Diphenhydramine may be a preventive medicine against cisplatin-induced kidney toxicity.

Cisplatin is widely used as an anti-tumor drug for the treatment of solid tumors. Unfortunately, it causes kidney toxicity as a critical side effect, limiting its use, given that no preventive drug against cisplatin-induced kidney toxicity is currently available. Here, based on a repositioning analysis of the Food and Drug Administration Adverse Events Reporting System, we found that a previously developed drug, diphenhydramine, may provide a novel treatment for cisplatin-induced kidney toxicity. To confirm this, the actual efficacy of diphenhydramine was evaluated in in vitro and in vivo experiments. Diphenhydramine inhibited cisplatin-induced cell death in kidney proximal tubular cells. Mice administered cisplatin developed kidney injury with significant dysfunction (mean plasma creatinine: 0.43 vs 0.15 mg/dl) and showed augmented oxidative stress, increased apoptosis, elevated inflammatory cytokines, and MAPKs activation. However, most of these symptoms were suppressed by treatment with diphenhydramine. Furthermore, the concentration of cisplatin in the kidney was significantly attenuated in diphenhydramine-treated mice (mean platinum content: 70.0 vs 53.4 µg/g dry kidney weight). Importantly, diphenhydramine did not influence or interfere with the anti-tumor effect of cisplatin in any of the in vitro or in vivo experiments. In a selected cohort of 98 1:1 matched patients from a retrospective database of 1467 patients showed that patients with malignant cancer who had used diphenhydramine before cisplatin treatment exhibited significantly less acute kidney injury compared to ones who did not (6.1 % vs 22.4 %, respectively). Thus, diphenhydramine demonstrated efficacy as a novel preventive medicine against cisplatin-induced kidney toxicity.

Deletion of H-ferritin in macrophages alleviates obesity and diabetes induced by high-fat diet in mice.

AIMS/HYPOTHESIS: Iron accumulation affects obesity and diabetes, both of which are ameliorated by iron reduction. Ferritin, an iron-storage protein, plays a crucial role in iron metabolism. H-ferritin exerts its cytoprotective action by reducing toxicity via its ferroxidase activity. We investigated the role of macrophage H-ferritin in obesity and diabetes. METHODS: Conditional macrophage-specific H-ferritin (Fth, also known as Fth1) knockout (LysM-Cre Fth KO) mice were used and divided into four groups: wild-type (WT) and LysM-Cre Fth KO mice with normal diet (ND), and WT and LysM-Cre Fth KO mice with high-fat diet (HFD). These mice were analysed for characteristics of obesity and diabetes, tissue iron content, inflammation, oxidative stress, insulin sensitivity and metabolic measurements. RAW264.7 macrophage cells were used for in vitro experiments. RESULTS: Iron concentration reduced, and mRNA expression of ferroportin increased, in macrophages from LysM-Cre Fth KO mice. HFD-induced obesity was lower in LysM-Cre Fth KO mice than in WT mice at 12 weeks (body weight: KO 34.6 ± 5.6 g vs WT 40.1 ± 5.2 g). mRNA expression of inflammatory cytokines and infiltrated macrophages and oxidative stress increased in the adipose tissue of HFD-fed WT mice, but was not elevated in HFD-fed LysM-Cre Fth KO mice. However, WT mice fed an HFD had elevated iron concentration in adipose tissue and spleen, which was not observed in LysM-Cre Fth KO mice fed an HFD (adipose tissue [µmol Fe/g protein]: KO 1496 ± 479 vs WT 2316 ± 866; spleen [µmol Fe/g protein]: KO 218 ± 54 vs WT 334 ± 83). Moreover, HFD administration impaired both glucose tolerance and insulin sensitivity in WT mice, which was ameliorated in LysM-Cre Fth KO mice. In addition, energy expenditure, mRNA expression of thermogenic genes, and body temperature were higher in KO mice with HFD than WT mice with HFD. In vitro experiments showed that iron content was reduced, and lipopolysaccharide-induced Tnf-α (also known as Tnf) mRNA upregulation was inhibited in a macrophage cell line transfected with Fth siRNA. CONCLUSIONS/INTERPRETATION: Deletion of macrophage H-ferritin suppresses the inflammatory response by reducing intracellular iron levels, resulting in the prevention of HFD-induced obesity and diabetes. The findings from this study highlight macrophage iron levels as a potential therapeutic target for obesity and diabetes.

Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mouse myoblast cells in vitro. In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of MAPK signaling pathways. In vitro, iron overload also suppressed the differentiation of C2C12 myoblast cells but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis.-Ikeda, Y., Satoh, A., Horinouchi, Y., Hamano, H., Watanabe, H., Imao, M., Imanishi, M., Zamami, Y., Takechi, K., Izawa-Ishizawa, Y., Miyamoto, L., Hirayama, T., Nagasawa, H., Ishizawa, K., Aihara, K.-I., Tsuchiya, K., Tamaki, T. Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells.

Arterial calcification, a risk factor of cardiovascular events, develops with differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase involved in cardiovascular diseases such as atherosclerosis and aortic aneurysms, and rho-associated protein kinase (ROCK) is involved in the pathogenesis of vascular calcification. CypA is secreted in a ROCK activity-dependent manner and works as a mitogen via autocrine or paracrine mechanisms in VSMCs. We examined the involvement of the ROCK-CypA axis in VSMC calcification induced by inorganic phosphate (Pi), a potent cell mineralization initiator. We found that Pi stimulated ROCK activity, CypA secretion, extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation, and runt-related transcription factor 2 expression, resulting in calcium accumulation in rat aortic smooth muscle cells (RASMCs). The ROCK inhibitor Y-27632 significantly suppressed Pi-induced CypA secretion, ERK1/2 phosphorylation, and calcium accumulation. Recombinant CypA was found to be associated with increased calcium accumulation in RASMCs. Based on these results, we suggest that autocrine CypA is mediated by ROCK activity and is involved in Pi-induced ERK1/2 phosphorylation following calcification signaling in RASMCs.

Irinotecan-induced neutropenia is reduced by oral alkalization drugs: analysis using retrospective chart reviews and the spontaneous reporting database.

PURPOSE: SN-38, an active metabolite of irinotecan, is reabsorbed by the intestinal tract during excretion, causing diarrhoea and neutropenia. In addition, the association between blood levels of SN-38 and neutropenia has been reported previously, and the rapid excretion of SN-38 from the intestinal tract is considered to prevent neutropenia. Oral alkalization drugs are used as prophylactic agents for suppressing SN-38 reabsorption. The relationship between oral alkalization drugs and neutropenia, however, has not been well studied. The aim of this study was to investigate the relationship between oral alkalization drugs and neutropenia in irinotecan-treated patients. METHODS AND RESULTS: Patients with cervical or ovarian cancer were administered irinotecan and investigated by medical chart reviews to determine whether oral alkalization drugs were effective at ameliorating irinotecan-induced neutropenia. The drug combination in the oral alkalization drugs-ursodeoxycholic acid, magnesium oxide, and sodium hydrogen carbonate-significantly improved neutrophil counts and reduced dose intensity compared with those of non-users. In the large-scale Japanese Adverse Drug Event Report database, the reporting odds ratio of irinotecan-induced neutropenia was significantly lower when irinotecan had been given in combination with oral alkalization drugs. CONCLUSIONS: These data indicate that oral alkalization drugs may reduce the frequency of neutropenia caused by irinotecan administration, making it possible to increase the dose safely.

The uremic toxin indoxyl sulfate interferes with iron metabolism by regulating hepcidin in chronic kidney disease.

Background: Hepcidin secreted by hepatocytes is a key regulator of iron metabolism throughout the body. Hepcidin concentrations are increased in chronic kidney disease (CKD), contributing to abnormalities in iron metabolism. Levels of indoxyl sulfate (IS), a uremic toxin, are also elevated in CKD. However, the effect of IS accumulation on iron metabolism remains unclear. Methods: We used HepG2 cells to determine the mechanism by which IS regulates hepcidin concentrations. We also used a mouse model of adenine-induced CKD. The CKD mice were divided into two groups: one was treated using AST-120 and the other received no treatment. We examined control mice, CKD mice, CKD mice treated using AST-120 and mice treated with IS via drinking water. Results: In the in vitro experiments using HepG2 cells, IS increased hepcidin expression in a dose-dependent manner. Silencing of the aryl hydrocarbon receptor (AhR) inhibited IS-induced hepcidin expression. Furthermore, IS induced oxidative stress and antioxidant drugs diminished IS-induced hepcidin expression. Adenine-induced CKD mice demonstrated an increase in hepcidin concentrations; this increase was reduced by AST-120, an oral adsorbent of the uremic toxin. CKD mice showed renal anemia, decreased plasma iron concentration, increased plasma ferritin and increased iron content in the spleen. Ferroportin was decreased in the duodenum and increased in the spleen. These changes were ameliorated by AST-120 treatment. Mice treated by direct IS administration showed hepatic hepcidin upregulation. Conclusions: IS affects iron metabolism in CKD by participating in hepcidin regulation via pathways that depend on AhR and oxidative stress.

Mechanisms of the pH- and Oxygen-Dependent Oxidation Activities of Artesunate.

Artemisinin was discovered in 1971 as a constituent of the wormwood genus plant (Artemisia annua). This plant has been used as an herbal medicine to treat malaria since ancient times. The compound artemisinin has a sesquiterpene lactone bearing a peroxide group that offers its biological activity. In addition to anti-malarial activity, artemisinin derivatives have been reported to exert antitumor activity in cancer cells, and have attracted attention as potential anti-cancer drugs. Mechanisms that might explain the antitumor activities of artemisinin derivatives reportedly induction of apoptosis, angiogenesis inhibitory effects, inhibition of hypoxia-inducible factor-1α (HIF-1α) activation, and direct DNA injury. Reactive oxygen species (ROS) generation is involved in many cases. However, little is known about the mechanism of ROS formation from artemisinin derivatives and what types of ROS are produced. Therefore, we investigated the iron-induced ROS formation mechanism by using artesunate, a water-soluble artemisinin derivative, which is thought to be the underlying mechanism involved in artesunate-mediated cell death. The ROS generated by the coexistence of iron(II), artesunate, and molecular oxygen was a hydroxyl radical or hydroxyl radical-like ROS. Artesunate can reduce iron(III) to iron(II), which enables generation of ROS irrespective of the iron valence. We found that reduction from iron(III) to iron(II) was activated in the acidic rather than the neutral region and was proportional to the hydrogen ion concentration.

Nitrosonifedipine, a Photodegradation Product of Nifedipine, Suppresses Pharmacologically Induced Aortic Aneurysm Formation.

BACKGROUND/AIMS: We have reported that nitrosonifedipine (NO-NIF), a photodegradation product of nifedipine, has strong antioxidant and endothelial protective effects, and can suppress several cardiovascular diseases in animal models. The objective of the present study was to investigate the effects of NO-NIF on aortic aneurysm formation. METHODS: The mice were infused with ß-aminopropionitrile for 2 weeks and angiotensin II for 6 weeks to induce aortic aneurysm formation. The oxidative stress was measured by dihydroethidium staining and nitrotyrosine staining. The expressions of inflammation-related genes were assessed by quantitative real-time PCR and immunohistochemical staining. To clarify the mechanisms of how NO-NIF suppresses vascular cell adhesion molecule (VCAM)-1, endothelial cells were used in in vitro system. RESULTS: NO-NIF suppressed pharmacologically induced the aortic aneurysm formation and aortic expansion without blood pressure changes. NO-NIF suppressed elastin degradation and matrix metalloproteinase-2 mRNA expression. NO-NIF suppressed the reactive oxygen species-cyclophilin A positive feedback loop. Upregulated mRNA expressions of inflammation-related genes and endothelial VCAM-1 were suppressed by NO-NIF co-treatment in aortae. CONCLUSION: NO-NIF has the potential to be a new, nifedipine-derived therapeutic drug for suppressing aortic aneurysm formation by directly improving aortic structure with its strong ability to reduce oxidative stress and inflammation.

Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation.

Renal anemia is a major complication in chronic kidney disease (CKD). Iron supplementation, as well as erythropoiesis-stimulating agents, are widely used for treatment of renal anemia. However, excess iron causes oxidative stress via the Fenton reaction, and iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CKD. EPO gene expression was suppressed in mice following direct iron treatment. Hypoxia-inducible factor-2 alpha (HIF-2α), a positive regulator of the EPO gene, was also diminished in the kidney of mice following iron treatment. Anemia-induced increase in renal EPO and HIF-2α expression was inhibited by iron treatment. In in vitro experiments using EPO-producing HepG2 cells, iron stimulation reduced the expression of the EPO gene, as well as HIF-2α. Moreover, iron treatment augmented oxidative stress, and iron-induced reduction of EPO and HIF-2α expression was restored by tempol, an antioxidant compound. HIF-2α interaction with the Epo promoter was inhibited by iron treatment, and was restored by tempol. These findings suggested that iron supplementation reduced EPO gene expression via an oxidative stress-HIF-2α-dependent signaling pathway.

Topical application of nitrosonifedipine, a novel radical scavenger, ameliorates ischemic skin flap necrosis in a mouse model.

Ischemic skin flap necrosis can occur in random pattern flaps. An excess amount of reactive oxygen species is generated and causes necrosis in the ischemic tissue. Nitrosonifedipine (NO-NIF) has been demonstrated to possess potent radical scavenging ability. However, there has been no study on the effects of NO-NIF on ischemic skin flap necrosis. Therefore, they evaluated the potential of NO-NIF in ameliorating ischemic skin flap necrosis in a mouse model. A random pattern skin flap (1.0 × 3.0 cm) was elevated on the dorsum of C57BL/6 mice. NO-NIF was administered by topical injection immediately after surgery and every 24 hours thereafter. Flap survival was evaluated on postoperative day 7. Tissue samples from the skin flaps were harvested on postoperative days 1 and 3 to analyze oxidative stress, apoptosis and endothelial dysfunction. The viable area of the flap in the NO-NIF group was significantly increased (78.30 ± 7.041%) compared with that of the control group (47.77 ± 6.549%, p < 0.01). NO-NIF reduced oxidative stress, apoptosis and endothelial dysfunction, which were evidenced by the decrease of malondialdehyde, p22phox protein expression, number of apoptotic cells, phosphorylated p38 MAPK protein expression, and vascular cell adhesion molecule-1 protein expression while endothelial nitric oxide synthase protein expression was increased. In conclusion, they demonstrated that NO-NIF ameliorated ischemic skin flap necrosis by reducing oxidative stress, apoptosis, and endothelial dysfunction. NO-NIF is considered to be a candidate for the treatment of ischemic flap necrosis.

Hypoxia-Inducible Factor-1α in Smooth Muscle Cells Protects Against Aortic Aneurysms-Brief Report.

OBJECTIVE: The purpose of this study was to determine the role of smooth muscle cell-derived hypoxia-inducible factor-1α (Hif-1α) in the pathogenesis of aortic aneurysms. APPROACH AND RESULTS: Control mice and smooth muscle cell-specific hypoxia-inducible factor-1α-deficient mice were infused with ß-aminopropionitrile for 2 weeks and angiotensin II for 6 weeks to induce aortic aneurysm formation. Mutant mice experienced increased levels of aneurysm formation of the thoracic or abdominal aorta with more severe elastin disruption, compared with control mice. Smooth muscle cell-specific hypoxia-inducible factor-1α deficiency did not affect matrix metalloproteinase-2 activity; however, the activity of lysyl oxidase and the levels of tropoelastin mRNA in the angiotensin II- and ß-aminopropionitrile-treated aortae, associated with elastin fiber formation, were suppressed. Furthermore, we observed reduced volumes of mature cross-linked elastin in the thoracoabdominal aorta after treatment with angiotensin II and ß-aminopropionitrile. CONCLUSIONS: Deficiency of smooth muscle cell-derived hypoxia-inducible factor-1α augments aortic aneurysms, accompanied by disruption of elastin fiber formation, but not changes of elastin fiber degradation.

Nitrite Activates 5'AMP-Activated Protein Kinase-Endothelial Nitric Oxide Synthase Pathway in Human Glomerular Endothelial Cells.

Recent studies have shown that orally supplied nitrates, which substantially exist in our daily diets, are reduced into nitrites and become significant sources of nitric oxide (NO) especially in hypoxic tissues. However, physiological significance of nitrites in normal tissues has not been elucidated though our serum concentrations of nitrites reach as high as micromolar levels. We investigated effects of nitrite on endothelial NO synthase (eNOS) using human glomerular endothelial cells to reveal potential glomerular-protective actions of nitrites with its underlying molecular mechanism. Here we demonstrate that nitrite stimulation evokes eNOS activation which is dependent on 5'AMP-activated protein kinase (AMPK) activation in accordance with ATP reduction. Thus, nitrites should facilitate AMPK-eNOS pathway in an energy level-dependent manner in endothelial cells. The activation of AMPK-eNOS signals is suggested to be involved in vascular and renal protective effects of nitrites and nitrates. Nitrites may harbor beneficial effects on metabolic regulations as AMPK activators.

HIF-2α/ARNT complex regulates hair development via induction of p21(Waf1/Cip1) and p27(Kip1).

The hypoxia-inducible factors HIF-1α or HIF-2α form heterodimeric complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1α/ARNT and HIF-2α/ARNT complexes activate hypoxia-inducible genes that play critical roles in angiogenesis, anaerobic metabolism, and other processes in response to O2 deprivation. HIF-2α is known to regulate the function and/or differentiation of stem cells by activating the POU domain transcription factor Oct4; however, the precise underlying mechanism is unknown. This study examined the role of HIF-2α/ARNT in hair development using conditional-knockout mice, in which Arnt was specifically deleted in keratinocytes. In wild-type mice, HIF-2α and ARNT were highly expressed in the precortex above the hair matrix, an area containing differentiating stem cells. An analysis of hair size and type in these mice showed that loss of ARNT decreased the production of zigzag hairs, corresponding to reduced expression of HIF-2α and induction of the mammalian cyclin-dependent kinase inhibitors p21(Waf1/Cip1) and p27 (Kip1). The results suggest that the HIF-2α/ARNT complex regulates hair follicle differentiation via induction of p21(Waf1/Cip1) and possibly p27(Kip1), as p27(Kip1) expression was not altered in ARNT knockout mice. The findings provide insight into a possible mechanism underlying hair growth disorders and can be useful for future studies on hair follicle response to insults, such as chemotherapy and ionizing radiation.

Angiotensin II alters the expression of duodenal iron transporters, hepatic hepcidin, and body iron distribution in mice.

PURPOSE: Angiotensin II (ANG II) has been shown to affect iron metabolism through alteration of iron transporters, leading to increased cellular and tissue iron contents. Serum ferritin, a marker of body iron storage, is elevated in various cardiovascular diseases, including hypertension. However, the associated changes in iron absorption and the mechanism underlying increased iron content in a hypertensive state remain unclear. METHODS: The C57BL6/J mice were treated with ANG II to generate a model of hypertension. Mice were divided into three groups: (1) control, (2) ANG II-treated, and (3) ANG II-treated and ANG II receptor blocker (ARB)-administered (ANG II-ARB) groups. RESULTS: Mice treated with ANG II showed increased serum ferritin levels compared to vehicle-treated control mice. In ANG II-treated mice, duodenal divalent metal transporter-1 and ferroportin (FPN) expression levels were increased and hepatic hepcidin mRNA expression and serum hepcidin concentration were reduced. The mRNA expression of bone morphogenetic protein 6 and CCAAT/enhancer-binding protein alpha, which are regulators of hepcidin, was also down-regulated in the livers of ANG II-treated mice. In terms of tissue iron content, macrophage iron content and renal iron content were increased by ANG II treatment, and these increases were associated with reduced expression of transferrin receptor 1 and FPN and increased expression of ferritin. These changes induced by ANG II treatment were ameliorated by the administration of an ARB. CONCLUSIONS: Angiotensin II (ANG II) altered the expression of duodenal iron transporters and reduced hepcidin levels, contributing to the alteration of body iron distribution.

Hypoxia decreases glucagon-like peptide-1 secretion from the GLUTag cell line.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from L cells located in the intestinal epithelium. It is known that intestinal oxygen tension is decreased postprandially. In addition, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), which accumulates in cells under hypoxic conditions, was significantly increased in the colons of mice with food intake, indicating that the oxygen concentration is likely reduced in the colon after eating. Therefore, we hypothesized that GLP-1 secretion is affected by oxygen tension. We found that forskolin-stimulated GLP-1 secretion from GLUTag cells, a model of intestinal L cells, is suppressed in hypoxia (1% O2). Forskolin-stimulated elevations of preproglucagon (ppGCG) and proprotein convertase 1/3 (PC1/3) mRNA expression were decreased under hypoxic conditions. The finding that H89, a protein kinase A (PKA) inhibitor, inhibited the forskolin-stimulated increase of ppGCG and PC1/3 indicated that the cAMP-PKA pathway is involved in the hypoxia-induced suppression of the genes. Hypoxia decreased hexokinase 2 mRNA and protein expression and increased lactate dehydrogenase A mRNA and protein expression. Concomitantly, lactate production was increased and ATP production was decreased. Together, the results indicate that hypoxia decreases glucose utilization for ATP production, which probably causes a decrease in cAMP production and in subsequent GLP-1 production. Our findings suggest that the postprandial decrease in oxygen tension in the intestine attenuates GLP-1 secretion.

Augmentation index does not reflect risk of coronary artery disease in elderly patients.

BACKGROUND: Augmentation index (AI) has been used as a clinical index of arterial stiffness and has been reported to be an independent predictor of cardiovascular events, but some investigators have reported that AI is not a useful marker to identify coronary artery disease (CAD) in elderly patients. The majority of CAD patients are elderly people, therefore the aim of this study was to examine whether AI is a useful marker to identify the risk of CAD. METHODS AND RESULTS: A total of 120 patients (69±10 years of age; 83 male) who underwent cardiac catheterization for suspected CAD were enrolled. Invasive central blood pressure (BP) was measured using a fluid-filled catheter. Non-invasive AI was calculated by the SphygmoCor (AtCor Medical) system at the end of catheterization. Subjects consisted of 99 patients with CAD and 21 patients without CAD. There was no significant difference in AI between the CAD and the non-CAD groups (24±10 vs. 24±14%). Non-invasive systolic central BP was lower than the invasive systolic central BP (115±18 vs. 130±23 mmHg, P<0.001) in all patients. Non-invasive diastolic central BP was greater than the invasive diastolic central BP (67±10 vs. 63±10 mmHg, P<0.001). CONCLUSIONS: In elderly patients, AI may not be a useful marker to identify CAD.

Albuminuria and Serum Tumor Necrosis Factor Receptor Levels in Patients with Type 2 Diabetes on SGLT2 Inhibitors: A Prospective Study.

INTRODUCTION: Large-scale clinical trials of sodium-glucose cotransporter 2 inhibitors (SGLT2i) demonstrate proteinuria-reducing effects in diabetic kidney disease, even after treatment with renin-angiotensin inhibitors. The precise mechanism for this favorable effect remains unclear. This prospective open-label single-arm study investigated factors associated with a reduction in proteinuria after SGLT2i administration. METHODS: Patients with type 2 diabetes (T2DM) who had glycated hemoglobin (HbA1c) levels ≥ 6.5% despite dietary and/or oral hypoglycemic monotherapy were recruited and administered the recommended daily dose of SGLT2i for 4 months. Dual primary outcomes were changes in the urine albumin-to-creatinine ratio (uACR) and urine liver-type fatty acid-binding protein (L-FABP)-to-creatinine ratio (uL-FABPCR) at month 4 from baseline. Changes in kidney injury, inflammation, and oxidative stress biomarkers were investigated as secondary endpoints to examine the effects of this treatment on the kidney. The correlation between renal outcomes and clinical indicators, including circulating tumor necrosis factor receptors (TNFR) 1 and 2, was evaluated using univariate and multivariate analyses. RESULTS: Participants (n = 123) had a mean age of 64.1 years (SD 13.4), with 50.4% being male. The median BMI was 25.8 kg/m2 (interquartile range (IQR) 23.1-28.9), and the median HbA1c level was 7.3% (IQR 6.9-8.3). After SGLT2i administration, the uACR declined from 19.2 mg/gCr (IQR 7.1-48.7) to 13.3 mg/gCr (IQR 7.5-31.6), whereas the uL-FABPCR was not influenced. In univariate analysis, the change in log-transformed uACR due to SGLT2i administration showed a positive correlation with the change in serum TNFR1 level (R = 0.244, p < 0.01). Multivariate regression analysis, including confounding factors, showed that the changes in serum TNFR1 level were independently associated with the changes in the log-transformed uACR (independent t = 2.102, p < 0.05). CONCLUSION: After the 4-month SGLT2i administration, decreased albuminuria level was associated with decreased serum TNFR level in patients with T2DM. TRIAL REGISTRATION NUMBER: UMIN000031947.

Previous studies have demonstrated the synergistic proteinuria-reducing effect of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in combination therapy with renin­angiotensin system blockers; however, the underlying mechanisms of this effect are poorly understood. This study was based on our hypothesis that the proteinuria-reducing effect is associated with the anti-inflammatory effects of SGLT2i beyond the effect on glycemic control. In total, 123 patients with type 2 diabetes mellitus (T2DM) were administered the recommended daily dose of SGLT2i for 4 months. Dual primary outcomes were changes in the urine albumin-to-creatinine ratio (uACR) and urine liver-type fatty acid-binding protein (L-FABP)-to-creatinine ratio (uL-FABPCR) as markers of glomerular and proximal tubular damage at 4 months from the baseline. Secondary outcomes included changes in kidney injury biomarkers, inflammation, and oxidative stress to examine the effects of treatment on the kidneys. The correlation between renal outcomes and clinical indicators, including circulating tumor necrosis factor receptors (TNFR) 1 and 2, was evaluated using univariate and multivariate analyses. We found that administration of SGLT2i decreased the urine albumin-to-creatinine ratio but did not affect the urine liver-type fatty acid-binding protein-to-creatinine ratio. Further, SGLT2i may exert a proteinuria-reducing effect dependent on the anti-inflammatory effect in patients with T2DM. The inflammation-reducing and renoprotective mechanisms of SGLT2i remain to be fully clarified, but this study provides novel evidence regarding the mechanism. The study findings can help in developing anti-inflammatory agents for metabolic diseases.

Heparin cofactor II, a serine protease inhibitor, promotes angiogenesis via activation of the AMP-activated protein kinase-endothelial nitric-oxide synthase signaling pathway.

We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII(+/-)) mice and male littermate wild-type (HCII(+/+)) mice at the age of 12-16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII(+/-) mice compared with that in HCII(+/+) mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII(+/-) mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII(+/-) mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCII-induced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency.

Dietary iron restriction inhibits progression of diabetic nephropathy in db/db mice.

Excess iron causes oxidative stress through hydroxyl-radical production via Fenton/Haber-Weiss reactions. Recently, body iron reduction has been found to ameliorate diabetes. In the present study, we examined the protective effect of dietary iron restriction against diabetic nephropathy in the db/db mouse model of diabetic nephropathy using db/m mice as controls. The db/db mice were divided into two groups and fed a normal diet (ND) or a low-iron diet (LID). Increasing urinary albumin excretion was observed in the ND db/db mice, but this was suppressed in db/db mice with LID. Histologically, the db/db mice in the ND group had increased glomerular volume and mesangial area compared with the LID group. Augmented deposition of extracellular matrixes was decreased in db/db mice with LID. In terms of oxidative stress, increased superoxide production observed in the kidneys of the ND db/db mice was diminished in the LID group. NADPH oxidase activity and renal expression of NADPH oxidase components p22(phox) and NADPH oxidase 4 (NOX4) were augmented in the ND group, and this was abolished by LID. There were no differences in expression of renal iron importers, transferrin receptor, or divalent metal transporter-1 between db/m mice and db/db mice. The level of ferroportin, an iron exporter, increased in the kidneys of the db/db mice. Urinary iron excretion was significantly higher in ND db/db mice and was reduced in the LID group. These findings suggest that dietary iron restriction exerts a preventive effect on the progression of diabetic nephropathy partly due to the reduction of oxidative stress.

Bovine milk-derived lactoferrin exerts proangiogenic effects in an Src-Akt-eNOS-dependent manner in response to ischemia.

Lactoferrin (LF) exerts a variety of biological effects, including the promotion of angiogenesis by increasing the expression of angiogenesis-related genes and reducing blood pressure via a nitric oxide-dependent mechanism. In this study, we investigated the effects of LF on angiogenesis using C57BL/6J mice that received daily unilateral treatment with or without bovine milk-derived LF (bLF) after unilateral hindlimb surgery. The analysis of laser speckle blood flow showed that bLF treatment promoted blood flow recovery in response to ischemic hindlimb. The capillary density of ischemic adductor muscles and the phosphorylation of Src, Akt, and endothelial nitric oxide synthase (eNOS) were also significantly higher in bLF-treated mice than in vehicle-treated mice. Furthermore, bLF increased the phosphorylation levels of Src, Akt, and eNOS in in vitro experiments using human aortic endothelial cells. The action of bLF on eNOS phosphorylation was abolished by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo [3,4-d]pyrimidine (PP2), an Src inhibitor. Similarly, bLF-induced acceleration of tube formation, cell proliferation, and cell migration in human aortic endothelial cells were inhibited by LY294002 or PP2. Thus, bLF promotes vascular endothelial cell function via an Src Akt eNOS-dependent pathway, thereby contributing to revascularization in response to ischemia.