Bromide supplementation exacerbated the renal dysfunction, injury and fibrosis in a mouse model of Alport syndrome

<div><p>A seminal study recently demonstrated that bromide (Br^-) has a critical function in the assembly of type IV collagen in basement membrane (BM), and suggested that Br^- supplementation has therapeutic potential for BM diseases. Because salts of bromide (KBr and NaBr) have been used as antiepileptic drugs for several decades, repositioning of Br^- for BM diseases is probable. However, the effects of Br^- on glomerular basement membrane (GBM) disease such as Alport syndrome (AS) and its impact on the kidney are still unknown. In this study, we administered daily for 16 weeks 75 mg/kg or 250 mg/kg (within clinical dosage) NaBr or NaCl (control) via drinking water to 6-week-old AS mice (mouse model of X-linked AS). Treatment with 75 mg/kg NaBr had no effect on AS progression. Surprisingly, compared with 250 mg/kg NaCl, 250 mg/kg NaBr exacerbated the progressive proteinuria and increased the serum creatinine and blood urea nitrogen in AS mice. Histological analysis revealed that glomerular injury, renal inflammation and fibrosis were exacerbated in mice treated with 250 mg/kg NaBr compared with NaCl. The expressions of renal injury markers (<i>Lcn2</i>, <i>Lysozyme</i>), matrix metalloproteinase (<i>Mmp-12</i>), pro-inflammatory cytokines (<i>Il-6</i>, <i>Il-8</i>, <i>Tnf-α</i>, <i>Il-1β</i>) and pro-fibrotic genes (<i>Tgf-β</i>, <i>Col1a1</i>, <i>α-Sma</i>) were also exacerbated by 250 mg/kg NaBr treatment. Notably, the exacerbating effects of Br^- were not observed in wild-type mice. These findings suggest that Br^- supplementation needs to be carefully evaluated for real positive health benefits and for the absence of adverse side effects especially in GBM diseases such as AS.</p></div

Long-term NaBr treatment does not affect the renal function in WT mice.

<p>(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.</p

ICP-MS measuring conditions for Br^-.

<p>ICP-MS measuring conditions for Br^-.</p

NaBr exacerbates the renal fibrosis in AS mice.

<p>(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 <i>Gapdh</i>. 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.</p

Bromide ion concentration.

<p>Bromide ion concentration.</p

NaBr exacerbates the renal inflammation.

<p>(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 <i>Gapdh</i>. 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.</p

NaBr induces the accumulation of type IV collagen.

<p>(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.</p

Primer sequence for real-time quantitative RT-PCR.

<p>Primer sequence for real-time quantitative RT-PCR.</p