BECLIN1 Is Essential for Podocyte Secretory Pathways Mediating VEGF Secretion and Podocyte-Endothelial Crosstalk

Abstract

Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier.

Keywords: BECLIN1; Golgi network; VEGF; autophagy; glomerulosclerosis; podocyte; secretory pathway.

Figure 1

Podocyte-specific knockout of Becn1 expression. (A) Schematic showing the generation of Becn1^Δpod mice by crossing Becn1^flox/flox mice with hNphs2-Cre mice; (B) Schematic showing the generation of fluorescence-labelled podocytes by crossing Becn1^flox/flox × hNphs2-Cre mice with Tomato/EGFP reporter mice; (C) Schematic showing the ex vivo isolation of podocytes after glomeruli isolation, digestion and mechanical shearing and subsequent fluorescence-activated cell sorting (FACS); (D) Western blotting was performed to determine the abundance of BECLIN1 in ex vivo isolated primary podocytes; (E) Densitometry data obtained from samples shown in (D) (*** ≤ 0.001); (F) PCR was performed to identify Becn1 deletion using a DNA template obtained from isolated Becn1^Δpod and WT podocytes. (G) Survival analysis of Becn1^Δpod and WT mice (n = 30 each genotype); (H) Body weight of Becn1^Δpod and WT mice through week 8 (** ≤ 0.01); (I) Urinary albumin/creatinine ratio in Becn1^Δpod and WT mice through week 8 (** ≤ 0.01 and *** ≤ 0.001, respectively); (J) Serum creatinine (mg/dL) in WT and Becn1^Δpod mice *** ≤ 0.001); (K) Serum urea (mg/dL) in WT and Becn1^Δpod mice (*** ≤ 0.001).

Figure 2

Podocyte-specific knockout of Becn1 leads to glomerular scarring. (A) Kidney sections obtained from 2-week-old, 4-week-old and 8-week-old WT and Becn1^Δpod mice stained with haematoxylin-eosin solution; (B) Glomeruli from kidney sections obtained from 2-week-old, 4-week-old and 8-week-old WT and Becn1^Δpod mice stained with haematoxylin-eosin solution; (C) Kidney sections and magnified glomeruli obtained from 4-week-old and 8-week-old WT and Becn1^Δpod mice stained with acid-fuchsin, orange-G, and aniline blue solution; (D) Glomerulosclerosis score calculated using the method of el Nahas in 4-week-old and 8-week-old WT and Becn1^Δpod mice (*** ≤ 0.001).

Figure 2

Podocyte-specific knockout of Becn1 leads to glomerular scarring. (A) Kidney sections obtained from 2-week-old, 4-week-old and 8-week-old WT and Becn1^Δpod mice stained with haematoxylin-eosin solution; (B) Glomeruli from kidney sections obtained from 2-week-old, 4-week-old and 8-week-old WT and Becn1^Δpod mice stained with haematoxylin-eosin solution; (C) Kidney sections and magnified glomeruli obtained from 4-week-old and 8-week-old WT and Becn1^Δpod mice stained with acid-fuchsin, orange-G, and aniline blue solution; (D) Glomerulosclerosis score calculated using the method of el Nahas in 4-week-old and 8-week-old WT and Becn1^Δpod mice (*** ≤ 0.001).

Figure 3

Becn1^Δpod mice displaying a high number of autophagosomes with an increased lysosomal compartment. (A) Representative image showing immunofluorescence staining for NEPHRIN (green) and LC3 (red) in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice; (B) Representative image showing immunofluorescence staining for NEPHRIN (green) and SQSTM1 (red) in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice; (C) Quantification of LC3 and SQSTM1 aggregates in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice (n = 6). (D) Schematic showing the generation of mice with podocyte-specific deletion of Becn1 and transgenic mice carrying fluorescence-labelled LC3 (GFP-LC3); (E) Representative immunofluorescence staining for NIDOGEN (red) and anti-GFP (green) in kidney sections obtained from 4-week-old GFP-LC3 and GFP-LC3 × Becn1^∆pod mice. Quantification of GFP-LC3 puncta (n = 6, *** ≤ 0.001); (F) Representative image showing immunofluorescence staining for NEPHRIN (green) and LAMP1 (red) in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice. Quantification of the LAMP1 signal (n = 8, *** ≤ 0.001).

Figure 4

Becn1^Δpod mice displaying aberrant vesicle accumulation and flattened podocyte foot processes. (A) Transmission electron microscopy (TEM) images showing glomeruli (a,b1,b2) and podocytes (c,d1,d2) in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice; (B) TEM images showing glomeruli (a,b1,b2) and podocytes (c,d1,d2) in kidney sections obtained from 8-week-old WT and Becn1^∆pod mice.

Figure 5

Beclin 1 localizes to the Golgi compartment and regulates Golgi size. (A) Schematic showing key interaction partners of the BECLIN1-core complex; (B) Immunofluorescence staining for GOLGIN 97 (green), BECLIN1, VPS15 and VPS34 (red) in human podocytes; (C) Schematic showing PI(4)P as the key upstream regulator of Golgi size and activity; (D) Representative image showing immunofluorescence staining for PODOCIN (green) and PI(4)P (red) in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice with the integrated density of the GOLGIN 97 signal quantified (n = 8, *** ≤ 0.001); (E) Representative image showing immunofluorescence staining for PODOCIN (green) and PI(4)P (red) in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice the integrated density of the GOLGIN 97 signal quantified (n = 8, ** ≤ 0.001).

Figure 6

(A) Immunofluorescence staining for GM-130 (red) in primary murine WT and Becn1^∆pod podocytes obtained from Becn1^flox/flox x Tomato/EGFP x hNphs2-Cre and WT x Tomato/EGFP x hNphs2-Cre mice (green, EGFP fluorescence after successful Cre-mediated recombination); (B) Schematic showing the generation of immortalized murine WT and Becn1^∆pod podocytes based on glomerular isolation of Becn1^flox/flox;Tomato/EGFP; hNphs2-Cre+ and WT;Tomato/EGFP; hNphs2-Cre+ with subsequent Adeno-SV40 transduction followed by fluorescence activated cell sorting (FACS); (C) Western blot showing the abundance of BECLIN1, ACTIN and LC3 in lysates of WT and Becn1-deficient immortalized murine podocytes; (D) Western blot showing the abundance of ß-COP, SEC23, ACTIN and RAB5 in lysates of WT and Becn1-deficient immortalized murine podocytes; (E) Western blot showing the abundance of CLATHRIN and ACTIN in lysates of WT and Becn1-deficient immortalized murine podocytes; (F) Representative image showing immunofluorescence staining for PODOCIN (green) and γ-ADAPTIN (red) in kidney sections obtained from 4-week-old WT and Becn1^∆pod mice; (G) Schematic showing the abundance of vesicle markers in Becn1-deficient cells indicating an accumulation of ß-COP and γ-ADAPTIN (red) and a reduction in CLATHRIN and RAB5 (blue).

Figure 7

Beclin1 is indispensable for glomerular endothelial integrity and VEGF secretion. (A) Transmission electron microscopy (TEM) images of kidney sections obtained from 4-week-old and 8-week-old WT and Becn1^flox/flox × hNphs2-Cre mice; (B) Western blot showing the abundance of BECLIN1, VEGF and ACTIN in lysates obtained from immortalized WT and Becn1-deficient murine podocytes; (C) Relative levels of VEGF in the supernatant of serum-starved WT and Becn1-deficient podocytes; (D) Relative levels of VEGF in the supernatant of serum-starved WT and Becn1-deficient podocytes after angiotensin II (AngII) stimulation (10⁻⁶ M at the time indicated); (E) Western blot showing the abundance of HSP47, ARF4 and GAPDH in lysates obtained from immortalized WT and Becn1-deficient murine podocytes with and without AngII stimulation (10⁻⁶ M); (F) Schematic showing the proposed role of BECLIN1 in orchestrating vesicle formation for anterograde Golgi trafficking and VEGF secretion.

Figure 7

Beclin1 is indispensable for glomerular endothelial integrity and VEGF secretion. (A) Transmission electron microscopy (TEM) images of kidney sections obtained from 4-week-old and 8-week-old WT and Becn1^flox/flox × hNphs2-Cre mice; (B) Western blot showing the abundance of BECLIN1, VEGF and ACTIN in lysates obtained from immortalized WT and Becn1-deficient murine podocytes; (C) Relative levels of VEGF in the supernatant of serum-starved WT and Becn1-deficient podocytes; (D) Relative levels of VEGF in the supernatant of serum-starved WT and Becn1-deficient podocytes after angiotensin II (AngII) stimulation (10⁻⁶ M at the time indicated); (E) Western blot showing the abundance of HSP47, ARF4 and GAPDH in lysates obtained from immortalized WT and Becn1-deficient murine podocytes with and without AngII stimulation (10⁻⁶ M); (F) Schematic showing the proposed role of BECLIN1 in orchestrating vesicle formation for anterograde Golgi trafficking and VEGF secretion.