Endocrinol Metab.  2022 Feb;37(1):96-111. 10.3803/EnM.2021.1305.

EW-7197 Attenuates the Progression of Diabetic Nephropathy in db/db Mice through Suppression of Fibrogenesis and Inflammation

• Ha KB
• Sangartit W
• Jeong AR
• Lee ES
• Kim HM
• Shim S
• Kukongviriyapan U
• Kim DK
• Lee EY
• Chung CH

• KMID: 2527141
• DOI: http://doi.org/10.3803/EnM.2021.1305

Abstract

Background
Diabetic nephropathy (DN) is characterized by albuminuria and accumulation of extracellular matrix (ECM) in kidney. Transforming growth factor-β (TGF-β) plays a central role in promoting ECM accumulation. We aimed to examine the effects of EW-7197, an inhibitor of TGF-β type 1 receptor kinase (ALK5), in retarding the progression of DN, both in vivo, using a diabetic mouse model (db/db mice), and in vitro, in podocytes and mesangial cells.
Methods
In vivo study: 8-week-old db/db mice were orally administered EW-7197 at a dose of 5 or 20 mg/kg/day for 10 weeks. Metabolic parameters and renal function were monitored. Glomerular histomorphology and renal protein expression were evaluated by histochemical staining and Western blot analyses, respectively. In vitro study: DN was induced by high glucose (30 mM) in podocytes and TGF-β (2 ng/mL) in mesangial cells. Cells were treated with EW-7197 (500 nM) for 24 hours and the mechanism associated with the attenuation of DN was investigated.
Results
Enhanced albuminuria and glomerular morphohistological changes were observed in db/db compared to that of the nondiabetic (db/m) mice. These alterations were associated with the activation of the TGF-β signaling pathway. Treatment with EW-7197 significantly inhibited TGF-β signaling, inflammation, apoptosis, reactive oxygen species, and endoplasmic reticulum stress in diabetic mice and renal cells.
Conclusion
EW-7197 exhibits renoprotective effect in DN. EW-7197 alleviates renal fibrosis and inflammation in diabetes by inhibiting downstream TGF-β signaling, thereby retarding the progression of DN. Our study supports EW-7197 as a therapeutically beneficial compound to treat DN.

Keyword

Diabetic nephropathies; Glomerular mesangial cells; Podocytes; Transforming growth factor beta; Activin receptor-like kinase 5

Figure

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  Fig. 1 EW-7197 has no effect on insulin tolerance but in kidney function. (A) Bodyweight was monitored throughout experimental period. (B) Intraperitoneal insulin tolerance test were performed after 10 weeks of EW-7197 treatment. (C) Urinary albumin/creatinine (ACR) ratios were measured 24-hour urine collection in metabolic cage. (D) Kidney weight/body weight in all experimental groups. Each group (n=6–8/group). aP<0.05 vs. db/m controls; bP<0.05 vs. db/db controls; cP<0.05 vs. db/db+EW5 mg/kg/day.

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  Fig. 2 EW-7197 reduces glomerular hypertrophy and fibrosis in db/db mice. (A, B) Glomerular hypertrophy was determined in hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) stained glomeruli from diabetic mice. (F) Changes in glomerular area, glomerular volume, Bowman’s space area, (G) percentage of mesangial matrix and mesangial expansion score. (C, D, H) Sirius red, trichrome staining were performed to check the degree of fibrosis of the glomerular (original magnification, 400×). (E) Ultrastructure of glomerular basement membrane (GBM). The GBM thickness, filtration silt pores, and foot process width of podocytes (I) were analyzed by transmission electron microscopy (TEM) at 15000-X magnification. An arrowhead indicates filtration slit pore; a up-down arrow shows GBM thickness (n=2/group). aP<0.05 vs. db/m controls; bP<0.05 vs. db/db controls; cP<0.05 vs. db/db+EW5 mg/kg/day.

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  Fig. 3 EW-7197 protects against nephrin loss from diabetic glomeruli and podocytes. (A) Immunohistochemistry of renal nephrin, (B) the percentage of positively stained area. Original magnification is 400× (scale bar=50 μm). (C, D) By Western blot protein expression of nephrin were analyzed from diabetic mice (n=4–5/group). (E, F) After treatment with high glucose (HG, 30 mM) and EW (500 nM) in podocytes, the protein expression of synaptopodin and nephrin was confirmed by Western blot. (G, H) F-actin stained with fluorescein isothiocyanate (FITC)-phalloidin and positive stained area. aP<0.05 vs. db/m controls; bP<0.05 vs. db/db controls; cP<0.05 vs. db/db+EW5 mg/kg/day; dP<0.05 vs. controls; eP<0.05 vs. high glucose.

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  Fig. 4 EW-7197 down-regulates the expression on fibrogenic mediators in diabetic mice. (A) Immunohistochemistry of renal collagen IV and fibronectin, (B) positively stained area. Original magnification is 400× (scale bar=50 μm). (C, D) By Western blot of renal cortex protein expression of col IV and fibronectin. (E, F) The expression of Smad2/3, p-Smad 2/3, vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) as proteins related to fibrosis was confirmed, respectively from diabetic mice (n=4–5/group). aP<0.05 vs. db/m controls; bP<0.05 vs. db/db controls; cP<0.05 vs. db/db+EW5 mg/kg/day.

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  Fig. 5 EW-7197 down-regulates the expression of fibrosis markers in podocyte and mesangial cell lines. (A, B) Podocyte fibrosis marker; fibronectin, transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor (VEGF) was detected by Western blots and quantified values. (C, D) Changes in extracellular matrix (ECM) through fibronectin, α-smooth muscle actin (α-SMA), and collagen type IV were detected by mesangial cell Western blot. (E, F) Immunofluorescence of fibronectin and collagen type IV and positive stained area. MES-13, mouse mesangial cells. aP<0.05 vs. controls; bP<0.05 vs. high glucose; cP<0.05 vs. controls; dP<0.05 vs. TGF-β.

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  Fig. 6 EW-7197 inhibits the inflammatory response and prevents cell death. (A, B) Protein expression in the renal cortex of diabetic mice, a marker related to the inflammatory response; phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB), NF-κB, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule 1 (ICAM-1). (C, D) Expression of proteins such as, Bcl2 Associated X (Bax) and caspase-3 were investigated using Western blot and fold change relative to control. Anti-apoptotic B-cell lymphoma/leukemia-2 (Bcl2) an apoptosis-related marker, in podocytes. (E, F, G, H) Expression of apoptosis-related proteins and immunofluorescence staining in mesangial cells. MES-13, mouse mesangial cells. aP<0.05 vs. db/m controls; bP<0.05 vs. db/db controls; cP<0.05 vs. controls; dP<0.05 vs. high glucose; eP<0.05 vs. controls; fP<0.05 vs. TGF-β.

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  Fig. 7 EW-7197 ameliorates renal reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. (A, B) Expression of ROS related proteins such as NAD(P)H oxidase 4 (NOX4) and ER stress markers; nuclear factor erythroid 2-related factor (Nrf2) and, transcription factor 6 alpha (ATF6α) in podocytes. (C, D) ROS measurements in podocytes with CM-H2DCF-DA. (E, F) Expression of NOX4 and ER stress-related proteins in mesangial cells. (G, H) ROS generation in mesangial cells by immunofluorescence staining with NOX4 and 8-hydroxydeoxyguanosine (8-OHdG). (I, J) The expression of Nrf2, heme oxygenase-1 (HO-1), and Lamin B was confirmed by extracting the protein from the nucleus. HG, high glucose; MES-13, mouse mesangial cells; TGF-β, transforming growth factor-β. aP<0.05 vs. controls; bP<0.05 vs. high glucose; cP<0.05 vs. controls; dP<0.05 vs. TGF-β.

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