Emodin Ameliorates Renal Damage and Podocyte Injury in a Rat Model of Diabetic Nephropathy via Regulating AMPK/mTOR-Mediated Autophagy Signaling Pathway

Figures & data

Figure 1 Experimental procedure.

Abbreviations: Sham, Sham operation + distilled water group; Vehicle, DN + distilled water group; LD-Emo, DN + low-dose emodin group; HD-Emo, DN + high-dose emodin group; DN, diabetic nephropathy; STZ, streptozotocin.

Figure 2 Effects of Emo on changes in body weight, kidney weight/body weight and renal appearance.

Notes: (A) body weight, (B) kidney weight/body weight, (C) renal appearance. (a) Sham group, (b) Vehicle group, (c) LD-Emo group, (d) HD-Emo group. Data are expressed as mean ± SEM. *P < 0.05, ^#P < 0.01.
Abbreviations: Emo, emodin; BW, body weight; KW/BW, kidney weight/body weight; LD-Emo, low-dose emodin; HD-Emo, high-dose emodin.

Figure 3 Effects of Emo on changes in urinary albumin/urinary creatinine ratio and blood glucose. Data are expressed as mean ± SEM. ^# P < 0.01.

Notes: (A) Urinary albumin/urinary creatinine ratio, (B) blood glucose.
Abbreviations: Emo, emodin; Urinary ACR, urinary albumin/urinary creatinine ratio; BG, blood glucose; LD-Emo, low-dose emodin; HD-Emo, high-dose emodin.

Table 1 Comparison of Blood Biochemical Parameters of Rats Among Groups at the end of 8 Weeks

Group	Sham (n = 5)	Vehicle (n = 7)	LD-Emo (n = 7)	HD-Emo (n = 7)
Alb (g/L)	28.06 ± 0.35	24.87 ± 0.60**	25.13 ± 0.30**	25.79 ± 0.49**
BUN (mmol/L)	5.36 ± 0.26	27.46 ± 1.86**	13.96 ± 1.64**,^#	10.13 ± 1.64*,^#
Scr (μmol/)	35.80 ± 1.16	43.57 ± 3.33*	42.71 ± 1.95	42.29 ± 2.33
UA (μmol/L)	49.00 ± 3.78	61.00 ± 9.43	54.57 ± 3.57	52.29 ± 6.20
TC (mmol/L)	1.25 ± 0.05	1.75 ± 0.15**	1.73 ± 0.02**	1.71 ± 0.04**
TG (mmol/L)	0.23 ± 0.02	0.40 ± 0.03**	0.37 ± 0.02**	0.36 ± 0.03**

Notes: *P < 0.05, **P < 0.01 vs Sham group. ^# P < 0.01 vs Vehicle group.

Abbreviations: Alb, albumin; BUN, blood urea nitrogen; Scr, serum creatinine; UA, uric acid; TC, total cholesterol; TG, triglyceride. Data are expressed as mean ± SEM.

Figure 4 Effects of Emo on the glomerular morphological changes.

Notes: (A) Light microscopy, periodic acid-Schiff (PAS) staining (magnification, ×400), (B) light microscopy, Masson staining (magnification, ×400), (C) the number of glomerular cells, (D) the rate of ECM/glomerular area, (E) the rate of collagen/glomerular area, (F) electron microscopy, 5000×. (a) Sham group, (b) Vehicle group, (c) LD-Emo group, (d) HD-Emo group. Data are expressed as mean ± SEM. ^#P < 0.01.
Abbreviations: Emo, emodin; ECM, extracellular matrix; LD-Emo, low-dose emodin; HD-Emo, high-dose emodin.

Figure 5 Effects of Emo on the glomerular and tubular epithelial cell apoptosis.

Notes: TUNEL staining (magnification, ×200). (A) Nucleus is blue by labeling with DAPI, (B) positive apoptosis cells are green, (C) merging of A and B, (D) quantification of the number of apoptotic cells. (a) Sham group, (b) Vehicle group, (c) LD-Emo group, (d) HD-Emo group. Data are expressed as mean ± SEM. ^#P < 0.01.
Abbreviations: Emo, emodin; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling; LD-Emo, low-dose emodin; HD-Emo, high-dose emodin.

Figure 6 Effects of Emo on the protein expression of Bax and Cleaved caspase-3 in the kidney.

Notes: (A) Western blot analysis of Bax, Cleaved caspase-3 and caspase-3 protein expression. (B and C) Quantitative analysis of Bax and Cleaved caspase-3 protein expression. Data are expressed as mean ± SEM. *P < 0.05, ^#P< 0.01.
Abbreviations: Emo, Emodin; LD-Emo, low-dose Emodin; HD-Emo, high-dose Emodin.

Figure 7 Effects of Emo on the protein expression of nephrin, LC3-II/I, Beclin-1, p62, p-AMPK, AMPK, p-mTOR and mTOR in the kidney.

Notes: (A) Western blot analysis of nephrin, LC3-II/I, Beclin-1 and p62 protein expression. (B–E) Quantitative analysis of nephrin, LC3-II/I, Beclin-1 and p62 protein expression. (F) Western blot analysis of p-AMPK, AMPK, p-mTOR and mTOR protein expression. (Gand H) Quantitative analysis of p-AMPK and p-mTOR protein expression. Data are expressed as mean ± SEM. ^#P < 0.01.
Abbreviations: Emo, Emodin; LD-Emo, low-dose Emodin; HD-Emo, high-dose Emodin; LC3, microtubule-associated protein 1 light-chain 3; P62 protein, p62/sequestosome-1 protein; AMPK, adenosine 5ʹ-monophosphate (AMP)-activated protein kinase; p-AMPK, phospho-AMPK; mTOR, mammalian target of rapamycin; p-mTOR, phospho-mTOR.

Figure 8 Effects of Emo on the autophagy of podocytes in rats with DN.

Notes: Electron microscopy (magnification, 15,000×). The white arrow indicates autophagosome. (A) Sham group, (B) Vehicle group, (C) LD-Emo group, (D) HD-Emo group.
Abbreviations: Emo, emodin; LD-Emo, low-dose emodin; HD-Emo, high-dose emodin.