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Deep-sea diving into the renal transcriptome of high-fat-fed mice nets unique catch.

According to recent estimates (2015) from the Center for Disease Control, approximately one-third of the US population is obese (5), i.e., has a body mass index 30. The prevalence for disease is highest in African-American females, at 57%, and considerably lower in Asian-Americans at just under 11% (5). A large proportion of obese subjects progress to cardiovascular disorders, including diabetes and the related diabetic nephropathy (DN). Overconsumption of calorie-rich, high-fat diets coupled with low exercise rates underlies the majority of cases. In this issue, Halperin Kuhns and Pluznick (2) report on their studies using deep-sequencing/transcriptomics to search for novel gene changes in the kidney that may have a role in DN using a well-defined model of diet-induced obesity. Male and female mice (C57Bl6/J) were fed a control (CT) or a high-fat diet (HFD; 60% of calories) from 6 to 15 wk of age. Male mice fed the HFD were significantly heavier at the time of euthanasia and had elevated nonfasting glucose levels and impaired glucose tolerance. These physiological effects of HFD were attenuated or absent in the females. Male mice on the HFD also had modestly increased glomerular filtration rate and enlarged glomeruli, changes indicative of early DN. After euthanasia, messenger RNA was purified from the kidney cortex, and libraries were prepared using standard approaches. RNASeq analysis identified 1,134 genes that were differentially expressed between control and HFD-fed male mice. Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis revealed that 353 of these genes clustered into 17 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG is a useful compilation of databases facilitating systematic integration and interpretation of genomic sequencing (11). These authors chose the most robust “differentially expressed genes” for further study. This list included 11 upregulated and 11 downregulated genes. They also conducted further analysis on any differentially expressed G proteins and sensory receptors, since this has been a focus of their laboratory for a number of years (7, 8). Gene changes were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) using a custom-made low-density array card. The majority of upregulated genes were confirmed in this manner; however, there was less agreement in the downregulated genes, although patterns of change (directionality) were predominantly preserved. For the most part, these gene changes appeared to be selective for the kidney cortex. The investigators evaluated liver, heart, and renal medulla and did not observe similar patterns. The genes with the highest fold increases included 1) Popeye domain-containing 3 (Popdc3), 2) synaptoporin (Synpr), 3) LIM homeobox 2 (Lhx2), and 4) ATPase H /K transporting nongastric 2-subunit (Atp12a) (Fig. 1). These genes were increased at minimum 20-fold; however, none are reportedly expressed at high levels in the kidney when examined as a “total cortex”, i.e., fragments per kilobase transcript per million mapped reads (FPKM) 10. On closer inspection, deep sequencing of microdissected tubule segments conducted by Lee et al. (4) revealed the presence of Synpr in glomeruli and Atp12a in thick ascending limb. Additional studies will need to be designed to better understand the impact of upregulation of these transcripts by HFD at these sites. Similarly, a number of low-expression level transcripts showed the greatest decrease with HFD. However, two downregulated genes are highly expressed in kidney, i.e., cytochrome P450, family 2, subfamily a, polypeptide (Cyp2a5), and glutathione S-transferase2 (GSTA2) with a FPKM 50. Cyp2a5 is induced by a number of toxins, including nicotine and phenobarbital (9). It has a fairly well-defined role in liver; however, its role in kidney is unclear. Similarly, GSTA2 has a detoxification role of primarily electrophilic compounds, including carcinogens. They further evaluated (by qRT-PCR) whether this same set of upand downregulated genes was significantly changed in the kidney cortex from HFD-fed female mice. These females did not substantially gain weight (relative to CT diet-fed females). Thus, a second set of female mice was started on the HFD earlier in life (at 3 wk of age). This set did gain weight but also failed to show a similar pattern of regulated genes (as compared with males or with the first group of females). There was one gene change in common with males; i.e., Synpr was increased 28.6-fold (by qRT-PCR) in males and 4.1-fold in females. It should be noted that deep sequencing was not conducted on female tissue, which likely would have revealed additional sets of regulated genes specific for females. Why female mice respond so differently to the HFD is not known. It provides yet another example of substantial sex differences in physiology. Part of it may be explained by lower weight gain and the putative anorexic actions of estrogen (1), at least in one group of females. Furthermore, structural differences are apparent between the sexes, at least in some species. This may affect the ratio of cell types found in the cortex. Oudar et al. (6) conducted comparisons in length and volume of portions of the kidney in rats and found that male Address for reprint requests and other correspondence: C. M. Ecelbarger, Dept. of Medicine, Georgetown University, 4000 Reservoir Rd., Washington, DC 20007 (e-mail: ecelbarc@georgetown.edu). Am J Physiol Renal Physiol 314: F879–F880, 2018; doi:10.1152/ajprenal.00607.2017.