ATRQβ-001 Vaccine Ameliorates Diabetic Renal Injury And The Mechanistic Study

Part I The effectiveness experiment of ATRQβ-001 vaccine in streptozotocin-induced diabetic nephropathyObjective:Recently, our group has developed a therapeutic hypertensive vaccine against angiotensin (Ang) Ⅱ type 1 receptor (AT1R) named ATRQβ-001. To explore the possibility of ATRQP-001 vaccine in ameliorating diabetic nephropathy (DN), the study was undertaken in Sprague Dawley rats treated with streptozotocin that developed renal injury similarities to human DN.Method:The ATR-001 peptide was covalently conjugated to Qβ-2aa VLPs using the Sulfo-SMCC cross-linker to produce the ATRQβ-001 vaccine. Experimental diabetes was induced by intraperitoneal injection of streptozocin (60 mg/kg). Sham-injected control animals (sodium citrate buffer) were followed concurrently. After 1 week, the diabetic rats were divided into 4 subgroups (each with 15 rats) for 14-week treatments with saline, olmesartan, ATRQβ-001 and Qβ VLP, respectively. Each week, rats were weighed and blood glucose levels were measured. ATR-001-specific antibody titers were detected in every two weeks. Every four weeks, systolic blood pressure (SBP) was determined by tail-cuff sphygmomanometry. Urine samples were collected using metabolic cages and the supernatant was used for examination of the 24-hour urinary protein. At the end of the experiment, the plasma samples were used for the measurement of glucose, lipids level, creatinine (Cr), blood urea nitrogen (BUN) and renal tissues for histopathology detection.Result:The rats generated high-titer antibody against ATR-001 peptide and successfully decreased SBP levels compared to VLP group, with a maximum decrease of 19.4 mmHg (129.9±2.9 versus 149.3±3.6 mmHg, P<0.01). Diabetic rats had significantly reduced body weight, and blood glucose as well as lipids level were elevated to a similar extent, irrespective of treatment. The ratio of kidney weight/body weight was increased while ATRQP-001 vaccine and olmesartan treatments decreased the level. ATRQβ-001 vaccine and olmesartan-treated rats reduced 24h urine volume and total protein excretion, blood urea nitrogen and creatinine levels compared to DN and VLP groups, and no significant differences were between these two groups. Reduced nephrin and podocin immunostaining in glomeruli were showed in diabetic rats and ATRQβ-001 vaccination decreased the decline. Additionally, glomerular mesangial expansion was also prevented detected by PAS staining and transmission electron microscopy.Conclusion:ATRQβ-001 vaccine attenuated the progression of streptozotocin-induced diabetic nephropathy. In addition to lower blood pressure, ATRQP-001 vaccination reduced biochemical parameters of renal dysfunction and ameliorated renal pathological changes, which showed similar renoprotection effect with olmesartan.Part Ⅱ The mechanistic study in renoprotection effect of ATRQβ-001 vaccineObjective:To investigate the potential mechanisms of ATRQβ-001 vaccine in ameliorating STZ-induced diabetic injury.Method:To determine whether blockade of ATIR influence the component of circulating and local renin-angiotensin system (RAS), we detected the plasma renin activity (PRA), AngⅡ and Ang(1-7) concentration as well as kidney Ang Ⅱ, Ang(1-7) concentration. PRA and Ang Ⅱ concentration were measured by radioimmunoassay (RIA) and Ang (1-7) was detected by high erformance liquid chromatography(HPLC). The RAS components were further examed by western blot and quantitative real-time PCR (qRT-PCR). The extend of interstitial expansion was quantified by Masson’s trichrome-staining. Glomerular and interstitial inflammation was quantified by the cell numbers of macrophages stained positively with CD68 antibody. Also, we detected transforming growth factor-β1(TGF-β1) expression and reactive oxygen species. The mRNA expressions of profibrotic and proinflammory factors in kidney cortex were measured by qRT-PCR.Result:Compared with olmesartan, the PRA and Ang Ⅱ concentration were not elevated in ATRQβ-001 vaccine-treated animals. Both of them decreased kidney Ang Ⅱ concentration and improved Ang (1-7) expression. ATRQβ-001 vaccination suppressed renal AngⅡ-AT1R activation and abrogated the downregulation of ACE2-Ang (1-7) with no feedback of RAS. Besides, renal fibrosis and macrophage infiltration as well as TGF-β1 expression and reactive oxygen species were also decreased by ATRQβ-001 vaccine treatment. Further, the mRNA expressions of profibrotic and proinflammory factors in kidney cortex showed similar changes as TGF-β1. In vitro, Anti-ATR-001 significantly decreased the expression of TGF-β1 and the phosphorylation levels of Smad3, as well as the downstream collagen Ⅳ and fibronectin, in rat mesangial cells (RMCs) stimulated by high glucose.Conclusion:The modulation of two major RAS axises and the inhibition of inflammation and fibrosis contribute to the renoprotction effect of ATRQβ-001 vaccine in STZ-induced diabetic injury.Part Ⅲ Safety evaluation of ATRQP-001 vaccineObjective:Safety considerations are paramount when developing any vaccine. This study was undertaken to evaluate the safety of ATRQβ-001 vaccine.Method:The ATR-001 peptide was covalently conjugated to Qβ-2aa VLPs using the Sulfo-SMCC cross-linker to produce the ATRQβ-001 vaccine. Sprague Dawley rats were immunized subcutaneously with 400μg on days 0,14 and 21. ATR-001-specific antibody titers were detected in every two weeks. Every four weeks, systolic blood pressure (SBP) was determined by tail-cuff sphygmomanometry. After 150 days, the blood samples were collected for serum complement C3a and C5a measurement. Heart, lung, kidney, liver and spleen tissues were used for hematoxylin-eosin (H&E) and CD 14, CD 19 immunohistochemistry. Renal histological changes were further observed by Masson’s trichrome staining, periodic acid-Schiff (PAS) staining and transmission electron microscopy (TEM).Result:The rats generated high-titer antibody against ATR-001 peptide and showed no effect on systolic blood pressure levels. The concentrations of serum complement C3a and C5a were similar with control rats. H&E and immunohistochemistry staining indicated there were no obvious immune-mediated tissue damages after ATRQβ-001 vaccination. Compared with control group, no obvious cell proliferation and pathological changes in the mesangial area were shown in vaccine group. Also, TEM demonstrated that no immune complexes were observed in the basement membrane, and the structure of the glomerulus was intact.Conclusion:No imumue-mediated injury was observed in vaccinated animals. ATRQβ-001 vaccine was found to be basically safe, although further assessments are needed to confirm this conclusion.