Stigmasterol-Mediated Targeting of Rho-Associated Coiled-Coil Protein Kinase 1 Ameliorates Diabetic Kidney Disease and Attenuating Renal Tubular Lipid Deposition

Figures

• 

  Fig. 1.. Schematic illustration of stigmasterol targeting the rho-associated coiled-coil protein kinase 1 (ROCK1)/p38 mitogen-activated protein kinase (MAPK)/peroxisome proliferator-activated receptor α (PPARα) axis for lipid metabolism regulation in diabetic kidney disease (DKD).

• 

  Fig. 2.. Rho-associated coiled-coil protein kinase 1 (ROCK1) is highly expressed in diabetic kidney disease (DKD) patients and mice. (A) Correlation analysis of ROCK1 expression in renal tubules with renal function. (B) Schematic diagram of an animal experiment. (C) Protein blotting assay for the expression of ROCK1. (D) Quantitative analysis of ROCK1 by protein blotting. (E) The messenger RNA (mRNA) levels of ROCK1 were analyzed using quantitative real-time polymerase chain reaction (qPCR). (F) Quantitative map of Oil Red O in kidney tissue. (G) Quantitative analysis of immunohistochemical (IHC) staining for ROCK1 in mouse kidney tissues. (H) Oil Red O staining of mouse kidney tissue (×200 magnification, bar = 60 μm/20 μm). (I) IHC staining of ROCK1 in mouse kidney tissues (×200 magnification, bar = 60 μm/20 μm). The data are presented as mean ± standard error of the mean (SEM) (n = 3/group). #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001, DKD vs. control; ns, not significant.

• 

  Fig. 3.. Molecular docking screened stigmasterol (ST) as a potential inhibitor of rho-associated coiled-coil protein kinase 1 (ROCK1). (A) The molecular docking table of ROCK1. (B) Docking scores of 50 natural compounds with ROCK1. (C) Binding mode of stigmasterol. (D) Western blot results from cellular thermal shift assay (CETSA). (E) CETSA thermogram showing ROCK1 thermal stability changes induced by ST. (F) Plot of the course of change in response units (RU) of ST and ROCK1. (G) Effect of ST on ROCK1 enzyme-linked immunosorbent assay (ELISA). (H) Quantitative analysis of Oil Red O staining. (I) Oil Red O staining of HK-2 cells (×200 magnification, bar = 200 μm/80 μm). (J) Messenger RNA (mRNA) levels of ROCK1, PPARα, PGC1α, and ACOX1. (K) mRNA levels of ANT1, CYPD, VDAC1, and VDAC2. (L) Mechanism of the cell model. (M) Detection of α-smooth muscle actin (α-SMA) and fibronectin protein in HK-2 cells by protein blotting. (N) Quantitative analysis of fibronectin by protein blotting. (O) Quantitative analysis of α-SMA by protein blotting. The data are presented as mean ± standard error of the mean (SEM) (n = 3/group). #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001, high-glucose…

• 

  Fig. 4.. Stigmasterol (ST) attenuates lipid accumulation in HK-2 cells via the rho-associated coiled-coil protein kinase 1 (ROCK1)/p38 mitogen-activated protein kinase (MAPK)/peroxisome proliferator-activated receptor α (PPARα) pathway. (A) Protein blotting assay for the expression of ROCK1, P-p38, p38, and PPARα in HK-2 cells. (B, C, and G) Quantitative analysis of ROCK1, P-p38/p38, and PPARα by protein blotting. (D to F) HK-2 cells were transfected with either a control small interfering RNA (siRNA) or ROCK1 siRNA. The messenger RNA (mRNA) levels of ROCK1 were analyzed using quantitative real-time polymerase chain reaction (qPCR). The protein levels of ROCK1 were determined by Western blotting. (H to K) ROCK1, PPARα, PGC1α, and ACOX1 mRNA levels. (L and M) Oil Red O staining (×200 magnification, bar = 200 μm/80 μm) and quantitative analysis of HK-2 cells with knockdown of the ROCK1 gene. The data are presented as mean ± standard error of the mean (SEM) (n = 3/group). #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001, high-glucose (HG) vs. control; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, HG vs. ST; ns, not significant.

• 

  Fig. 5.. Stigmasterol (ST) alleviated streptozotocin (STZ)-induced renal injury in diabetic kidney disease (DKD) mice. (A) Schematic diagram of an animal experiment. (B) Body weight changes during treatment. (C) Final body weight. (D) Final fasting blood glucose levels. (E) Fasting blood glucose changes during treatment. (F) Serum creatinine levels. (G) Blood urea nitrogen levels. (H) 24-h urinary protein levels. (I) Renal histopathology (hematoxylin–eosin [H&E], periodic acid–Schiff [PAS], and Masson staining; ×200 magnification, scale bar = 60 μm). The data are presented as mean ± standard error of the mean (SEM) (n = 6/group). #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001, DKD vs. control; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, DKD vs. ST; ns, not significant.

• 

  Fig. 6.. Stigmasterol (ST) alleviated fibrosis and mitochondrial damage caused by lipid accumulation in diabetic kidney disease (DKD) mice. (A) Total cholesterol levels in mouse serum. (B) Total triglyceride levels in mouse serum. (C) Serum low-density lipoprotein cholesterol levels in mice. (D) Quantitative map of Oil Red O in kidney tissue. (E) Oil Red O staining of mouse kidney tissue (glomeruli and tubules) (×200 magnification, bar = 60 μm/20 μm). (F and G) Immunohistochemical (IHC) staining of α-smooth muscle actin (α-SMA) and fibronectin for quantitative analysis. (H) IHC staining of α-SMA and fibronectin in mouse kidney tissue (×200 magnification, bar = 60 μm). (I and J) Quantitative analysis of α-SMA and fibronectin by protein blotting. (K) Protein blotting for the expression of α-SMA and fibronectin in renal tissues of each group. The data are presented as mean ± standard error of the mean (SEM) (n = 3/group). #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001, DKD vs. control; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, DKD vs. ST; ns, not significant.

• 

  Fig. 7.. Stigmasterol (ST) reduces mitochondrial dysfunction in diabetic kidney disease (DKD) mice. (A) Reactive oxygen species (ROS) staining of mouse kidney tissue (bar = 50 μm). (B) Quantification of ROS fluorescence intensity. (C to F) Messenger RNA (mRNA) levels of Rock1, Pparα, Pgc1α, and Acox1 in mouse kidney. (G) Effects of ST on ultrastructure in the kidneys of DKD mice (yellow arrows indicate podocyte injury, red arrows indicate lipid droplet accumulation, and blue arrows indicate mitochondrial swelling). The data are presented as mean ± standard error of the mean (SEM) (n = 3/group). #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001, DKD vs. control; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, DKD vs. ST; ns, not significant.

• 

  Fig. 8.. Stigmasterol inhibits the rho-associated coiled-coil protein kinase 1 (ROCK1)/p38 mitogen-activated protein kinase (MAPK)/peroxisome proliferator-activated receptor α (PPARα) signaling pathway in diabetic kidney disease (DKD) mice. (A) The mechanism of DKD mice. (B) Protein blotting assay for the expression of ROCK1, P-p38, p38, and PPARα in renal tissues. (C to E) Quantitative analysis of ROCK1, P-p38/p38, and PPARα by protein blotting. (F, H, and I) Quantitative analysis of immunohistochemical (IHC) staining for ROCK1, P-p38/p38, and PPARα. (G) IHC staining of mouse kidney tissues for ROCK1, P-p38, p38, and PPARα (×200 magnification, bar = 40 μm). The data are presented as mean ± standard error of the mean (SEM) (n = 3/group). ##P < 0.01, ###P < 0.001, and ####P < 0.0001, DKD vs. control; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, DKD vs. ST; ns, not significant.