Deletion of Klotho enhancer deepens sexual dimorphism of gene expression without impacting female resistance to kidney injury [ChIP]

Sexual dimorphism in kidney injury markers, such as Klotho, is frequently underestimated or disregarded, as most of the preclinical research is done in male animals. Klotho is well-linked to renal health and its deletion in mice results in a severe accelerated aging-like phenotype and premature death. Here, we identified candidate Klotho enhancers and discovered their function using mice carrying deletions in the enhancer loci. Candidate Klotho enhancers were deleted using CRISPR/Cas9 genome editing. Warm ischemia-reperfusion surgery (IRI) was performed bilaterally to induce acute kidney injury and unilaterally in a fibrosis model. Using ChIP-seq and RNA-seq, we analyzed renal chromatin features and gene expression. ELISA and colorimetric assays were used to measure serum FGF23 and serum component levels. Removal of the enhancer caused more substantial reduction in Klotho mRNA levels in female mice compared to males (90 vs. 50%), supported by gene promoter marks remaining more pronounced in males. Weight, lifespan, and fertility of mice lacking the enhancers were not impacted. Only male mutant mice displayed higher Havcr1 expression after IRI than controls (mean 1048 vs. 231.4, p=0.0016). 28 days after unilateral IRI, only males displayed increased fibrosis markers, but with no difference between genotypes. Our findings reveal sexual dimorphism of Klotho gene expression and its enhancer regulation. Only male mutant mice were more susceptible to acute injury and the enhancer deletion had no impact on fibrosis. Additional investigation into the mechanisms governing Klotho regulation is imperative to reassess its effectiveness as a kidney injury marker. Chromatin Immunoprecipitation and DNA Sequencing (ChIP-Seq) of histone modifications H3K27ac and H3K4me3 and transcription factor HNF1b in mouse kidneys was used to assess efficacy of deletion of candidate Klotho gene enhancers and to assess its effect on renal chromatin landscape.

以肾损伤标志物克洛索蛋白（Klotho）为代表的性别二态性（sexual dimorphism）常被低估或忽视，因多数临床前研究均以雄性动物为实验对象。克洛索蛋白与肾脏健康密切相关，在小鼠体内敲除该基因会引发严重的加速衰老样表型，并导致过早死亡。本研究通过构建增强子位点敲除小鼠，筛选得到候选Klotho增强子并阐明其功能：利用CRISPR/Cas9基因组编辑技术敲除候选Klotho增强子位点；通过双侧温热缺血再灌注手术（ischemia-reperfusion injury, IRI）构建急性肾损伤模型，单侧温热缺血再灌注手术则用于构建纤维化模型。采用染色质免疫沉淀测序（ChIP-seq）与RNA测序（RNA-seq）分析肾脏的染色质特征与基因表达水平，通过酶联免疫吸附实验（ELISA）与比色法检测血清FGF23及其他血清成分水平。实验结果显示，与雄性小鼠相比，增强子敲除后雌性小鼠的Klotho mRNA水平下降更为显著（降幅分别为90%与50%），该结论得到雄性小鼠体内基因启动子区域表观标记仍更为显著的佐证。增强子敲除小鼠的体重、寿命与生育能力均未受影响。仅雄性突变小鼠在IRI术后的Havcr1表达水平高于对照组（均值分别为1048与231.4，p=0.0016）。单侧IRI术后28天，仅雄性小鼠出现纤维化标志物水平升高，但不同基因型小鼠间无显著差异。本研究结果揭示了Klotho基因表达及其增强子调控的性别二态性：仅雄性突变小鼠对急性肾损伤更为易感，而增强子敲除对纤维化进程无显著影响。未来需进一步探究Klotho调控的分子机制，以重新评估其作为肾损伤标志物的应用价值。本研究通过对小鼠肾脏内组蛋白修饰H3K27ac、H3K4me3以及转录因子HNF1b进行染色质免疫沉淀测序（ChIP-seq），以验证候选Klotho基因增强子的敲除效果，并分析其对肾脏染色质调控图谱的影响。