*2.6. Augmentation Index*

The aortic augmentation index was identified from the late systolic portion of the arterial pressure wave as described previously [20]. The augmentation index was defined as the height from the augmentation point to the systolic peak of the pressure wave divided by the pulse pressure and was expressed as a percentage.

#### *2.7. Western Blot*

Fresh frozen kidneys were homogenized in RIPA lysis buffer supplemented with phosphatase inhibitor cocktail (Roche) and protease inhibitor cocktail (Roche). Kidney tissue homogenates were mixed with Laemmli sample buffer, fractionated on SDS-PAGE (20 μg/well), and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Immunoblots were incubated overnight at 4 ◦C with primary antibodies, including monoclonal rat anti-human Klotho (1:500, KO603, Trans Genic Inc., Tokyo, Japan), polyclonal rabbit anti-phospho-NCC (pThr53, 1:1000, Novus Biologicals, Littleton, CO, USA), and monoclonal mouse anti-β-actin (1:5000, Sigma, St. Louis, MS, USA) in 2% (*w*/*v*) bovine serum albumin (BSA, Sigma) and washed in TBS-T buffer (150 mM NaCl, 10 mM Tris/HCl pH 7.4, 0.2% *v*/*v* Tween-20). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Buckinghamshire, UK). Specific signal was visualized using ECL kit (Amersham Life Sciences, Arlington Heights, IL, USA). The protein bands were quantified by Image Studio Lite 5.2 software (LI-COR, Bad Homburg, Germany).

#### *2.8. Histological Evaluation*

Hearts were fixed in 4% paraformaldehyde, paraffin-embedded, and cut into 5 μm sections. Fibrotic tissue was visualized by staining with picrosirius red according to a standard protocol. Total collagen was quantified using ImageJ software and was expressed as the ratio of collagen-stained area to total muscle area of the left ventricle and septum. For the analysis of cardiomyocyte size, cardiac sections were stained with FITC-labeled wheat germ agglutinin. At least ten random areas of the heart were measured, and only cardiomyocytes with well-defined borders and visible nuclei were used. Images were obtained by the Zeiss LSM 880 Airyscan confocal microscope and analyzed using semiautomated Image J software. All histological images were analyzed by two independent investigators in a blinded manner.
