GoKawiilExperimental samples
Animals from ITP cohorts
Liver samples of mice from the ITP61 were acquired from the collections of University of Michigan Medical School (UM), University of Texas (UT) and The Jackson Laboratory (TJL), from animals of the 2015, 2016 and 2017 cohorts8,62,63,64. Female and male mice, 22 to 23 months old, were euthanized following interventions, including 17-DMAG (30 ppm, as in ref. 62), b-GPA (3,300 ppm, as in ref. 62), minocycline (300 ppm, as in ref. 62), mitoQ (100 ppm, as in ref. 62), rapamycin applied from 20 months (42 ppm, as in ref. 62), canagliflozin (180 ppm, as in ref. 8), candesartan cilexetil (30 ppm, as in ref. 64), geranylgeranyl acetone (600 ppm, as in ref. 64), 17-α-oestradiol applied to males from 20 months (14.4 ppm, as in ref. 64), 17-α-oestradiol applied to males from 16 months (14.4 ppm, as in ref. 64), MIF098 (240 ppm, as in ref. 64), nicotinamide riboside (1,000 ppm, as in ref. 64), 1,3-butanediol (100,000 ppm, as in ref. 63), captopril (180 ppm, as in ref. 63), leucine (40,000 ppm, as in ref. 63), PB125 (100 ppm, as in ref. 63), sulindac (5 ppm, as in ref. 63), syringaresinol (300 ppm, as in ref. 63), a combination of rapamycin and acarbose applied from 9 months (14.7 ppm and 1,000 ppm, as in ref. 63), and a combination of rapamycin and acarbose applied from 16 months (14.7 ppm and 1,000 ppm, as in ref. 63). All interventions continued until the mice were euthanized. In addition, livers were taken from control untreated male and female mice euthanized at 4–6 and 22 months of age. All mice were fed ad libitum with the same diet (Purina 5LG6) made in the same commercial diet kitchen (TestDiet). Mice represented genetically heterogeneous UM-HET3 strain, produced by crossing female CByB6F1/J and male C3D2F1/J mice. Therefore, each mouse in the cohort had a unique genetic background but shared the same inbred grandparents (C57BL/6J, BALB/cByJ, C3H/HeJ and DBA/2 J). Mice were housed in plastic ventilated cages under similar environmental conditions in all sites (21–23 °C temperature, 40–60% humidity, 12 h:12 h light:dark cycle). Details of the mouse housing and methods used for health monitoring are provided in refs. 61,65. All experiments were approved by the Institutional Animal Care and Use Committee of each site (The Jackson Laboratory in Bar Harbor (TJL), the University of Michigan at Ann Arbor (UM), and the University of Texas Health Science Center at San Antonio (UT)).
Klotho-KO mice
Eight-week-old male wild-type mice (C57BL/6J) and Klotho−/− knockout mice were purchased from CLEA Japan. Mice were housed under specific pathogen-free conditions, at a temperature of 22 ± 2 °C, with a relative humidity of 50 ± 10%, and on a 12 h:12 h light:dark cycle. Mice had free access to tap water and standard chow. The animal protocols were approved by the Tohoku University Institutional Animal Care and Use Committee. The animal procedures performed conform to the NIH guidelines (Guide for the Care and Use of Laboratory Animals).
RNA sequencing
Bulk RNA-seq profiling of mouse tissues
For RNA-seq profiling of liver samples from ITP cohorts, three biological replicates (animals) per sex were utilized for each intervention, except for canagliflozin (seven biological replicates per sex). Additional control samples included 24 old and 6 young mice per sex across different cohorts, resulting in 182 total liver samples (Supplementary Table 1a). For RNA-seq profiling of bulk kidney and gastrocnemius skeletal muscle samples from wild-type and Klotho-KO male mice, 6 biological replicates (animals) per group were utilized for every tissue (24 samples total). Samples selected for sequencing were randomly chosen from available animals within each group. No a priori power calculations were performed; sample sizes were determined by cohort availability and by sample sizes used in similar molecular signature and clock application studies15,21,54. RNA was extracted with PureLink RNA Mini Kit as described in the protocol and submitted for sequencing. Paired-end sequencing with 150 bp read length was performed on Illumina NovaSeq 6000.
Nuclei extraction from tissues of Klotho-KO mice
Brains and kidneys from one 8-week-old control mouse and one age-matched Klotho-KO animal were collected following cervical dislocation and flash frozen in liquid nitrogen. The cortical region of brain and a radial section of the kidney were dissected. Their nuclei were isolated with the Minute single nucleus isolation kit for neuronal tissue/cells and the Minute single nucleus isolation kit for tissue/cells (BN-020, SN-047, Invent Biotechnologies), following the manufacturer’s protocol with minor modifications. Specifically, the isolated nuclei were resuspended in 5% BSA solution with RNase inhibitor (1000494; 10X Genomics).
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