Rapamycin Promotes Podocyte Autophagy And Ameliorates Renal Injury In Diabetic Mice

Background：Diabetic nephropathy (DN), one of the most severe complications of diabetes, has beenconsidered as the main cause of end-stage renal disease. The pathological features of earlystage DN are mainly consisted of accumulation of mesangial matrix and diffused thickeningof glomerular basement membrane (GBM). To our knowledge, nodular glomerulosclerosisand renal interstitial fibrosis, served as the major histological hallmarkers, were commonlynoticed with the progression of DN. In clinical practices, DN is mainly featured by thegradual increase of urinary albumin excretion, which ultimately resulted in irreversiblerenal failure. Therefore, early intervention to diabetes-induced renal injury may contributeto the delay of DN progression.Podocyte is the major component that formed the filtration slit structure, whichprevents the escape of plasma protein from the glomerular circulation. Recently, increasingevidences indicate that a decrease in the number of podocyte is a predictor of renal diseasesuch as focal segmental glomerulosclerosis and progression of DN. Meanwhile, injury andsubsequent loss of podocytes can seriously affect the integrity of the glomerular filtrationbarrier, which eventually led to glomerulosclerosis. Subsequently, progressiveglomerulosclerosis contributes to the irreversible loss of renal function, and finally inducesend-stage renal failure. The decrease of podocyte number is associated with the limitedregenerative capacity. Stress factor such as DM can usually lead to accumulation ofdamaged protein and organelles, which finally resulted in cytotoxicity. Once these damagedsubstances could not be removed, apoptosis or necrosis may be triggered. This processcontributed to irreversible podocyte injury and functional disorder, which finally resulted inpodocyte defect. Thus, we speculated that a stable number of podocytes may represent aneffective method to delay the progression of DN.Autophagy, a process named by the self-phagocytosis of cells, is an importantmechanism for intracellular degradation that is highly conserved in higher eukaryotes to maintain the stability of the intracellular environment. During the autophagy, the damagedprotein and organelles were removed through the lysosomes, and transferred the degradedsubstances to cytoplasm for recycling. Under normal conditions, the autophagy in thepodocytes is comparatively high. However, studies indicated that autophagy defects inpodocytes are closely related with triggering proteinuria and glomerulosclerosis. Therefore,autophagy is important in stabilizing the podocyte count and attenuation of the podocyteinjury.Mammalian target of rapamycin (mTOR) is of vital importance for the autophagysignaling pathway. As a serine/threonine protein kinase, mTOR could inhibit the formationof autophagosome as a negative regulator. To date, several factors have been confirmed toaffect the autophagy through modulating the mTOR signal pathway. Recent studiesindicated that mTOR activation in podocytes in crucial for the initiation and developmentof DN. Accordingly, significant inhibition of DN progress is obtained through attenuatingthe activation of mTOR in podocytes. To our knowledge, no study has been carried out toinvestigate the association between mTOR, autophagy in podocytes and the progression ofDN. Additionally, the activation of mTOR that functioned as a negative regulator ofautophagy could inevitably lead to attenuated autophagy in podocytes. On this basis, wespeculate that the initiation of DN may be associated with the injury and defect ofpodocytes induced by decreased autophagy mediated by activation of mTOR signalpathway. Additionally, we hope to identify a potential target for treatment of DN throughmaintenance of autophagy balance and inhibition the mTOR activation.Rapamycin, an FDA approved drug that has been clinical used to inhibit rejection afterorgan transplantation, could specifically bind the mTOR and inhibit the activity of mTORprotein kinase, which subsequently induce the activation of autophagy. In this study, we aimto investigate the effects of rapamycin on podocytes autophagy and renal damage instreptozotocin (STZ)-induced type1diabetic mice. Our results show that intraperitonealinjection of rapamycin reduces urinary albumin excretion, ameliorates the podocyte injury,and delays the progression of DN, which may be related to the promotion of podocyteautophagy and the inhibition of podocyte apoptosis.Method：1. Healthy male Balb/c mice were divided into healthy control group (n=12); diabetes mellitus (DM) group (n=12), which were subjected to intraperitoneal injection of STZ; andDM+Rapa group (n=12), which received intraperitoneal injection of rapamycin(2mg/kg/48h) after induction of DM.2. The blood glucose and body weight were measured at0,2,4,8, and12weeks afterinjection of rapamycin. Levels of urinary albumin (UA), Urinary albumin creatinine(UACR), serum creatinine (Scr), blood urea nitrogen (BUN), and kidney weight/bodyweight were measured at week12.3. Renal pathologic changes, size and number of podocytes autophagy and podocyteinjury were investigated by PAS staining, transmission electron microscopy andimmunoflurescence staining, respectively at week12. Western blot was performed todetermine the expression of LC3(a podocyte autophagy marker), p-mTOR, p-p70S6K (atarget protein in the downstream of mTOR), bax and caspase-3protein.Immunofluorescence staining,Westernblot blot and immunohistochemistry wererespectively used to detect the expression changes of Nephrin, Podocin and WT-1(markersof podocyte) to evaluate the number of glomerular podocytes.Results：1. The blood gluose, UACR, kidney weight/body weight showed significant increasein DM mice compared with those of control group. Nevertheless, in the DM+Rapa group,significant decrease was noted in the urinary albumin and kidney weight/body weightcompared with the DM group. Meanwhile, no change of the blood glucose was noted in thediabetic mice after interference of rapamycin.2. PAS staining demonstrated thickening of glomerular basement membrane andexcessive accumulation of mesangial matrix in DM mice. Immunohistochemistry of WT-1indicated significant decrease of podocytes in the renal glomerulus in DM mice. Aftertreating with rapamycin for12weeks, remarkable attenuation was noted in theaccumulation of glomerular mesangial matrix, together with amelioration of podocyteinjury, increase of podocyte number, as well as improvement in the physiological changes.3. The fusion of podocytes foot processes, glomerular basement membranethickening，the mitochondrial vacuolization and decrease of autophagosomes in podocytesof the DM mice were observed by electron microscopy at week12.About12weeks afterinjection of rapamycin, significant attenuation was noted in the podocyte injury in DM mice together with increase of autophagosomes number.4. Western blot assay indicated LC3-Ⅱ/LC3-Ⅰ(a marker of autophagy activation)decreased in podocytes in DM group, and at the same time, significant upregulation ofp-mTOR and p-p70S6K protein. Immunoflurescence staining indicated remarkabledownregulation of LC3was noted in the podocytes in DM mice. However, thephenomenon was reversed after injection of rapamycin at week12, which indicated thatrapamycin could enhance autophagy in podocytes through inhibiting the activation ofmTOR signal pathway.5. Western blot assay indicated remarkable decrease was noted in the expression ofnephrin and podocin in the podocytes in DM mice. In addition, significant increase wasnoticed in the bax and caspase-3expression. Immunoflurescence staining revealeddownregulation of nephrin protein in podocytes. Immunohistochemistry assaydemonstrated remarkable decrease of WT-1postive podocytes. The phenomenonabovementioned was reversed after injection of rapamycin, which indicated that rapamycinplayed an important role in the maintenance of podocyte number and the inhibition ofpodocyte apoptosis.Conclusions1. Significant podocyte injury and loss was shown in mice with DM, among withreduced autophagy activity in podocytes2. Rapamycin can significantly alleviate urinary albumin excrtion, amelioratepodocyte injury, and delays the progression of DN. we believe that the effect of rapamycinon DN is not associated with the modulation of blood glucose.3. The protective effect of rapamycin against DN progression may be associated withthe increase of the autophagy activity and inhibition of apoptosis of podocytes.