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Adiponectin Protects the Kidney Through the Akt/Ampk/Enos Signalling Path- Way in the Blood Vessels of Diabetic Model Rats

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Adiponectin Protects the Kidney Through the Akt/Ampk/Enos Signalling Path- Way in the Blood Vessels of Diabetic Model Rats Acta Medica Mediterranea, 2019, 35: 307 ADIPONECTIN PROTECTS THE KIDNEY THROUGH THE AKT/AMPK/ENOS SIGNALLING PATH- WAY IN THE BLOOD VESSELS OF DIABETIC MODEL RATS TIAN MENG1,2#, KANG LILI2#, HAN HONGLING1* 1Tianjin Medical University General Hospital, Tianjin 300052 - 2Internal Medicine of Xianshuigu Hospital Jinnan District of Tianjin, Tianjin 300350, China #These authors contributed equally to this work as co-first author ABSTRACT Objective: To explore the mechanism by which adiponectin protects the kidney through the vascular Akt/AMPK/eNOS signalling pathway in diabetic model rats. Methods: Overall, 72 rats were randomly divided into the normal control group (NC group), the diabetes mellitus group (DM group), the pIRES2-EGFP-gAd plasmid transfected adiponectin interference group (ADPN group) and the pIRES2-EGFP transfected empty vector group (DP group). Except for the NC group, the animals were fed a high fat and high sugar diet for 4 weeks and streptozo- tocin was injected intravenously to establish a diabetic rat model. Rats in the ADPN group and DP group were intraperitoneally injected with the pIRES2-EGFP-gAd plasmid transfection complex and the pIRES2-EGFP transfection complex, respectively. The general situa- tion of rats was observed and the urine protein, blood glucose level and HbA1c were measured in each group after 8 weeks. The western blot assay was used to detect the relative expression of protein kinase B (Akt), adenosine monophosphate activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) in rat blood vessels. The pathologic condition of renal tissue sections of rats was observed by HE staining, and the levels of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the serum of rats were deter- mined by an enzyme-linked immunosorbent assay (ELISA). Results: Nine rats died during modelling. The levels of urine protein, blood glucose, HbA1c, TNF-α and IL-1β in the DM, DP and ADPN groups were significantly higher than those in the NC group, with a difference that is statistically significant (P<0.05). The levels of urine protein, blood glucose, HbA1c, TNF-α and IL-1β in the ADPN group were significantly lower than those in the DM and DP groups, with a difference that is statistically significant (P<0.05). Compared with the NC and ADPN groups, the expression level of Akt, AMPK and eNOS in the DM and DP groups was significantly lower than that in NC and ADPN groups, with a difference that is statisti- cally significant (P<0.05). Compared with the NC group, the glomerular diameter enlarged, the basement membrane thickened, diffuse hyperplasia occurred in the mesangium, renal tubular epithelial cells and microvilli fell off, and vacuolar degeneration was observed in the DM and DP groups. The pathological changes in the ADPN group were lighter than those in the DM group and the DP group. Conclusion: Adiponectin can regulate the blood glucose levels in diabetic model rats and protect the kidney by up-regulating the expression of proteins in the Akt/AMPK/eNOS pathway in the blood vessels of diabetic model rats. Keywords: Adiponectin, diabetic nephropathy, Akt, AMPK, eNOS. DOI: 10.19193/0393-6384_2019_1_49 Received July 17, 2018; Accepted Septemper 20, 2018 Introduction retinopathy, which can lead to organ and tissue damage. As one of the most harmful complications Diabetes mellitus is a chronic metabolic disor- in diabetic patients, the incidence of diabetic der characterised by disorder of glucose metabo- nephropathy (DN) is positively correlated with the lism and lipid metabolism. The main pathogenesis course of diabetes(2). It has been shown that patients of diabetes mellitus is the absolute or relative insuf- with diabetes for more than 10 years often experi- ficiency of insulin secretion(1). Because the body is ence DN, meaning that their kidneys are gradually in a high glucose state for a long time, diabetic irreversibly damaged, which seriously threatens patients develop microangiopathy, neuropathy and their life(3). 308 Tian Meng, Kang Lili et Al Recent studies have found that adiponectin 55±5% and the illumination time was maintained at (ADPN) decreased significantly in the plasma of 12 h/d. All rats were fed adaptively with the same diabetic patients, while the Akt/AMPK/eNOS path- common feedstuff and drinking water for one week. way promoted the proliferation of vascular Before the formal experiment, the peripheral blood endothelial cells(4, 5). glucose of all rats was normal. The rats were divid- In this study, the diabetic rat model was estab- ed into 4 groups by the random digital table lished to investigate the effects of adiponectin on method: a normal control group (NC group), a dia- protein kinase B (Akt), adenosine monophosphate- betes group (DM group), a pIRES2-EGFP-gAd activated protein kinases (AMPK) and endothelial plasmid-transfected adiponectin interference group nitric oxide synthase (eNOS), thereby exploring the (ADPN group), and a pIRES2-EGFP-transfected mechanism of its protective effect on the kidney. empty vector group (DP group), with 18 rats in each group. Materials and methods Except for the NC group, the animals were fed a high fat and high sugar diet for 4 weeks to induce Experimental animals the insulin resistance model. Then, after fasting and In this study, 72 healthy male Sprague Dawley prohibiting for 8h, 25mg/kg streptozotocin was (SD) rats (SPF grade) were selected from the injected intravenously and random blood glucose Experimental Animal Centre of Sun Yat-Sen levels in the rat tail vein were measured after 3 University, aged 11-12 weeks, with a body weight days. When glucose levels in venous blood were of 218±22 g. This study has been submitted to the higher than 16.7mmol/L and accompanied by poly- Medical Ethics Committee and the Animal dipsia, polyuria, increased appetite, weight loss and Protection Association for approval. so on, the diabetes model was considered to have been constructed successfully. Main drugs and reagents Rats in the ADPN group were injected The plasmid pIRES2-EGFP-gAd, which was intraperitoneally with the pIRES2-EGFP-gAd plas- constructed and saved by a laboratory in our hospi- mid transfection complex twice a week, and rats in tal, contained the full-length coding region of the the DP group were injected intraperitoneally with adiponectin spherical domain and could express the pIRES2-EGFP transfection complex twice a adiponectin in vivo. Antibodies to Akt, AMPK and week. Five weeks after the experiment, the rats eNOS (BioVision, USA) and kits for tumour necro- were placed in metal metabolic cages each week to sis factor-α (TNF-α) and Interleukin-1β (IL-1β) collect urine samples for 24h, and the competitive (Shenzhen Zike Biotechnology Co., Ltd.) were radioimmunoassay was used for the quantitative obtained. detection of proteins in the urine. At the end of the 8th week of the experiment, the rats were killed and Experimental instruments blood glucose and glycosylated haemoglobin Blood glucose test strips and the glucometer (HbA1c) were measured by a glucometer and the (Sinocare biosensor Inc), a low temperature and glycosylated haemoglobin apparatus, respectively. high speed centrifuge (Hunan Xiangxin instrument The relative expression level of proteins in rat Co., Ltd.), basic western blot electrophoresis appa- blood vessels was detected by the western blot ratus (Beijing Junyi electrophoresis Co., Ltd.), wet assay. The kidney specimens of rats were stained electric transmembrane apparatus (Guangzhou with H&E for pathological observation. The con- Ruiheng instrument Co., Ltd.), and a visualizer tent of inflammatory factors in the serum was deter- (Shanghai Jiabiao Testing instrument Co., Ltd.) mined by the enzyme-linked immunosorbent assay were used. (ELISA). Methods Observation index The observation index included the general All experimental animals were raised in the situation of the rats during the experiment, the independent ventilator cages of the animal laborato- quantification of urinary protein, blood glucose lev- ry in our hospital. There were five rats in each cage. els, HbA1c, relative expression of proteins in the During the experiment, the laboratory temperature blood (Akt, AMPK and eNOS), pathological was kept at 23±2°C, the humidity was kept at changes of the rat kidney and serum inflammatory Adiponectin protects the kidney through the Akt/AMPK/eNOS signalling pathway in the blood vessels... 309 factor (TNF-α and IL-1β). Comparison of the relative expression level of Akt, AMPK and eNOS in the four groups of Statistical treatment rats blood vessels In order to ensure the accuracy of the input, The expression levels of Akt, AMPK and the data of this research were all recorded with eNOS in the DM and DP groups were significantly double input; SPSS 21.0 statistical software was lower than in the NC and ADPN groups, with a dif- used to analyse the data. The mean±standard devi- ference that is statistically significant (P<0.05) ation (x±s) was used to express the measurement (Figure 1). data, and t test was used for the comparison between groups. P<0.05 was considered statisti- cally significant. Results Comparison of general condition, and uri- nary protein, blood glucose and HbA1c levels in the four groups of rats During the course of the experiment, only 9 rats died during modelling: 4 rats in the DM group, 2 rats in the DP group and 3 rats in the ADPN group. All of the rats except those in the NC group showed polydipsia, polyuria, increased appetite, weight loss and, retarded movement during the experiment. In the DM, DP and ADPN groups, uri- nary protein, blood glucose and HbA1c levels were significantly higher than those in the NC group with a difference that is statistically significant (P<0.05). The urinary protein, and blood glucose and HbA1c levels in the ADPN group were signifi- cantly lower than those in the DM and DP groups, with a difference that is statistically significant (P<0.05).
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