| Part I Changes of renin-angiotensin system in cultured mesangial cells by serum from renal ablated rats feeding low protein diet with a-keto acid supplementObjectiveLow protein diet with a-keto acid supplement was considered to reducing the 24-hours urinary protein excretion and delaying the progression of chronic kidney disease (CKD) and thus was widely used to treat CKD-patient in phase 3 and phase 4 with uncertain mechanism. Low protein diet with a-keto acid supplement was found to reduce the local RAS level in nephrectomized rats in our pre-work. This research aims to observe the changes of renin-angiotensin system in cultured mesangial cells by serum from renal abated rats feeding with low protein diet with a-keto acid with exclusion of hemodynamic effects.Methods1. Animal experiment30 male SD rats received 3/4 nephrectomy (Nx) were placed on 18% normal protein diet (Nx-NPD),6% low protein diet (Nx-LPD) or 5% low protein plus 1% keto-acid diet (Nx-LK.) one week after operation (n=10 in each).10 male SD sham-operated rats fed with Nx-NPD were used as control. All the rats were sacrificed 12 weeks later and sera from each groups were collected.2. Cell cultureMesangial cells were cultured in sera (10%) collected from animals treated with or without losartan (0.02mmol/l) for 48 hours. ELISA was applied to detect the level of AngⅡ, TGF-β1, fibronectin and laminin in cell culture medium. Immunoblot was used to determine the protein level of AT1 receptor and real-time PCR were used to detect the mRNA level of AT1, TGF-β1 and fibronectin.Results1. Experiment in vivo:Nutritional indices including body weight, total protein and albumin were comparable in each group. Serum creatinine were significantly increased in nephrectomized rats compared to sham group (P<0.05) but with little different in between. Blood urea nitrogen (BUN) and 24h urinary protein excretion were greatly increased in Nx-NPD group compared to sham group (P<0.05). Nx-LPD lowered these two criteria compared to Nx-NPD group and Nx-LK enhanced these effects (P<0.05).2. Experiment in vitro:Nx-NPD greatly increased the level of AngⅡ(Nx-NPD:12.70±0.12; sham: 8.04±0.62 ng/ug protein) in cell cultured medium and the level of AT1 receptor in mRNA level (Nx-NPD:0.5752±0.1603; sham:0.2179±0.0763) and protein expression level (Nx-NPD:44.73±1.93; sham:24.61±1.17), as well as the increased production (FN:Nx-NPD:0.2206±0.0203, sham:0.0881±0.0183; LN:Nx-NPD:0.1179±0.0309, sham:0.0686±0.0102; TGF-β1:Nx-NPD: 0.0372±0.0079, sham:0.0071±0.0016) and secretion (FN:Nx-NPD:39.84±0.06; sham:20.58±0.46 ug/ug protein, LN:Nx-NPD:91.31±4.52; sham:66.49±3.90ug/ug protein, TGF-β1:Nx-NPD:83.85±0.56; sham:10.12±1.32ng/ug protein) of fibronectin, laminin and TGF-β1 in cultured mesangial cell (P<0.05). Nx-LPD decreased these increment (P<0.05) and Nx-LK showed stronger inhibitory effect(P<0.05).Losartan application (0.02mmol/l) sharply reduced fibronectin (LOS(+):mRNA: 0.0416±0.0230; ELISA:20.22±1.63ug/ug protein) and TGF-β1 (LOS(+): mRNA:0.0024±0.0010; ELISA:6.21±0.67ng/ug protein) both in cell cultured medium and in mRNA expression in Nx-NPD serum treated cells (P<0.05) while in Nx-LPD serum treated cells, losartan application further reduced the production and secretion level of fibronectin and TGF-β1. In Nx-LK serum treated cells, losartan application only reduced the mRNA level of fibronectin and TGF-β1 (P<0.05) but not the secretion level.ConclusionsLow protein diet with a-keto acid supplement significantly reduced 24-hours urinary excretion with the maintenance of nutritional requirement. Meanwhile, serum from nephrectomized rats fed with low protein diet plus a-keto acid supplement directly inhibited the renin-angiotensin system in mesangial cells and therefore reduced the expression and secretion of FN, LN and TGF-β1 which may contribute to its beneficial effect on the kidney in patients with chronic kidney disease. Part II The proteomics and biochemical detection of nephrectomized rats fed with different protein dietsObjectiveLow protein diet with a-keto acid supplement was usually applied to patients with moderate to severe chronic kidney disease for its beneficial effect of delaying the progression. Our research in part I revealed that something in the serum from Nx rats fed with low protein diet plus a-keto acid can directly inhibit the RAS activation in mesangial cells and further reduced the expression and secretion of ECM and TGF-β1. This study screened the proteomics of sera and biochemical criteria to investigate the possible effector.Methods30 male SD rats received 3/4 nephrectomy (Nx) were placed on 18% normal protein diet (Nx-NPD),6% low protein diet (Nx-LPD) or 5% low protein plus 1% keto-acid diet (Nx-LK) one week after operation (n=10 in each).10 male SD sham-operated rats fed with Nx-NPD were used as control. All the rats were sacrificed 12 weeks later and sera, urea and tissue from each groups were collected. TG, CHO, HDL, LDL and FBG were detected through biochemical methods. Radioimmunoassay was applied to detect the fasting blood insulin level. The changes of protein in renal injured rats fed with different protein diets were detected by proteomics. MDA, SOD and GSH-Px were detected through colorimetric method and ELISA was used for the carbonyl level of protein in sera from rats.Results1. Proteomics328 kinds of protein were analyzed by proteomics, in which 37 kinds of protein changed, accounting for 11% of total proteins. Fifteen proteins including COL1A1, IGFALS, HBA1, AHSG, TF, SRRM2, FN1, PTPRB, ALB, NOS2A, GPX3, FASN, LAMB3, APOE and CES1 was showed with statistical differences which mainly contribute to the nutritional metabolism, carbonhydrate and lipid metabolisam and the regulation of oxidative and antioxidative balance. 2. Biochemical criteriaNutritional indices including body weight, total protein and albumin were comparable in each group, as well as the level of fasting blood glucose. Fasting insulin levels in Nx groups was significantly increased versus sham group (insulin:12.48±2.24 um/mL; HOMA-IR:4.51±0.61). Nx-LK (insulin:18.71±3.92 um/mL; HOMA-IR:10.23±2.02) greatly reduced these two criteria compared to both Nx-NPD (insulin:41.86±6.13 um/mL; HOMA-IR:19.93±1.52) and Nx-LPD (insulin:27.49±4.15 um/mL; HOMA-IR:14.68±0.10) (p< 0.05). LDL, CHO and TG also increased significantly in Nx groups compared with sham group (LDL:0.16±0.03; CHO:1.53±0.11; TG:0.71±0.16 mmol/L). little differences were found between Nx-NPD (LDL:0.33±0.12; CHO:2.89±0.65; TG:1.25±0.46 mmol/L) and Nx-LPD (LDL:0.28±0.10; CHO:2.56±0.51; TG:1.20±0.36 mmol/L), Nx-LK reduced CHO and TG (CHO:2.47±0.52; TG:0.92±0.33 mmol/L)greatly.Detection of SOD, MDA and GSH-Px in sera revealed that compared with sham group, Nx-NPD greatly enhanced the oxidative stress in rats presenting as significantly increased SOD (sham:46.51±2.41; Nx-NPD:15.21±1.91 U/ml) and GSH-Px (sham:332.25±33.82; Nx-NPD:63.08±4.85 U) which were anti-oxidative enzymes and increased secretion of MDA (sham:3.20±0.19; Nx-NPD:6.49±0.26 nmol/ml) as an product of lipid oxidation (P<0.05). Low protein diet greatly reversed these changes and LK further confirmed its effect. Protein carbonylation level in sera also decreased in parallel to the protein intake.Detection of SOD, MDA and GSH-Px in tissue homogenate showed that in nephrectomized rats, even normal dietary protein intake could significantly increase the oxidative stress in kidney, presenting as decreased secretion of SOD (sham:152.39±3.78; Nx-NPD:40.82±3.01 U/ml) and GSH-Px (ham:1214.70±1.63; Nx-NPD:589.50±21.25 U) which were anti-oxidative enzymes and increased secretion of MDA (ham:3.95±0.40; Nx-NPD:11.37±0.74 nmol/ml) as an product of lipid oxidation (P<0.05). Low protein diet ameliorated this phenomenon.Conclusion Restriction of protein intake was proved to maintain the stable nutritional status in nephrectomized rats and improve the disturbance of lipid and carbonhydrate metabolism and ameliorate insulin resistance, besides of these, low protein diet was revealed to reduce the oxidative stress in rats'kidney.
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