The Study Of Erythropoietin Receptor And Its Signaling Pathway In The Pathogenesis Of Renal Anemia

Renal anemia is secondary which occurs when the glomerular rate(GFR) falls below 30mL/min, roughly corresponding to a serum creatinine above 300μmol/L.It is one of the complications of CRF.The main cause of renal anemia is the deficency of EPO synthesis.However,the serum level of EPO in CRF patients is much higher than the normal.The high level of EPO,but the patients are anemia.What is the cause?Why do we use hrEPO in clinic for renal anemia patients?EPO is the regulatory factor of erythrocyte development. It can promote the erythrocyte series of bone marrow proliferation and differentiation by combination with the EPOR on the surface of erythrocyte. It also suppress the series apoptosis by delay the DNA division. The erythrocyte progenitor reach the summit in the CFU-E phase, about 1000EPOR/per cell. With the maturation process, the EPOR is decreasing and disappear in reticulocyte.EPO binding to the receptor changes the conformation of the EPO receptor (erythropoietin receptor, EPOR), which is necessary for JAK2 activation by a mechanism of self dimerization. Since the EPO-R, like other hematopoietic cytokine receptors, lacks intrinsic enzymatic activity, the receptor must associate with and activate protein tyrosine kinases in order to transmit a signal. EPO-induced intracellular signaling occurs through a rapid tyrosine phosphorylation of several proteins even though the EPO receptor does not possess endogenous tyrosine kinase activity. First, intracellular signaling occurs by activating the JAK2 tyrosine kinase, which is associated with the EPO receptor constitutively. JAK2 is associated with the EPO receptor in the transmembrane region. After EPO activates the receptor, eight tyrosine residues in the cytoplasmic domain of the EPO receptor are phosphorylated .Now EPOR structure and its function have been popular accepted, but most works are focus on some normal tissues and cell.There is little resear on the CRF patients.We are interesting whether there is EPOR and its signal pathway changes in bone marrow cell in CRF patiens.In our research, we discussed the tissue-specific EPOR expression.We use the CRF model of mice according to the international standard of 5/6 nephrectomy.The biochemical parameters were obviously increased after 12 weeks and the count of erythrocyte and Hb were decreased.There was significance between the normal , the persudo-operation group and the operation group.The more severe deficiency of renal function,the more severe of anemia could be found.On the basis of the former study,our research was divided into four parts.The serum level of EPO in CRF model was studied by radioimmunology. We found that the EPO level is much higher than the normal and persudo-operation group and there was significance. But there is no direct relation between the EPO level and anemia. To further exploer the mechanism of renal anemia, we observed the EPO mRNA level and EPO expression by RT-PCR and immunochemistry. We found there were no changes between the normal and the residul renal tissue. But we found the more expression in liver and suspected that liver maybe the main organ of EPO in CRF model.In the study of EPOR, we observed the affinity and count of EPOR by FACS and 125-I combination test. The results showed that the number of EPOR was decreased and negative relation with the renal function, right relation with the hemoglobin. We concluded that the severer of renal function, the severe of anemia. So improve the renal function is the main effect mesure. Our experiments also showed that the affinity of EPOR was much lower than the normal and persudo-operation group. There was significance. The results confirmed that the body took effect by strengthen the affinity of EPOR in face of the decreased number of EPOR and EPO level. We also observed the RNA and protein changes of EPOR. The signal transduction protein, Jak2 and Stat5 also were studied. The mRNA level and expression of EPOR were decreased. The phosphorylation of Jak2 and Stat5 was decreased. The results showed that the signal pathway was weakened.We also observed the erythrocyte series proliferation and apoptosis in CRF model. The number of BFU-E, CFU-E were decreased compared with the normal group in the same level of EPO.3-H combination test and flow cytometry confirmed that the proliferation weakened and apoptosis increased.On the basis of former study, we also observed the EPOR expression in renal, liver, brain, heart and lung. he results showed that the increasing expression could be found when CRF. Whether the phenomenon is reactive or the protective function in local region should be furthered studied. The relationship between the encephorypathy ,myopahy and lung injure should be explored in later. The former research set foundation for studying the EPOR in non-hematopoiesis organ.From our works we can conclude: 1. EPO is probably produced by the liver in CRF. 2. The number of EPOR was decreased and affinity ability improved of renal anemia. 3. The expression of EPO mRNA was increased liver in CRF. 4.Jak2-Stat5 pathway weakened in CRF. 5.The proliferation of erythrocyte decreased, especially in low level of EPO condition.6.The apoptosis of erythrocyte series of bone marrow increased in CRF condition.We first explored the EPOR and Jak2-Stat5 pathway in renal anemia, and observed the site of EPO synthesis ,the affinity of EPO and EPOR, the pathway involved in the process by molecular technichs. The research made a foundation for the renal anemia treatment. There is no similar report now.