Effects Of The High Calorie Intake And Calorie Restriction On Aging Renal Oxidative Damage

Objective:It has been confirmed that calorie restriction can reduce the production of ROS and oxidative damage in kidney to protect renal function.The decline of aging kidney clearance of free radicals may lead to reactive oxygen species(ROS) surge.Currently,whether high-calorie intake will increase the oxidative damage of aging kidney is rarely reported.The aim of this study is to explore the effects of high-calorie and calorie restriction to aging kidney of oxidative damage through in vivo and in vitro methods.Methods:In vivo:Thirty male Wistar rats(14-month-old,n=10) assigned randomly 3 groups:â‘ Control group:ab libitum-fed;â‘¡high-calorie group:rats were given ad libitum access to high-calorie diet,daily monitored;â‘¢Calorie restriction group:normal food intake restricted to 70%(Rats were given food and water with a standard of the control group,and rats appetite were mornitored weekly,adjusted at any moment).Blood and urine samples were tested to acquire the paramters of blood biochemistry baseline,renal function(Scr,BUN) and urine protein/creatinine.The weight changes in rats were monitored monthly till 24-month-old,mercy killed by ether anesthesia.Urine protein/creatinine was detected two days before euthanasia;Collecting blood through renal artery blood before an overnight fasting,thereafter blood biochemistry,renal function and other indicators were detected;Tissue of kidney was isolated,fixed in 10% buffered fomalin or in liquid nitrogen,embedded in paraffin,and sections were sliced to staine with PAS for renal pathological study;TUNEL assay was to detected the apoptosis of renal tubular epithelial cells;Colorimetry assaywere to detected hydrogen peroxide(H₂O₂),lipid and protein oxidative damage product of malondialdehyde(MDA),protein carbonyl and total superoxide dismutase (T-SOD).In vitro:To observe the effect of high glucose on human renal tubular epithelial cell line(human kidney proximal tubular epithelial cell line,HKC) causing oxidative stress and oxidative damage:â‘ the control group(5.5 mmol/L glucose);â‘¡mannite group(5.5 mmol / L glucose + 24.5 mmol / L mannite);â‘¢high glucose treatment group(30mmol / L glucose).Fluorescent(CM-H2DCFDA) staining at 0 h,0.5 h,1 h,6 h,12 h,24 h,48 h,flow cytometry and confocal microscope were used to detect HKC reactive oxygen species(ROS) fluorescence intensity at different time points in each group.Confocal microscope was used to detect mitochondrial membrane potential(MMP),and MDA and protein carbonyl content were determined by colorimetric assay.Body weight baseline in each group,blood biochemistry,renal function, urine protein/creatinine was no significant difference(P＞0.05).24 months later, the weight of high-calorie group was＞control group(P＜0.05),whereas caloric restriction group was＜the control group(P＜0.05).Compared with the control group,high-calorie group of serum albumin,blood glucose,Scr and BUN were no significant difference(P＞0.05);Triglyceride was significantly high(P＜0.05), while no significant difference in cholesterol(P＞0.05);Urinary protein/creatinine was significant difference(P＜0.05).Compared with control group,serum albumin,blood glucose,Scr and BUN were no significant difference(P＞0.05);Triglyceride,cholesterol and urinary protein/creatinine were significantly decreased(P＜0.05);Compared with the control group,PAS staining of renal tissue showed that glomerular lesions aggravated in high-calorie group and glomerular sclerosis index increased(P＜0.05),with a middle-severe mesangial proliferation,focal segment sclerosis of glomerular increasing,and most of the glomeruli can be seen middle podocytes foam degeneration.Compared with the control group,glomerular lesions were reduced significantly and glomerular sclerosis index were decreased in caloric restriction group(P＜0.05),which was mainly shown that glomerular mesangial proliferation were less,and focal segmental glomerular sclerosis were also decreased significantly.The tubules-interstitial changes in each group:in comparison with the control,high-calorie group of tubules-interstitial lesions were significantly increased,and tubules-interstitial score was rising(P＜0.05),which showed marked tubular basement membrane thickening,focal tubular atrophy,a large number of protein casts and interstitial fibrosis,and a large number of inflammatory cells infiltration accompanied;Compared with the control group,caloric restriction group tubulointerstitial lesions were significantly reduced,and tubules-interstitial score was decreased(P＜0.05),which showed no basement membrane thickening,occasional focal atrophy of tubules,with possession of a small amount of protein-based and less interstitial fibrosis,occasional inflammatory cell infiltration.Compared with the control group,high-calorie group of H₂O₂,MDA,protein carbonyl content of the three indicators were significantly higher(P＜0.05).In calorie restriction group,H₂O₂,protein carbonyl content were significantly decreased compared with the control group(P＜0.05),while MDA content was not significantly difference(P＞0.05).Compared with the control group,detection of SOD showed that high-calorie group and caloric restriction groups were not significant difference(P＞0.05).TUNEL assay found the number of apoptotic cells in high-calorie group was more than the control group(P＜0.05),while caloric restriction group less than the control group(P＜0.05).In vitro studies showed that cells began to rise at 0.5 h and reached a peak in ROS and MMP at 1 h after incubation of high glucose to HKC,where there is significant difference compared the control group and the mannitol group in the same time points(P＜0.05),thereafter a downward trend was shown.The lipid and protein oxidative damage of macromolecules in a short period of time(48h) was not significant changes.Conclusion:This study established high-calorie and caloric restriction aging rats model to explore theirs impact on renal oxidative damage,which found that high-calorie diet causing blood lipid levels,urine protein/creatinine,oxidative stress and oxidative damage raised,and glomerular sclerosis,tubulointerstitial damage,apoptosis in tubular epithelial cells increased,while caloric restriction can reduce the adverse effects of the above.It was confirmed that the stimulation of high glucose in the relatively short period could cause oxidative stress indicators of human renal tubular epithelial cells increase at the cellular level,but no lipid and protein molecules material damage.