ANGPTL4 Exacerbates Renal Injury in Diabetic Kidney Disease by Impairing Podocyte Lipophagy via Compromised Lysosomal Degradative Function

FIGURE 1. ANGPTL4 expression is upregulated and associated with disease‐severity indices in human diabetic kidney disease. (A) Representative images of Masson's trichrome staining and immunohistochemical staining for ANGPTL4 in renal tissues from DKD patients. From top to bottom: ANGPTL4 expression in glomeruli, renal tubules, interstitial microvessels, and fibrotic areas. Black double arrows: podocytes; white double arrows: tubular cells; black arrows: microvascular endothelial cells; white arrows: fibroblasts. Scale bar = 50 µm; insets = 10 µm. (B, C) Expression and colocalization of ANGPTL4 with the podocyte marker nephrin. (B) Immunofluorescence co‑staining of ANGPTL4 (green) and nephrin (red) in glomeruli; nuclei are counterstained with DAPI (blue). White arrows indicate sites of colocalization. (C) Quantitative analysis of ANGPTL4–nephrin colocalization in glomeruli from DKD and control groups. (D) Immunofluorescence staining of ANGPTL4 (green) in the tubulointerstitial compartment, with corresponding bright‑field images; double arrows highlight representative positive regions. (E) Quantification of ANGPTL4 fluorescence intensity in glomeruli and tubulointerstitium. (F)…

FIGURE 2. ANGPTL4 is associated with suppressed autophagy and lipid accumulation in podocytes in DKD. (A) Gene set enrichment analysis (GSEA) of the bulk RNA‑seq dataset (GSE30528) shows that ANGPTL4 expression is significantly associated with the suppression of multiple autophagy pathways and the activation of lipid oxidation‑related pathways. (B) Analysis of single‑nucleus RNA‑seq data (GSE211785) reveals downregulation of KEGG autophagy pathways specifically in podocytes, but not in tubular cells, from DKD patients compared with healthy controls and patients with hypertensive kidney disease (HKD). (C) KEGG and GO analyses further confirm an inverse correlation between ANGPTL4 expression and autophagy‑related pathways in podocytes. (D) Representative immunofluorescence images of LC3B (green) and SQSTM1/p62 (red) co‐staining in podocytes from DKD and control groups; nuclei are counterstained with DAPI (blue). Scale bar = 20 µm; insets = 5 µm. Single arrows indicate double‐positive puncta; double arrows indicate SQSTM1/p62‐only puncta. (E) Quantification of LC3B intensity, SQSTM1/p62 intensity, and LC3B‐SQSTM1/p62 colocalization in podocytes. (F) Transmission electron microscopy images…

FIGURE 3. HGPA induces autophagic dysfunction and PA dose‑dependently upregulates ANGPTL4 in podocytes. (A) Transmission electron microscopy images showing lipid droplets (white arrows) in serum‑starved human podocytes (HPCs) cultured under control, high glucose (HG), AGE‑BSA, or HGPA conditions. Scale bar = 500 nm. (B‐‑D) HPCs expressing the RFP‑GFP‑LC3B reporter were treated with control or HGPA medium, with or without chloroquine (CQ). (B) Representative fluorescence images and quantification of GFP and RFP puncta per cell. (C) Numbers of autophagosomes (yellow puncta in merged images) and autolysosomes (red‑only puncta) per cell. (D) Schematic summary of autophagic flux under the indicated conditions. (E‐‑G) Serum‑starved HPCs were treated with HG (30 mM) and increasing concentrations of palmitic acid (PA; 0–400 µM) for 24 h. (E) Western blot analysis of full‑length ANGPTL4, SQSTM1/p62, LC3B, and tubulin in cell lysates, and of full‑length and cleaved ANGPTL4 in conditioned media; Ponceau S staining serves as the loading control for secreted proteins. (F) Quantification of full‑length ANGPTL4/tubulin, SQSTM1/p62/tubulin, and LC3B‑II/LC3B‑I ratios in cell lysates. (G) Secreted levels of…

FIGURE 4. ANGPTL4 overexpression impairs lipophagic flux and promotes lipid accumulation in podocytes under diabetic conditions. (A) Podocytes were transfected with an ANGPTL4‑overexpressing plasmid or empty vector for 72 h, followed by HGPA treatment. Cells were then subjected to the indicated interventions: chloroquine (CQ, 50 µM, 4 h) or rapamycin (Rapa, 100 nM, 6 h) + CQ (50 µM, 4 h). Prior to live‑cell imaging, cells were transfected with LC3B‑RFP to label autophagosomes and stained with BODIPY 493/503 to visualize lipid droplets. Upper panels: confocal micrographs (scale bar = 20 µm; inset = 2 µm). Middle panels: fluorescence intensity profiles along the yellow line in the enlarged regions, showing the colocalization of autophagosomes (LC3B‑RFP, red) and lipid droplets (BODIPY, green). Lower panels: transmission electron microscopy images (scale bar = 500 nm). White arrows: lipid droplets (LDs); yellow double arrows: early endosomes; yellow arrows: lysosomes (Lys). (B) Quantitative analysis of lipid droplet parameters and autophagosome‑lipid droplet colocalization across the five experimental groups. From left to right: number of lipid droplets per cell, average lipid droplet area…

FIGURE 5. ANGPTL4 impairs autophagic flux and lysosomal function in podocytes. (A) Schematic of experimental treatments and representative immunofluorescence images. Podocytes were transfected with ANGPTL4‑overexpressing plasmid or empty vector for 72 h, followed by transfection with LC3B‑RFP. Cells were then treated with bafilomycin A1 (Baf A1, 100 nM, 4 h) or rapamycin (100 nM, 6 h). After fixation, cells were immunostained for LC3B (green) and SQSTM1/p62 (p62, red); nuclei were stained with DAPI (blue). Scale bar = 20 µm. (B) Quantification of total p62 puncta per cell (left) and the fraction of p62 colocalizing with LC3B (right) across six experimental groups. Data are mean ± SD (n ≥ 3). (C, D) ANGPTL4 knockdown rescues autophagy under HGPA stress. (C) Western blot analysis of ANGPTL4, SQSTM1/p62, LC3B, LAMP1, and β‑actin in podocytes transduced with scramble (shNC) or ANGPTL4‑targeting (shANGPTL4) shRNA followed by HGPA treatment. (D) Quantification of ANGPTL4/β‑actin, SQSTM1/β‑actin, and LC3B‑II/LC3B‑I ratios. Data are mean ± SD (n ≥ 3). (E, F) ANGPTL4 knockdown restores functional autophagic flux. (E) Representative confocal images of podocytes expressing the RFP‑GFP‑LC3B reporter…

FIGURE 6. ANGPTL4 overexpression impairs TFEB nuclear translocation and lysosomal function in podocytes. (A) Volcano plot of differentially expressed genes in podocytes treated with shANGPTL4 versus shNC under HGPA conditions (∣log2FC∣ > 1, padj < 0.05). Red and blue dots indicate significantly up‑ and down‑regulated genes, respectively. Top five genes in each direction are labeled. (B) Heatmap showing expression patterns of selected autophagy‑ and lysosome‑related genes in shANGPTL4+HGPA versus shNC+HGPA groups (n = 3 per group). (C) Representative immunoblots and quantitative analysis of TFEB phosphorylation at Ser211 in vector‐control and ANGPTL4‐overexpressing podocytes. Phosphorylated TFEB was normalized to total TFEB (n = 3 independent experiments). (D, E) Representative immunoblots and quantitative analyses of TFEB protein levels in the nuclear (D) and cytoplasmic (E) fractions of vector‐control and ANGPTL4‐overexpressing podocytes. Lamin B1 and tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 4). (F) Representative immunofluorescence images of TFEB localization in vector‐control and ANGPTL4‐overexpressing podocytes. TFEB is shown in…

FIGURE 7. Recombinant N‐terminal ANGPTL4 fragment impairs podocyte lipophagy. (A) Representative immunoblots and quantitative analyses of TFEB protein levels in the nuclear and cytoplasmic fractions of podocytes treated with vehicle (NC), recombinant human N‐terminal ANGPTL4 fragment (rhANGPTL4), rhANGPTL4 plus an ANGPTL4‐neutralizing antibody (α‐ANGPTL4 Ab), or rhANGPTL4 with TFEB‐S211A expression. Histone H3 and tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 4 independent experiments). (B) Representative immunofluorescence images and quantification of TFEB nuclear localization in the indicated groups. TFEB is shown in green, nuclei were counterstained with DAPI (blue), and merged images are shown. Images were acquired using a Leica STELLARIS 5 confocal microscope. Cell and nuclear boundaries are outlined in white. Scale bars, 10 µm. Each dot represents one cell. (C) Representative images of podocytes stained with LysoSensor Yellow/Blue DND‐160. Fluorescence was detected in the blue emission channel (450 ± 33 nm) and yellow emission channel (510 ± 20 nm), and merged images are shown. Images were acquired using a Leica STELLARIS 5…

FIGURE 8. ANGPTL4 promotes podocyte fibrotic remodeling. (A) Representative immunofluorescence images of collagen I, α‐smooth muscle actin, fibronectin, and collagen III in kidney biopsy specimens from patients with diabetic kidney disease (DKD) exhibiting high or low glomerular ANGPTL4 expression and from control subjects. The indicated extracellular matrix and profibrotic markers are shown in green, and nuclei were counterstained with DAPI (blue). Boxed regions are shown at higher magnification. Scale bars, 50 µm; insets, 5 µm. (B) Quantification of collagen I, α‐SMA, fibronectin, and collagen III fluorescence levels in the ANGPTL4high, ANGPTL4low, and control groups. Values were normalized to those of the control group. (C) Comparison of ANGPTL4, autophagy, and epithelial–mesenchymal transition (EMT) pathway activity scores between the shANGPTL4 + HGPA and shNC + HGPA groups (n = 3 per group). (D) Representative immunoblots of ANGPTL4, SQSTM1/p62, LC3B‐I/II, synaptopodin (SYNPO), and collagen III α1 chain (COL3A1) in vector‐control and ANGPTL4‐overexpressing podocytes. Tubulin was used as the loading control. (E) Densitometric quantification of ANGPTL4, SQSTM1/p62, the LC3B‐II/LC3B‐I…