Abstract Body: Background: Chronic kidney disease (CKD) has emerged as an independent risk factor for cerebral microhemorrhages (CMH). We examined molecular mechanisms of CMH formation in mouse models of CKD and hypertension (HTN) using RNA transcriptomics and mTOR complex 1 (mTORC1) inhibition.
Methods: Aged (17-month-old) mice were treated with either 0.2% adenine diet to induce CKD, or angiotensin II (ATII) via subcutaneous osmotic pumps to induce HTN and compared with control mice. A subset of CKD mice was treated with rapamycin to inhibit mTORC1 signaling. After 4 weeks, RNA sequencing of mouse brain was done via Illumina NovaSeq 6000 with downstream analysis using Hisat2, Salmon, and DESeq2. Differentially expressed genes (DEGs) and enriched pathways were identified using the MsigDB Hallmark 2020 database. Pathway analysis was further examined using qPCR and immunohistochemistry staining. CMH were quantified using Prussian blue histology.
Results: CKD mice had increased CMH number (1.12±0.10 vs 0.85±0.08 per cm² in CKD vs control mice, p<0.05), and whole brain RNA-seq showed significant alterations in the mTORC1 pathway, a critical regulator for cell growth and metabolism. Specifically, 18 DEGs were involved in mTORC1 signaling in CKD compared with control animals. qPCR and immunohistochemistry analysis confirmed CKD-associated upregulation of DNA damage-induced transcript 4 (DDIT4), an upstream component of the mTORC1 pathway. ATII-induced HTN more than doubled CMH formation (1.44±0.47 vs. 0.60±0.66 per cm² in HTN vs. control mice, p< 0.01), without significant alteration of the mTORC1 pathway. Treatment of CKD mice with the mTOR inhibitor rapamycin significantly reduced CMH burden (24% reduction in CMH number, p<0.05 and 56% reduction in CMH area, p<0.01).
Conclusions: Cerebral microhemorrhages were increased in both the CKD and HTN models. However, alteration of brain mTORC1 pathway was observed only in CKD animals, suggesting a specific molecular signature in the CKD brain. Our study supports mTORC1 inhibition as a novel approach to inhibit cerebral microhemorrhage formation in CKD.