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. 2011 Dec;179(6):2810–2821. doi: 10.1016/j.ajpath.2011.08.023

Figure 2.

Figure 2

GLO1-overexpressing aged rats showed morphological amelioration in senescence-associated alterations of kidney. A: Light micrographs of rat renal cortex with SABG staining showing senescent tubular cells in aged WT rats. The number of senescent tubules was reduced by overexpression of GLO1. Frozen tissue specimens (15 μm thick) were counterstained with Nuclear Fast Red. Original magnification: ×100. Scale bars: 100 μm. Blue indicates SABG-positive cells. B: Quantitative analysis of SABG-positive area. The positive rate was calculated by dividing the positive area by the total area in each field and is expressed as fold increase, relative to the level of young WT rats. *P < 0.01. C: Representative light micrographs of renal cortex of aged WT rat, costained with SABG (blue) and γ-GTP (pale red). Black arrows mark senescent proximal tubular cells; white arrows mark nonsenescent proximal tubular cells. Black arrowheads mark senescent nonproximal tubular cells; white arrowheads mark nonsenescent nonproximal tubular cells. The letter G marks the glomerulus. Frozen specimens were examined without counterstaining. Original magnification = ×200. Scale bar = 100 μm. D: Representative light micrographs of renal cortex of aged WT rat costained with SABG (blue) and calbindin-D-28K. Black arrows mark senescent distal tubules-connecting tubules; white arrows mark nonsenescent distal tubules-connecting tubules. Frozen specimens were counterstained with hematoxylin. Original magnification = ×200. Scale bar = 100 μm. E: Representative light micrographs of renal cortex of aged WT rat costained with SABG (blue) and AQP2. Black arrows mark senescent connecting tubules-collecting ducts; white arrows mark nonsenescent connecting tubule-collecting ducts. The letter G marks the glomerulus. Frozen specimens were counterstained with hematoxylin. Original magnification = ×200. Scale bar = 100 μm. F: Relative transcript levels of p53, p21WAF1/CIP1, and p16INK4A were determined by real-time quantitative RT-PCR. All were significantly elevated in aged WT. This age-dependent increase was significantly reduced by overexpression of GLO1. The values for young WT rats were set as 1. *P < 0.01; **P < 0.05 versus young WT rats; †P < 0.01 versus aged WT rats. G: Representative micrographs of immunohistochemistry staining for p53, p21WAF1/CIP1, and p16INK4A in the rat renal cortex. In each staining, aged WT tissue exhibited marked increases in positive area in tubules. The extent of positive areas was significantly decreased by overexpression of GLO1. Methyl Carnoy's fixed specimens (4 μm thick) were counterstained with hematoxylin. Original magnification = ×400. Scale bars: 50 μm. H: Protein immunoblot of p53 and p16INK4A of renal cortex lysate. Elevation of p53 and p16INK4A in aged WT rats was attenuated by overexpression of GLO1. Actin served as a control. I: Densitometric quantification of p53 and p16INK4A immunoblot. The values for aged WT rats were set as 1. *P < 0.05 versus young WT rats; †P < 0.05 versus aged WT rats. J: Protein immunoblot of CEL of renal cortex lysate indicates amelioration of carbonyl stress by GLO1 overexpression. Actin served as a control. Many bands were detected as CEL-modified protein, indicating that AGEs were accumulated in renal cortex.