Journal: British Journal of Pharmacology

Article Title: Genetic fusion of human FGF21 to a synthetic polypeptide improves pharmacokinetics and pharmacodynamics in a mouse model of obesity

doi: 10.1111/bph.13499

Figure Lengend Snippet: The characterization of FGF21 and fusion proteins. The unmodified FGF21 and fusion proteins were assayed by (A) 12% SDS‐PAGE and (B) Western blotting with a rabbit anti‐human FGF21 antibody (lane 1: FGF21; lane 2: PsTag200‐FGF21; lane 3: PsTag400‐FGF21; lane 4: PsTag600‐FGF21). (C) CD spectra of unmodified FGF21 and fusion proteins. (D) IEF of FGF21 and PsTag600‐FGF21 (lane 1: PsTag600‐FGF21; lane 2: FGF21). (E) MALDI‐TOF mass spectrometry of PsTag600‐FGF21 (M+ and M2 + refer to the singly and doubly charged ionic species of PsTag600‐FGF21 respectively. (F) SEC‐HPLC in the presence of 150 mmol·L−1 sodium phosphate buffer (pH 7.0) resulted in a single peak with decreasing elution time for PsTag fusion proteins with increasing number of amino acid residues. (G) Binding affinities of FGF21 and PsTag fusion proteins to human β‐klotho were examined by direct binding elisa. (H) Cellular glucose uptake stimulated by native FGF21 and PsTag fused FGF21 in 3 T3‐L1 cells. n = 3. *P < 0.05 versus vehicle control. (I) 3T3‐L1 cells were treated with vehicle (lane 1), 10 nmol·L−1 FGF21 (lane 2), 10 nmol·L−1 PsTag200‐FGF21 (lane 3), 10 nmol·L−1 PsTag400‐FGF21 (lane 4) and 10 nmol·L−1 PsTag600‐FGF21 (lane 5) for 10 min. Phospho‐specific antibody was used to determine phosphorylation of ERK. (J) Pharmacokinetic plasma profile of native FGF21 and PsTag fusion proteins intravenously injected in C57BL/6 mice (n = 10 per group).

Article Snippet: Binding affinities of FGF21 and PsTag fusion proteins to human β‐klotho (58 890 KB, R&D) were examined by direct binding elisa .

Techniques: SDS Page, Western Blot, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Injection

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Effect of Klotho on cellular viability in HUVECs. Cells were exposed to various concentrations of ox-LDL (25, 50, 100, and 200 μg/ml) for 24 h. a HUVEC morphology was observed under an inverted phase contrast microscope (×10) following 24 h of ox-LDL treatment. Typical cellular fragmentations, vacuoles, and debris are arrowed. (Bar = 50 μm) ( b ) Cellular viability was detected by MTT assay. c and d SOD enzymatic activity and MDA levels in HUVECs were analyzed using commercially available assay kits. e HUVECs were pretreated with 100, 200, 400, and 800 pM of Klotho for 1 h, and then incubated with ox-LDL (50 μg/ml) for 24 h. Cellular viability was detected by MTT assay. Data are shown as mean ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Microscopy, MTT Assay, Activity Assay, Incubation, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho inhibited ROS production induced by ox-LDL in HUVECs. HUVECs were pre-incubated with 200 pM of recombinant human Klotho for 1 h, then treated with ox-LDL (50 μg/mL) for another 24 h. Ox-LDL alone and Klotho alone were used as controls. a Images observed under an inverted fluorescence microscope. (Bar = 50 μm) ( b ) Output figure of the fluorescence intensity detected by flow cytometry. c Average fluorescence intensity: Mean = total area under the peak/the total number of cells. Data are shown as mean ± S.D. d Lipid peroxidation was assessed by measuring the MDA levels in HUVECs treated with ox-LDL and/or Klotho ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. blank control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Incubation, Recombinant, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: SOD, Cu/Zn-SODandgp91 phox in ox-LDL and Klotho-treated HUVECs. Cellular treatment was the same as described in Fig. . a Intracellular SOD activity was determined by the hydroxylamine method. b , c and d Western blotanalysis of Cu/Zn-SOD and gp91 phoxexpression in pre-treated HUVECs. Densitometry of the probed bands from the western blot were analyzed by Image J2x. Values are means ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Activity Assay, Western Blot, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho regulated NO production in HUVECs. a NO production in pre-treated HUVECs. b , c , d , and e mRNA levels of iNOS, eNOS, PI3K, and Akt were measured by real time PCR. Values are means ± S.D. ( n = 3). Statistical differences are expressed as # p < 0.05 vs . control; * p < 0.05 vs . ox-LDL. ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: Klotho deficiency is a common feature of mouse kidney with preexisting chronic kidney diseases or advanced age. ( A – E ) Representative Western blots show renal protein level of Klotho in mouse models of ischemia-reperfusion injury (IRI), unilateral ureteral obstruction (UUO), remnant kidney after 5/6 nephrectomy (5/6Nx), db/db with diabetic kidney disease (DKD) and advanced age (24 months old), respectively. ( F ) Quantitative data of Klotho protein levels in the kidney of various models as indicated are presented. ** p < 0.01 versus sham ( n = 6). ( G ) Representative micrographs of immunochemical staining show Klotho expression and localization in different models of mice as indicated. Arrows indicate positive staining. Scale bar, 50 µm.

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: Western Blot, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: Klotho-deficiency sensitizes mice to SARS-CoV-2 N protein-triggered tubular injury and apoptosis. (A) Experimental design. The blue arrow indicates the timing of injecting pcDNA3 empty vector or pSARS-CoV-2 N Protein (N) plasmid into normal (WT) or Klotho-deficient ( KL/KL ) mice, respectively. (B, C) Graphic presentation shows serum creatinine (SCr) and blood urea nitrogen (BUN) levels in different groups. ns, not significant. (D–H) Representative Western blot (D) and quantitative data show renal protein levels of N Protein (F) , KIM-1 (G) and NGAL (H) in different groups. * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). ( I – L ) Representative Western blot (I) and quantitative data show renal protein levels of cleaved PARP (J) , p53 (K) and FADD ( L ). * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). ( M ) Representative micrographs of immunochemical staining show renal expression of N protein and P53 are presented. Arrows indicate positive staining. Scale bar, 50 µm. ( N ) Quantitative data of N Protein and p53. ** p < 0.01, *** p < 0.001 ( n = 6).

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: Plasmid Preparation, Western Blot, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: KP1 ameliorates tubular cell injury and apoptosis induced by SARS-CoV-2 N protein in vitro . (A–C) Representative Western blot (A) and quantitative data show the expression of N Protein (B) and KIM-1 (C) . Cells were treated with KP1 or recombinant human Klotho for 48 h after transfecting SARS-CoV-2 N Protein plasmid. ** p < 0.01, *** p < 0.001 ( n = 6). (D–G) Representative Western blot (D) and quantitative data show the expression of PARP (E) , p53 (F) and FADD (G) . * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). (H, I) Representative TUNEL staining (H) and quantitative data (I) show that SARS-CoV-2 N protein induced HK-2 cell apoptosis, while KP1 or Klotho (KL) protected HK-2 cells from apoptosis. ** p < 0.01 ( n = 6). Arrow indicates apoptotic cell.

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: In Vitro, Western Blot, Expressing, Recombinant, Plasmid Preparation, TUNEL Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: KP1 ameliorates acute kidney injury induced by SARS-CoV-2 N protein and ischemia-reperfusion injury in vivo . (A) Experimental design. The blue arrow indicates the timing of injecting pcDNA3 empty vector or pSARS-CoV-2 N Protein plasmid (N). The green arrowheads indicate the timing of injecting KP1 at the concentration of 1 mg/day/kg. The black arrow indicates the timing of IRI. (B, C) Graphic presentations show SCr and BUN levels in different groups as indicated. * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). (D–H) Representative Western blot (D) and quantitative data show renal protein levels of Klotho (E) , N protein (F) , KIM-1 (G) and NGAL (H) . * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). (I, J) Representative micrographs of Periodic acid-Schiff (PAS) staining and immunochemical staining for N protein and KIM-1 are presented. Arrows indicate positive staining. Scale bar, 50 µm. Semi-quantification data are presented in Panel (J) . * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6).

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: In Vivo, Plasmid Preparation, Concentration Assay, Western Blot, Staining