Kochi Kakimoto

Effects of peroxisome proliferator‐activated receptor‐α and ‐γ agonist, JTT‐501, on diabetic complications in Zucker diabetic fatty rats

This study has investigated the effects of JTT‐501, a peroxisome proliferator‐activated receptor (PPAR)‐α and PPAR‐γ agonist, on the pathogenesis of diabetic complications in the Zucker diabetic fatty (ZDF) rats, a model of type 2 diabetes. Comparison is made with troglitazone, a PPAR‐γ agonist. The ZDF rats exhibited hyperglycaemia and hyperlipidaemia, and developed diabetic complications such as cataract, nephropathy, and neuropathy. Treatment with JTT‐501 from the prediabetic stage controlled glycaemia and lipidaemia, and prevented the development of diabetic complications. Troglitazone was less effective in controlling serum cholesterol and neuropathy. ZDF rats developed diabetic osteopenia with reduced bone turnover, and this was prevented by JTT‐501 and troglitazone, possibly mediated by increased bone turnover and bone formation. Since JTT‐501 controlled glycaemia and lipidaemia in ZDF rats and prevented several diabetic complications, it is suggested that treatment with JTT‐501, which activates both PPAR‐α and PPAR‐γ, could provide a valuable therapeutic approach against diabetic complications in type 2 diabetes.

Ferric Citrate Hydrate, a New Phosphate Binder, Prevents the Complications of Secondary Hyperparathyroidism and Vascular Calcification

Background/Aims: Ferric citrate hydrate (JTT-751) is being developed as a treatment for hyperphosphatemia in chronic kidney disease patients, and shows serum phosphorus-reducing effects on hyperphosphatemia in hemodialysis patients. We examined whether JTT-751 could reduce phosphorus absorption in normal rats and prevent the progression of ectopic calcification, secondary hyperparathyroidism and bone abnormalities in chronic renal failure (CRF) rats. Methods: Normal rats were fed a diet containing 0.3, 1 or 3% JTT-751 for 7 days. The effects of JTT-751 on phosphorus absorption were evaluated with fecal and urinary phosphorus excretion. Next, a CRF model simulating hyperphosphatemia was induced by feeding rats a 0.75% adenine diet. After 21 days of starting the adenine diet feeding, 1 or 3% JTT-751 was administered for 35 days by dietary admixture. The serum phosphorus levels and mineral parameters were measured. Calcification in the aorta was examined biochemically and histopathologically. Hyperparathyroidism and bone abnormalities were evaluated by histopathological analysis of the parathyroid and femur, respectively. Results: In normal rats, JTT-751 increased fecal phosphorus excretion and reduced phosphorus absorption and urinary phosphorus excretion. In CRF rats, JTT-751 reduced serum phosphorus levels, the calcium-phosphorus product and calcium content in the aorta. Serum intact parathyroid hormone levels and the incidence and severity of parathyroid hyperplasia were also decreased. JTT-751 reduced femoral bone fibrosis, porosity and osteoid formation. Conclusions: JTT-751 could bind with phosphate in the gastrointestinal tract, increase fecal phosphorus excretion and reduce phosphorus absorption. JTT-751 could prevent the progression of ectopic calcification, secondary hyperparathyroidism and bone abnormalities in rats.

Hypothesis for the mechanism for heat-induced antigen retrieval occurring on fresh frozen sections without formalin-fixation in immunohistochemistry

The mechanism involved in heat-induced antigen retrieval (AR) remains unproven but probably utilizes the breaking of formalin-induced cross-linkages. We investigated the effectiveness of heat-induced AR on immunohistochemistry and dot-blot analysis using rat uterus tissue sections and protein extracts without formalin-fixation. The unfixed frozen sections, which did not show immunostaining with nine antibodies, were clearly stained after heating the sections. In the dot-blot analysis, the immunoblot sensitivity of detection was greatly enhanced by heating the protein-blotted membrane. These results indicate that other mechanisms of breaking formalin-induced cross-linkages may be present. We propose that one of the other mechanisms for heat-induced AR is that accessibility to the target epitopes of antigenic proteins is limited by natural steric barriers even in the fresh state caused by the antigenic protein itself.

Expression of Prolactin mRNA in Rat Mammary Gland During Pregnancy and Lactation

We studied the expression of prolactin (PRL) mRNA in the mammary gland of resting, pregnant, lactating, and weanling rats using in situ and solution reverse transcriptase-polymerase chain reaction (RT-PCR). In mid- to late pregnancy and throughout lactation, PRL mRNA was detected in both in situ and solution RT-PCR. These PRL mRNA signals were clearly identified in the cytoplasm of alveolar and ductal mammary epithelial cells by the in situ RT-PCR method. In mid- to late pregnancy, such as at the initiating point of PRL mRNA expression, we confirmed in some cases a lack of PRL mRNA by solution RT-PCR. In addition, in the early weaning phase, no signals were detected by solution RT-PCR. However, slight focal signals were detected in some poorly vacuolated cytoplasm of regressing acinar cells by in situ RT-PCR. These findings suggest that PRL mRNA in rat mammary gland begins in mid- to late pregnancy in parallel with the development of the mammary gland, continues throughout lactation, and declines in the early phase of weaning, with regression of mammary epithelial cells.

Inhibitory Effects of Anti-VEGF Antibody on the Growth and Angiogenesis of Estrogen-induced Pituitary Prolactinoma in Fischer 344 Rats: Animal Model of VEGF-targeted Therapy for Human Endocrine Tumors

Estrogen-induced pituitary prolactin-producing tumors (PRLoma) in F344 rats express a high level of vascular endothelial growth factor (VEGF) associated with marked angiogenesis and angiectasis. To investigate whether tumor development in E2-induced PRLoma is inhibited by anti-VEGF monoclonal antibody (G6-31), we evaluated tumor growth and observed the vascular structures. With simultaneous treatment with G6-31 for the latter three weeks of the 13-week period of E2 stimulation (E2+G6-31 group), the following inhibitory effects on the PRLoma were observed in the E2+G6-31 group as compared with the E2-only group. In the E2+G6-31 group, a tendency to reduction in pituitary weight was observed and significant differences were observed as (1) reductions in the Ki-67-positive anterior cells, (2) increases in TUNEL-positive anterior cells, and (3) repair of the microvessel count by CD34-immunohistochemistry. The characteristic “blood lakes” in PRLomas were improved and replaced by repaired microvascular structures on 3D observation using confocal laser scanning microscope. These inhibitory effects due to anti-VEGF antibody might be related to the autocrine/paracrine action of VEGF on the tumor cells, because VEGF and its receptor are co-expressed on the tumor cells. Thus, our results demonstrate that anti-VEGF antibody exerted inhibitory effects on pituitary tumorigenesis in well-established E2 induced PRLomas.

A novel JAK inhibitor JTE-052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents

Janus kinases (JAKs) are required for several inflammatory cytokine signalling pathways and are implicated in the pathogenesis of chronic dermatitis, including atopic dermatitis and psoriasis. JAK inhibitors are therefore promising therapeutic candidates for chronic dermatitis. In this study, we evaluated the effects of the novel JAK inhibitor JTE‐052 on inflammatory responses associated with chronic dermatitis, and compared its profile with those of conventional therapeutic agents in rodent models of chronic dermatitis. JTE‐052 inhibited the Th1‐, Th2‐ and Th17‐type inflammatory responses of human T cells and mast cells in vitro. Oral administration of JTE‐052 inhibited skin inflammation in hapten‐induced chronic dermatitis in mice, associated with reduced levels of inflammatory cytokines in the skin and immunoglobulin (Ig) E in serum. In contrast, although ciclosporin partly inhibited skin inflammation, it did not reduce interleukin (IL)‐4 production in skin, and enhanced IgE production in serum. Oral administration of JTE‐052 also inhibited skin inflammation in mouse models of atopic dermatitis and psoriasis induced by a mite extract, thymic stromal lymphopoietin or IL‐23. The maximal efficacy of JTE‐052 in these dermatitis models was superior to the conventional therapeutic agents, ciclosporin and methotrexate. Topical application of JTE‐052 ointment ameliorated hapten‐induced chronic dermatitis in rats more effectively than tacrolimus ointment. Furthermore, JTE‐052 ointment did not cause the thinning of normal skin associated with topical corticosteroids. These results indicate that JTE‐052 is a promising candidate as an anti‐inflammatory drug for various types of chronic dermatitis, with a distinctly different profile from conventional therapy following either oral or topical application.

Contribution of hyperglycemia on diabetic complications in obese type 2 diabetic SDT fatty rats: effects of SGLT inhibitor phlorizin

The spontaneously diabetic torii (SDT) fatty rat is a new model of type 2 diabetes showing overt obesity, hyperglycemia and hyperlipidemia. With early onset of diabetes mellitus, diabetic microvascular complications, including nephropathy, peripheral neuropathy and retinopathy, are observed at young ages. In the present study, blood glucose levels of female SDT fatty rats were controlled with phlorizin, a non-selective SGLT inhibitor, to examine whether and how these complications are caused by hyperglycemia. Phlorizin treatment adequately controlled plasma glucose levels during the experiment. At 29 weeks of age, urinary albumin excretion considerably increased in SDT fatty rats. Glomerulosclerosis and tubular pathological findings also indicate diabetic nephropathy. These renal parameters tended to decrease with phlorizin; however, effects were partial. Sciatic nerve conduction velocities were significantly delayed in SDT fatty rats compared with Sprague-Dawley (SD) rats. Intraepidermal nerve fiber density, an indicator of subclinical small nerve fiber neuropathy, significantly decreased in SDT fatty rats. Retinal dysfunction (prolongation of peak latency for oscillatory potential in electroretinograms) and histopathological eye abnormalities, including retinal folding and mature cataracts were also observed. Both nerve and eye disorders were prevented with phlorizin. These findings indicate that severe hyperglycemia mainly causes diabetic complications in SDT fatty rats. However, other factors, such as hyperlipidemia and hypertension, may affect diabetic nephropathy. These characteristics of diabetic complications will become helpful in evaluating new drugs for diabetic complications using SDT fatty rats.

Physiological changes induced by salt intake in female Spontaneously Diabetic Torii-Leprfa (SDT fatty) rat, a novel obese type 2 diabetic model

Salt plays an important role in the control of blood pressure in obesity and diabetes mellitus. In this study, we investigated physiological changes such as blood pressure and renal function in salt-loaded female Spontaneously Diabetic Torii-Lepr(fa) (SDT fatty) rats. SDT fatty rats were given 1% NaCl in drinking water for 14 weeks, from 4 to 18 weeks of age. Significant salt-sensitive hypertension was observed in the salt-loaded SDT fatty rats. Moreover, the salt-loaded rats showed a decrease of creatinine clearance and deterioration on pathological renal findings, including glomerulosclerosis and tubular and interstitial lesions. Female SDT fatty rat is a useful model for investigating the mechanisms of high salt sensitivity in obesity and diabetes mellitus.

Ocular Inflammation in Uveal Tract in Aged Obese Type 2 Diabetic Rats (Spontaneously Diabetic Torii Fatty Rats)

We report uveitis observed in an obese type 2 diabetes rat model, Spontaneously Diabetic Torii Leprfa (SDT fatty) rats aged over 50 weeks. The eyes of SDT fatty rats (16 animals: 7 males and 9 females with 50 or 60 weeks of age) were examined histopathologically. Infiltration of inflammatory cells in the uveal tract was observed in 13 of 16 animals. One female showed severe inflammation affecting the entire uveal tract including the iris, ciliary body, and choroid with a variety of inflammatory cells (neutrophils, lymphocytes, and macrophages). Those changes clinically mimic the findings of diabetic iridocyclitis in diabetic patients. Uveitis associated with diabetes can occur in diabetic patients but the pathogenesis still remains unknown. Since increased extramedullary hematopoiesis in the spleen and abscess in the genital and lower urinary tracts were observed in some SDT fatty rats, increased susceptibility to infection, prolongation of inflammatory states, and disorders of the immune system were considered to be possible factors of the uveitis in aged SDT fatty rats. There have been few reports on how diabetes has influence on the development of uveitis associated with bacterial infection. The SDT fatty rat can be an animal model to investigate diabetes-associated uveitis.

Effects of Unilateral Nephrectomy on Renal Function in Male Spontaneously Diabetic Torii Fatty Rats: A Novel Obese Type 2 Diabetic Model

The Spontaneously Diabetic Torii (SDT) fatty rat is a new model for obese type 2 diabetes. The aim of the present study was to investigate the effect of 1/2 nephrectomy (Nx) on renal function and morphology and on blood pressure in SDT fatty rats. Male SDT fatty rats underwent 1/2 Nx or a sham operation (Sham). Subsequently, animals were studied with respect to renal function and histological alterations. Induction of 1/2 Nx in SDT fatty rats led to functional and morphological damage to the remnant kidney and to hypertension, which are considered main characteristics of chronic kidney disease, at a younger age compared with the sham group. In conclusion, the SDT fatty rat is useful in investigations to elucidate the pathogenesis of human diabetic nephropathy and in new drug discovery.