Immunohistochemical Expressions of Main PGE2 Biosynthesis-related Enzymes and PGE2 Receptor in Rat Nephrogenesis

Endogenous prostaglandin (PG) E2 plays important roles in renal homeostasis. Immunoexpressions of PGE2 biosynthesis-related enzymes, cyclooxygenase (COX)-2 and microsomal PGE2 synthetase (mPGES)-1 and EP4 (a PGE2 receptor), were investigated in renal development. Kidney tissues were obtained from fetuses on gestation days 18 and 21 and neonates on days 1 to 18. In fetuses and early neonates, the expressions of COX-2, mPGES-1 and EP4 were observed in developing renal tubules, indicating that COX-2 and its product, PGE2, play important roles in blastemal cell-derived renal tubular development via EP4. Cyclin D1 expression was seen in both the nucleus and cytoplasm of the developing tubules. These findings differed from the decreased COX-2 expression and exclusive nuclear expression of cyclin D1 seen in abnormal epithelial regeneration of injured renal tubules in cisplatin-treated rats in our previous articles. Collectively, PGE2, induced by COX-2, regulates renal tubular epithelial formation via EP4

Comparative Gene Expression Analysis in the Skeletal Muscles of Dysferlin-deficient SJL/J and A/J Mice

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted to determine whether or not there are interstrain or site-dependent differences in the gene expression profiles of skeletal muscles in SJL/J and A/J mice as dysferlinopathy models. Upon analysis by qRT-PCR, SJL/J mice showed a trend of increased gene expression level of uncoupling protein 2 in the rectus femoris and longissimus lumborum at 30 weeks of age when dystrophic lesions became histopathologically pronounced. Heme oxygenase 1 and S100 calcium binding protein A4 were upregulated in the rectus femoris, longissimus lumborum and abdominal muscles, in which dystrophic lesions occur more commonly in SJL mice. The gene expression levels of heat shock protein 70 in most muscles of A/J mice were lower than those of BALB/c mice as control. SJL/J mice exhibited a marked lowering of decay-accelerating factor 1/CD55 gene expression level in all studied muscles except for the heart at all ages compared with that of BALB/c mice. This study showed that there were some interstrain differences in the gene expres sion profiles of skeletal muscles between SJL/J and A/J mice. Further investigation is required to reveal whether these alterations of the expression levels are the cause of dystrophic changes or occur subsequent to muscle damage

Metabolic Fingerprinting in Toxicological Assessment Using FT-ICR MS

Detection of the toxicity of a candidate compound at an early stage of drug development is an emerging area of interest. It is difficult to determine all of the effects of metabolism of a compound using traditional approaches such as histopathology and serum biochemistry. The goal of a metabolomics approach is to determine all metabolites in a living system, with the potential to detect and identify biomarkers involved in toxicity onset. Here, we summarize the metabolic fingerprints for detection and identification of metabolic changes and biomarkers related to drug-induced toxicity using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS)

Thy-1 Expressing Mesenchymal Cells in Rat Nephrogenesis in Correlation with Cells Immunoreactive for α-Smooth Muscle Actin and Vimentin

Thy-1 expression may influence myofibroblast development. Through the epithelial-mesenchymal transition (EMT), injured renal epithelial cells undergo regression to the metanephric mesenchymal phenotype and then acquire a myofibroblastic nature (expressing α-smooth muscle actin; α-SMA). Because the metanephric blastema differentiates into mesenchymal and renal epithelial cells, we investigated Thy-1 immunoexpression during nephrogenesis in F344 rats in correlation with vimentin and α-SMA expressions. Kidney samples were obtained from fetuses on gestation days 18 and 21, neonates on days 1-18 and adults at 6 weeks of age. Mesangial cells in S-shaped bodies and immature and mature glomeruli continuously expressed both Thy-1 and α-SMA during early nephrogenesis (fetuses and neonates on days 1-9). During early nephrogenesis, loosely-arranged blastemal cell-derived mesenchymal cells in the cortex and medulla also exhibited Thy-1 and α-SMA, although the α-SMA expression was weaker than that of Thy-1. Vimentin expression coincided with that of Thy-1. These findings indicate that the derivation of α-SMA-expressing myofibroblastic cells may be related to mesangial or blastemal cells expressing both Thy-1 and α-SMA. Interestingly, there was a difference in Thy-1 expression between cortical and medullary tubulointerstitial cells from late nephrogenesis (neonates on days 12-18) and those from adults in that the cortical cells reacted faintly or negatively to Thy-1, whereas the medullary cells reacted strongly to Thy-1; additionally, bundle-arranged mesenchymal cells that were only observed in the neonates on days 1-12 reacted strongly to α-SMA, but faintly to Thy-1. Blastemal cell-derived mesenchymal cells seem to alter the immunoexpressions of Thy-1 and α-SMA, depending on the conditions which they develop. Thy-1 immunoexpression would be useful for investigation of reverse embryogenesis, which might occur in fibrotic kidneys

Relationship of Cell Proliferating Marker Expressions with PGE2 Receptors in Regenerating Rat Renal Tubules after Cisplatin Injection

Cisplatin, an anticancer drug, is well known to have nephrotoxicity as an adverse effect. We investigated the expressions of cell cycle markers and prostaglandin E2 (PGE2) receptors (EP) in the affected renal tubules in rats injected with a single dose (6 mg/kg body weight) of cisplatin. On days 1–3 after dosing, the affected renal epithelial cells were almost desquamated, showing necrosis. On day 5 onwards, the renal tubules were rimmed by flattened or cuboidal epithelial cells with basophilic cytoplasm; BrdU-immunopositive cells began to significantly increase, indicating regeneration. Simultaneously, TUNEL-positive apoptotic cells were also seen. On days 1–5, cyclin D1-immunopositive cells were decreased with an increased expression in p21 mRNA, indicating G1 arrest in the cell cycle. The affected renal epithelial cells began to react to EP4 receptor, but not to EP2 receptor. Some EP4 receptor-reacting epithelial cells gave a positive reaction to BrdU or cyclin D1. Collectively, the affected renal tubules underwent various alterations such as necrosis, apoptosis, regeneration and G1 arrest; the aspects might be influenced by endogenous PGE2 through EP4 receptor

A Rhabdomyosarcoma Arising in the Larynx of a Dog

A neoplastic nodular lesion, 2 × 3 cm in diameter, was found in the larynx of a 6-year-old spayed female dog. The tumor was ill-circumscribed, consisting histologically of large round cells with abundant cytoplasm interspersed with small round cells with less cytoplasm and occasional multinucleated cells (myotubes). Immunohistochemically, tumor cells were positive for myoglobin, desmin and vimentin in varying degrees, but negative for S-100 protein, GFAP or cytokeratin. Cytoplasmic myofilaments/myofibrils with a dense Z-line-like structure were seen, the fine structures of which were complemented by PTAH stain. Based on these findings, the tumor was diagnosed as a rhabdomyosarcoma, a very rare tumor in the larynx of dogs

Deficiency of the RIβ subunit of protein kinase A causes body tremor and impaired fear conditioning memory in rats

The RIβ subunit of cAMP-dependent protein kinase (PKA), encoded by Prkar1b, is a neuronal isoform of the type I regulatory subunit of PKA. Mice lacking the RIβ subunit exhibit normal long-term potentiation (LTP) in the Schaffer collateral pathway of the hippocampus and normal behavior in the open-field and fear conditioning tests. Here, we combined genetic, electrophysiological, and behavioral approaches to demonstrate that the RIβ subunit was involved in body tremor, LTP in the Schaffer collateral pathway, and fear conditioning memory in rats. Genetic analysis of WTC-furue, a mutant strain with spontaneous tremors, revealed a deletion in the Prkar1b gene of the WTC-furue genome. Prkar1b-deficient rats created by the CRISPR/Cas9 system exhibited body tremor. Hippocampal slices from mutant rats showed deficient LTP in the Schaffer collateral–CA1 synapse. Mutant rats also exhibited decreased freezing time following contextual and cued fear conditioning, as well as increased exploratory behavior in the open field. These findings indicate the roles of the RIβ subunit in tremor pathogenesis and contextual and cued fear memory, and suggest that the hippocampal and amygdala roles of this subunit differ between mice and rats and that rats are therefore beneficial for exploring RIβ function

IL-19 Contributes to the Development of Nonalcoholic Steatohepatitis by Altering Lipid Metabolism

Interleukin (IL)-19, a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. Nonalcoholic steatohepatitis (NASH) is a disease that has progressed from nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. We evaluated the functions of IL-19 in a NAFLD/NASH mouse model using a 60% high fat diet with 0.1% methionine, without choline, and with 2% cholesterol (CDAHFD). Wild-type (WT) and IL-19 gene-deficient (KO) mice were fed a CDAHFD or standard diet for 9 weeks. Liver injury, inflammation, and fibrosis induced by CDAHFD were significantly worse in IL-19 KO mice than in WT mice. IL-6, TNF-α, and TGF-β were significantly higher in IL-19 KO mice than in WT mice. As a mechanism using an in vitro experiment, palmitate-induced triglyceride and cholesterol contents were decreased by the addition of IL-19 in HepG2 cells. Furthermore, addition of IL-19 decreased the expression of fatty acid synthesis-related enzymes and increased ATP content in HepG2 cells. The action of IL-19 in vitro suppressed lipid metabolism. In conclusion, IL-19 may play an important role in the development of steatosis and fibrosis by directly regulating liver metabolism and may be a potential target for the treatment of liver diseases

A rapid, targeted, neuron-selective, in vivo knockdown following a single intracerebroventricular injection of a novel chemically modified siRNA in the adult rat brain

peer reviewedThere has been a dramatic expansion of the literature on RNA interference and with it, increasing interest in the potential clinical utility of targeted inhibition of gene expression and associated protein knockdown. However, a critical factor limiting the experimental and therapeutic application of RNA interference is the ability to deliver small interfering RNAs (siRNAs), particularly in the central nervous system, without complications such as toxicity and inflammation. Here we show that a single intracerebroventricular injection of Accell siRNA, a new type of naked siRNA that has been modified chemically to allow for delivery in the absence of transfection reagents, even into differentiated cells such mature neurons, leads to neuron-specific protein knockdown in the adult rat brain. Following in vivo delivery, targeted Accell siRNAs were incorporated successfully into various types of mature neurons, but not glia, for 1 week in diverse brain regions (cortex, striatum, hippocampus, midbrain, and cerebellum) with an efficacy of delivery of approximately 97%. Immunohistochemical and Western blotting analyses revealed widespread, targeted inhibition of the expression of two well-known reference proteins, cyclophilin-B (38–68% knockdown) and glyceraldehyde 3-phosphate dehydrogenase (23–34% knockdown). These findings suggest that this novel procedure is likely to be useful in experimental investigations of neuropathophysiological mechanisms