Safety assessment of a novel C-type natriuretic peptide derivative and the mechanism of bone- and cartilage-specific toxicity.

ASB20123, a C-type natriuretic peptide/ghrelin chimeric peptide, was designed as a novel peptide and demonstrated full agonistic activity for natriuretic-peptide receptor B and a significantly longer half-life in plasma compared with the native peptide. We researched the toxicological profile of ASB20123, the correlation between the morphological change of the epiphyseal plate and bone and cartilage toxicity, and biomarkers to detect the toxicity. ASB20123 was systemically administered to male and female rats at daily dose levels of 0.5, 1.5, and 5.0 mg/kg/day for 4 weeks. In this study, toxicity was observed as changes related to bone and cartilage tissues, and no other toxicological changes were observed in all animals. Next, ASB20123 was administered to 12-month-old rats with a little epiphyseal plate. The toxic changes related to bone and cartilage tissues were not observed in any animal with a closed epiphyseal plate, indicating that the toxic changes were triggered by the growth-accelerating effect on the bone and cartilage. Furthermore, we searched for the biomarker related to the bone and cartilage toxicity using rats treated with ASB20123 at doses of 0.005, 0.05, 0.5, and 5.0 mg/kg/day for 4 weeks. A close correlation between necrosis/fibrosis in the epiphysis and metaphysis and thickness of the epiphyseal plate in the femur was confirmed in this study. A decrease in the bone mineral density (BMD) of the femur also was associated with the appearance of bone toxicity. These results indicated that the toxicity of ASB20123 was limited to bone- and cartilage-specific changes, and these changes were triggered by an excessive growth accelerating effect. Furthermore, our data suggested that the thickness of the epiphyseal plate and BMD could be reliable biomarkers to predict bone toxicity.

Foramen magnum stenosis and midface hypoplasia in C-type natriuretic peptide-deficient rats and restoration by the administration of human C-type natriuretic peptide with 53 amino acids.

C-type natriuretic peptide (CNP)-knockout (KO) rats exhibit impaired skeletal growth, with long bones shorter than those in wild-type (WT) rats. This study compared craniofacial morphology in the CNP-KO rat with that in the Spontaneous Dwarf Rat (SDR), a growth hormone (GH)-deficient model. The effects of subcutaneous administration of human CNP with 53 amino acids (CNP-53) from 5 weeks of age for 4 weeks on craniofacial morphology in CNP-KO rats were also investigated. Skulls of CNP-KO rats at 9 weeks of age were longitudinally shorter and the foramen magnum was smaller than WT rats. There were no differences in foramen magnum stenosis and midface hypoplasia between CNP-KO rats at 9 and 33 weeks of age. These morphological features were the same as those observed in CNP-KO mice and activated fibroblast growth factor receptor 3 achondroplasia-phenotype mice. In contrast, SDR did not exhibit foramen magnum stenosis and midface hypoplasia, despite shorter stature than in control rats. After administration of exogenous CNP-53, the longitudinal skull length and foramen magnum size in CNP-KO rats were significantly greater, and full or partial rescue was confirmed. The synchondrosis at the cranial base in CNP-KO rats is closed at 9 weeks, but not at 4 weeks of age. In contrast, synchondrosis closure in CNP-KO rats treated with CNP-53 was incomplete at 9 weeks of age. Administration of exogenous CNP-53 accelerated craniofacial skeletogenesis, leading to improvement in craniofacial morphology. As these findings in CNP-KO rats are similar to those in patients with achondroplasia, treatment with CNP-53 or a CNP analog may be able to restore craniofacial morphology and foramen magnum size as well as short stature.

ASB20123: A novel C-type natriuretic peptide derivative for treatment of growth failure and dwarfism.

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1-22) and human ghrelin(12-28, E17D). Compared to CNP(1-22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1-22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety.

Exogenous C-type natriuretic peptide restores normal growth and prevents early growth plate closure in its deficient rats.

Signaling by C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B, is a pivotal stimulator of endochondral bone growth. We recently developed CNP knockout (KO) rats that exhibit impaired skeletal growth with early growth plate closure. In the current study, we further characterized the phenotype and growth plate morphology in CNP-KO rats, and the effects of exogenous CNP in rats. We used CNP-53, an endogenous form of CNP consisting of 53 amino acids, and administered it for four weeks by continuous subcutaneous infusion at 0.15 or 0.5 mg/kg/day to four-week old CNP-KO and littermate wild type (WT) rats. We demonstrated that CNP-KO rats were useful as a reproducible animal model for skeletal dysplasia, due to their impairment in endochondral bone growth. There was no significant difference in plasma bone-turnover markers between the CNP-KO and WT rats. At eight weeks of age, growth plate closure was observed in the distal end of the tibia and the calcaneus of CNP-KO rats. Continuous subcutaneous infusion of CNP-53 significantly, and in a dose-dependent manner, stimulated skeletal growth in CNP-KO and WT rats, with CNP-KO rats being more sensitive to the treatment. CNP-53 also normalized the length of long bones and the growth plate thickness, and prevented growth plate closure in the CNP-KO rats. Using organ culture experiment of fetal rat tibia, gene set enrichment analysis indicated that CNP might have a negative influence on mitogen activated protein kinase signaling cascades in chondrocyte. Our results indicated that CNP-KO rats might be a valuable animal model for investigating growth plate physiology and the mechanism of growth plate closure, and that CNP-53, or its analog, may have the potential to promote growth and to prevent early growth plate closure in the short stature.

Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death.

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.

(+)-Usnic acid-induced myocardial toxicity in rats.

(+)-Usnic acid (UA) has been known to be a strong uncoupler, and mitochondrial and endoplasmic reticulum (ER)-related stresses are suggested to be involved in the mechanism of hepatotoxicity. However, it has not been clarified whether UA causes toxicity in other mitochondria-rich organs such as the heart. We elucidated whether UA induces cardiotoxicity and its mechanism. UA was orally administered to rats for 14 days, and laboratory and histopathological examinations were performed in conjunction with toxicogenomic analysis. As a result, there was no alteration in blood chemistry, whereas cytoplasmic rarefaction of myocardium was observed microscopically. This finding corresponded to the swollen mitochondria observed ultrastructurally. Immunohistochemically, expression of prohibitin, indicating mitochondrial imbalance, increased in the sarcoplasmic area. Toxicogenomic analysis highlighted the upregulation of gene groups consisting of oxidative stress, ER stress, and amino acid limitation. Interestingly, the number of upregulated genes was larger in the amino acid limitation-related gene group than that in other groups, implying that amino acid limitation might be one of the sources of oxidative stress, not only mitochondria and ER-originated stresses. In conclusion, the heart was manifested to be one of the target organs of UA. Mitochondrial imbalance with complex stresses may be involved in the toxic mechanism.

Subcutaneous soft tissue sarcoma with rhabdoid features in a dog.

A nine-year-old male beagle dog had a white spherical mass in the subcutis of the left lumbar region. Microscopically, spindle to oval cells diffusely proliferated in the fibrous and myxoid stroma. Many neoplastic cells showed rhabdoid features or vacuolated cytoplasm. Immunohistochemically, the neoplastic cells were positive for vimentin and S100 and partly positive for neuron-specific enolase and glial fibrillary acidic protein but were negative for von Willebrand factor, desmin and α-smooth muscle actin. Ultrastructurally, the neoplastic cells had abundant cytoplasmic processes and desmosome-like structures. Cytoplasmic inclusions of rhabdoid-featured cells in HE sections were composed of aggregates of intermediate filaments, and cytoplasmic vacuoles were identified as an invagination of cytoplasm. Although malignant peripheral nerve sheath tumor was suggested according to these results, the present case was diagnosed as a soft tissue sarcoma with rhabdoid features due to a lack of identification of the basal lamina under electron microscopy.

The Process and Development Mechanism of Age-related Fibrosis in the Pancreatic Islets of Sprague-Dawley Rats: Immunohistochemical Detection of Myofibroblasts and Suppression Effect by Estrogen Treatment.

The mechanism of spontaneous islet fibrosis in Sprague-Dawley rats was investigated. Using sections of the pancreas in naive males aged 26 to 102 weeks old and 26-week-old males injected with ß-estradiol 3-benzoate (EB), the incidence of lesions and histological scores of fibrosis were examined in conjunction with immunohistochemistry for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-α (PDGFRα) and estrogen receptor-α (ERα). The incidence of islet fibrosis increased in 78-week-old animals compared to the 26-week-old animals, and the incidence of atrophy in the fibrotic islet increased in animals over 52 weeks old. α-SMA and PDGFRα were positively stained mainly in fibrotic/inflammatory islets, and the histological score of α-SMA in the fibrotic islet decreased age-dependently. Notably, α-SMA and PDGFRα were co-expressed in inflammatory islets with a high score at all ages. The positive index of ERα in the EB-treated group increased when compared with that of the naive group. However, it was independent of the existence of fibrosis. In contrast, the score of α-SMA and PDGFRα decreased in the EB-treated group. In conclusion, it was clarified that a part of age-related fibrosis in islets became atrophy with age, and α-SMA-positive myofibroblasts were considered to contribute to the development of fibrosis. Strong PDGFRα stainability in fibrotic/inflammatory islets may imply that myofibroblasts were stimulated by PDGF to produce an extracellular matrix. Although estradiol has been known to suppress fibrosis/inflammation in the islet, nuclear-located ER-dependent signaling was considered not to be involved in the suppression mechanism. EB possibly affected the inhibition of the appearance of myofibroblasts.

Inflammatory fibroid polyp in the duodenum of a common marmoset (Callithrix jacchus).

A 32-month-old male common marmoset had a firm and white-colored mass in the duodenal wall. The cut surface was smooth and grayish white in color. Histologically, the mass consisted of a proliferation of spindle cells with an oval to spindle-shaped nucleus and scant eosinophilic cytoplasm in a loose myxoid or fibrotic background. Most of the lesion displayed no specific growth pattern whereas some of the cells concentrated around the vessels and created an onion-bulb structure. Additionally, marked inflammatory cellular infiltration, mainly eosinophils, was observed throughout the lesion. Immunohistochemically, the spindle cells were positive for vimentin, α-smooth muscle actin, fascin, and cyclin D1, and negative for S-100, factor VIII-related antigen, and c-kit. These histological and immunohistochemical features did not meet any differential diagnoses such as gastrointestinal stromal tumor, inflammatory myofibroblastic tumor, solitary fibrous tumor/hemangiopericytoma, smooth muscle tumor, schwannoma, and hemangiosarcoma. Collectively, the authors diagnosed the mass as a lesion that corresponded to an inflammatory fibroid polyp (IFP) in humans. IFP is defined as a mesenchymal proliferation composed of spindle stromal cells, small blood vessels, and inflammatory cells, particularly eosinophils, and is currently classified as a nonneoplastic lesion. To the best of our knowledge, this is the first case of spontaneous IFP in nonhuman primates.

Effect of body-weight loading onto the articular cartilage on the occurrence of quinolone-induced chondrotoxicity in juvenile rats.

The effect of body-weight loading onto the articular cartilage on the occurrence of chondrotoxicity was investigated in male juvenile Sprague-Dawley rats given ofloxacin (OFLX) orally once at 900 mg/kg. Just after dosing of OFLX, hindlimb unloading was performed for 0, 2, 4, or 8 h by a tail-suspension method. Animals were sacrificed at 8h post-dose, and then the distal femoral articular cartilage was subjected to a histological examination and an investigation for gene expression of tumor necrosis factor receptor superfamily, member 12a (Tnfrsf12a); prostaglandin-endoperoxide synthase 2 (Ptgs2); plasminogen activator, urokinase receptor (Plaur); and matrix metalloproteinase 3 (Mmp3) by qRT-PCR analysis. As a result, cartilage lesions and up-regulations of these 4 genes that were seen in rats without the tail suspension were not observed in rats with the 8-h tail suspension, and a tendency to decrease in the incidence of the cartilage lesions and the gene expression was noted in a tail-suspension time dependent manner. Our results clearly indicate that body-weight loading onto the cartilage is necessary to induce cartilage lesions and gene expression of Tnfrsf12a, Ptgs2, Plaur, and Mmp3 in juvenile rats treated with OFLX.

Effect of Hypertension on the Occurrence of Micro-hemorrhage in the Pancreatic Islet of Dahl Salt-sensitive Rats.

The effect of hypertension on the occurrence of micro-hemorrhage in the pancreatic islet, known to be observed in Sprague-Dawley (SD) rats spontaneously, and endothelial markers were investigated in male Dahl-Iwai salt-sensitive (DIS, derived from SD rats), salt-resistant (DIR), and SD rats. DIS and DIR rats were fed 8% NaCl-containing diet to induce hypertension, with blood pressure measurement once a week, euthanized at 6, 8, or 12 weeks of age, and subjected to the measurement of plasma nitric oxide (NO) and von Willebrand factor (vWF) concentrations combined with histopathological examinations and immunohistochemical detections of vWF in the pancreas and kidney. As a result, hypertension was observed from 7 through 12 weeks of age in DIS rats. At 12 weeks of age, only DIS rats showed decreased plasma NO and increased vWF, indicating endothelial abnormality in the body. Histopathologically, micro-hemorrhage in the islet was observed with a similar incidence and severity in SD and DIS rats aged 12 weeks, and vWF was immunohistochemically localized in the islet endothelium with similar reactivity between age-matched SD rats. On the other hand, in the kidney, glomerular sclerosis was observed in DIS rats aged 12 weeks and accompanied broad stainability of vWF in the sclerotic glomerulus, including endothelium. In conclusion, there was no enhancement/exaggeration in the micro-hemorrhage in the pancreatic islet of hypertensive DIS rats in comparison with that in SD rats under the present experimental conditions. It is suggested that hypertension is not related to the occurrence of islet micro-hemorrhage, spontaneously observed in SD rats.

Chondrotoxicity and toxicokinetics of novel quinolone antibacterial agents DC-159a and DX-619 in juvenile rats.

Quinolone antibacterial agents are extensively utilized in antimicrobial chemotherapy. However, they have been reported to induce arthropathy in juvenile animals, and the mechanism has not been clarified. Recently, we have demonstrated that Dusp1, Tnfrsf12a, Ptgs2, Fos, Mt1a, Plaur, Mmp3, Sstr1 and Has2 genes change in the articular cartilage of juvenile rats with a single oral administration of ofloxacin (OFLX), suggesting that these genes are involved in the induction of OFLX-induced chondrotoxicity. In the present study, to compare the chondrotoxic potential between new synthesized quinolones DC-159a and DX-619, and OFLX, they were orally administered by gavage at a dose level of 300 or 900 mg/kg/day to male juvenile Sprague-Dawley (SD) rats, 3 weeks of age, for 7 days. Then the distal humerus and femur were subjected to microscopic examination. Moreover, concentrations of these quinolones in the femoral articular cartilage were measured in male juvenile SD rats following a single oral administration at 100, 300 or 900 mg/kg. Furthermore, gene expression of Dusp1, Tnfrsf12a, Ptgs2, Fos, Mt1a, Plaur, Mmp3, Sstr1 and Has2 was investigated in the articular cartilage of the distal femur in male juvenile SD rats treated with 900 mg/kg of DC-159a or DX-619 by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis. In a microscopic examination, no changes in the articular cartilage were observed in any animal administered DC-159a or DX-619. On the contrary, cavity formation and chondrocyte cluster in the cartilage of distal humerus and femur were noted in animals receiving OFLX at 300 mg/kg/day or more. In toxicokinetic analysis, the maximum concentration (C(max)) value in the articular cartilage (cartilage C(max)) of DC-159a or DX-619 at 900 mg/kg was lower than that of OFLX at 300 mg/kg. However, the area under the cartilage concentration-time curve (cartilage AUC)(0-24h) value of DC-159a or DX-619 at 900 mg/kg was higher than that of OFLX at 300 mg/kg. In qRT-PCR analysis, up-regulated Dusp1, Fos and Mt1a, and down-regulated Sstr1 and Has2 genes were seen in the femoral articular cartilage of rats given DX-619 or DC-159a at 900 mg/kg. However, Tnfrsf12a, Ptgs2, Plaur and Mmp3 genes, which were up-regulated in the distal femoral articular cartilage exposed to OFLX, did not increase or slightly increased. In conclusion, the penetration of DC-159a or DX-619 into the cartilage was low compared with that of OFLX, and no obvious changes in Tnfrsf12a, Ptgs2, Plaur and Mmp3 genes were observed in the articular cartilage of juvenile rats treated with DC-159a or DX-619, which was likely to be responsible for non-chondrotoxic potentials of DC-159a and DX-619.

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats.

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

Gene expression profiles in the articular cartilage of juvenile rats receiving the quinolone antibacterial agent ofloxacin.

Quinolone antibacterial agents are extensively utilized in antimicrobial chemotherapy. However, they have been reported to induce arthropathy in juvenile animals, and the mechanism has not been clarified. In the present study, to investigate the molecular details of the chondrotoxicity of the quinolone ofloxacin (OFLX), it was orally administered by gavage at a dose level of 900 mg/kg once to male juvenile Sprague-Dawley rats, 3 weeks of age. Then gene expression profiles in the articular cartilage of the distal femur were analyzed at 2, 4, 8 and 24h post-dose. In the GeneChip analysis, the expression of 134 gene probes in the OFLX-treated group showed statistically significant differences with at least 1.5-fold difference from the control. Among them, intracellular signaling cascade- and stress response-related genes changed at 2h post-dose; cell death- and inflammatory response-related genes at 4 and 8h post-dose; basic-leucine zipper transcription factor and stress response-related genes at 8 and 24h post-dose; stress response-, proteolysis- and glycoprotein-related genes at 24h post-dose. In a quantitative real-time reverse transcription-polymerase chain reaction analysis, up-regulated Dusp1 (intracellular signaling cascade-related gene), Tnfrsf12a (cell death-related gene), Ptgs2, Fos (inflammatory response-related genes), Mt1a, Plaur (stress response-related genes) and Mmp3 (proteolysis-related gene) and down-regulated Sstr1 and Has2 (glycoprotein-related genes) were observed with dose dependency in the articular cartilage of juvenile rats treated with OFLX at 100, 300 and 900 mg/kg. The expression of Tnfrsf12a, Ptgs2, Plaur and Mmp3 was also noted in chondrocytes around the cartilage lesions by in situ hybridization. In conclusion, our results suggest that cytokines, chemokines and/or proteases produced by up-regulation of cell death-, inflammatory response-, stress response- and proteolysis-related genes play a important role in the onset of OFLX-induced chondrotoxicity in juvenile rats.

Early pathophysiological features in canine renal papillary necrosis induced by nefiracetam.

To ascertain the early pathophysiological features in canine renal papillary necrosis (RPN) caused by the neurotransmission enhancer nefiracetam, male beagle dogs were orally administered nefiracetam at 300 mg/kg/day for 4 to 7 weeks in comparison with ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), at 50 mg/kg/day for 5 weeks. During the dosing period, the animals were periodically subjected to laboratory tests, light-microscopic, immunohistochemical, and electron-microscopic examinations and/or cyclooxygenase (COX)-2 mRNA analysis. In laboratory tests, a decrease in urinary osmotic pressure and increases in urine volume and urinary lactate dehydrogenase (LDH) level were early biomarkers for detecting RPN. Light-microscopically, nefiracetam revealed epithelial swelling and degeneration in the papillary ducts in week 7, while ibuprofen displayed degeneration and necrosis in the papillary interstitium in week 5. In immunohistochemical staining with COX-2 antibody, nefiracetam elicited a positive reaction within interstitial cells around the affected epithelial cells in the papillary ducts (upper papilla) in week 7, and ibuprofen positively reacted within interstitial cells adjacent to the degenerative and/or necrotic lesions in week 5. Ultrastructurally, nefiracetam exhibited reductions of intracellular interdigitation and infoldings of epithelial cells in the papillary ducts, whereas ibuprofen showed no changes in the identical portions. Thus, the early morphological change in the papilla brought about by nefiracetam was quite different from that elicited by ibuprofen. By the renal papillary COX-2 mRNA expression analysis, nefiracetam exceedingly decreased its expression in week 4, but markedly increased it in week 7, suggesting an induction of COX-2 mRNA by renal papillary lesions. These results demonstrate that the epithelial cell in the papillary ducts is the primary target site for the onset of RPN evoked by nefiracetam.

Investigation on urinary proteins and renal mRNA expression in canine renal papillary necrosis induced by nefiracetam.

The occurrence of renal papillary necrosis (RPN), seen only in dogs after repeated oral administration of nefiracetam, a neurotransmission enhancer, at a relatively high dose, is because of inhibition of renal prostaglandin synthesis by the nefiracetam metabolite M-18. In this study, analyses of urinary proteins and renal mRNA expression were performed to investigate the possible existence of a specific protein expressing the characteristics of RPN evoked by nefiracetam. In the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary proteins from male dogs given nefiracetam at 300 mg kg(-1) day(-1) over weeks 5-11, a protein of approximately 40 kDa, which was not seen in control urine, and protein of approximately 30 kDa emerged as distinct bands. Subsequently, clusterin precursor was identified in the former band and tissue kallikrein precursor in the latter by LC-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). By quantitative real-time RT-PCR analysis with renal morphological aspects, individual findings showed that renal clusterin mRNA was increased in dogs with severe renal injury, and renal tissue kallikrein also increased, presumably related to hemodynamics. These results demonstrate that changes in renal clusterin mRNA may reflect the progression or severity of RPN, whereas upregulation of tissue kallikrein mRNA may subsequently play a compensating role in the prevention of RPN.

Structure-phototoxicity relationship in Balb/c mice treated with fluoroquinolone derivatives, followed by ultraviolet-A irradiation.

We examined the structure-phototoxicity relationship for fluoroquinolone antimicrobial agents (quinolones) using female albino Balb/c mice. First of all, to obtain an optimum dosage level for induction of phototoxicity, the prototype phototoxicant sparfloxacin was intravenously administered once at 10 mg/kg, 30 mg/kg or 100 mg/kg to female mice, followed immediately by ultraviolet-A (UVA) irradiation for 4 h (21.6J/cm2). The auricular thickness was measured at pre-dose (0 h), 4, 24, 48, 72 and 96 h post-dose, and then the histopathological examination of the auricle was performed. As results, the auricular thickness increased from 30 mg/kg, in conjunction with edema, cellular infiltration, epidermal necrosis and focal loss of the auricle. On the basis of these information, ciprofloxacin, enoxacin, fleroxacin, gatifloxacin, lomefloxacin, norfloxacin and ofloxacin were given intravenously to mice at a fixed dose of 100 mg/kg to compare their potential phototoxicities. Certain quinolones caused the auricular lesions in the following rank order (from lowest to highest): vehicle control (non-phototoxicity)=gatifloxacin=ofloxacin

Comprehensive evaluation of canine renal papillary necrosis induced by nefiracetam, a neurotransmission enhancer.

The effects of nefiracetam, a neurotransmission enhancer, on renal biochemistry and morphology with toxicokinetic disposition were investigated in both in vivo and in vitro systems. In the in vivo studies with rats, dogs, and monkeys, only the dog exhibited renal papillary necrosis. Namely, when beagle dogs were orally administered with 300 mg/kg/day of nefiracetam over 11 weeks, decreased urinary osmotic pressure was noted from week 5, followed by increases in urine volume and urinary lactate dehydrogenase from week 8. The first morphological change was necrosis of ductal epithelia in the papilla in week 8. In toxicokinetics after 3 weeks of repeated oral administration to dogs, nefiracetam showed somewhat high concentrations in serum and the renal papilla as compared with rats and monkeys. As for metabolites, although metabolite-18 (M-18) concentration in the renal papilla of dogs was between that in rats and monkeys, the concentration ratios of M-18 in the papilla to cortex and papilla to medulla were remarkably high. In the in vitro studies, while nefiracetam itself showed no effects on the synthesis of prostaglandin E2 and 6-keto-prostaglandin F1alpha, a stable metabolite of prostaglandin I2, in canine renal papillary slices, only M-18 among the metabolites clearly decreased both prostaglandin syntheses. The basal prostaglandin synthesis in canine renal papillary slices was extremely low relative to those in rats and monkeys. Taken together, certain factors such as basal prostaglandin synthesis, M-18 penetration into the renal papilla leading to an intrarenal gradient, and inhibitory potential of M-18 on prostaglandin synthesis were considered to be crucial for the occurrence of renal papillary necrosis in dogs.

Effect of nefiracetam, a neurotransmission enhancer, on primary uroepithelial cells of the canine urinary bladder.

Repeated oral treatment of dogs with a high dose of nefiracetam is reported to induce hemorrhagic lesions in the urinary bladder. To delineate its pathogenesis, we established the primary culture of uroepithelial cells of the canine urinary bladder, and then explored the effect of nefiracetam on the cultured cells. Uroepithelial cells scraped from the connective tissues of the urinary bladder of naive dogs were suspended in the minimum essential medium containing dispase, and then resuspended in the keratinocyte medium to be 6.0-7.0 x 10(5) cells/ml. Afterward, they were added to an apical chamber with a 12-mm transwell filter, cultured for three days, and recultured in the keratinocyte medium containing 1 mM CaCl(2) for 20 days. Microscopically, these cultured cells consisted of three cell layers with high transepithelial electric resistance (TER; > 10,000 ohm-cm(2)). Immunofluorescence observations revealed ZO-1 and E-cadherin bands, and electron microscopic examinations displayed the superficial cells with the assembly of tight junctions. When the effect of nefiracetam and its five main metabolites (M-3, M-10, M-11, M-18, and M-20) on TER and the ZO-1 band was assessed using cultured cells, only M-18 significantly reduced TER in the coculture for 48 h or more. Both M-10 and M-18 exhibited a deformation of uroepithelial cells and a slight reduction of the ZO-1 band from 120 h later. In conclusion, this culture system possesses both functional and morphological features of the uroepithelium reflected in vivo, and M-18 may play a pivotal role in the impairment of uroepithelial cells, leading to the onset of the urinary bladder lesion in dogs due to nefiracetam.