Fig 2: Wnt/ß-catenin/RAS axis plays a central role in mitochondrial dysfunction and cellular senescence in renal tubular cells. (a–c) Representative (a) Western blots and graphical representations of (b) active ß-catenin, (c) phospho-PGC-1a, and TFAM protein expression levels in different groups are presented. HKC-8 cells were transfected with empty vector (pcDNA3) or Wnt1 expression plasmid (pHA-Wnt1) for 24 hr. Whole-cell lysates were analyzed by Western blot. *p < .05 versus pcDNA3 group (n = 3). (d–f) Representative (d) Western blots and graphical representations of (e) AT1 and (f) phospho-PGC-1a are presented. HKC-8 cells were transfected with empty vector (pcDNA3) or ß-catenin expression plasmid (pDel-ß-catenin) for 24 hr. Whole-cell lysates were analyzed by Western blot. *p < .05 versus pcDNA3 group (n = 3). (g–h) Graphical representations of the relative mRNA abundance of mitochondrial-encoded OXPHOS genes in two groups are presented. The mRNA levels of (g) TFAM and Cytb, as well as (h) COX1 and COX2 in two groups, were assessed. HKC-8 cells were transfected with empty vector (pcDNA3) or ß-catenin expression plasmid (pDel-ß-catenin) for 24 hr. Total RNA was extracted and analyzed by quantitative real-time PCR. *p < .05 versus pcDNA3 group (n = 3). (i, j) Representative (i) Western blots and graphical representations of (j) phospho-PGC-1a and TFAM are presented. HKC-8 cells were treated with AngII (10 nM) for 24 hr. Whole-cell lysates were analyzed by Western blot. *p < .05 versus the control group (n = 3). Ctrl, control. (k) Graphical representation shows AngII significantly induced mitochondrial superoxide production in HKC-8 cells. HKC-8 cells were treated with AngII (10 nM) for 12 hr and then incubated with mitochondrial superoxide indicator mitoSOX™ Red (5 µM) as manufacturer's instruction. The fluorescence was analyzed by flow cytometry. *p < .05 versus the control group (n = 3). Ctrl, control. (l–n) Representative (l) Western blots and graphical representations of (m) phospho-PGC-1a and (n) p16INK4A are presented. HKC-8 cells were pretreated with losartan (10 µM) for 1 hr and then transfected with Wnt1 expression plasmid for 24 hr. *p < .05 versus pcDNA3 group; †p < .05 versus pHA-Wnt1 group (n = 3). (o) An analysis of O2 consumption in renal tubular cells. HKC-8 cells were pretreated with losartan (10 µM) for 1 hr and then transfected with Wnt1 expression plasmid for 24 hr. OCR was first measured in approximately 3 × 104 cells of each group under basal condition. Those cells then were sequentially added oligomycin (1 µM), FCCP (0.5 µM), rotenone (0.5 µM), and antimycin A (0.5 µM) to determine different parameters of mitochondrial functions according to the manufacturer's instructions. (p) Graphical representations of basal OCR, maximal OCR, ATP-linked OCR, and reserve capacity. The average of four determinations for each group is shown. *p < .05 versus pcDNA3 group; †p < .05 versus pHA-Wnt1 group (n = 4). (q) Graphical representation of mitochondrial membrane potential (MMP). MMP was detected by JC-1 staining and analyzed by flow cytometry. After pretreatment with losartan (10 µM) for 1 hr, HKC-8 cells were transfected with Wnt1 expression plasmid for 24 hr and then stained with JC-1. The MMP is shown as the ratio of the fluorescence intensity at absorbance of 590 nm (JC-1 aggregate) to 520 nm (JC-1 monomer). *p < .05 versus pcDNA3 group; †p < .05 versus pHA-Wnt1 group (n = 3). (r) Graphical representation of ß-galactosidase-positive cells. After pretreatment with losartan (10 µM) for 1 hr, HKC-8 cells were transfected with Wnt1 expression plasmid for 24 hr and then stained for ß-galactosidase activity. *p < .05 versus pcDNA3 group; †p < .05 versus pHA-Wnt1 group (n = 3). (s) Graphical representation of mitochondrial mass determined by flow cytometry analysis of NAO fluorescence. The mean fluorescence intensities of 10,000 events were counted for each cell population. After pretreatment with losartan (10 µM) for 1 hr, HKC-8 cells were transfected with Wnt1 expression plasmid for 24 hr and then stained with NAO (5 µM). *p < .05 versus pcDNA3 group; †p < .05 versus pHA-Wnt1 group (n = 3). (t, u) Representative (t) Western blots and graphical representations of (u) p16INK4A and fibronectin are presented. HKC-8 cells were pretreated with resveratrol (50 µM) or mitoQ (100 nM) for 1 hr and then transfected with Wnt1 expression plasmid for 24 hr. Whole-cell lysates were analyzed by Western blot. *p < .05 versus pcDNA3 group; †p < .05 versus pHA-Wnt1 group (n = 3)

Fig 3: Klotho ameliorates age-related renal fibrosis in vivo. (a) Experimental design. Red arrows indicate the injections of pcDNA3 or pV5-sKlotho plasmid. Blue line indicates the timing of d-gal treatment. (b) Graphical representation of Klotho protein expression levels in different groups. *p < .05 versus control mice; †p < .05 versus d-gal-treated mice (n = 5–6). (c) Representative immunofluorescence micrographs show Klotho expression in kidneys. Scale bar, 25 µm. And representative photographs show Klotho reversed d-gal-induced growth retardation. Klo, Klotho. (d) Graphical representation shows Klotho increased body weight gain in d-gal-treated mice. *p < .05 versus control mice; †p < .05 versus d-gal-treated mice (n = 5–6). (e) Representative micrographs show Masson trichrome staining and collagen I expression in kidneys. Paraffin sections were used for Masson trichrome staining. Frozen sections were used for immunofluorescence staining of collagen I. Arrows indicate positive staining. Scale bar, 25 µm. (f–h) Representative (f) Western blots and graphical representations of (g) fibronectin and (h) a-SMA are presented. FN, fibronectin. *p < .05 versus control mice; †p < .05 versus d-gal-treated mice (n = 5–6). (i–j) Representative (i) Western blots and graphical representation of (j) Wnt10b in kidneys are presented. *p < .05 versus control mice; †p < .05 versus d-gal-treated mice (n = 5–6). (k–l) Representative micrographs show renal expression of (k) Wnt1 and (l) ß-catenin. Paraffin kidney sections were immunostained with an antibody against Wnt1. Frozen kidney sections were immunostained with an antibody against ß-catenin. Arrows indicate positive staining. Scale bar, 25 µm. (m, n) Representative (m) Western blots and graphical representation of (n) active ß-catenin protein expression in kidneys are presented. *p < .05 versus control mice; †p < .05 versus d-gal-treated mice (n = 5–6). (o–q) Representative (o) Western blots and graphical representations of (p) AGT and (q) AT1 protein expression levels in different groups are presented. *p < .05 versus control mice; †p < .05 versus d-gal-treated mice (n = 5–6). (r) Representative micrographs show renal expression of AGT, AT1, and ACE. Paraffin kidney sections were used for immunohistochemistry staining. Arrows indicate positive staining. Scale bar, 25 µm