The Renal Protective Effect Of Dynepo And Serum Aluminium Level Of CRF Patients In East China

Erythropoietin (EPO), a haematopoietic growth factor produced mainly from liver in fetus and kidneys in adult, regulates red cell production. It is widely used for the treatment of anemia in a variety of clinical situations, especially for renal anemia. EPO has recently been shown to, besides its effects on hematopoiesis, exert tissue protective effects, probably by activating anti-oxidant pathways. It has been proved that EPO have neuroprotective and cardiovascular protective effects. However, the effect of EPO in kidney is not clear although kidney is the main site of EPO production and secretion. In the present study, the protective effect of Dynepo (EPOetin delta, a human cell derived EPO molecule) was investigated in primary human renal tubular (phRTE) cells and chronic renal failure (CRF) rats.Chapter One the Protective Effect of Dynepo inphRTE CellsAimMimic biological oxidative stress by adding different concentrations of glucose oxidase (GO) in primary human renal tubular epithelial (phRTE) cells. Dynepo was used to intervene the oxidative stress and possible cell death. The anti-oxidative mechanisms were explored to provide clues and theoretical basis for clinical verification of Dynepo's renal protective effect.Methods:1. Normal human kidney tissue was obtained from renal cell carcinoma nephrectomy and phRTE cells were isolated and cultured under sterile conditions. Oxidative stress was induced by adding different concentrations of GO. The cellular oxidatives stress level, cell morphology, cell mortality rate and extracelluar LDH (Lactic dehydrogenase) activity were measured at different time points (60min, 120 min, 180 min and 240 min) after induction of oxidative stress.2. Different concentrations of Dynepo (10U/ml and 100IU/ml) and Eprex (100U/ml) were incubated with phRTE cells for different time periods (0h and 24h) to intervene the intracellar oxidative stress reactions to GO. The cellular oxidatives stress level, cell mortality rate and extracelluar LDH activity were measured at different time points (60min, 120 min, 180 min and 240 min) after induction of oxidative stress.3. Soluble EPO-R was added to see whether it would block Dynepo from bingding to membraneous EPO-R to stop Dynepo's cytoprotective effect.4. The expression level and sites of EPO-R were detected by fluorescent staining method 6, 12, 18 and 24 hours after incuation with Dynepo.5. The mRNA expression of Bcl-2, HO-1, CPM, DPP4 and Cygb was measured by real-time RT-PCR 30min, 60min, 120 min, 180 min and 240 min after induction of oxidative stress in phRTE cells pre-incubated with Dynepo (100IU/mL) or not.Results:1. GO could induce time- and concentration- dependent oxidative stress in phRTE cells, and the reactive oxygen species (ROS) level in phRTE cells was positively correlated with cell mortality rate and extracellular LDH activity.2. Dynepo could lower the intracellular ROS level, reduce cell mortality rate and keep the cell membrane stability. All these cytoprotective effects of Dynepo were positively correlated with Dynepo's concentration and incubation time with phRTE cells. Eprex had the same effect as Dynepo.3. Soluble EPO-R could stop the cytoprotective effects of Dynepo.4. Dynepo could induce the expression of EPO-R. With different incubation time with Dynepo, we could observe the process of EPO-R being transported from nucleus to cytoplasm and finally expressed on cell membrane.5. Dynepo could up-regulate the mRNA expression of Bcl-2, HO-1, CPM, DPP4 and Cygb in phRTE cells after induction of oxidative stress. These up-regulation effects co-existed with reduced cellular ROS level, increased cell survival rate and cell membrane stability.Conclusions:GO could induce a time- and concentration-dependent oxidative stress in phRTE cells. Dynepo incubation could significantly reduce GO-induced intracellular reactive oxygen concentration, keep cell membranes integrity, and decrease cell morbility. These cytoprotective effects of Dynepo were probably exerted by binding to cell membraneous EPO-R and followed upregulation of Bcl-2, HO-1, CPM, DPP4 and Cygb gene expression.Chapter Two the Protective Effect of Dynepo on CRF RatsAim:To study the protective effects of Dynepo on chronic renal failure (CRF) rats and to provide more clues and theoretical basis for clinical verification of Dynepo's renal protective effect.Methods:1. Male Wistar rats were randomly divided into four groups: fake operation group: rats went through all operative procedure without taking away their kidneys; simple CRF group: 5/6 nephrectomy was performed to set up CRF models. For the first two group, no Dynepo was administrated through tail vein except 0.4ml saline water. CRF+100 IU/kg×3/week group: 5/6 nephrectomy was performed to set up CRF models. Dynepo (100 IU/kg) was administrated three times a week through the tail vein. CRF+10 IU/kg×3/week group: 5/6 nephrectomy was performed to set up CRF models. Dynepo (10 IU/kg) was administrated three times a week through the tail vein.2. Measure the rats' weight, food intake, blood pressure and observe the rats' basic conditions every week.3. Take whole blood every four weeks for detecting of renal function indicators (serum creatinine (Scr), blood urea nitrogen (BUN), calcium, phosphorus, albumin), blood routine test (white blood cells, red blood cells, platelets, hemoglobin) and serum oxidative stress level (malondialdehyde (MDA) level and glutathione (GSH) level).4. Collect 24h urine of rats for detecting of 24h urine volume, urine creatinine and calcium phosphate excretion rate.5. Observe and calculate the rat mortality rate every week.Results:1. We successfully set up the CRF models as the serum creatinine, BUN and 24h urine protein excretion rate increased dramatically while creatinine clearance rate decreased dramatically one month after the 5/6 nephrectomy.2. Nutritional state: the basic condistions and nutritional state (weight, hemoglobin level and albumin level) of rats from simple CRF group was comparatively worse than rats from Dynepo intervened groups.3. Renal function: the renal function of rats from simple CRF group kept on decreasing and 24h urine volume, 24h urine protein excretion rate kept on increasing. Dynepo administration through tail vein could lessen the increasing of 24h urine volume, 24h urine protein excretion rate. Small dose of Dynepo (10IU/kg×3/week) could slow down the decreasing process of renal function while large dose of Dynepo (100IU/kg×3/week) did not exert this renal protective effect.4. Blood pressure: the blood pressure of simple CRF rats kept on increasing one month after the 5/6 nephrectomy and small dose of Dynepo had no obvious influence on the blood pressure of CRF rats while large dose of Dynepo could significant aggravate the hypertension of CRF rats.5. Calcium and phosphorus metabolism: the urine calcium excretion rate and serum phosphorus level increased and urine phosphorus excretion rate decreased in CRF rats. Dynepo intervention has no effect on calcium metabolism, and small dose of Dynepo could improve the phosphorus metabolic disturbance while large dose of Dynepo had no effect on phosphorus metabolic disturbance in CRF rats.6. Blood routine test: Dynepo intervention has no effect on accounts of white blood cells and platelets. Small dose of Dynepo administration could slightly improve the anemia of CRF rats while large dose of Dynepo could dramatically increase the hemoglobin level in CRF rats.7. Serum oxidative stress level: the serum oxidative stress level of CRF rats was significantly higher than that of simple CRF rats. Dynepo administration could decrease the serum oxidative stress level of CRF rats and the anti-oxidative effect of Dynepo was dose-dependent.8. Mortality rate: No rat died in fake operation group at the end of the observation. Rats began to die at week 3 in CRF group and CRF+Dynepo 100IU Group. Rats in CRF+ Dynepo 10IU group began to die at week 5. Since then the mortality rate of these three groups continued to increase. At the end of the observation, more rats in CRF+ Dynepo 100IU group died than simple CRF group, while less rats in CRF+ Dynepo 10IU group died than that of simple CRF group.Conclusion:Small dosage of Dynepo has protective effects on CRF rats as it could reduce urinary protein excretion rate, slow down the process of renal function decrease, increase urinary phosphorus discharges, decrease serum oxidative stress level and overall mortality rate in CRF rats without aggravate the hypertension. We assume that small dosage of Dynepo exert its protective effect on CRF rats through decreasing oxidative stress level and protecting renal tubular epithelial cells. While large dose of Dynepo could not decrease the final mortality rate of CRF rats although it could also improve the malnutrition state and trace elements metabolism disturbance, decrease urinary protein excretion and serum oxidative stress level. The possible reason is that large dose of Dynepo administration could bring severe side effects as aggravating hypertension and increasing the blood viscidity. Aluminum (Al) is the most abundant metal elements in the earth's crust and accumulation in human body can lead to toxic effects. However, commonly present of Al in the environment and widely used in our daily life, Al put people in great danger of aluminum exposure. Many studies and epidemiological surveys home and abroad have indicated that Al accumulation in human body could cause chronic neuronal damage, and lead to mental and cognitive abilities decline in learning and memory. Many nervous system diseases are related to Al accumulation in brain such as Alzheimer's disease (AD), dialysis dementia and Parkinson's disease (PD).For people with normal renal function, the intake of aluminum comes mainly from food additives, aluminum-containing drugs and products and absorbed through the respiratory tract and intestinal tract. Apart from the basal Al taken by normal people in daily life, chronic renal failure (CRF) patients take more Al by swallowing aluminum-containing drugs and direct contact with dialysis fluid. In addition, the way to excrete aluminum from kidneys in CRF patients was gradually blocked in the natural process of renal failure. It is not difficult to understand why CRF patients take more risks of Al accumulation than normal people. Aluminum is distributed unevenly in human body with most Al stored in bones, liver and spleen and few in heart, muscle, parathyroid glands and particularly low in brain. Although the aluminum content in brain is much lower than other organs, the slight accumulation of aluminum in brain was able to cause severe toxicity. Domestic and foreign scholars have confirmed Al's toxicity in Alzheimer-type senile dementia (AD) and dialysis dementia (Dialysis Encephalopathy, DE). Aluminum concentration in bone tissue can represent the total body load of aluminum and is significantly related with aluminum toxicity. Accurate measurement of aluminum load is a necessary prerequisite for diagnosis and treatment of aluminum overload. Serum Al reflects the recent exposure and is routinely used abroad although it is not the most reliable indicators of chronic Al intoxication.Every hemodialysis centers in more developed countries as Europe and America are equipped with the detection system for regular monitoring of serum aluminum in long-term hemodialysis patients and the concentration of aluminum in dialysis water. With the generalization of double-osmosis water treatment systems in China, acute Al poisoning is rarely seen in clinical practice. The inadequate dialysis of most CRF patients due to economic reasons, low quality drinking water coupled with special eating habits in rural area exposed partial domestic CRF patients at high risk of aluminum accumulation. However, there has not been a massive-survey of serum aluminum in CRF patients in China.Aim:To evaluate serum aluminum (Al) levels in chronic renal failure (CRF) patients undergoing either conservative or hemodialysis treatment in East China, and check its possible relationships with patients' age, sex, dialysis modalities, use of phosphate binders, EPO, VitD, blood transfusion, diet and other trace elements in serum.Method:The Al concentration was measured in serum samples from 281 CRF patients not yet in dialysis (CRF-NH patients) coming from 5 hospitals of 5 cities, and 319 hemodialysis patients (CRF-H patients) recruited from 8 hospitals of 6 cities in East China. Serum taken from 262 people with normal renal function from every hospital was used as normal controls. We also measured the Al content in local tap water and dialysate samples from each dialysis center. Serum Al levels in Chinese patients were also compared with those of CRF-H patients from other countries/continents that were available in the lab from previous and ongoing monitoring programmes. Al was measured by electrothermal atomic absorption Spectrometry with automatic sampling. Meticulous care was taken to avoid contamination during processes of sampling, storage, shipment and sample analysis. Results:1. The serum Al level of CRF patients in East China was significantly higher than that of normal controls (26.64±6.73μg/L versus 1.77±0.47μg/L, p<0.001). The serum Al level of CRF-H patients was significantly higher than that of CRF-NH patients (29.21±6.38μg/L versus 23.39±7.68μg/L, p<0.001). The serum Al level of CRF-H patients in East China was significantly higher than that of European countries (p<0.001) and no significant difference was observed when compared with that of CRF-H patients in Africa.2. The serum Al level of CRF patients was significantly positively correlated with age and time period of being ill, while not significantly correlated with sex.3. Significant differences in serum Al levels were noted between different dialysis centers under study, however the Al content in dialysis fluid or local tap water could not explain these differences.4. Some treatment procedures for CRF patients were significantly related with serum Al levels: patients who had received treatment of Al-containing phosphorus binders, VitD, EPO and blood transfusion had significantly higher serum Al levels.5. The serum Al level of CRF-NH patients was significantly correlated with serum creatinine level. The serum Al level of CRF patients was significantly positively correlated with serum calcium level, and not correlated with serum strontium level.6. Food preference had significant influence on serum Al levels in CRF-H patients and no significant influence on serum Al levels in CRF-NH patients. The percentage of fruits and vegetables in daily food was negatively correlated with serum Al levels (r = -0.231, p<0.05) and the percentage of meat was positively correlated with serum Al levels in CRF-H patients (r = 0.281, p<0.01).Conclusions:1. CRF patients in East China have a relatively high serum aluminum level, and the serum Al in CRF-H patients is higher than that of CRF-NH patients. Serum Al level of CRF-H patients in East-China is higher than European countries and similar with that of African countries. 2. Medical treatments and diet preference could significantly influence serum Al level in CRF patients.3. Age, serum creatinine, calcium, period of being ill is significantly correlated with serum Al level in CRF patients.In summary: Al accumulation still exists in chronic renal failure patients in East China despite the very low Al levels found in dialysis fluid. In the absence of an adequate renal function, uptake via food or medication seems to be a significant determinant of Al accumulation resulting in higher serum Al levels.