Rictor/mTORC2 signaling mediates TGFβ1-induced fibroblast activation and kidney fibrosis

The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases. The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases. Kidney interstitial fibrosis is one of the major pathological features for chronic kidney diseases. Fibroblasts in the kidneys have an essential role in maintaining the homeostasis of interstitial matrix and adjacent tissue under physiological condition. Although all cell types in the kidneys are involved in the pathogenesis of kidney fibrosis, fibroblasts which acquire the phenotype of myofibroblast and generate a large amount of interstitial matrix upon activation are the principal matrix-producing cells.1.Sharma S.K. Zou H. Togtokh A. et al.Burden of CKD, proteinuria, and cardiovascular risk among Chinese, Mongolian, and Nepalese participants in the International Society of Nephrology screening programs.Am J Kidney Dis. 2010; 56: 915-927Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 2.Liu Y. Cellular and molecular mechanisms of renal fibrosis.Nat Rev Nephrol. 2011; 7: 684-696Crossref PubMed Scopus (924) Google Scholar, 3.Duffield J.S. Cellular and molecular mechanisms in kidney fibrosis.J Clin Invest. 2014; 124: 2299-2306Crossref PubMed Scopus (425) Google Scholar, 4.Boor P. Ostendorf T. Floege J. Renal fibrosis: novel insights into mechanisms and therapeutic targets.Nat Rev Nephrol. 2010; 6: 643-656Crossref PubMed Scopus (469) Google Scholar The 'target of rapamycin' (TOR) is a serine/threonine kinase that controls cell metabolism, growth, proliferation, survival, and aging. Mammalian TOR kinase (mTOR) exists in two structurally and functionally distinct protein complexes, named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).5.Johnson S.C. Rabinovitch P.S. Kaeberlein M. mTOR is a key modulator of ageing and age-related disease.Nature. 2013; 493: 338-345Crossref PubMed Scopus (1114) Google Scholar, 6.Delgoffe G.M. Pollizzi K.N. Waickman A.T. et al.The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2.Nat Immunol. 2011; 12: 295-303Crossref PubMed Scopus (825) Google Scholar, 7.Laplante M. Sabatini D.M. mTOR signaling at a glance.J Cell Sci. 2009; 122: 3589-3594Crossref PubMed Scopus (1642) Google Scholar A number of studies demonstrated that mTORC1 signaling has an important role in several forms of kidney disease.8.Inoki K. 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Steinhardt F. et al.mTORC1 maintains renal tubular homeostasis and is essential in response to ischemic stress.Proc Natl Acad Sci USA. 2014; 111: E2817-E2826Crossref PubMed Scopus (68) Google Scholar Our and the other's studies report that mTORC1 signaling is linked to the fibrogenic process through promoting fibroblast activation.13.Jiang L. Xu L. Mao J. et al.Rheb/mTORC1 signaling promotes kidney fibroblast activation and fibrosis.J Am Soc Nephrol. 2013; 24: 1114-1126Crossref PubMed Scopus (63) Google Scholar,14.Goc A. Choudhary M. Byzova T.V. et al.TGFbeta- and bleomycin-induced extracellular matrix synthesis is mediated through Akt and mammalian target of rapamycin (mTOR).J Cell Physiol. 2011; 226: 3004-3013Crossref PubMed Scopus (45) Google Scholar MTORC2 consists of mTOR kinase, Rictor, mSIN1, Protor, mLST8, and DEPTOR. Among them, Rictor, acting as a scaffold protein, facilitates both the assembly of mTORC2 and the interaction of mTORC2 with its substrates and regulators. Ablation of Rictor diminishes mTORC2 signaling. MTORC2 may control cell survival, actin cytoskeleton organization, and other processes through phosphorylating several members of the AGC kinase family, including Akt, SGK1, and protein kinase C (PKC).6.Delgoffe G.M. Pollizzi K.N. Waickman A.T. et al.The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2.Nat Immunol. 2011; 12: 295-303Crossref PubMed Scopus (825) Google Scholar, 7.Laplante M. Sabatini D.M. mTOR signaling at a glance.J Cell Sci. 2009; 122: 3589-3594Crossref PubMed Scopus (1642) Google Scholar, 15.Cybulski N. Hall M.N. TOR complex 2: a signaling pathway of its own.Trends Biochem Sci. 2009; 34: 620-627Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 16.Goncharova E.A. Goncharov D.A. Li H. et al.mTORC2 is required for proliferation and survival of TSC2-null cells.Mol Cell Biol. 2011; 31: 2484-2498Crossref PubMed Scopus (101) Google Scholar, 17.Kocalis H.E. Hagan S.L. George L. et al.Rictor/mTORC2 facilitates central regulation of energy and glucose homeostasis.Mol Metab. 2014; 3: 394-407Abstract Full Text Full Text PDF Scopus (50) Google Scholar, 18.Facchinetti V. Ouyang W. Wei H. et al.The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C.EMBO J. 2008; 27: 1932-1943Crossref PubMed Scopus (410) Google Scholar, 19.Gu Y. Lindner J. Kumar A. et al.Rictor/mTORC2 is essential for maintaining a balance between beta-cell proliferation and cell size.Diabetes. 2011; 60: 827-837Crossref PubMed Scopus (121) Google Scholar Our previous study reports that Rictor/mTORC2 signaling is crucial for protecting against cisplatin-induced tubular epithelial cell death.20.Li J. Xu Z. Jiang L. et al.Rictor/mTORC2 protects against cisplatin-induced tubular cell death and acute kidney injury.Kidney Int. 2014; 86: 86-102Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar MTORC2 pathway is reported to be an essential downstream branch of TGFβ1 signaling and represents a responsive target to inhibit cancer cell epithelial-to-mesenchymal transition.21.Lamouille S. Connolly E. Smyth J.W. et al.TGF-beta-induced activation of mTOR complex 2 drives epithelial-mesenchymal transition and cell invasion.J Cell Sci. 2012; 125: 1259-1273Crossref PubMed Scopus (240) Google Scholar Blocking mTORC2 signaling with MLN0128, an active site dual mTOR inhibitor, could largely inhibit TGFβ1-induced lung fibroblast activation and lung fibrosis.22.Chang W. Wei K. Ho L. et al.A critical role for the mTORC2 pathway in lung fibrosis.PLoS One. 2014; 9: e106155Crossref PubMed Scopus (41) Google Scholar Together, it is highly possible that Rictor/mTORC2 signaling have an important role in mediating TGFβ1-induced renal fibroblast activation and kidney fibrosis. In this study, we found that TGFβ1 could induce Rictor/mTORC2 signaling activation in NRK-49F cells, and blocking this signaling inhibited TGFβ1-induced fibroblast activation. Mice with fibroblast-specific deletion of Rictor are phenotypic normal, and less kidney fibrosis could be detected in the knockout kidneys compared with those in the control littermates after unilateral ureter obstruction (UUO). NRK-49F cells, a rat kidney interstitial fibroblast cell line, were treated with TGFβ1 (2 ng/ml) for different time duration and dosage as indicated. As shown in Figure 1a and b, Rictor expression was induced at as early as 15 min, reached peak from 0.5 to 6 h and declined at 12 h after treatment. Following the induction of Rictor, the abundance of p-Akt (Ser473) began to increase at 3 h and reached peak from 6 to 24 h. Phosphorylated SGK1 was induced at a similar pattern with p-Akt (Ser473). The p-Akt (Thr308) abundance was continuously increased within 48 h after TGFβ1 treatment. NRK-49F cells were also treated with TGFβ1 at different dosage as indicated and harvested at 6 h after treatment. The western blotting analyses revealed that TGFβ1 could upregulate Rictor, p-Akt (Ser473), and p-Akt (Thr308) expression in a dose-dependent manner (Figure 1c). Immunofluorescent staining results showed that Rictor protein was increased in NRK-49F cells and mainly located in the cytosol at 6 h after TGFβ1 treatment (Figure 1d). TGFβ1 could induce the expression of Rictor mRNA at 0.5 and 1 h after treatment (Figure 1e). Together, it is concluded that TGFβ1 may activate mTORC2 signaling in kidney fibroblasts. The role for Rictor/mTORC2 signaling in TGFβ1-induced fibroblast activation was then investigated. NRK-49F cells were transfected with scramble or Rictor small interfering RNA (siRNA). As shown in Figure 2a, at 24 h after transfection, Rictor protein expression was downregulated about 55% in cells transfected with Rictor siRNA compared with those transfected with scramble siRNA. To investigate the role for Rictor on TGFβ1-induced Akt phosphorylation, NRK-49F cells transfected with scramble or Rictor siRNA were treated with TGFβ1 for 3 and 6 h. As shown in Figure 2b, TGFβ1 treatment could largely increase p-Akt (Ser473), p-Akt (Thr308), and p-S6 abundance in cells transfected with scramble siRNA, whereas in Rictor siRNA-transfected cells, the abundance for p-Akt (Ser473) but not p-Akt (Thr308) or p-S6 was largely diminished, suggesting that the induction of p-Akt (Ser473) was dependent on Rictor in NRK-49F cells. To further determine the induction of Rictor on TGFβ1-induced fibroblast activation, NRK-49F cells transfected with different sets of the siRNA were treated with TGFβ1 (2 ng/ml) for 48 h. As shown in Figure 2c and d, TGFβ1 could remarkably induce fibronectin (FN) and α-smooth muscle actin (α-SMA) expression in scramble siRNA-transfected cells, while Rictor siRNA transfection could markedly decrease their expression. Immunofluorescent staining further confirmed western blotting results for FN and α-SMA in NRK-49F cells (Figure 2e). We also detected the mRNA abundance of FN and α-SMA in NRK-49F cells by real-time PCR assay. The induction of FN and α-SMA mRNA expression after TGFβ1 treatment was decreased in Rictor siRNA-transfected cells (Figure 2f). Knocking down Rictor could not affect Smad3 phosphorylation induced by TGFβ1 treatment, suggesting that the profibrotic role of Rictor in fibroblast is independent of Smad3 phosphorylation (Supplementary Figure S1). Download .jpg (.06 MB) Help with files Supplementary Figure S1 Akt1 and Akt2 but not Akt3 mRNA are expressed in the kidneys,23.Tschopp O. Yang Z.Z. Brodbeck D. et al.Essential role of protein kinase B gamma (PKB gamma/Akt3) in postnatal brain development but not in glucose homeostasis.Development. 2005; 132: 2943-2954Crossref PubMed Scopus (334) Google Scholar and mTORC2 is able to regulate the activity of both Akt1 and Akt2.24.Kim E.K. Yun S.J. Ha J.M. et al.Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis.Oncogene. 2011; 30: 2954-2963Crossref PubMed Scopus (92) Google Scholar,25.Canaud G. Bienaime F. Viau A. et al.AKT2 is essential to maintain podocyte viability and function during chronic kidney disease.Nat Med. 2013; 19: 1288-1296Crossref PubMed Scopus (173) Google Scholar To decipher the role for Akt in TGFβ1-Induced fibroblast activation, NRK-49F cells were transfected with scramble, Akt1 or Akt2 siRNA. As shown in Figure 3a and d, Akt1 or Akt2 siRNA transfection could markedly downregulate Akt1 or Akt2 protein expression, respectively, compared with those transfected with scramble siRNA. NRK-49F cells were transfected with different sets of siRNA for 24 h, followed by TGFβ1 treatment for 48 h. Similar to Rictor siRNA transfection, Akt1 siRNA transfection could markedly downregulate TGFβ1-induced FN and α-SMA expression (Figure 3b and c). However, Akt2 siRNA transfection could only significantly decrease α-SMA expression induced by TGFβ1 treatment (Figure 3e and f). Immunofluorescent staining further confirmed the results of western blotting assay (Figure 3g). The mRNA abundance for α-SMA and FN in the cells transfected with Akt1 siRNA was also significantly downregulated compared with those transfected with scramble siRNA at 24 h after TGFβ1 treatment (Supplementary Figure S2). Thus it is clear that Rictor/mTORC2/Akt signaling activation has an important role in mediating TGFβ1-induced fibroblast activation. Download .jpg (.05 MB) Help with files Supplementary Figure S2 The activation of Rictor/mTORC2 signaling in myofibroblasts was further investigated in the fibrotic kidneys from mice with UUO nephropathy. Similar to the previous report,26.Yang J. Dai C. Liu Y. Hepatocyte growth factor suppresses renal interstitial myofibroblast activation and intercepts Smad signal transduction.Am J Pathol. 2003; 163: 621-632Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar the UUO kidneys developed severe interstitial fibrosis at 7 and 14 days after operation (data not shown). Western blotting analysis was employed to identify the activation of mTORC2 signaling. As shown in Figure 4a, pronounced induction of Rictor protein and p-Akt (ser473) was detected in the fibrotic kidneys at 7 and 14 days after UUO. To identify the cell types in which Rictor was induced, an immunohistochemical staining for Rictor was deployed. As shown in Figure 4c, the staining for Rictor protein was weak in both tubular cells and interstitial cells from the sham kidneys. However, in UUO kidneys, the staining for Rictor was markedly enhanced in both tubular cells and interstitial cells. The kidney sections were then co-stained with antibodies against α-SMA and Rictor to further identify the expression of Rictor in myofibroblasts in the UUO kidneys. The results showed that very weak expression for α-SMA or Rictor was detected in the interstitial cells from the sham kidneys, while in the UUO kidneys the induction of Rictor in α-SMA-positive cells could be detected, suggesting the induction of Rictor in myofibroblasts in the UUO kidneys. Thus these results suggest that Rictor/mTORC2 signaling is activated in the myofibroblasts from the fibrotic kidneys with UUO nephropathy. To further explore the role of Rictor/mTORC2 signaling in fibroblast activation and kidney fibrosis in vivo, we generated a mouse model with fibroblast-specific deletion of Rictor gene by utilizing the Cre-LoxP system. Figure 5a shows the breeding strategy for generating the knockout mice. Mice with fibroblast-specific ablation of Rictor were named as Fibro-Rictor-/- (Figure 5b, lane 1), whereas age and gender matched Rictor-floxed littermates were considered as controls (Fibro-Rictor+/+, Figure 5b, lane 3). Western blotting analysis and immunostaining were used to examine the expression of Rictor protein in the kidneys. Rictor protein abundance was markedly reduced in the UUO kidneys from Fibro-Rictor-/- mice compared with those from control littermates (Figure 5c and d). All mice were born normal with expected Mendelian frequency. No significant difference was observed in body weight, kidney/body weight ratio, urinary albumin excretion, blood urea nitrogen or serum creatinine level between the knockouts and control littermates within 2 months after birth, respectively (Figure 5e–i). In short, Fibro-Rictor-/- mice were phenotypic normal under physiological condition. The mice were subjected to UUO and the Akt phosphorylation was analyzed by western blotting assay. As shown in Figure 5j and k, in the UUO kidneys from control littermates, Akt kinase activation exhibited as Akt phosphorylation (Ser473 and Thr308) were largely induced at day 14, whereas in the Fibro-Rictor-/- kidneys p-Akt (Ser473) abundance was much less. In addition, p-Akt (Thr308) was also significantly diminished in the knockout kidneys after UUO. Phosphorylated Smad3 was upregulated in the UUO kidneys from control littermates and was slightly decreased in the knockout kidneys. Double staining of Abs against Fsp1 and p-Akt (Ser473) showed that phosphorylated Akt at Ser473 could be detected in Fsp1-positive cells in the kidneys with UUO nephropathy, whereas p-Akt (Ser473) was negative in Fsp1-positive cells in the knockout kidneys after UUO (Figure 5l). These results suggest that deletion of Rictor in fibroblasts diminishes Akt phosphorylation at Ser473 in the fibroblasts from the UUO kidneys. The above data demonstrated that deletion of Rictor in fibroblasts diminished Akt kinase phosphorylation in the kidneys with UUO nephropathy. Kidney sections from different groups were stained with periodic acid–Schiff, Masson, or Sirius red. In the sham groups, the kidney morphology was comparable between the knockouts and controls. In the kidneys with UUO nephropathy from control littermates, remarkably tubular atrophy and interstitial extracellular matrix deposition could be found, whereas in the knockout kidneys tubular damage as well as extracellular matrix deposition were largely alleviated (Figure 6a). As shown in Figure 6b, the fibrotic area was largely increased in the control littermates with UUO nephropathy, whereas specific deletion of Rictor in fibroblast could markedly ameliorate kidney fibrosis. The total collagen content within the kidneys was significantly increased at 1 or 2 weeks after UUO from control littermates, and in the knockout kidneys, total collagen content was diminished (Figure 6c). Fibronectin, α-SMA, and type I collagen expression in the kidneys were also examined by western blotting assay and immunostaining. As shown in Figure 7a and b, FN, α-SMA, and type I collagen protein abundance were remarkably increased after UUO in control littermates, and they were significantly decreased in the knockout kidneys compared with those in the control littermates. immunostaining for FN, α-SMA, and type I collagen further confirmed the results of western blotting assay (Figure 7c). Taken together, these results demonstrate that specific deletion of Rictor in fibroblasts ameliorates fibroblast activation and kidney fibrosis after UUO operation. Our previous study demonstrated that Rictor/mTORC2 signaling has a critical role in protecting against tubular cell death induced by cisplatin treatment.20.Li J. Xu Z. Jiang L. et al.Rictor/mTORC2 protects against cisplatin-induced tubular cell death and acute kidney injury.Kidney Int. 2014; 86: 86-102Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar To investigate cell survival status in the UUO kidneys from the knockouts, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling) staining was employed. As shown in Figure 8a and b, in the sham groups, few TUNEL-positive tubular or interstitial cells could be detected. In the UUO kidneys from control littermates, at day 14 after surgery the number for TUNEL-positive tubular or interstitial cells was significantly increased, while in the UUO kidneys from the knockouts both tubular cell and interstitial cell apoptosis was significantly ameliorated compared with those in the control littermates. These results suggest that fibroblast apoptosis in the knockout kidneys is not exacerbated under physiological condition or after UUO compared with those in control littermates. Kidney interstitial macrophage infiltration is one of the most important features of chronic kidney diseases. To evaluate the macrophage infiltration in the kidneys, immunostaining for F4/80 was employed, and F4/80+ cells were counted in at least five randomly selected fields ( × 400) for each sample. As shown in Figure 9a and b, few F4/80-positive cells were detected in the sham groups. The number of F4/80-positive cell was significantly increased in the kidneys with UUO nephropathy from control littermates, while in the knockout kidneys macrophage infiltration was much less. We performed co-immunostaining of anti-Fsp1 with anti-α-SMA or with anti-F4/80 on the kidney tissues with UUO nephropathy. Immunofluorescent staining results showed that 73.7% of the Fsp1-positive cells expressed α-SMA; about 16.4% of the Fsp1-postive cells were co-stained with F4/80; and only 5.8% of the F4/80-positive cells were Fsp1 positive (Figure 9c). Fibroblasts are regarded as the principal cells that generate a large amount of matrix components that accumulate within the interstitial space upon activation.2.Liu Y. Cellular and molecular mechanisms of renal fibrosis.Nat Rev Nephrol. 2011; 7: 684-696Crossref PubMed Scopus (924) Google Scholar, 27.Eddy A.A. 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Mechanisms of tubulointerstitial fibrosis.J Am Soc Nephrol. 2010; 21: 1819-1834Crossref PubMed Scopus (667) Google Scholar It has been reported that mTORC1 signaling mediates the fibrogenic process through promoting fibroblast activation and kidney interstitial fibrosis.8.Inoki K. Role of TSC-mTOR pathway in diabetic nephropathy.Diabetes Res Clin Pract. 2008; 82: S59-S62Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13.Jiang L. Xu L. Mao J. et al.Rheb/mTORC1 signaling promotes kidney fibroblast activation and fibrosis.J Am Soc Nephrol. 2013; 24: 1114-1126Crossref PubMed Scopus (63) Google Scholar, 37.Godel M. Hartleben B. Herbach N. et al.Role of mTOR in podocyte function and diabetic nephropathy in humans and mice.J Clin Invest. 2011; 121: 2197-2209Crossref PubMed Scopus (406) Google Scholar Here we demonstrated that Rictor/mTORC2 signaling activation contributes to TGFβ1-promoted fibroblast activation independent of mTORC1 signaling. In cultured NRK-49F cells, TGFβ1 treatment could induce both mTORC1 and mTORC2 signaling activation at a similar pattern and blocking either one of them could partially interfere with TGFβ1-induced fibroblast activation. It may be concluded that the activation of both signaling are required for the full activation of fibroblast after TGFβ1 treatment. In in vivo studies, we found that phosphorylated Smad3 was slightly decreased in the knockout kidneys after UUO operation compared with those in the control littermates, which suggest that long-term ablation of mTORC2 may reduce the activity of TGFβ1 itself. However, how does TGFβ1 induce these signaling activation in kidney fibroblasts is not clear? The crosstalk between two complexes is complicated and seems on a cellular context–dependent manner. MTORC1 may impair the activation of mTORC2 by phosphorylating insulin receptor substrate-1, Grb10, or Sin1.38.Hsu P.P. Kang S.A. 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MTORC2 may stimulate mTORC1 signaling activation through phosphorylated Akt at Ser473, an activator of mTORC1 through phosphorylation of TSC2. However, in this study, we found that knocking down Rictor expression could not affect TGFβ1-induced S6 phosphorylation, suggesting that the profibrotic role of mTORC2 is independent of mTORC1 signaling in kidney fibroblast (Figure 2b). Because of the overall complexity of the context-dependent regulation and function of the mTOR pathway, and most of the results are not obtained from kidney fibroblast, further work is needed to elucidate the interaction between mTORC1 and mTORC2 in kidney fibroblast activation and kidney fibrosis. Akt kinase controls many fundamental cellular processes, such as cell proliferation and survival.20.Li J. Xu Z. Jiang L. et al.Rictor/mTORC2 protects against cisplatin-induced tubular cell death and acute kidney injury.Kidney Int. 2014; 86: 86-102Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 24.Kim E.K. Yun S.J. Ha J.M. et al.Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis.Oncogene. 2011; 30: 2954-2963Crossref PubMed Scopus (92) Google Scholar, 42.Brunet A. Bon