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In May 2014, the National Institutes of Health (NIH) stated its intent to “require applicants to consider sex as a biological variable (SABV) in the design and analysis of NIH-funded research involving animals and cells.” Since then, proposed research plans that include animals routinely state that both sexes/genders will be used; however, in many instances, researchers and reviewers are at a loss about the issue of sex differences. Moreover, the terms sexand genderare used interchangeably by many researchers, further complicating the issue. In addition, the sex or gender of the researcher might influence study outcomes, especially those concerning behavioral studies, in both animals and humans. The act of observation may change the outcome (the “observer effect”) and any experimental manipulation, no matter how well-controlled, is subject to it. This is nowhere more applicable than in physiology and behavior. The sex of established cultured cell lines is another issue, in addition to aneuploidy; chromosomal numbers can change as cells are passaged. Additionally, culture medium contains steroids, growth hormone, and insulin that might influence expression of various genes. These issues often are not taken into account, determined, or even considered. Issues pertaining to the “sex” of cultured cells are beyond the scope of this Statement. However, we will discuss the factors that influence sex and gender in both basic research (that using animal models) and clinical research (that involving human subjects), as well as in some areas of science where sex differences are routinely studied. Sex differences in baseline physiology and associated mechanisms form the foundation for understanding sex differences in diseases pathology, treatments, and outcomes. The purpose of this Statement is to highlight lessons learned, caveats, and what to consider when evaluating data pertaining to sex differences, using 3 areas of research as examples; it is not intended to serve as a guideline for research design.

A physical interaction is shown between CRF1R and CRF2R, two class B G protein–coupled receptors that mediate stress and immune responses. Trafficking of CRF2R but not CRF1R is actin dependent, and coexpression of the two receptors alters actin-independent trafficking. Receptor cross-talk alters agonist binding and signaling.

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na+) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na+/H+exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na+/H+exchange activity by Na+-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na+/H+exchange activity by >30%. Moreover, the sgk2-mediated increase in Na+/H+exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na+transport through NHE3 in the proximal tubule.

Serum and glucocorticoid-regulated kinase-1 (SGK1) is a serine-threonine kinase that is regulated at the transcriptional level by numerous regulatory inputs, including mineralocorticoids, glucocorticoids, follicle-stimulating hormone, and osmotic stress. In the distal nephron, SGK1 is induced by aldosterone and regulates epithelial Na+channel-mediated transepithelial Na+transport. In other tissues, including liver and shark rectal gland, SGK1 is regulated by hypertonic stress and is thought to modulate epithelial Na+channel- and Na+-K+-2Cl−cotransporter-mediated Na+transport. In this report, we examined the regulation of SGK1 mRNA and protein expression and Na+currents in response to osmotic stress in A6 cells, a cultured cell line derived from Xenopus laevisdistal nephron. We found that in contrast to hepatocytes and rectal gland cells, hypotonic conditions stimulated SGK1 expression and Na+transport in A6 cells. Moreover, a correlation was found between SGK1 induction and the later phase of activation of Na+transport in response to hypotonic treatment. When A6 cells were pretreated with an inhibitor of phosphatidylinositol 3-kinase (PI3K), Na+transport was blunted and only inactive forms of SGK1 were expressed. Surprisingly, these results demonstrate that both hypertonic and hypotonic stimuli can induce SGK1 gene expression in a cell type-dependent fashion. Moreover, these data lend support to the view that SGK1 contributes to the defense of extracellular fluid volume and tonicity in amphibia by mediating a component of the hypotonic induction of distal nephron Na+transport.

The epithelial Na+channel (ENaC) constitutes the rate-limiting step for Na+transport across tight epithelia and is the principal target of hormonal regulation, particularly by insulin and mineralocorticoids. Recently, the serine-threonine kinase (SGK) was identified as a rapidly mineralocorticoid-responsive gene, the product of which stimulates ENaC-mediated Na+transport. Like its close relative, protein kinase B (also called Akt), SGK's kinase activity is dependent on phosphatidylinositol 3-kinase (PI3K), a key mediator of insulin signaling. In our study we show that PI3K is required for SGK-dependent stimulation of ENaC-mediated Na+transport as well as for the production of the phosphorylated form of SGK. In A6 kidney cells, mineralocorticoid induction of the phosphorylated form of SGK preceded the increase in Na+transport, and specific inhibition of PI3K inhibited both phosphorylation of SGK and mineralocorticoid-induced Na+transport. Insulin both augmented SGK phosphorylation and synergized with mineralocorticoids in stimulating Na+transport. In a Xenopus laevisoocyte coexpression assay, SGK-stimulated ENaC activity was also markedly reduced by PI3K inhibition. Finally, in vitro-translated SGK specifically interacted with the ENaC subunits expressed inEscherichia colias glutathione S-transferase fusion proteins. These data suggest that SGK is a PI3K-dependent integrator of insulin and mineralocorticoid actions that interacts with ENaC subunits to control Na+entry into kidney collecting duct cells.

The proto-oncogeneyesencodes a protein tyrosine kinase described as a member of the non-receptor protein tyrosine kinases family (NRPTK). We have identified two clones from the genomic library ofDrosophila melanogasterwhich hybridized to thev-yesoncogene (encoded by the Y73/Esh Avian Sarcoma Virus). These clones represent two different genomic sequences fromDrosophila.By RNA slot blot hybridization, we have demonstrated the presence ofyeshomologous sequences in the RNA of larvae (the third instar), pupae and an embryonic cell line ofDrosophila.On northern analysis of the cell line RNA, three transcripts (3.8, 2.0 and 1.0 kb) hybridized with theyessequence. Oncogeneyesis thus well conserved across widely separated phylogenetic lines like human, chicken andDrosophilaindicating involvement in essential biological functions.