Fig 2: The effects of LIPUS on renal histology and serum indoxyl sulfate levels in CKD mouse models following adenine administration (the mice were euthanized 28 days after adenine administration): (A) the gross appearance of both the left and right kidneys; (B) pathological changes in renal tissue sections from the left kidneys stained with Periodic acid–Schiff (PAS), which were analyzed using a light microscope, and histological score (scale bar: 50 µm); (C) serum indoxyl sulfate levels; (D) senescence-related molecules (p53, p21, and p16) in the left kidneys, determined by Western blot. Quantifications for the protein expression are also shown. Data are presented as mean ± SD (n = 5–9): * p < 0.05 versus the control group; # p < 0.05 versus the CKD-alone group.

Fig 3: Endothelial cells and macrophages are SASP-carrying cells in the diabetic mice kidney. A. Representative confocal microscope images of kidney sections stained for p16 (red) from control (Ctrl) and hyperglycaemic mice (HG-M). Nuclei are counterstained with DAPI (blue). Scale bar = 50 µm. B. Experimental design used to isolate macrophages, ECs, and non-macrophagic/non-endothelial cells from kidneys. C. Fold changes in sorted populations in expression levels of SA and SASP mRNAs (p16, p21, IL-6, TGF-ß, PAI-1 and MCP-1) (n=3 mice/group). Blue histograms = means of vehicle-treated mice (Ctrl); red histograms = means of STZ-treated hyperglycaemic mice (HG-M). For all diagrams error bars are ± SD and * t-test p < 0.05, ** t-test p < 0.01 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig 4: The effects of a high calcium/phosphate diet, of 1,25(OH)2D3, and of antioxidant supplementation on oxidative stress, DNA damage, and protein expression of oncogenes and tumor suppressive genes in 1a(OH)ase-/- mice. Mice from each group were treated as described in Figure 1. ROS levels in freshly isolated cells of (a) skin, (b) liver, and (c) kidney from 10-week-old wild-type (WT) and 1a(OH)ase-/- (KO) mice. Representative micrographs of skin sections stained immunohistochemically for (d) SOD2 and (f) ?-H2AX. Scale bars represent 50 µm in d and f. (e) The percentage of SOD2-positive cells of skin and (g) the percentage of ?-H2AX-positive cells of skin; (h) representative Western blots of skin extracts to determine Prdx I protein levels. ß-actin was used as loading control. (i) Skin Prdx I and Bmi1, p16, p53 and p21 protein levels in (j) skin and (k) liver relative to ß-actin protein levels were assessed by densitometric analysis and expressed as a percentage of the levels of vehicle-treated wild-type mice fed the normal diet (ND).Each value is the mean ± SEM of determinations in five mice of each group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice. # p < 0.05; ## p < 0.01 compared with ND KO mice. & p < 0.05; && p < 0.01 compared with RD KO mice

Fig 5: Hyperglycaemia promotes a SASP-like program in cell line-derived macrophages. THP-1-derived macrophages were exposed to HG for a week. A. Measurement of SA ß-gal activity. B. Representative images of SA ß-gal staining (detail showing magnification of 40x images) with percentage of SA ß-gal+ cells. C. Fold changes in expression levels of SASP mRNAs (IL-1a, IL-6, IL-8, PAI-1, TGF-ß, TNF-a, MCP-1, ICAM-1, and IGFBP6). D. Western blot analysis of SASP proteins expression (procaspase-1, caspase-1, p-p38, IL-1ß, and IL-8). The same experiment was performed in U937-derived macrophages. E. Percentage of SA ß-gal+ cells. F. Fold changes in expression levels of p16 and IL-6 mRNAs. G. Heatmap of RT-PCR assessment of selected M1/M2 mRNAs in THP-1-derived macrophages. Histograms show the means of cells treated as described (the treatment colour legend is reported in the figure). N = 3 (experiments) for all. For all graphs error bars are ± SD and * t-test p < 0.05, ** t-test p < 0.01.