption validated by Regulation (EU) 2021/808

In this work, an analytical method for the simultaneous detection and quantification of 11 macrolides (erythromycin A, gamithromycin, lincomycin, neospiramycin, pirlimycin, spiramycin, tildipirosin, tilmicosin, tulathromycin A and its metabolite CP-60300, and tylosin A) and 10 quinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid, and sarafloxacin) in muscle (bovine, ovine, porcine, equine, rabbit, poultry including laying hens, and fish) and kidney (bovine, ovine, porcine, equine) for human consumption was developed and validated according to Commission Implementing Regulation (EU) 2021/808. Sample preparation consisted of liquid extraction using a citrate–phosphate/EDTA buffer solution (McIlvaine buffer) followed by solid phase extraction for cleanup and UHPLC-MS/MS analysis. The developed method was comprehensively validated with regard to relative matrix effects (CVMF ≤ 20 %), linearity (R2 ≥ 0.98), accuracy (84–119 %), trueness (−16 to + 19 %), repeatability (2.1–20.0 %), reproducibility (3.0–25.0 %), selectivity, and robustness. The method limit of quantification values ranged from 2 to 40 µg kg−1, established as 0.1 × the lowest maximum residue limit (MRL) by species for the authorized antibiotics. Method applicability was demonstrated in six interlaboratory proficiency tests, and in the official control analyses of 1200 field samples of muscle and kidney.

The integrated effect of roflumilast and selenium nanoparticles on nephrotoxicity generated by cisplatin through the regulation of the antioxidant and apoptotic pathways

AimThe current investigations aimed to investigate the potential synergistic effect of Roflumilast (ROF) and Selenium nanoparticles (SeNPs) administration on Cisplatin (Cis) -induced nephrotoxicity. Materials and methodsFifty male rats were divided into five groups; Control group: animals were administered 0.9 % saline solution. Cis group: animals were injected with a single dose of 6 mg/kg. ROF group: Rats received a dosage of 1.2 mg/kg orally daily for 11 days. SeNPs group: animals orally received 0.5 mg/kg of ROF daily for 11 days. The ROF + SeNPs group was administered both after receiving a Cis injection for 11 days. Animals were sacrificed at 5 and 11 days, and the urine and blood samples were collected on day 5 and day 11 for chemical analysis, while kidney samples were obtained for molecular, histological, and immunohistochemical studies. ResultsThe levels of serum creatinine, Blood urea nitrogen (BUN), and total protein were elevated in the Cis group compared to the control group (p < 0.05). While the combination of ROF and SeNPs dramatically decreased these values after 5 and 11 days (p < 0.05). In addition, Cis caused renal oxidative stress by elevating MDA levels and suppressing the activities of SOD, GSH, and CAT. Similarly, these effects were modulated by ROF and SeNPs after 11 days (p < 0.05). Furthermore, the concurrent administration of ROF and SeNPs resulted in a significant increase in the expression of HO-1, Nrf2, and Bcl2, while decreasing the expression of BAX and IL-6 compared to the Cis group after 11 days (P < 0.05). ConclusionThe study showed that both ROF and SeNPs had significant therapeutic potential in reducing the pathological alterations caused by Cis.

The emergence of multidrug-resistant Proteus vulgaris infection in cage reared Pangasianodon hypophthalmus: Molecular characterization and host-pathogen response

The study investigates the causative agent responsible for massive mortality in Pangasianodon hypophthalmus cage farms. The infected pangasius were lethargic, not taking feed, and had exophthalmia, deep ulceration, and hemorrhage on the ventral body surface. Pathogens were isolated from infected pangasius tissue samples, and the strain was preliminarily identified as Proteus vulgaris based on morphology, biochemical tests, 16S rRNA PCR sequencing, and phylogenetic analysis. The microbiological analysis revealed that P. vulgaris was associated with the disease outbreak and mortality of P. hypophthalmus, as confirmed by the biofilm formation, swimming activity, and survival assay results. The isolated P. vulgaris was resistant to several antibiotics, including Doxycycline, Dicloxacillin, Polymyxin B, Chloramphenicol, and Imipenem, exhibiting multiple antibiotic-resistant phenotypes. Furthermore, a host-pathogen model was developed to investigate the in vivo effect of emerging P. vulgaris infection in striped catfish. Results showed that P. vulgaris infection exhibited varying degrees of cellular changes in the kidney, liver, and gill tissues of infected P. hypophthalmus samples. The transcription analysis further highlights that P. vulgaris modulates the antioxidants, immune activation, growth regulator, homeostasis, degradation of invading DNA, and DNA damage-related gene expression in liver, kidney, spleen, skin, and gill tissue samples of infected P. hypophthalmus. The lessons learned from the study could be critical in understanding the P. vulgaris infection patterns and pathobiology necessary to enable risk assessment and develop management measures to control the bacterium's virulence.

Effect of Pregnancy on Mercury Concentration in the Body of Free-living Small Rodents.

Relatively little information exists on the effects of mercury on terrestrial wildlife populations. We analyzed 38 free-living small rodent females (Myodes glareolus, Microtus agrestris, and Apodemus flavicolis), of which 11 were pregnant, for total mercury concentrations in combined liver and kidney samples. Using a single-purpose atomic absorption spectrometer for mercury determination, the measured mercury values ranged from 0.006 to 0.079 mg/kg. Pregnant females had significantly (P<0.041) higher mercury levels in liver and kidney than did nonpregnant females. Our results suggest that during mercury biomonitoring studies it is necessary to consider the pregnancy of the analyzed animals.

Obesity-related drug transporter expression alterations in human liver and kidneys.

Pathophysiological changes associated with obesity might impact various drug pharmacokinetics (PK) parameters. The liver and kidneys are the primary organs involved in drug clearance, and the function of hepatic and renal transporters is critical to efficient drug elimination (or reabsorption). Considering the impact of an increased BMI on the drug's PK is crucial in directing dosing decisions. Given the critical role of transporters in drug biodisposition, this study investigated how overweight and obesity affect the gene expression of renal and hepatic drug transporters. Human liver and kidney samples were collected post-mortem from 32 to 28 individuals, respectively, which were divided into the control group (lean subjects; 18.5 ≤ BMI < 25kg/m2) and the study group (overweight/obese subjects; BMI ≥ 25kg/m2). Real-time quantitative PCR was performed for the analysis of 84 drug transporters. Our results show significant changes in the expression of genes involved in human transporters, both renal and hepatic. In liver tissue, we found that ABCC4 was up-regulated in overweight/obese subjects. In kidney tissue, up-regulation was only observed for ABCC10, while the other differentially expressed genes were down-regulated: ABCA1, ABCC3, and SLC15A1. The observed alterations may be reflected by the differences in drug PK between lean and obese populations. However, these findings need further evaluation through the proteomic and functional study of these transporters in this patient population.

Detection of Mycobacterium bovis in Free-Ranging Sapajus nigritus, Argentina.

Mycobacterium bovis and Mycobacterium tuberculosis are the most relevant among pathogenic mycobacteria, both belonging to the M. tuberculosis complex (MTC). Samples of blood, liver, spleen, kidneys, lungs and caseous tubercles were collected from a free-ranging juvenile black capuchin monkey (Sapajus nigritus) showing non-specific signs of illness. Macroscopic findings included emaciation, a caseous lesion in a tooth and gingiva, disseminated nodules in both lungs and left kidney parenchyma and caseous nodules on the pleura and mesentery. The lesions suggested MTC infection, a diagnosis subsequently supported in the lung by bacilloscopy, immunochromatography and PCR. A multiplex PCR further validated the presence of M. bovis genes. Cases of tuberculosis in platyrrhine primates have only been reported in animals maintained in captivity. We describe for the first time the pathological and molecular findings of M. bovis infection in a free-ranging platyrrhine monkey within an area of intense human-wildlife interaction, which has important implications from a One Health perspective.

Active Role of Lactoferrin on Arsenic and Imidacloprid Toxicity in Broiler Chicks

Abstract This work aimed to evaluate the lactoferrin (LF) effect on arsenic (As) and imidacloprid (IMI) toxicity in broiler chicks. One-week old broiler chicks (n=105) were divided into seven groups (x15 each). The animals were orally supplemented with As, IMI, and/or LF for 4 weeks as follows: Control (G1) no supplements, G2 supplemented with As, G3 supplemented with IMI, G4 supplemented with As+IMI, G5 supplemented with As+LF, G6 supplemented with IMI+LF, G7 supplemented with As+IMI+LF. Body weight and weight gain were recorded on weekly interval. Blood, serum, liver, kidney, and muscle samples were collected at the end of the experimental period for biochemical and histopathological examination. Body weight performance, hematological, serum, and liver tissue biochemical analysis revealed adverse changes in G2, G3, and G4 compared to control, G5, G6, and G7. There was higher tissue residue of As and IMI in G4 and G5 compared to G5, G6, and G7. Liver histopathological changes in the groups supplemented with As and/or IMI were observed with necrosis, congestion, and inflammatory cell aggregates. The use of LF in broiler chicks improves weight gain performance and modulates the adverse effects of As and/or IMI toxicity.

The Haematotoxicity and Nephrotoxicity Effects of Diazepam Administration in Male Albino Rats

Objectives: This study aimed to assess the haematological and nephrotoxic effects of diazepam at varying doses in male albino rats. Materials and Methods: Rats were administered therapeutic (0.062 mg/kg/day), high (0.33 mg/kg/day), and extremely high (0.661 mg/kg/day) doses of diazepam orally for 14 and 28 days. Blood and kidney samples were collected for haematological and biochemical analysis.Results: Diazepam administration significantly reduced red blood cell count (RBC), packed cell volume (PCV), and platelet count (PLT), while other haematological indices were not notably affected. Plasma creatinine and urea levels increased, and total protein decreased across all doses. The treatment also elevated renal thiobarbituric acid reactive substances (TBARS) levels, while reducing antioxidant enzyme activity superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH). Conclusions: Diazepam poses significant risks of oxidative stress, haematotoxicity, and nephrotoxicity. While effective in managing anxiety, caution is necessary due to its potentially harmful effects on blood and renal function.

Molecular detection and genomic characterization of Samak Micromys paramyxovirus-1 and -2 in Micromys minutus, Republic of Korea

BackgroundThe discovery of viruses in small mammalian populations, particularly rodents, has expanded the family Paramyxoviridae. The overlap in habitats between rodents and humans increases the risk of zoonotic events, underscoring the importance of active surveillance. Rodent species, such as Apodemus agrarius, are natural hosts for Paramyxoviridae in the Republic of Korea (ROK). However, it is unknown whether Paramyxoviridae is present in Micromys minutus, another common rodent.MethodHere, we screened M. minutus collected from the Gangwon Province in the ROK for paramyxoviruses using nested polymerase chain reaction and confirm positive samples by next-generation metagenomic sequencing. Complete paramyxovirus genomes were further characterized by phylogenetic analysis, amino acid similarity, secondary structure, and cophylogeny.ResultOverall, 57 of 145 (39.3%) M. minutus kidney samples tested positive for paramyxoviruses. Among them, four whole genome sequences were identified and clustered within the genus Jeilongvirus. One sequence was determined as Samak Micromys paramyxovirus 1 (SMPV-1; 19,911 nucleotides long) and three sequences as Samak Micromys paramyxovirus 2 (SMPV-2; 18,199 nucleotides long). SMPV-1 has a smaller hydrophobic gene and a longer glycoprotein gene than SMPV-2. Cophylogenetic analysis suggests that SMPV-1 evolved through co-divergence, whereas SMPV-2 was inferred to have undergone transfer events.ConclusionThese findings highlight the prevalence of paramyxoviruses in the wild and the potential of M. minutus as a natural viral reservoir. The discovery of SMPV-1 and SMPV − 2 also reveals the genetic diversity and evolutionary history of the genus Jeilongvirus in the Paramyxoviridae.

Towards high throughput analysis using 96-well plate solid phase extraction to determine sedatives and β-blocker residues in food control monitoring

BackgroundVeterinary drugs are widely used in animal production to prevent infections and treat diseases but, this may cause a risk to consumers. Due to the high number of food samples required to monitor yearly, simple, fast, sensitive and selective analytical methods are needed in control laboratories to ensure consumers safety. Nevertheless, many analytical methodologies available in these laboratories include multiple steps and therefore are time-consuming and hinder the analysis throughput requiring significant amounts of solvents and reagents. ResultsThis work developed a 96-well plate solid phase extraction (SPE) method for the extraction of seven sedatives and a β-blocker in animal kidney by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC−MS/MS). The developed method was validated based on Implementing Regulation (EU) 2021/808 in kidney, meat and fish. The performance characteristics of the validation (1–5 μg kg−1) showed good linearity (R2 > 0.998) while decision limits (CCα) were between 1 and 1.2 μg kg−1. Trueness and precision were determined at three levels (n = 7) and the results showed values ranging from 85 to 103 % and from 1 to 9 %, respectively. The feasibility of the method was demonstrated for the residue control requirement established by EU. Method was applied to 201 samples of kidney, meat and fish. SignificanceThis study is the first to present an optimized and validated holistic method for sedatives and β-blocker using 96-well plate SPE and UHPLC−MS/MS. The method showed good performance in kidney, meat and fish samples being an universal method for any species along the same type of sample. The fast, easy, efficient, reliable and universal method showed high throughput and so reduced the analysis time by nine-fold and the required solvent amount by four times fulfilling the green chemistry principals.

Novel Megaptera novaeangliae (Humpback whale) haplotype chromosome-level reference genome

The sequencing of a kidney sample (KW2013002) from a stranded Megaptera novaeangliae (Humpback whale) calf is the first chromosome-level reference genome for this species1. The calf, a 457 cm and 2,500 lbs male, was found stranded in Hawai’i Kai, HI, in 2013 and was marked as abandoned/orphaned. In 2023, 1 g of kidney was sequenced with PacBio long-read DNA sequencing, chromatin conformation capture (Hi-C), RNA sequencing, and mitochondrial sequencing to comprehensively characterize the genome and transcriptome of M. novaeangliae. Data validation includes a synteny analysis, mitochondrial annotation, and a comparison of BUSCO scores (scaffold v. reference genome and Balaenoptera musculus (Blue whale) v. M. novaeangliae). BUSCO analysis was performed on an M. novaeangliae scaffold-level assembly to determine genomic completeness of the reference genome, with a scaffold BUSCO score of 91.2% versus a score of 95.4%. Synteny analysis was performed using the B. musculus genome as comparison to determine chromosome-level coverage and structure. Further, a time-based phylogenetic tree was constructed using the sequenced data and publicly available genomes.

Lack of Cannabinoid Type 2 Promoter Activity in Normal or Injured Kidneys Using a Cnr2-GFP Reporter Mouse.

Introduction: Although cannabinoid type 2 (CB2) receptor activity is known to promote diverse biological functions in the kidney, published data regarding CB2 receptor protein levels and cellular distribution within the kidney is inconsistent. The goal of the present study was to investigate the changes of CB2 in the kidney obtained from mice exposed to various forms of kidney injury using a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous cannabinoid receptor 2 (Cnr2) promoter. Materials and Methods: Kidney injury was established in a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous Cnr2 promoter. Kidney injury was initiated by either treatment with different chemicals [cisplatin or lipopolysaccharide (LPS)] or by unilateral ureteral obstruction (UUO). Changes in the detection of GFP were used as a proxy for CB2 levels and localization. Histological changes due to the injury stimuli were observed by time-related, morphological changes in kidney cytoarchitecture and blood parameters, such as serum creatinine levels. Cnr2 mRNA levels were detected by reverse transcription coupled to polymerase chain reaction (RT-PCR) while protein changes in the tissue lysates were measured by Western blot analysis. Cellular localization of GFP was detected by fluorescent microscopy. Results: Our data demonstrated that there was no band or a minimally detectable band for GFP using kidney lysates from vehicle- or cisplatin-treated mice. A similar lack of GFP was detected in the UUO kidney versus the contralateral control kidney. This is consistent with the low, albeit detectable levels of Cnr2 mRNA in the kidney samples from control or cisplatin treatment. In frozen kidney sections from vehicle and cisplatin-treated mice, GFP fluorescence was not detectable in tubular epithelia, glomeruli or blood vessels in the cortex. Instead, GFP was detected in rare cells within the interstitial space. A second chemical injury model using LPS found a similar lack of GFP protein levels and an absence of legitimate GFP fluorescence in the main cell types within the kidney. Conclusion: These findings suggest that Cnr2 promoter activity is minimally active in normal or injured kidneys, and that pharmacological manipulation of CB2 receptors may be associated with receptors being expressed in cells recruited to the kidney.