Klotho

Protein samples of 5 μg were dissolved in sample buffer (Laemmli buffer, Bio-Rad) containing DTT (dithiothreitol) and treated for 5 min at 95 °C. Protein samples were separated on a 4–12% CRIT XT BIS-TRIS GEL (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). Membranes were blocked with 5% fat-free milk in tris-buffered saline containing 0.1% Tween-20 for 1.5 h. Primary antibodies were added and membranes were incubated overnight at 4 °C. Hereafter, an adequate horseradish peroxidase (HRP)-conjugated secondary antibodies were added and incubated for 2 h at room temperature. Visualization was performed using Clarity ECL or Clarity ECL max (Biorad). Primary Antibodies: Klotho (MAB1819, 1:500, R&D), KIM-1 (#3809, 1:1000, ProSci, Poway, CA, USA), Pan-Actin antibody (#4968, dilution, Cell Signaling, Frankfurt am Main, Deutschland).

Methacarn-fixed, paraffin-embedded tissue sections were used for immunohistochemistry (using the avidin–biotin complex (ABC) method). For dewaxing, the sections were placed for 10 min in xylene, followed by a descending alcohol series and immersion with distilled water for 5 min. For KLOTHO and fibrinogen staining, specimens were heated in a 10 mM sodium citrate buffer (pH 6.0) in a microwave for 5 min. Endogenous peroxidase was inactivated by incubation with 3% hydrogen peroxide. After washing, the sections were incubated with a blocking solution (10% FCS, 1% BSA in 1× Tween–PBS) for 30 min at room temperature, and a primary antibody (goat anti-mouse KLOTHO, R&D Systems, AF1819; goat anti-mouse Fibrinogen, Santa Cruz, 18032) was added overnight at 4 °C diluted in 1% FCS plus 1× Tween–PBS. After incubation with a peroxidase-labeled secondary antibody (Jackson Immuno Research, 705-035-147, 1:500) for 1 h at room temperature, the sections were developed using a diaminobenzidine substrate and counterstained with a hematoxylin solution. Subsequent dehydration was achieved using ascending alcohol series (70% ethanol, 80% ethanol, 96% ethanol, and 100% ethanol). Finally, washing was performed twice for 5 min in Histo-Clear (Carl Roth, Karlsruhe, Germany). Embedding was performed with a ROTI-Histokitt II (Carl Roth, Karlsruhe, Germany), according to the manufacturer’s instructions.

Sprague Dawley pups assigned to normoxia (21% O₂) or hyperoxia (85% O₂), were randomly assigned to receive intraperitoneal (IP) injections of recombinant Klotho 30 mcg/kg ( Cat# 1819-KL; R&D Systems; Minneapolis, MN), or phosphate buffered saline as placebo (PL) every other day from postnatal day 1 to 21. This dose was based on pilot studies in our laboratory. Oxygen exposure was achieved in a Plexiglas chamber by a flow-through system and the oxygen level inside the chamber was monitored daily with a Maxtec Oxygen Analyzer (Model OM-25; Maxtec, Salt Lake City, Utah). Dams were rotated every 48 h between hyperoxia and normoxia chambers to prevent oxygen-induced damage to their lungs. Litter size was adjusted to 10–12 pups to control for the effect of litter size on nutrition and growth. The animals were recovered in normoxia for three additional weeks prior to sacrifice. Transthoracic echocardiography, lung morphometric and molecular studies were performed at postnatal day 42.

To study the effects of varying Pi concentrations on phosphatase expression, it was essential to control Pi concentration in the basal mineralizing medium. This ruled out the use of β-glycerophosphate (βGP) as the availability of Pi from βGP requires the action of TNAP (Huesa et al. 2015 (link)) which can itself be modulated by CKD-associated endocrine factors such as Pi, PTH, and FGF23 (Shalhoub et al. 2011 (link), Rendenbach et al. 2014 (link), Houston et al. 2016 (link)). Therefore, upon confluence (day 0), mineralization was induced by supplementing the growth medium (basal concentration: 1.8 mM Ca; 1 mM Pi) with 50 μg/mL l-ascorbic acid (AA) and 1.5 mM CaCl₂ to provide a final medium containing 3.3 mM Ca (Houston et al. 2016 (link)). Cultures were also supplemented with a range of Pi (1–5 mM), PTH (0–50 nM), and FGF23 (0–200 ng/mL) with or without klotho (50 ng/mL) (R&D Systems). Cells were maintained in a 5% CO₂ atmosphere at 37°C and mineralization media was changed every second/third day for 28 days.

C2C12 myoblasts were seeded and cultured as outlined above. Cultures were stimulated with 10 μg/ml heparin (Sigma, St. Louis, MO, USA) or heparin and 1 μg/ml Klotho (R&D Systems, Minneapolis, MN, USA) in a growth medium at 24‐ and 48‐h post‐plating. Following 48 h of stimulation, cells were collected in Trizol reagent for RNA isolation.

For the recombinant mouse Klotho (R&D Systems, Minneapolis, MN, USA) treatment, mice were administrated with a dose of 10 μg/kg intraperitoneally every other day, immediately post IR. For the MK-2206 (MedChem Express, Monmouth Junction, NJ, USA) treatment, mice were administrated with a dose of 4 mg/kg intraperitoneally every other day, immediately post IR.

Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO₂ atmosphere. TSC2-deficient ELT3V and TSC2-addback ELT3T cells are derived from Eker rat uterine leiomyoma, as described previously (46 (link)). TSC2-deficient and TSC2-addback cystadenoma 105K cell lines were obtained from Dr Elizabeth Henske’s laboratory (59 (link)). TSC2-deficient MEF infected with pBabe-Puro-Myr-Flag-AKT1, to generate TSC2-deficient MEF with myristoylated AKT1 (MEF-AKT1), and control TSC2-deficient MEF (MEF-EV) infected with EV were obtained from Dr Carmen Priolo’s laboratory (72 (link)). Myristoylation directs AKT to the membrane, keeping it constitutively phosphorylated at both Ser473 and Thr308 sites (72 (link), 73 (link)). Cells were serum starved overnight before treatment with indicated drugs/compounds, including losartan (100 nM (47 (link), 74 (link)); Tocris Bioscience), DMSO vehicle (Sigma-Aldrich), water (vehicle), or Klotho (100 ng/ml (55 , 56 ); R&D Systems) for indicated durations.