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

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.


Introduction
Renal interstitial fibrosis is the major pathological characteristic in progressive renal diseases, including nephropathy, eventually leading to end-point renal malfunction [1,2]. The key feature of renal interstitial fibrosis is the accumulation and deposition of extracellular matrix (ECM), which is thought to be produced mainly by myofibroblasts [2,3]. Over the past decade, Epithelialmesenchymal transition (EMT) of tubular epithelial cells, characterized by loss of epithelial cell characteristics, and gain of ECM-producing myofibroblast characteristics, is an important pathway in myofibroblast production and is a key event in the pathogenesis and progression of renal interstitial fibrosis [4,5,6]. Recent cell lineage tracking experiments showed that EMT did not contribute to myofibroblast formation in kidney [7,8]. These results suggest that EMT may not directly contribute to myofibroblast formation and fibrosis. However, loss of epithelial cells, namely EMT, may still contribute to myofibroblast formation and fibrosis indirectly as there are clear evidences of an EMT-like process occurs in renal epithelial cells [9]. For example, some studies have suggested that the loss of epithelial cells, or EMT, may indirectly contribute to interstitial fibrosis development through a paracrine signaling mechanism [9,10]. Despite the novel nature of this hypothesis and increasing supportive evidence, the molecular mechanism of EMT and fibrosis has not been fully characterized.
It is reported that the Akt signaling has a critical role in mediating tubular EMT [11]. The Akt/PKB family of kinases, a downstream effector of phosphatidylinositol 3-kinase pathway, plays an important key role in regulating growth, proliferation, survival, metabolism, and other cellular activities [12,13]. However, there are three major isoforms of Akt:Akt1, -2, and -3. Which one plays important role in the TGF-b1-induced EMT is not very clear. In previous in vitro study, we found that Akt2 activity is involved in TGF-b1-induced EMT in HK-2 cells, but whether Akt2 activity is involved in renal tubular EMT and renal fibrosis in vivo has not been reported.
Unilateral ureteral obstruction (UUO) in mice is a wellestablished experimental model resulting in tubulointerstitial fibrosis and tubular EMT in the obstructed kidney [14,15]. Hence, in present study, we explored the role of Akt2 in renal tubular EMT and renal interstitial fibrosis following UUO, we found that Akt2 and phosphor(p)-Akt levels were increased in the obstructed kidneys, knockout (KO) of Akt2 suppressed UUOinduced EMT, kidney fibrosis. These results provide a new insight into the role of Akt2 in the UUO-induced kidney fibrosis and EMT.

Animals and Ethics Statement
Akt2 knockout mice and wild-type littermates were purchased from the Jackson Laboratory. Mice were maintained under specific-pathogen-free conditions in the animal facility at the Beijing Heart Lung and Blood Vessel Diseases Institute. The mice were given a standard rodent chow and water ad libitum. All animal care and experimental protocols complied with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85- 23,1996) and were approved by the Institutional Animal Care and Use Committee of Capital Medical University.

UUO model in mice
Surgical procedures were performed in 2-month-old mice as we previously described [16] with minor modification. Six WT littermate and Akt2 KO mice (in C57/BL6 genetic background), of 20-30 g body weight, in each group were studied, UUO was carried out as described [16]. Mice were anesthetized with pentobarbital, the abdomen was opened, and the left ureter was ligated with 5-0 silk. Then the abdomen was closed with running sutures and the skin was closed with interrupted sutures. Shamoperated mice had their ureter exposed but not ligated. After surgery, the mice were maintained in a temperature-controlled room.

Histopathology and Immunohistochemistry
After 3 days, 5 days, and 7 days of surgery, mice were enthanized by an overdose of pentotarital (100 mg/kg). Renal morphology was examined in 10% neutral-buffered formalinfixed, paraffin-embedded tissue sections after they were stained with Masson's-modified trichrome. For immunohistochemical staining assay, kidneys were perfused as described [16]. After removing paraffin, sections were incubated for 30 min in 3% hydrogen peroxide (H 2 O 2 ) in methanol at room temperature (RT), washed with PBS, and heated in a microwave in 10 mM citrate buffer (pH 6.0) for 20 min. Sections were blocked using 10% goat serum (Vector Laboratories, Burlingame, CA, USA) for 30 min, incubated with anti-fibronectin, anti-Snail, anti-b-catenin, anti-p-Akt (Ser473), anti-p-GSK3b, anti-E-cadherin, anti-a-SMA (1:200 dilution). The staining protocol was performed according to the ABC kits (Vector Lab). The signals were visualized using a peroxidase substrate kit (Vector Lab). The pictures were recorded using a Nikon light microscope.

Western blot analysis
Total cells and tissues were homogenized in lysis buffer and centrifuged at 14,000 g for 30 min. Supernatant was recovered and proteins were quantified using the BCA protein assay kit. Lysates (60 mg per lane for tissue, 30 mg per lane for cells) were loaded onto 12% SDS-polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1.5 h at room time (RT) in fresh blocking buffer (0.1% Tween20 in Trisbuffered saline (TBS-T) containing 5% fat-free milk) and then incubated with the primary antibodies anti-p-Akt (Ser473), anti-p-Akt (Thr 308), anti-Akt1, anti-Akt2, anti-Akt3, anti-TGFb1, antip-Smad3, anti-vimentin, anti-E-cadherin, anti-a-SMA, anti-Snail, anti-b-catenin, anti-p-GSK3b, anti-t-GSK3b, anti-fibronectin, anti-collagen 1(1:1000 dilution), or GAPDH antibodies, overnight at 4uC, then secondary antibodies (1:5000 dilution, horseradish peroxidase-conjugated goat anti-rabbit IgG) for 1.5 h at RT. Membranes were washed three times with TBS-T and the signals were visualized and analyzed using Odyssey image system (Li-COR, Lincoln, NE, USA). Each experiment was repeated at least three times. The density of specific bands was measured and normalized to that of GAPDH.

Statistics
All data are representative of experiments done in triplicate and are expressed as the means 6 SD. Differences between groups were analyzed by one-way analysis of variance (ANOVA) using SPSS 13.0 software. A P,0.05 was considered statistically significant.

Akt2 deficiency decreases UUO-induced fibrosis
After 7 days of surgery, the obstructive kidney in WT mice showed the typical features of obstructive nephropathy: tubular dilatation and widespread renal tubulointerstitial damage and fibrosis, evidenced by the increase in collagen deposition as assessed by Masson's-modified trichrome staining ( Figure 1A) and western blot ( Figure 1C). In contrast, kidneys from Akt2 KO mice were protected against UUO-induced collagen deposition ( Figure 1 A and C). In addition, the effects of UUO on the levels of fibronectin protein expression were significantly reduced in Akt2 KO mice by western blot assay and immunohistochemistry (Figure 1 B and D). Although we found the level of fibronectin protein in the unobstructed kidneys from Akt2 KO mice is more than that in the unobstructed kidneys from WT mice, we speculated this due to individual variation. All the above results reveal that Akt2 depletion attenuates UUOinduced fibrosis.

Akt2 and Active Akt levels are increased in the obstructed kidneys
We examined active Akt levels in the obstructed kidneys from WT mice. As shown in Figure 2B, Western blot demonstrated similar results: p-Akt (Ser473) protein was significantly elevated in the obstructive kidneys as compared with that in the unobstructed, contra-lateral kidneys. This data was confirmed by immunohistochemical analysis as shown in Figure 2A, increased levels of p-Akt (Ser473) was present in obstructed kidneys compared with control kidneys, with staining predominantly located in the kidney tubules and interstitium. We also examined the expression of p-Akt (Thr308) protein in obstructed kidneys, we found that the level of p-Akt (Thr308) protein is not significantly increased following UUO (Fig. S1). It is known that Akt1 and Akt2 but not Akt3 mRNA is expressed in the kidneys [17]. Next, we examined the expression of Akt1 protein and Akt2 protein in the obstructive kidneys. Western blot analysis showed that Akt2 protein levels were elevated in the obstructed kidneys compared with that in the unobstructed kidneys from WT mice, whereas Akt1 protein levels were similar among groups ( Figure 2B). Thus Akt2 expression is enhanced in the obstructive kidneys, and that Akt signaling in obstructive kidneys may have been activated.
We also detected increases in protein levels of TGF-b1 and phosphor (p)-Smad3 in obstructed kidneys compared with control kidneys (Figure 2 C and D), suggesting that amplified TGF-b1 signaling was presented in obstructed kidneys. As TGF-b1 is a key inducer of EMT and fibrosis [18], we hypothesized that elevated levels of TGF-b1 would have consequences for EMT and fibrosis in the renal tubules.

Akt2 deficiency suppresses UUO-induced EMT
To further determine whether Akt2 contributes to EMT following UUO, we firstly examined the expression of Akt1 protein and Akt2 protein in the obstructed kidneys from WT mice or Akt2 knockout (KO) mice. As shown in Figure 3 A and B, the protein expression of Akt1 were presented in both WT mice and Akt2 KO mice, but the expression of Akt2 was presented in only WT mice, indicating that the expression of Akt1 is not impacted in Akt2 KO mice and Akt2 KO is specific. In order to see whether there is a complementary expression of Akt3 in Akt2 KO mice, we also test the expression of Akt3, as shown in Figure 3 C, there is no Akt3 expression in kidneys of WT and Akt2 KO mice. In addition, we detected that p-Akt (Ser473) expression is also increased in Akt2 KO mice, which is less than that in WT kidneys (Figure 3 D), this may explain the partially influence of Akt2 KO on the fibrosis following UUO.
As the TGF-b1/Smad3 signaling is the classical pathway in EMT, so we investigated the effect of Akt2 KO on the expression of p-Smad3, as shown in Figure 4 A, the expression level of p-Smad3 protein was markedly increased as compared with that in unobstructed kidneys of WT mice, p-Smad3 expression in the obstructed kidneys of Akt2 KO mice was less as compared with kidneys from obstructed WT mice, suggesting that Akt2 KO may affect EMT following UUO. Next, we measured protein expression of EMT markers vimentin (Figure 4 B) and E-cadherin (Figure 4 C, and E) using western blotting and immunohistochemical staining. As shown in Figure 4 B, C and E, after 7 days of surgery, the expression level of vimentin protein was markedly increased and the expression level of E-cadherin protein was significantly decreased as compared with that in unobstructed kidneys of WT mice. However, Increased vimentin expression and decreased E-cadherin expression were less in the obstructed kidneys of Akt2 KO mice (Figure 4 B, C and E), indicating that Akt2 KO could affect EMT following UUO. Next, we measured the expression of a-smooth-muscle actin (a-SMA) in the kidneys of WT and Akt2 KO mice, as shown in Figure 4 D and F, the expression level of a-SMA protein was markedly increased as compared with that in unobstructed kidneys of WT mice, but this increase is lower in Akt2 KO mice than in WT mice (Figure 4 D and F). These data suggest that UUO-induced EMT and fibrosis is suppressed in Akt2 KO mice.

Akt2 depletion suppresses UUO-induced the expression of Snail and b-catenin in the obstructed kidneys
Snail is a key transcriptional repressor of E-cadherin, contributes to EMT [19], we therefore assessed the expression of Snail in UUO kidneys. As shown in Figure 5 A and C, after 7 days of surgery, the expression level of Snail protein was increased as compared with that in unobstructed kidneys of WT mice. However, Snail expression in the obstructed kidneys of Akt2 KO mice was less increased as compared with kidneys from obstructed WT mice. Something similar occurs with b-catenin expression, which increased significantly after UUO in WT mice, but not in Akt2 KO mice (Figure 5 B and D). The above findings suggest that Akt2 partially mediates the expression of Snail and bcatenin induced by UUO.
UUO activated Akt2 promotes the expression of Snail and b-catenin depends on GSK3b deactivation in the obstructive kidneys To determine how activated Akt2 promotes expression of Snail and b-catenin following UUO. We examined the effect of Akt2 KO on the expression of p-GSK3b. GSK3b, as a downstream target of PI3K and Wnt pathways, is necessary for the maintenance of epithelial architecture [20]. As shown in Figure 6, after 7 days of surgery, the expression of phosphor (p) GSK3b was increased as compared with that in unobstructed kidneys of WT mice, which is suppressed in Akt2 KO obstructed kidneys.
In order to examine the role of GSK3b in the expression of Snail and b-catenin in HK-2 cells, we used TGF-b1 or LiCl to treat cells. As shown in Figure 7, TGF-b1-induced expression of Snail and b-catenin (Figure 7). Inhibition of GSK3b with LiCl 2 (the inhibitor of GSK3b) also increased the expression of Snail and b-catenin (Figure 7), indicating that GSK3b deactivation is involed in expression of Snail and b-catenin in the tubule epithelial cells.

Discussion
In this study, we reported that UUO induced lower interstitial fibrosis, in Akt2 KO than in WT mice. All the fibrogenic hallmarks of renal fibrosis were attenuated in the obstructed kidneys from the Akt2 KO mice, including the less of fibronectin and collagen I accumulation. Indicating that Akt2 is involved in the development of renal fibrosis. EMT is thought to be a significant component of renal fibrosis. We found the levels of TGF-b1 and p-Smad3 protein were both increased in obstructed kidneys ( Figure 2 C and D). These data suggested that amplified TGF-b signaling was present in kidneys, which would have consequences for epithelial cells integrity in the renal tubules. Furthermore, we also found that p-Akt (Ser473) was increased in UUO (Figure 2 A and B). Indicating us that Akt activation may be Akt2 Partially Contributes to Epithelial-Mesenchymal Transition (EMT) PLOS ONE | www.plosone.org elicited by TGF-b1 and involved in loss of tubular epithelial cells in UUO. Firstly, we found p-Smad3 expression in the obstructed kidneys of Akt2 KO mice was less as compared with kidneys from obstructed WT mice, suggesting that Akt2 KO may affect EMT following UUO. Secondly, the level of vimentin, E-cadherin and a-SMA was detected and we found that Akt2 KO suppressed increases in vimentin and a-SMA, while loss of E-cadherin was prevented by Akt2 KO (Figure 4). Although Akt2 KO abolished the UUO-induced increase in vimentin and a-SMA, UUOinduced alteration in E-cadherin were only partially recovered, suggesting that Akt2 signaling is not the only pathway involved in the EMT following UUO.
The present observations further provide insight into the Akt2dependent mechanisms leading to EMT in vivo. Snail, a zinc finger transcription factor, has been characterized as a key regulator EMT. Many studies have shown that Snail binds to specific DNA sequences called E-boxes in the promoter of the E-cadherin gene to repress transcription of E-cadherin [16]. E-cadherin is a major gene in the epithelium, which is an important determinant in maintaining the epithelial phenotype [21]. In our study, we found that UUO increased Snail accumulation of kidney tubule cells ( Figure 5A), and the influence of UUO on the expression of Snail was at least partially mediated by Akt2, as the expression of Snail after UUO was blocked in kidneys of Akt2 KO mice ( Figure 5 A and C). These results are consistent with our vitro data that TGF-b1 induced the expression of Snail, the effect of TGF-b1 on Snail expression was sensitive to the presence of Akt2 in HK-2 cells. Furthermore, Villagrasa P, et al. reports that Akt2 interacts with Snail on its target promoters and regulates transcription [22], which is consistent with our findings. b-catenin signal cascade, a principal mediator of canonical Wnt signaling, plays a fundamental role in regulating various biologic processes such as organ development, tissue homeostasis, and pathogenesis of human disease [23,24]. It is reported that b-catenin signaling is activated in a wide variety of fibrotic CKDs such as obstructive nephropathy. Consistent with previous studies [16], we also found that a marked increase of bcatenin protein in WT obstructed kidneys, which was blocked in Akt2 KO obstructed kidneys (Figure 5 B and D). Our findings are supported by a recent evidence that elevated Akt2 levels in the Irs2-/-kidney lead to increased GSK3b phosphorylation and increased b-catenin levels [25] In addition to Wnt, b-catenin activation is also regulated by other signal pathways such as TGF-b1. In cultured HK-2 cells, we observed that TGF-b1-induced bcatenin activation ( Figure 7B).
To determine how activated Akt2 promotes expression of Snail and b-catenin following UUO. We examined the effect of Akt2 KO on the expression of p-GSK3b. GSK3b, as an active kinase of Snail, can bind to Snail and facilitate its proteasomal degradation [26]. In addition, GSK3b, as a downstream target of the PI3K/ Akt and Wnt pathways, is necessary for the maintenance of epithelial architecture [27]. According to our observations, UUO increased GSK3b phosphorylation in kidneys ( Figure 6), which was attenuated in obstructed Akt2 KO mice. Our findings are consistent with the previous studies [25]. Importantly, inhibiting  GSK3b with LiCl increased Snail and b-catenin expression in HK-2 cells. The above results indicated that Akt2 partially mediated UUO-induced Snail and b-catenin expression, but it rather effective owing to its influence of GSK3b.
Taken together our results show that, after UUO, the kidneys of Akt2 KO mice showed a lower interstitial fibrosis and EMT than those of WT animals due to a increase of GSK3b activity and a decrease of Snail and b-catenin expression. Our data show a direct role of the Akt2 signaling pathway in the development of renal tubule EMT and fibrosis and point to its inhibitors as potential antifibrotic drugs.