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Protein kinase B/Akt activity is involved in renal TGF-beta1-driven epithelial-mesenchymal transition in vitro and in vivo.

ABSTRACT: The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

SUBMITTER: Kattla JJ

PROVIDER: S-EPMC2494512 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Protein kinase B/Akt activity is involved in renal TGF-beta1-driven epithelial-mesenchymal transition in vitro and in vivo.

Kattla Jayesh J JJ Carew Rosemarie M RM Heljic Mediha M Godson Catherine C Brazil Derek P DP

American journal of physiology. Renal physiology 20080521 1

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment o ...[more]

PMID: 18495798

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Project description:BACKGROUND: Understanding how mesenchymal cells arise from epithelial cells could have a strong impact in unveiling mechanisms of epithelial cell plasticity underlying kidney regeneration and repair. In primary human tubular epithelial cells (HUTEC) under different TGF beta 1 concentrations we had observed epithelial-to-mesenchymal transition (EMT) but not epithelial-myofibroblast transdifferentiation. We hypothesized that the process triggered by TGFbeta 1 could be a dedifferentiation event. The purpose of this study is to comprehensively delineate genetic programs associated with TGF beta 1-driven EMT in our in vitro model using gene expression profile on large-scale oligonucleotide microarrays. RESULTS: In HUTEC under TGF beta 1 stimulus, 977 genes were found differentially expressed. Thirty genes were identified whose expression depended directly on TGF beta 1 concentration. By mapping the differentially expressed genes in the Human Interactome Map using Cytoscape software, we identified a single scale-free network consisting of 2630 interacting proteins and containing 449 differentially expressed proteins. We identified 27 hub proteins in the interactome with more than 29 edges incident on them and encoded by differentially expressed genes. The Gene Ontology analysis showed an excess of up-regulated proteins involved in biological processes, such as "morphogenesis", "cell fate determination" and "regulation of development", and the most up-regulated genes belonged to these categories. In addition, 267 genes were mapped to the KEGG pathways and 14 pathways with more than nine differentially expressed genes were identified. In our model, Smad signaling was not the TGF beta 1 action effector; instead, the engagement of RAS/MAPK signaling pathway seems mainly to regulate genes involved in the cell cycle and proliferation/apoptosis. CONCLUSION: Our present findings support the hypothesis that context-dependent EMT generated in our model by TGF beta 1 might be the outcome of a dedifferentiation. In fact: 1) the principal biological categories involved in the process concern morphogenesis and development; 2) the most up-regulated genes belong to these categories; and, finally, 3) some intracellular pathways are involved, whose engagement during kidney development and nephrogenesis is well known. These long-term effects of TGF beta 1 in HUTEC involve genes that are highly interconnected, thereby generating a scale-free network that we named the "TGF beta 1 interactome", whose hubs represent proteins that may have a crucial role for HUTEC in response to TGF beta 1.

Project description:Salvianolic Acid B (Sal B) is a water-soluble component from Danshen (a traditional Chinese herb widely used for chronic renal diseases) with anti-oxidative and cell protective properties. Sal B also has potential protective effects on renal diseases. Tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT), which plays an important role in the pathogenesis of renal interstitial fibrosis (RIF) and is mainly regulated by TGF-beta1/Smads pathway. The aims of the study are to investigate the effect of Sal B on tubular EMT in vivo and in vitro, and to elucidate its underlying mechanism against EMT related to TGF-beta1/Smads pathway.For in vivo experiments, RIF was induced in rats by oral administration of HgCl2 and prophylaxised with Sal B and vitamin E. The protein expression of E-cadherin was down-regulated, while the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3 and the activity of matrix metalloproteinase-2 (MMP-2) were up-regulated in kidneys of model rats when compared with those of normal rats. In contrast, Sal B and vitamin E significantly attenuated the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3, and MMP-2 activity, but increased E-cadherin expression. For in vitro experiments, HK-2 cells were incubated with TGF-beta1 to induce EMT, and the cells were co-cultured with 1 and 10 microM Sal B or SB-431542 (a specific inhibitor of TbetaR-I kinase). TGF-beta1 induced a typical EMT in HK-2 cells, while it was blocked by Sal B and SB-431542, as evidenced by blocking morphologic transformation, restoring E-cadherin and CK-18 expression, inhibiting alpha-SMA expression and F-actin reorganization, and down-regulating MMP-2/9 activities in TGF-beta1 mediated HK-2 cells. Furthermore, Sal B and SB-431542 profoundly down-regulated the expressions of TbetaR-I and p-Smad2/3 but prevented the decreased expression of Smad7 in TGF-beta1 stimulated HK-2 cells.Sal B can prevent tubular EMT in the fibrotic kidney induced by HgCl2 as well as HK-2 cells triggered by TGF-beta1, the mechanism of Sal B is closely related to the regulation of TGF-beta1/Smads pathway, manifested as the inhibition of TGF-beta1 expression, suppression of TbetaR-I expression and function, down-regulation of Smad2/3 phosphorylation, and restoration of the down-regulation of Smad7, as well as inhibition of MMP-2 activity.

Dataset Information

Protein kinase B/Akt activity is involved in renal TGF-beta1-driven epithelial-mesenchymal transition in vitro and in vivo.

Publications

Protein kinase B/Akt activity is involved in renal TGF-beta1-driven epithelial-mesenchymal transition in vitro and in vivo.

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