Roles And Mechanisms Of MTOR Pathway In Acute Renal Injure And Spermatogenesis

The mechanistic target of rapamycin(mTOR)is a highly conserved Ser/Thr protein kinase that forms two distinct functional complexes,mTORCl and mTORC2.Many reports showed that the mTOR pathway plays a critical role in cell growth,proliferation,survival,and metabolism,but the physiological function of mTOR in a variety of tissue cells in vivo is far from elucidated.Here,we generated tissue/cell specifically conditional knockout mice by the Cre-loxP system and studied the role and mechanism of mTOR pathway in the physiology of testis and kidney.This study was divided into two parts.The first part is to research the role and mechanism of DEPTOR,an inhibitory component of mTORC1 and mTORC2,in the renal physiology and acute renal injury.Renal proximal tubules are the important site of reabsorption and major damage site of acute kidney injury(AKI).AKI often resulting from ischemia/reperfusion,sepsis,and nephrotoxins is a major kidney disease characterized by rapid loss of renal function,leading to accumulation of metabolic wastes and imbalance of electrolytes and body fluid.DEPTOR,found in 2009,binds mTORC1 and mTORC2 and inhibits their activity,and its role in renal proximal tubule physiology and AKI has not been reported.In this study,we first analyzed the expression of DEPTOR in proximal tubules and cisplatin-induced AKI by immunohistochemistry(IHC)and western blotting.Then mouse with specifically knockout DEPTOR in the proximal tubule by PEPCK-Cre was generated to study the role of DEPTOR in AKI.IHC and western blotting results showed that DEPTOR expression was low in proximal tubules but significantly increased at late stage of cisplatin-induced AKI.No significant phenotype in development and physiology was found in mice with DEPTOR deletion in the proximal tubule.However,knockout mice indicated reduced histological damage,improved renal function,and decreased cell apoptosis in cisplatin-induced AKI,suggesting that DEPTOR knockout protects against cisplatin-induced AKI.Interestingly,IHC,immunofluorescence(IF)and western blotting showed that DEPTOR deletion in proximal tubule activated only slightly mTORC1 and mTORC2,but inhibited p-p38 MAPK expression under physiological conditions and after cisplatin treatment.p38 MAPK was reported activating and increasing renal injury by promoting TNF-α production and inflammation in AKI.Studies in knockout mice and human proximal tubule cell lines(HK-2)and HEK293 cells demonstrated DEPTOR knockout inhibited p38 MAPK activation and downstream TNF-α production.In conclusion,our results suggest that DEPTOR knockout protects against cisplatin-induced AKI by inhibition of p38 MAPK and TNF-α production.The second part was is to explore the role and mechanism of mTORC1 by deleting TSC1,a upstream inhibitor of mTORC1,in testicular development and spermatogenesis.Spermatogenesis is a continuous,cyclical and complex process accompanied by a series of cell differentiation and morphological changes,in which spermatogonia first differentiate into spermatocytes,then undergo meiosis,and finally become sperms by certain morphological changes.The proliferation and differentiation of spermatogonia are the basis of spermatogenesis,in which the role of mTORC1 is still unclear.In this study,we first analyzed the activity of mTORC1 in testis and spermatogonia during the postnatal development of mouse testes by IHC and IF,and then constructed the spermatogonia-specific Tsc1 knockout mouse model by Stra8-Cre to study the effect of mTORC1 activation in vivo on spermatogonial cell differentiation and testicular spermatogenesis.The results showed that spermatogonia display stage-dependent mTORCl activity during their postnatal development,with extremely low activity in undifferentiated spermatogonia and high activity in differentiated spermatogonia.Mutant mice with activated mTORC1 in spermatogonia by conditionally deleting Tsc1 showed decreased testicular weight,testicular developmental defects,partial spermatogenic arrest,excessive germ cell loss,sperm count reduction and subfertility.Importantly,mTORC1 activation promoted spermatogonial differentiation at the expense of germline maintenance,inducing the early depletion of germ cells,and thus impairing spermatogenesis.In summary,our study defines the critical roles of TSC/mTORC1 in the maintenance of the spermatogonial population and functions and spermatogenesis.