Under general anesthesia, the left ureter was exposed through a midline abdominal incision and double-ligated with 4–0 silk. Sham operation was performed on the contralateral kidney. WT, Trpc6−/− and Trpc3/6-DKO mice were subjected to UUO for 10 days. After surgery, kidneys were harvested for histological analysis of fibrosis by trichrome staining and for measurement of mRNA expression by quantitative real-time PCR. In separate experiments, WT mice underwent UUO and received of BTP2 injection (2 mg/kg daily by i.p.) or vehicle (polypropylene glycol) (Sigma-Aldrich, St Louis, MO) for 7 days. To test the efficacy of klotho in renal fibrosis after UUO injury, recombinant human klotho consisting of the entire klotho ectodomain (R&D Systems Inc. Minneapolis, MN; at 10 ug/kg in 0.1 ml of 10 mM phosphate buffered saline) was administered via intraperitoneal injection to mice immediately after ureteral ligation and then administered every other day until 7 days after surgery. PBS-treated animals were used as controls.
Recombinant human klotho
Manufactured by R&D Systems
Sourced in United Kingdom
Recombinant human klotho is a protein produced using recombinant DNA technology. Klotho is a type-I membrane protein that functions as an essential cofactor for the fibroblast growth factor 23 (FGF23) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Polypropylene glycol, by Merck Group (1 mentions)
Klotho, by R&D Systems (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
Hasmc, by Lonza (1 mentions)
Ang 2, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Tunicamycin, by Bio-Techne (1 mentions)
4 protocols using recombinant human klotho
1
Ureteral Obstruction Model Fibrosis
2
MTT Assay for ox-LDL and Klotho
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Cellular viability was measured by the MTT uptake method. Briefly, HUVECs were plated at a density of 6.0 × 103 cells/well in a 96-well plate. 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. Continuously, the intracellular MTT purple formazan was solubilized with 150 μl of DMSO (Life, USA). The absorbance was detected at optical density (OD) 490 nm with auniversal microplate reader (ELx800, BioTeK, USA). Each experiment was performed three times.
3
Vascular Smooth Muscle Cell Culture
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HASMC (Lonza Group, Basel, Switzerland) were cultured in Dulbecco's Modified Eagle Medium (Sigma-Aldrich, St. Louis, MO) containing 10% (v/v) fetal calf serum, 1% (v/v) L-glutamine and penicillin/streptomycin (100 U ml−1/100 μg ml−1) in a 5% CO2/95% humidified air incubator at 37 °C as described before.20 (link),21 (link) Cells were passaged with trypsin and all experiments were performed between passages 6 and 10. Cells were treated with either recombinant human klotho (1 nM) (R&D systems, Abingdon, UK) or AngII (100 or 200 nΜ) (Sigma-Aldrich, St. Louis, MO), for the indicated periods of time.
4
Podocyte Stress Response Assay
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Mouse podocytes were cultured as previously described61 (link). Briefly, podocytes were grown under permissive conditions at 33 °C in RPMI 1640 media containing 10% FBS, 10 U/ml interferon-γ. The cells were treated with 5 μg/ml Tunicamycin (#3516, Tocris Bioscience) and/or 0.05 μg/ml recombinant human Klotho (#5334-KL, R&D Systems) for 16 h prior to lysis using RIPA lysis buffer (ThermoFisher Scientific) containing protease and phosphatase inhibitors (Roche).
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