Protective Effects Of Melatonin In Chronic Renal Failure Rats And Its Mechanism Study

Objective: To investigate the effects of melatonin on morphological changes in the kidney and renal function in 5/6 nephrectomy rat model.Methods: The SD rats (250-300g) were assigned into three groups. The abdominal cavity of control(CTL) group was opened and closed after renal envelope had been peeled off. The kidney of subtotal nephrectomy(STN) group was subjected to 5/6 subtotal nephrectomy(SNx) by a two-step produre. The rat of melatonin (MEL) was given melatonin to SN_x rats. The levels of renal function and urinary protein, the rates of renal and body weigh and the changes of renal pathology by light and electron microscope were observed at eight different periods of time in 40 weeks after operation finished.Results: The levels of plasma creatinine and blood urea nitrogen were predominatly elevated in 1st week and were given into the period of renal failure in 40th week of STN group. The pathological changes of the kidney in STN group were swell of glomerulis, slight proliferation of mesangial cells and slight dilation of renal tubule in 2nd week;Severe proliferation of mesangial cells, inflammation cells infiltrated and expand in renal insterstitium in 8th week; Focal segmental glomerular sclerosis and renal interstitial fibrosis in 12th week; With the renal pathological changes mentioned above further aggravated in 40th week, massive glomeruli sclerosis and fibrosis, severe fibrosis in renal interstitium occured. The melatonin could partly ameliorated renal function, predominantly reduced urinary protein excretion and retarded the appearance of renal failure. It could reduce pathological damages.Conclusion: The renal development of 5/6 nephrectomy rat is an ideal model of chronic renal failure caused by glomerular sclerosis and renal interstitial fibrosis. Melatonin can protect renal function and morphological change in the kidney.Part II The effect and significance of melatonin on oxidative stress,inflammation, proliferation and apoptosis in chronic renal failure ratsObjective: To study the effects of long-term administration of melatonin on oxidativestress, inflammation, proliferation and apoptosis in subtotally nephrectomized rats andto explore the mechanism of its function.Method: By established a model of subtotal (5/6) nephrectomy and by taken melatonin(5mg. Kg^-1.d^-1) for 40 weeks. The changes of superoxide dismutase(SOD),malondialdehyde(MDA), proliferating cell nucler antigen (PCNA), apoptotic cells byterminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) andinflammatory cells infiltration(ED-1) at 1st, 2nd, 4th, 8th, 12th, 16th, 26th and 40thweek after operation have been observed.Result: The activity of SOD decreased and the level of MDA increased on the tissues ofremnant kidney in STN group. The indexs of proliferation and apoptosis were higherthan that of CTL group. The proliferation and apoptosis rate of glomerulus and tubules were finally reduced to a point lower than CTL group, while the rate of interstitial cells sustained at a higher level. The melatonin could reduce the level of MDA, which had statistic difference after 8 weeks (P<0.05, P<0.01). Although it could increase SOD, but there was no significant difference (P>0.05). The infiltration of inflammatory cells (macrophages) in renal interstitiun were lessened remarkably (P<0.05, P<0.01). The main roles of melatonin were to decrease the apoptotic cells in tubules after 16 weeks, and increase apoptosis and decrease proliferation in intertitium before 8 weeks. The correlation analysis showed inflammation and oxidative stress had much to do with proliferation and apoptosis.Conclusion: The imbalance of oxidative stress, inflammation, proliferation and apoptosis play an important role in chronic renal failure. The apoptotic cells in glomeruli and tubules keep increased and the excessive proliferation of the interstitium cells were lead to glomerular sclerosis and renal interstitial fibrosis. Melatonin has therapeutic effect on reducing oxidative stress and apoptosis of tubular epithelial cells, inhibiting inflammtion in interstitium and promoting inflammatory cells to apoptosis. Melatonin can improve the renal function and protect pathological morphology with methods mentioned above.Part III Changes of apoptosis-related genes and signal pathways in chronicrenal failure rats by melatoninObjective: To investigate the dynamic role and chemanisms of apoptosis-related genes in 5/6 nephrectomy rat model, and to research the regulation and its signal pathway on remnant kidney by melatonin.Method: The SD rats were divided into three groups: CTL group( sham-operation), STN group(subtotal nephrectomy group), MEL group(subtotal nephrectomy and treat with melatonin). The protein level of Fas and CytC were detected by immunhistochemistry staining, the activity of caspase-8, -9 were determined by colorimetic assay and the mRNA and protein level of caspase-3, -8, -9 were found by RT-PCR and Western-blot in 1st, 2nd, 4th, 8th, 12th, 16th, 26th and 40th weeks after the operation. The role of melatonin on apoptosis and on signal pathways were analyzed. Results: The STN group compared with the CTL group, the expression of Fas and CytC were progressively increased in remant kidney, the activities of caspase-8, -9 were increased, moreover, the up-regulated activity of caspase-9 became remarkably higher than that of caspase-8, the changes of mRNA and protein level of caspase-3, -8, -9 had the same tendency and up-regulated wavily in the rat model. The expression of Fas and CytC in MEL group were weakened compared with STN group, caspase-8, -9 activity were lessened, the increased degree of caspase-3, -8, -9 were reduced. Conclusion: Both death recepator and mitochondria signal pathways participate in chronic renal failure. Through the two signal pathways, Melatonin can reduce the apoptosis of renal parenchyma cells, with the mitochondria signal pathways playing the primary role to protect renal.