PEDF Alleviates Diabetic Renal Fibrosis by Degrading Kidney Ectopic Fat Deposition and Inhibiting Metabolic Reprogramming of Renal Tubular Epithelial Cells

Figure 1. Body weight, blood glucose and food intake of mice in each group (n=10). PEDF improved renal function and blood lipid of db/db mice, and alleviated renal tubule injury and urinary albumin of db/db mice. (A) Schematic diagram of the mice experiment. (B) The expression of PEDF protein in mice renal cortex was detected by Western blot. (C) Histogram quantization results of PEDF/β-actin gray values. (D) Body weight. (E) Blood glucose. (F) Food intake. (G) Serum creatinine (Cr). (H) Urinary albumin/creatinine ratio (ACR). (I) Urine N-acetyl-beta-glucosaminase (NAG). (J) Total blood cholesterol (TC). (K) Blood triglyceride (TG). Data are expressed as mean±SEM. “*” compared with db/m group, **P<0.01, ***P<0.001, ****P<0.0001, “#” compared with db/db group, #P<0.05, ##P<0.01.

Figure 2. PEDF alleviated renal fibrosis in db/db mice. (A–D) PAS staining of mice kidney. (E–H) Sirius red staining of mice kidney. (I) Statistical analysis of collagen area of mice kidney stained with Sirius red. (J) The expressions of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) protein, the indicators of renal cortical fibrosis in mice, were detected by Western blot. (K) Histogram quantized analysis results of TGF-β1/β-actin gray value. (L) Histogram quantized analysis results of α-SMA/β-actin gray values. Data are expressed as mean±SEM. “*” compared with db/m group, ***P<0.001, ****P<0.0001, “#” compared with db/db group, #P<0.05, ##P<0.01, ####P<0.0001.

Figure 3. PEDF inhibited renal ectopic fat deposition in db/db mice. (A) Mice kidney sections were stained with oil red, and electron microscopy revealed the presence of lipid droplets (indicated by black arrows). Immunohistochemical staining for adipose differentiation associated protein (ADRP) was also performed. (B) Quantitative statistical analysis of the gray value of mice kidney oil red stained fat drops. (C) Statistical analysis of mean optical density (AOD) of mice kidney ADRP staining. Data are expressed as mean±SEM. “*” compared with db/m group, *P<0.05, ****P<0.0001, “#” compared with db/db group, #P<0.05.

Figure 4. PEDF increased renal protein expression of peroxisomal and fatty acid β oxidation in db/db mice. (A) Mice renal peroxisome membrane protein ATP-binding cassette sub-family D member 3 (ABCD3) immunofluorescence staining. (B) Analysis of the average fluorescence intensity of ABCD3 immunofluorescence staining. (C) The expression of peroxidase acyl-CoA oxidase-1 (ACOX1) protein in renal cortex was detected by Western blot. (D) Histogram quantized analysis results of ACOX1/β-actin gray values. Data are expressed as mean±SEM. “*” compared with db/m group, **P<0.01, ****P<0.0001, “#” compared with db/db group, #P<0.05, ###P<0.001.

Figure 5. PEDF increased renal protein expression of mitochondrial and fatty acid β oxidation in db/db mice. (A) Immunofluorescence staining of kidney cytochrome C oxidase IV subtype (COX IV). (B) Analysis of mean fluorescence intensity of COX IV immunofluorescence staining. (C) The expression of carnitine palmitoyl transferase 1A (CPT1A) protein in renal cortex was detected by Western blot. (D) Histogram quantized analysis results of CPT1A/β-actin gray values. Data are expressed as mean±SEM. “*” compared with db/m group, ***P<0.001, ****P<0.0001, “#” compared with db/db group, ##P<0.01, ####P<0.0001.

Figure 6. PEDF increased the expression of ATGL-PPARα pathway in kidney of db/db mice and inhibited the expression of HIF1α-HK2 pathway. (A) The mRNA expression of hypoxia-inducible factor-1 α (HIF-1α) was detected using in situ hybridization. (B) Analysis of mean fluorescence intensity of HIF-1α mRNA expression. (C) The expressions of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), peroxisome proliferator-activated receptor-α (PPARα), adipotriglyceride lipase (ATGL) and hexokinase-2 (HK2) proteins in renal cortex of mice were detected by Western blot assay. (D) Histogram quantized analysis results of HK-2 /β-actin gray values. (E) Histogram quantized analysis results of ATGL/β-actin gray values. (F) Histogram quantized analysis results of PPARα/β-actin gray values. (G) Histogram quantized analysis results of PGC1α/β-actin gray values. Data are expressed as mean±SEM. “*” compared with db/m group, **P<0.01, ***P<0.001, ****P<0.0001, “#” compared with db/db group, #P<0.05, ###P<0.001, ####P<0.0001.

Figure 7. PEDF alleviated lipid deposition and transdifferentiation of human proximal renal tubular epithelial cells (HK-2) induced by high glucose and high palmitic acid via ATGL-PPARα pathway. (A) Oil red staining of HK-2 cells in each group. (B) Quantitative statistical analysis of the gray value of HK-2 cell oil red stained lipid drops. (C) The expression of ATGL protein in HK-2 cells was detected by Western blot. (D) Histogram quantized analysis results of ATGL/β-actin gray values. (E) The expressions of TGF-β1, E-cadherin and α-SMA in HK-2 cells were detected by Western blot. (F) Histogram quantized analysis results of TGF-β1/β-actin gray value. (G) Histogram quantized analysis results of gray values of E-Cadherin/β-actin. (H) Histogram quantized analysis results of α-SMA/β-actin gray values. HGPA stands for high sugar and high palmitic acid, Atglistatin is an ATGL inhibitor, GW6471 is a PPARα antagonist. Data are expressed as mean±SEM. “*” compared with HGPA group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, “#” compared with HGPA + PEDF group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001.

Figure 8. PEDF increased fatty acid β oxidation in mitochondria and peroxisome of HK-2 cells cultured with high glucose and palmitic acid through ATGL-PPARα pathway. (A) RT-qPCR was used to detect the expression of mitochondrial fatty acid β oxidation related genes in HK-2 cells. (B) The expression of peroxisome fatty acid β oxidation related genes in HK-2 cells was detected by RT-qPCR. (C) Western blot assay was used to detect the expression of PGC1α, PPARα, ACOX1 and CPT1A in HK-2 cells. (D) Histogram quantized analysis results of PGC1α/β-actin gray values. (E) Histogram quantized analysis results of PPARα/β-actin gray values. (F) Histogram quantized analysis results of ACOX1/β-actin gray values. (G) Histogram quantized analysis results of CPT1A/β-actin gray values. HGPA stands for high sugar and high palmitic acid, Atglistatin is an ATGL inhibitor, GW6471 is a PPARα antagonist. Data are expressed as mean±SEM. “*” compared with HGPA group, *P<0.05, **P<0.01, ***P<0.001, “#” compared with HGPA + PEDF group, #P<0.05, ##P<0.01.

Figure 9. PEDF inhibited metabolic reprogramming in HK-2 cells induced by high glucose and high palmitic acid. (A) HK-2 cell HIF-1α immunofluorescence staining. (B) Oxygen consumption rate (OCR) of HK-2 cells was measured by Seahorse Energy metabolism analyzer. (C) The extracellular acidification rate (ECAR) of HK-2 cells was determined by Seahorse Energy metabolism analyzer. (D) Basal respiration of OCR. (E) Analysis results of ATP production for OCR oxidative phosphorylation. (F) Results of OCR maximal oxygen consumption (maximal respiration) analysis. (G) OCR spare respiratory capacity analysis results. (H) Basal glycolysis analysis of ECAR. (I) Results of ECAR Glycolytic capacity analysis. HGPA stands for high sugar and palmitic acid. Data are expressed as mean±SEM. “*” compared with HGPA group, nsP means no significant difference, *P<0.05, **P<0.01, ***P<0.001.

Figure 10. PEDF inhibited glycolysis of HK-2 cells cultured with high glucose and palmitic acid through HIF-1α. (A) The expression of glycolysis-related genes in HK-2 cells was detected by RT-qPCR. (B) The expressions of HIF-1α and HK-2 protein in HK-2 cells were detected by Western blot. (C) Histogram quantized analysis results of HIF-1α/Lamin A gray value. (D) Histogram quantized analysis results of HK2/β-actin gray values. (E) Extracellular lactic acid content. (F) The expression of PEDF protein in HK-2 cells was detected by Western blot. (G) Histogram quantization results of PEDF/β-actin gray values. (H) Western blot assay was used to detect HIF-1α and HK-2 protein expression in HK-2 cells. (I) Histogram quantized analysis results of HIF-1α/Lamin A gray value. (J) Histogram quantized analysis results of HK2/β-actin gray values. (K) Extracellular lactic acid content. HGPA stands for high sugar and palmitic acid, FG4592 is an HIF-α prolyl hydroxylase inhibitor that can stabilize the activity of HIF, and Echinomycin is an HIF-1α inhibitor. Data are expressed as mean±SEM. “*” compared with HGPA group, *P<0.05, **P<0.01, ***P<0.001, “#” compared with HGPA + PEDF group, ##P<0.01, ###P<0.001,…

Figure 11. A summary of the proposed effects of PEDF for alleviating diabetic renal fibrosis. PEDF facilitates the degradation of ectopically accumulated fat in the diabetic kidney, leading to the generation of free fatty acids via its receptor ATGL. On one hand, PEDF promotes β-oxidation of fatty acids within mitochondria and peroxisomes through the ATGL-PPARα pathway; on the other hand, PEDF inhibits glycolysis via HIF-1α. This mechanism hinders metabolic reprogramming of renal tubular epithelial cells and mitigates diabetic renal fibrosis. (Blue arrows denote process flows, red upward arrows indicate promoting effects, and red downward arrows signify inhibiting effects.).