Shunji Nakatsuji

Proliferative and Nonproliferative Lesions of the Rat and Mouse Urinary System

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the urinary tract of rats and mice. The standardized nomenclature of urinary tract lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for urinary tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Immunohistochemical Study of Rat Renal Interstitial Fibrosis Induced by Repeated Injection of Cisplatin, with Special Reference to the Kinetics of Macrophages and Myofibroblasts:

Progressive renal interstitial fibrosis occurs following tissue injury, resulting in chronic renal failure. In the fibrogenesis, macrophages are speculated to induce myofibroblasts producing matrix protein. The kinetics of these cells in renal fibrosis induced in rats by repeated injection of cisplatin (CDDP) (2 mg/kg body weight, once weekly) was investigated immunohistochemically. During the 10-wk injection period, epithelial damage of the proximal renal tubules in the corticomedullary junction was seen, followed by cystic dilation of the affected tubules. During the 8-wk recovery period following the seventh injection, the size of the dilated lumina was diminished and atrophic tubules lined by regenerating epithelial cells appeared. Morphometrical analysis revealed that fibrosis began to develop around the dilated renal tubules in the injection period and was more advanced in the following recovery period. Coinciding with development of fibrotic tissues, the number of α-smooth muscle actin-positive myofibroblasts was significantly increased in the areas compared to that of controls. In the injection period, despite a significant increase in myofibroblast number, an elevation of ED-1 (primary antibody)-positive macrophage number was not observed. In the recovery period, however, a significant elevation of macrophage number was noticed in markedly advanced interstitial fibrosis. This suggests that rapid expansion of the macrophage population, probably resulting from release from myelosuppression due to CDDP, might contribute in part to development of myofibroblasts, leading to the augmented fibrosis in the recovery period.

Comparison of the course of biomarker changes and kidney injury in a rat model of drug-induced acute kidney injury

Objective: To aid in evaluating the performance of biomarkers, we measured kidney injury biomarkers in rat models of drug-induced acute kidney injury. Methods and results: Rats were treated with site-specific nephrotoxins, puromycin, gentamicin, cisplatin, or 2-bromoethylamine. Fifteen biomarkers (β-2-microglobulin, calbindin, clusterin, cystatin-C, KIM-1, GST-α, GST-μ, NGAL, osteopontin, EGF, TIMP-1, VEGF, albumin, RPA-1, and urinary total protein) were examined in comparison with BUN, serum creatinine, and NAG. Some biomarkers, which were different depending in each nephrotoxin, showed ability to detect the prodromal stage of drug-induced kidney injury. Characteristic changing patterns of biomarkers were also found depending on the specific lesion site in the kidney. Conclusion: These data suggested that establishment of a suitable biomarker panel would facilitate detection of site-specific kidney injury with high sensitivity.

In vivo responses of macrophages and myofibroblasts in the healing following isoproterenol-induced myocardial injury in rats

To clarify the relation between macrophage and myofibroblast involvement in various myocardial diseases, the authors investigated the kinetics of these cells in the healing (scar tissue formation) following isoproterenol-induced myocardial injury in rats. Alphasmooth muscle actin (α-SMA) expressing myofibroblasts were seen at the border of the affected area and appeared in the greatest numbers on days 3–7 post-injection, followed by a gradual decrease by day 35. The peak on day 3 was consistent with the timing of the highest proliferative activity of myofibroblasts. The number of ED1-positive macrophages began to increase as early as day 1, reaching a peak on day 3 within the injured myocardium. The expansion of EDI-positive macrophages preceded an increased number of α-SMA-positive myofibroblasts suggesting that myofibroblast proliferation and activation may be mediated by factors released by ED1-positive mcrophages in response to myocardial injury. The number of ED2-positive tissue-fixed, resident macrophages gradually increased from day 3 post-injection, and peaked on day 14, but the number of ED2-positive macrophages was consistently fewer than that of ED1-positive macrophages during the 35 day-observation period after the injection. The labelling index of the ED2-positive cells was maximal on day 14, indicative of local proliferation of resident macrophages. In the healing process after myocardial injury, EDI-positive macrophages increase markedly in the early stages; ED2-positive macrophages appear later.

Immunohistochemical Analysis of Macrophages, Myofibroblasts, and Transforming Growth Factor-β Localization during Rat Renal Interstitial Fibrosis Following Long-Term Unilateral Ureteral Obstruction

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-β (TGF-β, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of α-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-β was found in infiltrating macrophages or myofibroblasts. However, TGF-β was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-β may be renal epithelia. However, the staining intensity for TGF-β on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-β to the advanced stages of UUO-induced renal fibrosis remains to be determined.

Characteristics of a rat fibrosarcoma-derived transplantable tumour line (SS) and cultured cell lines (SS-P and SS-A3-1) showing myofibroblastic and histiocytic phenotypes.

Abstract A transplantable tumour line (SS) was established in syngeneic rats from a spontaneous fibrosarcoma that had arisen in the submandibular salivary gland of a 24-month-old male F344 rat. A cell line (SS-P) was induced from SS, and a cloned cell line (SS-A3-1) was isolated from SS-P. The primary tumour consisted of oval to spindle-shaped cells arranged in bundles with abundant collagen fibres; ultrastructurally, neoplastic cells exhibited fusiform morphology with prominent rough endoplasmic reticulum. SS tumours showed marked interlacing fascicle and herring-bone growth patterns. SS-P and SS-A3-1 were simmilar morphologically to each other, consisting of oval, spindle or polygonal cells and occasional multinucleated giant cells. Tumours induced by SS-P and SS-A3-1 were histologically similar to SS tumours. Immunohistochemically, all cells in the primary tumour, SS tumours and tumours induced both by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures gave a positive reaction to vimentin. Interestingly, neoplastic cells reacting to ED1 (rat macrophage/histiocyte-specific antibody) and α-smooth muscle actin (α-SMA) appeared in SS tumours and tumours induced by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures. Cells with histiocytic fine structures and myofibroblastic cells with cytoplasmic actin-like microfilaments were also observed by electron microscopy. The present rat fibrosarcoma-derived transplantable tumour line (SS) and cell lines (SS-P and SS-A3-1) might express myofibroblastic and histiocytic phenotypes, probably depending on the surrounding conditions. These cell lines may prove useful for studying the mechanisms of phenotypic plasticity in neoplastic fibroblasts.

Abdominal cysticercosis in a cynomolgus monkey.

Cysticercus tenuicollis, the larval form of Taenia hydatigena, was observed in a 5-year-old male cynomolgus monkey used in a toxicity study for a safety assessment of a pharmaceutical. The animal was born and raised in a primate colony in China. A pale yellow cyst filled with more than 100ml of pale yellow fluid was found in the abdominal cavity in the autopsy. The cyst was found attached to the greater omentum, and it was double layered. Histopathologically, the outer layer was a part of the greater omentum, and the inner layer was the bladder wall of a cysticercus with a well developed scolex. A partial sequence of mitochondrial NADH dehydrogenase subunit 1 showed a high homology to the same region of Taenia hydatigena (1.5-3.3%).

Lysozyme-Containing Renal Tubular Hyaline Droplets in F344 Rats Bearing a Rat Fibrosarcoma-Derived Transplantable Tumor

Renal tubular hyaline droplets developed in male and female F344 rats bearing a rat fibrosarcoma-derived transplantable tumor (SS). The droplets accumulated exclusively in the proximal renal tubular epithelia as eosinophilic granules of various sizes in hematoxylin and eosin-stained sections. The granules stained bright red with azan-Mallory stain. Immunohistochemically, the droplets were positive for lysozyme to various degrees but were negative for a2u-globulin, albumin, and a 1-antitrypsin. These findings indicated the involvement of lysozyme, a low-molecular-weight protein, in the droplet formation. The morphological and immunohistochemical findings of the hyaline droplets bore a close resemblance to those reported in rats as a secondary lesion to spontaneous histiocytic sarcomas. Others have speculated that renal tubular hyaline droplets in histiocytic sarcoma-bearing rats are formed in lysosomes through cellular overload of lysozyme secreted excessively by the tumor cells. However, neoplastic cells of SS tumors were negative to lysozyme. The pathogenesis of renal hyaline droplets appearing in SS tumor-bearing rats remains to be investigated.

Altered gene expression profile in ovarian follicle in rats treated with indomethacin and RU486

It is well-known that indomethacin (the cyclooxygenase 1 & 2 inhibitor) and RU486 (or mifepristone, the progesterone receptor antagonist) block follicular rupture in rats. To characterize genetic alterations in unruptured follicles, gene expression profiles in ovarian follicle were analyzed in indomethacin- and RU486-treated female Sprague-Dawley rats. Ovaries are collected at 22:00 on the proestrus day and 10:00 on the following estrus day after a single dose of indomethacin and RU486. Histopathologically, changes depicting responses to LH surge were observed in ovaries, uteri and vagina. Total RNA was extracted from pre-ovulatory follicles or unruptured follicles collected by laser microdissection and analyzed by Genechip(®). Among genes showing statistically significant changes compared to control groups, following changes were considered relevant to induction of unruptured follicles. In indomethacin-treated rats, Wnt4 was down-regulated, suggesting effect on tissue integrity and steroid genesis. In RU486-treated rats, Adamts1, Adamts9, Edn2, Ednra, Lyve1, Plat, and Pparg were down-regulated. These changes suggest effects on proteolysis for extra cellular matrix or surrounding tissue (Adamts1 & 9, and Plat), constriction of smooth muscle surrounding follicles (Edn2, Ednra, and Pparg), follicular fluid (Lyve1), and angiogenesis (Pparg). Down-regulation of angiogenesis related genes (Angpt2, Hmox1, and Vegfa) was observed in both treatment groups. Here, we clarify genetic alterations induced by the inhibition of cyclooxygenase or progesterone receptor.

Transitional Gene Expression Profiling in Ovarian Follicle during Ovulation in Normal-Cycle Rats

Evaluation of ovarian toxicity requires an understanding of the physiological changes related to the estrous cycle in the ovary. The authors investigated the transitional gene expression profile of ovulatory follicles in rats that show normal estrous cyclicity. Ovaries were collected at 10:00 and 22:00 on the proestrus day and at 10:00 on the estrus day. Ovarian follicles or early corpora lutea were isolated using laser microdissection, and extracted total RNA was analyzed using microarray technology. Clustering analysis revealed four different expression patterns: transient up- or down-regulation only at 22:00 on the proestrus day (pattern 1), up- or down-regulation only at 10:00 on the estrus day (pattern 2), continuous increase at 22:00 on the proestrus day and at 10:00 on the estrus day (pattern 3), and up- or down-regulation at 22:00 on the proestrus day and level maintenance at 10:00 on the estrus day (pattern 4). In addition, these probe sets were functionally categorized in each pattern using the Ingenuity Pathways Analysis database. These data will aid in understanding the physiology of ovulation and may be useful in assessing ovarian toxicity and its mechanism, such as in investigations of chemical-induced ovulatory impairment.