Empagliflozin Attenuates Vascular Calcification in Mice with Chronic Kidney Disease by Regulating the NFR2/HO-1 Anti-Inflammatory Pathway through AMPK Activation.

Vascular calcification (VC) is associated with increased cardiovascular risks in patients with chronic kidney disease (CKD). Sodium-glucose cotransporter 2 inhibitors, such as empagliflozin, can improve cardiovascular and renal outcomes. We assessed the expression of Runt-related transcription factor 2 (Runx2), interleukin (IL)-1ß, IL-6, AMP-activated protein kinase (AMPK), nuclear factor erythroid-2-related factor (Nrf2), and heme oxygenase 1 (HO-1) in inorganic phosphate-induced VC in mouse vascular smooth muscle cells (VSMCs) to investigate the mechanisms underlying empagliflozin's therapeutic effects. We evaluated biochemical parameters, mean artery pressure (MAP), pulse wave velocity (PWV), transcutaneous glomerular filtration rate (GFR), and histology in an in vivo mouse model with VC induced by an oral high-phosphorus diet following a 5/6 nephrectomy in ApoE-/- mice. Compared to the control group, empagliflozin-treated mice showed significant reductions in blood glucose, MAP, PWV, and calcification, as well as increased calcium and GFR levels. Empagliflozin inhibited osteogenic trans-differentiation by decreasing inflammatory cytokine expression and increasing AMPK, Nrf2, and HO-1 levels. Empagliflozin mitigates high phosphate-induced calcification in mouse VSMCs through the Nrf2/HO-1 anti-inflammatory pathway by activating AMPK. Animal experiments suggested that empagliflozin reduces VC in CKD ApoE-/- mice on a high-phosphate diet.

Association between Serum Soluble Urokinase-Type Plasminogen Activator Receptor Level and Arterial Stiffness in Chronic Hemodialysis Patients.

Cardiovascular diseases (CVDs) remain a significant cause of death in hemodialysis (HD) patients. To explore their associations, we examine the role of soluble urokinase-type plasminogen activator receptor (suPAR) in arterial stiffness in chronic HD patients. From June to August 2020, we recruited 135 chronic HD patients. The arterial stiffness group included patients with a carotid-femoral pulse-wave velocity (cfPWV) of >10 m/s. Fifty-five HD patients (40.7%) were in the arterial stiffness group. They had a higher prevalence of diabetes (p = 0.001) and hypertension (p = 0.039), were older (p = 0.007) and had higher aortic systolic blood pressure (p = 0.034), brachial systolic blood pressure (p = 0.025), glucose (p = 0.019), C-reactive protein (p = 0.039), and AIx75 (p = 0.003) and suPAR (p < 0.001) levels than the control group. After we performed multivariable logistic regression analysis, except age and glucose, serum suPAR (odds ratio [OR]: 2.05; 95% confidence interval [CI]: 1.48-2.70, p < 0.001) was independently associated with arterial stiffness in chronic HD patients. In the multivariable linear regression analysis, suPAR positively correlated with cfPWV (ß = 0.475, p < 0.001) and could serve as a biomarker for arterial stiffness development in patients undergoing HD.

Correlation between Serum 25-Hydroxyvitamin D Level and Peripheral Arterial Stiffness in Chronic Kidney Disease Stage 3-5 Patients.

Vitamin D deficiency and high brachial-ankle pulse wave velocity (baPWV) are each independently associated with higher incidence of mortality and cardiovascular (CV) disease or CV events, respectively. This study aimed to evaluate the relationship between serum 25-hydroxyvitamin D levels and baPWV in non-dialysis patients with stage 3−5 chronic kidney disease (CKD). We enrolled 180 CKD patients. A commercial enzyme-linked immunosorbent assay was used to measure 25-hydroxyvitamin D levels. BaPWV values were measured using an automatic pulse wave analyzer. Either left or right baPWV > 18.0 m/s was considered indicative of peripheral arterial stiffness (PAS). In this study, 73 (40.6%) patients were found to have PAS. Compared to those without PAS (control group), patients with PAS were older and had higher incidence of diabetes mellitus, higher systolic and diastolic blood pressure, higher levels of intact parathyroid hormone, and C-reactive protein, and lower levels of 25-hydroxyvitamin D. Multivariate logistic regression analysis found 25-hydroxyvitamin D levels (odds ratio [OR]: 0.895, 95% confidence interval [CI] 0.828−0.968, p = 0.005) and old age (OR: 1.140, 95% CI 1.088−1.194, p < 0.001) to be independently associated with PAS in patients with stage 3−5 CKD. Lower serum 25-hydroxyvitamin D levels and older age were associated with PAS in these patients.

Dapagliflozin Ameliorates Lipopolysaccharide Related Acute Kidney Injury in Mice with Streptozotocin-induced Diabetes Mellitus.

Sepsis, which is a serious medical condition induced by infection, has been the most common cause of acute kidney injury (AKI) and is associated with high mortality and morbidity. Sodium-glucose cotransporter 2 (SGLT2) inhibitor is a new oral antidiabetic drug that has greatly improved the cardiovascular and renal outcomes in patients with type 2 diabetes independent of its sugar lowering effect, possibly by attenuation of the inflammatory process. We investigated the effect of the SGLT2 inhibitor dapagliflozin on lipopolysaccharide (LPS)-induced endotoxic shock with AKI in streptozotocin-induced diabetic mice. Endotoxin shock with AKI was induced by intravenous injection of 10 mg/kg LPS in C57BL6 mice with streptozotocin-induced diabetic mellitus with or without dapagliflozin treatment. Observation was done for 48 hours thereafter. In addition, NRK-52E cells incubated with LPS or dapagliflozin were evaluated for the possible mechanism. Treatment with dapagliflozin attenuated LPS-induced endotoxic shock associated AKI and decreased the inflammatory cytokines in diabetic mice. In the in vitro study, dapagliflozin decreased the expression of inflammatory cytokines and reactive oxygen species and increased the expressions of AMP-activated protein kinase (AMPK), nuclear factor erythroid-2-related factor, and heme oxygenase 1. These results demonstrated that dapagliflozin can attenuate LPS-induced endotoxic shock associated with AKI; this was possibly mediated by activation of the AMPK pathway.

Linagliptin Protects against Endotoxin-Induced Acute Kidney Injury in Rats by Decreasing Inflammatory Cytokines and Reactive Oxygen Species.

Septic shock can increase pro-inflammatory cytokines, reactive oxygen species (ROS), and multiple organ dysfunction syndrome (MODs) and even lead to death. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been proven to exert potential antioxidant and anti-inflammatory effects. We investigated the effects of linagliptin on endotoxic shock and acute kidney injury (AKI) in animal and cell models. In the cell model, linagliptin attenuated ROS by activating the AMP-activated protein kinase (AMPK) pathway, restoring nuclear-factor-erythroid-2-related factor (Nrf2) and heme oxygenase 1 (HO-1) protein, and decreasing pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß)). In the animal model, 14-week-old conscious Wistar-Kyoto rats were randomly divided into three groups (n = 8 in each group). Endotoxin shock with MODs was induced by the intravenous injection of Klebsiella pneumoniae lipopolysaccharide (LPS, 20 mg/kg). Linagliptin improved animal survival without affecting hemodynamic profiles. In the histopathology and immunohistochemistry examinations of the rat kidneys, linagliptin (10 mg/kg) suppressed nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inducible nitric oxide synthase (iNOS), decreased injury scores, and preserved E-cadherin expression from LPS damage. In conclusion, linagliptin ameliorated endotoxin-shock-induced AKI by reducing ROS via AMPK pathway activation and suppressing the release of TNF-α and IL-1ß in conscious rats.

Suppression of multiple processes relevant to cancer progression by benzyl isothiocyanate may result from the inhibition of Aurora A kinase activity.

Cruciferous vegetables are good sources of phytochemicals that have the potential to prevent cancer. Benzyl isothiocyanate (BITC) is the hydrolysis product of glucosinolates that are especially abundant in cruciferous vegetables. The anti-cancer activities of BITC have been studied for decades. The mechanisms of reducing the incidence of cancer by BITC involve multiple pathways, including the inhibition of proliferation, induction of apoptosis and inhibition of angiogenesis. One of the major common phenotypes induced by BITC in previous studies is G2/M cell cycle arrest. Therefore, interference of mitosis progression is likely to be an important anti-tumor mechanism of BITC. Using immunofluorescence staining, we show that BITC induces cell arrest in mitosis by perturbation of mitotic spindles. The abnormal mitotic spindles were resulted from the inactivation of Aurora A by BITC. The fact that BITC inhibits the activation of Aurora A and disrupts mitotic spindles provides one of the possible explanations why BITC is able to arrest cells in the G2/M phase and induce apoptosis in many previous studies. Besides, Aurora A is an essential player of mitosis and also has non-mitotic functions in tumorigenesis. As an inhibitor of Aurora A, BITC not only is an antimitotic agent but also inhibits tumor progression via many pathways.

Association of Low Serum l-Carnitine Levels with Aortic Stiffness in Patients with Non-Dialysis Chronic Kidney Disease.

l-carnitine (LC) is a co-factor in fatty acid metabolism; its role with respect to aortic stiffness (AS) associated with chronic kidney disease (CKD) was unclear. Our aim was to investigate associations between serum LC levels and AS in patients with non-dialysis CKD stage 3-5. The AS patients were those with carotid-femoral pulse wave velocities (cfPWV) >10 m/s; those with cfPWV ≤10 m/s were included as controls. Serum LC was measured by liquid chromatography and mass spectrometry. Of 136 CKD patients, the 44 (32.4%) with AS were older, exhibited higher rates of diabetes, and had elevated diastolic and systolic blood pressures (SBP), elevated fasting glucose levels and lower levels of serum LC compared to controls. Multivariable logistic regression revealed that serum LC (odds ratio [OR] = 0.949, 95% confidence interval [CI] 0.911-0.988, p = 0.011) and age (OR = 1.055, 95% CI 1.013-1.099, p = 0.009) were independent predictors of AS. Multivariable stepwise linear regression revealed significant positive (age and SBP) and negative (serum LC) correlations with cfPWV. The area under the curve of serum LC as a means to predict AS in CKD patients was 0.657 (95% CI 0.571-0.736, p = 0.0009). We concluded that low serum LC is a significant predictor of AS in patients diagnosed with CKD.

Vanadocene dichloride inhibits cell proliferation by targeting Aurora B.

Vanadocene dichloride (VDC) was shown to exhibit antitumor properties against a wide spectrum of tumor cell lines. Many studies have been carried out to reveal the bioactivities of VDC and the interaction mechanism of VDC with biological molecules in test tubes. One of the bioactivities of VDC is to arrest the cell cycle at the G2/M phase. However, its underlying mechanisms of action and cytotoxicity profile are still not fully understood. HeLa cells were used in this study, and the IC50 value of VDC was 8.61 µM after a 24-hour treatment. We used an immunofluorescence staining method to analyze the morphology of cells in the mitosis stage to elucidate what defects caused cell arrest in mitosis. Chromosomal misalignment was found to be the major phenotype. One of the proteins responsible for chromosome alignment at the metaphase is Aurora B kinase. Results of immunoblotting assay showed that Aurora B kinase activity was inhibited by VDC treatment. More than 50% of the Aurora B activity was inhibited when cells were treated with VDC at a concentration of 6.25 µM. That VDC was able to induce defects in chromosomal alignment at the metaphase by inhibiting the activity of Aurora B kinase is an important mechanism of VDC to be developed as an antitumor agent.

Anticancer activity of Kalanchoe tubiflora extract against human lung cancer cells in vitro and in vivo.

Uncontrolled cell proliferation is a common feature of human cancer. Some of herbal extract or plant-derived medicine had been shown as an important source of effective anticancer agents. We previously reported that an n-BuOH-soluble fraction of Kalanchoe tubiflora has antiproliferative activity by inducing mitotic catastrophe. In this study, we showed that the H2 O-soluble fraction of Kalanchoe tubiflora (KT-W) caused cell cycle arrest, and senescence-inducing activities in A549 cells. We used 2 dimensional PAGE to analyze the protein expression levels after KT-W treatment, and identified that the energy metabolism-related proteins and senescence-related proteins were disturbed. In vivo experiments showed that the tumor growths in A549-xenografted nude mice were effectively inhibited by KT-W. Our findings implied that KT-W is a putative antitumor agent by inducing cell cycle arrest and senescence. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1663-1673, 2016.

Euphorbia formosana root extract induces apoptosis by caspase-dependent cell death via Fas and mitochondrial pathway in THP-1 human leukemic cells.

Acute myeloid leukemia (AML), a very rare type of cancer, generally affects patients over 50 years old. While clinical drugs to treat advanced stages of AML exist, the disease becomes increasingly resistant to therapies. Euphorbia formosana Hayata (EF) is a native Taiwanese medicinal plant used to treat rheumatism, liver cirrhosis, herpes zoster, scabies, and photoaging, along with tumor suppression. However, the mechanisms by which it suppresses tumors have not been explored. Here, we provide molecular evidence that a hot-water extract of Euphorbia formosana (EFW) selectively inhibited the growth of human leukemic cancer cells more than other solid human cancer cell lines. Most importantly, the plant extract had limited toxicity toward healthy peripheral blood mononuclear cells (PBMCs). After THP-1 leukemic cells were treated with 50-100 µg/mL EFW for one day, the S phase DNA content of the cells increased, while treatment with 200-400 µg/mL caused the cells to accumulate in the G0/G1 phase. Notably, EFW did not affect A-549 lung cancer cells. The effectiveness of EFW against THP-1 cells may be through caspase-dependent apoptosis in leukemic cells, which is mediated through the Fas and mitochondrial pathways. The potent antileukemic activity of EFW in vitro warrants further investigation of this plant to treat leukemias and other malignancies.

Kalanchoe tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus.

BACKGROUND: Kalanchoe tubiflora (KT) is a succulent plant native to Madagascar, and is commonly used as a medicinal agent in Southern Brazil. The underlying mechanisms of tumor suppression are largely unexplored. METHODS: Cell viability and wound-healing were analyzed by MTT assay and scratch assay respectively. Cell cycle profiles were analyzed by FACS. Mitotic defects were analyzed by indirect immunofluoresence images. RESULTS: An n-Butanol-soluble fraction of KT (KT-NB) was able to inhibit cell proliferation. After a 48 h treatment with 6.75 µg/ml of KT, the cell viability was less than 50% of controls, and was further reduced to less than 10% at higher concentrations. KT-NB also induced an accumulation of cells in the G2/M phase of the cell cycle as well as an increased level of cells in the subG1 phase. Instead of disrupting the microtubule network of interphase cells, KT-NB reduced cell viability by inducing multipolar spindles and defects in chromosome alignment. KT-NB inhibits cell proliferation and reduces cell viability by two mechanisms that are exclusively involved with cell division: first by inducing multipolarity; second by disrupting chromosome alignment during metaphase. CONCLUSION: KT-NB reduced cell viability by exclusively affecting formation of the proper structure of the mitotic apparatus. This is the main idea of the new generation of anti-mitotic agents. All together, KT-NB has sufficient potential to warrant further investigation as a potential new anticancer agent candidate.

Preferential promotion of apoptosis of monocytes by Lactobacillus casei rhamnosus soluble factors.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is characterized by dense infiltrates of and defective apoptosis by mucosal cell populations. Some probiotics inhibit monocytes' expansion, although mechanisms remain unknown. Supernatants of Lactobacillus strains were investigated for inducing apoptosis of monocytes. METHODS: Secreted factors produced by Lactobacillus strains were tested on human lymphocytes, monocytes and a human monocytic leukemia-cell line (THP-1). Cell death mechanisms were investigated by a variety of methods. Lipopolysaccharide (LPS)-induced proinflammatory cytokines (IL-1beta, IL-6, IL-8, TNF-alpha) and anti-inflammatory TGF-beta1 were determined. RESULTS: Soluble factor(s) from Lactobacillus casei rhamnosus strain supernatants (LcrS) effectively induced apoptosis of immune cells. These were mainly soluble proteins (MW 5-30 kDa; LcrS(5-30)). For immune cells, but not human colonic epithelial carcinoma cells (HT-29), pretreatment with LcrS(5-30) significantly promoted apoptosis via a mitochondrial pathway. LcrS(5-30) suppressed pro-inflammatory cytokines and induced anti-inflammatory TGF-beta1. CONCLUSIONS: Probiotic Lcr produced heat-stable molecules (MW range 5-30 kDa) that promoted immune cell apoptosis without affecting intestinal epithelial cells. LcrS(5-30) triggered apoptosis by a mitochondrial pathway, but not via TGF-beta signaling pathway. LcrS(5-30) also inhibited LPS-induced inflammatory cytokines by immune cells. Thus, LcrS(5-30) promotes apoptosis of immune cells, and suggests probiotics-based regimens for prevention of IBD.