Takehiko Koji

In situ hybridization of interleukin 6 in diabetic nephropathy

Increased mesangial expansion is one of the most characteristic histological changes in diabetic nephropathy (DN). Although the pathogenesis of DN remains unclear, recent studies associate interleukin (IL) 6 with mesangial proliferative glomerulonephritis. To elucidate the expression and localization of IL-6 mRNA in renal tissues of patients with DN, a high-resolution in situ hybridization using digoxigenin-labeled oligonucleotide was performed. Patients were divided into three groups based on light microscopy findings: mild (group 1), moderate (group 2), and severe (group 3) mesangial expansion. The relationship between the expression of IL-6 mRNA and the degree of glomerular mesangial expansion in DN was examined. Individual cells positive for IL-6 mRNA were observed in glomeruli. These cells were mesangial cells, glomerular epithelial cells, and Bowmans capsule. The signal intensity was strongest in tissues from group 2 but was weak in those from groups 1 and 3. Most cells in the area of mesangial proliferation were strongly stained for IL-6 mRNA, and few positive cells were found in the Kimmelstiel-Wilson nodular lesion. In the interstitium, some tubules, particularly atrophic tubules, and some infiltrating cells were positively stained for IL-6 mRNA. The interstitial expression of IL-6 mRNA correlated significantly with the degree of interstitial injury and was remarkable in tissues from groups 2 and 3. We conclude that IL-6 mRNA is expressed by glomerular resident cells and interstitial cells in the renal tissue of patients with DN and that its expression may be associated with mesangial proliferation and may be involved in the tissue injury of DN.

17β-Estradiol Protects against Oxidative Stress-induced Cell Death through the Glutathione/Glutaredoxin-dependent Redox Regulation of Akt in Myocardiac H9c2 Cells

The GSH/glutaredoxin (GRX) system is involved in the redox regulation of certain enzyme activities, and this system protects cells from H2O2-induced apoptosis by regulating the redox state of Akt (Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003) J. Biol. Chem. 278, 50226–50233). Estrogens, such as 17β-estradiol (E2), play an important role in development, growth, and differentiation and appear to have protective effects on oxidative stress mediated by estrogen receptor α (ERα). However, the role of the ERβ-mediated pathway in this cytoprotection and the involvement of E2 in the redox regulation are not well understood. In the present study, we demonstrated that E2 protected cardiac H9c2 cells, expressing ERβ from H2O2-induced apoptosis concomitant with an increase in the activity of Akt. E2 induced the expression of glutaredoxin (GRX) as well as γ-glutamylcysteine synthetase, a rate-limiting enzyme for the synthesis of GSH. Inhibitors for both γ-glutamylcysteine synthetase and GRX and ICI182,780, a specific inhibitor of ERs, abolished the protective effect of E2 on cell survival as well as the activity of Akt, suggesting that ERβ is involved in the cytoprotection and redox regulation by E2. Transcription of the GRX gene was enhanced by E2. The promoter activity of GRX was up-regulated by an ERβ-dependent element. These results suggest that the GRX/GSH system is involved in the cytoprotective and genomic effects of E2 on the redox state of Akt, a pathway that is mediated, at least in part, by ERβ. This mechanism may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy.

Transcription switch of two phosphoglycerate kinase genes during spermatogenesis as determined with mouse testis sections in situ

In order to elucidate the mechanistic interpretations underlying differential expression of the two phosphoglycerate kinase (PGK) genes during mammalian spermatogenesis, localization of its mRNAs in mouse testis sections was determined by in situ hybridization. MRNA for nonsperm-type PGK-1 was identified in nongerminal Leydig and Sertoli cells, spermatogonia, and spermatocytes, but was not detected in spermatids. In contrast, mRNA for sperm-type PGK-2 was notable in leptotene spermatocytes, becoming most abundant in pachytene spermatocytes. It was amply present in spermatids only up to step 10, completely disappearing after step 12. It is possible to assume that a transcription switch of the two PGK genes ensued following the onset of meiosis. These findings taken together with previous observations indicate that differential expression of the two PGK genes during mammalian spermatogenesis is regulated at the transcriptional and post-transcriptional levels.

Detection of nuclear factor-κB in IgA nephropathy using Southwestern histochemistry

BACKGROUNDnThe transcription factor nuclear factor-kappaB (NF-kappaB) is involved in inflammatory and immune responses through induction of various cytokines and growth factors. The aim of this study is to examine the correlation between NF-kappaB expression and severity of tissue injury in immunoglobulin A (IgA) nephropathy and the mechanism of such correlation.nnnMETHODSnThe study included 43 renal tissue samples from 28 patients, including 28 samples of IgA nephropathy, 5 samples of non-IgA mesangial proliferative glomerulonephritis (non-IgA nephropathy), and 10 samples with nonproliferative glomerulonephritis (membranous nephropathy [MN] n = 5; minimal change nephrotic syndrome [MCNS]; n = 5). Tissue sections were examined by Southwestern histochemistry and immunohistochemistry for monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and intercellular cell adhesion molecule-1 (ICAM-1), which are regulated by NF-kappaB. Normal portions of surgically resected kidney with adenocarcinoma served as controls.nnnRESULTSnIn normal kidney, MCNS, and MN sections, NF-kappaB expression was detected in a few mesangial cells and tubular epithelial cells. In IgA nephropathy and non-IgA nephropathy samples, NF-kappaB was expressed in mesangial, glomerular endothelial and epithelial cells, tubular epithelial cells, and infiltrating cells. Expression in both glomeruli and interstitium correlated with progression of tissue injury. In IgA nephropathy samples, MCP-1 and GM-CSF expression was increased in both glomeruli and interstitium and correlated with progression of tissue injury. Glomerular ICAM-1 expression was weaker in severe lesions, whereas interstitial expression correlated with progression of tissue injury.nnnCONCLUSIONnOur results indicate that NF-kappaB is involved in the progression of tissue injury in IgA nephropathy through the induction of transcriptionally regulated genes.

Localizationin situ ofc-myc mRNA andc-myc protein in adult mouse testis

SummaryIt has been suggested that c-myc, one of the proto-oncogenes, plays a role in normal somatic cell proliferation and differentiation. To define whether c-myc is only expressed during somatic cell division or is also expressed during meiotic cell division, the production of c-myc mRNA and protein were investigated in the mouse testis by usingin situ hybridization with non-radioactive DNA probes and enzyme immunohistochemistry respectively. Forin situ hybridization, T-T dimerized DNA probes were used and DNAs hybridizedin situ were detected immunohistochemically using specific antibody against T-T dimer. The results indicate that c-myc mRNA and protein are expressed in a cell-cycle-dependent manner only in spermatogonia and not in spermatocytes and spermatids.

Intraglomerular synthesis of complement C3 and its activation products in IgA nephropathy

Background: Complement activation is thought to be pathologically important in IgA nephropathy (IgAN). Although C3 deposition in the mesangium is found in IgAN, the origin of C3 is not clear. We recently demonstrated intraglomerular C3 synthesis in the human kidney; however, the activation and pathological role of locally synthesized C3 remains unclear. Here we performed nonradioactive in situ hybridization for C3 mRNA and immunohistochemistry for C3 and its activation products, such as C3d and membrane attack complex (MAC), to determine whether locally produced C3 in glomeruli was activated in IgA nephropathy. Methods: Renal samples from 14 patients with IgAN and 5 with minimal change nephrotic syndrome (MCNS) were examined. Uninvolved portions of surgically removed kidneys with tumors served as normal controls. Results: C3 mRNA was not detected in glomeruli in control tissue and MCNS, but was strongly expressed in resident glomerular cells of IgAN, including mesangial cells, glomerular epithelial cells and the cells of Bowman’s capsule. Examination of serial sections disclosed that more than 70% of cells positive for C3 mRNA were also stained for C3 protein, C3d, and MAC. Double staining for in situ hybridization and immunohistochemistry also revealed that those C3 mRNA signals were present in intraglomerular cells positive for C3. The expression of C3 mRNA and MAC in glomeruli correlated significantly with the degree of mesangial matrix expansion. Conclusions: Our results demonstrated that locally synthesized C3 is activated in the glomeruli of IgAN and that its expression correlated with the severity of mesangial matrix expansion. These findings suggest that activation of C3 may be involved in tissue injury in IgAN through the formation of membrane attack complex.

Identification of type VI collagen synthesizing cells in human diabetic glomerulosclerosis using renal biopsy sections

Although the role of extracellular matrices in the development of glomerulosclerosis has been discussed widely, the cellular origin of type VI collagen in diabetic nephropathy (DN) has remained relatively unexplored. This study reports the distribution and cellular origin of type VI collagen in DN. Type VI collagen‐specific oligonucleotide probes and monoclonal antibody were used to assess the relative expression of mRNA for alpha1 (VI) chain and its translated protein in paraffin‐embedded renal biopsy sections of DN. By immunohistochemistry, compared to the control, increased deposition of type VI collagen was noted in the diffuse and nodular lesions of diabetic glomeruli. For cellular localization of type VI collagen mRNA, paraffin‐embedded renal sections of the control and DN were hybridized in situ with digoxigenin (Dig)‐labeled antisense oligo‐DNA probe complementary to a part of alpha1 (VI) mRNA. In comparison to the control kidney sections, increased numbers of intraglomerular cells (both mesangial and epithelial cells) were positive for α1 (VI) mRNA in renal biopsy sections of DN. From the results, we conclude that overexpression of type VI collagen by intraglomerular cells with its increased deposition might significantly contribute to the glomerulosclerosis found in DN.

Animal Models of Middle Ear Cholesteatoma

Middle ear acquired cholesteatoma is a pathological condition associated with otitis media, which may be associated with temporal bone resorption, otorrhea and hearing loss, and occasionally various other complications. Cholesteatoma is characterized by the enhanced proliferation of epithelial cells with aberrant morphologic characteristics. Unfortunately, our understanding of the mechanism underlying its pathogenesis is limited. To investigate its pathogenesis, different animal models have been used. This paper provides a brief overview of the current status of research in the field of pathogenesis of middle ear acquired cholesteatoma, four types of animal models previously reported on, up-to-date cholesteatoma research using these animal models, our current studies of the local hybrid ear model, and the future prospect of new animal models of middle ear cholesteatoma.

Recombinant human erythropoietin attenuates renal tubulointerstitial injury in murine adriamycin-induced nephropathy

BACKGROUNDnErythropoietin (EPO) has been found to provide cytoprotection against acute ischemic and toxic renal tubulointerstitial injury. This study aimed to elucidate the mechanism(s) underlying EPO protection while examining whether EPO provides tubulointerstitial protection in a mouse model with adriamycin (ADR)-induced tubulointerstitial injury.nnnMETHODSnAdriamycin nephropathy (AN) was induced by a single injection of ADR in the 2 experimental groups on day 0. The saline-control group and the AN-saline group were administered saline at days 7, 14, and 21, while the EPO-control group and the AN-EPO group were administered EPO at days 7, 14, and 21. Kidneys were harvested at days 14 and 28 after ADR injection to measure the expression levels of the EPO receptor (EPO-R), CD34, and phosphorylated Akt by immunohistochemistry; to determine the extent of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and active caspase-3 staining; and to map the hypoxic area by pimonidazole staining.nnnRESULTSnEPO-R was detected in glomerular, tubular epithelial, and endothelial cells. EPO administration significantly improved tubulointerstitial injury, decreased the number of TUNEL-positive and active caspase-3-positive cells, and increased the phosphorylated-Akt-positive area in the tubulointerstitial area without increasing the hemoglobin or hematocrit levels.nnnCONCLUSIONSnEPO provides renoprotection against AN by reducing apoptotic cell death and preserving peritubular capillaries, possibly by exerting pleiotropic effects independently of its hemopoietic effects.

KGFR as a possible therapeutic target in middle ear cholesteatoma.

Abstract Conclusion: We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma. Objectives: Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. Methods: Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection. Results: The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.