Sexual dimorphism and epigenetic control of the kidney disease marker Klotho

Abstract

BackgroundPreclinical studies investigating physiological and pathophysiological processes in the kidney are mainly performed in males. This is due to a higher susceptibility to kidney injury and subsequent disease progression in male rodents. In consequence, sexual dimorphism of kidney injury markers like Klotho is often overlooked. Klotho is well-linked to renal health and its deletion in mice results in severe phenotype and premature death. Here, we identified putative Klotho enhancers and investigated their functions in males and females using mice with deletions in the Klotho-associated enhancers. Experimental designWe generated mutant mice carrying deletion of putative Klotho enhancers using CRISPR/Cas9 gene editing. Warm ischemia-reperfusion surgery was performed bilaterally to induce acute kidney injury and unilaterally in a fibrosis model. Using ChIP-seq and RNA-seq, we analyzed chromatin features and gene expression in mouse kidney. ELISA assay was utilized to measure serum FGF23 levels. ResultsWe detected Klotho gene expression being twice as high in males compared to females at baseline. Enhancer deletion decreased Klotho mRNA levels more effectively in female then in male mice (90 vs. 50%). ChIP-seq data suggest additional regulatory elements present only in male mice, as promoter marks remain more pronounced in males even after deletion. Weight, lifespan and fertility of the knockout mice was not impacted. Baseline serum FGF23 level was significantly higher only in female enhancer knockouts (192.4 vs. 599.6pg/ml, p=0.0003). Severe bilateral ischemia resulted in similar creatinine increase in male and female WT and knockout mice, but only male knockout mice displayed higher Havcr1 expression after injury than controls (1048 vs. 231.4, p=0.0016). 28 days after unilateral renal ischemia, fibrosis as measured by Acta2 and Tgfb expression and Masson Trichrome staining was not significantly changed regardless of genotype. ConclusionsOur results demonstrate sexual dimorphism of Klotho gene expression and its enhancer regulation. Despite having a larger effect in female mice, including changes in baseline FGF23 levels, only male knockout mice are more susceptible to acute injury and the deletion has no impact on the fibrosis model. Further dissection of the mechanisms of Klotho regulation is necessary to reexamine its efficacy as a kidney injury marker.