Mi Ra Yu

Histone deacetylase-2 is a key regulator of diabetes- and transforming growth factor-β1-induced renal injury

Excessive accumulation of extracellular matrix (ECM) in the kidneys and epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells contributes to the renal fibrosis that is associated with diabetic nephropathy. Histone deacetylase (HDAC) determines the acetylation status of histones and thereby controls the regulation of gene expression. This study examined the effect of HDAC inhibition on renal fibrosis induced by diabetes or transforming growth factor (TGF)-beta1 and determined the role of reactive oxygen species (ROS) as mediators of HDAC activation. In streptozotocin (STZ)-induced diabetic kidneys and TGF-beta1-treated normal rat kidney tubular epithelial cells (NRK52-E), we found that trichostatin A, a nonselective HDAC inhibitor, decreased mRNA and protein expressions of ECM components and prevented EMT. Valproic acid and class I-selective HDAC inhibitor SK-7041 also showed similar effects in NRK52-E cells. Among the six HDACs tested (HDAC-1 through -5 and HDAC-8), HDAC-2 activity significantly increased in the kidneys of STZ-induced diabetic rats and db/db mice and TGF-beta1-treated NRK52-E cells. Levels of mRNA expression of fibronectin and alpha-smooth muscle actin were decreased, whereas E-cadherin mRNA was increased when HDAC-2 was knocked down using RNA interference in NRK52-E cells. Interestingly, hydrogen peroxide increased HDAC-2 activity, and the treatment with an antioxidant, N-acetylcysteine, almost completely reduced TGF-beta1-induced activation of HDAC-2. These findings suggest that HDAC-2 plays an important role in the development of ECM accumulation and EMT in diabetic kidney and that ROS mediate TGF-beta1-induced activation of HDAC-2.

High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production.

Background/Aim: Increased nitric oxide (NO) generation and action have been suggested to be associated with glomerular hyperfiltration and increased vascular permeability early in diabetes. However, previous studies have primarily focused on the constitutive nitric oxide synthase (cNOS) pathway present in endothelial cells, and the role of the inducible NOS (iNOS) pathway in diabetic nephropathy has remained unclear. This study examined whether high glucose modulates NO synthesis by the iNOS pathway in rat mesangial cells. In addition, the effect of inhibition of the iNOS pathway on fibronectin production was determined to examine the role of the iNOS pathway in high glucose-induced extracellular expansion by mesangial cells. Methods: NO synthesis by the iNOS pathway was evaluated by nitrite and iNOS mRNA and protein productions. The effects of protein kinase C (PKC) inhibitor and aldose reductase inhibitor on the iNOS mRNA expression and aminoguanidine, a relatively specific inhibitor of the iNOS on fibronectin protein production were examined. Results: High 30 mM glucose concentration led to significant increases in nitrite production of rat mesangial cells upon stimulation with lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) compared with control 5.6 mM glucose concentration. Mesangial iNOS mRNA expression and protein production also increased significantly in response to high glucose. The addition of calphostin C, a PKC inhibitor, and 6-bromo-1,3-dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid, an aldose reductase inhibitor, significantly suppressed the enhancement of iNOS mRNA expression in high glucose concentration. High glucose also significantly increased fibronectin protein production of mesangial cells upon stimulation with LPS plus IFN-γ compared to control glucose. Aminoguanidine reversed this high glucose-induced fibronectin production at dose inhibiting iNOS mRNA expression. Conclusions: These results indicate that high glucose enhances cytokine-induced NO production by rat mesangial cells, and that the activation of PKC and aldose reductase pathway may play a role in this enhancement. In addition, high glucose-induced NO production by the iNOS pathway may promote extracellular matrix accumulation by mesangial cells under certain condition.

Uremia induces functional incompetence of bone marrow-derived stromal cells

BACKGROUND Chronic kidney disease (CKD) is associated with increased risk for cardiovascular diseases (CVD). We hypothesized that inadequate angiogenic response in uremic patients could result from dysfunction of bone marrow-derived stromal cells [mesenchymal stem cells (MSCs)]. METHODS We investigated whether MSCs are functionally competent in uremia induced by partial kidney ablation in C57Bl/6J mice. RESULTS Uremic MSCs showed decreased expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1 and stromal cell-derived factor (SDF)-1α, increased cellular senescence, decreased proliferation, defects in migration in response to VEGF and SDF-1α and in vitro tube formation. Interestingly, the expression of fibroblast-specific protein-1 was higher in uremic MSCs. Uremia decreased hypoxia-inducible factor-1α, VEGF and VEGFR1 expression under hypoxia and Akt phosphorylation in both basal and VEGF-stimulated states. A diminished mitogenic effect on endothelial proliferation was observed in conditioned media from uremic MSCs. In addition, intravital microscopic analysis showed decreased angiogenesis in uremic MSCs. CONCLUSION These results clearly demonstrate the functional incompetence in MSCs under uremic conditions and may significantly contribute to the disproportionately high risk for CVD in patients with CKD.

Positive Feedback Loop between Plasminogen Activator Inhibitor-1 and Transforming Growth Factor-Beta1 during Renal Fibrosis in Diabetes

Background/Aims: Plasminogen activator inhibitor (PAI)-1 is increasingly recognized as a profibrotic factor but the mechanisms are not entirely clear. The present study examined the profibrotic mechanism of PAI-1 focusing on its effect on transforming growth factor (TGF)-β1 in experimental diabetes. Methods: PAI-1 knockout (KO) mesangial cells cultured under high glucose (HG) in addition to streptozotocin-induced diabetic PAI-1 KO mice were used. Results: PAI-1 deficiency did not affect plasma glucose significantly but reduced the fractional mesangial area, fibronectin and collagen I expression in the renal cortex after 20 weeks of diabetes as well as in HG-stimulated mesangial cells along with suppression of TGF-β1 mRNA expression. PAI-1 deficiency also reduced HG-induced βig-h3, a TGF-β1-induced gene product, mRNA expression. All these losses-of-function in PAI-1 KO mesangial cells were effectively gained by recombinant PAI-1. Recombinant PAI-1-induced fibronectin and collagen I expression was abrogated by TGF-β1 receptor inhibitor or anti-TGF-β antibody suggesting that the effect of PAI-1 was mediated by TGF-β1. In a similar context, recombinant PAI-1 stimulated TGF-β1 promoter activity to the same extent as TGF-β1 itself. Conclusion: Since TGF-β1 is well known to stimulate the PAI-1 promoter, we suggest that TGF-β1 and PAI-1 together constitute a positive feedback loop in the development of renal fibrosis in diabetes.

Uremic Toxin p‐Cresol Induces Akt‐Pathway‐Selective Insulin Resistance in Bone Marrow‐Derived Mesenchymal Stem Cells

We reported a functional incompetence in mesenchymal stem cells (MSCs) under uremia, but the mechanisms have not been explored. To study the mechanisms of dysfunctional MSCs induced by uremia, we characterized insulin signaling in MSCs and investigated the effect of uremic toxin, p‐cresol, on the proangiogenic actions of insulin. In MSCs, insulin induced hypoxia‐inducible factor (HIF)−1α, vascular endothelial growth factor, and stromal cell‐derived factor 1α expressions via PI3K/Akt‐dependent pathway. MSCs treated with p‐cresol exhibited altered insulin signaling in a selective manner for insulin receptor substrate‐1/PI3K/Akt pathway, whereas ERK pathway remained active. The insulin‐induced increase of HIF‐1α was blunted by p‐cresol treatment. This Akt‐selective insulin resistance was also observed in MSCs isolated from chronic kidney disease (CKD) mice. In mice model of hindlimb ischemia, blood flow recovery, capillary density, and local production of angiogenic factors in the ischemic limb treated with CKD MSCs were significantly inferior to those promoted by control MSCs. However, modifying CKD MSCs by overexpression of HIF‐1α restored all of these changes. Taken together, these data suggest that p‐cresol contributes to insulin resistance in a selective manner for Akt pathway. This might be a biological explanation for the functional incompetence of MSCs under uremia through defects in the insulin‐induced elevation of HIF‐1α protein expression. Stem Cells 2014;32:2443–2453

Beta 2-adrenergic receptor agonists are novel regulators of macrophage activation in diabetic renal and cardiovascular complications

Macrophage activation is increased in diabetes and correlated with the onset and progression of vascular complications. To identify drugs that could inhibit macrophage activation, we developed a cell-based assay and screened a 1,040 compound library for anti-inflammatory effects. Beta2-adrenergic receptor (β2AR) agonists were identified as the most potent inhibitors of phorbol myristate acetate-induced tumor necrosis factor-α production in rat bone marrow macrophages. In peripheral blood mononuclear cells isolated from streptozotocin-induced diabetic rats, β2AR agonists inhibited diabetes-induced tumor necrosis factor-α production, which was prevented by co-treatment with a selective β2AR blocker. To clarify the underlying mechanisms, THP-1 cells and bone marrow macrophages were exposed to high glucose. High glucose reduced β-arrestin2, a negative regulator of NF-κB activation, and its interaction with IκBα. This subsequently enhanced phosphorylation of IκBα and activation of NF-κB. The β2AR agonists enhanced β-arrestin2 and its interaction with IκBα, leading to downregulation of NF-κB. A siRNA specific for β-arrestin2 reversed β2AR agonist-mediated inhibition of NF-κB activation and inflammatory cytokine production. Treatment of Zucker diabetic fatty rats with a β2AR agonist for 12 weeks attenuated monocyte activation as well as pro-inflammatory and pro-fibrotic responses in the kidneys and heart. Thus, β2AR agonists might have protective effects against diabetic renal and cardiovascular complications.