THE ROLE OF BETA-KLOTHO PROTEIN IN NAFLD PATHOGENESIS

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent and progressive liver condition characterized by the accumulation of fat in more than 5% of hepatocytes in the absence of excessive alcohol consumption. Considered a spectrum of liver disorders, NAFLD ranges from simple steatosis, where fat buildup is the primary feature, to Non-Alcoholic Steatohepatitis (NASH), a more severe form associated with inflammation and liver cell damage. As a burgeoning public health concern, NAFLD poses risks of progression to advanced liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma and its evolution is closely linked to metabolic risk factors such as obesity, insulin resistance, and dyslipidemia. In addition, a genetic burden strongly underpins NAFLD pathogenesis, and several at-risk genetic variations have been found to predispose patients to the onset of liver disease. In this concern, our group previously reported that the rs17618244 G>A variant in the β-Klotho (KLB) gene dampened the KLB hepatic/plasma levels, leading to inflammation, ballooning, and fibrosis in NAFLD patients. KLB protein, functioning as coreceptor for fibroblast growth factor receptors 4 (FGFR4), mediates in the liver the hormonal activity of FGF19 and plays a central role in the regulation of lipid metabolism and in bile acids’ biosynthesis. Thus, a stable KLB full knock-out model in HepG2 hepatoma cells (KLB-/-) has been generated by using Crispr/Cas9 technology, with the aim to reproduce in in vitro the effects of the KLB rs17618244 variant. Then, in a cohort of 1311 NAFLD patients, among which 261 were children, the new intronic KLB rs12152703 T>G variant was associated with NAFLD traits and the hepatic/plasma levels of KLB were determined. Our results outlined that KLB-/- cells have altered cell homeostasis and reported the impairment of several hepatocyte typical features. Indeed, KLB-/- cells showed the downregulation of important genes involved in lipid metabolism and in cholesterol production and uptake. Furthermore, an increase in the endoplasmic reticulum (ER) and oxidative stress were observed in KLB-/- cells. Cell homeostasis was further affected by the enhancement of cell apoptosis and by the impairment of the autophagic flux, mirroring in in vitro the progression to severe liver damage. Accordingly, in our model the release of pro-inflammatory cytokines was increased and a panel of of 586 genes associated to inflammation revealed the upregulation of important mediators related to the inflammatory response. Additionally, tumorigenic features were acquired after KLB depletion, resulting in increased cell growth, proliferation and invasiveness. Interestingly, in KLB depleted cells a reduction in the response to anti-cancer therapy was observed, along with the establishment of a pro-tumorigenic environment, supported by the upregulation of several oncogenes and by the increasing of cell self-renewal activity. We further reported that the absence of KLB in in vitro may mediate the transition to a mesenchymal phenotype through the downregulation of important epithelial markers and the upregulation of mesenchymal mediators. Finally, in patients we evaluated a new variant, the KLB rs12152703, showing its possible protective action against advanced liver disease in term of reduction of transaminases levels, NAFLD activity score (NAS) and predisposition to developing NASH. This action may be mediated by an increase in circulating and hepatic KLB mutated protein, as demonstrated in a pediatric patient with the rs12152703 TT homozygous genotype we evaluated. In conclusion, our results outlined that KLB KO induced in HepG2 cells may drive important changes in liver cells metabolism and homeostasis, as consequence of the altered KLB signaling. In these cells, lipid and cholesterol metabolism were the mainly affected, thus possibly speculating that KLB depletion may lead to the loss of important hepatocyte-specific proprieties. Moreover, KLB-/- cells showed features resembling the switching towards progressive forms of liver damage observed in patients, pinpointing the possible involvement of KLB in NAFLD progression. Moreover, the acquisition of several pro-carcinogenic traits, such as enhanced proliferation, invasiveness and acquisition of stemness features may indicate that the absence of KLB further contributes to the shift towards a more aggressive cancerous phenotype. As for the translational approach, we determined for the first time the potential protective role of another variant of KLB, the rs12152703, on liver disease which is correlated to an increase in KLB levels, observed in those patients carrying the TT mutation. These evidence highlights the potentiality of KLB as novel clinical targets to refine the NAFLD prognosis. Moreover, the recombinant isoforms of KLB could be used for the treatment of NASH and might have stronger effects in those patients reporting KLB downregulation.