Ras homolog enriched in brain (Rheb) is a small GTPase that regulates cell growth, differentiation, and survival by upregulating mammalian target of rapamycin complex 1 (mTORC1) signaling. The role of Rheb/ mTORC1 signaling in the activation of kidney fibroblasts and the development of kidney fibrosis remains largely unknown. In this study, we found that Rheb/mTORC1 signaling was activated in interstitial myofibroblasts from fibrotic kidneys. Treatment of rat kidney interstitial fibroblasts (NRK-49F cell line) with TGFb1 also activated Rheb/mTORC1 signaling. Blocking Rheb/mTORC1 signaling with rapamycin or Rheb small interfering RNA abolished TGFb1-induced fibroblast activation. In a transgenic mouse, ectopic expression of Rheb activated kidney fibroblasts. These Rheb transgenic mice exhibited increased activation of mTORC1 signaling in both kidney tubular and interstitial cells as well as progressive interstitial renal fibrosis; rapamycin inhibited these effects. Similarly, mice with fibroblast-specific deletion of Tsc1, a negative regulator of Rheb, exhibited activated mTORC1 signaling in kidney interstitial fibroblasts and increased renal fibrosis, both of which rapamycin abolished. Taken together, these results suggest that Rheb/mTORC1 signaling promotes the activation of kidney fibroblasts and contributes to the development of interstitial fibrosis, possibly providing a therapeutic target for progressive renal disease.

The mammalian target of rapamycin (mTOR) plays a critical role for cell growth and survival in many cell types. While substantial progress has been made in understanding the abnormal activation of mTORC1 in the pathogenesis of kidney disease, little is known about mTORC2 in kidney disease such as acute kidney injury (AKI). To study this, we generated a mouse model with tubule-specific deletion of Rictor (Tubule-Rictor-/-). The knockouts were born normal and no obvious kidney dysfunction or kidney morphologic abnormality was found within 2 months of birth. However, ablation of Rictor in the tubular cells exacerbated cisplatin-induced AKI compared to that in the control littermates. As expected, tubular cell apoptosis, Akt phosphorylation (Ser473), and autophagy were induced in the kidneys from the control littermates by cisplatin treatment. Less cell autophagy or Akt phosphorylation and more cell apoptosis in the kidneys of the knockout mice were identified compared with those in the control littermates. In NRK-52E cells in vitro, Rictor siRNA transfection sensitized cell apoptosis to cisplatin but with reduced cisplatin-induced autophagy. Metformin, an inducer of autophagy, abolished cell death induced by Rictor siRNA and cisplatin. Thus, endogenous Rictor/mTORC2 protects against cisplatin-induced AKI, probably mediated by promoting cell survival through Akt signaling activation and induction of autophagy.

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Podocytes play a key role in the formation of cellular crescents in experimental and human diseases. However, the underlying mechanisms for podocytes in promoting crescent formation need further investigation. Here, we demonstrated that mammalian target of rapamycin complex 1 (mTORC1) signaling was remarkably activated and hypoxia-inducible factor (HIF) 1α expression was largely induced in cellular crescents from patients with crescentic glomerular diseases. Specific deletion of Tsc1 in podocytes led to mTORC1 activation in podocytes and kidney dysfunction in mice. Interestingly, 33 of 36 knockouts developed cellular or mixed cellular and fibrous crescents at 7 wk of age (14.19 ± 3.86% of total glomeruli in knockouts vs. 0% in control littermates, n = 12–36, P = 0.04). All of the seven knockouts developed crescents at 12 wk of age (30.92 ± 11.961% of total glomeruli in knockouts vs. 0% in control littermates, n = 4–7, P = 0.002). Most notably, bridging cells between the glomerular tuft and the parietal basement membrane as well as the cellular crescents were immunostaining positive for WT1, p-S6, HIF1α, and Cxcr4. Furthermore, continuously administrating rapamycin starting at 7 wk of age for 5 wk abolished crescents as well as the induction of p-S6, HIF1α, and Cxcr4 in the glomeruli from the knockouts. Together, it is concluded that mTORC1 activation in podocytes promotes cellular crescent formation, and targeting this signaling may shed new light on the treatment of patients with crescentic glomerular diseases.

Cyclosporin A (CsA) is a calcium antagonist and can enhance the efficacy of some protein drugs, but its mechanism remains unknown. In this study, MAP30, a ribosome-inactivating protein reported to have apoptotic effects on cancer cells, was fused with S3, an epidermal growth factor receptor (EGFR)-targeting peptide. In addition, CsA was used to investigate whether it can further promote the apoptotic effects of S3 fused MAP30 (MAP30-S3). Our result showed that the internalization of FITC-labeled MAP30-S3 was increased significantly by S3 in HeLa cells. Unexpectedly, MAP30-S3 only showed a minor decrease in the viability of EGFR-overexpressing cancer cells, including HeLa, SMMC-7721, and MGC803 (IC50>5 μmol/l). However, 2 μmol/l CsA significantly increased the cytotoxicity of MAP30-S3, especially for HeLa cells (IC50=40.3 nmol/l). In comparison, CsA did not further decrease the cytotoxicity of MAP30-S3 on MRC-5, an EGFR low-expressing cell line from normal lung tissue, indicating that CsA did not affect the cancer-targeting specificity of MAP30-S3. Our results also showed that CsA further increased the apoptotic activity of MAP30-S3 in HeLa cells. CsA could promote the endosomal escape of FITC-MAP30-S3 with a diffused pattern in the cytoplasm. Five endocytic inhibitors were used to investigate the cellular uptake mechanism of MAP30-S3, and the results showed that the endosomal escape-enhancing effect of CsA on MAP30-S3 may be associated with the clathrin-dependent endocytic pathways. Our study suggested that CsA could be a novel endosomal escape enhancer to potentiate the intracellular release of anticancer protein drugs, resulting in their improved therapeutic efficacy.