Attenuation of diabetic nephropathy in streptozotocin-induced diabetic rats by Punica granatum Linn. leaves extract

Abstract

With an objective to develop Complementary and Alternative Medicine for the treatment of diabetic nephropathy, the present study investigated the protective effects of methanolic extract of Punica granatum leaves (MPGL) in streptozotocin-induced diabetic nephropathy. Diabetic nephropathy has become a leading cause of end stage renal failure worldwide. P. granatum, due to its anti-diabetic, anti-inflammatory and antioxidant activities may retard the progression of diabetic nephropathy. In this study, diabetes was induced by a single injection of streptozotocin (STZ, 45 mg/kg, i.p.) in rats. STZ-diabetic rats were treated with oral doses of MPGL (100, 200 and 400 mg/kg) for 8 weeks. At the end of the experimental period, body and kidney weight and blood glucose levels were determined. Serum and urine parameters were investigated. Antioxidant enzymes and lipid peroxide levels were determined in the kidney along with histopathological examination of the same. MPGL significantly increased body weight, lowered blood glucose levels and ameliorated kidney hypertrophy index in the STZ-diabetic rats. The extract also decreased the levels of creatinine, blood urea nitrogen, total cholesterol, triglycerides, advanced glycation end products and albumin in serum and urine, respectively. MPGL significantly increased the antioxidant parameters in the kidney. Histological evaluation revealed that MPGL treated STZ-diabetic rats demonstrated reduced vacuolar degeneration of tubules; periodic acid Schiff base (PAS) positivity staining intensity in glomeruli and basement membrane thickening. Present findings provide experimental evidence that MPGL has potential antioxidant, antihyperglycemic and anti-glycation activities which might be helpful in slowing the progression of diabetic nephropathy.

Keywords: AGEs, advanced glycation end products; Antihyperglycemic; Antioxidant; BUN, blood urea nitrogen; CAT, catalase; CPCSEA, Committee for the Purpose of Control and Supervision of Experiments on Animals; DTNB, 5-5-dithiobis (2-nitrobenzoic acid); Diabetic nephropathy; GAE, gallic acid equivalent; GSH, reduced glutathione; H&E, hematoxylin and eosin; IAEC, Institutional Animal Ethics Committee; MDA, malondialdehyde; MPGL, methanolic extract of Punica granatum leaves; PAS, periodic acid Schiff base; Punica granatum; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; Streptozotocin.

Graphical abstract

Fig. 1

TLC analysis of methanolic extract of Punica granatum leaves (MPGL). (A) Visualization of MPGL and Luteolin standard at 254 nm (Rf value: 0.37). (B) Visualization of MPGL and Luteolin standard at 366 nm (Rf value: 0.37). (C) Visualization of MPGL and β-sitosterol standard at 366 nm (Rf value: 0.46).

Fig. 2

Effect of Punica granatum leaves extract on antioxidant parameters in STZ-diabetic rats. (A) GSH in kidney homogenate of control, STZ-diabetic rats and MPGL treated rats. (B) SOD in kidney homogenate of control, STZ-diabetic rats and MPGL treated rats. (C) Catalase in kidney homogenate of control, STZ-diabetic rats and MPGL treated rats. (D) Lipid peroxidation in kidney homogenate of control, STZ-diabetic rats and MPGL treated rats. * Treatment group was compared with diabetic control, p < 0.05; ** Treatment group was compared with diabetic control, p < 0.01; *** Treatment group was compared with diabetic control, p < 0.001; ### Diabetic group was compared with normal control, p < 0.001, using one-way ANOVA with Dunnett's test.

Fig. 3

Photomicrographs of kidney (Hematoxylin and Eosin staining under a light microscope at 400× magnification). A: Control rats; B: STZ-diabetic rats; C: 100 mg/kg MPGL treated group; D: 200 mg/kg MPGL treated group; E: 400 mg/kg MPGL treated group. G: glomeruli, T: tubules, GS: glomerular space, VD: vacuolar degeneration.

Fig. 4

Photomicrographs of kidney (Periodic Acid Schiff staining under light microscope at 400× magnification). A: Control rats; B: STZ-diabetic rats; C: 100 mg/kg MPGL treated group; D: 200 mg/kg MPGL treated group; E: 400 mg/kg MPGL treated group. PP: PAS positivity, BM: Basement membrane thickening, G: glomeruli, +: mild change, ++: moderate change.