Plasma kisspeptin levels in lactational amenorrhea.

The kisspeptin is a neuropeptide to play physiological roles in regulating gonadotropin-releasing hormone secretion in the hypothalamus. In human plasma, the kisspeptin concentration is measured, but gonadotropin-releasing hormone is not. This study aims to understand the physiological roles of the circulating kisspeptin in lactational amenorrhea in humans because prolactin reduces the kisspeptin expression and luteinizing hormone secretion resulting in anovulations in rodent brains. Plasma kisspeptin levels were measured in 11 subjects in lactational amenorrhea and in four cases with pathological amenorrhea by different etiologies for comparison using the enzyme immunoassay specific for human kisspeptin. The plasma kisspeptin levels in the 11 women with lactational amenorrhea were 15.2 ± 2.5 fmol/mL (mean ± SD) which were not significantly different as compared with 16.5 ± 4.8 fmol/mL (mean ± SD) in four age-matched women with menstrual cycles as we reported previously. In the four cases with pathological amenorrhea, their plasma kisspeptin levels were from 5.8 to 13.7 fmol/mL. This study demonstrated that the plasma kisspeptin levels were not totally reduced in lactational or pathological amenorrhea. These results suggest the physiological roles of the circulating kisspeptin are different from the role in the brain.

Effect of Posterior Decompression on the Spinal Cord of Thoracic Ossification of the Posterior Longitudinal Ligament: A Finite Element Analysis.

OBJECTIVE: Thoracic ossification of the posterior longitudinal ligament (T-OPLL) causes myelopathy. Although posterior decompression for T-OPLL has shown positive results, patients with kyphotic curvatures often endure poor outcomes. Posterior decompression with fusion (PDF) has demonstrated better results compared to posterior decompression alone. This study aims to evaluate the effects of the posterior procedures for T-OPLL. METHODS: A 3-dimensional finite element model of the C2-T12 spine, created from medical images, was used to develop the following T3-T4 OPLL compression models: an intact model (no surgery), 25% canal occupancy ratio (COR) OPLL, a discontinuous 25% COR OPLL, a continuous 50% COR OPLL, and a discontinuous 50% COR OPLL. These models were analyzed to evaluate the effects of posterior decompression (laminectomy [LN]) with varied fixation lengths (LN T3-T4, PDF T3-T4, LN T2-T5, and PDF T2-T5) in neutral, flexion, and extension positions. RESULTS: Increased discontinuity in OPLL led to increased stress on the spinal cord. Posterior decompression reduced spinal cord stress in the neutral posture. However, in flexion and extension, spinal cord stress increased for LN T3-T4, LN T2-T5, and PDF T3-T4 compared to the neutral posture. Notably, PDF T2-T5 prevented an increase in spinal cord stress during these motions. CONCLUSIONS: Effective management of intervertebral mobility and the appropriate length of decompression are crucial for addressing the thickness and mobility of T-OPLL.

A Case of Delayed Airway Stenosis Due to Retropharyngeal Hematoma Caused by Low Energy Trauma.

Airway narrowing due to trauma-induced retropharyngeal hematoma is rare. However, it is dangerous to overlook this lesion because it can lead to airway obstruction and even death. In this article, we report a case of a patient who developed pharyngeal pain and dysphagia two days after bruising on the forehead due to a fall and required intubation management. A 52-year-old man fell while walking and bruised his forehead two days before visiting our hospital. He had a sore throat and dysphagia two days after the injury and came to our hospital three days after the injury. The swelling was observed in the anterior neck, and stenotic sounds were heard in the upper airway. Cervical CT and MRI of the cervical spine showed extensive hyperabsorption areas in the ventral side of the cervical spine that appeared to be hematomas. No fracture of the cervical spine was observed. The patient has been placed on emergency tracheal intubation due to concerns about airway stenosis caused by the hematoma. Although pneumonia was observed during treatment, it resolved with antimicrobial therapy, and the hematoma tended to shrink, so the patient was extubated on the 15th day of admission. However, the patient was intubated again on the 17th day of hospitalization due to poor oxygenation. A tracheostomy was performed on the 26th day of hospitalization due to suspected narrowing of the upper airway caused by hematoma or sputum. On day 59 of hospitalization, the cannula was removed, and the patient was discharged home on the 68th day after hospitalization. Low-energy trauma tends to be underrecognized as producing anterior cervical hematomas that can lead to fatal airway narrowing. Care should be taken because fatal anterior cervical hematomas are not often part of the differential diagnosis due to their often delayed onset. More caution is needed if an underlying disease may cause coagulation abnormalities.

Usefulness of subclassification of adult diabetes mellitus among inpatients in Japan.

AIMS/INTRODUCTION: We aimed to replicate a new diabetes subclassification based on objective clinical information at admission in a diabetes educational inpatient program. We also assessed the educational outcomes for each cluster. METHODS: We included diabetes patients who participated in the educational inpatient program during 2009-2020 and had sufficient clinical information for the cluster analysis. We applied a data-driven clustering method proposed in a previous study and further evaluated the clinical characteristics of each cluster. We investigated the association between the clusters and changes in hemoglobin A1c level from the start of the education program. We also assessed the risk of re-admission for the educational program. RESULTS: We divided a total of 651 patients into five clusters. Their clinical characteristics followed the same pattern as in previous studies. The intercluster ranking of the cluster center coordinates showed strong correlation coefficients with those of the previous studies (mean ρ = 0.88). Patients classified as severe insulin-resistant diabetes (cluster 3) showed a more pronounced progression of renal dysfunction than patients classified as the other clusters. The patients classified as severe insulin-deficient diabetes (cluster 2) had the highest rate of reduction in hemoglobin A1c level from the start of the program (P < 0.01) and a tendency toward a lower risk of re-admission for the education program (hazard ratio 0.47, P = 0.09). CONCLUSION: We successfully replicated the diabetes subclassification using objective clinical information at admission for the education program. In addition, we showed that severe insulin-deficient diabetes patients tended to have better educational outcomes than patients classified as the other clusters.

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells.

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.

A case of type B insulin resistance syndrome treated with low-dose glucocorticoids.

SUMMARY: Type B insulin resistance syndrome is characterized by the presence of autoantibodies to the insulin receptor. We present a 57-year-old male admitted to a hospital due to body weight loss of 16 kg and hyperglycemia of 13.6 mmol/L. He was diagnosed with type B insulin resistance syndrome because the anti-insulin receptor antibodies were positive. We informed him that some hyperglycemic cases of this syndrome had been reported to be spontaneously remitted in 5 years, and he did not agree to be treated with high-dose glucocorticoids and/or immunosuppressive agents due to his concern for their adverse effects such as hyperglycemia and immunosuppression. He chose to be treated with insulin and voglibose, but fair glucose control could not be obtained. Six years later, he agreed to be treated with low-dose glucocorticoids practicable in outpatient settings. One milligram per day of betamethasone was tried orally and reduced gradually according to the values of glycated hemoglobin. After 30 months of glucocorticoid treatment, the anti-insulin receptor antibodies became undetectable and his fasting plasma glucose and glycated hemoglobin were normalized. This case suggests that low-dose glucocorticoids could be a choice to treat type B insulin resistance syndrome in outpatient settings. LEARNING POINTS: Type B insulin resistance syndrome is an acquired autoimmune disease for insulin receptors. This case suggested the possibility of long-lasting, low-dose glucocorticoid therapy for the syndrome as an alternative for high-dose glucocorticoids or immunosuppressive agents. Since the prevalence of autoimmune nephritis is high in the syndrome, a delay of immunosuppressive therapy initiation might result in an exacerbation of nephropathy.

Antifungal spectrum characterization and identification of strong volatile organic compounds produced by Bacillus pumilus TM-R.

To obtain biocontrol agents for suppression of food-deteriorating fungi during storage of agricultural products, bacteria producing volatile organic compounds (VOCs) with strong antifungal activity were screened and isolated from various environmental samples. Among 136 bacterial isolates, strain TM-R showed the strongest and broadest antifungal activity. Based on physiological and genetical characterization, the bacterium was identified as Bacillus pumilus. The effects of VOCs produced by the bacterium, which was grown on four types of agar media (nutrient, Trypto-Soya, Luria-Bertani, and TM Enterprise), were examined against six species of fungi (Alternaria alternata, Aspergillus niger, Cladosporium cladosporioides, Curvularia lunata, Fusarium oxysporum, and Penicillium italicum) in both small- and large-scale tests (plate and 12-L tests, respectively). In the plate test, the bacterium markedly suppressed the mycelial growth of five fungi (Alternaria alternata, Cladosporium cladosporioides, Curvularia lunata, F. oxysporum, and P. italicum) but promoted the growth of Aspergillus niger. In the 12-L test, the degree of growth inhibitiondecreased; however, the bacterium grown on TMEA still exhibited the strongest inhibition, especially against P. italicum (growth inhibition rate of 93%). Surprisingly, the growth of Aspergillus niger was promoted even more strongly (-36%) by the bacterium on TMEA than in the plate test (-9%). Twenty-two of 32 VOCs detected by GC-MS were identified using three databases (NIST 2011, AromaOffice, and AroChemBase). The species and concentration of detected VOCs differed greatly among growth media. To identify causative antifungal VOCs, we estimated the correlation between growth inhibition of P. italicum by the bacterium grown on each of the four media vs. the relative abundance of individual VOCs. As a result, four VOCs (methyl isobutyl ketone, ethanol, 5-methyl-2-heptanone, and S-(-)-2-methylbutylamine) were determined to be the predominant antifungal VOCs. To the best of our knowledge, this study is the first to specify causative antifungal VOCs using such an approach.

In vitro differentiation of Runx3-/- p53-/- gastric epithelial cells into intestinal type cells.

We have reported that a lack of RUNX3 function is causally associated with gastric carcinogenesis. We have also presented evidence that loss of Runx3 may be related to the genesis of intestinal metaplasia because expression of RUNX3 is reduced in some intestinal metaplasias, and some Runx3(-/-)p53(-/-) gastric epithelial cells differentiate into intestinal type cells in vivo. Recently several reports have indicated that blood cells play important roles in the gastric carcinogenesis. In the present study, we therefore examined whether Runx3(-/-)p53(-/-) gastric epithelial cells differentiate autonomously into intestinal type cells, or whether the presence of other cells is necessary for the differentiation in vitro. When Runx3(-/-)p53(-/-) gastric epithelial cells were cultured with collagen gels, they did not exhibit any morphogenesis and differentiated poorly. When cultured with fetal mouse gastric mesenchymes, the cells formed glandular structures and differentiated into surface mucous cells, but differentiation of intestinal type cells was never observed. When cultured with Matrigel, the cells formed glandular structures, and some cells differentiated into intestinal type cells in vitro. Reverse transcription-polymerase chain reaction analysis showed that the cells expressed stomach-specific genes, and their levels decreased gradually during the culture. The cells expressed some intestine-specific genes weakly at the start of culture, and their levels were increased with time in culture. We therefore conclude that Runx3(-/-)p53(-/-) gastric epithelial cells differentiate into intestinal type cells in combination with Matrigel in the absence of other cell types. Extracellular matrix, not blood cells, may play a role in the genesis of intestinal metaplasia.

The unified hypothesis of interactions among the bone, adipose and vascular systems: 'osteo-lipo-vascular interactions'.

Epidemiological evidence has established a link among hyperlipidemia, visceral obesity, osteoporosis, and cardiovascular diseases (CVD). We here propose the hypothesis that the associations of those disorders are based on interaction of the three organs, i.e. the bone, adipose, and vascular tissues, possibly through multiple interactions among several humoral factors and/or transcription factors. The unified hypothesis of three organs, which we call 'osteo-lipo-vascular interactions', may be explained by the common origin of the cells in each organ. The mesenchymal stem cells are capable of differentiating into osteoblasts, vascular smooth muscle cells, and adipocytes. Alternatively, macrophages may evolve into osteoclasts or infiltrate both the vascular and adipose tissues, thereby leading to chronic inflammation. This unified concept of three organs may provide insights into the development of a new drug for the treatment of osteoporosis, obesity, hyperlipidemia or CVD.

[Anesthetic management of renal transplantation for a 15-year-old girl with cerebral palsy].

A 15-year-old girl with severe cerebral palsy underwent renal transplantation. It was difficult to anastomose blood vessels because her inferior limbs were contracted. The clamp time was 67 minites. After unclamping, blood pressure fell down from 120/60 to 80/50 mmHg, and CVP fell down from 6 to 3 mmHg. First flow of urine from the donor kidney was noticed 9 hours after unclamping. We regarded difficulty of the operation for cerebral palsy and insufficient infusion as the cause of the late first flow of urine. Two weeks later, there was enough urine flow, and renal function became better. Recently, it is thougt optimal to perform renal transplantations of children who have chronic renal insufficiency and end-stage renal disease. However, there are few reports of renal transplantations for children with cerebral palsy, and there is no guideline for them. Therefore, we anesthesiologists, also have to further examine anesthetic management for each case.

Inflammation-Related Tumor Progression in Murine Fibrosarcoma Exhibited Over-expression of Sex-determining Region Y-box 2 (Sox2) Compared to Parental Regressor Cells.

BACKGROUND/AIM: Tumor progression is one of the most serious issues to overcome cancer disease. As a model of inflammation-induced tumor progression, we used the regressive murine fibrosarcoma cell clone QR-32 and the progressive malignant clone QRsP-11, that was derived from QR-32. Heat shock protein beta-1 (Hspb1) is a molecular chaperone. Hspb1 plays roles in not only cell protection but also chemo-resistance, tumorigenicity and protection from apoptosis. In a recent study, we showed that Hspb1 was up-regulated in QRsP-11 compared to QR-32. MATERIALS AND METHODS: We compared the expression levels of Hspb1, Hsf1 and Sox2 in QR-32 and QRsP-11 cells by means of western blotting. RESULTS: Hsf1, a transcription factor for Hspb1 was not increased in QRsP-11. Sex determining region Y-box 2 (Sox2) is a transcription factor, reported to interact with Hspb1. Sox2 was up-regulated in QRsP-11 compared to QR-32. CONCLUSION: These results suggest that Sox2-Hspb1 signaling is a possible pathway responsible to tumor progression of QRsP-11.

Expression and role of angiotensin II type 2 receptor in the kidney and mesangial cells of spontaneously hypertensive rats.

Angiotensin II type 2 (AT2) receptor is developmentally regulated and exerts antiproliferative and proapoptotic actions. Genetic ablation of this receptor in mice affects regulation of blood pressure, but the involvement of the AT2 receptor in the pathogenesis of hypertension remains unknown. In the present study, we examined developmental changes of angiotensin receptor subtypes in the kidney of stroke-prone spontaneously hypertensive rats (SHRSP), and compared them with those in normotensive Wistar-Kyoto rats (WKY). We also investigated the regulation and functional role of the AT2 receptor in cultured mesangial cells. Receptor binding and Northern blot analyses revealed that AT2 receptor expression is significantly lower in the SHRSP kidney than in the WKY kidney during the perinatal period, while AT1 receptor expression is not different between them. In WKY mesangial cells, AT2 receptor stimulation exerted a potent antiproliferative effect; this effect was not observed in SHRSP cells lacking the AT2 receptor expression. The expression of interferon regulatory factor (IRF)-1 paralleled the growth-dependent induction of AT2 receptor in WKY mesangial cells, and transfection of IRF-1 antisense oligonucleotide significantly suppressed AT2 receptor expression, indicating IRF-1-dependent regulation of AT2 receptor expression in mesangial cells. However, this induction was inefficient in SHRSP cells. Thus, we found impaired AT2 receptor expression in the SHRSP kidney in vivo and in mesangial cells in vitro. The unbalanced expression of renal angiotensin receptor subtypes with exaggerated AT1 receptor signaling during early life in SHRSP may play a role in the programming for hypertension and related renal injury.

Serum levels of IgG and IgG4 in Hashimoto thyroiditis.

Although IgG4-related disease is characterized by extensive infiltration of IgG4-positive plasma cells and lymphocytes of various organs, the details of this systemic disease are still unclear. We screened serum total IgG levels in the patients with Hashimoto thyroiditis (HT) to illustrate the prevalence of IgG4-related thyroiditis in HT. Twenty-four of 94 patients with HT (25.5%) had elevated serum IgG levels and their serum IgG4 was measured. Five of the 24 cases had more than 135 mg/dL of IgG4, which is the serum criterion of IgG4-related disease. One was a female patient who was initially treated as Graves' disease and rapidly developed a firm goiter and hypothyroidism. The biopsy of her thyroid gland revealed that follicular cells were atrophic with squamous metaplasia, replaced with fibrosis, which was compatible with the fibrous variant of HT. Immunohistochemical examination revealed diffuse infiltration of IgG4-positive plasma cells, and the serum IgG4 level was 179 mg/dL. The levels of IgG and IgG4 were positively correlated with the titers of anti-thyroglobulin antibody or anti-thyroid peroxidase antibody. In conclusion, at least a small portion of patients with HT with high titers of anti-thyroid antibodies may overlap the IgG4-related thyroiditis.

Integrated network systems and evolutionary developmental endocrinology.

Endocrine system has been considered to be a linear one, but the 'real world endocrine system' is a complex system, which is difficult to investigate using conventional strategies, such as single nucleotide polymorphism, genome-wide analysis, or gene targeting in animals. Here we propose a new strategy to comprehend the endocrine system as a complex network system. We introduced several novel concepts, such as complex system, network analysis, systems biology and evolutionary medicine, into the comprehension of endocrine system as a whole complex network system. This system is considered to be a scale-free network with key molecules such as acetyl CoA, NAD or ATP as 'hubs'. This system is robust against simple mutations, but various complex diseases may attack hubs. The system is also 'fractals', since there exist similar network systems among cells, proteins, and transcription factors in the lower levels, and there are similar ones among disease and social network in the higher levels. We propose to call this model 'Integrated Network Systems and Evolutionary DEvelopmental ENdocrinology (INS-EDEN)'. This novel framework will facilitate us to develop a new approach for understanding and treatment of various complex diseases related to endocrinology, and identify a unified theory of complex diseases.

Reversible posterior leukoencephalopathy in a patient with Wegener granulomatosis.

A 14-year-old girl with rapidly progressive glomerulonephritis was transferred to our hospital because of acute renal failure. A diagnosis of Wegener granulomatosis was made according to the symptom triad of a renal biopsy demonstrating crescentic glomerulonephritis, severe sinusitis, and serological findings of raised proteinase 3 anti-neutrophil cytoplasmic antibody level. In spite of combination therapy with methylprednisolone, cyclophosphamide, and plasma exchange, her renal function gradually deteriorated. Thereafter, she suffered a severe headache and generalized seizures. Brain computed tomography (CT) scan revealed bilateral low-density areas in the parieto-occipital lobes. Magnetic resonance imaging (MRI) disclosed a high-intensity area on T2-weighted images and a low-signal intensity area on T1-weighted images in the same lesion. Follow-up brain CT scan 3 weeks and MRI 2 months after the first studies showed complete resolution of the abnormal lesions, which indicated reversible posterior leukoencephalopathy syndrome. In addition to renal failure, hypertension, and cyclophoshamide, the primary disease may have played a role in the development of this uncommon syndrome in our patient.

Overexpression of brain natriuretic peptide in mice ameliorates immune-mediated renal injury.

One of major causes of end-stage renal disease is glomerulonephritis, the treatment of which remains difficult clinically. It has already been shown that transgenic mice that overexpress brain natriuretic peptide (BNP), with a potent vasorelaxing and natriuretic property, have ameliorated glomerular injury after subtotal nephrectomy. However, the role of natriuretic peptides in immune-mediated renal injury still remains unknown. Therefore, the effects of chronic excess of BNP on anti-glomerular basement membrane nephritis induced in BNP-transgenic mice (BNP-Tg) were investigated and the mechanisms how natriuretic peptides act on mesangial cells in vitro were explored. After induction of nephritis, severe albuminuria (approximately 21-fold above baseline), tissue damage, including mesangial expansion and cell proliferation, and functional deterioration developed in nontransgenic littermates. In contrast, BNP-Tg exhibited much milder albuminuria (approximately fourfold above baseline), observed only at the initial phase, and with markedly ameliorated histologic and functional changes. Up-regulation of transforming growth factor-beta (TGF-beta) and monocyte chemoattractant protein-1 (MCP-1), as well as increased phosphorylation of extracellular signal-regulated kinase (ERK), were also significantly inhibited in the kidney of BNP-Tg. In cultured mesangial cells, natriuretic peptides counteracted the effects of angiotensin II with regard to ERK phosphorylation and fibrotic action. Because angiotensin II has been shown to play a pivotal role in the progression of nephritis through induction of TGF-beta and MCP-1 that may be ERK-dependent, the protective effects of BNP are likely to be exerted, at least partly, by antagonizing the renin-angiotensin system locally. The present study opens a possibility of a novel therapeutic potential of natriuretic peptides for treating immune-mediated renal injury.

Role of prostaglandin E receptor EP1 subtype in the development of renal injury in genetically hypertensive rats.

One of the major causes of end-stage renal diseases is hypertensive renal disease, in which enhanced renal prostaglandin (PG) E2 production has been shown. PGE2, a major arachidonic acid metabolite produced in the kidney, acts on 4 receptor subtypes, EP1 through EP4, but the pathophysiological importance of the PGE2/EP subtypes in the development of hypertensive renal injury remains to be elucidated. In this study, we investigated whether an orally active EP1-selective antagonist (EP1A) prevents the progression of renal damage in stroke-prone spontaneously hypertensive rats (SHRSP), a model of human malignant hypertension. Ten-week-old SHRSP, with established hypertension but with minimal renal damage, were given EP1A or vehicle for 5 weeks. After the treatment period, vehicle-treated SHRSP showed prominent proliferative lesions in arterioles, characterized by decreased alpha-smooth muscle actin expression in multilayered vascular smooth muscle cells. Upregulation of transforming growth factor-beta expression and tubulointerstitial fibrosis were also observed in vehicle-treated SHRSP. All these changes were dramatically attenuated in EP1A-treated SHRSP. Moreover, EP1A treatment significantly inhibited both increase in urinary protein excretion and decrease in creatinine clearance but had little effect on systemic blood pressure. These findings indicate that the PGE2/EP1 signaling pathway plays a crucial role in the development of renal injury in SHRSP. This study opens a novel therapeutic potential of selective blockade of EP1 for the treatment of hypertensive renal disease.

Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.

Connective tissue growth factor (CTGF) is one of the candidate factors mediating downstream events of transforming growth factor-beta (TGF-beta), but its role in fibrogenic properties of TGF-beta and in tubulointerstitial fibrosis has not yet been clarified. Using unilateral ureteral obstruction (UUO) in rats, we analyzed gene expression of TGF-beta1, CTGF, and fibronectin. We further investigated the effect of blockade of endogenous CTGF on TGF-beta-induced fibronectin expression in cultured rat renal fibroblasts by antisense oligodeoxynucleotide (ODN) treatment. After UUO, CTGF mRNA expression in the obstructed kidney was significantly upregulated subsequent to TGF-beta1, followed by marked induction of fibronectin mRNA. By in situ hybridization, CTGF mRNA was detected mainly in the interstitial fibrotic areas and tubular epithelial cells as well as in parietal glomerular epithelial cells in the obstructed kidney. The interstitial cells expressing CTGF mRNA were also positive for alpha-smooth muscle actin. CTGF antisense ODN transfected into cultured renal fibroblasts significantly attenuated TGF-beta-stimulated upregulation of fibronectin mRNA and protein compared with control ODN transfection, together with inhibited synthesis of type I collagen. With the use of a reporter assay, rat fibronectin promoter activity was increased by 2.5-fold with stimulation by TGF-beta1, and this increase was abolished with antisense CTGF treatment. Thus CTGF plays a crucial role in fibronectin synthesis induced by TGF-beta, suggesting that CTGF blockade could be a possible therapeutic target against tubulointerstitial fibrosis.