Grape seed proanthocyanidins protect against streptozotocin‑induced diabetic nephropathy by attenuating endoplasmic reticulum stress‑induced apoptosis

Abstract

Diabetic nephropathy (DN) is by far the most common cause of end‑stage renal disease (ESRD) in industrial countries, accounting for ~45% of all new ESRD cases in the United States. Grape seed proanthocyanidin extracts (GSPE) are powerful antioxidants, with an antioxidant ability 50‑fold greater than that of vitamin E and 20‑fold greater than that of vitamin C. The present study investigated whether GSPE can protect against streptozotocin (STZ)‑induced DN and aimed to elucidate a possible mechanism. Male Sprague Dawley rats were randomly divided into three groups: Control group (N), diabetes mellitus group (DM) injected with 40 mg/kg STZ, and the GSPE treatment group (intragastric administration of 250 mg/kg/day GSPE for 16 weeks after diabetes was induced in the rats). Blood and kidney samples were collected after treatment. The renal pathological changes were determined with periodic acid‑Schiff (PAS) staining, while the protein expression levels of glucose‑regulated protein 78 (GRP78), phosphorylated‑extracellular signal‑regulated kinase (p‑ERK) and Caspase‑12 were determined by western blotting and immunohistochemical staining. Apoptosis was determined with a terminal deoxynucleotidyl transferase dUTP nick‑end labeling (TUNEL) assay. Compared with the DM group, the GSPE group had no significant changes in the blood urea nitrogen (BUN) level and serum creatinine (Scr) level, but showed a significant decline in the renal index (RI) level and 24‑h urinary albumin level (P<0.05). The histopathology results indicated very little pathological damage in the GSPE group. Compared with the DM group, the GSPE group had a significantly reduced number of TUNEL‑positive cells (P<0.05), and the GSPE group had an obvious reduction in the protein expression of GRP78, p‑ERK, and Caspase‑12 (P<0.05). In this study, the results indicated that GSPE can protect renal function and attenuate endoplasmic reticulum stress‑induced apoptosis via the Caspase‑12 pathway in STZ‑induced DN.

Figure 1.

GSPE protects Streptozotocin-induced diabetic nephropathy. Animals were divided into three group: The control group (N), the DM group (D), the GSPE group (G), Sections were stained with PAS and were examined by microscopy. Original magnification, ×400. The glomerular hypertrophy, thickness of the glomerular basement membrane in the D group can be found. In G group, the change of glomerular was significant reduced. The histogram means the relative ECM accumulation of each group to the N group, N group was set to 1. The ECM in the D group is 5.46±0.61, and in the G group is 1.04±0.09. Higher ECM accumulation was observed in the kidneys of D group rats compared with that of the N group, Lower ECM accumulation was observed in the kidneys of the G group rats compared with the D group. *P<0.05 compared with the N group; ^#P<0.05 compared with the D group. DM, diabetes mellitus; GSPE, grape seed proanthocyanidin extracts; PAS, periodic acid-Schiff; ECM, extracellular matrix.

Figure 2.

GSPE inhibits apoptosis in DM group rats. Arrows are representative of TUNEL-positive cells. Original magnification, ×400. Animals were divided into three group: The control group (N), the DM group (D), the GSPE group (G). Data are presented as means ± SD. *P<0.05 compared with the N group; ^#P<0.05 compared with the D group. DM, diabetes mellitus; GSPE, grape seed proanthocyanidin extracts; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Figure 3.

GSPE inhibits the expression of GRP78, p-ERK and Caspase-12. Western blotting analysis for GRP78, p-ERK, and Caspase-12, and quantification of corresponding protein levels. Animals were divided into three group: The control group (N), the DM group (D), the GSPE group (G). Data in the expression of GRP78, p-ERK, ERK and Caspase-12 are expressed as mean ± SD levels relative to β-actin. The histogram means the relative protein expression of each group to the N group, N group was set to 1. In the relative expression of ERK, D group was 1.01±0.02, G group was 0.98±0.01. No significant difference was observed between the two groups (P>0.05). In the relative expression of p-ERK, D group was 1.59±0.09, G group was 0.92±0.08. This indicates that p-ERK levels changed without any effect on the level of normal ERK. In the relative expression of GRP78, D group was 2.22±0.21, G group was 1.44±0.19. In the relative expression of Caspase-12, D group was 1.60±0.03, G group was 0.70±0.01. Compared with the N group, the expressions of GRP78, p-ERK and Caspase-12 in the D group were significantly increased. Compared with the D group, the expression of GRP78, p-ERK and Caspase-12 were significantly decreased in the G group. *P<0.05 compared with the N group; ^#P<0.05 compared with the D group. DM, diabetes mellitus; GSPE, grape seed proanthocyanidin extracts; p-ERK, phosphorylated-extracellular signal-regulated kinase; GRP78, glucose-regulated protein 78.

Figure 4.

GSPE inhibits the expression of GRP78, p-ERK and Caspase-12. Immunohistochemical staining of GRP78, p-ERK and Caspase-12 of kidney and their measurement of the intensity of corresponding protein in the immunohistochemical staining. Animals were divided into three group: the control group (N), the DM group (D), the GSPE group (G). The grown granules reprehensive the positive cells. Original magnification, ×1,000. The expression of GRP78, p-ERK and Caspase-12 were significantly increased in D group compared with N group and decreased in G group compared with D group. Data are presented as means ± SD. The histogram means the relative protein expression of each group to the N group, N group was set to 1. In the relative expression of GRP78, D group was 4.85±0.24, G group was 1.57±0.11. In the relative expression of p-ERK, D group is 23.3±1.60, G group is 1.70±0.06. In the relative expression of Caspase-12, D group was 8.90±1.10, G group was 1.30±0.09. *P<0.05 compared with the N group; ^#P<0.05 compared with the D group. DM, diabetes mellitus; GSPE, grape seed proanthocyanidin extracts; p-ERK, phosphorylated-extracellular signal-regulated kinase; GRP78, glucose-regulated protein 78.