Fig 2: CaN inhibitors protect from LPS-induced podocyte injury. (A, B) CsA and FK506 decreased LPS-induced urinary protein in mice. (C) PAS staining of CsA or FK506 pretreatment in LPS-induced mice. Scale bar = 20 µm. (D) Transmission electron microscopy analysis of CsA or FK506 pretreatment in LPS-induced mice. Scale bar = 2 µm. (E) Immunofluorescence staining of synaptopodin (red) and podocin (red) in CsA or FK506 pretreated mice at 24 h after LPS induction. Scale bar = 10 µm. (F) Immunofluorescence staining of F-actin (green) and synaptopodin (red) in CsA or FK506 pretreated cultured podocytes at 24 h after LPS induction. Scale bar = 50 µm. (G) Western blot analysis of synaptopodin in CsA or FK506 pretreated cultured podocytes at 24 h after LPS induction. (H) Schematic diagram indicates LPS-induced podocyte Angptl4 is mediated by TLR4/MyD88/NF-?B and calcineurin/NFAT signal pathways. All values were presented as mean ± SD, n = 3. *P < 0.05 versus LPS group, **P < 0.01 versus LPS group, ***P < 0.001 versus LPS group, ****P < 0.0001 versus LPS group using one-way ANOVA followed by Turkey's method.

Fig 3: LPS induces the expression of Angptl4 in podocytes in vivo. (A) Immunofluorescence staining of synaptopodin (red) and podocin (red) in normal and LPS-treated mouse glomerulus. Scale bar = 20 µm. (B) Transmission electron microscopy analysis of foot process fusion in control and LPS-induced mice. Scale bar = 2 µm. (C) mRNA detection of kidney Angptl4 at 24 h after LPS induction. (D, E) ELISA detection of serum and urine Angptl4 in LPS-induced mice. (F) mRNA detection of Angptl4 in separated mouse glomerulus and tubules at 24 h after LPS induction. (G) Western blot analysis and quantification of Angptl4 expression in LPS-induced glomerulus. All values were presented as mean ± SD, n = 3. *P < 0.05 versus control group, **P < 0.01 versus control group, ***P < 0.001 versus control group, ****P < 0.0001 versus control group using a two-tailed Student's t test.

Fig 4: LPS increases podocyte Angptl4 in vitro. (A) Immunofluorescence staining of F-actin (green) and synaptopodin (red) in control and LPS-treated podocytes. Scale bar = 50 µm. (B) Western blot detection and quantification of synaptopodin in normal and LPS-treated podocytes. Comparison between control and LPS group was made using a two-tailed Student's t test. (C) mRNA detection of Angptl4 at 24 h after 0, 5, 10 or 20 µg/ml LPS induction. (D, E) Western blot detection and quantification of supernatant (CM: conditioned medium) and cellular (cell lysis) Angptl4 at 24 h after 0, 5, 10 or 20 µg/ml LPS induction. (F) mRNA detection of Angptl4 at 0 h, 3 h, 6 h, and 24 h after 20 µg/ml LPS induction. (G, H) Western blot detection and quantification of supernatant and cellular Angptl4 at 0 h, 3 h, 6 h and 24 h after 20 µg/ml LPS induction. All values were presented as mean ± SD, n = 3. Data from multiple groups were made using one-way ANOVA followed by Turkey's method. *P < 0.05 versus control group, **P < 0.01 versus control group, ****P < 0.0001 versus control group.

Fig 5: Angptl4 deteriorates LPS-induced podocyte injury. (A, B) mRNA and western blot quantification of Angptl4 in EV and Angptl4-OE podocytes. (C) Western blot quantification of synaptopodin in EV and Angptl4-OE podocytes before or after LPS induction. (D) Immunofluorescence staining of F-actin (green) and synaptopodin (red) in EV and Angptl4-OE podocytes before or after LPS induction. Scale bar = 50 µm. (E, F) mRNA and western blot quantification of Angptl4 in NC and Angptl4-KD podocytes. (G) Western blot quantification of synaptopodin in NC and Angptl4-KD podocytes before or after LPS induction. (H) Immunofluorescence staining of F-actin (green) and synaptopodin (red) in NC and Angptl4-KD podocytes before or after LPS induction. Scale bar = 50 µm. All values were presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using one-way ANOVA followed by Turkey's method.