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. 2021 Aug;32(8):1974–1986. doi: 10.1681/ASN.2021020231

Figure 1.

Figure 1.

Rest deletion accelerates podocyte injury and proteinuria. (A) Quantitative RT-PCR (RT-qPCR) of REST mRNA in kidney extracts from young 8-week-old (n=6) and aged 52 weeks (n=5) C57bl/6 mice. (B) Double-labeling for REST and nestin in young (n=6) and aged (n=5) C57bl/6 mice. Scale bar, 10 μm. Insets show higher-magnification images of representative podocytes. (C) REST mRNA in kidney extracts of Sham-operated (Sh) (n=6) and 75% nephrectomized (Nx) (n=10) C57BL/6 mice at 2 months after surgery. (D) Immunolabeling of REST in Sh (n=6) and Nx (n=10) C57bl/6 mice at 2 months after surgery. Insets show higher-magnification images of representative podocytes. (E) Double labeling for REST and GFP in primary isolated GFP-podocytes from 8- and 52-week-old C57BL/6 mice. (F) Western blot analysis of REST in cytoplasmic (n=4) and nuclear fractions (n=4) of primary isolated GFP-podocytes from 52-week-old C57bl/6 mice. (G) Immunofluorescence of REST and Nestin in podocyte-specific Rest mutant (RESTΔpod) (n=5) and Rest floxed (RESTflox/flox) (n=5) 52-week-old C57BL/6 mice. Scale bar, 10 μm. Insets show higher-magnification image of representative podocytes. (H) Western blot of REST in laser microdissected glomerular extracts from kidneys of RESTflox/flox (n=4) and RESTΔpod (n=4) 52-week-old C57bl/6 mice. (I) Albumin to creatinine ratio (mg/mmol) in urine samples from C57bl/6 RESTflox/flox and RESTΔpod 52-week-old mice (n=5 for both conditions). (J)–(L) Morphology and lesion scores of kidneys from RESTflox/flox (n=4) and RESTΔpod (n=4) 52-week-old C57bl/6 mice. Scale bar, 10 μm. (M–O) Cleaved caspase-3 (CC3) immunolabeling (M), terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling assay (N), and WT1 immunolabeling and quantification (lower panel) of kidneys from RESTflox/flox and RESTΔpod 52-week-old C57BL/6 mice. Scale bar, 10 μm. (P) Western blot and quantitative analysis of CC3 in kidney extracts from RESTflox/flox (n=4) and RESTΔpod (n=4) 52-week-old C57bl/6 mice. Scale bar, 10 μm. All data are shown as the mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 between groups, Mann–Whitney or t test.