Left ventricular hypertrophy (LVH) is a common complication in patients with CKD and an independent risk factor for death. Changes in the levels of uremic solutes or Klotho have been reported to be related to CKD, whereas the relationships between these factors and CKD-associated LVH remain unclear. Here, we investigated the interaction between Klotho and indoxyl sulfate (IS), a typical uremic solute, in CKD-associated LVH. In a survey of 86 patients with CKD, a negative relationship was found between serum levels of IS and Klotho (r=20.59, P,0.001). Furthermore, serum levels of IS and Klotho were independently associated with LVH (for IS: r=0.69, P,0.001; for Klotho: r=20.49, P,0.001). In normal mice, intraperitoneal injection of IS for 8 weeks induced LVH accompanied by substantial downregulation of renal Klotho. Notably, IS-induced LVH was more severe in heterozygous Klotho-deficient (kl/+) mice. In vitro, treatment with Klotho strongly inhibited IS-induced cardiomyocyte hypertrophy by blocking oxidative stress and inhibiting p38 and extracellular signal-regulated protein kinase 1/2 signaling pathways. In a mouse model of CKD-associated LVH, the renal expression of Klotho was lower and the level of serum IS was higher than in healthy controls. Moreover, treatment of CKD mice with Klotho protein significantly restrained the development of LVH. Taken together, these results suggest that Klotho is an endogenous protector against IS-induced LVH, and the imbalance between Klotho and IS may contribute to the development of LVH in CKD.

Renal fibrosis is the main pathological characteristic of chronic kidney disease (CKD), whereas the underlying mechanisms of renal fibrosis are not clear yet. Herein, we found an increased expression of microRNA-34a (miR-34a) in renal tubular epithelial cells of patients with renal fibrosis and mice undergoing unilateral ureteral obstruction (UUO). In miR-34a À/À mice, miR-34a deficiency attenuated the progression of renal fibrosis following UUO surgery. The miR-34a overexpression promoted epithelial-to-mesenchymal transition (EMT) in cultured human renal tubular epithelial HK-2 cells, which was accompanied by sharp downregulation of Klotho, an endogenous inhibitor of renal fibrosis. Luciferase reporter assay revealed that miR-34a downregulated Klotho expression though direct binding with the 3 0 UTR of Klotho. Conversely, overexpression of Klotho prevented miR-34a-induced EMT in HK-2 cells. Furthermore, results showed that miR-34a was induced by transforming growth factor b1 (TGF-b1) through p53 activation, whereas dihydromyricetin could inhibit TGF-b1-induced miR-34a overexpression. Accordingly, dihydromyricetin administration dramatically restored the aberrant upregulation of miR-34a and Klotho reduction in obstructed kidney, and markedly ameliorated renal fibrosis in the Adriamycin nephropathy and UUO model mice. These findings suggested that miR-34a plays an important role in the progression of renal fibrosis, which provides new insights into the pathogenesis and treatment of CKD.

Increased basic fibroblast growth factor-2 (FGF2) and reduced Klotho have both been reported to be closely associated with renal fibrosis. However, the relationship between Klotho and FGF2 remains unclear. We demonstrate that FGF2 induced tubulo-epithelial plasticity in cultured HK-2 cells, accompanied by a reduction in Klotho expression, whereas recombinant Klotho protein could inhibit the action of FGF2. The FGF2 effects required extracellular signal-regulated protein kinase 1/2 activation, which was suppressed by Klotho. Moreover, Klotho also restrained FGF2-induced fibroblast proliferation and activation. The inhibitory effect of Klotho on the activity of FGF2 was likely due to its potent ability to compete with FGF2 binding to FGF receptor 1. Unilateral ureteral obstruction (UUO)-induced renal fibrosis was associated with an increase in FGF2 and a reduction in Klotho expression in wild-type mice, whereas FGF2(-/-) mice largely preserved Klotho expression and developed only mild renal fibrosis after obstructive injury. Furthermore, administration of Klotho protein in UUO mice significantly reduced renal fibrosis, concomitant with a marked suppression of FGF2 production and signalling. These studies demonstrate a feedback loop between Klotho depletion and FGF2 activation in renal fibrosis. Our results also suggest that Klotho treatment reduces renal fibrosis, at least in part, by inhibiting FGF2 signalling.