Application And Mechanism Of Low Protein Diet With Keto-acids Supplement In Maintenance Hemodialysis And Chronic Kidney Disease

PARTâ… The effects of short-time application of low protein diet supplement withα-keto acidson refractory hyperphosphatemia in maintenance hemodialysis patientsObjectiveHyperphosphatemia is one of the most common complications in maintenance hemodialysis(MHD) patients, which closely relates to cardiovascular complications and renalosteodystrophy disease. Protein restriction will certainly bring to reduced phosphrousintake, but there is little experience of treating hyperphosphatemia in hemodialysis patientswith low protein diet (LPD) because of worring about malnutrition. Ketosteril^?, a mixtureofα-ketoanalogs, is often combined with LPD to add essential amino acids whichdeficiency in chronic kidney disease (CKD) patients, as well as be used as a potentphosphrous binder. The purpose of the study is to evaluate the effects of short-timeapplication of low protein diet supplement withα-keto acids on refractoryhyperphosphatemia in MHD patients.MethodsAll planned menu was designed and individualized by the dietitian according to body massindex (BMI) with total calorie intake of 30～35kcal/kg/d, protein intake of 0.8g/kg/d.α-ketoacids were administered at the dose of 12 pills per day throughout the 8-week study. Thenutrition status was evaluated through the three-day-diaries (recorded at week 1, 2, 4 and 8),MNA score (minimal nutritional assessment), somatometric measurement indices (armmuscle circumference, triceps skin-fold thickness, dry weight, body mass index) andbiochemical indices (serum creatinine, protein catabolic rate, total protein, albumin, plasmaamino acids profile). High sensitivity C reactive protein (hs CRP), urea clearance rate (Kt/V)and CO₂ combinding power (CO₂-CP) before and after the trial were measured to observethe inflammation, dialysis adequacy and metabolic acidosis, respectively. Serum calcium,phosphrous and intact parathyroid hormone (iPTH) were detected before and after the trialto estimate the effects on calcium and phosphrous metabolism. ResultsThe average calories intake, protein intake and phosphate intake during the study were30.10±3.70kcal/kg/d, 0.86±0.15g / kg/d and 669.55±134.51 mg/d, respectively. There is nosignificant difference in somatometric measurement indices and biochemical indicesbetween pre-trial and post-trial (p＞0.05). No difference was observed in hs CRP and Kt/Vbefore and after the trial (p＞0. 05), while CO₂-CP was significantly higher after the trial thanbefore [ (25.34±2.81 vs. 18.49±1.77)mmol/L, p＜0.001]. Serum phosphate level and calciumphosphateproduct were significantly decreased in the end of the study compared to thosebefore the trial [P: (5.59±1.20vs7.26±1.42)mg/dl, p＜0.001; Ca×P: (52.94±12.80 vs 70.60±12.39) mg²/dl², p＜0.001]。There was no marked change of serum calcium and iPTH afterthe trial [Ca: (9.44±1.04vs9.80±1.00) mg/dl;iPTH: (454.23±36.51vs531.28±48.00) pg/ml](p＞0.05).ConclusionLow protein diet supplement withα-keto acids could be a safe and efficient therapy tomanage the refractory hyperphosphatemia of the MHD patients. A large scaled, multicenters,randomized controlled clinical trial is needed to confirm whether a long-termapplication of such treatment has the similar benefits. PARTâ…¡In vivo study of the renal protective mechanisms oflow protein diet withα-keto acids supplement1. Study of the effects of low protein diet withα-keto acids supplementon renal function of 5/6 nephrectomized rats and its mechanismsObjectiveLow protein diet withα-keto acids supplement therapy is often been used in advancedchronic kidney disease patients to slow the progression of the disease, but it has been usedwith controversary on its safty and little known mechanisms. The purpose of the study is toobserve the effects of low protein diet withα-keto acids supplement on renal function of 5/6nephrectomized rats and its possible mechanisms.MethodsChronic renal failure model was established by 5/6 nephrectomy (Nx) in 30 male Sprague-Dawleyrats, then the animals were randomly assigned to the following diet groups: normalprotein group (NPD:18% casein protein), low protein group (LPD:6% casein protein) andsupplemented low protein group (LK: 5% casein protein+1%α-keto acids). Ten maleSprague-Dawley sham-operated rats giving 18% casein protein served as control group(Sham). All rats were killed at the end of the 12^th week with blood and urine samplescollected. Serum albumin (Alb), total protein (TP), blood urea nitrogen (BUN), serumcreatinine (Scr), triglyceride (TG), cholesterol (CHO), high density lipoprotein(HDL), lowdensity lipolprotein (LDL) and fasting blood glucose (FBG) were measured by routinebiochemistry. Fasting serum insulin was detected by radioimmunoassay. 24h urine proteinexcretion was detected with coomassie brilliant blue combined techniques. Electronicmicroscope was used to observe the structure of glomerular filtration membrane.Proteomics was used to identify the differentially-expressed protein in serum among thefour groups. The assays of malonaldehyde (MDA), superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) in serum were measured by colorimetric method.Immunohistochemistry and western blot were used to detect the protein expression ofTGF-β₁ in residual kidney. Pathological changes of the residual kidney were investigatedwith periodic acid schiff (PAS) staining. Results1. General status: nutritional indices including weight, Alb and TP were not significantdifferent among the four groups (p＞0.05). Scr was significantly higher in the Nx ratsthan in Sham rats [(NPD: 58.67±4.80vs.LPD:59.40±3.65 vs.LK:58.60±4.56 vs.Sham:34.67±5.47) umol/L, p＜0.05], but was of no difference among the three Nx groups(p＞0.05). BUN was obviously lower in LPD (7.26±1.29) and LK group (6.63±2.20)than in NPD group (11.03±2.45), (p＞0.05). Proteinuria level of Nx groups wasmarkedly higher than that of the Sham group [(17.70±8.10)mg/24h], and that of the LKgroup [(38.30±5.50)mg/24h] was lower than those of NPD [(94.23±20.15)mg/24h] andLPD [(58.70±8.18)mg/24h] groups(p＜0.05). The glomerular filtration membrane ofNPD group manifested foot process fusion and endothelial cell fenestrationdisappearance, but it was kept almost normal in LPD and LK group;2. 328 kinds of serological protein in four groups were analyzed by proteomics. When theNx groups were compared to Sham group, there were five kinds of protein expressionelevated, while eight kinds of protein expression declined (eg: anti-oxidative stressrelated protein GSH-Px, et al). Five types of protein expressed more in LPD group thanin NPD group (eg: GSH-Px, et al), while ten types of protein expressed less in LPDgroup (eg: acute reactive protein ITIH4, et al). There were fourteen kinds of elevatedprotein (eg: nutrition related protein serotrasferrin and anti-oxidative stress relatedprotein GSH-Px, et al) and seven kinds of reduced protein (eg: ITIH4 and ApoE, et al)in LK group compared to NPD group. Compared with LPD group, nine types of proteinlevel (eg: GSH-Px, et al) raised up while six kinds of protein level (eg: ApoE andfibrosis related protein laminin) stepped down in LK group;3. Fat and carbohydrate metabolism: the FBG was of no difference among the fourgroups(p＞0.05), while fasting serum insulin and HOMA-IR in Nx groups weresignificantly higher than in Sham group [Insulin:(12.48±2.24);HOMA-IR:(4.51±0.61)],but those in LK group [Insulin: (18.71±3.92); HOMA-IR:(10.23±2.02)] weresignificantly lower than in LPD [Insulin: (27.49±4.15); HOMA-IR:(14.68±0.10)] andNPD [Insulin:(41.86±6.13); HOMA-IR: (19.93±1.52)] groups(p＜0,05); There was nosignificantly differences of HDL in the four goups (p＞0.05), while TG (NPD:1.25±0.46;LPD:1.20±0.36; LK:0.92±0.33), CHO(NPD:2.89±0.65; LPD: 2.56±0.51; LK:2.47±0.52) and LDL (NPD:0.33±0.12; LPD:0.28±0.10; LK: 0.25±0.08) in Nx groups weresignificantly higher than in Sham group[TG (0.71±0.16); CHO(1.53±0.11 ); LDL(0.16±0.03)], but only TG and CHO in LK group were significantly lower than in LPD and NPD groups (p＜0.05);4. Circualr oxidative stress: Serological MDA of the Nx groups was significantly higherthan that of Sham group (3.1992±0.1781) (p＜0.05), but was reduced lower in LK(4.6056±0.1217) and LPD group (5.2752±0.1572) than in NPD group (6.4898±0.2621),which was reduced further lower in LK group than in LPD group (p＜0. 05).SerologicalSOD and GSH-Px of the Nx groups were notably lower than those of Sham group [SOD:(46.5054±2.4142); GSH-Px:(332.2500±33.8189)], (p＜0.05), but they were elevatedhigher in LK [SOD: (28.0876±1.0904); GSH-Px:(173.7770±4.0926)] and LPD group[SOD: (22.4170±1.2948); GSH-Px:(117.5703±5.4685)] than in NPD group [SOD:(15.2142±1.9010); GSH-Px:(63.0785±4.8479)], which were elevated further higher inLK group than in LPD group (p＜0. 05);5. Renal fibrosis: TGF-β₁ protein expression in NPD group (0.6459±0.0442) wasremarkably higher than that in Sham group (0.0671±0.0172), which was significantlydown-regulated in LPD (0.3763±0.0314) and LK group (0.3128±0.0296), (p＜0.05).Glomerular sclerosis index (GSI) and excelluar matrix score of the Nx groups weresignificantly higher than those of Sham group, but improved lessen in LPD and LKgroup than in NPD group [GSI (NPD: 37.50±6.31 vs. LPD: 18.32±3.22 vs. LK: 14.91±2.33 vs. Sham:2.71±1.02); ECM score (NPD: 0.176±0.05 vs LPD: 0.078±0.02 vs LK:0.056±0.02 vs Sham: 0.032±0.01); p＜0. 05].ConclusionsLow protein diet withα-ketoacids supplement could keep stable nutritional status as well asameliorate the lipids metabolism disturbance and insulin resistance in 5/6 nephrectomizedrats. The therapy could also exhibit renal protective effects of lessening azotemia, reducingfoot process fusion and urinary protein excretion at the same time. Improvement of circularoxidative stress and renal fibrosis change may both be involved. 2. Effects of low protein diet withα-keto acids supplement onrenin-angiotensin system(RAS) in 5/6 nephrectomized ratsObjectiveThe activation of renal local RAS, one of key factors contributing to the progression ofchronic kidney disease, may lead to enhanced oxidative stress and aggravated renal fibrosis.It has been proved that high protein diet will result in increased renin secretion. The purposeof the study is to observe the effects of low protein diet withα-keto acids supplement onrenin-angiotensin system in 5/6 nephrectomized rats.MethodsExperimental animal modeling composition and grouping were the same as the part 1. Therenin and angiotensinâ…¡in tissue were measured by radioimmunoassay, whileangiotensinâ…¡in plasma were detected with the ELISA kit. Immunohistochemistry andwestern blot were used to locate and quantitate the protein expression of renin and AT₁.Real-time PCR was used to detect the gene expression of renin and AT_1a, the main subtypesof AT₁ receptor. The assays of malonaldehyde (MDA), superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and catalase (CAT) in tissue were measured bycolorimetric method.Results1. Change of RAS: the Angâ…¡level both in plasma and in tissue were higher in the Nxgroups than in Sham group [plasma:(0.073±0.001)pg/ml;tissue:(20.48±2.34) pg/ml,p＜0.05], but only in tissue the Angâ…¡level was lower in LPD group (38.57±5.87) and LKgroup (30.23±4.23) than in NPD group (68.92+10.23) (p＜0.05).The renin level in tissuewas lower in LK group (0.16±0.01) than in NPD group (0.20±0.01) (p＜0.05). The reninprotein expressed much less in LPD group (0.4824±0.0640) and LK group (0.3363±0.0267) than that in NPD group (0.7805±0.0905) (p＜0.05). Renin mRNA level in LKgroup (0.64±0.10) was significantly lower than that in NPD group (1.11±0.18) and LPDgroup (1.06±0.15) (p＜0. 05). The AT₁ protein expression in LPD group (0.14±0.02) andLK group (0.13±0.01) was much lesser than that in NPD group (0.19±0.02) (p＜0.05).AT_1a mRNA level in LK group (0.66±0.17) was significantly lower than that in NPDgroup (1.16±0.30) and LPD group (0.91±0.29) (p＜0.05). 2. Renal local oxidative stress: MDA in tissue of the Nx groups was significantly higherthan in Sham group (3.9458±0.3953), which was reduced lower in LK (5.7220±0.2922)and LPD group (8.9340±0.2363) than in NPD group(11.3703±0.7423), and was reducedeven lower in LK group than in LPD group (p＜0.05). SOD, GSH-Px and CAT in tissueof the Nx groups were significantly lower than in Sham group[SOD:(152.3860±3.7769);GSH-Px: (88.5448±2.7205); CAT: (1189.2600±95.4250)], (p＜0.05), while those wereelevated higher in LK [SOD:(88.5448±2.7205); GSH -Px: (860.9982±18.0659); CAT:(917.6600±30.1156)] and LPD [SOD:(62.7137±2.3850); GSH-Px:(755.9942±17.3367);CAT:(715.0025±22.9055)] group than in NPD group [SOD: (40.8243±3.0096); GSH-Px:(589.5017±21.2464); CAT: (464.0778±20.6500)] (p＜0.05), which were elevated evenhigher in LK group than in LPD group (p＜0.05).3.Correlation analysis: MDA in tissue (r=0.892), TGF-β₁ expression (r=0.716), GSI(r=0.807) and ECM score (r=0.673) were positively related to Angâ…¡level in tissue (p＜0.001), but SOD (r=-0.978), GSH-Px (r=-0.965) and CAT (r=-0.891) in tissue werenegatively related to Angâ…¡level in tissue (p＜0.001).ConclusionsLow protein diet withα-keto acids supplement therapy may exhibit renal protective effectsof improving oxidative stress and renal fibrosis pathology through inhibition the acitivity oflocal renin-angiotensin system in 5/6 nephrectomized rats.