Comparison of changes in urinary and blood levels of biomarkers associated with proximal tubular injury in rat models.

To investigate useful biomarkers associated with proximal tubular injury, we assessed changes in levels of a focused set of biomarkers in urine and blood. Male rats administered a single dose or four doses of gentamicin (GM, 240 mg/kg/day) or a single dose of cisplatin (CDDP, 5 mg/kg) were euthanized on days 2 (the day after initial dosing) 5, or 12. At each time point, histopathological examination of the kidney and immunohistochemistry for biomarkers, kidney injury molecule-1 (Kim-1), lipocalin (NGAL), clusterin (CLU), cystatin C (CysC) and ß2-microglobulin (ß2M) were performed. Biomarker levels were measured in urine and blood. In both treatment groups, degenerated/necrotic proximal tubules and regenerated tubules were mainly observed on days 5 and 12, respectively. At the same time as these tubular injuries, urinary Kim-1, CysC and ß2M levels were increased. Moreover, urinary levels of CysC and ß2M in GM-treated animals and Kim-1 in CDDP-treated animals increased (on day 2) prior to tubular injury on day 5. This was considered to reflect the characteristics of drug toxicity. Although almost all of the biomarkers in blood were not sufficiently sensitive to detect proximal tubular injury, urinary and plasma ß2M levels simultaneously increased. Therefore, in addition to urinary Kim-1, CysC and ß2M levels, plasma ß2M levels were also considered useful for detecting proximal tubular injury.

Th2 cytokine-mediated methimazole-induced acute liver injury in mice.

Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical practice. The pathogenesis of DILI usually involves the participation of the parent drug or metabolites that either affect cellular function or elicit an immune response. However, the mechanisms leading to DILI are unknown in most cases. Methimazole (MTZ) is used as an antithyroid drug and is well known to have induced liver injuries such as cholestatic hepatitis in a small number of human cases. Immune-mediated reactions were also suggested to play a role in MTZ-induced acute liver injury, but the mechanism underlying this process has not been elucidated. To address this issue, we measured plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, hepatic glutathione levels, hepatic expression of CD4⁺ Th cell-related transcriptional factors, cytokines and chemokines, plasma interleukin (IL)-4 levels and histopathological changes in the liver following MTZ (450 mg kg⁻¹ , p.o.) administration in mice. The hepatic expression of mRNA for Th2 cell-related factors, such as GATA-binding protein, macrophage inflammatory protein-2 (MIP-2) and plasma IL-4 levels, as well as plasma AST and ALT levels, was significantly increased in mice treated with MTZ. These changes were markedly enhanced by pre-treatment with L-buthionine sulfoximine (3 mmol kg⁻¹, i.p.) and MTZ (15 mg kg⁻¹, p.o.). Neutralization of IL-4 using a monoclonal anti-mouse IL-4 antibody (100 µg/mouse, single i.p.) suppressed the hepatotoxic effect of MTZ. In conclusion, this report is the first to demonstrate that Th2 cytokine-mediated immune responses are involved in MTZ-induced acute liver injury in mice.

Diet modification and its influence on metabolic and related pathological alterations in the SHR/NDmcr-cp rat, an animal model of the metabolic syndrome.

SHR/NDmcr-cp (SHR/NDcp) rats, which carry a nonsense mutation of the leptin receptor gene, are known to spontaneously develop hypertension, obesity and hyperlipidemia, and have therefore found use as an animal model of the metabolic syndrome and type 2 diabetes. However, some recent studies on SHR/NDcp rats revealed only mild elevation of blood glucose levels. To investigate whether metabolic factors including blood glucose and histopathological alterations of SHR/NDcp rats deteriorate with a diabetogenic diet, biochemical and histopathological examinations were conducted with animals fed normal or diabetogenic diets for 20 weeks. SHR/NDcp rats receiving the normal diet displayed obesity, hypertension, hyperlipidemia, and mild elevation of blood glucose and HbA1c levels. Urinary glucose excretion was noted in only 1 out of 6 animals. Histologically, macro- and micro-vesicular steatosis in the liver, glomerular and tubular damages in the kidney and islet hyperplasia mainly of beta cells in the pancreas were characteristically noted. In SHR/NDcp rats fed the diabetogenic diet, obesity was more severe, with higher blood glucose and HbA1c levels, increased numbers of animals with urinary glucose excretion, and more pronounced hepatic steatosis and renal tubular changes. However, elevation of blood glucose levels and urinary glucose excretion proved transient. These observations indicate that the diabetic state and associated histopathological alterations in SHR/NDcp rats are exacerbated by feeding a diabetogenic diet, but the effects are limited. Elevated islet function with compensative insulin secretion might be related to amelioration of the hyperglycemic state. Further diet modification could be needed to induce a more prominent and persistent diabetic state in SHR/NDcp rats.

A Hepatocellular Adenoma in a Diet-induced Obese Mouse.

A hepatic nodule was noted in a C57BL/6J mouse with diet-induced obesity at 53 weeks of age. Macroscopically, a protruding yellowish white nodule was observed on the visceral surface of the left lateral lobe. Light microscopy demonstrated clear demarcation from the compressed adjacent parenchyma, with loss of the distinct lobular pattern. The proliferating cells of the lesion varied in shape and showed cellular atypia and prominent nucleoli along with vacuoles of various sizes. Some of the cells contained various-sized eosinophilic inclusion bodies in their cytoplasm, and electron microscopy revealed the presence of lipid droplets in the rough endoplasmic reticulum. Eosinophilic inclusions were observed as electron dense granular material in the rough endoplasmic reticulum, with one or a few low density central cores. A diagnosis of hepatocellular adenoma was made based on these findings.

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).

Urinary metabolic fingerprinting for alpha-naphthylisothiocyanate-induced intrahepatic cholestasis in rats using Fourier transform-ion cyclotron resonance mass spectrometry.

Urinary metabolic fingerprinting with Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) was performed to monitor metabolic changes in an alpha-naphthylisothiocyanate (ANIT)-induced rat model of intrahepatic cholestasis and to investigate the relationships among metabolic changes, histopathology, and blood chemistry. ANIT was administered orally as a single dose of 100 mg/kg. Urine samples were collected predose (-31 to -24 hours) and postdose at 0-7, 7-24, 24-31, 31-48, 48-55, 55-72, and 72-96 hours, and serum samples were collected on days 1, 2, and 4 postdose. Increased levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin were seen on day 2. The negative ion profiles for urine samples collected after 7-24, 24-31, 31-48, and 48-55 hours differed from the predose profile based on principal component analysis. Onset of recovery was observed after 24-31 hours, when the urinary composition reverted toward the predose position. In conclusion, it is possible to monitor the progression of and recovery from drug-induced hepatotoxicity by urinary metabolic fingerprinting with FT-ICR MS and to search for potential biomarkers involved in intrahepatic cholestasis.

Urinary metabolic fingerprinting for thioacetamide-induced rat acute hepatic injury using fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS), with reference to detection of potential biomarkers for hepatotoxicity.

In this study, we performed urinary metabolic fingerprinting using Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) in the thioacetamide (TAA)-induced rat model of acute hepatic injury to search for useful biomarkers involved in the acute hepatic toxicity. TAA was intraperitonealy administered a single dose of 300 mg/kg, and urine sample and livers were collected on predose, and days 1, 3, 5, and 7 postdose (Days 1, 3, 5, and 7). Histopathologically, infiltration of macrophages occurred in the TAA-induced centrilobular injured area on Days 1 and 3, and the injured liver recovered on Days 5 and 7. On the scores plot of principal component analysis (PCA), the ion profiles of Days 1 and 3 were different from those of the predose, Days 5 and 7. The loading plot revealed that the metabolites causing PCA results were m/z 266.05390, 401.20737, and 429.23882. The ion at m/z 266.05390 was identified as a potassium ion adduct of deoxycytidine (dCyt). Because the appearance of urinary dCyt was corresponding to macrophage infiltration in the rat-injured liver, it was considered that the urinary dCyt might be released from infiltrated macrophages. dCty might be a biomarker for the acute hepatotoxicity in rats.

Systemic candidiasis in a dog, developing spondylitis.

A 4-year-old male Shiba dog initially presented with pain of an undetermined origin and hypersensitivity to touch. Seven days later, the dog developed ataxia, hind-leg weakness and knuckling. The dog died on 20 days after presentation. Postmortem examination revealed a mass in the body of thoracic vertebra. Histopathologically, the mass consisted of granulomatous inflammation, including fungal organisms that were immunohistochemically positive for Candida albicans. Similar granulomatous lesions were observed in the systemic lymph nodes, kidneys, pancreas, spleen, prostate gland, thyroid glands and heart. This case was diagnosed as systemic candidiasis with spondylitis.

Cisplatin-induced renal interstitial fibrosis in neonatal rats, developing as solitary nephron unit lesions.

Cisplatin (CDDP)-induced renal lesions in rats prove a useful model for analysis of the pathogenesis of post-tubular injury-renal interstitial fibrosis. This study investigated the histopathological changes in 10-day-old neonatal rats induced by a single injection of CDDP (4.5 mg/kg). Compared with age-matched controls, on postinjection (PI) days 1 to 6, the number of apoptotic cells, demonstrable with TUNEL method, was significantly increased in CDDP-treated neonates, and there was no marked epithelial necrosis nor fibrotic lesions. Fibrotic lesions began to be developed solitarily around some nephrons with dilated ducts in the corticomedullary junction on PI day 10 and the lesions became more prominent until PI day 20. The alpha-SMA-positive myofibroblastic cells were seen exclusively in the fibrotic lesions. Additionally, the numbers of macrophages reacting with EDI (specific for exudate macrophages), ED2 (for resident macrophages), and OX6 (recognizing MHC class II antigens expressed in antigen-presenting macrophages/dendritic cells) were significantly increased around the affected renal tubules. A greater immunoreaction for TGF-beta1 was seen mostly in the renal epithelial cells of CDDP-treated neonates. These findings indicated that macrophage populations and myofibrolastic cells as well as TGF-beta1 may be responsible for the production of neonatal renal interstitial fibrosis. Compared with CDDP-injected adult rats that develop extensive interstitial fibrosis (Yamate et al., J Comp Pathol, 1995), the formation of fibrotic lesions was delayed, and the lesions were limited to the area around the affected nephrons; this could be attributable to differences in renal morphology between neonates and mature kidney of adult rats.

Effects of lipopolysaccharide on the appearance of macrophage populations and fibrogenesis in cisplatin-induced rat renal injury.

Macrophages play an important role in renal interstitial fibrosis via production of transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alpha (TNF-alpha); these fibrogenic factors mediate induction of myofibroblastic cells capable of producing extracellular matrices. We investigated the effects of lipopolysaccharide (LPS), a macrophage activator, on the appearance of macrophage populations and subsequent fibrogenesis in cisplatin (CDDP)-induced rat renal lesions. In keeping with the progression of interstitial fibrosis, alpha-smooth muscle actin (alpha-SMA)-immunopositive myofibroblastic cell number began to increase on day 4 and continued gradually until day 16 after CDDP injection. Cells immunoreactive for ED1 (for exudate macrophages), ED2 (for resident macrophages) and ED3 (for activated resident macrophages) showed the highest number on day 4 or day 7, and thereafter, the numbers were gradually decreased up to day 16. On the other hand, the number of cells immunoreactive for OX6 (rat MHC class II-recognizing antibody) was increased on day 7 and remained elevated up to day 16. LPS was injected on day 7 after CDDP injection when the greatest number of ED1-positive macrophages were present. In CDDP/LPS-injected rats, the numbers of macrophages reacting to ED1, ED2, ED3, and OX6 were higher than those in CDDP-injected rats during the observation period between days 7 and 16; ED3- and OX6-positive cells were more prominently increased than ED1- and ED2-postive cells. By RT-PCR analysis, the expression of TGF-beta1 and TNF-alpha mRNAs in CDDP/LPS-injected rats on day 7 was markedly increased in contrast to those in CDDP-injected rats. These findings indicate that LPS treatment enhanced the macrophage expression of fibrogenic factors. However, there was no marked difference in the fibrogenesis between CDDP/LPS- and CDDP-injected rats. These findings suggest that the macrophage populations appearing in CDDP-induced rat renal lesions should be investigated further, to address the complicated pathogenesis of renal interstitial fibrosis.

Establishment of a transplantable rat pulmonary carcinoma-derived cell line (IP-B12) as a new model of humoral hypercalcemia of malignancy and bone metastasis.

A cloned cell line (IP-B12) derived from a transplantable rat pulmonary carcinoma (IP), of which neoplastic cells produce parathyroid hormone-related protein (PTHrP), was established. Tumors induced in syngeneic F344 rats by intraperitoneal injection of IP-B12 cells had features of pulmonary adenocarcinomas, consisting of neoplastic cells immunopositive to PTHrP. The IP-B12 tumor-bearing rats developed severe emaciation and hypercalcemia, with a marked elevation of plasma PTHrP level; there was an increase in osteoclastic areas of the femur and calcium depositions in systemic organs, indicating progression to humoral hypercalcemia of malignancy (HHM) in the tumor-bearing rats. In addition, the injection of IP-B12 cells into the left cardiac ventricle of syngeneic rats resulted in osteolytic skeletal metastases in the long bones and vertebrae. In the metastatic lesions, histologically, neoplastic cells showed an immunopositive reaction to PTHrP, and a prominent osteoclastic activity was seen; bone lesions, including osteolysis, fracture, and nerve compression as well as replacement of bone marrow cells by proliferated tumor cells were similar to those reported in human cancer patients with bone metastases. IP-B12 is a new animal model for HHM and osteolytic bone metastases, and will become a useful tool for studies on the pathogenesis and therapeutic strategies for such conditions.

Immunophenotypical changes of neoplastic cells and tumor-associated macrophages in a rat dendritic cell sarcoma-derived transplantable tumor line (KB-D8).

Basically, dendritic cell-derived sarcomas are characterized by expression of major histocompatibility complex class-II molecules, but the biological properties of the tumor cells remain to be elucidated. Recently, we established a novel transplantable cell line (KB-D8) from a dendritic cell sarcoma found in an F344 rat. In the present study, we investigated immunophenotypical changes of KB-D8 tumor cells and tumor-associated macrophages (TAMs) appearing in relation to tumor development in syngeneic F344 rats. A number of neoplastic cells in 0.5-cm-diameter KB-D8 tumors showed immunoreactions to OX6 (specific for rat antigen-presenting cells), ED1 (for rat exudate macrophages), and ED2 (for rat resident macrophages), and 72% and 11% of the OX6+ cells were double-immunostained with ED1 and ED2, respectively. Interestingly, the immunoreactions to these antibodies were gradually reduced with increasing size of KB-D8 tumors of 1-, 2-, and 3-cm diameter. These findings indicated that immunophenotypes of dendritic cell-derived sarcomas may be changeable depending on microenvironmental conditions in vivo. Many TAMs seen outside KB-D8 tumors reacted to OX6, ED1, and ED2; the numbers of TAMs immunopositive for these antibodies also decreased as the tumor grew. Similarly, the earlier temporary increase and subsequent gradual decrease in ED2+ and OX6+ cell numbers were observed in the spleen and liver of KB-D8-bearing rats. The reverse-transcription polymerase chain reaction showed mRNA expressions of granulocyte/macrophage-colony stimulating factor, monocyte-chemoattractant protein-1, and osteopontin in KB-D8 tumor tissues. Although the functional roles (biphasic roles: suppressing or promoting) of these factors should be investigated further in relation to tumor development, the factors might be partially responsible for the TAM reactions. KB-D8 would be a useful experimental model to investigate the biological characteristics of dendritic cell sarcomas and tumor immunology in the host.

Establishment and characterization of transplantable tumor line (KB) and cell lines (KB-P and KB-D8) from a rat thymus-derived dendritic cell sarcoma.

A transplantable tumor line (KB) was established in syngeneic rats from a naturally occurring sarcoma that had arisen in the thymus of a 24-month-old male F344 rat. Further, a cell line (KB-P) was induced from KB and a cloned cell line (KB-D8) was isolated from KB-P. The primary thymic tumor and KB tumors showed heterogeneous histological growth patterns such as sheet-like, ill-defined bundle, fascicular and interwoven fashions, consisting of spindle cells, oval cells and histiocytic large round cells. Immunohistochemically, neoplastic cells in KB tumors and KB-P and KB-D8 cultures reacted to vimentin and were labeled with antibodies of OX6 (for rat major histocompatibility complex class-II antigens), ED5 (for rat follicular dendritic cells; FDCs) and RED-1 (for interdigitating dendritic cells) in varying degrees, indicating that neoplastic cells exhibited the immunophenotypes of rat dendritic cells. In addition, neoplastic cells were immunoreactive to ED1 (for rat monocytes/macrophages) and ED2 (for rat tissue macrophages), and also showed positive reactions to histiocytic lysosomal enzymes such as acid phosphatase and non-specific esterase. Ultrastructurally, neoplastic cells had cell surface projections, cisterna-like structures and variously developed lysosomes in the cytoplasm. Based on these findings, the present tumor was regarded as dendritic cell-derived sarcoma capable of expressing macrophage-like and histiocytic nature. A reverse-transcription polymerase chain reaction method revealed that the addition of lipopolysaccharide dose dependently increased the expression of mRNA of transforming growth factor-beta1, a proinflammatory factor, in KB-D8 cells. The transplantable line (KB) and cell lines (KB-P and KB-D8) may become useful tools for studying the histogenesis and pathobiological functions of dendritic cells.

Establishment of a transplantable tumor line (IP) derived from rat pulmonary carcinoma, developing humoral hypercalcemia of malignancy in IP-bearing rats.

A transplantable tumor line (IP) was established in syngeneic rats from a spontaneous pulmonary carcinoma found in a male F344 rat aged 25 months. A tissue fragment of IP grew into a nodule, 2-3 cm in diameter, 2-3 weeks after implant, and IP has been serially passed through 26 generations. Lined by cuboidal or columnar epithelial cells, the primary tumor consisted of completely alveolar architecture. However, IP tumors developed various growth patterns such as glandular, acinar, trabecular, cord, and solid, and consisting of epithelial cells showing cellular atypia. Ultrastructurally, neoplastic cells had microvilli, basement membranes, and desmosomes, and occasional cells possessed dense cytoplasmic granules. Interestingly, it was shown by the immunohistochemistry, the RT-PCR method, and immunoradiometric assay that IP tumor cells produce parathyroid hormone-related protein (PTHrP). During a 3-week observation period after implant, IP-bearing rats showed severe emaciation, hypercalcemia, and hypophosphatemia, as well as an increase in osteoclastic areas and a decrease in shaft thickness of the femurs. These were considered to be due to a marked elevation of plasma PTHrP levels. Furthermore, IP-bearing rats developed calcification in various organs including the kidneys, lungs, and heart. These findings in IP-bearing rats were similar to those of humoral hypercalcemia of malignancy (HHM) reported in human cancer patients. PTHrP plays a central role in the development of HHM, but the mechanisms of HHM remain poorly understood. IP may become a useful model for studying the pathogenesis of HHM and the pathophysiological role of PTHrP.