Multiomics Analyses Reveal Sex Differences in Mouse Renal Proximal Subsegments

Abstract

Significance Statement Sex-dependent differences in kidney function are recognized but the underlying molecular mechanisms are largely unexplored. Advances in genomics and proteomic technologies now allow extensive characterization of differences between the same cell types of males and females. Multiomics integrating RNA-seq, ATAC-seq, and proteomics data to investigate differences in gene expression, chromatin accessibility, and protein expression in proximal tubules of male and female mice identified many sex-biased genes and proteins associated with kidney functions, including metabolic and transport processes. Sex differences may also arise from variations of the interaction between transcription factors and accessible chromatin regions. A comprehensive web resource is provided to advance understanding of sex differences in cells of the proximal tubule. Background Sex differences have been increasingly recognized as important in kidney physiology and pathophysiology, but limited resources are available for comprehensive interrogation of sex differences. Methods RNA-seq and ATAC-seq of microdissected mouse proximal tubules and protein mass spectrometry of homogenized perfused mouse kidneys reveal differences in proximal tubule cells of males and females. Results The transcriptomic data indicated that the major differences in the proximal tubules between the sexes were in the S2/S3 segments, and most of the sex-biased transcripts mapped to autosomes rather than to the sex chromosomes. Many of the transcripts exhibiting sex-biased expression are involved in monocarboxylic acid metabolic processes, organic anion transport, and organic acid transport. The ATAC-seq method on microdissected tubules captured chromatin accessibility. Many of the more than 7000 differentially accessible DNA regions identified were in distal regions. Motif analyses revealed a lack of direct involvement of estrogen receptors or the androgen receptor (absence of canonical hormone response elements), suggesting an indirect regulatory role of sex hormones. Instead, analyses identified several transcription factors (TFs) (Tead1, Nfia/b, and Pou3f3) whose interplay with proximal tubule-specific TFs (e.g., Hnf1b, Hnf4a) may contribute to sex differences. Finally, the whole-kidney proteome was correlated with the transcriptome, and many sex-biased proteins (e.g., Cyp2e1, Acsm2/3) were identified. Conclusions Sex-dependent cis-regulatory elements interact with TFs in ways that lead to sex-biased gene expression in proximal tubule cells. These data are provided as a user-friendly web page at https://esbl.nhlbi.nih.gov/MRECA/PT/.

Sex differences in renal electrolyte transport

“…Their analysis of the many sex differences along the proximal tubule is provided in the very useful Kidney Cell Explorer web resource (https://cello.shinyapps.io/kidneycellexplorer/). Recently, Chen et al [4 ▪▪ ] conducted a transcriptomic and proteomic analysis of microdissected proximal tubule S1, S2, and S3 segments and found that sex differences are primarily evident in the S2–S3 segments of the proximal tubule, in agreement with previous reports [2,3]. They also reported correlation between proteome and transcriptome in sex-biased proteins; information is provided at https://esbl.nhlbi.nih.gov/MRECA/PT/.…”

“…Importantly, transporter mRNA levels are not effective predictors of transporter abundance or activity because of the prominent differences in rates of protein synthesis and degradation, posttranslational modifications, and subcellular trafficking of transporters. A combination of mRNA and protein analyses [4 ▪▪ ] can theoretically provide novel information about relative turnover rates [30].…”

“…The female kidney adapts to maintain homeostasis through these normal life cycle challenges. During the past 6 years, a substantial body of new information has revealed sex-specific differences in renal transporter expression [1][2][3]4 && ], relative abundance [5][6][7][8], activity and regulation [5,6,9,10,11 && ,12 && , [13][14][15][16]]. Herein we discuss these reports, their implications, gaps, and future opportunities.…”

Differences in chromatin accessibility between renal cortex and inner medulla correlate with spatial differences in gene expression and are modulated by NFAT5 function

“…Meanwhile several studies have been performed using single cells, renal tissues or nephron segments to correlate gene expression with differences in chromatin accessibility using ATACseq [49][50][51] . With these and other studies, it was possible to identify a nephron segment or cell type specific gene expression pattern that might be associated with epigenetic modifications.…”

Sex as a biological variable in acute kidney injury

“…Recently, Chen et al demonstrated sex-biased gene expression in autosomes resulting in sex differences in the S2/S3 segments of male and female mice [22]. Novel animal models enable the investigation of sex hormones, sex chromosomes, and autosomes on sex biases [23].…”