p16 INK4a , BRCA1, and GATA4 protein expression in human kidneys from individuals with and without CKD, and effects of bilateral BIRI or AA to induce fibrosis in mice with proximal tubule–targeted Brca1 exon 11 deletions. (A–C) IHC staining of representative human kidney sections with antibodies to BRCA1, p16INK4a, and GATA4, comparing kidneys from patients with either CKD or minimal change disease without CKD. Arrows indicate the nuclear staining of BRCA1. Scale bars, 50 μm. Adjacent graphs indicate fold changes in CKD relative to non-CKD (n = 5, non-CKD; n = 5, CKD). Each dot represents one subject. (D–M) Fold changes in mRNA (RT-PCR) in C57BL/6J mice kidneys for Fn1, Col1a1, Acta2, Tgfb1, and Havcr1 (Kim-1) mRNA/Gapdh mRNA (D–H) normalized to sham mice 28 days after BIRI or (I–M) normalized to control injection at 14 days after AA. n = 4 mice per group. (N) FISH for Brca1 exon 11 mRNA (red) and DAPI staining in Brca1+/+, Brca1+/Δ11, and Brca1Δ11/Δ11 mice at 28 days after BIRI and 14 days after AA injection. Scale bars = 50 μm. Quantitation of mean fluorescence intensity (MFI) is represented as fold change normalized to sham, n = 3 per group. (O) Co-immunostaining with FITC-conjugated LTL (green) and FISH for Brca1 exon 11 mRNA (red) and merged in the Brca1+/+, Brca1+/Δ11, and Brca1Δ11/Δ11 mice. Scale bars = 50 μm. The percentage of LTL-labeled proximal tubules that were positive for Brca1 exon 11 mRNA probes were compared with the total number of LTL-positive tubules among Brca1+/+, Brca1+/Δ11, and Brca1Δ11/Δ11 mice, n = 3 per group. (P and R) Picrosirius red staining of mouse kidneys cortex and outer medulla 28 days after BIRI and 14 days after AA injection and its quantitation plotted as fold change gray value of Picrosirius red (PSR) (n = 5 mice per group). Scale bars = 50 μm. (Q and S) IFTA scores of the mice kidneys 28 days after BIRI and 14 days after AA injection, n = 4 per group. (T and U) Co-immunostaining and quantitation of Fn (green) and α-SMA (red) in mouse kidneys 14 days after AA injection and 28 days after BIRI, n = 4 per group. Scale bars = 50 μm. (V) Western blot analysis of Fn, α-SMA, and p16INK4a from kidney cortices from mice at 28 days after BIRI and its quantitation normalized to ERK2 expression, n = 3 per group. All data are represented as mean ± SD and repeated twice. (D–V) Each dot represents one mouse. Unpaired t tests were performed using Welch’s correction for all statistical analyses. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.