**2. The Wnt**/β**-Catenin Cell Signaling Pathway**

The Wnt pathway is highly conserved in the evolution of animal life. It is classified into several sub-pathways called canonical and non-canonical. The non-canonical Wnt pathways are not dependent on the β-catenin-T-cell factor/lymphoid enhancer-binding factor (TCF/LEF), such as the Wnt/Ca2<sup>+</sup> pathway and the non-canonical Wnt planar cell polarity [9]. The canonical Wnt pathway involves the nuclear translocation of β-catenin and the activation of the target genes via TCF/LEF transcription factors (Figure 1). The activation of the canonical Wnt pathway requires the binding of the Wnt ligands to the receptors of the Frizzled family, and the interaction with co-receptors lipoprotein-receptor related protein 5 (LRP5) and LRP6. The binding of ligand and receptor stimulates the sequestration of Axin protein by the Disheveled protein, which prevents the formation of the complex necessary for the degradation of β-catenin. In this setting, β-catenin is not phosphorylated, became stabilized, and is translocated into the nucleus. Into the nucleus, it activates the transcription of the Wnt target genes through the interaction with the transcription factors TCF/LEF [10] (Figure 1A).

In the absence of soluble Wnt protein ligands, the protein Axin forms a complex with the proteins adenomatous polyposis coli (APC), Casein kinase 1 isoform α (CK1α), and glycogen synthase kinase 3α (GSK3α). Axin and APC act as scaffold proteins for GSK3β that binds and phosphorylates β-catenin, which is degraded by the proteasome (Figure 1B).

Some proteins regulate the Wnt/β-catenin pathway by blocking the Wnt ligands and co-receptors. The members of the secreted Frizzled-related protein (sFRP) are proteins that contain a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins, which inhibit Wnt activation (Figure 1B). Other proteins as sclerostin (the product of the SOST gene) and Dkk1 interact with LRP5/6, and they function as Wnt signaling inhibitors. Sclerostin binds the LRP5/6 receptors, impairs the LRP5/6-Frizzled interaction, and the interaction of the Wnt signaling proteins with the receptors [11,12]. Dkk1 also binds the LRP5/6 receptor and prevents the activation of the Wnt/β-catenin pathway [13] (see Figure 1).

Although there is not much evidence about the direct interaction of FGF23 or Klotho with Wnt elements, it has been shown that the extracellular domain of Klotho binds to multiple Wnt ligands, inhibiting their ability to activate Wnt signaling [14,15]. It is also known that there is a reciprocal relationship between Klotho, FGF23, and Wnt signaling; thus, Wnt signaling dysregulation affects to FGF23 and Klotho levels and vice versa. There is data suggesting potential crosstalk between Wnt/β-catenin signaling and the regulation of Klotho and FGF23. In CKD patients, uremic toxins, phosphate overload, sclerostin, Dkk1, and inflammation may affect Wnt signaling, thus contributing to the progression of CKD-associated comorbidities [16]. This subject will be exposed in the following sections.

**Figure 1.** Simplified scheme of the Wnt/β-catenin signaling pathway. (**A**) Wnt ligand interaction with Frizzled protein and LRP5/6. Disheveled (DVL) protein binds the Frizzled receptor and sequester the protein complex CK1a-GSK3-Axin-APC blocking β-catenin phosphorylation and degradation. β-catenin activates TCF/LEF transcription factor in the nucleus. (**B**) Interference of Wnt ligand–Frizzled protein interaction by sFRP, SOST, or DKK1. Disheveled (DVL) protein does not bind to the Frizzled receptor. Protein complex GSK3-Axin-APC phosphorylates β-catenin. Phosphorylated β-catenin is led to proteasomal degradation. Abbreviations: GSK3β: glycogen synthase kinase 3; APC: adenomatous polyposis coli; TCF/LEF: T-cell factor/lymphoid enhancer-binding factor; sFRP: secreted Frizzled-related proteins; SOST: sclerostin; DKK1: Dickkopf-related proteins.

### **3. Klotho-FGF23 and Wnt in Chronic Kidney Disease**

### *3.1. Regulation of Klotho Expression in the Kidney: The E*ff*ect of the Tubular Load of Phosphate*

Our group has studied the factors associated with a reduction of renal Klotho expression in rats. Administration of recombinant FGF23 (rFGF23) produced phosphaturia and reduced renal Klotho expression in healthy rats [6]. In 5/6 nephrectomized rats, circulating levels of FGF23 were markedly increased, and Klotho was found to be reduced. In these rats, the administration of anti-FGF23 antibodies further reduced the renal Klotho expression. These results sugges<sup>t</sup> that the increased tubular load of phosphate causes a reduction in Klotho expression. In vitro, HEK-293 cells incubated in high phosphate medium produced nuclear translocation of β-catenin that was followed by a reduction in Klotho expression [6]. We concluded that high phosphate levels decreased renal Klotho expression via activation of the Wnt/β-catenin pathway (Figure 2). The administration of calcitriol to cultured HEK-293 cells prevented Klotho reduction induced by high phosphate.

**Figure 2.** Schematic representation of FGF23/Klotho interactions with the Wnt/β-catenin pathway in the bone, kidney, and heart.
