| Objectives: Renal fibrosis, especially tubulointerstitial fibrosis, which isa complicated progress that involves a series of associated processes, such asthe injury of tubulointerstitium, the generation of cytokines and inflammatorymediators, the infiltration of inflammatory cells, the proliferation of fibroblasts,the accumulation of extra cell matrix, is the common final outcome of almostall progressive chronic kidney disorders. Renal fibrosis is also a reliablepredictor of prognosis and a major determinant of renal insufficiency.Autophagy, that is,‘self-eating’ in Greek, which exists among all eukaryotescells from yeast to humans. Autophagy is a large-scale mechanism ofintracellular degradation that seeks to maintain homeostasis. The activation ofautophagy is maintained at a relatively low basic level under the physicalcircumstance, which allows the body to clear and degrade the injuredorganelles and large molecules, such as mitochondria and protein. However,when the function is deficient or injured, injury and aging are likely to occur,research has revealed that autophagy may be related to varied human disordersand senility. In the recent years, the role of autophagy in kidney graduallycaught our attention, a series of research showed that autophagy is associatedwith several renal disorders. It seems that increasing the activity of autophagyusually contribute to renal function protection, while under certaincircumstances, autophagy overload may be harmful and even cause cell end inapoptosis or death. Recent researches mostly focus on renal epithelial cell,mesangial cell, and researches on renal tubular cells usually talk about theischemia-reperfusion injury and drug induced kidney damages, while reportson association between obstructive nephrology and autophagy are little.It is commonly agreed that the induction of autophagy is regulated by the mammalian target of rapamycin (mTOR), which is an atypical serine kinase. Itis a revolutionarily conserved protein, which is widely existed among all kindsof mammals. It is located at the human chromosome lp36.2, as a biologicalmacromolecule, it is responsible for the regulation of cell proliferation, growth,as well as autophagy. mTOR will be activated when the cell get enoughnutrion or when the cell is lack of stress signals, and autophagy is inhibited;however, once the cell is under starvation or stress, the activation of mTORwill decrease or be inhibited, thus, autophagy is activated. The purpose ofautophagy induced during starvation is to degrade intracellular protein. As aresult, the amino acids remaining during starvation are supplied to cells as anew source of energy again. Initiation of autophagy is started from thenucleation of autophagosome membrane. When the activation of mTORdecreases, autophagy related protein (Atg)13executes dephosphorylation,allowing the production of active polymeride, that is, Atg1, which initiates theformation of autophagosome membrane, then the autophagosome membraneextends and eventually wraps the components to be degraded. The extensionof the autopagosome membrane involves a lot of Atg proteins, the mostimportant one of which is the protein light chain (LC)3, the express level ofwhich is commonly applied in all kinds of laboratory methods as the label ofautophagy.Transforming growth factor-β1(TGF-β1) is recognized as a centralmediator in the pathological process of renal tubulointerstitial fibrosis. Itincreases the formation of extra cellular matrix, promotes the activation ofinflammatory cell and their chemotaxis activity, mediates the transformationof renal tubular epithelial cells to mesenchymal cells, contributes to theglomerular sclerosis and tubulointerstitial fibrosis, and eventually leads to endstage renal deficiency.Rapamycin, a macrolide antibiotic which is produced by streptococcus, ismainly applied to the anti-rejection after organ transplant. It is an inhibitor ofthe mammalian target of rapamycin protein signal pathway, researchesconducted during recent years realved that it contributes to the delay of renal interstitial fibrosis progress, and it may be related to autophagy. However, noevidence has yet revealed the effect of rapamycin on the autophagy activity inobstructive renal injury, how much it is, and what kind of changes happen asthe time goes by. Thus, this study builds up unilateral urethral obstructionmodels on SD rats, and use rapamycin as an intervention, combining withimmunohistochemistry methods, and observe the different express levels ofLC3, mTOR and TGF-β1over time, in order to examine the protective role ofrapamycin plays in the renal tubulointerstitial fibrosis, explore a noveltherapeutic target of renal tubulointerstitial fibrosis, apply theory evidence forclinical renal tubulointerstitial fibrosis treatment.Methods:54healthy male SD rats weighed200±20grams of SPF levelwere involved. They were randomly divided into3groups after1weekadaptive feeding. The3groups were: group A, group B, group C. Group A iscontrol group, group B is model group, group C is rapamycin therapy group.Each group consisted of18rats.3,7,14days after operation, respectively,harvested the obstructed kidneys in each group and divided into N3, N7,N14group(n=6). The rats in group B and group C received Chloral hydrate(0.1ml/kg) intraperitoneal injection of anesthesia, as well as the2%lidocainehydrochloride subcutaneous injection of anesthesia. Conduct the surgery understerile conditions. Left ureter was exposed via a left abdominal incision. Themid-ureter was obstructed by two point ligation with silk sutures, and then cutthe ureter between the ligated two points. The sham operated rat underwentthe same surgery procedure except for the obstruction and cut of the left ureter.Each group administrated orally one day before the surgery, once a day, revealrat’s weight per week, and adjust the administration according to their weight.The treatment group were given rapamycin2mg/kg/d orally, the control groupand model group were gavaged with isometric saline. The rats were allowed toeat and drink free. Randomly select6rats from each group respectively, andharvest their obstructed kidneys on day3,7,14after the surgery. Slice thekidneys and place the slices into fixation solution, embed them in paraffin.The pathological changes were determined in the kidney tissues underwent hematoxylin eosin (HE) staining, Masson staining, as well asimmunohistochemistry to detect the expression of LC3, mTOR and TGF-β1.Conduct semi-quantitative analysis of the results with image analysis system.Statistical analysis software SPSS20.0was applied for statistical analysis ofexperimental data which was analyzed with one-way ANOVA, and displayedas mean±standard deviation, P <0.05was considered statistically significant.Results:1HE staining under light microscopy: in group A, no abnormalwas detected; in group B, mild expansion of renal tubules and a small amountof inflammatory cells infiltration were found3days after UUO, on the7thdayafter UUO, the renal tubules dilated obviously, tubular epithelial cells werefaced with degeneration and necrosis, large quantities of inflammatory cellsinfiltration occurred in renal interstitium, interstitial width was significantlyincreased, on the14thday, visible tubular atrophy was more, diffuse thickeningof tubular basement membrane, shrinkage, with varying degrees of faultoccurred; in group C, things became worse as time went by, however, theywere still better than those in group B, but they were worse than those ingroup A.2Masson staining: in group A, there was no abnormal signs; ingroup B, slight tubular expansion and increased collagenous fibers presentedon the3rdday after UUO, and these phenomenon got worse as time went by;in group C, things became worse as time went by, however, they were stillbetter than those in group B, but they were worse than those in group A.3Immunohistochemistry results⑴TGF-β1: in group A, no or only traceamount of TGF-β1expression was detected; in group B, the expression levelof TGF-β1went up in a time dependent manner, and there was a significantdifference(P<0.05) between group A and group B on the same time point; ingroup C, the expression level of TGF-β1was not as much as that in groupB(P<0.05), but it was still much higher than that of group A(P<0.05).⑵LC3: in group A, a basic expression level of LC3existed in tubular cells; ingroup B, LC3significantly increased on the3rdand the7thday compared tothe group A(P<0.05), while the expression level on7thday was lower than thaton the3rdday(P<0.05), and then it was decreased towards the basal level on the14thday after UUO; in group C, high level of expression was also detectedon the3rdand7thday after UUO, and it was even higher than that in group B,on the14thday, it decreased, but it was still higher than that of group B.⑶mTOR：in group A, a basal level of expression was presented in tubular cells;in group B, the level of expression was decreased significantly compared togroup A(P<0.05), while on the7thand the14thday, it increased in a timedependent manner(P<0.05); in group C, it expressed significantly less than ingroup A(P<0.05), as well as in group B, on the7thand the14thday, itincreased in a time dependent manner, but the expression level was still lowerthan that in group B.Conclusions:(1) In the early stage of UUO, the level of autophagy increased obviouslyin the kidney, while in the late stage of UUO, it decreased obviously, and thelevel of mTOR was just the reverse. These indicate that when the tubular cellsare lightly injured, the mTOR may be inhibited, and the autophagy activityincreased to fight with the injury caused by the obstruction to protect theobstructed kidney. In the late stage of obstruction, the tubular cells may beinjured too much to call up the autophagy, the inhibition of the mTOR may berelieved, the autophagy activity decreased, and the cells may failed inpreventing the severe damages from happening.(2) After intervention with rapamycin, the activity of autophagy increasedwhile the expression of mTOR and TGF-β1decreased which indicated thatrapamycin may helped increase the autophagy activity and relieve the renalfibrosis of the UUO rats by blocking the mTOR pathway. |
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