Unilateral ureteral obstruction (UUO)-induced kidney fibrosis was attenuated in Akt2 knockout (KO) mice.

<p>(A) Sections from control and obstructed kidneys from WT and Akt2 KO mice were stained with Masson's-modified trichrome. The blue color shows extracellular collagen deposition. (B) Immunohistochemistry for Fibronectin in unobstructed and obstructed kidneys from WT and Akt2 KO mice. Magnification: ×400. (C and D) Western blot analysis of Fibronectin and Collagen I protein expression in obstructed and unobstructed kidneys of WT and Akt2 KO mice. GAPDH was used as internal loading control. Band intensities were calculated using Scion Image software. Values are means ± SD (n = 6). ^##<i>P</i><0.01 compared to the. nonobstructive kidney from WT mice; ^*<i>P</i><0.05, ^**<i>P</i><0.01 compared to the obstructive kidney from WT mice.</p

Deactivation of GSK3β mediated activation of Snail and β-catenin in tubular epithelial cells.

<p>(A and B) Cells were treated with TGF-β or GSK3 inhibitor (LiCl 40 mmol/L). Prptein was then extracted and blotted with antibodies against Snail and β-Catenin. GAPDH was used as internal loading control. Band intensities were calculated using Scion Image software. Data are shown as mean ± SD (n = 3). ^##<i>P</i><0.01 compared to the control group; ^**<i>P</i><0.01 compared to the TGF-β1 treated group.</p

Klf4 transactivates the TGF-β1 promoter.

<p>A, ChIP assays of Klf4 binding to the −184∼−180-bp site (Klf BS1) and −45∼−41-bp site (Klf BS1) of the TGF-β1 promoter. A non-target region (GAPDH exon 2) was used as a negative control. B, normalization of each ChIP DNA fraction to the input DNA fraction using densitometric units (n = 3) was shown. Data are the mean ± SEM. *P<0.05, **P<0.01 vs. Ang II untreated and Klf4 non-overexpression. ^##P<0.01 vs. Ang II treatment. C, pGL3-luciferase reporter driven by the wild-type TGF-β1 promoter or mutant (at −184∼−180-bp and −45∼−41-bp promoter sites) in which the potential Klf4-binding site was constructed. D, promoter-reporter analysis for Klf4-depedent transactivation of the TGF-β1. The pGL3-TGF-β1 WT or mutant vector and CMV-renilla luciferase vector were cotransfected. Luciferase activation driven by the wild-type TGF-β1 promoter or mutant promoter and were normalized to renilla luciferase (n = 4). Data are the mean ± SEM. E-F, effects of neutralizing TGF-β1 on the cardiac fibroblast differentiation activity. An antibody against TGF-β1 (1 μg/ml) was added to the conditional medium. E, the effects of Klf4-dependent α-SMA mRNA expression was assessed by qPCR after Ang II treatment for 0 or 6 hrs (n = 4). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. Klf4 non-overexpression; ^#P<0.05, ^##P<0.01 vs. Klf4 overexpression. F, levels of α-SMA and p-Smad3 were assessed by Western blot after Ang II treatment for 24 hrs.</p

UUO-induced expression of Snail and β-catenin was suppressed by Akt2 knockout (KO) in vivo.

<p>(A and B) Immunohistichemistry for Snail and β-catenin in unobstructed and obstructed kidneys from WT and Akt2 KO mice. Magnification ×400. (C and D) Western blot analysis of Snail and β-catenin protein expression in unobstructed and obstructed kidneys from WT and Akt2 KO mice. GAPDH was used as internal loading control. Band intensities were calculated using Scion Image software. Data are expressed as mean ± SD (n = 6). ^##<i>P</i><0.01 compared to the nonobstructive kidney from WT mice; ^*<i>P</i><0.05 compared to the obstructive kidney from WT mice.</p

Unilateral ureteral obstruction (UUO)-stimulated epithelial-mesenchymal transition (EMT) was blocked by Akt2 knockout (KO).

<p>(A and B) Western blot analysis of p-Smad3 and vimentin protein expression in unobstructed and obstructed kidneys from WT and Akt2 KO mice. (C and D) Immunohistochemistry for E-cadherin and α-smooth-muscle actin (α-SMA) in unobstructed and obstructed kidneys from WT and Akt2 KO mice. Magnification: ×200. (E and F) Western blot analysis of E-cadherin and α-SMA protein expression in unobstructed and obstructed kidneys from WT and Akt2 KO mice. GAPDH was used as internal loading control. Band intensities were calculated using Scion Image software. Data are shown as mean ± SD (n = 6). ^#<i>P</i><0.05, ^##<i>P</i><0.01 compared to the nonobstructive kidney from WT mice; ^*<i>P</i><0.05 compared to the obstructive kidneys from WT mice.</p

Akt2 Is Involved in Loss of Epithelial Cells and Renal Fibrosis following Unilateral Ureteral Obstruction

<div><p>Obstructive nephropathy is an aggressive form of chronic kidney disease (CKD), which is characterized by an epithelial-to-mesenchymal transition (EMT) and interstitial fibrosis. However, the molecular mechanisms of EMT and fibrosis are complex and not fully understood. In this study, we investigated the contribution of Akt2 to experimental renal EMT and fibrosis using the well-established model of unilateral ureteral obstruction (UUO). We found that Akt2 and phosphor (p)-Akt protein levels were increased in the obstructed kidneys. UUO induced activation of transforming growth factor-β1 (TGF-β1) signaling. Importantly, knockout of Akt2 suppressed UUO-induced EMT, kidney fibrosis, increased GSK3β activity, and decreased expression of Snail and β-catenin. Inhibition of GSK3β with LiCl (the inhibitor of GSK3β) increased the expression of Snail and β-catenin in cultured kidney epithelial cells. Our findings suggest that Akt2 partially contributes to interstitial fibrosis following UUO and that inhibition of this signaling pathway may provide a novel approach of prevent progression of renal fibrosis.</p></div

Klf4 controls Ang II-induced TGF-β1 production.

<p>A, Klf4 and TGF-β1 mRNA levels were assessed by qPCR in Ang II-treated cardiac fibroblasts and were normalized to β-tubulin (n = 3). B, Klf4 and TGF-β1 protein levels were assessed by Western blot in Ang II-treated cardiac fibroblasts. GAPDH was the loading control (n = 3). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. Ang II treatment for 0 hr. C-F, cardiac fibroblasts were co-infected with Ad-tTA and Ad-Klf4 and treated with Ang II. C, TGF-β1 mRNA levels were assessed by qPCR after Ang II treatment for 0, 6 or 12 hrs and normalized to β-tubulin (n = 4). D, TGF-β1 and phosphorylated Smad3 (p-Smad3) protein levels were assessed by Western blot after Ang II treatment for 0, 24 or 48 hrs. E, level of quantification of TGF-β1 and p-SMAD3 as a ratio of GAPDH in densitometric units was presented. n = 4. F, ELISA was used to analyze active TGF-β1 levels in the supernatant after Ang II treatment for 0, 24 or 48 hrs (n = 5). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. control. ^#P<0.05, ^##P<0.01 vs. control and Ang II treatment for 0 hr.</p

Elevated Klf4 promotes Ang II-induced fibroblast differentiation and collagen synthesis.

<p>Cardiac fibroblasts were exposed to co-infection of Ad-tTA and Ad-Klf4 for 2 hrs. After the viruses were washed off, infected cells were further incubated for the indicated time in the presence or absence of Tet (tetracycline, 0.1 μg/ml) for 24 hrs, then the cells were treated with Ang II (1 μmol/L). A, immunofluorescence analysis of myofibroblast differentiation by staining with a α-smooth muscle actin (α-SMA) antibody after Ang II treatment for 0, 24 or 48 hrs. Scale bars: 10 μm. B, Quantitative analyses of α-SMA positive cells are presented (n = 4). C, α-SMA mRNA levels were assessed by quantitative real-time PCR (qPCR) after Ang II treatment for 0, 6 or 12 hrs and normalized to β-tubulin (n = 4). D, α-SMA protein levels were assessed by Western blot after Ang II treatment for 0, 24 or 48 hrs. GAPDH was a loading control. E, Level of quantification ofα-SMA as a ratio of GAPDH in densitometric units was presented. n = 4. F, Col1α1, Col1α2, Col3α1 mRNA levels were assessed by quantitative real-time PCR (qPCR) after Ang II treatment for 0, 6 or 12 hrs and normalized to β-tubulin. n = 4. Data are the mean ± SEM. *P<0.05, **P<0.01, ***P<0.01 vs. control. ^#P<0.05, ^##P<0.01 vs. control and Ang II treatment for 0 hr.</p

Akt isoforms and p-Akt (Ser473) protein expressions in the kidneys from wild type (WT) and Akt2 knockout (KO) mice.

<p>Akt2, Akt1, Akt3 and p-Akt(Ser473) protein expression in the obstructed kidneys isolated from WT and Akt2 KO mice 5 day after UUO were detected by the western blotting. (A) The expression levels of Akt2 protein in WT mice and Akt2 KO mice by western blotting analysis. (B) The expression levels of Akt1 in WT mice and Akt2 KO mice by western blotting analysis. (C) The expression levels of Akt3 protein in WT mice and Akt2 KO mice by western blotting analysis. (D) The expression levels of p-Akt (Ser473) protein in WT mice and Akt2 KO mice by western blotting analysis. GAPDH was used as internal loading control.</p

siKlf4 weakens Ang II-induced fibroblast differentiation and collagen synthesis.

<p>The effect of Klf4 depends on cytokine secretion. A, semiquantitative PCR of the mRNA levels of Klf4 showed siRNA-mediated knockdown in cardiac fibroblasts. B, Klf4 mRNA levels were assessed by quantitative real-time PCR (qPCR) after siKlf4 transfection (n = 3). C, α-SMA mRNA levels were assessed by quantitative real-time PCR (qPCR) after Ang II treatment for 0, 6 or 12 hrs in siRNA transfected cardiac fibroblasts and normalized to β-tubulin (n = 3). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. scrambled siRNA. D, cultured cardiac fibroblasts were incubated with supernatants collected from cultured cardiac fibroblasts that overexpressed Klf4 or control. Immunofluorescence analysis was performed after staining with α-SMA antibody. E, quantitative analyses of α-SMA positive cells are presented (n = 4). Data are the mean ± SEM. *P<0.05 vs. control supernatant.</p