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 streptozotocin 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 situation 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 statistically 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 statistically significant.

Results

Comparison of general condition, and urinary 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, urinary 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 significantly lower than those in the DM and DP groups, with a difference that is statistically significant (P<0.05). There was no statistically significant difference of the indexes between the DM and DP groups (P>0.05) (Table 1).

Urine protein quantitation Blood glucose Group HbA1c (%) (mg/24h) (mmol/L)

NC group (n=18) 10.52±2.71 5.67±1.24 5.70±1.91 Figure 1: Comparison of the relative expression levels DM group (n=14) 49.79±9.02a 24.69±4.16a 10.68±1.98a of Akt, AMPK and eNOS in the four groups of rats. DP group (n=16) 45.08±6.73a 25.32±6.27a 10.07±2.03a Renal pathology in the four groups of rats

ADPN group (n=15) 36.94±8.17abc 19.58±4.09abc 8.49±1.62abc Pathological sections of the rat kidney were observed under an optical microscope. The renal F 109.02 77.85 22.93 tissue structure of the NC group was normal, with P <0.001 <0.001 <0.001 no changes in the glomerular and renal tubule ultra- Table 1: Comparison of urinary protein, and blood glu- structure observed. In the DM and DP groups, the cose and HbA1c levels in the 4 groups of rats at the end glomerular diameter enlarged, basement membrane of the 8th week. thickened, diffuse hyperplasia occurred in the Note: Compared with the NC group, aP<0.05; compared with mesangium, renal tubular epithelial cells and the DM group, bP<0.05; compared with the DP group, microvilli fell off and vacuolar degeneration was c P<0.05. observed. There was no significant difference between the two groups with regard to renal pathological changes. 310 Tian Meng, Kang Lili et Al

Compared with the DM group, the glomerular toms of diabetes are not obvious, the difficulty in diameter reduced, basement membrane thickened, diagnosis and treatment increases, and the blood the degree of mesangial hyperplasia was lower, and glucose levels of patients cannot be effectively con- the degree of renal tubule lesion was lower in the trolled. Vascular endothelial cells have a variety of ADPN group (Figure 2). physiological functions in vivo, not only playing an important role in maintaining the permeability and proper tension of blood vessels, but also participating in important physiological processes such as synthesis, secretion, metabolism and immunity(6). Due to the elevated levels of blood glucose in diabetic patients for a long time, the continuous stimu- lation of vascular endothelial cells leads to a dysfunction of endothelial cells, which is mainly caused by the impaired bioavailability of nitric oxide (NO), leading to DN, diabetic foot, iritis, glaucoma and other complications(7, 8). ADPN is a kind of adipocyte factor that is negatively related to obesity. ADPN can regulate lipid metabolism, reduce serum total cholesterol by Figure 2: Results of H&E staining in pathological sections of the kidney in the 4 groups of rats (×200). specifically binding with receptors on the cell mem- A: NC group; B: DM group; C: DP group; D: ADPN group brane, protect blood vessels, promote vascular endothelial cell regeneration, increase insulin sensi- Comparison of serum inflammatory factors tivity and reduce blood glucose levels(9, 10). Urine in the 4 groups of rats protein quantification is mainly used in the diagno- The levels of TNF-α and IL-1β in the DM, DP sis of urinary system diseases and is used to evalu- and ADPN groups were significantly higher than ate renal function(11). HbA1c is a combination of those in the NC group, with a difference that is sta- haemoglobin and blood glucose and is a stable tistically significant (P<0.05). The contents of TNF- index, reflecting the blood glucose level of the sub- α and IL-1β in the ADPN group were significantly jects in the last 8-12 weeks(12). lower than those in the DM and DP groups, with a eNOS is an isozyme produced by vascular difference that is statistically significant (P<0.05). endothelial cells, which can interact with oxygen to There was no statistically significant difference produce NO and L-citrulline using L-arginine as between the DM and DP groups (P>0.05) (Table 2). substrate. NO has a protective effect on nerve and vascular endothelial cells(13). Wang Yunshan et al. Group TNF-α (ng/L) IL-1β (ng/L) have found that the decreased activity of eNOS NC group (n=18) 7.11±0.41 92.27±4.33 could induce the down-regulation of NO secretion, DM group (n=14) 10.18±0.85a 142.42±20.06a which leads to the dysfunction of endothelial cells (14) DP group (n=16) 9.92±0.49a 138.31±14.52a with abnormal vasodilation function . As a mem- ber of the serine/threonine protein kinase family, ADPN group (n=15) 7.54±0.35abc 105.46±7.85abc Akt can increase the ability of anti-apoptosis by F 133.94 60.02 enhancing the signalling of the phosphatidylinosi-

P <0.001 <0.001 tol-3 kinase pathway, and can also regulate the metabolism of proteins and glucose, participating in Table 2: Comparison of serum inflammatory factors in inflammatory reactions(15). the 4 groups of rats at the end of the 8th week. AMPK is widely distributed in eukaryotic Note: Compared with the NC group, aP<0.05; compared with the DM group, bP<0.05; compared with the DP group, cells and plays an important role in regulating ener- cP<0.05. gy metabolism in vivo. It has been shown by Zhang Dacheng et al. that AMPK can regulate glucose Discussion transport and lipid metabolism in vivo to ensure energy supply(16). Ford et al. also found that AMPK At present, the incidence of diabetes is could increase insulin sensitivity and effectively increasing year by year. Because the early symp- improve insulin resistance(17). TNF-α and IL-1β are Adiponectin protects the kidney through the Akt/AMPK/eNOS signalling pathway in the blood vessels... 311 inflammatory cytokines produced by macrophages. 5) Zhou H, Liu J, Hou W, Wang Y, Zhang W, et al. Low concentrations of TNF-α and IL-1β can acti- Protective Effect of Berberine on Oleic Acid-Induced Injury in Human Aortic Endothelial Cells via AMPK- vate immune cells and regulate the immune func- eNOS Activation. Food Sci 2018; 39: 213-219. tion of the body. When the concentrations of TNF-α 6) Zhao JL, Li MH. Research progress of P120 catenin and IL-1β increase abnormally, TNF-α and IL-1β and Kaiso signalling in regulating endothelial cell func- stimulate the hypothalamus to cause fever, promote tion. Guangdong Med J 2016; 37: 2498-2501. the uptake of amino acids by hepatocytes, synthe- 7) Liu MM, Li AL, Xiu RJ. Research progress on mechanism of impaired function of diabetic microvascular sise a large number of acute phase proteins such as endothelial cells. Chin J Diabetes Mellitus 2016; 8: C-reactive protein and a2 globulin, induce inflam- 439-442. matory reactions, and cause injury to tissues and 8) Zhou CT, Wang CH. Impact of inhalation of exogenous organs(18, 19). nitric oxide on vascular endothelial cell function, platelet function and thrombosis of patients with acute In this study, it was shown that the levels of ischaemic stroke. Pract J Cardiac Cerebr Pneumal Vasc urine protein quantification, blood glucose, HbA1c, Dis 2016; 24: 63-65. TNF-α and IL-1β were significantly increased in 9) Huang YJ, Chen JS. The research progress of diabetic rats compared with the control group. adiponectin in China and overseas. Chin J Integrat Med However, the levels of urine protein quantification, Cardio Cerebrovasc Dis 2014; 12: 234-236. 10) Kishida K, Funahashi T, Shimomura I. Adiponectin as blood glucose, HbA1c, TNF-α and IL-1β in rats of a routine clinical biomarker. Best Pract Res Clin the ADPN group with adiponectin transfection were Endocrinol Metab 2014; 28: 119-30. significantly lower than those in the DM and DP 11) Wang F, Zhang YH. Relationship of cystatin C, fibrino- groups. The expression of Akt, AMPK and eNOS in gen, and 24-hour urinary protein with renal pathological grade in children with Henoch-Sch(o)nlein purpura the blood vessels of the ADPN group was signifi- nephritis. Chin J Contemp Ped 2016; 18: 233-237. cantly higher than that in the DM and DP groups. 12) Zhao W, Katzmarzyk PT, Horswell R, Wang Y, Johnson Also, the degree of pathological changes of J, et al. HbA1c and Coronary Heart Disease Risk glomerular diameter, basement membrane, mesan- Among Diabetic Patients. Diabetes Care 2014; 37: 428. gial and renal tubules in the ADPN group were 13) Philippova M, Joshi M B, Pfaff D, Kyriakakis E, Maslova K, et al. T-cadherin attenuates insulin-depen- lower than those in the DM and DP groups. It was dent signalling, eNOS activation, and angiogenesis in suggested that ADPN can regulate blood glucose vascular endothelial cells. Cardiovasc Res 2017; 93: levels and protect the kidneys of diabetic model 498-507. rats, with a protective effect that is achieved by up- 14) Wang YS, Zhang H, Zhang XC. Effects of Jatrorrhizine on the Akt/AMPK/eNOS signalling pathways in blood regulating the expression of the Akt/AMPK/eNOS vessel of diabetes rats. Herald Med 2017; 10: 1107- signalling pathway in the blood vessels. 1111. In conclusion, ADPN can regulate the blood 15) Tian HY, Hong-Li LI, Liu YQ, Zhang CJ, Yin CG. glucose levels of diabetic model rats. By up-regu- GAB2 Promotes Migration of MCF-7 Breast Cancer lating the expression of Akt, AMPK and eNOS in Cells in Culture. Chin J Biochem & Mol Biol 2016; 32: 80-84. the blood of diabetic rats, ADPN can enhance sig- 16) Liu J, Liang G, Wang H. Research Progress of nalling of the Akt/AMPK/eNOS pathway, thereby Adenosine Monophosphate-activated Protein Kinase protecting the kidneys. and Insulin Resistance in Myocardium. Me Recapitulate 2017; 23: 1883-1887. 17) Smith BK, Steinberg GR. AMP-activated protein kinase, fatty acid metabolism, and insulin sensitivity. References Curr Opin Clin Nutr Metab Care 2017; 20: 178-180. 18) Guo J, Li J, Zhao J, Yang S, Wang L, et al. MiRNA-29c 1) Puchulu FM. Definition, Diagnosis and Classification regulates the expression of inflammatory cytokines in of Diabetes Mellitus. Diabet Med 2018; 15: 539-553. diabetic nephropathy by targeting tristetraprolin. Sci 2) Yang Y, Hong S, Sun Z. Advanced glycation end prod- Rep 2017; 7: 2314. ucts (AGEs) increase renal lipid accumulation: a patho- 19) Wei H, Zhao G, Liu H, Wang H, Ni W, et al. A simple genic factor of diabetic nephropathy (DN). Lipids and efficient method for the extraction and separation Health Dis 2017; 16: 126. of menaquinone homologs from wet biomass of 3) Gong WY, Liu FN, Hu B, Yin LH. Prevalence and cor- Flavobacterium. Bioprocess Biosyst Eng 2018; 41: relation factors of cardiovascular damage in patients 107. with diabetic nephropathy and non-diabetic nephropathy. Chin J Nephrol 2017; 33: 510-516. ______4) Xing JD. The Influence of Serum Adiponectin and Corresponding author Inflammatory Cytokones to Fat Patient's Nephropathy Han Hongling with Type 2 Diabetes Mellitus. J Shanxi Datong Univ Email: [email protected] (Nat Sci Ed) 2017; 33: 48-49. (China)