Effects Of RHuEPO On High Glucose Induced Mesangial Cells And Relationship Between Hepcidin And RHuEPO Resistance In Hemodialysis Patients

Background and objectiveDiabetic nephropathy(DN) is one of the most common chronic complications of diabetes mellitus and the major cause of end-stage renal disease(ESRD). It is widely accepted for the time being that therapeutic principles for DN are as follows:the control of hyperglycemia, blood pressure and diet as well as renal function protection. Renal function would decline quickly and progress into ESRD when clinical proteinuria appears in diabetic patients. Thus the expenditure on dialysis and kidney transplantation increases every year and causes heavy economical burdens on the patients and society. It is considered traditionally that an interaction of metabolic and hemodynamic factors involved in the development of DN. However, the recent study has demonstrated that inflammation is an important cause in chronic progressive renal disease in diabetic patients. Mesangial cell (MCs) proliferation and extracellular matrix accumulation are the main causes of diabetic nephropathy. MCs secrete cytokines and extracellular matrix, and induces glomerulosclerosis. So studying MCs proliferation and secretion is important in the therapy of proliferating glomerular disease.Erythropoietin is a glycoprotein hormone produced in adults primarily in the kidneys. It was originally understood to be an erythropoietic stimulator, regulating the differentiation and maturation of erythroid progenitor cells and preventing apoptosis of developing erythroid progenitor cells. The recombinant forms of the hormone have been used to effectively treat anemia in many diseases such as chronic inflammation, cancer especially renal insufficiency. The hormone's effects are signaled through the erythropoietin receptor and increasingly understood transduction intermediates. Recently studies have suggested that erythropoietin could act as tissue cell and organ protective molecule. The erythropoietin receptor has been found not only on hematopoietic tissues but also on many none-hematopoietic tissues. It is well established that EPOR has been found in kidney tissue, including mesangial and renal tubular cells and endothelial cell. Many studies confirmed that EPO plays a significant renoprotective when administered to animal models with acute renal injury, ischemia reperfusion injury and cisplatin-induced kidney damage. Further, some studies showed that EPO may have protective effects for chronic kidney disease such as DN, but whether EPO can affect MCs under high glucose is still unclear.In the present study, by cck-8, real time RT-PCR, Western blot and FCM, we investigated the potential effect of recombinant human erythropoietin on high glucose induced renal mesangial cell damage, the morphological change of renal mesangial cell, inflammation marks, apoptosis index and expression of Bcl-2and Caspase-3protein expression.Methods1. Rat MCs were cultured with the medium which contains of normal glucose (NC), different concentrations of high glucose and30mmol/L high glucose (HG) with1,10,50,100,1000IU/ml of rHuEPO (E1-E5). Cell proliferation was tested with CCK-8and observed by spectrometry24,48and72h later.2. The concentration of MCP-1and TGF-β1in supernatant were measured by ELISA. The mRNA expressions of MCP-1and TGF-β1in cells were determined by real-time RT-PCR.3. MCs apoptosis index were measured by flow cytometry with fluorescene-activated cell sorter analysis. 4. Apoptosis associated gene Bcl-2and Caspase-3expressions in MCs were detected by Western blot.Results1. Glucose and rHuEPO on proliferation of MCsThe MCs proliferation presented a time-and dose-dependent feature under30mmol/L glucose, and got to the highest point at the concentration of30mmol/L for72hours.40mmol/L and50mmol/L glucose could inhibit the proliferation of MCs. RHuEPO (I,10,50,100IU/ml) could inhibit the high glucose induced proliferation in a time-and dose-dependent feature. RHuEPO of1000IU/ml could have cytotoxicity on high glucose induced MCs.2. MCP-1,TGF-β1protein and mRNA expression in MCsThere had low level MCP-1and TGF-β1protein and mRNA expression in normal MCs. High glucose could stimulate the synthesis of MCP-1and TGF-β1protein and mRNA. Different concentrations from1-100IU/ml of rHuEPO could inhibit MCP-1and TGF-β1protein and mRNA expression significantly.3. Apoptosis in MCsThe percentage of apoptotic MCs cell stimulated by30mmol/L glucose significantly increased compared with control. When the cells were incubated with30mmol/L glucose and rHuEPO (1,10,50,100IU/ml) for24,48,72h, the percentage of apoptotic MCs cell decreased significantly.4. High glucose (30mmol/L) significantly decreased Bcl-2mRNA and protein expression and increased Caspase-3protein.The presence of rHuEPO, dose (1,10,50,100IU/ml) dependently prevented high glucose induced decreases in Bcl-2and increases in Caspase-3.Conclusion1. High concentrations of glucose can stimulate MCs proliferation with a time-and dose-dependent feature. Large dose of rHuEPO (1000IU/ml) has cytotoxic effect on high glucose induced MCs.(1-100IU/ml) rHuEPO can promote high glucose induced proliferation of MCs also in time-and dose-dependent way. 2. High glucose contributes to the upregulation of MCP-1and TGF-βprotein and mRNA in rat MCs.1-100IU/ml rHuEPO can inhibit expressions of MCP-1and TGF-Pprotein and mRNA in high glucose induced MCs. These findings provide experimental evidence for further study of the possibly protective effect of rHuEPO against DN.3. High glucose can promote apoptosis in MCs. RHuEPO can suppress apoptosis of high glucose induced MCs. The effects of rHuEPO on MC apoptosis perhaps correlate with upregulation of Bcl-2and downregulation of Caspase-3. BackgroundAnemia is among the most frequent complications associated with HD. Anemia can lead to a reduction in tissue oxygenation, increase in cardiac output, left ventricular hypertrophy, congestive heart disease, fatigue, reduction in exercise capacity and immunodeficiency, and eventually a higher mortality. The incidence of renal anemia has reduced significiantly in maintenance hemodialysis (MHD) patients due to rHuEPO use. Many patients can be treated successfully with recombinant human erythropoietin (rHuEPO). However, the responses to EPO varies among individual patients. According to the literature,90-95%of renal anemia responded in a dose-dependent manner to EPO, whereas the remaining5-10%of patients had a blunted or no response to EPO, despite high-dose therapy. These patients are rHuEPO-resistant. Various factors have been reported to be related to this hypo-responsiveness including iron deficiency, inflammation, infection, blood loss, hyperparathyroidism, aluminum toxicity, and vitamin B12and folate deficiencis. The main cause of resistance is iron deficiency and inflammation status, but rHuEPO-resistant anemia persists in some HD patients after exclusion of these factors.A recent study showed that a newly discovered mediator of innate immunity, hepcidin, is a key regulator of iron homeostasis. Hepcidin is a defensin-like peptide of25amino acids that is produced mainly in liver cells and can be detected in human urine and ultrafiltrates. Hepcidin production is enhanced by iron overload, inflammation and IL-6, and suppressed by anemia, hypoxemia and rHuEPO treatment. Hepcidin binds to ferroportin, a cellular iron exporter that is highly expressed in duodenal and liver cells and macrophages. Therefore, hepcidin suppresses extracellular release of iron by decreasing ferroportin. It has been proposed that hepcidin is a primary factor in the pathogenesis of anemia of chronic diseases (ACD).It has been reported that cytokine-induced inflammation suppressed bone marrow erythropoiesis in HD patients and is a possible cause of anemia. Panichi et al. observed a higher level of CRP in HD patients. It is therefore suggested that HD patients have a chronic inflammatory status and ACD may be one of the causes of rHuEPO-resistant anemia in these patients. Hepcidin synthesis is regulated by inflammation in HD patients, being enhance in non-responders compared with responders. Moreover, increased levels of prohepcidin have been reported in HD patients.However, the relationship between rHuEPO-resistant anemia, inflammation and hepcidin in HD patients has not been studied. In the present study, we investigated whether prohepcidin is involved in rHuEPO-resistant anemia in HD patients.Objective1. To determine serum pro-hepcidin in rHuEPO resistant anemia, ESRJD patients and healthy controls.2. To analyze the relationship between rHuEPO resistant anemia,inflammation markers and pro-hepcidin.3. To predict the value of pro-hepcidin in rHuEPO resistant anemia by ROC curve.Methods1. Forty HD patients and twenty healthy controls were enrolled in the study. Among HD patients,20were hyporesponsive to rHuEPO therapy and20were normal responsive to rHuEPO therapy.2. Blood sample collection was performed in all HD patients immediately before the start of HD. Blood sample collection was performed in healthy controls on an empty stomach.3. Complete blood red cell count, Hb concentration, hematocrit and serum ferritin (SF), Serum iron (SI), total ironbinding capacity (TIBC), saturation rate of transferrin (TSAT), transferrin (TF), hyper-sensitive C-reactive protein(hs-CRP), pro-hepcidin were measured in all patients and controls.Hematometry including Red blood cell(RBC), hematocrit, Hb concentration, hematological indices were performed with a Sysmex2100automatic analyzer (Japan) and biochemical analyses were performed with a Hitachi7060automatic analyzer (Tokyo, Japan), Post-dialysis blood urea nitrogen (BUN) levels were measured to calculate the delivered dialysis dose (Kt/V) using the Daugirdas method. TSAT was calculated as [(serum iron/TIBC)×100].Plasma concentrations of pro-hepcidin were determined using an enzyme-linked immunosorbent assay. The plasma concentrations of high sensitive CRP (hs-CRP) was determined using turbidimetry, according to the procedure recommended by the manufacturer. Serum pro-hepcidin and hs-CRP were measured using a Pro-hepcidin ELISA kit (Competition ELISA; DRG International Inc, Germany) and Bekman automatic chemical analyzer respectively. Serum ferritin, transferrin concentration and total iron binding capacity were determined using Bekmen AccEss automatic immune analyzer according to the procedure recommended by the manufacturer. Serum iron concentration was mearsured using colorimetric method.ResultsSerum levels of SF, pro-hepcidin and hs-CRP were higher in HD patients than in controls. While Serum TF was lower in HD patients than in controls. Serum levels of SF, pro-hepcidin and hs-CRP were high in rHuEPO resistant patient. Serum pro-hepcidin levels correlated positively with SF and hs-CRP in HD patients and HD1patients.ConclusionA close interaction exists between inflammation, iron status and prohepcidin serum levels that ultimately regulate intracellular iron availability. Prohepcidin and SF, together with hs-CRP, may prove to be good markers of resistance to rHuEPO therapy in HD patients.