PMC

(A-B) Kidney sections of 22-week-old mice were stained with H&E (A) or immunostained with F4/80 (B). Scale bar, 50 μm. (C) F4/80-positive area was quantified based on the immunostained images using Bio-Revo imaging and analysis software. (D) Total RNA was isolated from renal tissues of 22-week-old mice. Quantitative RT-PCR was performed to analyze the expression of the indicated pro-inflammatory cytokines. The data were normalized to Gapdh. Bars indicate the mean ± S.D. (n = 4–6). *P<0.05; **P<0.01 versus NaCl (250 mg/kg)-treated AS mice; #P<0.05; versus NaBr (75 mg/kg)-treated AS mice. P values were assessed by Tukey-Kramer test. n.s., not significant.

(A) Representative images of PAS-stained renal sections from 22-week-old mice AS mice and age-matched WT mice. Scale bar, 50 μm. (B) Glomerular injury score was evaluated based on the PAS-stained sections. (C) Total RNA was isolated from renal tissues of 22-week-old mice. Quantitative RT-PCR was performed to analyze the expression of the indicated renal injury markers such as Lcn2 and Lysozyme. The data were normalized to Gapdh. Bars indicate the mean ± S.D. (n = 4–6). *P<0.05; **P<0.01 versus NaCl (250 mg/kg)-treated AS mice; #P<0.05; versus NaBr (75 mg/kg)-treated AS mice. P values were assessed by Tukey-Kramer test.

(A) Schematic diagram of the experimental design for NaCl (250 mg/kg) or NaBr (75 mg/kg, 250 mg/kg) treatment in WT mice. (B) Body weight was measured every two weeks until mice were 32 weeks old. (C) Serum creatinine in 32-week-old mice was measured. (D) Total RNA was isolated from renal tissues of 32-week-old mice. Quantitative RT-PCR was performed to evaluate the expression of the indicated genes. Bars indicate the mean ± S.D. (n = 4). P values were assessed by Tukey-Kramer test. n.s., not significant.

(A) Representative images of Masson-Trichrome (MT)-stained renal sections from 22-week-old AS mice are shown. Scale bar, 50 μm. (B) Tubulointerstitial fibrosis score was evaluated by measuring the fibrotic region in MT-stained section. (C-E) Total RNA was isolated from renal tissues of 22-week-old mice. Quantitative RT-PCR was performed to analyze the expression of the indicated pro-fibrotic genes (C), matrix metalloproteinase (D) and anti-fibrotic genes (E). The data were normalized to Gapdh. Bar graphs indicate the mean ± S.D. (n = 4–6). *P<0.05; **P<0.01 vs NaCl (250 mg/kg)-treated AS, #P<0.05; ##P<0.01 versus NaBr (75 mg/kg)-treated AS mice. P values were assessed by Tukey-Kramer test. n.s., not significant.

(A) Schematic diagram of the experimental design for NaCl or NaBr treatment in AS mice. (B) Body weight was measured every two weeks. (C) Urine samples were collected every four weeks, and urinary protein was measured by Bradford method. Proteinuria score was calculated based on urinary protein and creatinine concentrations in NaCl- or NaBr-treated AS mice (mean±S.D. n = 6). (D) Serum creatinine was measured by Jaffe’s method in WT, NaCl- or NaBr-treated AS mice at 22 weeks. (E) BUN score of each group in 22-week-old mice was measured. Bars indicate the mean ± S.D. *P<0.05; **P<0.01 versus NaCl (250 mg/kg)-treated AS; #P<0.05; ##P<0.01 versus NaBr (75 mg/kg)-treated AS mice. P values were assessed by Tukey-Kramer test. n.s., not significant.

(A) Lysates were extracted from mouse kidneys of the indicated groups, and subjected to immunoblotting analysis. Protein expressions of type IV collagen, phosphorylated Smad1/5/8 and γ-tubulin were visualized by chemiluminescence. γ-Tubulin was used as loading control. (B-C) Blot intensities were quantified using Image J software (Fujifilm, Japan). Values were normalized to γ-tubulin and presented as relative expression. (D) Kidney sections of 22-week-old mice were immunostained with type Ⅳ collagen. Scale bar, 100 μm. (E) Type Ⅳ collagen-positive area was quantified based on the immunostained images using Bio-Revo imaging and analysis software. Bars indicate the mean ± S.D. (n = 3–4). *P<0.05; **P<0.01 versus NaCl (250 mg/kg)-treated AS, #P<0.05 versus NaBr (75 mg/kg)-treated AS mice. P values were assessed by Tukey-Kramer test. n.s., not significant.