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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE243944 | GEO | 2024/04/04

REPOSITORIES: GEO

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Project description:Fibroblasts are present in every organ. While the role fibroblasts in chronic diseases such as fibrosis or tumor expression has been extensively explored, little is known about their physiological role. The kidney possesses a unique capacity to recover from even severe acute injury. We study molecular mechanisms of this intrinsic repair capacity in the mouse model of ischemia-reperfusion (IR). In this model, the renal artery and vein are clamped for 45 min, leading to acute kidney injury. The kidney spontaneously recovers from such IR injury within 14 days. IR kidney injury is associated with a transient accumulation of fibroblasts in the diseased tissue. We hypothesized that fibroblasts aid the repair of acute IR injury in the kidney. To elucidate how FSP1+ fibroblasts may contribute to the repair of kidney injury, we undertook a global unbiased approach to compare gene expression profiles of fibroblasts isolated from kidneys post-IRI and from control kidneys by FACS sorting. To investigate the role fibroblasts may play in the repair of kidney inhury, we performed ischemia reperfusion injury surgery on transgenic mice in which the FSP-1 promoter drives EGFP expression. Kidney injury peaks at day 3 post-IRI, followed by spontaneous regeration that restores nearly perfect kidney architecture and health by day 10. Fibroblasts are thought to possibly play a role in this process, as they are normally rare in the healthy kidney, acute kidney injury is associated with a transient accumulation of interstitial fibroblasts, but whether they may help repair the acute kidney injury or in fact could contribute to the damage is not known. To compare the gene expression profiles of normal fibroblasts and those from post-ischemic kidneys, we sacrificed control FSP1-GFP mice and the FSP1-GFP mice three days post-IRI. We generated single-cell suspensions from both the post-IRI and control kidneys, and then isolated FSP1-GFP+ cells by FACS sorting that, when cultured on plastic, displayed typical fibroblast morphology. Total RNA was immediately extracted from the sorted cells and amplified to produce enough for a array. We biotinylated five of the samples from post-ischemic kidneys and three of the control (non-ischemic) kidneys and used Affymetrix 3' Arrays to examine differences in gene expression profiles between the two groups that may she some light on what role, if any, fibroblasts play in the spontaneous healing of the kidney after acute kidney injury. We performed ischemia reperfusion surgery in FSP1-GFP mice, and at day 3, we sacrificed the mice, isolated FSP1-GFP positive cells from both IR and normal control kidneys by FACS sorting, extracted total RNA from the isolated FSP1-GFP cells and used Affymetrix Mouse Expression Array 430 2.0 microarrays to perform gene expression profiling of the samples. All told, we performed the FACS Sorting, RNA extration, and hybridization with 5 ischemic kidneys and 3 normal kidneys. Fibroblasts, acute kidney injury, repair, comparative gene expression profiling, microarrays, FACS sorting, role in healing

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

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