Role And Mechanisms Of Rictor/mTORC2 In Acute And Chronic Kidney Diseases

Objective:The mammalian target of rapamycin (mTOR) plays a critical role in cellular growth, proliferation, protein synthesis, autophagy, metabolism and cell survival in many cell types. While substantial progress has been made in understanding the abnormal activation of mTORC1 in the pathogenesis of kidney diseases, little is known about mTORC2 in renal diseases. Recent studies showed the downstream targets of mTORC2 contribute to the regulation of diverse cellular processes including cell survival, metabolism and cell differentiation,which play an important role in a number of kidney diseases. The aims of our study were to explore the role and mechanisms of Rictor/mTORC2 in kidney diseases including acute kidney injury (AKI) and chronic kidney disease(CKD).Methods:(1)To clarify the role and mechanisms of mTORC2 signaling in kidney tubular epithelial cell survival and AKI, NRK-52E cells were treated with cisplatin for different time, western blotting was used to detect the activation of mTORC2 pathway. NRK-52E cells were transfected with rictor siRNA to downregulate rictor expression.Western blot assay for anti-cleaved caspase 3 and TUNEL or anti-cleaved caspase3 staining were used to identify the apoptotic cells in NRK-52E cells. Cells were co-transfected with scramble or Rictor siRNA and GFP-LC3 expression plasmid to detect autophagosome formation in NRK-52E cells during cisplatin treatment.We used western blot assay to detect LC3-II abundance in NRK-52E cell. To study the role of Rictor/mTORC2 in vivo, first, we examined the expression of Rictor in the kidney after cisplatin injection in mice. A mouse model with tubule specific deletion of Rictor (Tubule-Rictor-/-) was generated. To induce the AKI model in vivo, Tubule-Rictor-/-or Tubule-Rictor+/+mice were injected with a single dose of cisplatin intraperitoneally. We examined the kidney function or kidney histological change from the control and knockouts under normal physiological conditions. We measured the blood urea and creatinine level between Tubule-Rictor-/-and their control littermates at day 3 after cisplatin injection. PAS staining was also used to evaluate the histological change between the two groups. TUNEL staining and anti-cleaved caspase 3 immunohistochemical staining were used to identify the apoptotic cells in the kidney tissue. Anti-PCNA and Ki67 immunohistochemical staining were used to identify cell proliferation in the kidney tissue. We stained kidney tissue with anti-ly6b to identify neutrophil and anti-F4/80 to identify macrophage in the kidney tissue. We examined p-Akt (Ser473) expression in the kidney tissue between Tubule-Rictor-/- and their control littermates at day 2 after cisplatin injection with western blot assay and immunofluorescent staining. We stained the kidney tissue with anti-LC3β to detect autophagy activation between the two groups, transmissive electronic microscropy was also used to further assess the autophagosome formation.(2) To clarify the role and mechanisms of mTORC2 signaling in kidney fibroblasts activation and CKD, NRK-49F cells were treated with TGFβ1 for different time or dosage. Western blotting and immunofluorescent staining were used to detect the activation of Rictor/mTORC2 pathway. NRK-49F cells were pretreated with scramble or Rictor siRNA, followed by TGF β 1 treatment for different time. We used western blot assay or immunofluorescent staining to detect the expression of FN,a-SMA,p-Akt(Ser473) and p-S6. NRK-49F cells were pretreated with scramble, Aktl or Akt2 siRNA for 24 h, followed by TGF β1 treatment. We used western blot assay and immunofluorescent staining to detect the expression of FN and a-SMA. Western blotting and immunostaining were used to detect the induction of Rictor/mTORC2 pathway in the kidneys with unilateral ureter obstruction (UUO) nephropathy. We generated a mouse model with fibroblast-specific deletion of Rictor gene by utilizing the Cre-LoxP system. We detected kidney function or kidney histological change from the control and knockouts under normal physiological conditions. We examined the expression of p-Akt (Ser473), p-Akt (Thr308) and p-Smad3 with western blot assay in the Fibro-Rictor+/+or Fibro-Rictor-/-kidneys at day 14 after UUO. Periodic acid-Schiff (PAS),Masson and Sirius red staining were also used in the kidneys at day 14 after UUO. Fibronectin, a-SMA, and type I collagen expression were examined by western blot assay and immunostaining. TUNEL staining was used to identify apoptotic cells in kidney tissue. We stained kidney tissue with anti-F4/80 to identify macrophage in the kidney tissue.Results:(1) In cultured NRK-52E cells, cisplatin could activate mTORC2 pathway. Rictor siRNA transfection sensitized cell apoptosis to cisplatin while reduced cisplatin-induced autophagy. Metformin could induce autophagy induction and abolish cell death induced by Rictor siRNA and cisplatin administration. In vivo study showed that rictor was slightly increased but did not reach statistical difference in kidney tissue at day 2 after cisplatin injection. The knockouts were born normal and no obvious kidney dysfunction or kidney morphologic abnormality was found within 2 months after birth. Cisplatin-induced AKI was generated in the mice and ablation of Rictor in the tubular cells exacerbated cisplatin-induced AKI compared to those in the control littermates. Tubular cell apoptosis, Akt phosphorylation (Ser473) as well as autophagy were induced in the kidneys from the control littermates with cisplatin-induced AKI. Less cell autophagy or Akt phosphorylation and more cell apoptosis in the knockout kidneys were identified compared with those in the control littermates.(2) Here, we found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured NRK-49F cells, a rat kidney fibroblast cell line, with a time and dose-dependent manner. Blocking Rictor/mTORC2 signaling with either Rictor, Akt1 or Akt2 small interfering RNA transfection markedly inhibited TGFβ1-induced fibronection and a-SMA expression. In addition, Western blotting or immuno-staining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureter obstruction (UUO) nephropathy. To further investigate the role of Rictor/mTORC2 signaling in fibroblast activation and kidney fibrosis, a mouse model with fibroblast-specific deletion of Rictor was generated with Cre/LoxP system.The knockouts were born normal and no obvious kidney dysfunction or kidney morphologic abnormality was found within 2 months after birth. However, kidney interstitial fibrosis and inflammatory cell infiltration were markedly diminished in the knockouts at 1 and 2 weeks after UUO compared to those in their control littermates.Conclusion:(1) Rictor/mTORC2 signaling has an important role in regulating tubular cell survival and AKI. Which is probably mediated by promoting cell survival through Akt signaling activation and autophagy induction.(2)Rictor/mTORC2 signaling has an important role in mediating TGFβ1-induced fibroblast activation and kidney fibrosis. Targeting this signaling pathway may provide a new strategy for inhibiting the progression of chronic kidney diseases.