Discovering mechanisms of altered kidney development using snRNA-seq in young HSRA rats, a model of low nephron number associated with CKD

Low nephron (the functional unit in the kidney) numbers are linked to the development of hypertension and chronic kidney disease (CKD), which are both significant health problems. Our lab has established a novel inbred genetic animal model (HSRA rat) to study the genetic and physiological impact of low nephron number. In the HSRA model, ~75% of offspring are born with a single kidney (HSRA-S) compared to normal two-kidney littermates (HSRA-C). HSRA-S rats also exhibit altered development in the solitary kidney leading to ~20% less nephrons vs. individual kidney from the HSRA-C. Although the underlying genetic causes in the HSRA remains unknown, we hypothesize that there are genetic variants in the HSRA-S that promote altered expression of specific genes during kidney development leading to failure of one kidney to develop and reduced nephrons (compared to HSRA-C). To better understand the genes/pathways that may be involved in the altered kidney development, single nuclei (sn)RNA-seq was performed on kidneys of 4-week-old male HSRA rats (n=4 -S and -C kidneys). Nuclei were isolated from frozen kidney samples using 10X Chromium Nuclei Isolation Kit and processed through the 10X Genomics Chromium Single Cell 3’ protocol v3.1 per manufactures’ instruction. Libraries were sequenced on the Illumina NextSeq 2000 per 10X generating on average 40,000 reads per cell, >30,000 cells. FASTQ were processed using Cellranger mkfastq(v6.1.1)(10x Genomics) and for differential expression between the samples, the output files from each count were combined using Cellranger aggr(v6.1.1) to generate a combined cloupe file and visualized in Loupe browser 6. The cell specific clusters were grouped based on canonical cell-specific markers. The preliminary analysis of snRNA-seq of HSRA-S vs. HSRA-C kidneys identified distinct clusters of kidney-specific cell types, including podocytes, proximal tubule (PTCs1-3), Loop of Henle, and endothelial cells, among others. Differentially expressed genes (DEGs) were identified between -S and -C for most cell types, with proximal tubule cells accounting for largest number (n=41). Interestingly, a top downregulated DEG in the –S was leukemia inhibitory factor receptor (LIFR), which has recently been associated with renal malformations. In addition, studies are underway involving timed breeding of HSRA rats and subsequent isolation of fetal kidney at different days of gestation (GD14.5-17.5) for multi-omics analysis, including snRNA-seq, smallRNA-seq, and reduced-representation bisulfite sequencing. To date, we’ve collected n=23 fetal kidneys from E17.5 [2K= 8 (5m/3f); SK=15 (8m/7f)] and n=19 at E16.5 [2K= 10 (8m/2f); SK=9 (4m/5f)]. The analysis of snRNA-seq data in young HSRA and at early developmental points will provide insight into genes/pathways involved in kidney development that can increase predisposition to hypertension and CKD later in life. Graduate School of Health Sciences at UMMC; NIH R01HL137673; P20GM103476; P20GM104357 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.