Renal Protection Of Erythropoietin In Diabetic Rats

With the improvement of living standards and changes in the structure of the daily diet, the incidence of diabetes has been increasing in our country. Meanwhile, as a common microvascular complications of diabetes, diabetic kidney disease(DKD) has become one of the main cause of ESRD. The incidence of DKD mainly caused by renal hemodynamic changes, oxidative stress and abnormal expression of relative cytokines, which induced by high blood sugar in diabetes. Now, the pathogenesis of DKD has not known well, and there is also no effective therapy to DKD. Once patients enter the stage of clinical DKD, the condition is irreversible development. Currently, there are many studies focus on the role of oxidative stress on DKD. Hyperglycemia in diabetes can lead to excessive production of ROS in tubular epithelial cells, mesangial cells, capillary endothelial cells and other cells, and reduce antioxidant capacity of the body, resulting increased level of oxidative stress. With the reactive chemical characteristics of ROS, they can made damage directly to macromolecules such as DNA, proteins, lipids and carbohydrates, etc. In addition, ROS can also activate a series of cellular stress sensitivity pathway leading to cell damage when served as signaling molecules. Some studies also confirmed that positive blood glucose control and ARB can play a role in the treatment of DKD, at least partly through their anti-oxidative stress effect.Erythropoietin, a glycoprotein hormone secreted by type I mesenchymal cells in renal cortex, stimulate erythropoiesis through combination of its specific erythropoietin receptor. However, recent studies show that some non-hematopoietic tissue of the body also express erythropoietin receptor, such as neurons, gastric epithelial cells, leydig cells and kidney mesangial cells, etc. Further studies confirmed that erythropoietin also play a physiological role to these tissue cells with no hematological effects. It is interesting, type I mesenchymal cells as the mainly erythropoietin secreting cells in kidney contact directly with proximal and distal renal tubular epithelial cells in the basal aspect. Because of the anatomical relationship between renal cells to I mesenchymal cells, We have reasons to believe that erythropoietin is more conducive to play the role of the endocrine and paracrine in kidney. So, we detect whether rat proximal tubular epithelial cells express erythropoietin receptor, and whether erythropoietin have renal protective effect on diabetic rat model in this experiment. In vitro, we found that rat renal proximal tubular epithelial cells express erythropoietin receptor, and high glucose can increase the expression of erythropoietin receptor. Further study confirmed that high glucose can elevate the levels of oxidative stress in renal tubular epithelial cells and induce apoptosis. However, erythropoietin can alleviate the oxidative stress induced by high glucose, and inhibits renal tubular epithelial cells apoptosis. Then we study the protective effect of erythropoietin on the kidney of diabetic rats induced by streptozotocin. The results show that renal tubular cells, mesangial cells and other kidney cells express erythropoietin receptor, but the expression of erythropoietin receptor was no difference between diabetic group and normal control group. This is inconsistent with the results in vitro, considering the possibility of the complex physiological environment in vivo. Further experiments confirmed that diabetic rats showed an elevated level of oxidative stress, TGF-β1and FN protein in the kidney. And low doses of erythropoietin can reduce the level of oxidative stress, inhibit the expression of TGF-β1and FN protein, alleviate pathological and functional damages of the kidney in diabetic rats.In summary, as one kind of glycoprotein hormones primarily secreted by type I mesenchymal cells in renal cortex, erythropoietin can inhibit high glucose-induced oxidative stress and apoptosis of tubular cells, and erythropoietin play a kidney protective effect through inhibition of oxidative stress and TGF-β1and FN protein expression in streptozotocin-induced diabetic rats.This increases our traditional understanding of the physiological role of erythropoietin, but also give us new ideas for the treatment of DKD. However, we must recognize that animal experiments can not completely simulate complex human environment. In particular, erythropoietin may cause hypertension, promote tumor growth and other adverse effect. So, the renal protective effect of erythropoietin has yet to be confirmed by a large number of clinical trials. Part1. The expression of erythropoietin receptor in renal tubular epithelial cellsObjective To detect the expression of erythropoietin receptor in rat renal proximal tubular epithelial cells, and to study the effects of high glucose on the expression of erythropoietin receptor.Methods Subcultured rat renal proximal tubular epithelial cells (NRK-52E), divided into normal control group (NC group), osmolarity control group (OC group), high glucose group (HG group). We detect the expression of erythropoietin receptor through immunofluorescence in NRK-52E cells. And detect the effects of high glucose on the mRNA and protein expression of erythropoietin receptor through RT-PCR and western blot.Results Immunofluorescence show that the expression of erythropoietin receptor was detected in the membrane and cytoplasm of renal tubular epithelial cells. RT-PCR and western blot found that the mRNA and protein expression of erythropoietin receptor was increased by high glucose.Conclusion Rat proximal tubular epithelial cells express erythropoietin receptor, and high glucose can increase the expression of erythropoietin receptor in renal tubular epithelial cells. Part2. Effects of erythropoietin on renal tubular cells apoptosis induced by high glucoseObjective To investigate whether erythropoietin can inhabit the pro-apoptotic effect of high glucose on rat proximal tubular epithelial cells, and the possible mechanisms through which erythropoietin exerts its anti-apoptotic role. Methods Rat proximal tubular epithelial cells (NRK-52E) were divided into5groups: normal control group, osmolarity control group, high glucose group, high glucose with erythropoietin (50U/ml) group and high glucose with erythropoietin (100U/ml) group. The rate of apoptosis was evaluated by flow cytometry Annexin V-FITC/PI double stains. The intracellular ROS was detected using fluorescent probe CM-H2DCFDA. The bcl-2/bax and Caspase-3mRNA expressions were examined by reverse transcription-polymerase chain reaction (RT-PCR). The bcl-2/bax protein expression were examined by western blot.Caspase-3activity was detected by Capases-3activity assay kit.Results High glucose induced NRK-52E cells apoptosis, and mannitol with the same osmotic concentration of high glucose did not significantly induce apoptosis. Early and late apoptosis rate of E1and E2groups was significantly lower than the HG group [E1(5.7%±0.8%)/(2.9%±0.5%), E2group (3.5%±0.7%)/(1.5%±0.3%) than the HG group (7.9%±1.0%/(5.5%±0.6%), P<0.05]. High glucose increased intracellular ROS production of NRK-52E cells. EPO can reduce the productions of ROS induced by high glucose. High glucose reduced the expression of bcl-2mRNA, increased the expression of Caspase-3and bax mRNA, enhanced Caspase-3activity in NRK-52E cells. EPO can inhibit these effects induced by high glucose.Conclusion Erythropoietin inhabits NRK-52E cells apoptosis induced by high glucose through attenuate oxidative stress,up-regulate the expression of bcl-2mRNA and down-regulate the expression of bax and Caspase-3mRNA, reduce Caspase-3activity. Part3. Renal protection of erythropoietin and its mechanism in diabetic ratsObjective To investigate the renoprotective effect of erythropoietin in streptozotocin-induced diabetic rats and explore the possible mechanism.Methods The SD rats were randomly divided into there groups as follows: normal controlrats, diabetic rats, diabetic rats treated with erythropoietin. The rats were sacrificed after8weeks treatment. Renal morphology was observed by light microscopy. The expression of erythropoietin receptor in kidney was detected by immunofluorescence and western blot. The expression of P47phox, TGF-β1and FN protein in kidney was detected by immunohistochemistry and western blot. And the activity of antioxidants including T-AOC, SOD, GSH-Px and the level of MDA in kidney were also measured.Results Erythropoietin treatment notably attenuated renal pathologic and functional changes. The expression of EPOR was detected in kidney, but there was no difference between every groups (P>0.05). Compared with normal rats, diabetic rats showed an elevated expression of P47phox,TGF-β1, FN proteins and MDA levels in kidney as well as reduced activities of SOD, GSH-Px and T-AOC (P<0.01). Compared with diabetic rats, erythropoietin could decrease the protein expression of P47phox,TGF-β1and FN in kidney (P<0.05, P<0.01). Meanwhile, elevated MDA levels in the kidney were decreased as well as decreased SOD, GSH-Px, T-AOC activities were significantly remitted in treatment group (P<0.01).Conclusion Our results suggest that erythropoietin can ameliorate renal damage via the inhibition of oxidative stress and TGF-β1and FN protein expression in streptozotocin-induced diabetic rats.