Denis Feliers

Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase

Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5–10 min) but delayed activation of phosphoinositide 3‐ki‐nase (PI3‐K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3‐K inhibitors or by the introduction of a dominant negative PI3‐K, indicating that in MCs, PI3‐K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase‐, lipoxyogenase‐, and cy‐tochrome P450‐dependent metabolism did not influence AA‐induced Akt/PKB activation. However, the antioxidants N‐acetylcysteine and diphenylene iodo‐nium inhibited both AA‐ and Ang II‐induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3‐K, inhibited Ang II‐induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox‐dependent pathway and Akt/PKB activation independent of PI3‐K.

Hydrogen Sulfide Inhibits High Glucose-induced Matrix Protein Synthesis by Activating AMP-activated Protein Kinase in Renal Epithelial Cells

Background: Whether hydrogen sulfide regulates protein synthesis is not known. Results: In kidney cells, hydrogen sulfide inhibited high glucose-induced synthesis of proteins including matrix proteins by activating AMP-activated protein kinase and inhibiting events in mRNA translation. Conclusion: Hydrogen sulfide reduces high glucose stimulation of matrix protein synthesis in renal cells. Significance: Hydrogen sulfide induction may inhibit kidney matrix protein accumulation in diabetes. Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H2S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

High Glucose, High Insulin, and Their Combination Rapidly Induce Laminin-β1 Synthesis by Regulation of mRNA Translation in Renal Epithelial Cells

Laminin is a glycoprotein that contributes to renal extracellular matrix expansion in diabetes. We investigated regulation of laminin-β1 synthesis in murine renal proximal tubular epithelial cells by 30 mmol/l glucose (high glucose), 1 nmol/l insulin (high insulin), and their combination (high glucose+high insulin), simulating conditions observed during progression of type 2 diabetes. Compared with 5 mmol/l glucose and no insulin (control), high glucose alone, high insulin alone, or high glucose+high insulin together increased laminin-β1 chain protein synthesis within 5 min, lasting for up to 60 min with no change in laminin-β1 mRNA levels. Cycloheximide, but not actinomycin-D, abrogated increased laminin-β1 synthesis. High glucose, high insulin, and high glucose+high insulin stimulated phosphorylation of 4E-BP1, a repressor binding protein for eukaryotic initiation factor 4E (eIF4E), that was dependent on activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin. High glucose, high insulin, and high glucose+high insulin also promoted release of eIF4E from 4E-BP1, phosphorylation of eIF4E, and increase in eIF4E association with eIF4G, critical events in the initiation phase of mRNA translation. High glucose, high insulin, and high glucose+high insulin increased Erk phosphorylation, which is an upstream regulator of eIF4E phosphorylation, and PD098059, which is a MEK inhibitor that blocks Erk activation, abolished laminin-β1 synthesis. This is the first demonstration of rapid increment in laminin-β1 synthesis by regulation of its mRNA translation by cells exposed to high glucose, high insulin, or high glucose+high insulin.

mRNA Translation: Unexplored Territory in Renal Science

Ambient protein levels are under coordinated control of transcription, mRNA translation, and degradation. Whereas transcription and degradation mechanisms have been studied in depth in renal science, the role of mRNA translation, the process by which peptide synthesis occurs according to the genetic code that is present in the mRNA, has not received much attention. mRNA translation occurs in three phases: Initiation, elongation, and termination. Each phase is controlled by unique eukaryotic factors. In the initiation phase, mRNA and ribosomal subunits are brought together. During the elongation phase, amino acids are added to the nascent peptide chain in accordance with codon sequences in the mRNA. During the termination phase, the fully synthesized peptide is released from the ribosome for posttranslational processing. Signaling pathways figure prominently in regulation of mRNA translation, particularly the phosphatidylinositol 3 kinase-Akt-mammalian target of rapamycin pathway, the AMP-activated protein kinase-tuberous sclerosis complex protein 1/tuberous sclerosis complex protein 2-Rheb pathway, and the extracellular signal-regulated kinase 1/2 type mitogen-activated protein kinase signaling pathway; there is significant cross-talk among these pathways. Regulation by mRNA translation is suggested when changes in mRNA and protein levels do not correlate and in the setting of rapid protein synthesis. Ongoing work suggests an important role for mRNA translation in compensatory renal growth, hypertrophy and extracellular matrix synthesis in diabetic nephropathy, growth factor synthesis by kidney cells, and glomerulonephritis. Considering that mRNA translation plays an important role in cell growth, development, malignancy, apoptosis, and response to stress, its study should provide novel insights in renal physiology and pathology.

Regulation of mRNA translation in renal physiology and disease

Translation, a process of generating a peptide from the codons present in messenger RNA, can be a site of independent regulation of protein synthesis; it has not been well studied in the kidney. Translation occurs in three stages (initiation, elongation, and termination), each with its own set of regulatory factors. Mechanisms controlling translation include small inhibitory RNAs such as microRNAs, binding proteins, and signaling reactions. Role of translation in renal injury in diabetes, endoplasmic reticulum stress, acute kidney injury, and, in physiological adaptation to loss of nephrons is reviewed here. Contribution of mRNA translation to physiology and disease is not well understood. Because it is involved in such diverse areas as development and cancer, it should prove a fertile field for investigation in renal science.

Apelin retards the progression of diabetic nephropathy

Apelin and its receptor APJ have pleiotropic effects in mice and humans and play a protective role in cardiovascular diseases at least partially by inhibiting oxidative stress. Our objective was to study the effect of apelin on the progression of kidney disease in mice with established type 1 diabetes. Ove26 mice with type 1 diabetes received daily subcutaneous injections of apelin for 2 or 14 wk. APJ localizes in the glomeruli and blood vessels of kidneys. Renal APJ expression was reduced in diabetic mice but increased after treatment with apelin. Apelin treatment did not affect glycemia, body weight, or blood pressure in diabetic mice. Whole kidney and glomerular hypertrophy, as well as renal inflammation, including monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression, NF-κB activation, and monocyte infiltration, was inhibited after short and long treatment with apelin. Apelin administration significantly reduced albuminuria at 6 mo. Short treatment with apelin was sufficient to reverse the downregulation of the antioxidant enzyme catalase. Expression of angiotensin II and angiotensin type 1 receptor (AT1) in kidneys from diabetic mice treated was not affected by apelin. These findings show for the first time that apelin exerts a protective effect on the diabetic kidney. Short administration is sufficient to reduce kidney and glomerular hypertrophy as well as renal inflammation, but prolonged treatment is required to improve albuminuria. This effect was independent of the activation of the renin angiotensin system but correlated with upregulation of the antioxidant catalase. Apelin may represent a novel tool to treat diabetic nephropathy.

Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1-AMPK axis.

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells.

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin‐like growth factor‐I (IGF‐I) and IGF binding protein (IGFBP) activity. IGF‐I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF‐I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP‐1 through IGFBP‐6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF‐I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP‐2 through IGFBP‐6 mRNAs and release detectable levels of IGFBP‐2 through IGFBP‐5. Because IGF‐I, platelet‐derived growth factor‐BB (PDGF‐BB), and transforming growth factor‐β (TGF‐β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF‐I induced IGFBP‐3 and IGFBP‐5 proteins in a time‐dependent manner without an increase in the corresponding mRNAs. IGFBP‐4 protein levels decreased in response to IGF‐I. TGF‐β stimulated IGFBP‐3 mRNA and protein but decreased IGFBP‐5 mRNA and protein. In contrast, PDGF‐BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP‐3 (rhIGFBP‐3) was then tested for its effect on IGF‐I‐induced mitogenesis in HSCs. rhIGFBP‐3 inhibited IGF‐I‐stimulated DNA synthesis in a dose‐dependent manner, with a peak effect observed at 25 nM IGFBP‐3. Because TGF‐β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP‐3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral‐induced chronic active hepatitis. In the majority of these samples, IGFBP‐3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF‐I and TGF‐β differentially regulate IGFBP‐3, IGFBP‐4, and IGFBP‐5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF‐I. J. Cell. Physiol. 174:240–250, 1998.

Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation

Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel–Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22phox-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22phox-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22phox-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22phox reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

Molecular events in matrix protein metabolism in the aging kidney

We explored molecular events associated with aging‐induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle‐aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age‐phase‐specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle‐aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ‐regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR‐21 and miR‐200c, but not miR‐192, miR‐200a, or miR‐200b, was increased with aging. Increased miR‐21 and miR‐200c contents were associated with reduced expression of their targets, Sprouty‐1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Age‐phase‐specific regulation of matrix protein synthesis occurs and involves matrix protein–specific transcriptional and post‐transcriptional mechanisms.