Figure 4: PBA protects from proteinuria-induced tubular lesion development and renal dysfunction by modulating LCN2 expression.

(a–e) WT1^+/mut mice and wild-type littermates were treated with PBA or vehicle for 2 weeks. As no significant difference between vehicle and PBA-treated WT1^+/+ mice was observed, only one group (Control) is represented. (a) Renal tubular morphology (left panels, periodic acid–Schiff (PAS) staining; scale bar, 100 μm) and quantification (right panel) in control (n=6), vehicle-treated WT1^+/mut (n=6) and PBA-treated WT1^+/mut (n=11) mice. (b) Interstitial fibrosis (left panels, Sirius red staining; scale bar, 100 μm) and quantification (right panel) in control (n=6), vehicle-treated WT1^+/mut (n=6) and PBA-treated WT1^+/mut (n=6) mice. (c) Plasma creatinine levels in control (n=6), vehicle-treated WT1^+/mut (n=5) and PBA-treated WT1^+/mut (n=9) mice. (d) Tubular cell apoptosis evaluated using TUNEL (left panel; scale bar, 100 μm) and quantification (right panel; n=6 per group). (e) LCN2 protein (scale bar, 100 μm; insert scale bar, 10 μm) and (f) mRNA expression in control (n=6), vehicle-treated WT1^+/mut (n=6) and PBA-treated WT1^+/mut (n=10) mice. (g,h) mIMCD-3 cells were exposed to 1% albumin for 24 h in the presence of either PBA or the vehicle (n=3). (g) Western blotting of ATF4 and LCN2 (left panel), and quantification (right panel; n=3). (h) Lcn2 mRNA expression (n=3). (i) LCN2 immunohistochemistry in the renal biopsy of a proteinuric patient (before PBA administration) and a control (scale bar, 100 μm). (j) Evolution of urinary LCN2 excretion (black line) and proteinuria (red line) before and during PBA treatment (arrow indicates the beginning of the treatment) in the proteinuric patient. Data are mean±s.e.m. Statistical analysis: one way analysis of variance followed by Tukey–Kramer test; *P<0.05, **P<0.01, ***P<0.001 versus controls: ^#P<0.05, ^##P<0.01 versus vehicle-treated WT1^+/mut mice or cells.