Increased levels of serum pigment epithelium-derived factor aggravate proteinuria via induction of podocyte actin rearrangement

Abstract

Purpose: To assess the role of serum pigment epithelium-derived factor (PEDF) in the occurrence and development of proteinuria and renal dysfunction and determine its relevant signaling pathway.

Methods: We analyzed serum PEDF, creatinine, the urinary albumin-to-creatinine ratio, and renal morphology of normal or streptozotocin (STZ)-induced diabetic mice, before and after treatment with PEDF. In vitro, podocytes were stimulated with PEDF under normal or high-glucose conditions; permeability was measured by the transwell assay with fluorescein isothiocyanate (FITC)-dextran; and F-actin cytoskeleton was analyzed by phalloidin staining. Apoptosis was assessed by flow cytometry. RhoA activity and ROCK1, ZO-1, nephrin, and podocin levels were detected by Western blotting.

Results: Diabetic mice exhibited a high serum PEDF level. In vivo, elevated serum PEDF led to proteinuria, increased serum creatinine, and podocyte foot process fusion in normal or diabetic mice. In vitro, both high-glucose and PEDF stimulation activated the RhoA/ROCK1 pathway in podocytes and promoted cell permeability, F-actin rearrangement, and apoptosis. Inhibition of RhoA/ROCK1 alleviated the damage from these effects.

Conclusions: Elevated serum PEDF aggravates the development of proteinuria and renal dysfunction by inducing F-actin arrangement, foot process fusion, and apoptosis of podocytes in both normal and diabetic mice, and this effect may be mediated by activation of the RhoA/ROCK1 pathway.

Keywords: Actin; Diabetic kidney disease; PEDF; Proteinuria; RhoA/ROCK1.

Fig. 1

Elevated serum PEDF was positively correlated with proteinuria in diabetic mice. a Serum PEDF levels, b urinary ACR levels, and c serum creatinine levels in different groups. n = 15 in NC 5w, DM1 5w, and DM2 5w; n = 8 in NC 10w, DM1 10w, and DM2 10w. d–f Representative microscopy images of HE staining of glomeruli in different groups. Original magnification 400×, scale bar = 25 µm. g Representative transmission electron microscopy images of podocyte foot process in different groups (foot processes indicated by black arrows). Original magnification 2000×, scale bar = 1000 nm. The data are shown as the mean ± SEM. ***P < 0.001, *P < 0.05. N.S. no significant difference

Fig. 2

PEDF promoted proteinuria development in both normal and diabetic mice. a Serum PEDF levels, b urinary ACR levels, and c serum creatinine levels in different groups. n = 6 in each group. d–f Representative microscopy images of HE staining of glomeruli in different groups. Original magnification 400×, scale bar = 25 µm. g Representative transmission electron microscopy images of podocyte foot process in different groups (foot processes indicated by black arrows). Original magnification 2000×, scale bar = 1000 nm. The data are shown as the mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05

Fig. 3

PEDF led to actin rearrangement and apoptosis of podocytes. a Paracellular permeability was measured 1 h after the addition of FITC-dextran. Podocytes were treated with PEDF at concentrations of 100, 200, 500, and 1000 ng/ml at different hours. b Podocyte paracellular permeability was measured in different groups. c, d Western blot was used to detect ZO-1 expression of cultured podocytes in different groups. e Podocytes were stained for F-actin (red). Scale bars = 10 µm. f, g Annexin V-Cy3/SYTOX double-staining labeled podocytes in each group and flow cytometry was used to determine the podocyte apoptosis rate. Cells in the early stage of apoptosis are Annexin V+/SYTOX−, while cells in late apoptosis or necrotic cells are Annexin V+/SYTOX+. h, i Western blot was used to detect nephrin and podocin expression of cultured podocytes in different groups. The data are shown as the mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05

Fig. 4

The RhoA/ROCK signaling pathway mediated actin rearrangement in PEDF-treated podocytes. a, b RhoA pull-down assay and western blot was used to detect RhoA activity and total RhoA expression of cultured podocytes in different groups. c, d Western blot was used to detect ROCK1 expression of cultured podocytes in different groups. e Paracellular permeability was measured 1 h after the addition of FITC-dextran in each group. f Podocytes were stained for F-actin (red). Scale bars = 10 µm. g, h Flow cytometry was used to determine the apoptosis of Annexin V/SYTOX double-staining labeled podocytes in each group. i–l Western blot was used to measure ZO-1 (i, j), nephrin, and podocin (k, l) expression of cultured podocytes in different groups. The data are shown as the mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05, N.S. no significant difference