Thinning factors influence on custom-made mouthguards thermoforming.

The aim of this study was to clarify and quantify factors influencing thinning during a thermoforming using a special simulation model that has three different flat surfaces such as 0 degree, 45 degree and 90 degree against a pressurizing force. Air pressure type samples were made by EVA and acrylic resin blank. Vacuum type samples were also made by EVA. Thickness gauge was employed to measure the thickness. As results, pressure forming showed significantly larger thinning at 45 and 90 degree surfaces and smaller thinning at 0 degree surface, 36% in thinning rate by vacuum forming and 66% by the pressure forming at 90 degree surface, and 17% and 20% at 45 degree surface, and 11% and 2% at 0 degree surfaces. Thinning was increased with the increase in distance from the centre in 0 degree surface and increased with the decrease in height in the vertical surface significantly. The air pressure, the material thickness in EVA (Drufosoft) and difference in material colour did not affect thinning rate. An acrylic resin material showed approximately 10% smaller thinning than EVA (Drufosoft). To retain enough thickness of 3 mm on 90 degree surface corresponding to an incisal labial aspect for pressure laminate type, over 55% reduction is taken into consideration and at least two 3-mm-thickness materials should be laminated. 0 degree surface showed at most 2 % reduction in pressure lamination; post thermoforming occlusal thickness became almost 6 mm with a usual 3 mm plus 3 mm lamination. Therefore, careful occlusal adjustment in an actual mouthguard fabrication to achieve an appropriate 2 mm thickness on this surface should be requested.

Macrophages are essential contributors to kidney injury in murine cryoglobulinemic membranoproliferative glomerulonephritis.

Mice transgenic for thymic stromal lymphopoietin (TSLP), under regulation of the lymphocyte-specific promoter Lck, develop cryoglobulinemia and membranoproliferative glomerulonephritis (MPGN) similar to the disease in patients. To determine whether infiltrating macrophages, a hallmark of this disease, are deleterious or beneficial in the injury process, we developed Lck-TSLP transgenic mice expressing the human diphtheria toxin receptor (DTR) under control of the monocyte/macrophage-restricted CD11b promoter (Lck-TSLP;CD11b-DTR). Treatment with DT resulted in a marked reduction of monocytes/macrophages in the peritoneal cavity of both CD11b-DTR and Lck-TSLP;CD11b-DTR mice and marked reduction of macrophage infiltration in glomeruli of Lck-TSLP;CD11b-DTR mice. Lck-TSLP;CD11b-DTR mice, with or without toxin treatment, had similar levels of cryoglobulinemia and glomerular immunoglobulin deposition as Lck-TSLP mice. Lck-TSLP;CD11b-DTR mice, treated with toxin, had reduced mesangial matrix expansion, glomerular collagen IV accumulation, expression of the activation marker α-smooth muscle actin and transforming growth factor-ß1 in mesangial cells, and proteinuria compared with control mice. Thus, macrophage ablation confers protection in this model and indicates a predominately deleterious role for macrophages in the progression of kidney injury in cryoglobulinemic MPGN.

Metallothionein is upregulated by hypoxia and stabilizes hypoxia-inducible factor in the kidney.

Recent studies underscore that chronic hypoxia in the tubulointerstitium is a final common pathway to progression to end-stage renal failure regardless of etiology. We used microarray analysis of rat kidneys made hypoxic by unilateral renal artery stenosis to measure transcriptomic events and clarify pathophysiological mechanisms of renal injury induced by chronic hypoxia. Many genes were upregulated in the kidney by chronic hypoxia, but we focused on metallothionein due to its antioxidative properties. Using tubular epithelial cells transfected with a reporter construct of luciferase, driven by the hypoxia-responsive elements (HRE), we found that addition of metallothionein to the culture media increased luciferase activity. This was associated with upregulation of the target genes of hypoxia-inducible factor (HIF), such as vascular endothelial growth factor and glucose transporter-1. Stimulation of the HIF-HRE pathway by metallothionein was confirmed by metallothionein overexpression. Hypoxia and exogenous metallothionein increased HIF-1alpha protein without changes in its mRNA levels, suggesting protein stabilization. Upregulation of the HIF-HRE system by metallothionein was associated with phosphorylation of ERK but not Akt. MEK inhibition and rapamycin decreased metallothionein-induced HIF activity. Our study shows that upregulation of metallothionein expression by hypoxia activates the HIF-HRE system through the ERK/mTOR pathway and may be a novel defense against hypoxia.

Hemoglobin is expressed by mesangial cells and reduces oxidant stress.

Hemoglobin (Hb) serves as the main oxygen transporter in erythrocytes, but it is also expressed in nonhematopoietic organs, where it serves an unknown function. In this study, microarray and proteomic analyses demonstrated Hb expression in the kidney. Rat kidneys were perfused extensively with saline, and glomeruli were isolated by several techniques (sieving, manual dissection, and laser capture-microdissection). Reverse transcriptase-PCR revealed glomerular alpha- and beta-globin expression, and immunoblotting demonstrated expression of the protein. In situ hybridization studies showed expression of the globin subunits in the mesangium, and immunostaining confirmed this localization of Hb. Furthermore, globin mRNA expression was detected in primary cultures of rat mesangial cells but not in cultured glomerular endothelial or epithelial cells. For investigation of Hb function in mesangial cells, the SV40-MES13 murine mesangial cell line was transfected with a vector expressing alpha- and beta-globins; this overexpression reduced production of hydrogen peroxide-induced intracellular radical oxygen species and enhanced cell viability against oxidative stress. In summary, Hb is expressed by rat mesangial cells, and its potential functions may include antioxidative defense.

Hypoxia and expression of hypoxia-inducible factor in the aging kidney.

Renal senescence is characterized by interstitial fibrosis and loss of peritubular capillaries. In this study, we provided evidence of tubulointerstitial hypoxia and the operation of hypoxia-inducible factor (HIF) in the aging kidney. Using two distinct methods, pimonidazole immunostaining and the expression of the "hypoxia-responsive" reporter of the transgenic rats, we identified the age-related expansion of hypoxia in all areas of the kidney. Expansion was most prominent in the cortex. Clusters of hypoxic tubules were observed in the superficial cortical zones, areas adjacent to the outer nephrons and expanded in the medullary rays. The degree of hypoxia was positively correlated with the age-related tubulointerstitial injury (R(2) = 0.88, p <.01), which was associated with the upregulation of HIF-regulated genes, such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT1) (real-time polymerase chain reaction). These findings point to the involvement of hypoxia and highlight the pathological relevance of HIF and its target genes in the aging kidney.

[Mouthguard effect of tooth distortion during clenching].

PURPOSE: A mouthguard can protect stomatognathic systems from traumatic damage. However, severe occlusal wear of teeth and loss of teeth have often been found in players clinically. These problems might originate in strong clenching during sports. Although it is thought that a mouthguard may be effective for these types of clenching, the relation between mouthguards and clenching has not been sufficiently examined. In this study, the effect of a mouthguard (Drufosoft 3mm, EVA) on tooth distortion caused by clenching was measured and examined at three different clenching strengths. METHODS: As a test tooth, a lower first molar was selected. A strain gauge applied to the outer surface of the buccal cusp was used to measure the distortion. A muscle balance monitor (GC) was used to regulate clenching strengths (10, 50, and 100%). The maximum-effort clenching without a mouthguard was assumed to be the 100% clenching strength. Measurements were conducted with or without mouthguard. A maximum value during clenching was assumed to be date of distortion by using analytical software AcquKnowledge (BIOPAC System Inc.). Statistical analysis software SPSS (SPSS Japan Inc.) was used for the Mann-Whitney test. RESULTS: 1. The tooth distortion by clenching, regardless of the presence of the mouthguard, increased as clenching power strengthened, from 10, 50 to 100%. 2. The tooth distortion, regardless of strength of clenching, was decreased by wearing the mouthguard in all subjects. At 50 and 100% clenching, it was decreased significantly by the mouthguard in all subjects. CONCLUSIONS: Mouthguards decreaseed the tooth distortion caused by clenching. Therefore, a mouthguard may prevent not only traumatic injuries in contact sports but also damage to teeth and periodontal tissues and so on, which occur due to frequent strong clenching in many sports.

Glyoxalase I overexpression ameliorates renal ischemia-reperfusion injury in rats.

Methylglyoxal (MG), a highly reactive carbonyl compound generated by carbohydrate oxidation and glycolysis, is the major precursor of protein glycation and induces cytotoxicity leading to apoptosis. Although recent studies have emphasized that MG accumulates in not only chronic oxidative stress-related diseases but also acute hypoxic conditions, the pathogenic contribution of MG in acute diseases is unclear. MG is efficiently metabolized by the glyoxalase system, namely, glyoxalase I. We investigated the pathophysiological role of glyoxalase I as an MG detoxifier in rat renal ischemia-reperfusion (I/R) injury. I/R-induced tubulointerstitial injury was associated with a deterioration in renal glyoxalase I activity independent of its cofactor, GSH, as well as an increase in renal MG level. In in vitro studies, knockdown of glyoxalase I by small interference RNA transfection in rat tubular cells exacerbated cell death by hypoxia-reoxygenation compared with control cells. We also examined whether glyoxalase I overexpression prevented renal I/R damage in rats overexpressing human glyoxalase I with enzyme activity in the kidney 17-fold higher than in wild-type. The histological and functional manifestations of I/R in these rats were significantly ameliorated in association with a decrease in intracellular MG adduct accumulation, oxidative stress, and tubular cell apoptosis. In conclusion, glyoxalase I exerts renoprotective effects in renal I/R injury via a reduction in MG accumulation in tubular cells.

Chronic hypoxia aggravates renal injury via suppression of Cu/Zn-SOD: a proteomic analysis.

Accumulating evidence suggests a pathogenic role of chronic hypoxia in various kidney diseases. Chronic hypoxia in the kidney was induced by unilateral renal artery stenosis, followed 7 days later by observation of tubulointerstitial injury. Proteomic analysis of the hypoxic kidney found various altered proteins. Increased proteins included lipocortin-5, calgizzarin, ezrin, and transferrin, whereas the decreased proteins were alpha(2u)-globulin PGCL1, eukaryotic translation elongation factor 1alpha(2), and Cu/Zn superoxide dismutase (SOD1). Among these proteins, we focused on Cu/Zn-SOD, a crucial antioxidant. Western blot analysis and real-time quantitative PCR analysis confirmed the downregulation of Cu/Zn-SOD in the chronic hypoxic kidney. Furthermore, our laser capture microdissection system showed that the expression of Cu/Zn-SOD was predominant in the tubulointerstitium and was decreased by chronic hypoxia. The tubulointerstitial injury estimated by histology and immunohistochemical markers was ameliorated by tempol, a SOD mimetic. This amelioration was associated with a decrease in levels of the oxidative stress markers 4-hydroxyl-2-nonenal and nitrotyrosine. Our in vitro studies utilizing cultured tubular cells revealed a role of TNF-alpha in downregulation of Cu/Zn-SOD. Since the administration of anti-TNF-alpha antibody ameliorated Cu/Zn-SOD suppression, TNF-alpha seems to be one of the suppressants of Cu/Zn-SOD. In conclusion, our proteomic analysis revealed a decrease in Cu/Zn-SOD, at least partly by TNF-alpha, in the chronic hypoxic kidney. This study, for the first time, uncovered maladaptive suppression of Cu/Zn-SOD as a mediator of a vicious cycle of oxidative stress and subsequent renal injury induced by chronic hypoxia.

Protective role of hypoxia-inducible factor-2alpha against ischemic damage and oxidative stress in the kidney.

Central to cellular responses to hypoxic environment is the hypoxia-inducible factor (HIF) transcriptional control system. A role for HIF-2alpha was investigated in a model of renal ischemia-reperfusion injury (IRI) associated with oxidative stress using HIF-2alpha knockdown mice. In these mice, HIF-2alpha expression was approximately one half that of wild-type mice, whereas HIF-1alpha expression was equivalent. HIF-2alpha knockdown mice were more susceptible to renal IRI, as indicated by elevated blood urea nitrogen levels and semiquantitative histologic analysis. Immunostaining with markers of oxidative stress showed enhanced oxidative stress in the kidney of HIF-2alpha knockdown mice, which was associated with peritubular capillary loss. Real-time quantitative PCR analysis showed decreased expression of antioxidative stress genes in the HIF-2alpha knockdown kidneys. Studies that used small interference RNA confirmed regulation of the antioxidative stress genes in cultured endothelial cells. Although HIF-2alpha knockdown mice were anemic, serum erythropoietin levels were not significantly increased, reflecting inappropriate response to anemia as a result of HIF-2alpha knockdown. Experiments that used hemodiluted mice with renal ischemia demonstrated that anemia of this degree did not affect susceptibility to ischemia. Knockdown of HIF-2alpha in inflammatory cells by bone marrow transplantation experiments demonstrated that HIF-2alpha in inflammatory cells did not contribute to susceptibility to renal IRI. Restoration of HIF-2alpha in endothelium by intercrossing with Tie1-Cre mice ameliorated renal injury by IRI, demonstrating a specific role of endothelial HIF-2alpha. These results suggest that HIF-2alpha in the endothelium has a protective role against ischemia of the kidney via amelioration of oxidative stress.

Novel drugs and the response to hypoxia: HIF stabilizers and prolyl hydroxylase.

Tissue hypoxia occurs when local metabolism is disturbed by an imbalance between oxygen supply and consumption. This condition can lead to a variety of serious ischemic disorders, including a number of important cardiovascular diseases. In the search for therapeutic approaches, focused modalities which specifically target hypoxia have been particularly sought. These efforts would profit from the ability to utilize the mechanisms by which cells adjust to hypoxic conditions. At the center of the cellular response to hypoxia is hypoxia-inducible factor, HIF. This factor is composed of two subunits, an oxygen-sensitive HIF-alpha subunit and a constitutively expressed HIF-beta subunit. Intracellular accumulation of HIF induces the coordinated expression of a number of adaptive genes against hypoxic insult. Because activation of HIF is a promising therapeutic modality for ischemic cardiovascular disease, recent studies have focused on the development of HIF stimulators. HIF levels are regulated by prolyl hydroxylation and asparaginyl hydroxylation of the HIF-alpha subunit. To date, a single HIF asparaginyl hydroxylase has been identified, factor inhibiting HIF (FIH), whereas the mammalian genome encodes three closely related proteins that have HIF prolyl hydroxylase activity, PHD1, PHD2 and PHD3. Recent patents have disclosed methods for identifying modulators of HIF or PHD as well as novel compounds with properties of HIF modulation or prolyl hydroxylase inhibition. This review highlights the identification of novel HIF stabilizers as specific molecularly targeted therapies against cardiovascular disease.

Cobalt promotes angiogenesis via hypoxia-inducible factor and protects tubulointerstitium in the remnant kidney model.

Tubulointerstitial hypoxia has been implicated in a number of progressive renal diseases, and several lines of evidence indicate that the administration of angiogenic growth factors ameliorates tubulointerstitial injury. We hypothesized that induction of hypoxia-inducible factors (HIF) mediates renoprotection by their angiogenic properties. At 5-9 weeks after subtotal nephrectomy, cobalt was administered to rats to activate HIF. Histological evaluation demonstrated that the tubulointerstitial injury was significantly ameliorated in animals that received cobalt (score: 2.51+/-0.12 (cobalt) vs 3.21+/-0.24 (vehicle), P<0.05). Furthermore, animals receiving cobalt had fewer vimentin- and TdT-mediated dUTP nick-end labeling (TUNEL)-positive tubular cells. The renoprotective effect of cobalt was associated with the preservation of peritubular capillary networks (rarefaction index: 13.7+/-0.4 (cobalt) vs 18.6+/-0.9 (vehicle), P<0.01). This improvement in capillary networks was accompanied by an increased number of proliferating (PCNA-positive) glomerular and peritubular endothelial cells. The angiogenesis produced by this method was not accompanied by an increase in vascular permeability. Furthermore, in vitro experiments clarified that HIF-1 in tubular epithelial cells promotes proliferation of endothelial cells and that HIF-2 overexpressed in renal endothelial cells mediates migration and network formation. Collectively, these findings demonstrate a renoprotective role of HIF through angiogenesis and provide a rationale for therapeutic approaches to target HIF for activation.

Induction of protective genes by cobalt ameliorates tubulointerstitial injury in the progressive Thy1 nephritis.

BACKGROUND: We previously demonstrated that chronic hypoxia has pivotal roles in the progression of tubulointerstitial injury from the early stage of the uninephrectomized Thy1 nephritis model. We have also shown that pretreatment of cobalt confers renoprotection in the ischemia/reperfusion (I/R) injury, in association with the up-regulation of hypoxia-inducible factor (HIF)-regulated genes. Here, we tested the hypothesis that cobalt administration not only attenuates acute ischemic insult, but also ameliorates tubulointerstitial injury secondary to chronic hypoxia. METHODS: We applied sustained cobalt treatment to the uninephrectomized Thy1 nephritis model at 3 to 5 weeks, when tubular hypoxia appeared. Histologic evaluation, including glomerular and peritubular capillary networks, was made at 8 weeks. HIF activation was confirmed by real-time polymerase chain reaction (PCR) analyses for HIF-regulated genes, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), and heme oxygenase 1 (HO-1). Up-regulation of HIF-1alpha and HIF-regulated genes was also verified by Western blotting analysis. To elucidate responsible mechanisms of cobalt in the amelioration of tubuloniterstitial injury, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was conducted at 5 weeks. A combination therapy with angiotensin receptor blocker (ARB), olmesartan, was also challenged. RESULTS: Although the intervention did not change glomerular structural damage or urinary protein excretion rate, tubulointerstitial injury was improved in cobalt-treated animals when compared with the vehicle-treated group. The amelioration was associated with the parallel up-regulation of renoprotective, HIF-regulated gene expression. TUNEL staining revealed that the number of apoptotic cells was reduced in the cortex by cobalt administration, suggesting that renoprotection was achieved partly through its antiapoptotic properties. Furthermore, it was demonstrated that cobalt treatment exerts additional renoprotective effects with the ARB treatment in this model. CONCLUSION: Maneuvers to activate HIF in the ischemic tubulointerstitium will be a new direction to future therapeutic strategies.

Hypoxia-inducible factor modulates tubular cell survival in cisplatin nephrotoxicity.

Hypoxia-inducible factor (HIF)-1 is a transcription factor mediating cellular response to hypoxia. Although it is expressed in tubular cells of the ischemic kidney, its functional role is not fully clarified in the pathological context. In this study, we investigated a role of HIF in tubular cell apoptosis induced by cisplatin. HIF-1alpha was expressed in tubular cells in the outer medulla 3 days after cisplatin (6 mg/kg) administration. With the in vivo administration of cobalt to activate HIF, the number of apoptotic renal tubular cells became much smaller in the outer medulla, compared with the vehicle group. We also examined the functional role of HIF-1 in vitro using immortalized rat proximal tubular cells (IRPTC). In hypoxia, IRPTC that express dominant-negative (dn) HIF-1alpha showed impaired survival in cisplatin injury at variable doses (25-100 microM, 24 h), which was not obvious in normoxia. The observed difference in cell viability in hypoxia was associated with the increased number of apoptotic cells in dnHIF-1alpha clones (Hoechst 33258 staining). Studies on intracellular signaling revealed that the degree of cytochrome c release, dissipation of mitochondrial membrane potentials, and caspase-9 activity were all more prominent in dnHIF-1alpha clones than in control IRPTC, pointing to the accelerated signaling of mitochondrial pathways. We propose that HIF-1 mediates cytoprotection against cisplatin injury in hypoxic renal tubular cells, by reducing the number of apoptotic cells through stabilization of mitochondrial membrane integrity and suppression of apoptosis signaling. A possibility was suggested that activation of HIF-1 could be a new promising therapeutic target for hypoxic renal diseases.

Protection of endothelial cells by dextran sulfate in rats with thrombotic microangiopathy.

The characteristic features of thrombotic microangiopathy (TMA) include glomerular and peritubular capillary endothelial cell injury in association with loss of heparan sulfate proteoglycans on the cell surface and thrombus formation, followed by subsequent ischemic tubulointerstitial damage. It therefore was hypothesized that dextran sulfate (DXS) may protect the kidney against endothelial damage in a model of TMA. TMA was induced in rats by renal artery perfusion of an antiglomerular endothelial antibody, followed by the administration of DXS or vehicle. Renal damage was assessed by histologic analysis and measurements of blood urea nitrogen and creatinine. Whereas control rats developed severe renal failure with extensive glomerular and tubular injury, administration of DXS significantly protected renal function and preserved the glomerular endothelium and peritubular capillaries. The beneficial effect of DXS could be attributed to the ability of DXS to protect endothelial cells from coagulation and complement activation, as demonstrated by the histologic analysis. In addition, binding of the administered DXS to the surface of the glomerular endothelium was confirmed in TMA rats, suggesting that DXS acts as a "repair coat" of injured glomerular endothelium. In conclusion, DXS protects the kidney from experimental TMA. This protection may be mediated by DXS's binding directly to the surface of glomerular endothelium and amelioration of coagulation, complement activation, and cellular matrix loss.

A biologic role of HIF-1 in the renal medulla.

BACKGROUND: Activation of hypoxia-inducible factor-1 (HIF-1) is the primary defensive mechanism against hypoxia. HIF-1 activation generally occurs in pathologic disruption of tissue oxygenation. However, a biologic role of HIF-1 in the medulla of the kidney, which is considered perpetually hypoxic under physiologic conditions due to its unique circulation, remains to be elucidated. METHODS: The expression of HIF-1alpha was detected by immunohistochemical analysis. Functional studies of HIF in medulla were carried out by gene transfer of various plasmids by retrograde injection via ureter. RESULTS: Our immunohistochemical analysis detected HIF-1alpha in the inner stripe and the inner medulla of normal rats. Water deprivation increased the number of HIF-1alpha-positive cells, which may be mediated by an increase in medullar workload and a decrease in local blood flow. To perform functional studies, we performed gene transfer. Efficient expression of the transgene was confirmed using an enhanced green fluorescent protein (E-GFP) expressing vector. Our histologic and immunoblotting analysis detected the transgene product at the inner medulla and the inner stripe 48 hours after injection. Administration of negative-dominant HIF induced severe damage in the medulla of normal rats. In contrast, gene transfer of constitutively active HIF (HIF/VP16) induced expression of various HIF-regulated genes and protected the medulla against ischemic insults. CONCLUSION: Our studies demonstrated a crucial role of HIF in the renal medulla under normal and hypoxic circumstances.

Time course of change in calcium x phosphorus product after percutaneous ethanol injection therapy.

BACKGROUND: Percutaneous ethanol injection therapy (PEIT) effectively suppresses PTH secretion, but the change in the serum calcium and phosphorus product (Ca x P) after PEIT has not been fully evaluated. METHODS: Twenty-seven haemodialysis patients with severe secondary hyperparathyroidism (2HPT) were divided into two groups according to their intact PTH (i-PTH) concentrations 6 months after PEIT: (i). effective (E) group, i-PTH concentration <360 pg/ml; and (ii). non-effective (N) group i-PTH > or = 360 pg/ml. The changes in serum calcium and phosphorus concentrations and the Ca x P were recorded for the following 2 years under post-PEIT medical treatment with oral calcitriol or intravenous 22-oxacalcitriol (OCT). RESULT: In the E group, the i-PTH concentrations decreased to <300 pg/ml 1 year after PEIT (801+/-302 to 280+/-134 pg/ml), then increased to 435+/-201 pg/ml at 2 years. Serum calcium concentration did not show any significant change except for a transient reduction at 1 month after PEIT. The Ca x P decreased for 1 year (from 66.3+/-15.3 to 56.2+/-10.3 mg(2)/dl(2); P<0.05), in agreement with the course of phosphorus concentration, and continued to be <60 mg(2)/dl(2) up to 2 years after PEIT. The Ca x P tended to decrease more with OCT than oral calcitriol. In the N group, calcium and Ca x P increased significantly at 6 months after PEIT and remained at a high value. CONCLUSION: Treatment with PEIT suppresses serum PTH concentration as well as Ca x P in the long term.