Effect of spirogyra neglecta extract on renal organic anion transporter 1 and 3 function in type 2 diabetic rats

Abstract

Spirogyra neglecta (SN) has been widely grown in Nan River, Northern Thailand. In vivo studies indicated that this species has several beneficial effects, including anti-gastric ulcer, anti-inflammatory, anti-hyperglycemic and anti- hyperlipidemic actions. Recently, antioxidant effect of SN extract was shown in vitro and suggested that SN may subsequently be useful for applying as therapeutic drugs and/or food supplements. The aims of this study were to determine the effect of Spirogyra neglecta extract on rat renal organic anion transporter 1 and 3 (rOat1 and rOat3) functions, and identify its effect on regulatory mechanisms of rOat1 and rOat3 functions in type 2 diabetic (T2DM) rats. Thirty Male Wistar rats were equally divided into five groups, which were normal control (NC), normal with SN supplement (NC+SN1000), type 2 diabetes (DMC), diabetes supplemented with SN (DM+SN1000) and diabetes supplemented with ascorbic acid (DM+vit.C). T2DM rats were induced by high-fat diet with low single dose of streptozotocin injection. The supplementations by SN extract (1000 mg/kg BW) and vitamins C (200 mg/kg BW) were subsequently administrated by daily oral gavage for 12 weeks. At the end of experiment, the animals were sacrificed. Blood and tissue samples were collected for biochemical analyses, and renal lipid peroxidation was determined. Organic anion transport functions by rOat1 and 3 were obtained using para-aminohippurate (PAH) and estrone sulfate (ES). The up- regulation of these two transporters by insulin stimulation was also determined. Oxidative stress gene expressions were analyzed using semi-quantitative polymerase chain reaction. Rat Oat3 and regulatory protein expressions were identified by western blotting analyses. T2DM rats were shown hyperglycemia, hypertriglyceridemia and insulin resistance indicated by HOMA index. However, theplasma glucose, triglyceride, and HOMA index were markedly reduced in DM+SN1000 compared to that of DM. Moreover, the significant increase in lipid peroxidation indicated by malondiadehyde (MDA) level in DM rat kidneys was apparently reduced by both SN extract and vitamin C supplementations, suggesting that antioxidant activity in SN extract had reduced hyperglycemic-induced lipid peroxidation in T2DM rat kidneys similarly to that of vitamin C. The basal uptake of PAH and ES in renal slices mediated by rOat1 and rOat3 were not different among experimental groups, paralleling with no changes in rOat3 protein expression. However, up-regulation of rOat1 and 3 functions by insulin stimulation were completely blunted in DM rats, and this defect was completely restored in DM supplemented with both SN extract and vitamin C. The result implies that the impairment of intracellular signaling proteins in T2DM rat kidneys could be recovered by antioxidant activity of SN extract. Although oxidative stress gene expressions, including glutathione peroxidase, superoxide dismutase and catalase, did not show significant differences among experimental groups, the activation of p65NFB and PKCα were significantly increased in DM rats. Likewise, SN extract was able to deactivate these oxidative stress protein markers. In addition, SN supplementation induced the activation and translocation of PKC from cytosol to the membrane in DM rat kidneys. Thus, these findings indicated that T2DM experimental rats were linked to hyperglycemia and hypertriglyceridemia induced oxidative stress through changing intracellular signaling protein expressions and functions, leading to down-regulated rOat1 and 3 functions. SN supplementation, according to this study, had ability to attenuate these consequences. Therefore, SN extract could be potential candidate for nutritional supplement for preventing diabetic complications.