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P2X2 receptors are ligand-gated cation channels activated by extracellular ATP that modulate neural transmission in various neuronal systems. Although the function and distribution of P2X2 receptors in the cochlea portion of the inner ear are well established, their physiological role in the vestibular portion is still not understood. Therefore, we investigated P2X2 receptor localization in the peripheral vestibular portion, and assessed their physiological function in vivo using P2X2 receptor knock out (P2X2-KO) mice. Histological analysis revealed that P2X2 receptors were localized on the epithelial surface of supporting and transitional cells of the vestibular end organs. To examine vestibular function in P2X2-KO mice, we conducted behavioral tests and tested the vestibulo-ocular reflex (VOR) during sinusoidal rotations. P2X2-KO mice exhibited significant motor balance impairment in the balance beam test. VOR gain in P2X2-KO mice was significantly reduced, with no decrease in the optokinetic response. In conclusion, we showed that P2X2 receptors are mainly localized in the supporting cells of the vestibular inner ear, and the loss of P2X2 receptors causes mild vestibular dysfunction. Taken together, our findings suggest that the P2X2 receptor plays a modulatory role in vestibular function.

The study of inner ear specific transcripts has revealed novel information about hereditary hearing loss and a mechanism of normal hearing. In this study, by analyzing a published cDNA library, we focused on Epiphycan (Epyc), a member of the small leucine-rich repeat proteoglycan family, whose transcript is enriched in the inner ear. Epyc mRNA was expressed abundantly and specifically in adult mice cochleae and was localized in supporting cells within the organ of Corti of both neonatal and adult mice. To examine the function of Epyc, we generated Epyc knockout (KO) mice using the CRISPR/Cas9 system. Epyc KO mice cochleae exhibited normal morphology. However, measurement of the auditory brain-stem response in Epyc KO mice revealed an elevated hearing threshold above 16 kHz frequency. This study suggests that Epyc is necessary for normal auditory function.

Objectives Some patients with sudden sensorineural hearing loss (SSNHL) are frustrated by residual tinnitus even after accomplishment of the treatment for SSNHL. In the present prospective study, we examined patients’ backgrounds of sex, laterality and age together with changes in hearing level and the tinnitus score after the onset of SSHNL to determine the prognostic factors of residual tinnitus after the final day of medical treatment for SSNHL. Methods Forty-four patients with SSNHL were all treated with systemic administration of steroids for 2 weeks and oral intake of vasoactive drugs and vitamin B12 for 6 months before accomplishment of the treatment for SSNHL. The hearing improvement rate (HIR) was determined by comparing the hearing level before and 6 months after the start of treatment. Tinnitus was subjectively evaluated by the tinnitus scoring questionnaire before, 6 and 24 months after the start of treatment. The score of a five-step evaluation of subjective tinnitus feelings, “loudness”, “duration” and “annoyance”, was recorded. Results HIR was significantly correlated with tinnitus score improvement (TSI) in “duration” at 6 months after the start of treatment compared with before treatment. The tinnitus score of all 3 items was significantly improved 6 months after the start of treatment compared with that before treatment but it was not significantly changed between 6 and 24 months after the start of treatment. TSI in “duration” between 6 and 24 months was significantly correlated with the patients’ age and HIR using multiple regression analysis. Conclusion According to the tinnitus scoring questionnaire, “duration” is the most reliable item for subjective evaluation of tinnitus accompanied by SSNHL. Generally, subjective feelings for residual tinnitus 6 months after the start of treatment for SSNHL are supposed to be almost the same, even at the 24th post-treatment month. Especially, younger patients with better hearing improvement are predicted to achieve further improvement of tinnitus between 6 and 24 months after the start of treatment.

TNP-470, an angiogenesis inhibitor, suppresses the progression of peritoneal fibrosis in mouse experimental model. Background In patients on long-term peritoneal dialysis (PD), angiogenesis and vasculopathy are observed in the peritoneum, and the degree of vascularization correlates with the area of fibrotic tissue, suggesting the involvement of angiogenesis in the progression of peritoneal fibrosis. The aim of the present study was to evaluate the effect of TNP-470, an anti-angiogenic compound, on the development of peritoneal fibrosis induced by chlorhexidine gluconate (CG). Methods Peritoneal fibrosis was induced by injection of CG into peritoneal cavity of Institute for Cancer Research (ICR) mice. TNP-470 was injected subcutaneously with CG. Mice were sacrificed, and peritoneal tissues were dissected out at days eight and 16 after CG and TNP-470 injection. The expression patterns of CD31 (as a marker of endothelial cells), vascular endothelial cell growth factor (VEGF), α-smooth muscle actin (as a marker of myofibroblasts), heat shock protein 47 (HSP47), type III collagen, F4/80 (as a marker of mice macrophages), proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (Cdk2) were examined by immunohistochemistry. Results CG-injected mice showed thickening of the submesothelial zone and increased number of vessels, myofibroblasts, and infiltrating macrophages. The expression levels of VEGF, type III collagen, and HSP47 were increased, and a large number of PCNA-positive cells and Cdk2-expressing cells were observed in the thickened submesothelial area. Treatment with TNP-470 suppressed the submesothelial zone thickening and reduced collagen III expression as well as angiogenesis. TNP-470 also decreased the number of VEGF-expressing cells, myofibroblasts, macrophages, PCNA-positive cells, and Cdk2-expressing cells. Conclusion Our results indicate the involvement of angiogenesis in the progression of peritoneal fibrosis, and suggest that TNP-470 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of angiogenesis and suppression of myofibroblast proliferation.

Background: The transcription factor nuclear factor-κB (NF-κB) 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-κB expression and severity of tissue injury in immunoglobulin A (IgA) nephropathy and the mechanism of such correlation. Methods: The 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-κB. Normal portions of surgically resected kidney with adenocarcinoma served as controls. Results: In normal kidney, MCNS, and MN sections, NF-κB expression was detected in a few mesangial cells and tubular epithelial cells. In IgA nephropathy and non-IgA nephropathy samples, NF-κB 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. Conclusion: Our results indicate that NF-κB is involved in the progression of tissue injury in IgA nephropathy through the induction of transcriptionally regulated genes.

Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization. Background Anaphylatoxin C5a mediates inflammatory responses through interaction with a specific C5a receptor (C5aR), the expression of which is thought to be restricted to peripheral blood leukocytes. Although the presence of C5aR on cultured mesangial cells and tubular epithelial cells has recently been documented, the tissue distribution of C5aR in diseased kidney has not yet been determined. Methods Immunohistochemistry and nonradioactive in situ hybridization for C5aR were performed in 34 tissue samples of kidneys from patients with various renal diseases, including 4 with minimal change nephrotic syndrome (MCNS), 5 with membranous nephropathy (MN), and 25 with mesangial proliferative glomerulonephritis (mesGN; 15 patients with IgA nephropathy, 5 with non-IgA mesGN, and 5 with lupus nephritis). Normal portions of surgically resected kidney served as the control. Results In normal kidneys, C5aR protein was detected in tubular epithelial cells, while C5aR mRNA was detected in a few glomerular cells, tubular epithelial cells, and vascular endothelial and smooth muscle cells. In MCNS, the distribution of C5aR protein and mRNA was similar to that in normal kidneys. In MN and mesGN, C5aR protein and mRNA were detected in mesangial cells, glomerular epithelial and endothelial cells, Bowman's capsule cells, tubular cells, infiltrating cells, and vascular endothelial and smooth muscle cells. The glomerular expression of C5aR mRNA and protein correlated positively with the degree of mesangial hypercellularity and mesangial matrix expansion in mesGN. In the tubulointerstitium, interstitial expression of C5aR mRNA correlated positively with the degree of tubular atrophy and interstitial broadening in mesGN. Furthermore, the interstitial expression of C5aR mRNA correlated positively with the level of serum creatinine. Conclusions Our results indicate that renal cells produce C5aR and that activation of C5a/C5aR pathway on renal cells may be involved in tissue injury in mesGN.

Expression of decay accelerating factor mRNA and complement C3 mRNA in human diseased kidney. Background Decay accelerating factor (DAF), a product of mesangial cells in vitro , is expressed on the surface of cells and is a candidate for the focal suppression of complement activation. It is not clear at present whether the levels of expression of DAF and intrarenal C3 synthesis correlate with the level of tissue injury. Methods Immunohistochemistry for DAF and C3 and nonradioactive in situ hybridization with digoxigenin-labeled oligonucleotide probe for DAF and C3 mRNA were performed in 22 tissue samples of kidneys from patients with IgA nephropathy (IgAN), 6 with membranous nephropathy (MN), 6 with lupus nephritis (LN), and five normal kidneys. Results In the normal kidney, DAF was confined to the juxtaglomerular apparatus and little or no C3 was detected; however, a few glomerular cells were positive for DAF mRNA but no C3 mRNA positive cells were detected. In diseased kidneys, DAF and C3 as well as their mRNAs were detected in mesangial cells, tubular cells and infiltrating cells. Glomerular epithelial cells and Bowman's capsule cells contained little or no DAF and C3 but were positive for their mRNAs. The mean percentages of mesangial cells positive for DAF and C3 mRNAs were 49.3 ± 11.5% and 50.7 ± 10.3% in IgAN, and 17.0 ± 6.3% and 19.4 ± 9.0% in MN, respectively. The percentage of mesangial cells positive for DAF and C3 mRNAs among intraglomerular cells correlated positively with the degree of mesangial proliferation and glomerular sclerosis in IgAN. In contrast, in LN the percentage of glomerular cells positive for DAF mRNA correlated negatively with the degree of glomerular injury, while the percentage of cells positive for C3 mRNA did not change with the progression of the disease. The ratio of C3 mRNA/DAF mRNA of glomerular cells correlated with the degree of glomerular injury in both IgAN and LN. In the tubulointerstitium, the percentage of cells expressing mRNA, and C3 mRNA/DAF mRNA ratio correlated with the degree of tubular atrophy and interstitial broadening in both IgAN and LN. Conclusions We conclude that DAF and C3 mRNAs are synthesized in human diseased kidneys, and that a balance between locally synthesized DAF and C3 may be important in the progression of glomerulonephritis.

Forms of hemophagocytic syndrome,which affects mainly children, vary from mild to very severe and often fatal. Wedescribe an adult patient with hemophagocytic syndrome in whom severe liver dysfunction developed.The condition continued to deteriorate despite treatment with plasma exchange,high-dose gamma globulin, and corticosteroid therapy. Treatment with cyclosporine (2.3 mg/kg/day) dramatically improved the conditionand normalized liver function. Cyclosporine reduced the serum levels of ferritin, interferon-T,interleukin-6, and soluble interleukin-2 receptor.These findings suggest that hemophagocytic syndrome accompanied with severe liver dysfunction results from hypercytokinemia,and cyclosporine is useful in preventing a fatal outcome during the acute phase.