By generating conditional knockout mouse model in which β-catenin is selectively ablated in renal tubules, the present study represents the first attempt to elucidate the function of endogenous β-catenin in normal renal physiology and in regulating tubular cell damage/survival after acute injury. Our data indicate that activation of β-catenin in the setting of AKI is advantageous and renal protective, as tubule-specific loss of endogenous β-catenin results in higher mortality rate, elevated serum creatinine and worsened morphologic lesions after folic acid and ishemia/reperfusion injury ( and ). Furthermore, loss of β-catenin also leads to an increased tubular cell apoptosis after AKI, which is associated with an increased renal expression of p53 and Bax, and decreased Akt phosphorylation and survivin expression ( and ). It should be pointed out that the difference in renal injury and kidney dysfunction between control and Ksp-β-cat−/− groups is mostly likely underestimated, because more mice with the most severe AKI died selectively in the Ksp-β-cat−/− group, compared to the controls. These results clearly indicate that activation of β-catenin in renal tubules after AKI is a defensive response of the kidneys in an attempt to protect against the catastrophic damage to tubule cells.
Kidney tubular cells are susceptible to various toxic and metabolic injuries to undergo apoptotic cell death, mainly by a mitochondria-dependent pathway. In AKI induced by folic acid or IRI, tubular cell apoptosis is a major pathogenic mechanism leading to acute renal failure.3, 22
Because β-catenin is a survival factor in vitro
for tubular epithelial cells (),35
loss of β-catenin would eradicate an endogenous survival mechanism that normally safeguards renal tubules, leading to an exaggerated apoptosis after injury. This notion is substantiated experimentally in two models of AKI induced by folic acid () and IRI (). However, we cannot exclude the possibility that endogenous β-catenin may also affect other forms of tubular cell death such as necrosis and necroptosis after injury.3, 36
Comparing to renal medulla, only a fraction of proximal tubules in the cortical region in Ksp-β-cat−/− kidneys exhibited an increased TUNEL staining and Bax induction (). This is probably related to the unique expression pattern of Cre recombinase in Ksp-Cre mice,37, 38
which closely imitates endogenous Ksp-cadherin, a tissue-specific member of the cadherin family of cell adhesion molecules.37, 39
Although Cre is expressed in all segments of the nephron and collecting ducts in Ksp-Cre mice, not all proximal tubules express it.37, 38
In fact, earlier studies using reporter gene indicate that only 21% of proximal tubular cells, but more than 92% of distal tubular and collecting duct cells, actually express sufficient Cre to mediate the deletion of a floxed DNA segment.38
This could result in an incomplete ablation of β-catenin in selective proximal tubules, thereby contributing to the discriminatory apoptosis and Bax induction in the cortical region of the Ksp-β-cat−/− kidneys.
The present study indicates that activation of β-catenin may promote tubular epithelial cell survival by a multitude of mechanisms in vivo
. Loss of β-catenin results in Bax induction in renal tubules after injury, suggesting that Bax, a pro-apoptotic member of Bcl-2 family proteins, may play a critical role in mediating pro-apoptotic effect of β-catenin ablation in vivo
. This notion is in line with previous in vitro
and is substantiated by the observations that either activation of endogenous β-catenin by expressing Wnt1 or ectopic expression of exogenous β-catenin effectively prevents Bax induction and tubular epithelial cell apoptosis after incubation with staurosporine, an apoptosis inducer (). It is conceivable that p53, a tumor suppressor with pro-apoptotic activity, could be an upstream regulator that is responsible for Bax induction, as it is induced in Ksp-β-cat−/− kidneys as well after folic acid injury (). It has been shown that p53, as a transcription factor, regulates Bax by controlling its transcription.40
Furthermore, p53 also exerts its pro-apoptotic activity in a transcription-independent fashion by interacting with Bax, which results in promotion of Bax activation as well as its insertion into the mitochondrial membrane.40, 41
Loss of β-catenin also leads to reduction of Akt phosphorylation and survivin expression, two pro-survival signals. Akt, a protein kinase activated by phosphatidylinositol 3-kinase (PI3K), is an essential regulator of cell survival/apoptosis.42
Although β-catenin might activate Akt through stimulating its upstream PI3K and/or inducing its expression,35
our results indicate that β-catenin ablation inhibits Akt phosphorylation, but does not affect Akt abundance in vivo
(). As Akt can phosphorylate Bax leading to its inactivation, a reduced Akt phosphorylation would enhance the pro-apoptotic activity of Bax. Similarly, survivin, a member of the IAPs family proteins that promotes cell survival by inhibiting caspase activity, is a direct transcription target of β-catenin.31, 43
Therefore, loss of β-catenin inevitably reduces the expression of this survival gene. Taken together, as summarized in , deletion of β-catenin in a tubule-specific fashion leads to an increased apoptosis after AKI by multiple mechanisms. On one hand, β-catenin ablation causes p53 induction and Akt inhibition, resulting in Bax induction and activation, respectively, which lead to subsequent activation of caspases (). On the other hand, loss of β-catenin reduces survivin expression, thereby effectively eliminating the negative inhibitor of caspases (). Undoubtedly, these effects resulted from loss of β-catenin would make tubular cells extremely vulnerable to injury, leading to an enhanced tubular cell apoptosis. In addition, as Akt is also involved in promoting tubular cell proliferation,44
reduced Akt activation in β-catenin-deficient tubules may contribute to AKI via an impaired renal regeneration.
It is interesting to point out that β-catenin is also important in mediating cell-cell adhesion, and is a constituent of adherens junctions where it links E-cadherin to the actin cytoskeleton. However, tubule-specific ablation of β-catenin seems not to cause any phenotypic abnormality, suggesting that β-catenin is dispensable for maintaining the tubular integrity and homeostasis in adult kidneys. This observation is also in line with recent reports demonstrating that genetic deletion of β-catenin in various tissues including glomerular podocytes, hepatocytes and cardiomyocytes does not cause significant pathologic lesions.13, 45–47
Although β-catenin is a component of cell adherens junction complex, our studies indicate that its function as a structural protein can be substituted by γ-catenin, also known as plakoglobin, a structurally related protein that also binds to E-cadherin ().46, 47
In summary, we report herein that β-catenin is induced in mouse models of AKI induced by folic acid or IRI, and loss of endogenous β-catenin in a tubule-specific fashion aggravates kidney injury by promoting apoptosis via multiple mechanisms. These findings suggest that renal activation of β-catenin after AKI is a protective response in attempt to minimize cell damage. This seems in sharp contrast to the setting of chronic kidney diseases, in which sustained activation of β-catenin after chronic injury is shown to lead to tubular epithelial-mesenchymal transition (EMT) and renal fibrogenesis.17, 19, 20
Therefore, a better understanding of the role and mechanism of endogenous β-catenin signaling in different settings would be essential for designing rational strategies for therapeutic interventions. Clearly, more studies are needed in this area.