Role of C/EBP-α in Adriamycin-induced podocyte injury

Abstract

Podocytes are terminally differentiated epithelial cells in the kidney glomeruli that act as a key component of the glomerular filtration barrier. Although the inciting injury to the podocyte may vary between various glomerular diseases, the inevitable consequence of podocyte injury results in their loss, leading to progressive kidney disease. Here, we report that the expression of CCAAT/enhancer binding protein-α (C/EBP-α), a transcription factor known to interact with and activate PPAR-γ and NF-κB, is suppressed in the glomerular cells, particularly in podocytes, in human kidneys with focal segmental glomerulosclerosis. Genetic ablation of C/EBP-α in podocytes resulted in increased proteinuria, increased podocyte foot process effacement, and to decreased podocyte number in the setting of Adriamycin (ADR)-induced nephropathy. Overexpression of C/EBP-α in human podocytes in vitro led to an inhibition of MCP-1 and IL-6 expression in response to TNF-α and IL-1β treatments. Conversely, augmented production of MCP-1 and IL-6 was observed in the glomeruli of C/EBP-α knockout mice and was associated increased infiltration of macrophages in vivo. Together, our data suggest that C/EBP-α mediates anti-inflammatory effects in podocytes to confer protection against podocyte injury and loss that may contribute to worsening glomerulosclerosis.

Figure 1. C/EBP-α expression in human kidney.

(a) Immunohistochemical staining for C/EBP-α performed on healthy donor nephrectomy specimens shows a nuclear distribution in glomerular cells (including endothelial, parietal, and podocytes) and in tubular cells (left). C/EBP-α expression is significantly reduced in kidney biopsy specimens from patients with FSGS (middle) and no staining is detected in no primary antibody negative control (right). Representative images of three subjects in each group are shown (original magnification x400, scale bar: 50 μm). (b) Semi-quantification of glomerular C/EBP-α expression in (a) is shown. Glomerular region was selected, and optical density (OD) was measured and quantified as a relative fold change to healthy donor specimens (n = 3, *p < 0.001 vs healthy donor).

Figure 2. Podocyte-specific knockout of C/EBP-α in mice.

(a) Western blot analysis of C/EBP-α in the lysates of primary podocytes isolated from WT and KO mice (n = 3 mice in each group). The blots were then stripped and reprobed for β-actin as loading controls. (b) Immunostaining of C/EBP-α and podocyte marker synaptopodin shows decreased C/EBP-α in podocytes in KO kidney sections in comparison to WT mice.

Figure 3. Worsened proteinuria in KO-ADR mice.

(a) Body weight of mice post-injection of vehicle (WT and KO) or ADR (WT-ADR and KO-ADR) are shown. Both ADR-WT and ADR-KO mice experienced similar weight loss following ADR injection. (b) Development of proteinuria in ADR-injected mice was assessed by urinary albumin to creatinine ratio. (c) The 12 h urinary albumin excretion at 4 weeks among these mice. (n = 6, *p < 0.001 vs WT and KO, ^#p < 0.05 vs WT-ADR).

Figure 4. Increased mesangial and glomerular area in KO-ADR mice.

(a) Representative images of periodic acid–Schiff (PAS)-stained kidneys at 4 weeks post-injection. Glomerular hypertrophy and mesangial area are significantly increased in KO-ADR in comparison to WT-ADR (Original magnification x200 and x400). (b) Quantification of glomerular area at 4 weeks post-injection. Values are mean ± SEM (n = 60 glomeruli per group of 6 mice, **p < 0.01 vs other groups). (c) Quantification of area of mesangial fraction in the glomeruli at 4 weeks post-injection (n = 6, **p < 0.01 and ***p < 0.001 vs WT, ^#p < 0.05 vs WT-ADR).

Figure 5. Increased podocyte foot process effacement in KO-ADR mice.

(a) Representative electron micrograph from WT, KO, WT-ADR, and KO-ADR mice are shown with both low (x2K) and high (x10K) magnifications (scale bar: 5 μm). Arrows point to examples of effaced foot processes in ADR-injected mice. (b) Quantification of average foot process width in each group of mice is shown. Values are mean ± SEM (n = 60 glomeruli per group of 6 mice, ***p < 0.01 vs WT an KO, ^#p < 0.05 vs WT-ADR).

Figure 6. Increased podocyte loss in KO-ADR mice.

(a) Representative images of WT1 (red) immunostaining and DAPI (blue) of glomeruli are shown (n = 6, original magnification x400, scale bar: 50 μm). (b, c) WT1-positive cell number per 1000 µm2 glomerular tuft area (b) and WT1-positive cell number/glomerular cross-section (c) were quantified and expressed as relative fold changes to WT mice. (d) mRNA levels of Wt1 mRNA were measured using real-time PCR. (60 glomeruli per group; n = 6, *p < 0.05, **p < 0.01, ***p < 0.001 vs WT and KO, ^#p < 0.05 compared to WT-ADR).

Figure 7. Expression of nephrin and podocin is reduced in KO-ADR mice.

(a,d) Representative images of immunostaining of nephrin (green, a) or podocin (red, d) in glomeruli are shown. (b,e) Semi-quantification of nephrin or podocin expression is shown. (c,f) mRNA levels of nephrin or podocin were measured using real-time PCR. (60 glomeruli per group; n = 6, ***p < 0.001 vs WT and KO, ^#p < 0.05 WT-ADR, original magnification x400, scale bar: 50 μm).

Figure 8. Effect of C/EBP-α overexpression on MCP-1 and IL-6 Expression in podocytes treated with TNF-α.

(a) MCP-1 mRNA expression in TNF-α-stimulated podocytes. (b) MCP-1 production in TNF-α-stimulated podocytes. (c) IL-6 mRNA expression in TNF-α-stimulated podocytes. (d) IL-6 production in TNF-α-stimulated podocytes. (*p < 0.05,**p < 0.01,***p < 0.001).

Figure 9. Effect of C/EBP-α overexpression on MCP-1 and IL-6 Expression in podocytes treated with IL-1β.

(a) MCP-1 mRNA expression in IL-1β-stimulated podocytes; (b) MCP-1 production in IL-1β-stimulated podocytes (c) IL-6 mRNA expression in IL-1β-stimulated podocytes (d) IL-6 production in IL-1β-stimulated podocytes. (*p < 0.05,**p < 0.01,***p < 0.001).

Figure 10. Increased activation of NF-κB and production of MCP-1 and IL-6 in KO-ADR glomeruli.

(a) Representative images of phosphorylated p65 NF-κB (Ser536) immunostaining are shown. (b) Semi-quantification of phospho-p65 NF-κB immunostaining is shown as relative fold change in optical density (OD). (c,d) mRNA levels of MCP-1 (C) and IL-6 (d) in isolated glomeruli from each group of mice were measured using real-time PCR (60 glomeruli per group; n = 6, ***p < 0.001 vs WT and KO, ^##p < 0.01 vs WT-ADR).

Figure 11. Increased infiltrating macrophages in KO-ADR kidneys.

(a) Representative images of immunostaining for macrophage marker F4/80 in WT, KO, WT-ADR, and KO-ADR mice at 4 weeks after ADR injection at x200 and x400 magnifcation. (b) Quantification of F4/80-positive area between ADR mice and non-ADR mice is shown as fold change relative to WT and KO (n = 6, ***p < 0.001 vs WT and KO, ^#p < 0.05 vs WT-ADR).