RESUMO
Aminoacyl-tRNA synthetases are essential enzymes for the accurate translation of genetic information. IARS1 and IARS2 are isoleucyl-tRNA synthetases functioning in the cytoplasm and mitochondria, respectively, with genetic mutations in these enzymes causing diverse clinical phenotypes in specific organs and tissues. Mutations in IARS1 and IARS2 have recently been linked to mitochondrial diseases. This review aims to explore the relationship between IARS1 and IARS2 and these diseases, providing a comprehensive overview of their association with mitochondrial diseases. Mutations in IARS1 cause weak calf syndrome in cattle and mitochondrial diseases in humans, leading to growth retardation and liver dysfunction. Mutations in IARS2 are associated with Leigh syndrome, craniosynostosis and abnormal genitalia syndrome. Future research is expected to involve genetic analysis of a larger number of patients, identifying new mutations in IARS1 and IARS2, and elucidating their impact on mitochondrial function. Additionally, genetically modified mice and the corresponding phenotypic analysis will serve as powerful tools for understanding the functions of these gene products and unraveling disease mechanisms. This will likely promote the development of new therapies and preventive measures.
Assuntos
Isoleucina-tRNA Ligase , Doenças Mitocondriais , Mutação , Humanos , Animais , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Isoleucina-tRNA Ligase/genética , Bovinos , Mitocôndrias/genética , CamundongosRESUMO
Several artificial intelligence (AI) systems have been developed for glomerular pathology analysis in clinical settings. However, the application of AI systems in nonclinical fields remains limited. In this study, we trained a convolutional neural network model, which is an AI algorithm, to classify the severity of Tensin 2 (TNS2)-deficient nephropathy into seven categories. A dataset consisting of 803 glomerular images was generated from kidney sections of TNS2-deficient and wild-type mice. Manual evaluations of the images were conducted to assess their glomerular injury scores. The trained AI achieved approximately 70% accuracy in predicting the glomerular injury score for TNS2-deficient nephropathy. However, the AI achieved approximately 100% accuracy when considering predictions within one score of the true label as correct. The AI's predicted mean score closely matched the true mean score. In conclusion, while the AI model may not replace human judgment entirely, it can serve as a reliable second assessor in scoring glomerular injury, offering potential benefits in enhancing the accuracy and objectivity of such assessments.
RESUMO
The Adriamycin (ADR) nephropathy model, which induces podocyte injury, is limited to certain mouse strains due to genetic susceptibilities, such as the PrkdcR2140C polymorphism. The FVB/N strain without the R2140C mutation resists ADR nephropathy. Meanwhile, a detailed analysis of the progression of ADR nephropathy in the FVB/N strain has yet to be conducted. Our research aimed to create a novel mouse model, the FVB-PrkdcR2140C, by introducing PrkdcR2140C into the FVB/NJcl (FVB) strain. Our study showed that FVB-PrkdcR2140C mice developed severe renal damage when exposed to ADR, as evidenced by significant albuminuria and tubular injury, exceeding the levels observed in C57BL/6J (B6)-PrkdcR2140C. This indicates that the FVB/N genetic background, in combination with the R2140C mutation, strongly predisposes mice to ADR nephropathy, highlighting the influence of genetic background on disease susceptibility. Using RNA sequencing and subsequent analysis, we identified several genes whose expression is altered in response to ADR nephropathy. In particular, Mmp7, Mmp10, and Mmp12 were highlighted for their differential expression between strains and their potential role in influencing the severity of kidney damage. Further genetic analysis should lead to identifying ADR nephropathy modifier gene(s), aiding in early diagnosis and providing novel approaches to kidney disease treatment and prevention.
Assuntos
Modelos Animais de Doenças , Doxorrubicina , Nefropatias , Animais , Doxorrubicina/efeitos adversos , Camundongos , Nefropatias/induzido quimicamente , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Predisposição Genética para Doença , Podócitos/metabolismo , Podócitos/patologia , Podócitos/efeitos dos fármacosRESUMO
Patients with inflammatory bowel disease (IBD) who experience long-term chronic inflammation of the colon are at an increased risk of developing colorectal cancer (CRC). Mitotic spindle positioning (MISP), an actin-binding protein, plays a role in mitosis and spindle positioning. MISP is found on the apical membrane of the intestinal mucosa and helps stabilize and elongate microvilli, offering protection against colitis. This study explored the role of MISP in colorectal tumorigenesis using a database, human CRC cells, and a mouse model for colitis-induced colorectal tumors triggered by azoxymethane (AOM)/dextran sodium sulfate (DSS) treatment. We found that MISP was highly expressed in colon cancer patient tissues and that reduced MISP expression inhibited cell proliferation. Notably, MISP-deficient mice showed reduced colon tumor formation in the AOM/DSS-induced colitis model. Furthermore, MISP was found to form a complex with Opa interacting protein 5 (OIP5) in the cytoplasm, influencing the expression of OIP5 in a unidirectional manner. We also observed that MISP increased the levels of phosphorylated STAT3 in the JAK2-STAT3 signaling pathway, which is linked to tumorigenesis. These findings indicate that MISP could be a risk factor for CRC, and targeting MISP might provide insights into the mechanisms of colitis-induced colorectal tumorigenesis.
Assuntos
Colite , Neoplasias Colorretais , Animais , Humanos , Camundongos , Azoximetano/efeitos adversos , Carcinogênese , Transformação Celular Neoplásica , Colite/patologia , Neoplasias Colorretais/patologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Janus Quinase 2/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fuso Acromático/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Euthanasia agents should rapidly induce death and loss of consciousness without causing pain or distress. Various methods exist for the euthanasia of laboratory animals, and injectable anesthetics, particularly barbiturate derivatives, are widely used due to the rapid onset of unconsciousness induced by these agents. Moreover, pharmaceutical-grade drugs should be used to eliminate undesirable side effects as much as possible. However, in Japan, the sale of pharmaceutical-grade pentobarbital sodium (PB) ended in 2019, and that of secobarbital sodium (SB) ended in 2023, leading to a demand for new pharmaceutical-grade injectable euthanasia drugs. This study evaluates thiamylal sodium (TM), a barbiturate derivative that is available domestically, as a euthanasia agent for mice. The results showed that when administered at dosages of 200 mg/kg or more, TM exhibited effects equivalent to those of PB and SB. In addition, the impact of TM administration on hematological characteristics was examined. In female mice administered TM, decreased blood chloride and calcium levels and increased aspartate aminotransferase and alanine aminotransferase levels, which are markers of liver damage, were observed. These findings suggest that high concentrations of TM may affect renal and liver function. This study revealed that TM is effective as a euthanasia agent at dosages of 200 mg/kg or more. However, considering the potential risks of renal and liver damage due to TM administration, it may be preferable to use alternative euthanasia drugs when these risks could affect the objectives or outcomes of the research.
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Eutanásia Animal , Animais , Feminino , Camundongos , Masculino , PentobarbitalRESUMO
Accurately and promptly assessing pain in experimental animals is extremely important to avoid unnecessary suffering of the animals and to enhance the reproducibility of experiments. This is a key concern for veterinarians, animal caretakers, and researchers from the perspectives of veterinary care and animal welfare. Various methods including ethology, immunohistochemistry, electrophysiology, and molecular biology are used for pain assessment. However, the grimace scale, which was developed by taking cues from interpreting pain through facial expressions of non-verbal infants, has become recognized as a very simple and practical method for objectively evaluating pain levels by scoring changes in an animal's expressions. This method, which was first implemented with mice approximately 10 years ago, is now being applied to various experimental animals and is widely used in research settings. This review focuses on the usability of the grimace scale from the "cage-side" perspective, aiming to make it a more user-friendly tool for those involved in animal experiments. Differences in facial expressions in response to pain in various animals, examples of applying the grimace scale, current automated analytical methods, and future prospects are discussed.
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Animais de Laboratório , Expressão Facial , Medição da Dor , Animais , Medição da Dor/métodos , Animais de Laboratório/fisiologia , Camundongos , Bem-Estar do Animal , Dor/diagnóstico , Dor/veterinária , Dor/fisiopatologia , Reprodutibilidade dos TestesRESUMO
Proper administration of anesthesia is indispensable for the ethical treatment of lab animals in biomedical research. Therefore, selecting an effective anesthesia protocol is pivotal for the design and success of experiments. Hence, continuous development and refinement of anesthetic agents are imperative to improve research outcomes and elevate animal welfare. "Balanced anesthesia" involves using multiple drugs to optimize efficacy while minimizing side effects. The medetomidine, midazolam, and butorphanol, called MMB, and medetomidine, alfaxalone, and butorphanol, called MAB, are popular in Japan. However, the drawbacks of midazolam, including its extended recovery time, and the narrow safety margin of MAB, have prompted research for suitable alternatives. This study replaced midazolam in the MMB combination with remimazolam (RMZ), which is noted for its ultra-short half-life. The resulting combination, called MRB, was effective in providing a wider safety margin compared to MAB while maintaining an anesthesia depth equivalent level to that of MMB in mice. Notably, MRB consistently exhibited better recovery scores after antagonist administration in contrast to MMB. Furthermore, the re-sedation phenomenon observed with MMB was not observed with MRB. The rapid metabolism of RMZ enables reliable anesthesia induction, circumventing the complications linked to MAB. Overall, MRB excelled in providing extended surgical anesthesia and swift post-antagonist recovery. These results highlight the potential of RMZ for broader animal research applications.
Assuntos
Butorfanol , Medetomidina , Animais , Medetomidina/administração & dosagem , Medetomidina/farmacologia , Butorfanol/administração & dosagem , Butorfanol/farmacologia , Camundongos , Masculino , Anestesia/métodos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/farmacologia , Benzodiazepinas/administração & dosagem , Benzodiazepinas/farmacologia , Anestésicos Combinados/administração & dosagem , Midazolam/administração & dosagem , Midazolam/farmacologiaRESUMO
The global incidence of ureteroliths in humans is increasing, particularly posing a problem in developed countries. The most common stone type is calcium oxalate, which is associated with a high recurrence rate. In veterinary medicine, stones are the most common cause of ureteral obstruction in cats, accounting for 72-87% of cases. In cats, stones cause irreversible ureteral damage, necessitating stone treatment as well as ureteral therapy. However, the mechanisms underlying the ureteral damage caused by stones remain unclear. Therefore, this study aimed to create a mouse model suitable for studying the ureteral fibrosis caused by oxalate stones by artificially embedding calcium oxalate in the ureter. Pathological tissue analysis was used to compare normal ureters without ligation and ureters with sham or oxalate bead implantation. The ureters of the sham and oxalate bead groups showed granulation tissue formation, transitional epithelium exfoliation, and densely packed connective tissue in the proprietary and muscle layer regions. Particularly in the oxalate bead group, infiltration of degenerated neutrophils, presence of foreign body giant cells, and hyperplasia of the transitional epithelium were observed. The proportion of fibrosis was higher in the oxalate group than in the sham group. Overall, this mouse model created using oxalate bead implantation has the potential to efficiently induce ureteral obstruction. This mouse model is expected to be used for elucidating the molecular mechanisms of ureteral fibrosis and evaluating therapeutic drugs in future.
Assuntos
Ureter , Obstrução Ureteral , Humanos , Camundongos , Gatos , Animais , Ureter/patologia , Oxalato de Cálcio/análise , Obstrução Ureteral/complicações , Camundongos Endogâmicos C57BL , Oxalatos , FibroseRESUMO
Adriamycin (ADR) nephropathy is the most widely used nephropathy model to study the pathophysiological mechanisms of chronic kidney disease (CKD) in mice. However, its application is limited to a few mouse strains such as the BALB/c strain; the standard strain, C57BL/6J (B6J), does not develop ADR nephropathy. Nevertheless, Arif et al. reported that C57BL/6N (B6N), another standard strain, is ADR-susceptible. Since then, no follow-up reports or other studies have been published on ADR nephropathy in B6N mice. Therefore, the goal of this study was to determine whether B6N mice are indeed susceptible to ADR nephropathy and whether there are differences in ADR susceptibility among the substrains of C57BL/6NCrl (NCrl) and C57BL/6NJcl (NJcl). NCrl mice showed marked albuminuria and mesangial cell proliferation, which are associated with mild ADR nephropathy, confirming that NCrl mice were susceptible to ADR nephropathy. On the other hand, NJcl mice did not exhibit these symptoms. ADR nephropathy models are usually generated by administering ADR through the tail vein, but Arif et al. administered ADR through the orbital vein. Therefore, we investigated the effect of the route of administration on ADR nephropathy. The degree of ADR nephropathy was found to vary based on the route of administration: more severe nephropathy was observed upon administration through the tail vein than through the orbital vein. Therefore, we conclude that NCrl mice are susceptible to ADR nephropathy, and the severity of ADR-induced nephropathy through orbital vein administration is relatively lower than that through the tail vein.
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Doxorrubicina , Nefropatias , Camundongos , Animais , Doxorrubicina/efeitos adversos , Camundongos Endogâmicos C57BL , Nefropatias/induzido quimicamente , Albuminúria/induzido quimicamenteRESUMO
Adriamycin (ADR) is an effective chemotherapy drug for various cancers but has serious side effects. ADR-induced liver damage is a common problem during therapy, but the underlying mechanism remains to be fully understood. In contrast, ADR-induced glomerular damage is well studied in rodents, and sensitivity to ADR-induced nephropathy is because of the R2140C polymorphism of Prkdc gene. To investigate whether strain differences or sensitivity to ADR-induced liver damage are related to Prkdc polymorphism, this study compared the sensitivity to ADR-induced liver damage among C57BL/6J (B6J), B6-PrkdcR2140C, and BALB/c mice. Although B6J exhibits resistance to ADR-induced liver injury, BALB/c and B6-PrkdcR2140C are more susceptible to liver injury, which is exacerbated by the presence of R2140C mutation in PRKDC.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Nefropatias , Animais , Camundongos , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/veterinária , Doxorrubicina/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/veterinária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polimorfismo GenéticoRESUMO
Circadian rhythms are endogenous oscillators that regulate 24 h behavioral and physiological processes. Our previous investigation demonstrated that bromobenzene metabolite (4-bromocatechol: 4-BrCA) exhibited chronotoxicity (i.e., the nephrotoxicity induced by 4-BrCA was observed during the dark phase, while not observed at light phase in mice). However, the molecular mechanism is still unknown. The aim of the present study is to investigate the cellular molecule(s) involved in the 4-BrCA-induced nephrotoxicity using mouse renal cortex tubular cell lines (MuRTE61 cells). We found that 4-BrCA showed dose dependent (0.01-1 mM) cell proliferation defect in MuRTE61 cells. By treating with 0.03 mM 4-BrCA, we demonstrated that major clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) were significantly downregulated. Interestingly, the expression levels of two genes, Bmal1 and Clock, continued to decrease after 3 h of treatment with 4-BrCA, while Cry1, Per1, and Per2 were unchanged until 24 h of treatment. Moreover, BMAL1 and CLOCK levels are higher at light phase. We speculated that BMAL1 and CLOCK might function defensively against 4-BrCA-induced nephrotoxicity since the expression levels of Bmal1 and Clock were rapidly decreased. Finally, overexpression of Bmal1 and Clock restored 4-BrCA-induced cell proliferation defect in MuRTE61 cells. Taken together, our results suggest that Bmal1 and Clock have protective roles against 4-BrCA-induced nephrotoxicity.
Assuntos
Fatores de Transcrição ARNTL , Bromobenzenos , Camundongos , Animais , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Ritmo Circadiano/genética , Regulação da Expressão GênicaRESUMO
Most mitochondrial diseases are hereditary and highly heterogeneous. Cattle born with the V79L mutation in the isoleucyl-tRNA synthetase 1 (IARS1) protein exhibit weak calf syndrome. Recent human genomic studies about pediatric mitochondrial diseases also identified mutations in the IARS1 gene. Although severe prenatal-onset growth retardation and infantile hepatopathy have been reported in such patients, the relationship between IARS mutations and the symptoms is unknown. In this study, we generated hypomorphic IARS1V79L mutant mice to develop an animal model of IARS mutation-related disorders. We found that compared to wild-type mice, IARSV79L mutant mice showed a significant increase in hepatic triglyceride and serum ornithine carbamoyltransferase levels, indicating that IARS1V79L mice suffer from mitochondrial hepatopathy. In addition, siRNA knockdown of the IARS1 gene decreased mitochondrial membrane potential and increased reactive oxygen species in the hepatocarcinoma-derived cell line HepG2. Furthermore, proteomic analysis revealed decreased levels of the mitochondrial function-associated protein NME4 (mitochondrial nucleoside diphosphate kinase). Concisely, our mutant mice model can be used to study IARS mutation-related disorders.
Assuntos
Hepatopatias , Doenças Mitocondriais , Gravidez , Feminino , Humanos , Criança , Animais , Bovinos , Camundongos , Proteômica , Isoleucina-tRNA Ligase/genética , Genoma , Hepatopatias/genética , Doenças Mitocondriais/genética , MutaçãoRESUMO
Inflammatory bowel disease (IBD) is classified into two types: Crohn's disease and ulcerative colitis. In IBD, the imbalance between the pro-inflammatory and anti-inflammatory cytokines prevents recovery from the inflammatory state, resulting in chronic inflammation in the colon. The mitotic spindle positioning protein (MISP) is localized to the apical membrane in the colon. In this study, we observed increased expression of MISP in the intestinal epithelial cells in dextran sulfate sodium (DSS)-induced colitis in mice. MISP-deficient mice receiving DSS showed significant exacerbation of colitis (e.g., weight loss, loss of the crypts). The intestinal epithelial cells of the MISP-deficient mice showed a trend towards decreased cell proliferation after DSS treatment. Reverse transcription followed by quantitative polymerase chain reaction revealed that the expression levels of Tgfb1, an anti-inflammatory cytokine, were significantly reduced in the colon of MISP-deficient mice compared with the wild-type mice regardless of DSS treatment. These findings indicate that MISP may play a role in the recovery of the colon after inflammation through its anti-inflammatory and proliferative activities, suggesting that MISP may be a new therapeutic target for IBD.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Anti-Inflamatórios/uso terapêutico , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colite/veterinária , Colo/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Sulfato de Dextrana/uso terapêutico , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/veterinária , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/veterinária , Camundongos Endogâmicos C57BL , Fuso Acromático/metabolismoRESUMO
Animal models of podocytopathy and chronic kidney diseases (CKD) help elucidate these pathologies. Adriamycin (ADR)-induced nephropathy is a common rodent model of podocytopathy. BALB/c mice are sensitive to ADR, whereas C57BL/6 (B6) mice, the most commonly used strain, are resistant to ADR. Therefore, mouse strains with the B6 genetic background cannot be used as an ADR nephropathy model. We previously generated DNA-dependent protein kinase catalytic subunit (Prkdc) mutant B6 mice (B6-PrkdcR2140C) carrying the R2140C mutation that causes ADR nephropathy. However, whether ADR nephropathy in the novel strain progresses to CKD after ADR administration has not been evaluated. Therefore, we examined whether the B6-PrkdcR2140C mice develop CKD after ADR administration. We also evaluated whether differences existed in the genetic background in ADR nephropathy by comparing the B6-PrkdcR2140C mice with BALB/c mice. Our findings demonstrated that B6-PrkdcR2140C progresses to CKD and is resistant to nephropathy compared with the BALB/c mice. The B6-PrkdcR2140C and BALB/c mice differed in the expression of genes related to inflammatory mediators, and further analysis is required to identify factors that contribute to resistance to nephropathy.
Assuntos
Nefropatias , Insuficiência Renal Crônica , Camundongos , Animais , Doxorrubicina/efeitos adversos , Camundongos Endogâmicos C57BL , Nefropatias/genética , Camundongos Endogâmicos BALB CRESUMO
Cystinuria is an autosomal metabolic disorder caused by mutations in the SLC3A1 and SLC7A9 genes, encoding the amino acid transporter proteins rBAT and b0,+AT, respectively. Based on the causative gene, cystinuria is classified into 3 types: type A (SLC3A1), type B (SLC7A9), and type AB (SLC3A1 and SLC7A9). Patients with cystinuria exhibit hyperexcretion of cystine and dibasic amino acids in the urine and develop cystine crystals due to its low solubility in the urine, often resulting in calculus formation. In this study, we present an inbred strain FVB/NJcl mice affected with cystinuria. In the affected mouse kidney, Slc7a9 expression was completely abolished because of a large sequence deletion in the promoter region of the Slc7a9 mutant allele. Slc7a9-deficient mice with FVB/NJcl genetic background developed cystine calculi in the bladder with high penetrance, as compared to the previously reported mouse models of cystinuria. This model may be useful to understand the determinants of crystal aggregation, affecting calculus formation.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Diamino Aminoácidos , Cálculos , Cistinúria , Camundongos , Animais , Cistinúria/genética , Cistinúria/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Cistina/metabolismo , Mutação , Modelos Animais de Doenças , Diamino Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Neutros/genéticaRESUMO
Tensin 2 (TNS2), a focal adhesion protein, is considered to anchor focal adhesion proteins to ß integrin as an integrin adaptor protein and/or serve as a scaffold to facilitate the interactions of these proteins. In the kidney, TNS2 localizes to the basolateral surface of glomerular epithelial cells, i.e., podocytes. Loss of TNS2 leads to the development of glomerular basement membrane lesions and abnormal accumulation of extracellular matrix in maturing glomeruli during the early postnatal stages. It subsequently results in podocyte foot process effacement, eventually leading to glomerulosclerosis. Histopathological features of the affected glomeruli in the middle stage of the disease include expansion of the mesangial matrix without mesangial cell proliferation. In this review, we provide an overview of TNS2-deficient nephropathy and discuss the potential mechanism underlying this mechanosensitive nephropathy, which may be applicable to other glomerulonephropathies, such as CD151-deficient nephropathy and Alport syndrome. The onset of TNS2-deficient nephropathy strictly depends on the genetic background, indicating the presence of critical modifier genes. A better understanding of molecular mechanisms of mechanosensitive nephropathy may open new avenues for the management of patients with glomerulonephropathies.
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Nefropatias , Podócitos , Animais , Predisposição Genética para Doença , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/patologia , Humanos , Rim , Nefropatias/genética , Podócitos/metabolismoRESUMO
Adriamycin (ADR)-induced nephropathy is frequently utilized in rodent models of podocytopathy. However, the application of this model in mice is limited to a few strains, such as BALB/c mice. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-PrkdcR2140C, that possess the R2140C mutation. After administration of ADR, B6-PrkdcR2140C mice exhibited massive proteinuria and glomerular and renal tubular injuries. In addition, there was no significant difference in the severity between B6-PrkdcR2140C and BALB/c. These findings demonstrated that B6-PrkdcR2140C show ADR-induced nephropathy susceptibility at a similar level to BALB/c, and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In future studies, ADR-induced nephropathy may become applicable to various kinds of genetically modified mice with a B6 background by mating with B6-PrkdcR2140C.
Assuntos
Substituição de Aminoácidos , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Nefropatias/induzido quimicamente , Albuminúria/induzido quimicamente , Albuminúria/complicações , Animais , Sequência de Bases , Biomarcadores , Sistemas CRISPR-Cas , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Nefropatias/complicações , Nefropatias/metabolismo , Nefropatias/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/complicações , Insuficiência Renal/metabolismo , Insuficiência Renal/patologiaRESUMO
Mast cell-deficient mice are helpful for understanding the roles of mast cells in vivo. To date, a dozen mouse models for mast cell deficiency have been reported. However, mice with a specific depletion of all populations of mast cells have not been reported. We generated knock-in mice, termed Mcpt5/Cma1DTR mice, expressing human diphtheria toxin A (DT) receptor under the endogenous promoter of Mcpt5 (also known as Cma1), which encodes mouse mast cell protease-5. Flow cytometry and histological analysis showed that intraperitoneal injection of DT induced almost complete depletion of mast cells in heterozygote Mcpt5/Cma1DTR/+ mice. The deletion rates of mast cells in peritoneal cavity, mesentery, abdominal skin, ear skin, and glandular stomach were 99.9%, 100%, 98.7%, 97.7%, and 100%, respectively. Passive cutaneous anaphylaxis reaction also revealed mast cell deficiency in ear skin after DT treatment. Other than mast cells, a small percentage of marginal zone B cells in Mcpt5/Cma1DTR/+ mice were killed by DT treatment. In conclusion, the Mcpt5/Cma1DTR/+ mouse model is valuable for achieving conditional depletion of all populations of mast cells without inducing a marked reduction in other cells.
Assuntos
Separação Celular/métodos , Quimases/genética , Mastócitos/citologia , Modelos Animais , Animais , Células do Tecido Conjuntivo/citologia , Feminino , Humanos , Injeções Intraperitoneais , Camundongos , Mucosa/citologia , Regiões Promotoras Genéticas/genéticaRESUMO
The number of patients with chronic kidney disease (CKD) is growing continuously globally. In order to study pathogenesis and mechanisms, many animal models have been developed, including spontaneous, genetic, and induced models. Although each type of CKD shows disease-specific tissue changes in the early stages, tubular disorder and interstitial fibrosis histologically occur in the course of progression to end-stage renal failure. Therefore, the quantification of tubular disorder and interstitial fibrosis in CKD research using animal models is essential for measuring the degree of CKD severity and, thus, efficacy of therapeutic agents. Several strategies have been used to quantify interstitial fibrosis. Among scoring factors, renal tubular flattening can be quantitatively evaluated easily and inexpensively. However, the diagnostic value of renal tubular flattening evaluation has not been investigated previously. Therefore, in this study, we investigated the correlation between renal tubular flattening and interstitial fibrosis or renal tubular injury markers. We observed a strong correlation between the degree of tubular injury/interstitial fibrosis and renal tubular flattening in three types of mouse renal disease model. This is advantageous because rapidly advancing technologies such as artificial intelligence and image processing can be easily applied; hence, a more precise, objective, and quantitative diagnosis should be possible in the future.
Assuntos
Insuficiência Renal Crônica , Doenças dos Roedores , Animais , Inteligência Artificial , Modelos Animais de Doenças , Fibrose , Rim/patologia , Túbulos Renais/patologia , Camundongos , Insuficiência Renal Crônica/veterinária , Doenças dos Roedores/patologiaRESUMO
Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on lymphocytes. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. In our previous studies, we have developed anti-bovine PD-L1 monoclonal antibodies (mAbs) and reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Furthermore, we found that blocking antibodies that target PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway for chronic equine diseases, including tumors, remains unclear. In this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 mAbs against EqPD-L1 using in vitro assays. In addition, we evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a considerable identity and similarity with homologs from non-primate species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Of note, immunohistochemistry confirmed the PD-L1 expression in EMM tumor tissues. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These findings showed that our anti-PD-L1 mAbs would be useful for analyzing the equine PD-1/PD-L1 pathway. Further research is warranted to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horses.