| Research Background:Deterioration of kidney disease progress more development for end-stage renal disease (ESRD), namely the end stage of chronic renal failure, uremia, treatments for it can only rely on hemodialysis, peritoneal dialysis and renal transplantation. Treatment cost is higher, the quality of life is lower. Renal source shortage becomes the bottleneck of renal transplantation, anti-rejection drug taking results in the decrease of immunity, easily complicated with infection. And patients with advanced renal failure often with a variety of complications. H igher incidence of complications of cardiovascular system is the first fatal complications. To prevention and control the death event in the patients with advanced renal failure greatly increase the difficulty and treatment costs. Most of all ended in death, seriously threat to renal failure patient’s life, reduce their quality of life. Late at present a lot of research on chronic renal failure prevention and treatment of cardiovascular complications, including research on these patients’aortic fatty deposits and blood fat of spectral density change is more, but the effect is low, the cost is higher. We should focus on the prevention and treatment of cardiovascular complications in patients with early-stage renal failure. Its’ effect is better than late and low cost. But such research is rare. The ideas of more present research are less clear. The comprehensive research is less. They only discuss its preliminary mechanism, which is not enough. There are a few studies reported on the decorin gene therapy in rats with chronic renal failure has certain curative effect, but no further study on the mechanism of concrete in addition to kidney failure of kidney damage. Whether are there certain influence to the cardiovascular complications, has not been further in-depth study.Therefore, this study envisaged, will the early renal failure rats lipid deposition and spectral density change in the blood lipid, the incidence of complications of cardiovascular system development, and decorin gene therapy for renal failure, delay glomerular sclerosis, renal interstitial fibrosis and slow the progression of renal failure, which affects its spectral density change in fat deposition, blood lipid, slow progress in cardiovascular complications, so as to find out early to prevent kidney failure and cardiovascular complications. To explore the new effective means and way of development and deterioration.Objective:This study was to investigate the aortic lipid deposits and blood lipid spectral concentration change and its mechanism of cardiovascular complications in rat with early stage renal failure. It was to explore the efficacy of treating chronic renal failure by decorin gene therapy at the molecular level, and whether or not they can control concurrent lipid metabolic disorders, cardiovascular complications. It was also to explore the possible mechanism.Methods:1. Experimental animals and groupA total of 186 adult SD rats (96 male,90, female)were purchased from Shandong university laboratory animal center, weight about 150g-200g.Under aseptic conditions using two-step,5/6 renal resection surgery, renal failure model in rats was manufactured. The sham operation rats were only with incision and suture on abdominal wall. Their kidneys were kept. 168 rats were used in two experiments. The other remanent 18 rats were ready for supplementary use when some rats were dead accidentally. They were made to corresponding animal model in the same experimental condition.2. Test of relevant indexes2.1 The first part of the experiment.2.1.1 Experimental groupThe experimental group, the real two-step 5/6 renal resection surgery made renal failure rats model, n= 24.The control group only accepted open surgical incision and then suture of abdominal skin, but their kidneys were kept, n= 24.Six rats were sacrificed on the 0, the 10th, the 30th and the 60th day respectively. Aortic arch were resected to test the lipid deposition. Blood was sampled only and no rats were sacrificed on the 40th day.2.1.2 The dynamic changes of plasma lipid and lipid in erythrocyte membrane and aortic archThe phospholipids in erythrocyte membrane were sampled and tested on the 0, the 10th, the 20th, the 40th and the 60th day to show the dynamic changes.The plasma TG, T-Ch, T-LP and lipid in aortic arch were sampled and tested on the 0, the 10th, the 30th and the 60th day to show the dynamic changes.Measurements of TG, T-Ch and T-PL levels were made using the glycerol-3-phosphate oxidase-phenol-aminophenazone (PAP), cholesterol oxidase-PAP and ammonium molybdate reduction methods, respectively. Lipids in aortic arch and erythrocyte membrane were extracted by the Rose-Gottlieb extraction method and dried with nitrogen. T-Ch and TG levels were measured with the same method utilized to measure the levels in the plasma. The T-PL level was determined using the coefficient between inorganic phosphorus and T-PL. The level of inorganic phosphorous was measured using the molybdenum blue colorimetric method following acid digestion. Measurements of urea and creatinine (Cr) were made using the PAP rate method.2.2 The second part of the experiment2.2.1 Experimental groupGroup A(control group,n=30), only by sham operation, the kidneys were kept. All rats after surgery can be free in diet. Until the model of renal failure was made in true operating rats successfully, they were further randomly divided into 1) group B, operation control, did not accept any treatment, n= 30, Blank group,2) group C, only accepted fibroblasts transfected by blank vector[FB (LXSN) cells] treatment, n= 30,3) D group, treatment group, accepted fibroblast transfected by vector with decorin[FB (LDCNSN) cells] treatment, n= 30. Six rats were sacrificed on the 0, the 10th, the 20th, the 30th and the 60th day respectively. Aortic arch were resected to test the lipid deposition.2.2.2 Changes in renal pathology were observed by microscopeHematoxylin and eosin (HE), Periodic Acid Schiff and Masson staining were used to observe the changes of renal pathology under light microscopy on the 0, the 30th and the 60th day respectively.The glomerular and tubulointerstitial lesions were then subjected to semi-quantitative grading, as follows:0, normal; Ⅰ, lesion scope ≤30%; Ⅱ, lesion scope 31-60%; and Ⅲ, lesion scope2561%.2.2.3 The culture and injection of fibroblastsFB (LDCNSN) and FB (LXSN) cells were cultured in Dulbecco’s modified Eagle’s medium containing 300 ng/ml G418 and 10% fetal calf serum. The cells were then digested and collected using EDTA-trypsin, and washed three times with high-pressure sterilized normal saline. The cell concentration was adjusted to 1×106/ml. The FB (LXSN) and FB (LDCNSN) cells were administered to the renal medulla in the control and treatment groups, respectively, via multi-point injection during the 5/6 nephrectomy. The injection points were distributed uniformly across five to six sites, with 1x106 cells/kidney in total.2.2.4 Testing of plasma TG, T-Ch, Scr and BUNThe phospholipids in erythrocyte membrane were sampled and tested on the 0,1 Oth, the 20th,30th and 60th day to show the dynamic changes.The plasma TG, T-Ch, T-LP and lipid in aortic arch were sampled and tested on the 0, 10th,30th and 60th day to show the dynamic changes.Measurements of TG, T-Ch and T-PL levels were made using the glycerol-3-phosphate oxidase-phenol-aminophenazone (PAP), cholesterol oxidase-PAP and ammonium molybdate reduction methods, respectively. Lipids in aortic arch and erythrocyte membrane were extracted by the Rose-Gottlieb extraction method and dried with nitrogen. T-Ch and TG levels were measured with the same method utilized to measure the levels in the plasma. The T-PL level was determined using the coefficient between inorganic phosphorus and T-PL. The level of inorganic phosphorous was measured using the molybdenum blue colorimetric method following acid digestion. Measurements of urea and creatinine (Cr) were made using the PAP rate method.Measurements of TG, T-Ch and T-PL levels were made using the glycerol-3-phosphate oxidase-phenol-aminophenazone (PAP), cholesterol oxidase-PAP and ammonium molybdate reduction methods, respectively. Measurements of urea and creatinine (Cr) were made using the PAP rate method.2.2.5Testing of DCN and TGF-P 1 in renal tissueThe En VisionTM immunohistochemistry system was used to detect the changes in DCN and TGF-(31 expression in the renal tissues on the 0, the 30th and the 60th day respectively.Following conventional deparaffinization, microwave-induced antigen retrieval was performed for 10 min and a drop of 3% H2O2 was added to each slice. Incubation was then carried out for 20 min at room temperature. The primary antibody, rabbit anti-rat monoclonal anti-TGFpi was added at a 1:200 dilution followed by rabbit anti-rat monoclonal anti-DCN at a 1:200 dilution, prior to incubation for 2 h at room temperature. Following incubation, the slice was rinsed with PBS three times for 5 min, avidin was added and the slice was further incubated for 20 min at room temperature. The slice was then rinsed again with PBS three times for 3 min, and horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added prior to incubation for 30 min at room temperature. Following incubation, the slice was rinsed with PBS three times for 5 min,3,3’-diaminobenzidine was added and the slice was examined under a light microscope for 5 min. The slices were restained with hematoxylin and 0.1% hydrochloric acid and washed with water, following which they turned blue. They were subsequently dehydrated in gradient ethanol, vitricated by dimethylbenzene, mounted with neutral balsam (mounting medium) and examined subsequent to air drying.The distribution of staining in the tubulointerstitial area was graded into levels, as follows: 0, no staining; 1, occasional staining; 2, focal staining; and 3, diffuse staining.3. Statistical analysisAll statistical analysis was performed by using SPSS 17.0 software. The Student’s t-test or ANOVA was applied for intergroup comparisons and liner correlation analysis was applied. P<0.05 was considered to indicate a statistically significant difference.Results:1. The results of the first experimental partIn experimental group, blood urea nitrogen (BUN) and serum creatinine (Scr) increased on the 10th day to the top (25.56±2.16 mmol/l,308.50±15.73 μmol/1), on the 20th day began to decline, on the 40th day to minimum (16.22±3.57 mmol/1,193.45±20.32 μmol/1), but was higher than the control group(7.89±2.58 mmol/1,91.22±19.73 μmol/1),P< 0.01, BUN and Scr began to increase on the 60th day again.The triglyceride, total cholesterol and phospholipids in aortic arch of the experimental group began to rise on the 10th day. TG was significantly higher than the control group on the 30th and the 60th day(P< 0.05, P< 0.01).T-Ch and T-LP began to rise on the 10th day, and was significantly higher than the control group on the 60th day (P< 0.01, P< 0.05). The TG and T-Ch levels in the plasma were significantly positively correlated with those in aortic tissue (r=0.944 and r=0.937), while the T-PL level in the plasma was significantly negatively corre-lated with that in aortic tissue (r=-0.832).The TG and T-Ch levels in the plasma of the experimental group rats started to increase from the 10th day and continuously rose; the levels were significantly higher than those of the control group on the 10th,30th,40th and 60th days (PO.05). The T-PL levels increased significantly on the 10th day, then decreased on the 20th and 30th days, and slightly increased again on the 60th day.The T-Ch level in the erythrocyte membranes began to increase on the 10th day and was significantly higher compared to that of the control group on the 40th and 60th days (PO.05). The T-PL level in the erythrocyte membranes was slightly lower than that of the control group on the 20th (P<0.05), and significantly decreased on the 40th and 60th days (P<0.01).The T-Ch level in the erythrocyte membrane was significantly positively correlated with those in plasma(r=0.946).2. The results of the second experimental partAfter the treatment by DCN, the BUN and Scr levels in Group D were significantly lower than those in Group B and C on the 10th day,the 20th day,the 30th and the 60th day (P<0.05). And a slight increase was observed in group D compared with group A for the values on the 10th day,the 20th day,the 30th and the 60th day (P<0.05). The expression of DCN in group D in the renal tissues increased significantly than group B and C on the he 30th and the 60th day (P<0.01). The expression of TGF-β1 in Group D decreased significantly than in Group B and Group C on the 30th and the 60th day (P<0.05). But increased significantly than in Group A(P<0.01).After the intervention by using decorin, although the lipid levels increased in group D, but they were lower than group B and C on the 30th and 60th day (P<0.05).And they were higher than group A, but the difference was less than compared with group B and C(P<0.05). The change of lipid of aortic arch was same as the plasma lipid. The T-Ch of erythrocyte membrane in group D began to increase on the 20th day, and increased obviously on the 30th and 60th day, but they were lower than group B and C(P<0.05). The T-LP of erythrocyte membrane in group D began to decrease on the 20th day, and decreased markedly on the 30th and 60th day, but they were higher than group B and C(P<0.05).Conclusion:The plasma lipid and lipid deposition in aortic arch increased in the rats with early-stage renal failure. It was liable to atherosclerosis. The increased T-Ch and decreased T-PL levels in the erythrocyte membrane increased the rigidity of the erythrocyte and decreased erythrocyte deformability.After the treatment by using decorin not only delayed the renal failure but also mitigated the abnormal lipid metabolic disorder of the rat with early-stage renal failure. Further, it could delay the atherosclerotic complications and improve the prognosis. |
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