γ-irradiation induces senescence in PTEC and leads to increased Cyclin D1 expression.

<p>PTEC were isolated and grown for 6 days in culture before being exposed to 10 Gy γ-irradiation. After y-irradiation, cells were split and grown for 10 days and tested for senescence markers. Controls were grown for 6 days, split and grown for another 10 days. (A) Representative immunoblots for Lamin B1 and cell cycle regulators p16<i>^INK4a</i>, p21, p53, and Cyclin D1. (B) Representative photographs of SA-β-Gal, γH2AX and BrdU; original magnification 400×. Quantification of (C) SA-β-Gal, (D) γH2AX⁺/Ki-67⁻, (E) BrdU, (F) TUNEL, and (G) cleaved caspase 3 positive cells in cultures of control and γ-irradiated PTEC. (H) Representative photographs of epithelial markers ZO-1, Aqp-2, and E-Cadherin in γ-irradiated PTEC; original magnification 400×. Data are mean values ± SEM. **P<0.01; ***P<0.001.</p

Baseline expression of cell cycle protein Cyclin D1 is higher in tubular cells of old kidneys than tubular cells of young kidneys.

<p>(A) Representative photographs of Cyclin D1 (red) immunostaining of kidney sections from young and old mice with or without lead acetate treatment. LTL (green) stains the brush border membrane of the proximal tubule; original magnification 400×. (B) Quantification of tubular cells with Cyclin D1 positive nuclei. (C) Analysis of Cyclin D1 mRNA expression. (D) Quantification of Cyclin D1 positive cells in renal transplant implantation biopsies (n = 36) and healthy renal tissue from nephrectomised patients (n = 22) shows a significant positive correlation between tubular Cyclin D1 expression and chronological age. (E) Representative photographs of Cyclin D1 immunostaining of kidney sections from a younger (36 years) and an older (76 years) human kidney; original magnification 400×. n = 5 for mice. Data are mean values ± SEM. *P<0.05.</p

Lead acetate induces tubular cell proliferation in young but not old kidneys.

<p>Old and young mice were sacrificed 36/100 g body weight lead acetate. (A) Representative Ki-67 (red) and LTL (green) immunostaining of kidney sections from young and old mice with or without lead acetate treatment; original magnification 400×. (B) Quantification of Ki-67 positive cells; (C) representative immunostaining of Ki-67 showing the segments of the kidney (C represents cortex, OM outer medulla and IM inner medulla). n = 5, data are mean values ± SEM. ***P<0.001.</p

Administration of lead acetate does not cause damage to kidney tissue.

<p>Young and old mice were injected with 10/100 g body weight and sacrificed 36 hours later; alternatively mice underwent kidney ischemia/reperfusion injury by clamping of the renal pedicles and were sacrificed 24 hours thereafter. (A) Haemotoxylin-eosin staining of kidney sections from young and old mice with or without lead acetate treatment show no difference in renal microstructure (G represents glomerulus, T represents tubule); original magnification 400×. Quantitative PCR for damage markers (B) Kim-1 and (C) NGAL in control young and old mice as well as young and old mice exposed to lead acetate or after IR damage. (D) LTL damage score of young and old mice injected with lead acetate shows no difference in brush border damage. (E) Quantification of cleaved caspase 3 positive cells; n = 5, data are mean values ± SEM. ***P<0.001</p

<i>In vitro</i> culturing of primary tubular epithelial cells (PTEC) induces SCS-associated changes.

<p>PTEC were isolated from young and old mice and harvested on day 0, day 3, or day 6 of culture. Quantitative PCR for (A) p16<i>^INK4a</i> (B) p15<i>^INK4b</i>, and (C) p19<i>^ARF</i> in PTEC. (D) Quantification of SA-β-GAL staining on day 3 and 6 of PTEC culture. (E) Quantification of cells stained positive for BrdU on day 3 and 6 of PTEC culture. (F) BrdU uptake after lead acetate treatment in PTEC from young and old mice on day 6 of culture. n =  at least 4 separate mice, data are mean values ± SEM. *P<0.05; **P<0.01; ***P<0.001.</p

Lead acetate treatment does not alter senescence markers in cells in the kidneys of old mice.

<p>(A) Representative double immunostainings for phospho-γH2AX (red) and Ki-67 (green) in kidney sections from young and old mice with or without lead acetate treatment. Nuclei with more than 4 foci were considered positive (arrowheads) while double positive cells were not considered senescent (asterisk); red staining in the interstitial space is due to secondary antibody binding to native IgG; original magnification 400×. (B) Quantification of γH2AX positive and Ki-67 negative nuclei. (C) Quantification of SA-β-GAL positive cells. (D) Quantitative PCR for p16<i>^INK4a</i>. (E) Quantitative PCR for p21. n = 5, Data are mean values ± SEM. *P<0.05; **P<0.01; ***P<0.001.</p