A CPEB2/ANGPTL3 feedback loop promotes the progression of podocyte injury in nephrotic syndrome

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  Fig. 1. Aberrant expression of CPEB2 in podocyte injury. (A) Venn diagram showing the number of overlapping DEGs down regulated in the injured podocytes from GEO datasets. (B) Venn diagram showing the number of overlapping DEGs up regulated in the injured podocytes from GEO datasets. (C) Cell viability. The MPC-5 cells were treated with adriamycin (ADR) at various dosages as indicated for 24 h. Data were presented as mean ± SD. (D) mRNA levels of CPEB2. The cells were treated as described in (C). Data were presented as mean ± SD. (E) mRNA levels of CPEB2. The MPC-5 cells were transfected with CPEB2 siRNAs for 24 h, followed by treatment with ADR (1 μm) for additional 24 h. Data were presented as mean ± SD. (F) Cell viability. The cells were treated as described in (E). Data were presented as mean ± SD. (G) Cell viability. The MPC-5 cells were treated with puromycin aminonucleoside (PAN) at various dosages as indicated for 24 h. Data were presented as mean ± SD. (H) mRNA levels of CPEB2. The cells were treated as described in (G). Data were presented as mean ± SD. (I) mRNA levels of CPEB2. The MPC-5 cells were transfected with CPEB2 siRNAs for 24 h, followed by treatment with PAN (1 mM)…

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  Fig. 2. Ectopic overexpression of CPEB2 induced podocyte injury through ANGPTL3. (A) Cell viability. The MPC-5 cells were transfected with CPEB2 plasmids for 24 h and 48 h. Data were presented as mean ± SD. (B) Representative images of CPEB2 and ANGPTL3 double staining in MPC-5 cells. The MPC-5 cells were transfected with CPEB2-GFP plasmids (green) for 24 h, and the expression of ANGPTL3 (red) was determined by immunofluorescence staining. Scale bar = 100 μm. Three experiments were performed that showed similar results. (C) Cell viability. The MPC-5 cells were transfected with CPEB2 plasmids and ANGPTL3 siRNAs for 48 h. Data were presented as mean ± SD. (D) Representative immune blots of CPEB2 and ANGPTL3. Data were presented as mean ± SD. (E) Cell apoptosis. Data were presented as mean ± SD. (F) Cell migration. Scale bar = 100 μm. Data were presented as mean ± SD. (G) Rearrangement of F-actin. F-actin rearrangement is characterized by the formation of lamellipodia (arrow). Scale bar = 20 μm. Data were presented as mean ± SD.

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  Fig. 3. CPEB2 promoted the translation of ANGPTL3 by interacting with the 3’ UTR of ANGPTL3 mRNA transcripts. (A) mRNA levels of ANGPTL3. The MPC-5 cells were transfected with CPEB2 plasmids for 24 h and 48 h. Data were presented as mean ± SD. (B) Polysomal profiles. The MPC-5 cells were transfected with CPEB2 plasmids for 48 h. Left panel: Global distribution of 40 S subunit, 60 S subunit, 80 S subunit, and polysomes as indicated. Right panel: Relative distribution ANGPTL3 transcripts in each gradient fraction. Data were presented as mean ± SD. (C) Nascent protein synthesis of ANGPTL3. The cells were treated as described in (A). Newly translated AMBRA1 protein levels were determined using CLICK-iT protein synthesis Data were presented as mean ± SD. (D) RNP-IP assays. The cells were harvested 48 h after transfection, and the association of ANGPTL3 mRNA with EIF3D and EIF3G was measured by RNP-IP. Data were presented as mean ± SD. (E) Representative immunoblots of ANGPTL3 following EIF3D depletion. The MPC-5 cells were harvested 48 h post transfection. Data were presented as mean ± SD. (F) Luciferase assay. Left panel: schematic representation of the constructed fragments. Right panel:…

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  Fig. 4. The CPEB2/ANGPTL3 feedback loop responding to ADR exposure. (A) Representative immune blots of CPEB2 and ANGPTL3. The MPC-5 cells were treated with ADR at various dosages as indicated for 24 h. Data were presented as mean ± SD. (B) Representative immune blots of CPEB2 and ANGPTL3. The MPC-5 cells were treated with ADR (1 µM) for 12 h and 24 h. Data were presented as mean ± SD. Data were presented as mean ± SD. (C) Representative immune blots of CPEB2 and ANGPTL3. The MPC-5 cells were transfected with ANGPTL3 siRNAs for 24 h, followed by treatment with ADR (1 µM) for additional 24 h. Data were presented as mean ± SD. (D) mRNA levels of CPEB2. The cells were treated as described in (C). Data were presented as mean ± SD. (E) mRNA stability of CPEB2 transcripts. The cells were treated as described in (C). Afterwards, the cells were incubated with the RNA polymerase inhibitor actinomycin D (Act D) for the indicated periods (0, 3, 6, 9 and 12 h), and the remaining CPEB2 mRNA levels were analyzed by qPCR.

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  Fig. 5. Knockdown of CPEB2 decreased renal ANGPTL3 expression and alleviated ADR-induced podocyte injury in vivo. (A) Urinary excretion of albumin. Data were presented as mean ± SD (n = 5/group). (B) Serum levels of albumin. Data were presented as mean ± SD (n = 5/group). (C) Serum levels of triglycerides Data were presented as mean ± SD (n = 5/group). (D) Serum levels of cholesterols. Data were presented as mean ± SD (n = 5/group). (E) Representative TEM images and podocyte foot processes. Scale Bar = 2 μm (upper panel) and 1 μm (lower panel). Data were presented as mean ± SD (n = 5/group). (F) Representative images of periodic acid–Schiff (PAS) staining (upper). Arrows indicate glomerular-capsule adhesions. IHC staining of ANGPTL3 (lower). Arrows indicate ANGPTL3-positive cells. Scale Bar = 20 μm. (G) Urinary excretion of ANGPTL3. Data were presented as mean ± SD (n = 5/group). (H) Representative immune blots of CPEB2 and NPHS2. Data were presented as mean ± SD (n = 5/group).