Efficacy of ensitrelvir against SARS-CoV-2 in a delayed-treatment mouse model.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) and a devastating worldwide health concern. Development of safe and effective treatments is not only important for interventions during the current pandemic, but also for providing general treatment options moving forward. We have developed ensitrelvir, an antiviral compound that targets the 3C-like protease of SARS-CoV-2. In this study, a delayed-treatment mouse model was used to clarify the potential in vivo efficacy of ensitrelvir. METHODS: Female BALB/cAJcl mice of different ages were infected with the SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021) or mouse-adapted SARS-CoV-2 MA-P10 and then 24 h post-infection orally administered various doses of ensitrelvir or vehicle. Viral titres and RNA levels in the lungs were quantified using VeroE6/TMPRSS2 cells and RT-qPCR, respectively. Body weight loss, survival, lung weight, cytokine/chemokine production, nucleocapsid protein expression and lung pathology were evaluated to investigate the in vivo efficacy of ensitrelvir. RESULTS: Based on infectious viral titres and viral RNA levels in the lungs of infected mice, ensitrelvir reduced viral loads in a dose-dependent manner. The antiviral efficacy correlated with increased survival, reduced body weight loss, reduced pulmonary lesions and suppression of inflammatory cytokine/chemokine levels. CONCLUSIONS: This was the first evaluation of the in vivo anti-SARS-CoV-2 efficacy of ensitrelvir in a delayed-treatment mouse model. In this model, ensitrelvir demonstrated high antiviral potential and suppressed lung inflammation and lethality caused by SARS-CoV-2 infection. The findings support the continued clinical development of ensitrelvir as an antiviral agent to treat patients with COVID-19.

Suspected spontaneous hyperadrenocorticism in a young experimental beagle dog.

A 6-month-old female beagle dog, assigned to the low-dose group in a toxicity study, was evaluated for compound toxicity, and spontaneous hyperadrenocorticism was suspected. The animal had an externally apparent distended abdomen on clinical examination upon arrival. Pre-dose clinical pathology showed slightly higher erythroid parameters and stress leukogram on hematology; plasma biochemistry showed higher total protein, gamma-glutamyl transferase, total cholesterol, and triglyceride levels than the reference data. On necropsy, a prominent increase in adipose tissues of the subcutis and abdomen and increased weight of the adrenal gland and liver were observed. Histopathology revealed diffuse hyperplasia of adrenocortical cells in the zona fasciculata and reticularis, cortical atrophy of the thymus, and abundant glycogen accumulation in the hepatocytes. These findings were incidental and not test-substance-related. Electron microscopy of the adrenocortical cells in the zona fasciculata revealed decreased typical translucent lipid droplets, increased electron-dense lipid droplets, and abundant smooth endoplasmic reticulum and lysosomes. Additionally, increased numbers of various sizes and forms of mitochondria with tubular, vesicular, or lamellar cristae compared to that of normal animals were observed. These ultrastructural characteristics of the adrenocortical cells suggested hyperfunction. The pre-dose plasma cortisol levels were slightly higher than those of other females assigned to the toxicity study, while plasma adrenocorticotropic hormone levels were within the normal range. These findings indicate that hyperadrenocorticism is a possible cause of the systemic changes in this case.

Adrenal dysplasia in a young SD rat.

The present report describes an adrenal dysplasia in which developmental abnormality was observed in the adrenal gland of a six-week-old male Crl:CD(SD) rat. Microscopically, a localized lesion composed of mildly vacuolated adrenal fasciculata cells with a slightly disturbed cord structure and containing areas with high cell density was observed in a unilateral adrenal gland; no macroscopical changes were detected in the organ. The areas with high cell density consisted of two cell types. One type included small cells with a round nucleus and acidophilic cytoplasm, and the cells were positive for steroidogenic factor-1 (SF-1) but negative for nestin. The other type of cells had a spindle to polygonal shape, clear nucleus, and a cytoplasm with an obscure boundary; the cells were positive for nestin but negative for SF-1, neuronal nuclear antigen, and chromogranin A. These results suggested that the former type of cells were adrenal cortex cells and that the latter were immature neuronal cells. Considering that immature adrenal cortex cells and neural crest cells (future adrenal medulla) are mixed during a stage in rat adrenal gland development, we concluded that the observed lesion was caused by developmental abnormality. To the best of our knowledge, this is the first report to describe dysplasia in rat adrenal glands.

Comparison of Acute Gene Expression Profiles of Islet Cells Obtained via Laser Capture Microdissection between Alloxan- and Streptozotocin-treated Rats.

To identify the molecular profiles of islets from alloxan (ALX)- and streptozotocin (STZ)-treated rats, a microarray-based global gene expression analysis was performed on frozen islets isolated via laser capture microdissection. At 6 weeks old, rats were injected with ALX (40 mg/kg) or STZ (50 or 100 mg/kg) and then euthanized 24 hr later. Histopathological analysis showed ß-cell necrosis, macrophage infiltration, and islet atrophy. The extent of these changes was more notable in the STZ groups than in the ALX group. Transcriptome analysis demonstrated a significant up- or downregulation of cell cycle arrest-related genes in the p53 signaling pathway. Cyclin D2 and cyclin-dependent kinase inhibitor 1A, mediators of G1 arrest, were remarkably altered in STZ-treated rats. In contrast, cyclin-B1 and cyclin-dependent kinase 1, mediators of G2 arrest, were remarkably changed in ALX-treated rats. Genes involved in the intrinsic mitochondria-mediated apoptotic pathway were upregulated in the ALX and STZ groups. Moreover, heat-shock 70 kDA protein 1A ( Hspa1a), Hsp90ab1, and Hsph1 were upregulated in ALX-treated rats, suggesting that ALX treatment injures ß cells via endoplasmic reticulum stress. These results contribute to a better understanding of gene expression in the pathogenesis of islet toxicity.

Distribution analysis of epertinib in brain metastasis of HER2-positive breast cancer by imaging mass spectrometry and prospect for antitumor activity.

Epertinib (S-222611) is a potent, reversible, and selective tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), human EGFR2 (HER2), and human EGFR4. We developed experimental brain metastasis models by intraventricular injection (intraventricular injection mouse model; IVM) of HER2-positive breast cancer (MDA-MB-361-luc-BR2/BR3) or T790M-EGFR-positive lung cancer (NCI-H1975-luc) cells. After a single oral administration, epertinib and lapatinib concentrations in brain metastatic regions were analyzed by quantitative imaging mass spectrometry. In the NCI-H1975 lung cancer IVM, the concentration of epertinib in brain metastasis was comparable to that of lapatinib. However, in the MDA-MB-361 breast cancer IVM, the concentration of epertinib in brain metastasis was >10 times higher than that of lapatinib. Furthermore, the epertinib tumor-to-normal brain ratio was ~4 times higher than that of lapatinib. Blood-tumor barrier (BTB) permeability was assessed in each brain metastatic region. In the lung cancer model, fluorescently labeled dextran was more highly detected in brain metastatic regions than in brain parenchyma. However, in breast cancer models, dextran fluorescence intensity in brain metastatic regions and brain parenchyma were comparable, suggesting that the BTB remained largely intact. Epertinib would be promised as a therapeutic agent for HER2-positive breast cancer with brain metastasis.

Characterization of pancreatic islet cell tumors and renal tumors induced by a combined treatment of streptozotocin and nicotinamide in male SD rats.

We herein investigated the histopathological features, including proliferative activity and immunoexpression, of pancreatic islet cell tumors (ICTs) in male SD rats induced by streptozotocin (STZ) and nicotinamide (NA), and discussed their relevance to biological behaviors and prognoses. A total of 70 and 43% of rats developed ICTs 37-45 weeks after the treatment with STZ (50 or 75mg/kg, i.v.) and NA (350mg/kg, twice, p.o.), respectively. Among the islet tumors observed in the STZ/NA-treated groups, 75% were adenomas, while 25% were carcinomas. Most STZ/NA-induced carcinomas were characterized by well-differentiated tumor cells with/without local invasion into the surrounding tissues, and weak proliferative activity. No outcome such as distance metastasis and death was noted. All of the ICTs strongly expressed insulin, part of which had hormone productivity; however there were no hypoglycemia-related clinical signs such as convulsion in these rats 36 weeks after the treatment. These results suggested that rat ICTs induced STZ/NA have small impact on biological activity or prognosis. STZ/NA treatment significantly increased of focal proliferative lesions in the kidney, liver and adrenal glands other than pancreatic islets. Of the STZ/NA-induced kidney tumors, more than 60% were renal cell adenomas, and many of them were basophilic type. The incidence of eosinophilic or clear cell type of tumors was less than 10%, respectively. Immunohistochemical analyses revealed that many of the STZ/NA-induced basophilic type of renal tumors were derived from proximal tubules, whereas the clear cell and eosinophilic types were derived from collecting tubules.

Cutaneous mastocytosis with a mutation in the juxtamembrane domain of c-kit in a young laboratory beagle dog.

Cutaneous mastocytosis, which resembles a subset of urticaria pigmentosa in humans, is rare in dogs. We herein report unrepresentative neoplastic proliferation of mast cells in ventral skin removed routinely from a nine-month-old female laboratory beagle dog at necropsy. A histological examination revealed diffuse extensive cellular infiltration from the superficial to deep dermis in most parts of the skin around the fourth and fifth mammary papilla without nodule formation. Tumor cells were fairly monomorphic, well-differentiated mast cells with round nuclei of small distinct nucleoli and moderate to abundant, slightly eosinophilic and granular cytoplasm. A perivascular arrangement of mast cells was noted at the margin of the lesions. Infiltration of eosinophils and degeneration of collagen were not observed in the dermis. Cutaneous mastocytosis was diagnosed based on these features. A sequence analysis of lesions revealed the deletion of Gln555 to Ile570 within the juxtamembrane domain of c-kit (exon 11).

Diagnostic and predictive performance and standardized threshold of traditional biomarkers for drug-induced liver injury in rats.

Traditional biomarkers such as alanine and aspartate aminotransferase (ALT, AST) and total bilirubin (TBIL) have been widely used for detecting drug-induced liver injury (DILI). Although the Food and Drug Administration (FDA) proposed standardized thresholds for human as Hy's law, those for animals have not been determined, and predictability of these biomarkers for future onset of hepatic lesions remains unclear. In this study, we investigated these diagnostic and predictive performance of 10 traditional biomarkers for liver injury by receiver-operating characteristic (ROC) curve, using a free-access database where 142 hepatotoxic or non-hepatotoxic compounds were administrated to male rats (n=5253). Standardization of each biomarker value was achieved by calculating the ratio to control mean value, and the thresholds were determined under the condition of permitting 5% false positive. Of these 10 biomarkers, AST showed the best diagnostic performance. Furthermore, ALT and TBIL also showed high performance under the situation of hepatocellular necrosis and bile duct injury, respectively. Additionally, the availability of the diagnostic thresholds in difference testing facility was confirmed by the application of these thresholds to in-house prepared dataset. Meanwhile, incorrect diagnosis by the thresholds was also observed. Regarding prediction, all 10 biomarkers showed insufficient performance for future onset of hepatic lesions. In conclusion, the standardized diagnostic thresholds enable consistent evaluation of traditional biomarkers among different facilities, whereas it was suggested that novel biomarker is required for more accurate diagnosis and prediction of DILI.

Plasma miR-208 as a useful biomarker for drug-induced cardiotoxicity in rats.

Cardiotoxicity is one of the major safety concerns in drug development. Therefore, detecting and monitoring cardiotoxicity throughout preclinical and clinical studies is important for pharmaceutical companies. The present study was conducted in order to explore a plasma miRNA biomarker for cardiotoxicity in rats. As organ specificity is an important factor for a biomarker, we analyzed the miRNA microarray dataset in 55 organs/tissues in normal male rats. Based on this analysis, 5 miRNAs consisting of miR-208 (heart-specific), miR-1, miR-133a, miR-133b (heart and skeletal muscle-specific) and miR-206 (skeletal muscle-specific) were selected. Next, we evaluated the usefulness of those 5 miRNAs as circulating biomarkers in rats administered with single-dose isoproterenol or doxorubicin. Plasma miR-208 was consistently increased through 24 h after dosing in rats administered with isoproterenol, whereas plasma concentrations of cardiac troponin (cTn) showed transient elevation. In contrast, the plasma levels of miR-1, miR-133a, miR-133a and miR-206 were elevated after treatment with doxorubicin, probably as a result of skeletal muscle toxicity. Additionally, the plasma miR-208 level was elevated even after repeat-dose administration (once daily for 7 days) of isoproterenol under which the pathological condition proceeded to the sub-chronic phase such as fibrosis. Thus, our data suggest that miR-208 is a promising plasma biomarker for cardiotoxicity in rats. Monitoring of plasma miR-208 levels in rats may lead to more accurate evaluation of cardiotoxicity in preclinical studies.

Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure.

The kidney has a capacity to recover from ischemic or toxic insults that result in cell death, and timely tissue repair of affected renal tubules may arrest progression of injury, leading to regression of injury and paving the way for recovery. To investigate the roles of neutrophil gelatinase-associated lipocalin (NGAL/lcn2) and osteopontin (OPN/spp1) during renal regeneration, the expression patterns of NGAL and OPN in the cisplatin-induced rat renal failure model were examined. NGAL expression was increased from day 1 after injection; it was seen mainly in the completely regenerating proximal tubules of the cortico-medullary junction on days 3-35; however, the expression was not seen in abnormally dilated or atrophied renal tubules surrounded by fibrotic lesions. On the other hand, OPN expression was increased from day 5 and the increased expression developed exclusively in the abnormal renal tubules. NGAL expression level well correlated with the proliferating activity in the regenerating renal epithelial cells, whereas OPN significantly correlated with the α-smooth muscle actin-positive myofibroblast appearance, expression of transforming growth factor (TGF)-ß1, and the number of CD68-positive macrophages. Interestingly, rat renal epithelial cell line (NRK-52E) treated with TGF-ß1 decreased NGAL expression, but increased OPN expression in a dose-dependent manner. Because increases of TGF-ß1, myofibroblasts and macrophages contribute to progressive interstitial renal fibrosis, OPN may be involved in the pathogenesis of fibrosis; on the contrary, NGAL may play a role in tubular regeneration after injury. Expression analysis of NGAL and OPN would be useful to investigate the tubule damage in renal-toxicity.

Genomic biomarkers for cardiotoxicity in rats as a sensitive tool in preclinical studies.

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver-operating characteristic (ROC) analysis using more cardiotoxic and non-cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi-gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi-gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi-gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs.

Effect of PPARß/δ agonist on the placentation and embryo-fetal development in rats.

BACKGROUND: The present study was conducted to evaluate the developmental toxicity in the endometrium and placenta due to GW501516 administration by gavage to pregnant rats. METHODS: GW501516 was orally administered repeatedly to pregnant rats from gestation day (GD) 6 to 17 at a dose of 0, 30, and 100 mg/kg/day. In next study, GW501516 was also orally administered to pregnant rats on GD 7, 8, 9, 10, or 11 at a single dose of 275 or 350 mg/kg. In these studies, caesarean section was performed to examine the pregnancy outcome on GD21. Additionally, GW501516 was orally administered to pregnant rats on GD 10 at a single dose of 275 mg/kg. Placentae were subjected for temporal histological examinations on GD 11, 13, 15, or 17. RESULTS: Placental malformation was induced by repeated administration of GW501516 at a dose of 100 mg/kg/day. Single oral administration of GW501516 at a dose of 275 and/or 350 mg/kg on GD 8, 9, 10, or 11 induced placental malformation, whereas GW501516 administered on GD 10 was the most effective for increasing placental malformation. Histopathologically, single oral administration of GW501516 on GD 10 induced cystic degeneration associated with cellular lysis of glycogen cells started from GD 15 in the basal zone. CONCLUSIONS: High frequency of placental malformation was observed by the administration of GW501516. From GD 8 to 11, especially GD 10, is more sensitive period to induce the placental malformation.

Expression patterns of heat shock protein 25 in carbon tetrachloride-induced rat liver injury.

Heat shock protein 25 (Hsp25) is a molecular chaperone playing roles in cytoprotection. We investigated the distribution and localization of Hsp25 expression in CCl(4)-induced rat hepatic lesions; liver samples were obtained from 3 h to 10 days after a single oral administration of CCl(4). Immunohistochemically, Hsp25-positive hepatocytes started to appear in the perivenular area at 6 h after CCl(4) administration. Their number and strength increased till day 1. Expression of Hsp25 mRNA significantly increased after 3 h and proceeded to increase with time till day 1. Apoptotic hepatocytes were detected around the perivenular area after 6 h. The area where Hsp25-positive hepatocytes were observed till day 1 corresponded to the area where apoptotic hepatocytes were seen. On days 2 and 3, degenerative and/or necrotic hepatocytes in the perivenular area were replaced by macrophages reacting to ED1 (for CD68) and ED2 (for CD163); Hsp25 expression was seen in hepatocytes around the perivenular area and there was a close relationship of reactive macrophages with Hsp25-positive hepatocytes, suggesting a potential role for Hsp25 in suppressing injury by inflammation. The mRNA expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and osteopontin, which can be produced by infiltrating macrophages, corresponded to that of Hsp25 from day 1 to day 3; these factors might be related to the induction of Hsp25 expression. The shift of the Hsp25 expression pattern in the liver lesion might have depended on microenvironmental conditions evoked by interactions between necrobiotic hepatocytes and infiltrating macrophages. Thus, Hsp25 expression analyses should be beneficial for evaluations of hepatotoxicants.

Underestimation of urinary biomarker-to-creatinine ratio resulting from age-related gain in muscle mass in rats.

Recent efforts have been made to identify useful urinary biomarkers of nephrotoxicity. Furthermore, the application of urine to the other toxicities as new biomarker source has been recently expanded. Meanwhile, correction of urinary biomarker concentrations according to fluctuations in urine flow rate is required for adequate interpretation of the alteration. The urinary biomarker-to-creatinine ratio (UBCR) is widely used because of the convenience, while the urinary biomarker-excretion rate is regarded as the gold standard corrective method. Because creatinine is a catabolite in energy production in muscles, we hypothesized that altered muscle mass could affect creatinine kinetics, ultimately affecting UBCR. However, no study has examined this hypothesis. In this study, we examined the influence of muscle mass gain on UBCR, using male Sprague-Dawley rats during the growth phase, 6-12-week old. Both plasma creatinine and excretion of urinary creatinine (Ucr excretion) showed increases with muscle mass gain in rats, in which the alterations of UBCR were lowered. The renal mRNA level of the organic cation transporter-2 (Oct2), a creatinine transporter, showed an age-related increase, whereas the mRNA level of multidrug and toxin extrusions-1 (Mate1) remained constant. Multiple regression analysis showed that the increase in creatinine clearance highly contributed to the age-related increase in Ucr excretion compared to the mRNA levels of Oct2 and Mate1. This suggested that the age-related increase in Ucr excretion may be attributable to the increased transglomerular passage of creatinine. In conclusion, the results suggest that muscle mass gain can affect creatinine kinetics, leading to underestimation of UBCR. Therefore, it is important to understand the characteristics of the corrective method when using urinary biomarker, the failure of which can result in an incorrect diagnosis.

Biomarker panel of cardiac and skeletal muscle troponins, fatty acid binding protein 3 and myosin light chain 3 for the accurate diagnosis of cardiotoxicity and musculoskeletal toxicity in rats.

Cardiotoxicity and musculoskeletal toxicity can be life-threatening, and thus have strong impact on both the development and marketing of drugs. Because the conventional biomarkers such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) have low detection power, there has been increasing interest in developing biomarkers with higher detection power. The current study examined the usefulness of several promising biomarkers, cardiac and skeletal muscle troponins (cTnI, cTnT and sTnI), fatty acid binding protein 3 (FABP3) and myosin light chain 3 (MYL3), and compared the obtained data to AST, LDH and CK in rat models treated with various myotoxic and non-myotoxic compounds (isoproterenol, metaproterenol, doxorubicin, mitoxantrone, allylamine, cyclosporine A, cyclophosphamide, aminoglutethimide, acetaminophen, methapyrilene, allylalcohol and α-naphthylisothiocyanate). These promising biomarkers were found to be superior to the conventional biomarkers, as they had a specific and abundant distribution within the heart and/or skeletal muscles; exhibited a positive correlation between the amplitude of increases and the degree of pathological alterations; had higher diagnostic accuracy for detecting pathological alterations; and had the additive effect of improving the diagnostic accuracy of conventional biomarkers. However, these promising biomarkers have several drawbacks including a rapid clearance, the fact that they are affected by renal dysfunction, and different reactivity to the mode of action of individual myotoxicants. In conclusion, the promising biomarkers cTnI, cTnT, FABP3, MYL3, and sTnI demonstrated sensitivity and specificity for cardiac and skeletal myotoxicity that was superior to those of conventional biomarkers, while we should pay attention to the drawbacks of these biomarkers when used in toxicity studies.

Predictive genomic biomarkers for drug-induced nephrotoxicity in mice.

The present study aimed to establish candidate biomarker genes for the early detection of nephrotoxicity in mice, with a particular focus on nephrotoxicity caused by polyene macrolides. Comprehensive gene expression changes were evaluated using microarrays in a mouse model in which acute nephrotoxicity was induced by amphotericin B deoxycholate, trade name Fungizone. The upregulated genes identified through microarray analysis of kidney tissue of Fungizone-treated mice included several genes that have been reported as nephrotoxicity biomarkers in rats, and 14 genes were selected as candidate nephrotoxicity biomarkers. The usefulness of these genes as nephrotoxicity biomarkers in mice was evaluated further through expression profiling under several experimental conditions using real time RT-PCR. Expression of genes encoding kidney injury molecule 1, lipocalin 2, tissue inhibitor of metalloproteinase 1, and secreted phosphoprotein 1 was highly upregulated by Fungizone, nystatin, natamycin, amphotericin B methyl ester, and liposomal amphotericin B, and their area under the ROC curve values were more than 0.95. These genes were more sensitive at detecting nephrotoxicity than traditional clinical chemistry and histopathology parameters. This study provides novel evidence that these nephrotoxicity biomarker genes identified are translatable to mice, and that they are useful for early and sensitive detection of nephrotoxicity.

Glaucoma in a New Zealand White Rabbit Fed High-cholesterol Diet.

Goniodysgenesis, malformation of the filtration angle, was observed in a New Zealand white rabbit supplied with 100 g/day rabbit chow containing 0.2% cholesterol for 10 months. Histopathology revealed cupping of the optic disc, atrophy of the retina and hyalinization of the ciliary body in the bilateral eyeballs. These findings corresponded with histopathological features caused by glaucoma. On the basis of these findings, we diagnosed this lesion as glaucoma, and classified it as primary glaucoma because of the presence of developmental defects of the filtration angle. In this case, hypercholesterolemia-induced changes, such as aggregation of lipid-laden macrophages and cholesterin clefts in the sclera or choroid, might cause deterioration of the lesions in glaucoma.

Decrease in urinary creatinine in acute kidney injury influences diagnostic value of urinary biomarker-to-creatinine ratio in rats.

Recent research has revealed several useful urinary biomarkers of renal dysfunction such as acute kidney injury (AKI). For adequate evaluation of altered urinary biomarkers, it is necessary to consider the influence of varied urine flow rate (UFR). Calculation of the excretion rate of a urinary biomarker (UFR-correction) is the gold standard for the correction of UFR variation. An alternative method that is widely used is to calculate the ratio of the biomarker level to urinary creatinine (Ucr-correction). To date, the equivalence between these two methods has been examined only in a steady state situation such as diabetic nephropathy, and the urinary biomarkers examined have been limited to proteinuria and albuminuria. Therefore, we comprehensively addressed the relationship between Ucr-correction and UFR-correction of ten urinary biomarkers N-acetyl-ß-d-glucosaminidase (NAG), lactate dehydrogenase (LDH), total protein, albumin, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, ß(2)-microglobulin, cystatin-c and glutathione S-transferase-α in non-steady state situations such as AKI. All ten urinary biomarkers showed larger amplitude increases in AKI by Ucr-correction than by UFR-correction in linear regression analysis. Moreover, receiver operating characteristic curves analysis suggested that, at least for the biomarkers NAG and LDH, Ucr-correction had higher diagnostic power than UFR-correction. We observed a decrease in the Ucr excretion in AKI that was accompanied by a reduction in creatinine clearance and reduced mRNA expression of the renal organic cation transporter-2, which is known to function as a transporter for creatinine. These results may provide a mechanistic explanation for the phenomena obtained in Ucr-correction. In conclusion, while Ucr-correction could overestimate the degree of AKI, it could also provide higher diagnostic power for AKI than UFR-correction. We should take into consideration of these backgrounds when using the Ucr-correction.

Expression of peroxisome proliferator-activated receptor isoforms in the rat uterus during early pregnancy.

Peroxisome proliferator-activated receptors (PPARs) play an important role in different compartments of the female reproductive system in rodents and humans. However, expressional profiles and physiological functions of PPARs in the endometrium prior to the placentation are not well understood. In this study, we determined expressional profiles of the PPARs during early pregnancy. Immunocytochemistry revealed that both PPARα and PPARß/δ were strongly detected in the endometrial stroma on days 4.5-6.5 of pregnancy, which is just a starting time of implantation. Delayed implantation animal model showed that the expressions of PPARα and PPARß/δ occurred after the initiation of implantation in the endometrial stroma. Moreover, an in vitro decidualization model further revealed that the expression of PPARα increased in the cultured rat endometrial stromal cells at 24 h after the decidualization treatment, but the expression of PPARß/δ was delayed and increased at 48 h after the treatment. PPARγ was expressed in the endometrial stroma and its expression decreased significantly at 2.5 days post-coitum and maintained a low level of expression during the period of implantation. These results indicate that PPARα is expressed and induced by the initiation of implantation, prior to the expression of PPARß/δ in decidualized endometrium. Increasing expression of PPARγ during fertilization and its decline during the period of implantation further suggest that PPARs may play important roles during early pregnancy.

A toxicogenomic approach for identifying biomarkers for myelosuppressive anemia in rats.

Myelosuppressive anemia is a serious side effect associated with several drugs. Thus, there is an increasing demand for sensitive biomarkers for the early detection of myelosuppressive anemia during toxicological studies. We applied a toxicogenomic approach to identify useful biomarker genes reflecting myelosuppressive anemia in the rat liver. Expression of the hemoglobin beta chain complex (Hbb), aminolevulinic acid synthase 2 (Alas2), and cell division cycle 25 homolog B (Cdc25b) genes changed as a result of anemia induced by the myelosuppressive agents linezolid, cisplatin, and carboplatin, suggesting that these genes may be suitable biomarkers. Moreover, evaluation of perfused and unperfused livers indicated that changes in the expression of these genes originate in circulating reticulocytes in the liver. Erythroid differentiation-associated changes in expression of the Hbb, Alas2, and Cdc25b genes were confirmed in vitro using Friend leukemia cells. In conclusion, our current research provides novel evidence that gene expression in circulating reticulocytes contained in the liver changes dramatically under myelosuppressive conditions. While further large-scale validation studies are needed, our results indicate that the genes we identified might be useful biomarkers for the sensitive detection of myelosuppressive anemia in rats.