Single-nucleus RNA and ATAC sequencing reveals marked changes and sex differences in the kidney collecting duct principal cell in response to mild dehydration
Authors: Huynh Nha , Rehage Cassidy , Hyndman Kelly 
Published: May 2023

The kidney principal cells in the connecting tubule (CNTPCs) and collecting duct (CDPCs) function to fine-tune water reabsorption and concentrate the urine upon activation of arginine vasopressin (AVP) receptor-2. Our study aimed to determine the kidney single-nucleus transcriptome and DNA accessibility in response to dehydration, a potent physiological stimulus for AVP secretion. We hypothesized that dehydrated male and female mice would have different responses in the PCs.Male and female C57BL/6J mice at 11 weeks old were subjected to either ad libitum (ad lib) or 9hr of dehydration (n = 1 per group) with samples taken at midnight. 10X Genomics Chromium Next GEM Single Cell Multiome ATAC+Gene Expression Kit was used. The fastq files were processed using CellRanger ARC pipeline and downstream analyses completed with the R packages Seurat and Signac. Gene set enrichment analyses were performed with ToppGene Suite.A total of 22,821 nuclei (median fragments per nucleus = 12,961 ± 336) were sequenced and 34 clusters identified with 586 and 129 nuclei annotated as CDPC and CNTPC, respectively. We found more DEGs in CDPC than in CNTPC (106 vs. 13). The common top 5 most highly expressed genes in both CDPC and CNTPC in the dehydrated state compared to ad lib were Grem2, Pde10a, Pappa, Fam129a, Scg5 (adjusted P values < 0.01). Grem2 encodes a cytokine known to antagonize the bone morphogenic protein family (log2FC adlib/dehydrated for CDPC = -2.18 and CNTPC -1.96). Pde10a is a gene related to cAMP-mediated signaling (log2FC for CDPC = -2.17 and CNTPC -2.02). Pappa encodes a metalloproteinase (log2FC for CDPC = -1.36 and CNTPC -1.24). Fam129a encodes a protein that regulates protein phosphorylation (log2FC for CDPC = -1.17 and CNTPC -1.20). Finally, Scg5 encodes a chaperone protein that regulates pituitary hormone secretion (log2FC for CDPC = -0.84 and CNTPC -0.92). As expected, Aqp2 and Aqp3 were significantly higher in CDPCs from the dehydrated male and female mice. When stratifying by sex, we found more DEGs in female CDPCs than in males (79 vs. 54, χ2 p = 2.68E-8), but in CNTPC there were less in females than in males (1 vs 14, Fisher’s Exact p = 0.016). Pathway analyses determined that in dehydrated male DEGs that were highly expressed were enriched in circulatory system development and response to cAMP signaling pathways. In dehydrated female highly expressed DEGs were enriched in pathways related to renal development. Overall, chromatin analysis showed that changes in gene expression were not driven by changes in accessibility of chromatin.In conclusion, these findings suggest that CDPCs are more responsive to dehydration and that although there are common DEGs in dehydrated male and female mice, unique genes are enriched in different biological processes. NIDDK 1R01DK128001 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.