Transforming Growth Factor β1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis

Abstract

TGF-␤ is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-␤1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-␤1-induced apoptosis in immortalized podocytes. TGF-␤1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-␤ receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTOR⌬ augmented TGF-␤1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-␤1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-␤1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-␤1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-␤1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-␤1-induced ERK1/2-mTOR activation. Our data suggest that TGF-␤1 increases translation ofNox4throughtheSmad-ERK1/2-mTORC1axis,whichisindependent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic kidney diseases.Podocyte damage is one of the key determinants of the majority of diabetic and non-diabetic glomerular diseases, leading to chronic kidney diseases and end-stage renal disease (1). Leakage of plasma proteins in the urine indicates the onset of renal diseases that are associated with podocyte injury (2-4). Recent evidence has shown that mammalian target of rapamycin (mTOR) 3 signaling activation is an important mediator of diabetic nephropathy in mice and humans (5, 6). Increased mTOR activity has been reported in different glomerular diseases, and mTORC1 inhibition by rapamycin and everolimus shows beneficial effects against diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranous nephropathy, etc. (7-10). But contrasting evidence also indicates that mTORC1 inhibition by the immunosuppressive drug rapamycin leads proteinuria and podocyte apoptosis and develops primary focal segmental glomerulosclerosis in renal transplant recipients (11). Therefore, it might be important to investigate the detailed molecular mechanisms to...

The MEK Inhibitor Trametinib Ameliorates Kidney Fibrosis by Suppressing ERK1/2 and mTORC1 Signaling

“…This was further corroborated by decreased phospho-S6 immunostaining observed in the kidneys of trametinib-treated animals ( Figure 3, E and 3F). In agreement with previous studies reporting Nox4 upregulation downstream of mTORC1 and ERK1/2, [40][41][42] Western blotting revealed marked induction of Nox4 protein in UUO kidneys ( Figure 3G). Trametinib significantly blunted Nox4 upregulation by 51% ( Figure 3, G and H).…”

“…Trametinib Blocked UUO-Induced mTORC1 Activation and Nox4 Overexpression mTORC1 activation downstream of TGF-b1 promotes kidney fibrosis 19 and contributes to injury by upregulating Nox4. [40][41][42] As previously reported, 19 significant phosphorylation of the key downstream mTORC1 effectors p70S6 kinase (P70S6K) and eIF4E binding protein-1 (4E-BP1) 43 was observed upon UUO ( Figure 3, A-C). In contrast, in trametinib-treated animals mTORC1 activation was significantly suppressed, as exemplified by the reduction of phospho-P70S6K and phospho-4E-BP1 levels by 80% and 81%, respectively ( Figure 3, A-C).…”

“…Nox4 is a major superoxide source during kidney pathology 55 and is induced by TGF-b1 in kidney fibroblasts 41 and podocytes. 40,42 Trametinib ameliorated Nox4 upregulation in vivo ( Figure 3G). This supports our finding that ERK1/2 inhibition suppressed mTORC1 activation in the UUO kidney because Nox4 overexpression occurs downstream of mTORC1 activation.…”

“…19,53 ERK1/2 can activate mTORC1, probably by phosphorylating upstream regulators in cells, 23 including kidney podocytes. 40 However, whether ERK1/2 modulates mTORC1 activity in kidney fibroblasts had not been previously reported. The effect of trametinib on mTORC1 is unlikely to be due to offtarget effects because it is specific for MEK1/2, with minimal activity against a panel of 180 other kinases, including mTORC1.…”

AT2R deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein (Hhip) gene expression

“…Evidence of both increased oxidative stress (as ‘read’ by DHE staining and Nox4 expression) and activation of TGFβ1 signaling was present in the kidneys of AT 2 RKO mice, as reported elsewhere . Although the mechanisms of ROS‐induced apoptosis and/or TGFβ1‐related epithelial–mesenchymal transition (EMT) have been well recognized in the pathogenesis of podocyte loss/dysfunction , and TGFβ1 can upregulate mitochondrial Nox4 in cultured podocytes, promoting apoptosis , it is unclear whether AT 2 R deficiency directly or indirectly mediates increased ROS and TGFβ1 activation, resulting in the podocyte phenotypes seen in AT 2 RKO mice. Previously, we established that the ROS, Hhip, and TGFβ1 signaling cross‐talk mediates impaired renal development in the progeny of diabetic dams .…”