Journal: Cell Death & Disease

Article Title: Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

doi: 10.1038/s41419-018-0324-4

Figure Lengend Snippet: a The changes of [Ca 2+ ]i in HGECs exposed to cinacalcet and high-glucose media. To determine whether the addition of cinacalcet might modulate [Ca 2+ ]i in HGECs, FURA-2AM-loaded HGECs were stimulated using different concentrations (15, 100 nM) of cinacalcet in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) media. The area under curve (AUC) was estimated from the baseline of normalized data (at the point of injection) to a fluorescence level and between time points of injection (0 min) and 10 min. The peak of the curve was measured as highest value of the curve. The peak amplitude and AUC of [Ca 2+ ]i were significantly increased by cinacalcet in dose-dependent manners in both LG and HG media. In Fig. 1a, the arrow denotes the administration of cinacalcet (15 and 100 nM, respectively) ( n = 6 independent experiments in each experiments). * p < 0.05; ** p < 0.01 compared with LG and HG. b The changes of intracellular signaling in HGECs exposed to cinacalcet and high-glucose media. Representative immunofluorescent ( n = 6 independent experiments in each experiments) and western blot analyses ( n = 4 independent experiments in each experiments) of CaSR, CaMKKα/β, phospho-Ser 428 LKB1, and phospho-Thr 172 AMPK in the cultured HGECs in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (15 nM) and the quantitative analyses of the results are shown. * P < 0.05; ** P < 0.01 and # P < 0.001 compared with other groups

Article Snippet: Human glomerular endothelial cells (HGECs) were purchased from Anigio-Proteomie (Boston, MA) and subcultured in endo-growth media (Angio-Proteomie, Boston, MA).

Techniques: Injection, Fluorescence, Western Blot, Cell Culture

Journal: Cell Death & Disease

Article Title: Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

doi: 10.1038/s41419-018-0324-4

Figure Lengend Snippet: The effect of cinacalcet on intracellular signaling for AMPK-eNOS oxidative stress and apoptosis in the HGECs cultured in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (1, 5, 15 nM) ( a–d ). Representative Western blot analyses and quantitative analyses of total AMPK, phosphor-Thr 172 AMPK, total eNOS, phospho-Ser 1177 eNOS ( a , * P < 0.05 and ** P < 0.01 compared with LG control), SOD1 and SOD2 ( b , * P < 0.05 compared with other groups), dihydroethidium expression (as an oxidative stress marker; c , * P < 0.05 and # P < 0.001 compared with other groups), Bcl-2, Bax, and TUNEL-positive HGECs ( e , * P < 0.05 and ** P < 0.01 compared with other groups), and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown ( n = 4 independent experiments in each experiments). d The effect of BAPTA-AM (25 μM) on cinacalcet-indueced in the HGECs cultured in low-glucose or high-glucose (HG; 30 mmol/l D-glucose) with or without cinacalcet treatment (15 nM). Representative Western blot analyses and quantitative analyses of CaMKKβ, phospho-LKB1, and total LKB1 ( n = 4 independent experiments in each experiments). * P < 0.05 and ** P < 0.01 compared with LG control. f The changes of intracellular signaling related to autophagy in HGECs exposed to cinacalcet and high-glucose media. Representative Western blot analyses and quantitative analyses of beclin-1, LC3-II/LC3-I ratio, and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown ( n = 4 independent experiments in each experiments). Representative immunofluorescent analyses of LC3 punctae in HGECs and the quantitative analyses of the results are shown ( n = 6 independent experiments in each experiments). ** P < 0.01 compared with other groups

Article Snippet: Human glomerular endothelial cells (HGECs) were purchased from Anigio-Proteomie (Boston, MA) and subcultured in endo-growth media (Angio-Proteomie, Boston, MA).

Techniques: Cell Culture, Western Blot, Control, Expressing, Marker, TUNEL Assay

Journal: Cell Death & Disease

Article Title: Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

doi: 10.1038/s41419-018-0324-4

Figure Lengend Snippet: Immunoblot for CaMKKβ, LKB1, phospho-AMPK, SIRT1 and phospho-Ser 1177 eNOS in AMPKα1 siRNA, AMPKα2 siRNA, or SIRT1 siRNA knock-down HGECs in a high-glucose environment with cinacalcet treatment ( a and b ). The cultured HGECs were transfected with a final concentration of 50 nM CaMKKβ and LKB1, α1 and α2-AMPK, SIRT1 siRNAs for 24-h by transfection reagent and treated with cinacalcet (15 nM) in high-glucose media. Representative Western blot analyses of CaMKKβ and phospho-Ser 428 LKB1 ( a ), as well as phospho-Thr 172 AMPK, total AMPK, SIRT1, phospho-Ser 1177 eNOS ( b ) and β-actin levels and the quantitative analyses of the results are also shown ( a and b , respectively) ( n = 4 independent experiments in each experiments).* P < 0.05, ** P < 0.01 compared with control siRNA with HG

Article Snippet: Human glomerular endothelial cells (HGECs) were purchased from Anigio-Proteomie (Boston, MA) and subcultured in endo-growth media (Angio-Proteomie, Boston, MA).

Techniques: Western Blot, Knockdown, Cell Culture, Transfection, Concentration Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: Apelin-13 alleviates diabetic nephropathy by enhancing nitric oxide production and suppressing kidney tissue fibrosis

doi: 10.3892/ijmm.2021.5008

Figure Lengend Snippet: Apelin-13 alleviates the higher glucose-induced injury of endothelial cells of glomerular vessels. (A) Cell viability was detected with the Cell Counting Kit-8 assay. (B) hRGECs were detected with the lumen formation experiments (magnification, x4). (C) The expression of AT1R, eNOS, APJ, TGBR, E-cadherin and α-SMA in these cells was determined with the western blotting. * P<0.05, ** P<0.01 and *** P<0.001. AT1R, Ang II type 1 receptor; eNOS, endothelial nitric oxide synthase; APJ, angiotensin domain type 1 receptor-associated proteins; TGBR, transforming growth factor β receptor; α-SMA, α-smooth muscle actin.

Article Snippet: A human renal glomerular microvascular endothelial cell line (hRGEC) was obtained from the BioVector NTCC, Inc. and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS) in humidified air at 37°C with 5% CO 2 .

Techniques: Cell Counting, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Apelin-13 alleviates diabetic nephropathy by enhancing nitric oxide production and suppressing kidney tissue fibrosis

doi: 10.3892/ijmm.2021.5008

Figure Lengend Snippet: Apelin-13 relieves the injury of endothelial cells of glomerular vessels by suppressing fibrosis. (A) Cell viability of endothelial cells of glomerular vessels was determined with Cell Counting Kit-8 assay. (B) hRGECs were detected with the lumen formation experiments (magnification, x4). (C) The levels of AT1R, eNOS, APJ, TGBR, E-cadherin and α-SMA in these cells were determined with western blotting. * P<0.05, ** P<0.01 and *** P<0.001. AT1R, Ang II type 1 receptor; eNOS, endothelial nitric oxide synthase; APJ, angiotensin domain type 1 receptor-associated proteins; TGBR, transforming growth factor β receptor; α-SMA, α-smooth muscle actin.

Article Snippet: A human renal glomerular microvascular endothelial cell line (hRGEC) was obtained from the BioVector NTCC, Inc. and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS) in humidified air at 37°C with 5% CO 2 .

Techniques: Cell Counting, Western Blot