Immunologic Mechanism Of Renal Matrix In Diabetes Mellitus Rats Mediated By α₁-adrenergic Receptor Antibodies

BACKROUND:Diabetes mellitus is a complicated endocrine disease, which has become the third most serious disease that threaten peoples’health after cancer and cardiovascular and cerebrovascular disease. At present there are1.5billion patients all over the world, and it will increase4billion by2030, China will become the largest country suffering from diabetes, and has the tendency of younger morbidity. Many kinds of complications that lead by diabetes have been effected the life of the patients seriously and is also the main cause of disability and death. The organs and visceras of the patients are under different damages, especially the diabetic nephropathy has the most perniciousness. The conventional therapies include control blood glucose strictly and use angiotensin system inhibitor to control blood pressure. However, in the clinical practice, even though control the blood pressure and blood glucose with the application of the above therapies, some of the patients can’t achieve the prospective purpose of renoprotection. And it is discovered that it is not sufficient to explain the DN and diabetes vessel complications using blood dynamics disorder and hyperglycosemia. The disorder of immunologic mechanism also has an important place in the pathogenesis of its complications.It is discovered in recent years that even the blood pressure and the blood glucose of the patients with positive autoantibody are controlled within normal limits, the liability rate and pathogenesis progression of DN is far outclass the patients with negative antoantibody. Therefore, we predict that the development of DN disease is probably related with autoantibody. But the mechanism that this antibody participate the DN pathogenic caused by kidney changed is not clear. Researcheres suggest that regard DN as a inflammation caused by metabolic disorder. Eigen cells of the kidney can develop inflammatory factors such as NF-κB, OPN, TGF-β1, TNF-a in pathological state. These factors make the inflammation effect expand by autocrine and paracrine secretion, which cause the cascade reaction of inflammation. NF-κB plays the main accommodation. Activated NF-κB not only activate TGF-α TL-1、 MCP-1itself, which leads to kidney glomerulus hypertrophy, increase the filtration of kidney glomerulus, thicken the kidney glomerulus basement membrane, intercapillary cells proliferation of kidney glomerulus, sedimentation of inflammatory cells, sedimentation of EMC, etc. Activated NF-κB also induce cascade reaction to expand inflammation, by leading generating the OPN、TGF-β1inflammatory factors. OPN itself can also induce the reconstruction of kidney groundmass. For instance prophase renal parenchyma damage cause mass proteinuria filter through kidney glomerulus, entering kidney tubules induce kidney interstitial substance inflammatory infiltration by OPN. TGF-β1is acknowledged one of the most important fibration cytokine, which can promote the synthesis of ECM in many ways, refrain the degradation of ECM, leading to excess conglomeration of ECM, eventually cause tissues fibration. Under normal circumstances, NF-κB as conformation of heterodimers by P50-P65combination of Arrestin IKBa almost exist in all hyalomitome. Only P65in intranuclear hardly express maintain normal activities. While with the activation of factors such as TGF-β1PKC, P65shift to nucleus, leading to the NF-κB mass expression. Protein Kinase C (PKC) is the downstream acceptor as the signal transduction element of G-protein-coupled-receptor (GPCR). a1-Adrenergic receptor antibodies (a1-R Ab) is the antibody of GPCR.GPCR is a membranes surfaces glucoprotein link-coupled with GTP. It is a transmembrane subunit composed by7polypeptide chains, form a space conformation of three cell exterior and cell endocyclic. The a1-R Ab is IgM or IgG immunoglobulin, mainly aim at amino acid sequence of a1receptor the second dipeptide1922218. It includes acceptor AT1、a1、β1、M2, etc, referring the largest family of signal transduction membrane receptor. The signals they mediated refer to the kidney functions and immune responses. When acceptors are stimulated again and again, they will produce autoantibody by internalization mechanism. The autoantibody of acceptor possess pathological activation, causing pathological effect by corresponding receptors, which leads to immunereaction itself, and cause corresponding tissue damage. It is discovered that the autoantibody of GPCR can also simulate normal physiological signals (Angiotensin Ⅱ, adrenalin), activate corresponding GPCR, have the semblable affects with Angiotensin Ⅱ, Adrenalin. Some people think that among the patients who is a1-R Ab positive,2/3of the patient are aimed at the first peptide outside the cells,1/3of the patients are aimed at the third peptide. This receptor antibody combines with acceptor can activate the signal channel mediated by GPCR, such as the a1-R Ab→GPCR→Diacyl Glycerol(DAG)→PKC→NF-KB→TGF-β1or the a1-R Ab→GPCR→DAG→PKC→NF-kB→OPN→TGF-β1, regulate the responses controlled by cell multiplication, cell differentiation and metabolism. Therefore, we design this experiment, mainline the ai-R Ab to the tail of the mouse, study after mediating the receptor antibody, by activating GPCR and sensitizing NF-kB, eventually activating both TGF-β1and OPN inflammatory factors make the inflammation effect expand unceasingly, which cause the cascade reaction of inflammation. It is not reported that immunologic mechanism linked with inflammatory factors lead to the reconstruction of the renal groundmass and blocking Agents interfere of doxazosin in the development of DN. Diabetic rats were modelinged successively in these experiments, and to investigate the effects of the a1-R Ab on the expression of TGF-β1and OPN and the effect of doxazosin in diabetic wistar rats and its mechanism of tubulointerstitial lesions were to be studied. So it is meaningful to explore the relationships among the development of DN, the a1-R Ab, TGF-β1, OPN and doxazosin in DM patients which high of expression of Autoantibodies.Chart I The effects of a1-adrenergic receptor autoantibodies on the expression of TGF-β1in renal cortex of DM ratsObject:To investigate the effects of a1-adrenergic receptor autoantibodies (ai-R Ab) on the expression of transforming growth factor-β1(TGF-β1) in diabetic wistar rats and its mechanism of renal cortex lesions were to be studied.Methods:1. High fat feed production:after mixing10%lard,10%sucrose,2.5%cholesterol,0.4%Sodium Cholate and77%basis forage, compressing it.2. DM rat model’s preparation and grouping laboratory animals:Wistar rats were feeding adaptability by a week, weigh rats, deliver into the group of healthy rats blank control group, healthy rats a1-R Ab-mediated group, DM rats non-mediated group, DM rats a1-R Ab-mediated group. DM rats were fed with high glucose and high fat food by four weeks, fasting12h before making model. Peritoneal injection is under well configuration of sterile Citric acid-streptozotocin (STZ) mixed liquor by the weight of0.5mL/100g [use0.1mol/L sodium citrate buffer solution (pH=4.3) to match the concentration of20g/L before using], getting blood from rats tail vein to measure fasting blood glucose after72h, it’s success of making model by measuring fasting blood glucose>16.7mmol/L. After the success of making model, the healthy rats a1-R Ab-mediated group and DM rats a1-R Ab-mediated group are syringe ai-R Ab by rats’tails vein at0,4,8,12,16week,100μg/100g by weight. After the DM rats were successful of making model, change into normal feeding till the end of the experiment. The control groups were feeding completely by general feeding. During the experiment, Keep room temperature18-20℃, humidity69%, lighting in turn by12h, the rats drinking and eating freely.3. Observation and detection:(1) Effects on the general conditions:activity, pelage, ingestion, drinking, urination and defecation, the body weight (BW).(2) Effects on24-hour urine protein (Radioimmunoassay) and SCr (picric acid method).4. Oberveing the distributions of TGF-β1on normal and DM rats with immunohistochemical method.5. Oberveing the change of proximal convoluted tubule, distal convoluted tubule and aglomerular on normal and DN rats with electronic microscope.Results:1.32rats were used to established the DM models and the rate of successful models was100%(32/32). And the death rate of DM model rats was zero(0/32).2. After mediating in16weeks, healthy rats’weight is increased gradually than before (P=0.000), DM rats’weight is significantly less than before (P=0.000) 3. After mediating in16weeks, the level of24hUpro increased gradually (P=0.011or P=0.000). The level of SCr increased gradually in DM rats (P=0.000) and there is no significant changes in healthy rats (P=0.383).4. The expression level of TGF-β1was evaluated by PU and average luminosity. The TGF-β1was exanmined in renal cortex in both healthy rats group and DM rats group mediated by ai-R Ab and DM rats group was significantly higher than healthy rats group (P=0.000or P=0.001or P=0.006or P=0.010)5. Results in electron microscope:Anormaly had not been seen in the healthy blank control group. Anormaly slightly had been seen in the healthy group mediated by a1-R Ab. Anormaly had been seen in the non-mediated DM rats group. Serious destruction had been seen in the DM rats group mediated by a1-R Ab.Conclusion:The expression of TGF-β1in DM rats which mediated by al-R Ab is increased than in healthy rats group. The expression of TGF-β1is related to immunity reaction.Chart Ⅱ The effects of a1-R Ab on the expression of OPN in renal cortex of DM ratsObject:To investigate the effects of a1-R Ab on the expression of OPN in diabetic wistar rats and its mechanism of renal cortex lesions were to be studied.Methods:The same with above.Results:The expression level of OPN was evaluated by PU and average luminosity. The OPN was exanmined in renal cortex in both healthy rats group and DM rats group mediated by al-R Ab and DM rats group was significantly higher than healthy rats group(P=0.000). The remaining results same with above.Conclusions:The expression of OPN in DM rats which mediated by a1-R Ab is increased than in healthy rats group. The expression of OPN is related to immunity reaction.Chart III The effects of a1-R Ab on the expression of TGF-β1inhibitted by doxazosin in renal cortex of DM ratsObject:To investigate the effects of a1-R Ab on the expression of TGF-β1inhibitted by doxazosin in diabetic wistar rats and its mechanism of renal cortex lesions were to be studied. In order to illustrate the a1-R Ab whether can reduce the expressing of the desmoplastic factor TGF-β1and to lessen the remodeling renal matrix, thus topostpone the generate of DN. Methods:1. High fat feed production:The same with above.2. Production of intragastric administration drug:doxazosin (4mg/slice), dispensing the drug according to0.36mg/kg dosage and administer10ml/kg per rat capacity after grinding into powder.3. DM rat model’s preparation and grouping laboratory animals:Wistar rats were feeding adaptability by a week, feeding high glucose and high fat food by four weeks, fasting12h before making model. Peritoneal injection is under well configuration of sterile Citric acid-streptozotocin (STZ) mixed liquor by the weight of0.5mL/100g [use0.1mol/L sodium citrate buffer solution (pH=4.3) to match the concentration of 20g/L before using], getting blood from rats tail vein to measure fasting blood glucose after72h, it’s success of making model by measuring fasting blood glucose>16.7mmol/L. After the success of making model, weigh rats, deliver into the group of DM rats non-mediated group, DM rats a1-R Ab-mediated group, DM rats both a1-R Ab-mediated and doxazosin intervention group and DM rats doxazosin intervention group. The DM rats a1-R Ab-mediated group, the DM rats both a1-R Ab-mediated and doxazosin intervention group are are syringe a1-R Ab by rats’tails vein at0,4,8,12,16week,100μg/100g by weight; The DM rats both a1-R Ab-mediated and the doxazosin intervention group and DM rats doxazosin intervention group are giving0.36mg/kg dosage,10ml/kg per rat capacity through intragastric administration. The DM rats none mediated group and the DM rats a1-R Ab-mediated group are giving the corresponding volume of distilled water,1/d, successive16weeks. After the DM rats were successful of making model, change into normal feeding till the end of the experiment. The control groups were feeding completely by general feeding. During the experiment, keep room temperature18-20℃, humidity69%, lighting in turn by12h, the rats drinking and eating freely.4. Observation and detection:The same with above.Results:1.48rats were used to established the DM models and the rate of successful models was100%(48/48). And the death rate of DM model rats was zero (0/48).2. After mediating in16weeks, DM rats’weight were significantly lower than before (P=0.000).3. After mediating in16weeks, the level of24hUpro and SCr in a1-R Ab-mediated group is higher than non-mediated DM rats(P<0.01). The level of24hUpro and SCr in the DM rats both a1-R Ab-mediated and doxazosin intervention group is obviously lower than the DM rats a1-R Ab-mediated group(P<0.01). The level of24hUpro in DM rats doxazosin intervention group is lower than the non-mediated DM rats (P<0.01) and there was no changes in the level of SCr(P=0.249).4. The expression level of TGF-β1was evaluated by PU and average luminosity. The TGF-β1was exanmined in renal cortex in both α1-R Ab-mediated group and α1-R Ab non-mediated group. The expression of TGF-β1in a1-R Ab-mediated groups is significantly higher than a1-R Ab non-mediated groups (P<0.01). Both a1-R Ab-mediated and doxazosin intervention group is significantly lower than a1-R Ab-mediated group (P<0.01). Doxazosin intervention group is significantly lower thana1-R Ab non-mediated group (P<0.01).5. Results in electron microscope:Anormaly slightly had been seen in the non-mediated DM rats. Serious destruction had been seen in the DM rats a1-R Ab-mediated group, and it had been taken a turn for the better in the DM rats both a1-R Ab-mediated and doxazosin intervention group. Anormaly had been seen in the DM rats intervented by doxazosin.Conclusions:The expression of TGF-β1is related to autoimmune.The expression of TGF-β1was inhibitted by doxazosin in a1-R Ab-mediated DM rats.