Methylseq, single-nuclei RNAseq, and discovery proteomics identify pathways associated with nephron-deficit CKD in the HSRA rat model.

Andrew R Milner, Ashley C Johnson, Esinam M Attipoe, Wenjie Wu, Lavanya Challagundla, Michael R Garrett
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Abstract

Low nephron numbers are associated with an increased risk of developing chronic kidney disease (CKD) and hypertension, which are significant global health problems. To investigate the impact of nephron deficiency, our laboratory developed a novel inbred rat model (HSRA rat). In this model, ∼75% of offspring are born with a single kidney (HSRA-S), compared with two-kidney littermates (HSRA-C). HSRA-S rats show impaired kidney development, resulting in ∼20% fewer nephrons. Our previous data and current findings demonstrate that nephron deficit (failure of one kidney to form and altered development in the remaining kidney) predisposes HSRA-S to CKD late in life (with increased proteinuria by 18 mo of age in HSRA-S = 51 ± 3.4 vs. HSRA-C = 8 ± 1.5 mg/24 h). To understand early molecular mechanisms contributing to the increased predisposition to CKD, Methylseq using reduced representation bisulfite sequencing, single-nuclei (sn)RNAseq, and discovery proteomics were performed in kidneys of 4-wk-old HSRA rats. Methylation analysis revealed a small number of differences, including five differentially methylated cytosines and six differentially methylated regions between groups. The snRNAseq analysis identified differentially expressed genes in most kidney cell types, with several hundred genes dysregulated depending on the analysis method (Seurat vs. DESeq2). Notably, many genes are involved in kidney development. Discovery proteomic analysis identified 366 differentially expressed proteins. A key finding was dysregulation of Deptor/DEPTOR and Amdhd2/AMDHD2 across omics layers, suggesting a potential role in compensatory mechanisms or the genetic basis of altered kidney development. Further understanding of these mechanisms may guide interventions to preserve nephron health and slow kidney disease progression.NEW & NOTEWORTHY The HSRA rat is a novel model of nephron deficiency and provides a unique opportunity to study the association between nephron number and chronic kidney disease (CKD). Previous work characterized the impact of age, hypertension, and diabetes on the development of CKD in HSRA animals. This study examined early changes in epigenetics, cell-type specific transcriptome, and proteomic changes in the kidney that likely predispose the model to CKD with age.

肾小球数量少与慢性肾脏病(CKD)和高血压的发病风险增加有关,而这两种疾病都是全球性的重大健康问题。为了研究肾小球缺乏的影响,我们实验室开发了一种新型近交系大鼠模型(HSRA 大鼠)。在该模型中,与双肾同窝鼠(HSRA-C)相比,约 75% 的后代出生时只有一个肾脏(HSRA-S)。HSRA-S 大鼠的肾脏发育受损,导致肾小球数量减少约 20%。我们之前的数据和目前的研究结果表明,肾小球缺失(一个肾脏无法形成,其余肾脏的发育发生改变)使 HSRA-S 大鼠在生命晚期易患慢性肾脏病(HSRA-S 大鼠在 18 个月大时蛋白尿增加(51± 3.4)毫克/24 小时,而 HSRA-C 大鼠为(8± 1.5)毫克/24 小时)。为了了解导致CKD易感性增加的早期分子机制,研究人员对4周大的HSRA大鼠的肾脏进行了甲基化测序(使用还原表征亚硫酸氢盐测序)、单核糖核酸测序(sn)和发现蛋白质组学研究。甲基化分析发现了少量差异,包括组间 5 个不同的甲基化胞嘧啶和 6 个不同的甲基化区域。snRNAseq 分析确定了大多数肾细胞类型中的差异表达基因,根据分析方法(Seurat 与 DESeq2)的不同,有几百个基因表达失调。值得注意的是,许多基因参与了肾脏的发育。发现蛋白质组分析确定了 366 种差异表达的蛋白质。一个重要发现是Deptor/DEPTOR和Amdhd2/AMDHD2在各omics层中的失调,这表明它们在肾脏发育改变的代偿机制或遗传基础中可能发挥作用。对这些机制的进一步了解可能会为保护肾小球健康和减缓肾脏疾病进展的干预措施提供指导。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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