Methylseq, Single-nuclei RNAseq, and Discovery Proteomics Identifies Pathways Associated with Nephron Deficit CKD in the HSRA Rat Model [scRNA-Seq]

Low nephron numbers are associated with increased risk of developing chronic kidney disease (CKD) and hypertension, both significant global health problems. To investigate the impact of nephron deficiency, our lab developed a novel inbred rat model (HSRA rat). In this model, approximately 75% of offspring are born with a single kidney (HSRA-S), compared to two-kidney littermates (HSRA-C). HSRA-S rats show impaired kidney development, resulting in ~20% fewer nephrons. Our previous data and current findings demonstrate nephron deficit (failure of one kidney to form and altered development in the remaining kidney) predispose HSRA-S to CKD late in life (increased proteinuria by 18 months of age in HSRA-S=51± 3.4 versus HSRA-C = 8± 1.5 mg/24hours). 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-week-old HSRA rats. Methylation analysis revealed a small number of differences, including 5 differentially methylated cytosines and 6 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. snRNASeq: Nuclei were isolated from archived kidneys using a central section/biopsy (cortex and medulla were included) collected at 4 weeks of age from HSRA-S (n=3) and HSRA-C (n=4) per 10X Chromium Nuclei Isolation Kit (PN-1000493). Single nuclei samples were processed through the 10X Genomics Chromium Single Cell 3’ protocol v3.1 per manufacturers’ instruction to capture 5000 cells per sample.