The Protective Effects of Cobra Venom from Naja naja atra on Acute and Chronic Nephropathy

Abstract

This study investigated the effects of Naja naja atra venom (NNAV) on acute and chronic nephropathy in rats. Rats received 6 mg/kg adriamycin (ADR) once to evoke the chronic nephropathy or 8 ml/kg 50% v/v glycerol to produce acute renal failure (ARF). The NNAV was given orally once a day starting five days prior to ADR or glycerol injection and continued to the end of experiments. The animals were placed in metabolic cages for 24 h for urine collection for urinary protein determination. The kidney function-related biochemical changes and index of oxidative stress were determined with automatic biochemistry analyzer or colorimetric enzyme assay kits. The pathomorphological changes were observed using light and transmission electron microcopies. The levels of inflammatory cytokines and NF- κ B activation were determined using ELISA kits, Western blot analysis, or immunofluorescence. The results showed that NNAV relieved ADR-induced chronic nephropathy and glycerol-triggered acute renal failure syndromes including proteinuria, hypoalbuminemia, hyperlipidemia, serum electrolyte unbalance, renal oxidative stress, and pathological damages. NNAV reduced kidney levels of TNF- α and IL-1 β , but it increased the levels of I κ B- α and inhibited NF- κ B p65 nuclear localization. These findings suggest that NNAV may be a valuable therapeutic drug for acute and chronic kidney diseases.

Figure 1

The effects of NNAV on urinary protein excretion. Wistar rats were treated with oral administration of NNAV at a dose of 30, 90, and 270 μg/kg (a) or 20, 40, and 80 μg/kg (b) once a day starting 5 days prior to adriamycin (ADR) injection. ADR (6 mg/kg) was administered by tail vein injection. Wistar rats were treated with oral administration of NNAV (20, 40, and 80 μg/kg) once a day starting 5 days prior to glycerol injection (c). Glycerol (50% v/v, 8 mL/kg) was administered by intramuscular injection. Urine was collected for determination of proteinuria before and after ADR administration and 72 h after glycerol injection. ***P < 0.001 compared with “saline + saline” group; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 compared with “adriamycin (or glycerol) + saline” group; n = 7–10.

Figure 2

The effects of NNAV on kidney coefficient. Wistar rats were treated as described in the legend of Figure 1. Rats were killed at the end of experiment, and kidneys were dissected for weight measurement. Kidney coefficient was derived by the weight of kidney divided by the total body weight of rat. ***P < 0.001 compared with “saline + saline” group; ^# P < 0.05 compared with “glycerol + saline” group; n = 7–10.

Figure 3

The effects of NNAV on levels of serum creatinine (SCr) and blood urea nitrogen (BUN) in acute and chronic nephropathy rats. Wistar rats were treated as described in the legend of Figure 1. At the end of the experiment, rats were killed and blood samples were collected for determination of serum levels of SCr and BUN in adriamycin ((a) and (b)) and glycerol ((c) and (d)) models. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with “saline + saline” group; ^# P < 0.05 and ^### P < 0.001 compared with “adriamycin (or glycerol) + saline” group; n = 7–10.

Figure 4

The effects of NNAV on levels of serum Cystatin C in adriamycin and glycerol-injected rats. Wistar rats were treated as described in the legend of Figure 1. At the end of experiment, rats were killed and blood samples were collected for determination of serum levels of cystatin C in adriamycin (a) and glycerol (b) models. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with “saline + saline” group, n = 7–10.

Figure 5

The effects of NNAV on levels of serum potassium and phosphorus in adriamycin-injected rats. Wistar rats were treated as described in the legend of Figure 1. At the end of experiment, rats were killed and blood samples were collected for determination of serum levels of potassium (a) and phosphorus (b). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with “saline + saline” group, n = 7–10.

Figure 6

The effects of NNAV on levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in adriamycin and glycerol-injected rats. Wistar rats were treated as described in the legend of Figure 1. At the end of the experiment, rats were killed and blood samples were collected for determination of serum levels of SOD in adriamycin (a) and glycerol (c) models, and renal cortical tissues were dissected for determination of the levels of SOD in adriamycin (b) and glycerol (d) models; meanwhile, the MDA level of renal cortical tissues in glycerol model was measured (d). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with “saline + saline” group; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 compared with “adriamycin (or glycerol) + saline” group; n = 7–10.

Figure 7

The effects of NNAV on renal pathology in adriamycin-injected rats. Wistar rats were treated as described in the legend of Figure 1. At the end of the experiment, rats were killed and kidneys were dissected and fixed for hematoxylin and eosin ((a)–(e)), Masson's trichrome ((f)–(j)), and periodic acid-Schiff ((k)–(o)) staining. Morphological analysis of renal pathology was performed with a light microscopy. It showed the glomerular deformation and damage (yellow arrows, (a)–(e)), tubular dilatation (green arrows, (a)–(e)), tubule interstitial collagen proliferation (yellow arrows, (f)–(j)), and glomerular basement membrane and mesangial expansion (yellow arrows, (k)–(o)) in the model group. NNAV reduced these pathological changes to a varying degree. Scale bar: 25 μm.

Figure 8

The effects of NNAV on renal pathology in glycerol-injected rats. Wistar rats were treated as described in the legend of Figure 1. At the end of the experiment, rats were killed and kidneys were dissected and fixed for hematoxylin and eosin (HE) staining. Morphological analyses of renal pathology of both cortex ((a)–(e)) and medulla ((f)–(j)) were performed with a light microscopy. It showed the tubular dilatation and necrosis (green arrows) and glomerulus deformation (yellow arrows) in the model group. However, NNAV markedly ameliorated these pathological magnifications. Scale bar: 25 μm.

Figure 9

The effects of NNAV on renal pathology in adriamycin-injected rats. Wistar rats were treated as described in the legend of Figure 1. At the end of the experiment, rats were killed and kidneys were dissected and fixed for electron microscopic examination. Morphological analysis of renal pathology was performed with a transmission electron microscopy. (a)–(e) represented control, model, and Naja naja atra venom in doses of 20, 40, and 80 μg/kg groups, respectively. It showed foot process effacement (arrows) in model group, but, in Naja naja atra venom-treated groups, the foot process lesions (arrows) were significantly reduced. Scale bars: (a) 2.5 μm; (b) 2 μm; (c) 2 μm; (d) 1 μm; (e) 0.5 μm, respectively.

Figure 10

The effects of NNAV on the levels of proinflammatory cytokines. Wistar rats were treated as described in the legend of Figure 1. At the end of the experiment, rats were killed, and the levels of TNF-α (a) and IL-1β (b) in kidney tissue homogenates were determined using ELISA kits. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with “saline + saline” group; ^## P < 0.01 compared with “adriamycin + saline” group; n = 7–10.

Figure 11

The effects of NNAV on the levels of P-IKK-α and IκB-α. Wistar rats were treated as described in the legend of Figure 1. At the end of experiment, rats were killed, and levels of P-IKK-α (a) and IκB-α (c) in kidney tissue homogenates were determined using with Western blot analyses (n = 6 experiments). Quantitative analysis of P-IKK-α (b) and IκB-α (d) were performed with Image J software and normalized to the protein levels of β-actin. *P < 0.05 and **P < 0.01 compared with “saline + saline” group.

Figure 12

The effects of NNAV on NF-κB activation. Wistar rats were treated as described in the legend of Figure 1. At the end of the experiment, rats were killed, and the nuclear translocation of NF-κB p65 was determined with immunofluorescence with kidney paraffin sections. Nuclei were stained with DAPI ((a)–(e)), and NF-κB p65 was stained with FITC-conjugated donkey anti-mouse IgG ((f)–(j)). Overlay of the images (p65 with DAPI, (k)–(o)) indicated the nuclear translocation of NF-κB p65 (yellow arrows) in some tubular cells. Scale bar: 5.5 μm.