Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Tryptase-Positive Mast Cells Promote Adipose Fibrosis in Secondary Lymphedema through PDGF.

Lymphedema is a chronic and progressive condition that causes physical disfigurement and psychological trauma due to the accumulation of lymphatic fluid in the interstitial space. Once it develops, lymphedema is difficult to treat because it leads to the fibrosis of adipose tissue. However, the mechanism behind this remains unclear. The purpose of this study was to investigate the involvement of mast cells (MCs) in the adipose tissues of patients with lymphedema. We found that fibrosis spread through blood vessels in the adipose tissues of lymphedema patients, and the expression of the collagen I and III genes was significantly increased compared to that of those in normal adipose tissue. Immunostaining of vimentin and α-smooth muscle actin showed that fibroblasts were the main cellular components in severely fibrotic regions. Toluidine blue staining confirmed a significant increase in the number of MCs in the adipose tissues of lymphedema patients, and immunostaining of serial sections of adipose tissue showed a significant increase in the number of tryptase-positive cells in lymphedema tissues compared with those in normal adipose tissues. Linear regression analyses revealed significant positive correlations between tryptase and the expressions of the TNF-α, platelet-derived growth factor (PDGF)-A, and PDGFR-α genes. PDGF-A-positive staining was observed in both fibroblasts and granules of tryptase-positive MCs. These results suggest that MC-derived tryptase plays a role in the fibrosis of adipose tissue due to lymphedema directly or in cooperation with other mediators.

Increased Chymase-Positive Mast Cells in High-Grade Mucoepidermoid Carcinoma of the Parotid Gland.

It has long been known that high-grade mucoepidermoid carcinoma (MEC) has a poor prognosis, but the detailed molecular and biological mechanisms underlying this are not fully understood. In the present study, the pattern of chymase-positive mast cells, as well as chymase gene expression, in high-grade MEC was compared to that of low-grade and intermediate-grade MEC by using 44 resected tumor samples of MEC of the parotid gland. Chymase expression, as well as chymase-positive mast cells, was found to be markedly increased in high-grade MEC. Significant increases in PCNA-positive cells and VEGF gene expression, as well as lymphangiogenesis, were also confirmed in high-grade MEC. Chymase substrates, such as the latent transforming growth factor-beta (TGF-ß) 1 and pro-matrix metalloproteinase (MMP)-9, were also detected immunohistologically in high-grade MEC. These findings suggested that the increased chymase activity may increase proliferative activity, as well as metastasis in the malignant condition, and the inhibition of chymase may be a strategy to improve the poor prognosis of high-grade MEC of the parotid gland.

Design, synthesis, and evaluation of new vanin-1 inhibitors based on RR6.

Fourteen novel vanin-1 inhibitors coded OMP-# were designed from RR6 and successfully synthesized by a nucleophilic addition-elimination reaction of the pantetheinic acid-derived Weinreb amide as a key step under Barbier conditions. The synthesized OMP compounds exhibited inhibitory activity against human serum vanin-1 in vitro. Among the synthesized compounds, OMP-7, which possesses a trifluoromethoxy group at the para-position on the phenyl ring, exhibited the most potent activity, approximately 20 times that of the mother compound RR6. OMP-7 was further subjected to an in vivo assay using a normal hamster. More potent activity was observed than that of RR6 against both serum and renal vanin-1. The activity lasted for 4 h after injection against serum vanin-1 and 1 h after injection against renal vanin-1, whereas RR6 did not show the desired activity.

Activation of Mast-Cell-Derived Chymase in the Lacrimal Glands of Patients with IgG4-Related Ophthalmic Disease.

The purpose of this present study was to investigate the distribution and expression of chymase in the lacrimal glands (LGs) of patients afflicted with IgG4-related ophthalmic disease (IgG4-ROD). LGs from patients with severe canalicular obstruction were considered the control group. Toluidine blue staining confirmed a significant increase in the number of mast cells in the LGs obtained from the IgG4-ROD patients. In addition, immunostaining of serial sections from the LGs showed a significant increase in the number of chymase-positive cells and tryptase-positive cells in the IgG4-ROD LGs compared to the normal control LGs. The mRNA expression of chymase, tryptase, TGF-ß1, and collagen-I tended to increase in the IgG4-ROD LGs. Immunostaining of vimentin and α-smooth muscle actin (α-SMA) showed that myofibroblasts were the main cellular components in severely fibrotic regions of LGs in patients with IgG4-ROD. Linear regression analyses on the number of mast cells, chymase-positive cells, and tryptase-positive cells revealed significant positive correlations between those respective cells. Our findings suggest that chymase may play a role in the fibrotic disorder of IgG4-ROD LGs through the regulation of TGF-ß1 activation and collagen-I deposition, and that it may be a therapeutic target for patients afflicted with IgG4-ROD.

Decreased Presence of Mast Cells in the Bursa Premacularis of Proliferative Diabetic Retinopathy.

INTRODUCTION: We previously reported that the intravitreal activities of chymase and tryptase were more increased in the patients with macular hole (MH) and epiretinal membrane (ERM) than in those with proliferative diabetic retinopathy (PDR) and that the source of these serine proteases might be mast cells in the bursa premacularis (BPM). The purpose of this study was to compare the density of mast cells in BPM samples obtained from MH, ERM, and PDR patients. METHODS: BPM and vitreous core samples were first collected during vitrectomy from eyes afflicted with vitreoretinal diseases (MH: 6 eyes, ERM: 3 eyes, and PDR: 9 eyes), and then were stained with hematoxylin, toluidine blue, antibodies against chymase and tryptase, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay kit. RESULTS: Hematoxylin nuclear staining showed fewer positive-staining cells in the BPM samples obtained from PDR patients than in those obtained from MH and ERM patients. Toluidine blue staining of the BPM revealed metachromasia in the mast cells of the patients with MH and ERM, but not those of the patients with PDR. In addition, immunostaining using anti-chymase and anti-tryptase antibodies showed that the BPM samples were more intensely stained than the vitreous core samples from the patients with MH and ERM and that both tissue samples were poorly stained in the patients with PDR. The apoptotic cells were more frequently observed in the BPM samples from patients with MH than in those from patients with PDR. CONCLUSIONS: These findings indicated that lower activities of chymase and tryptase in the vitreous of PDR patients appeared to be attributable to the decreased presence of mast cells in the BPM. The lack of mast cells in the BPM might be related to the pathogenesis of PDR.

In Vivo and In Vitro Evaluation of Urinary Biomarkers in Ischemia/Reperfusion-Induced Kidney Injury.

Oxidative stress plays an important role in the pathophysiology of acute kidney injury (AKI). Previously, we reported that vanin-1, which is involved in oxidative stress, is associated with renal tubular injury. This study was aimed to determine whether urinary vanin-1 is a biomarker for the early diagnosis of AKI in two experimental models: in vivo and in vitro. In a rat model of AKI, ischemic AKI was induced in uninephrectomized rats by clamping the left renal artery for 45 min and then reperfusing the kidney. On Day 1 after renal ischemia/reperfusion (I/R), serum creatinine (SCr) in I/R rats was higher than in sham-operated rats, but this did not reach significance. Urinary N-acetyl-ß-D-glucosaminidase (NAG) exhibited a significant increase but decreased on Day 2 in I/R rats. In contrast, urinary vanin-1 significantly increased on Day 1 and remained at a significant high level on Day 2 in I/R rats. Renal vanin-1 protein decreased on Days 1 and 3. In line with these findings, immunofluorescence staining demonstrated that vanin-1 was attenuated in the renal proximal tubules of I/R rats. Our in vitro results confirmed that the supernatant from HK-2 cells under hypoxia/reoxygenation included significantly higher levels of vanin-1 as well as KIM-1 and NGAL. In conclusion, our results suggest that urinary vanin-1 might be a potential novel biomarker of AKI induced by I/R.

Glaucocalyxin A Ameliorates Hypoxia/Reoxygenation-Induced Injury in Human Renal Proximal Tubular Epithelial Cell Line HK-2 Cells.

Ischemia-reperfusion injury is one of the major causes of acute kidney injury (AKI), which is increasingly prevalent in clinical settings. Glaucocalxin A (GLA), a biologically ent-kauranoid diterpenoid, has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. In this study, the effect of GLA on AKI and its mechanism were studied in vitro. HK-2 human renal tubular epithelial cells were exposed to hypoxia/reoxygenation (H/R), which were established as an in vitro AKI model. Subsequently, the mRNA expressions of inflammatory and antioxidant factors were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Reactive oxygen species (ROS) production and cell death were detected by fluorescence-activated cell sorting. GLA pre-treatment improved the cell viability of HK-2 cells exposed to H/R. GLA suppressed the H/R-induced ROS production in HK-2 cells. GLA also elevated the activities of superoxide dismutase of HK-2 cells exposed to H/R. Moreover, GLA prevented H/R-induced cell death in HK-2 cells. Furthermore, GLA ameliorated the activation of the protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in HK-2 cells exposed to H/R. Our findings suggested that GLA protected HK-2 cells from H/R-induced oxidative damage, which was mediated by the Akt/Nrf2/HO-1 signaling pathway. These results indicate that GLA may serve as a promising therapeutic drug for AKI.

Increase in Chymase-Positive Mast Cells in Recurrent Pleomorphic Adenoma and Carcinoma Ex Pleomorphic Adenoma of the Parotid Gland.

Incomplete excision of pleomorphic adenoma (PA) may result in recurrent pleomorphic adenoma (RPA). Furthermore, long-term neglected PA may become carcinoma ex pleomorphic adenoma (CXPA). In the present study, the relationships between mast cell-derived chymase and these tumors were examined. The tumor tissues of PA consisted of either or both glandular and fibrotic structures. Histological features of RPA were almost similar to those of PA, except that they showed multinodular structures. CXPA is composed of a mixture of PA and carcinoma. The main stromal cells in PA were myofibroblasts, whereas fibroblasts constituted the main cellular portion in the stromal tissue of RPA. Cancer-associated fibroblasts (CAFs) were present abundantly in CXPA. With increased VEGF expression, neovascularization tended to increase in RPA or CXPA. Compared with PA, chymase-positive mast cells, as well as chymase gene expression, were increased in the tumor tissues from patients with RPA or CXPA. SCF, TGFß1, and PCNA-positive staining was widely observed in these tumor tissues. The above results suggest that mast cell-derived chymase through its direct or cooperative effects with other mediators may participate in the pathophysiology of RPA and CXPA.

Chymase as a Novel Therapeutic Target in Acute Pancreatitis.

Acute pancreatitis is still a life-threatening disease without an evidenced therapeutic agent. In this study, the effect of chymase in acute pancreatitis and the possible effect of a chymase inhibitor in acute pancreatitis were investigated. Hamsters were subcutaneously administered 3.0 g/kg of L-arginine to induce acute pancreatitis. Biological markers were measured 1, 2, and 8 h after L-arginine administration. To investigate the effect of a chymase inhibitor, a placebo (saline) or a chymase inhibitor TY-51469 (30 mg/kg) was given 1 h after L-arginine administration. The survival rates were evaluated for 24 h after L-arginine administration. Significant increases in serum lipase levels and pancreatic neutrophil numbers were observed at 1 and 2 h after L-arginine administration, respectively. Significant increases in pancreatic neutrophil numbers were observed in the placebo-treated group, but they were significantly reduced in the TY-51469-treated group. A significant increase in the pancreatic tumor necrosis factor-α mRNA level was observed in the placebo-treated group, but it disappeared in the TY-51469-treated group. Chymase activity significantly increased in the placebo-treated group, but it was significantly reduced by treatment with TY-51469. The survival rate significantly improved in the TY-51469-treated group. A chymase inhibitor may become a novel therapeutic agent for acute pancreatitis.

Expression of Lymphatic Markers in the Berger's Space and Bursa Premacularis.

We previously reported that the bursa premacularis (BPM), a peculiar vitreous structure located above the macula, contains numerous cells expressing markers of lymphatic endothelial cells, such as podoplanin and LYVE-1. Herein, we examined the expression of lymphatic markers in the Berger's space (BS), BPM, and vitreous core (VC). BS, BPM, and VC specimens were selectively collected in macular hole and epiretinal membrane patients during vitrectomy and were then immunostained with antibodies for podoplanin, LYVE-1, and fibrillin-1 and -2. By visualization using triamcinolone acetonide, the BS was recognized as a sac-like structure with a septum located behind the lens as well as BPM. Those tissues adhered to the lens or retina in a circular manner by means of a ligament-like structure. Immunostaining showed intense expression of podoplanin and LYVE-1 in the BS. Both BS and BPM stained strongly positive for fibrillin-1 and -2. The VC was faintly stained with antibodies for those lymph-node markers. Our findings indicate that both BS and BPM possibly belong to the lymphatic system, such as lymph nodes, draining excess fluid and waste products into lymphatic vessels in the dura mater of the optic nerve and the ciliary body, respectively, via intravitreal canals.

The novel mechanism of valproate to prevent peritoneal adhesion formation.

PURPOSE: A novel pharmacological mechanism of valproate was analyzed using a hamster model of adhesion. METHODS: Valproate or placebo was administered just after cecal injury and adhesion severity scores and histological were analyzed. RESULTS: The adhesion severity scores in the placebo- and valproate-treated groups were 2.67 ± 0.42 and 1.0 ± 0.37, respectively, with a significant difference between the groups. A significant increase in mast cell numbers was observed in the placebo-treated group vs. the sham-operated group; however, the mast cell number in the adhesive lesion was significantly lower in the valproate-treated group than in the placebo-treated group. The number of cells positive for chymase, an enzyme in mast cells, in the adhesive lesion was significantly higher in the placebo-treated group, but its increase was attenuated significantly by treatment with valproate. The myeloperoxidase gene expression level in the cecum was significantly higher in the placebo-treated group than in the sham-operated group, but there was no significant difference in the myeloperoxidase gene expression level between the sham-operated and valproate-treated groups in. In an in vitro experiment, valproate inhibited purified human and hamster chymases dose-dependently. CONCLUSION: The chymase inhibitory effect of valproate may contribute to prevent adhesion formation after abdominal injury.

Chymase as a Possible Therapeutic Target for Amelioration of Non-Alcoholic Steatohepatitis.

The development and progression of non-alcoholic steatohepatitis (NASH) are linked to oxidative stress, inflammation, and fibrosis of the liver. Chymase, a chymotrypsin-like enzyme produced in mast cells, has various enzymatic actions. These actions include activation of angiotensin II, matrix metalloproteinase (MMP)-9, and transforming growth factor (TGF)-ß, which are associated with oxidative stress, inflammation, and fibrosis, respectively. Augmentation of chymase activity in the liver has been reported in various NASH models. Generation of hepatic angiotensin II and related oxidative stress is upregulated in NASH but attenuated by treatment with a chymase inhibitor. Additionally, increases in MMP-9 and accumulation of inflammatory cells are observed in NASH but are decreased by chymase inhibitor administration. TGF-ß and collagen I upregulation in NASH is also attenuated by chymase inhibition. These results in experimental NASH models demonstrate that a chymase inhibitor can effectively ameliorate NASH via the reduction of oxidative stress, inflammation, and fibrosis. Thus, chymase may be a therapeutic target for amelioration of NASH.

Mechanism of Albuminuria Reduction by Chymase Inhibition in Diabetic Mice.

Chymase has several functions, such as angiotensin II formation, which can promote diabetic kidney disease (DKD). In this study, we evaluated the effect of the chymase inhibitor TY-51469 on DKD in diabetic db/db mice. Diabetic mice were administered TY-51469 (10 mg/kg/day) or placebo for 4 weeks. No significant difference was observed in body weight and fasting blood glucose between TY-51469- and placebo-treated groups. However, a significant reduction in urinary albumin/creatinine ratio was observed in the TY-51469-treated group compared with the placebo-treated group. In the renal extract, chymase activity was significantly higher in placebo-treated mice than in non-diabetic db/m mice, but it was reduced by treatment with TY-51469. Both NADPH oxidase 4 expression and the oxidative stress marker malondialdehyde were significantly augmented in the placebo-treated group, but they were attenuated in the TY-51469-treated group. Significant increases of tumor necrosis factor-α and transforming growth factor-ß mRNA levels in the placebo-treated group were significantly reduced by treatment with TY-51469. Furthermore, the expression of nephrin, which is a podocyte-specific protein, was significantly reduced in the placebo-treated group, but it was restored in the TY-51469-treated group. These findings demonstrated that chymase inhibition reduced albuminuria via attenuation of podocyte injury by oxidative stress.

Possible Roles of Periostin in the Formation of Hemodialysis Vascular Access Stenosis after Polytetrafluoroethylene Graft Implantation in Dogs.

Periostin, a recently found matricellular protein, has been implicated in neointima formation after balloon injury. However, the relationship between periostin and hyperplastic intima formation after PTFE graft implantation is unclear. Under mixed anesthesia, PTFE grafts were implanted between the canine carotid artery and jugular vein, and PTFE graft samples were harvested 1, 2, and 4 months after implantation. Intima formation started on the luminal surface of PTFE grafts at the venous anastomotic region 1 month after implantation. Thereafter, the increase in intimal volume was not only observed in the venous and arterial anastomotic regions, but also in the middle region of the PTFE grafts. In accordance with the increased intimal formation, time-dependent increases in mRNA expressions of periostin and transforming growth factor beta 1 (TGF-ß1), as well as a strong positive correlation between periostin and TGF-ß1, were observed. These findings suggest that periostin may play a very important role in the pathogenesis of hemodialysis vascular access stenosis through the acceleration of intimal formation. Thus, periostin may be a very important therapeutic target for the treatment of vascular access graft dysfunction in hemodialysis patients.

Involvement of the Retinal Pigment Epithelium in the Development of Retinal Lattice Degeneration.

Lattice degeneration involves thinning of the retina that occurs over time. Here we performed an immunohistological study of tissue sections of human peripheral retinal lattice degeneration to investigate if retinal pigment epithelium (RPE) cells are involved in the pathogenesis of this condition. In two cases of retinal detachment with a large tear that underwent vitreous surgery, retinal lattice degeneration tissue specimens were collected during surgery. In the obtained specimens, both whole mounts and horizontal section slices were prepared, and immunostaining was then performed with hematoxylin and antibodies against glial fibrillary acidic protein (GFAP), RPE-specific protein 65 kDa (RPE65), pan-cytokeratin (pan-CK), and CK18. Hematoxylin staining showed no nuclei in the center of the degenerative lesion, thus suggesting the possibility of the occurrence of apoptosis. In the degenerative lesion specimens, GFAP staining was observed in the center, RPE65 staining was observed in the slightly peripheral region, and pan-CK staining was observed in all areas. However, no obvious CK18 staining was observed. In a monkey retina used as the control specimen of a normal healthy retina, no RPE65 or pan-CK staining was observed in the neural retina. Our findings suggest that migration, proliferation, and differentiation of RPE cells might be involved in the repair of retinal lattice degeneration.

Impaired function of aorta and perivascular adipose tissue in IL-18-deficient mice.

IL-18 is ubiquitously produced by both hematopoietic and non-hematopoietic cells. The present study examined the thoracic aorta, including the surrounding perivascular adipose tissue (PVAT), of IL-18KO mice from functional and histological perspectives. IL-18KO mice exhibited raised blood pressure compared with wild-type mice. Echocardiographic examination showed a thickened vascular wall and narrowed vascular diameter of the aorta. Examination by the Magnus test demonstrated dysfunction of endothelial cells (ECs) in the IL-18KO thoracic aorta and impairment of the anticontractile function of IL-18KO PVAT. Histological examination showed no inflammatory lesions in the aorta but indicated progressive fibrosis in the vessel and conversion of PVAT from brown adipose tissue-like features to white adipose tissue-like features. Electron microscopic observation suggested several deformed mitochondria in the aorta and vacuole-like structures in ECs from IL-18KO mice. In addition, activity of complex IV was lower and production of reactive oxygen species was augmented in the mitochondria of IL-18KO aorta. Although expression of LC3 B was higher, rapamycin-induced autophagy flux was impaired in the IL-18KO PVAT. Moreover, Western blot analysis revealed that LAMP 1/2 was increased in IL-18KO PVAT, and measurement of cathepsin-D activity indicated decreased levels in IL-18KO PVAT. The IL-18KO thoracic aorta thus showed defects in physiological functions related to histological alterations, and the inflammasome/IL-18 system was suggested to play a protective role in cardiovascular cells, probably through quality control of mitochondria via promotion of autophagosome/autophagolysosome formation.NEW & NOTEWORTHY IL-18 deficiency caused aortic abnormalities in terms of morphology and functions in parallel with an accumulation of damaged mitochondria and anomalous turnover of protein complexes, such as PGC-1 and LAMP1 and -2 in PVAT. These findings suggested that IL-18 plays roles in maintaining the homeostasis of vessels and PVAT around the aorta, possibly by promoting autophagy.

Involvement of Vanin-1 in Ameliorating Effect of Oxidative Renal Tubular Injury in Dahl-Salt Sensitive Rats.

In salt-sensitive hypertension, reactive oxygen species (ROS) play a major role in the progression of renal disease partly through the activation of the mineralocorticoid receptor (MR). We have previously demonstrated that urinary vanin-1 is an early biomarker of oxidative renal tubular injury. However, it remains unknown whether urinary vanin-1 might reflect the treatment effect. The objective of this study was to clarify the treatment effect for renal tubular damage in Dahl salt-sensitive (DS) rats. DS rats (six weeks old) were given one of the following for four weeks: high-salt diet (8% NaCl), high-salt diet plus a superoxide dismutase mimetic, tempol (3 mmol/L in drinking water), high-salt diet plus eplerenone (100 mg/kg/day), and normal-salt diet (0.3% NaCl). After four-week treatment, blood pressure was measured and kidney tissues were evaluated. ROS were assessed by measurements of malondialdehyde and by immunostaining for 4-hydroxy-2-nonenal. A high-salt intake for four weeks caused ROS and histological renal tubular damages in DS rats, both of which were suppressed by tempol and eplerenone. Proteinuria and urinary N-acetyl-ß-D-glucosaminidase exhibited a significant decrease in DS rats receiving a high-salt diet plus eplerenone, but not tempol. In contrast, urinary vanin-1 significantly decreased in DS rats receiving a high-salt diet plus eplerenone as well as tempol. Consistent with these findings, immunohistochemical analysis revealed that vanin-1 was localized in the renal proximal tubules but not the glomeruli in DS rats receiving a high-salt diet, with the strength attenuated by tempol or eplerenone treatment. In conclusion, these results suggest that urinary vanin-1 is a potentially sensitive biomarker for ameliorating renal tubular damage in salt-sensitive hypertension.

Effects of Regorafenib, a Multi-Kinase Inhibitor, on Conjunctival Scarring in a Canine Filtration Surgery Model in Comparison with Mitomycin-C.

Regorafenib eye drops were developed for treating age-related macular degeneration. This study aimed to investigate the effects of this multi-kinase inhibitor on intraocular pressure (IOP), bleb formation, and conjunctival changes in a canine filtration surgery model. Glaucoma filtration surgery models were created in 24 eyes of 24 beagles. In experiment 1 (Ex 1), regorafenib eye drops (regorafenib group: n = 6) or a vehicle (control group, n = 6) were instilled twice daily for 4 weeks postoperatively. In experiment 2 (Ex 2), regorafenib eye drops were instilled as in Ex 1 (regorafenib group: n = 6) for 12 weeks while conventional intraoperative mitomycin-C (MMC) was utilized (MMC group: n = 6), In Ex 1, only the regorafenib group showed significant IOP reduction with a significantly higher bleb score. Subconjunctival area, collagen density, vessels, and cells showing proliferation and differentiation were lower in subconjunctival tissue in the regorafenib group. In Ex 2, no significant difference was found in IOP reduction and bleb formation between the regorafenib and MMC groups; bleb walls were significantly thicker and collagen density and vessels were higher in the regorafenib group; and no differences were observed in the above-mentioned cells. Thus, regorafenib might be a better alternative to MMC for creating thicker and less ischemic blebs in glaucoma filtration surgery.

Early urinary biomarkers of renal tubular damage by a high-salt intake independent of blood pressure in normotensive rats.

Dietary sodium intake has been associated with progression to chronic kidney disease (CKD) as well as hypertension. A high-salt intake causes renal damage independent of hypertension. Because traditional renal biomarkers are insensitive, it is difficult to detect renal injury induced by a high-salt intake, especially in normotensive patients. Here, we investigated whether newly developed renal biomarkers could be detected earlier than traditional biomarkers under a high-salt intake, in normotensive rats. Male Wistar Kyoto rats (WKY) received a regular (0.8% NaCl) or salt-loaded (2, 4, and 8% NaCl) diet from 9 to 17 weeks of age. A urine sample was obtained once a week and urinary vanin-1, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (Kim-1) were measured. At 17 weeks of age, 8% salt-loaded WKY showed histopathological renal tubular damage and elevated Rac1 activity in renal tissues. Although there was no significant increase in serum creatinine, urinary albumin, N-acetyl-ß-D-glucosaminidase (NAG), or Kim-1 during the study period among the groups, urinary vanin-1 and NGAL significantly increased in 8% salt-loaded WKY from 10 to 17 weeks of age. These results suggest that urinary vanin-1 and NGAL, which might be induced by salt per se, are potentially earlier biomarkers for renal tubular damage in normotensive rats under a high-salt intake.