Biliverdin Reductase Regulates Hypoxia Induced Renal Tubular Epithelial To Mesenchymal Transition Via PI3K/Akt Pathway

Background and objective: In the past, biliverdin reductase(BVR) was solely considered as a reductase that catalyzes biliverdin to bilirudin. Recent studies reveal that BVR functions as a serine/threonine/tyrosine kinase, a transcription factor, or an antioxidant. BVR can be induced by reactive oxygen species(ROS) and was identified to have PKB/Akt-like activity. Akt is a key mediator of epithelial to mesenchymal transition(EMT) process. However, whether BVR is induced by hypoxia and whether BVR is involved in EMT, especially hypoxia induced EMT, is unclear. This study investigates the effect of BVR on fibrogenesis in hypoxic HK-2 cells and kidney of chronic renal failure rats, and possible correlation with intracellular PI3K/Akt signaling pathway. Methods: (1)Chronic renal failure was induced by 5/6 nephrectomy.Immunohistochemical SABC method was used to detect the expression of BVR at 12 weeks, and the proteins of BVR and hypoxia inducible factor 1 alpha (HIF-1α) were detected by Western Blot. In vivo, HIF-1αand BVR in HK-2 cells under hypoxia for 12, 24 and 48 h were also detected by Western blot. (2)BVR plasmids were transfected into HK-2 cells. The phenotypic changes associated with EMT in BVR transfected HK-2 cells were detected by Western blot or confocal microscope, as demonstrated by alterations in epithelial and mesenchymal protein E-cadherin, Vimentin andα-SMA. Wound healing test were performed to observe the cell migration of BVR transfected HK-2 cells. To further test the BVR-induced EMT related to the activition of PI3K pathway, BVR transfected cells were treated with Ly294002 and Wortmannin respectively, and the phenotypic changes associated with EMT were also determined by Western blot or confocal microscope.(3) The phenotypic changes associated with EMT in hypoxic HK-2 cells were detected by Western blot or confocal microscope, as demonstrated by alterations in epithelial and mesenchymal protein E-cadherin, Vimentin andα-SMA. To further test the BVR-induced EMT related to the activation of PI3K pathway, hypoxic HK-2 cells were treated with Ly294002 or Wortmannin respectively, and the phenotypic changes associated with hypoxic EMT were also determined by Western blot or confocal microscope. Immunohistochemistry of BVR and p-AKT was performed using serial sections in order to clarify co-localization of BVR and p-AKT in the kineys of both sham and 5/6 nephrectomized rats. The BVR specific siRNA were transfected into hypoxic HK-2 cells, and the phenotype changes associated with hypoxic EMT were further determined by Western blot or confocal microscope. Intracellular ROS was detected by means of an oxidation-sensitive fluorescent probe (DCFH-DA).Result: (1)The expression of BVR and HIF-1αin the renal epithelium of 5/6 nephrectomized rats was increased in contrast with sham group at 12 weeks, and there are significant correlations between BVR and HIF-1α. In hypoxic HK-2 cells, the proteins BVR was also significantly upregulated as hypoxic exposure, which was consistent with the increase of HIF-1α. (2)Transfection of BVR into HK-2 cells caused phenotypic changes associated with EMT. The cobblestone-like epithelial cells were turned into elongated cells in shape. E-cadherin, a cell-cell adhesion molecule present in the plasma membrane of most epithelial cells, was reduced and formed zipper-like patterns at cell borders in BVR- transfected cells. Vimentin andα-SMA, markers of mesenchymal cells, were induced in BVR-transfected cells. With treatment of Ly294002 or Wortmannin, the phosphorylated Akt was decreased and the lost E-cadherin was re-expressed and re-localized at cell borders in BVR-transfected cells, on the other hand Vimentin andα-SMA were deceased. (3)Hypoxia induced phenotypic changes associated with EMT in HK-2 cells. E-cadherin, a cell-cell adhesion molecule present in the plasma membrane of most epithelial cells, was reduced and formed zipper-like patterns at cell borders in hypoxic HK-2 cells. Vimentin andα-SMA, markers of mesenchymal cells, were induced in hypoxic HK-2 cells. With treatment of Ly294002 or Wortmannin, the phosphorylated Akt was decreased and the lost E-cadherin was re-expressed and re-localized at cell borders in hypoxic HK-2 cells, on the other hand Vimentin andα-SMA were deceased. Because chronic hypoxia contributed to the development of fibrosis in the 5/6 nephrectomy model, to further confirm the hypoxia-induecd EMT related with activation of Akt phosphorylation by BVR, we immunolocalized BVR and p-Akt on serial sections in the kidney of both sham and 5/6 nephrectomized rats. In sham rats, although a little weak, the distribution pattern of p-Akt in renal epithelium most closely corresponded to that of BVR locally expressed in some renal epithelial tubular cells. In 5/6 nephrectomized rats, along with the incease of BVR, the staining of p-Akt was widely distributed in glomerulus, renal epithelium and interstitium, and the distribution patterns of p-Akt and BVR were highly congruent in renal tubules. SiRNA experiments were performed to decrease the activity of BVR in hypoxic HK-2 cells. The control hypoxic cells and hypoxic control siRNA cells developed similarly renal EMT after 48h hypoxia, E-cadherin downregulated and Vimentin de novo expressed. The HK-2 cells transfected with BVR-specific siRNA displayed a greater inhibition to hypoxia induced EMT. E-cadherin relocalized at cells borders which changed from zipper-like structures to linear, and it's protein levels were inceased when compared with control hypoxic cells or hypoxic control siRNA cells. Similar results were observed on the other EMT hallmark Vimentin. Transfection of BVR siRNA into HK-2 cells inhibited de novo expression of Vimentin to chronic hypoxia stimulation. To explore the mechanism further, we detected the status of Akt phosphorylation. The p-Akt was decreased by BVR RNAi knockdown no matter hypoxia or not, compared with control cells, while the amount of total Akt protein remained unchanged. This result further confirmed that BVR is the upstream activator of Akt. In normonic HK-2 cells, although phosphorylation of Akt was inhibited by BVR specific siRNA, the mock transfected, control siRNA transfected cells and BVR siRNA knockdown cells all displayed the similar positive expression of E-cadherin and negative expression of Vimentin after the whole 96h incubation. Intracellular ROS was detected by FACScan flow cytometer. Exposure of HK-2 cells to hypoxia led to a remarkable increase in intracellular ROS compared with normoxic HK-2 cells. with BVR siRNA knockdown, increased ROS level was aggravated in the hypoxic cells. However, in hypoxic BVR-transfected cells the intracellular ROS was reduced significantly.Conclusion: Those results suggest BVR plays as an inducer in hypoxia-mediated tubular EMT and the mechanism is involved in a PI3K/Akt dependent pathway. BVR acts as a two-edged sword, not only a powerful antioxidant but also a potential accelerator of renal fibrosis in remnant kidneys.