P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease that can compromise the functioning of various organs, including the salivary glands (SG). The purinergic system is one of the most important inflammatory pathways in T2DM condition, and P2X7R and P2X4R are the primary purinergic receptors in SG that regulate inflammatory homeostasis. This study aimed to evaluate P2X7R and P2X4R expression, and morphological changes in the submandibular gland (SMG) in T2DM. Twenty-four 5-week-old mice were randomly assigned to control (CON) and diabetes mellitus (DM) groups (n = 12 each). Body weight, diet, and blood glucose levels were monitored weekly. The histomorphology of the SMG and the expression of the P2X7R, and P2X7R was evaluated by immunohistochemistry (IHC) staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 11 and 13 weeks of age. Our findings indicate a significant increase in food consumption, body weight, and blood glucose levels in the DM group. Although a significant increase in P2X7R and P2X4R expression was observed in the DM groups, the receptor location remained unchanged. We also observed a significant increase in the acinar area in the DM13w group, and a significant decrease in the ductal area in the DM11w and DM13w groups. Targeting purinergic receptors may offer novel therapeutic methods for diabetic complications.

Eps15/Pan1p is a master regulator of the late stages of the endocytic pathway.

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases-including actin nucleation promoting factors-and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.

Osmotic diuresis by SGLT2 inhibition stimulates vasopressin-induced water reabsorption to maintain body fluid volume.

Most of the filtered glucose is reabsorbed in the early proximal tubule by the sodium-glucose cotransporter SGLT2. The glycosuric effect of the SGLT2 inhibitor ipragliflozin is linked to a diuretic and natriuretic effect that activates compensatory increases in fluid and food intake to stabilize body fluid volume (BFV). However, the compensatory mechanisms that are activated on the level of renal tubules remain unclear. Type 2 diabetic Goto-Kakizaki (GK) rats were treated with vehicle or 0.01% (in diet) ipragliflozin with free access to fluid and food. After 8 weeks, GK rats were placed in metabolic cages for 24-hr. Ipragliflozin decreased body weight, serum glucose and systolic blood pressure, and increased fluid and food intake, urinary glucose and Na+ excretion, urine volume, and renal osmolar clearance, as well as urine vasopressin and solute-free water reabsorption (TcH2O). BFV, measured by bioimpedance spectroscopy, and fluid balance were similar among the two groups. Urine vasopressin in ipragliflozin-treated rats was negatively and positively associated with fluid balance and TcH2O, respectively. Ipragliflozin increased the renal membrane protein expression of SGLT2, aquaporin (AQP) 2 phosphorylated at Ser269 and vasopressin V2 receptor. The expression of SGLT1, GLUT2, AQP1, and AQP2 was similar between the groups. In conclusion, the SGLT2 inhibitor ipragliflozin induced a sustained glucosuria, diuresis, and natriuresis, with compensatory increases in fluid intake and vasopressin-induced TcH2O in proportion to the reduced fluid balance to maintain BFV. These results indicate that the osmotic diuresis induced by SGLT2 inhibition stimulates compensatory fluid intake and renal water reabsorption to maintain BFV.

[Retrosternal Giant Aortic Aneurysm;Report of Two Cases].

This is a 2-case report of successful aortic repair surgery for the retrosternal giant aortic aneurysm. Our surgical strategy is "deep hypothermia and left ventricular( LV) unloading under cardiopulmonary bypass before approaching to the aortic aneurysm" in case of possible catastrophic bleeding. Case 1, a 64-year-old woman, had a retrosternal pseudoaneurysm (80 mm) at the distal anastomosis of a Dacron graft used to replace the ascending aorta 7 years before. An LV vent tube was cannulated via the right upper pulmonary vein through an inferior T-shaped ministernotomy. Case 2, an 86-year-old woman, had a retrosternal chronic aortic dissecting aneurysm (66 mm). An LV vent cannula was inserted via the LV apex through a left minithoracotomy. Arch replacement and ascending aorta replacement were performed in Case 1 and 2, respectively, without cardiac, neurological, or any other complications. This strategy is safe and useful in a case with complex aortic disease.

Unmasking a sustained negative effect of SGLT2 inhibition on body fluid volume in the rat.

The chronic intrinsic diuretic and natriuretic tone of sodium-glucose cotransporter 2 (SGLT2) inhibitors is incompletely understood because their effect on body fluid volume (BFV) has not been fully evaluated and because they often increase food and fluid intake at the same time. Here we first compared the effect of the SGLT2 inhibitor ipragliflozin (Ipra, 0.01% in diet for 8 wk) and vehicle (Veh) in Spontaneously Diabetic Torii rat, a nonobese type 2 diabetic model, and nondiabetic Sprague-Dawley rats. In nondiabetic rats, Ipra increased urinary excretion of Na+ (UNaV) and fluid (UV) associated with increased food and fluid intake. Diabetes increased these four parameters, but Ipra had no further effect, probably because of its antihyperglycemic effect, such that glucosuria and, as a consequence, food and fluid intake were unchanged. Fluid balance and BFV, determined by bioimpedance spectroscopy, were similar among the four groups. To study the impact of food and fluid intake, nondiabetic rats were treated for 7 days with Veh, Ipra, or Ipra+pair feeding+pair drinking (Pair-Ipra). Pair-Ipra maintained a small increase in UV and UNaV versus Veh despite similar food and fluid intake. Pair-Ipra induced a negative fluid balance and decreased BFV, whereas Ipra or Veh had no significant effect compared with basal values. In conclusion, SGLT2 inhibition induces a sustained diuretic and natriuretic tone. Homeostatic mechanisms are activated to stabilize BFV, including compensatory increases in fluid and food intake.

Delayed perforation occurring on the 24th day after endoscopic submucosal dissection for early gastric cancer.

Delayed perforation occurs after 0.5% of endoscopic submucosal dissection (ESD) procedures for early gastric cancer (EGC). This complication can occur within a few hours or days after ESD. There are few reports in the English literature concerning patients who developed delayed perforation after ESD for EGC. An 81-year-old woman was referred to the emergency department of our hospital on the 24th day after ESD because of abdominal pain. We diagnosed her with delayed perforation with peritonitis after ESD for EGC using computed tomography (CT) and esophagogastroduodenoscopy (EGD). We performed primary closure with interrupted sutures covered via pedicled omentoplasty. The patient was discharged 13 days after surgery without any postoperative complications. Delayed perforation is generally treated with conservative, surgical, or endoscopic methods. Several benefits of endoscopic clipping have been reported. However, in the present case, we performed emergency surgery while considering possible fatal complications, such as severe peritonitis. It is important to recognize delayed perforation in the differential diagnosis. The decision to perform surgery should be made after carefully considering the degree of perforation based on EGD, CT findings, and patient conditions.

MicroRNA expression profiling in peritoneal fibrosis.

Peritoneal fibrosis (PF) is an intractable complication leading to peritoneal membrane failure in peritoneal dialysis (PD). The aim of this study was to identify microRNAs (miRNAs) involved in PF. Peritoneal tissue from a PF rat model was screened for miRNA expression using microarray analysis. The expression levels of differentially expressed miRNAs were evaluated in serum and drained dialysate and associated with peritoneal membrane functions, as measured by the peritoneal equilibrium test in 33 PD patients. Furthermore, an miRNA inhibitor (anti-miRNA-21-5p locked nucleic acid (LNA): anti-miRNA-21-LNA) was intraperitoneally injected to PF model mice to investigate its effects on PF. The initial profiling study of PF rat peritoneal tissue identified 6 miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-34a-5p, and miRNA-327) whose expression was increased more than 2-fold and no miRNAs whose expression was decreased more than half. Among them, serum levels of miRNA-21-5p, miRNA-221-3p, and miRNA-327 and drained dialysate levels of miRNA-221-3p and miRNA-34a-5p were significantly correlated with peritoneal membrane functions in PD patients. Anti-miRNA-21-LNA significantly inhibited miRNA-21-5p expression in the PF mouse peritoneum, inhibited peritoneal fibrous thickening, and maintained peritoneal membrane functions. These results suggest that several miRNAs are involved in PF and that they may be useful as novel diagnostic biomarkers and therapeutic targets for PF.

Delivery of microRNA-146a with polyethylenimine nanoparticles inhibits renal fibrosis in vivo.

Renal fibrosis is the final common pathway leading to end-stage renal disease. Although microRNA (miR) was recently shown to be involved in the development of renal fibrosis, few studies have focused on the effects on renal fibrosis of exogenous miR delivered in an in vivo therapeutic setting. The study reported here investigated the effects of miR-146a delivery using polyethylenimine nanoparticles (PEI-NPs) on renal fibrosis in vivo. PEI-NPs bearing miR-146 or control-miR (nitrogen/phosphate ratio: 6) were injected into the tail vein of a mouse model of renal fibrosis induced by unilateral ureteral obstruction. PEI-NPs bearing miR-146 significantly enhanced miR-146a expression in the obstructed kidney compared with the control group, while inhibiting the renal fibrosis area, expression of alpha-smooth muscle actin, and infiltration of F4/80-positive macrophages into the obstructed kidney. In addition, PEI-NPs bearing miR-146a inhibited the transforming growth factor beta 1-Smad and tumor necrosis factor receptor-associated factor 6-nuclear factor kappa B signaling pathways. Control-miR-PEI-NPs did not show any of these effects. These results suggest that the delivery of miR-146a attenuated renal fibrosis by inhibiting pro-fibrotic and inflammatory signaling pathways and that the delivery of appropriate miRs may be a therapeutic option for preventing renal fibrosis in vivo.

siRNAs targeted to Smad4 prevent renal fibrosis in vivo.

Renal fibrosis is the final common pathway leading to decreased renal function. No therapy has been established to prevent it. In order to establish a therapeutic approach and target molecule for renal fibrosis, we investigated the effects of Smad4 knockdown by siRNAs on renal fibrosis in vivo. Renal fibrosis mice were produced by single intraperitoneal injection of folic acid. siRNAs targeted to Smad4 (Smad4-siRNAs) (5 nmol) were injected into each mouse by systemic tail vein injection three times per week. Non-targeted siRNAs (control-siRNAs) were injected in the same way for a control group. The siRNAs were delivered to the interstitial fibrous area and tubules. Smad4-siRNAs significantly knocked down Smad4 expression and inhibited renal fibrosis. They also inhibited α-SMA-positive myofibroblasts. Control-siRNAs did not show these effects. The results of this study suggest that Smad4 knockdown is one of the crucial therapeutic options for the prevention of renal fibrosis in vivo.

Action of irbesartan on blood pressure and glucose/lipid metabolism, in hemodialysis patients with hypertension.

BACKGROUND: Irbesartan has been reported to have beneficial effects on glucose/lipid metabolism in addition to an antihypertensive effect; however, such effects have not been clarified in hemodialysis (HD) patients. We investigated the effects of irbesartan on blood pressure (BP) as well as glucose/lipid metabolism, in HD patients with hypertension. METHODS: Seventeen HD patients with hypertension, aged 62.7 ± 12.5 years, were treated with daily oral administration of 50 to 100 mg of irbesartan for 12 weeks. Then, the changes of BP as well as glucose metabolism (random serum glucose level and serum glycosylated hemoglobin [HbA1c] level) and lipid metabolism (serum low-density lipoprotein cholesterol [LDL-chol] level, serum high-density lipoprotein cholesterol [HDL-chol] level, and serum triglyceride [TG] level) were evaluated. RESULTS: Irbesartan significantly reduced systolic BP (154.9 ± 12.8 to 139.4 ± 13.1 mmHg (P < 0.01) and diastolic BP (78.9 ± 9.1 to 72.2 ± 9.7 mmHg, P < 0.01). It also reduced LDL-chol (77.6 ± 19.1 to 72.0 ± 18.6 mg/dL, P < 0.05), whereas it did not significantly affect random serum glucose (129.3 ± 46.9 mg/dL to 130.6 ± 47.2 mg/dL), HbA1c (5.58% ± 1.41% to 5.49% ± 1.11%), TG (104.3 ± 65.8 mg/dL to 100.2 ± 59.9 mg/dL), or HDL-chol (44.8 ± 17.1 mg/dL to 45.7 ± 15.6 mg/dL). CONCLUSION: Irbesartan is effective for BP control and may have beneficial effects on lipid metabolism in HD patients.

Long-term effects of aliskiren on blood pressure and the renin-angiotensin-aldosterone system in hypertensive hemodialysis patients.

OBJECTIVE: The long-term effects of aliskiren in hypertensive hemodialysis patients remain to be elucidated. DESIGN: In this post hoc analysis, we followed up 25 hypertensive hemodialysis patients who completed 8-week aliskiren treatment in a previous study for 20 months to investigate the blood pressure-lowering effect and inhibitory effect on the renin-angiotensin-aldosterone system. RESULTS: Among the 25 patients, eleven patients continued with aliskiren treatment. Blood pressure (± standard deviation) decreased from 175 ± 18/80 ± 11 mmHg at baseline to 156 ± 20/76 ± 9 mmHg at month 20. Plasma renin activity, angiotensin I, and angiotensin II decreased from baseline to month 20 (plasma renin activity (ng/mL/h): 2.3 ± 2.6 to 0.3 ± 0.4 (P < 0.05), angiotensin I (pg/mL): 909.1 ± 902.5 to 41.5 ± 14.8 (P < 0.05), angiotensin II (pg/mL): 41.5 ± 45.8 to 11.0 ± 4.9 (P < 0.05)). CONCLUSION: Long-term treatment with aliskiren provides effective blood pressure lowering and inhibition of the renin-angiotensin-aldosterone system, which are sustained over 20 months in hypertensive hemodialysis patients.

Level of 8-OHdG in drained dialysate appears to be a marker of peritoneal damage in peritoneal dialysis.

PURPOSE: Peritoneal dialysis (PD) is a successful renal replacement therapy; however, long-term PD leads to structural and functional peritoneal damage. Therefore, the monitoring and estimation of peritoneal function are important in PD patients. Oxidative stress has been implicated as one possible mechanism of peritoneal membrane damage. The aim of this study was to evaluate the association between an oxidative stress marker, 8-hydroxydeoxyguanosine (8-OHdG), and peritoneal damage in PD patients. METHODS: The authors evaluated 8-OHdG in drained dialysate by enzyme immunoassay to investigate the association between 8-OHdG and solute transport rate estimated by peritoneal equilibration test and matrix metalloproteinase-2 (MMP-2) level in 45 samples from 28 PD patients. RESULTS: The 8-OHdG level was significantly correlated with dialysate:plasma creatine ratio (r = 0.463, P < 0.05) and significantly inversely correlated with D/D0 glucose (where D is the glucose level of peritoneal effluents obtained 4 hours after the injection and D0 is the glucose level obtained immediately after the injection) (r = -0.474, P < 0.05). The 8-OHdG level was also significantly correlated with MMP-2 level (r = 0.551, P < 0.05), but it was not correlated with the age of subjects, the duration of PD, or blood pressure. CONCLUSION: The level of 8-OHdG in drained dialysate may be a useful novel marker of peritoneal damage in PD.

Establishment of acute kidney injury mouse model by 0.75% adenine ingestion.

BACKGROUND: Acute kidney injury (AKI) has a profound impact on the morbidity and mortality of patients. Tubular obstruction is an important mechanism of AKI development because many molecules will cause tubular obstruction, leading to AKI; however, few AKI animal models by tubular obstruction have been established. The aim of this study was to establish AKI model mice due to tubular obstruction. METHOD: C57BL/6 male mice, aged 10 weeks, 20-30 g body weight, were ingested 0.75% adenine compound food. The mice were sacrificed from day 1 to 5 for blood, urine, and histological analysis. RESULTS: All mice (50/50 mice) developed AKI and had died by day 6 of uremia after ingesting 0.75% adenine. Histological examination of the kidneys of AKI mice showed that dilatation of the tubular lumen in the medullary papilla resulted from the occupation by adenine casts followed by atrophy, scattering, and flattening of the epithelial cells of tubules. The urine neutrophil gelatinase-associated lipocalin (NGAL) level significantly increased in AKI mice on day 1 after ingesting 0.75% adenine. CONCLUSION: We established a very simple and reproducible AKI mouse model due to tubular obstruction by ingesting 0.75% adenine.

The interaction of LFA-1 on mononuclear cells and ICAM-1 on tubular epithelial cells accelerates TGF-ß1-induced renal epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) of renal epithelial cells (RTECs) has pivotal roles in the development of renal fibrosis. Although the interaction of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes and its ligand, intracellular adhesion molecule 1 (ICAM-1), plays essential roles in most inflammatory reactions, its pathogenetic role in the EMT of RTECs remains to be clarified. In the present study, we investigated the effect of the interaction of LFA-1 on peripheral blood mononuclear cells (PBMCs) and ICAM-1 on HK-2 cells after stimulation with TGF-ß(1) on the EMT of RTECs. ICAM-1 was highly expressed in HK-2 cells. After TGF-ß(1) stimulation, the chemokines CCL3 and CXCL12 increased on HK-2 cells. After co-culture of PBMCs and HK-2 cells pre-stimulated with TGF-ß(1) (0.1 ng/ml) (HK-2-TGF-ß(1) (0.1)), the expression of the active form of LFA-1 increased on PBMCs; however, total LFA-1 expression did not change. The expression of the active form of LFA-1 on PBMCs did not increase after co-culture with not CCL3 but CXCL12 knockdown HK-2-TGF-ß(1) (0.1). The expression of epithelial cell junction markers (E-cadherin and occludin) further decreased and that of mesenchymal markers (vimentin and fibronectin) further increased in HK-2-TGF-ß(1) (0.1) after co-culture with PBMCs for 24 hrs (HK-2-TGF-ß(1) (0.1)-PBMCs). The phosphorylation of ERK 1/2 but not smad2 and smad3 increased in HK-2-TGF-ß(1) (0.1)-PBMCs. The snail and slug signaling did not increase HK-2-TGF-ß(1) (0.1)-PBMCs. Although the migration and invasion of HK-2 cells induced full EMT by a high dose (10.0 ng/ml) and long-term (72-96 hrs) TGF-ß(1) stimulation increased, that of HK-2-TGF-ß(1) (0.1)-PBMCs did not increase. These results suggested that HK-2 cells stimulated with TGF-ß(1) induced conformational activation of LFA-1 on PBMCs by increased CXCL12. Then, the direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK1/2 signaling to accelerate the part of EMT of HK-2 cells induced by TGF-ß(1).

Thermo-responsive polymer brushes as intelligent biointerfaces: preparation via ATRP and characterization.

PIPAAm-brush grafted glass substrates with various graft densities and chain lengths were prepared via surface-initiated ATRP. Temperature-dependent physicochemical properties of the surfaces were characterized by means of ATR/FT-IR spectroscopy, XPS, AFM, and contact angle measurements. ATRP conditions influence the amount of grafted PIPAAm and the surface wettability and roughness of the substrate. Fibronectin adsorption and EC adhesion increased with decreasing density of PIPAAm brushes. EC adhesion was diminished with increasing PIPAAm graft length. Thus, the preparation of PIPAAm brush surface with various graft densities and chain lengths using the surface-initiated ATRP is an effective method for modulating thermo-responsive properties of surfaces.