Results from this investigation support a role for Ang II in eliciting PTC injury and mTOR-associated loss of PTC adhesion and tubulointerstitial fibrosis. Our current observations extend evidence in smooth muscle cells, cardiac tissue, and skeletal muscle cells that Ang II engages mTOR/S6K1 through a PI3-K/Akt-dependent pathway [8
]. Herein, we report that in the transgenic Ren2 model, a model of increased tissue Ang II and proteinuria, there were increases in renal tissue levels of NADPH oxidase and dependent increases in reactive oxygen species (ROS). Furthermore, it also displayed PT brush border injury with increases in KIM-1 and reductions in neprilysin with parallel increases in p-mTOR, mTOR and S6K1. Contemporaneous with these findings, there were associated reductions in the PT-specific adhesion molecule N-cadherin and ultrastructural findings of PT remodeling consistent with EMT and tubulointerstitial fibrosis. Collectively, our findings were largely improved with AT1
R blockade, supporting a role for AT1
R-mediated signaling through mTOR/S6K1 in PT injury and tubulointerstitial fibrosis.
Blockade of the AT1
R reduces NADPH oxidase activity and the Nox subunit Rac1, a small GTP-ase [10
]. Rac1 is a critical G-protein that not only contributes to activation of the enzyme complex NADPH oxidase and generation of oxidative stress in PT cells, but also functions as an important regulator of endocytosis of albumin in the PT [17
]. Our finding that AT1
R blockade reduced subunits Rac1, p47phox
and NADPH oxidase-dependent generation of 3-NT in the Ren2 further supports a role for Ang II engagement in NADPH oxidase generation of oxidant stress in the PT.
Recent data also highlight a potential role for Ang II in regulating the mTOR pathway and in PT albumin endocytosis that is mediated, in part, via regulation of megalin [11
]. Megalin is involved in albumin reabsorption through a retrieval mechanism wherein albumin is transported through an endosomal/lysosomal degradation pathway and degraded into its constituent polypeptides and amino acids [37
]. In this context, our data extend findings from PT culture models in recent studies [17
] into an in vivo model (e.g. the Ren2), where Ang II-dependent signaling through the AT1
R reduces megalin expression that occurs in conjunction with ultrastructural findings of reduced endosomal lysosomes and increases in proteinuria [25
]. Although the exact mechanism remains unclear, our data would suggest that this reduction in megalin expression may be mediated, in part, through an AT1
R-dependent signaling pathway.
In this investigation, renal cortical KIM-1 levels were increased in Ren2 rats and normalized with AT1
R blockade. Although KIM-1 was originally described to be induced in the post-ischemic kidney, tubular KIM-1 induction has also been reported recently in models of proteinuric and polycystic kidney disease [28
]. Recent data suggest that KIM-1 expression is increased in the Ren2 rats and attenuated following treatment with AT1
R blockade or mitogen-activated protein kinase inhibition [28
]. This is a particularly important finding in that mitogen-activated protein kinase is known to converge with mTOR signaling through phosphorylation of S6K1. Our data further highlight a role for Ang II in PT injury as the observed improvement in KIM-1 following AT1
R blockade was temporally related to increases in neprilysin (e.g. neutral endopeptidase), a brush border enzyme responsible for processing peptides, such as Ang II, that is reduced during injury to the PT [33
]. Our collective findings suggest that increased KIM-1 and reduced neprilysin expression represent markers of brush border injury and targets for Ang II-dependent generation of ROS and altered mTOR/S6K1 signaling in PT injury.
Targeting reductions in mTOR/S6K1 activity with rapamycin treatment improves tubulointerstitial fibrosis and proteinuria in rodent models of diabetic nephropathy and polycystic kidney disease [18
]. The activity of mTOR appears to be highly regulated under conditions of high nutrient/energy levels, hypoxia, or various hormones such as Ang II, as suggested by our study, which allow mTOR kinase activity to be turned on promoting cell phenotype transition and fibrosis. Ser2448
p-mTOR serves as a marker for downstream activation of S6K1 as well as mTOR inhibition. While our study did not include a direct inhibitor of Ser2448
p-mTOR such as rapamycin, our finding that blockade of the AT1
R in this transgenic RAS model inhibits total and Ser2448
phosphorylated mTOR further support a role for Ang II in mTOR/S6K1 signaling in the kidney.
Our observation that AT1
R blockade in the Ren2 rat also led to improvements in tubulointerstitial fibrosis temporally related to improvements in p-mTOR further support a role for Ang II in fibrosis. Ang II actions on the AT1
R have been shown to regulate collagen synthesis and extracellular matrix protein synthesis as well as through generation of ROS [29
]. Under steady state conditions, PTCs are attached to each other and to the basement membrane through specialized junctional complexes (adherens junctions) that are susceptible to ROS and include molecules such as cadherin. Recent data suggest that the cadherin present in the kidney [23
] and specific to the PT is N-cadherin [23
]. N-cadherin in the PT has been shown to bind cytoskeletal components that provide a structural foundation for adherens junctions. Of note, cadherins not only function as static structural components of adherens junctions but also play a role in cell-signaling pathways [42
] suggesting that cadherins may be a target of Ang II and mTOR signaling. Our finding that AT1
R blockade improves the PT-specific N-cadherin in the Ren2, support a role for Ang II activation of mTOR/S6K1 and loss of N-cadherin in association with EMT and tubulointerstitial fibrosis.
Recent evidence suggests EMT as a potential initial mechanism for tubulointerstitial fibrosis in human and other species [1
]. Our findings that loss of N-cadherin occurred contemporaneous with ultrastructural remodeling of the basilar region of the PT in the Ren2 rats is consistent with structural changes of EMT and fibrosis. The finding that these changes were improved with AT1
R blockade supports a potential role for Ang II. Although our findings are ultrastructural in nature and not related to the alterations in EMT transcriptome. Ultrastructural changes of early-stage EMT are characterized by loss of basal polarity and basement membrane thickening on ultrastructural analysis [2
]. Our observations in the Ren2 kidney of reductions in basilar lysosomes with parallel loss in basal polarity, mitochondrial fragmentation, basement membrane thickening and canalicular infoldings of the plasma membrane with improvements following AT1
R blockade support a direct role for Ang II actions on loss of N-cadherin and promotion of structural changes consistent with EMT and tubulointerstitial fibrosis.
Thereby, this preclinical investigation highlights a novel mechanism in eliciting tubulointerstitial fibrosis. The clinical/translational importance of our study resides in the identification of an AT1R-mediated mechanism at an early juncture in the development of tubulointerstitial fibrosis, a process the dictates progressive kidney disease.