In many species including humans, aging reduces female fertility. Intriguingly, some animals preserve fertility longer under specific environmental conditions. For example, at low temperature and short day-length, Drosophila melanogaster enters a state called adult reproductive diapause. As in other stressful conditions, ovarian development arrests at the yolk uptake checkpoint; however, mechanisms underlying fertility preservation and post-diapause recovery are largely unknown. Here, we report that diapause causes more complete arrest than other stresses yet preserves greater recovery potential. During dormancy, germline stem cells (GSCs) incur DNA damage, activate p53 and Chk2, and divide less. Despite reduced niche signaling, germline precursor cells do not differentiate. GSCs adopt an atypical, suspended state connected to their daughters. Post-diapause recovery of niche signaling and resumption of division contribute to restoring GSCs. Mimicking one feature of quiescence, reduced juvenile hormone production, enhanced GSC longevity in non-diapausing flies. Thus, diapause mechanisms provide approaches to GSC longevity enhancement.

The diverse functions of each nephron segment rely on the coordinated action of specialized cell populations that are uniquely defined by their transcriptional profile. In the collecting duct, there are two critical and distinct cell populations: principal cells and intercalated cells. Principal cells play key roles in the regulation of water, Na+, and K+, while intercalated cells are best known for their role in acid-base homeostasis. Currently, there are no in vitro systems that recapitulate the heterogeneity of the collecting ducts, which limits high-throughput and replicate investigations of genetic and physiological phenomena. Here, we have demonstrated that the transcription factor Foxi1 is sufficient to alter the transcriptional identity of M-1 cells, a murine cortical collecting duct cell line. Specifically, overexpression of Foxi1 induces the expression of intercalated cell transcripts including Gpr116, Atp6v1b1, Atp6v1g3, Atp6v0d2, Slc4a9, and Slc26a4. These data indicate that overexpression of Foxi1 differentiates M-1 cells towards a non-A, non-B type intercalated cell phenotype and may provide a novel in vitro tool to study transcriptional regulation and physiological function of the renal collecting duct.

G protein‐coupled receptors (GPCRs) are a diverse family of integral membrane proteins that have significant roles in numerous physiological systems. We previously reported that GPR116 (ADGRF5), an adhesion‐class GPCR, is a critical regulator of vacuolar‐type H+‐ATPase (V‐ATPase) surface expression in A‐type intercalated cells (AICs) in mouse kidney cortical collecting ducts. The V‐ATPase is a multi‐subunit proton pump that localizes to the plasma membrane in specialized acid‐secreting epithelial cells. FOXI1 is a transcription factor that regulates V‐ATPase expression in ICs and other mitochondria‐rich cells. Recently, FOXI1 was identified as a key regulator of CFTR‐rich pulmonary ionocytes which express both V‐ATPase and GPR116. Therefore, we hypothesized that FOXI1 is a transcription factor in AICs that upregulates V‐ATPase as well as GPR116. To test this hypothesis, we cloned FOXI1 from whole mouse kidney into a pME18s expression vector (pFOXI1). Transfection with pFOXI1 significantly increased GPR116 expression (qPCR) in M‐1 mouse cortical collecting duct cells (n=3, ΔCt ±SEM: untransfected = 13.8±0.6, pFoxi1 = 10.7±0.2, p<0.05), but not in HEK293T cells (n=3, not significant). RNA‐scope hybridization on murine kidney sections and pFOXI1‐transfected M‐1 cells revealed colocalization of FOXI1 and GPR116 mRNA in the same cell. Treating pFOXI1‐transfected M‐1 cells with a synthetic agonist peptide for GPR116 results in an increase of [Ca2+]iin a subset of cells (n=3, ΔF340/380 ±SEM: pFoxi1 = 0.296±0.017). Internal calcium mobilization is never seen with the agonist peptide in untransfected control cells, indicating that pFOXI1 is sufficient for the production of physiologically responsive GPR116 on the cell surface (n=3, ΔF340/380±SEM: untransfected = 0 ±0.0009). Moreover, only M‐1 cells transfected with pFOXI1 express GPR110 (ADGRF1), an adhesion‐GPCR with unknown function in the kidney, as well as three V‐ATPase subunits, ATP6V1B1, ATP6V1G3, ATP6V0D2, indicating a larger transcriptional program initiated by pFOXI1 (n=3, untransfected: Ct ~ 40 for all genes, pFoxi1 ΔCt±SEM: ADGRF1 = 13.2±0.21, ATP6V1B1 = 11.4±0.3, ATP6V1G3 = 10±0.1, ATP6V0D2 = 12.1±0.03). Additionally, these pFOXI1 cells also upregulate the expression of SLC26A4 (pendrin), a known marker of B‐type intercalated cells, suggesting that FOXI1‐driven regulation is upstream of AIC fate determination (n=3, ΔCt ± SEM: untransfected = 19.3±0.7, pFoxi1 = 16.1±0.2, p<0.01). These data reveal that FOXI1 upregulates both GPR116 and V‐ATPase in M‐1 cells, generating IC‐like cells. Finally, our study suggests that GPR116 may be a universal and genetically‐coded regulator of V‐ATPase surface expression in FOXI1‐positive cells.

G protein-coupled receptors (GPCRs) are seven transmembrane domain receptors that comprise the largest class of proteins in the mammalian genome. GPR39 is part of the ghrelin subfamily of GPCRs, and is well-expressed in the kidney but has no reported function in renal physiology. To probe the renal role of GPR39, we first sought to localize it; in the absence of a validated immunofluorescence (IF) antibody, we used RNAscope. We found that Gpr39 localizes to nephron segments in the cortex and medulla which are positive for DBA (Dolichos Biflorus Agglutinin), a collecting duct marker. RNAScope expression of Gpr39 was highest in the inner medullary collecting duct, with lower levels in the outer medullary and cortical collecting ducts. Within the collecting duct, Gpr39 transcripts colocalized with Aqp2, indicating that Gpr39 is expressed specifically in principal cells. To address whether GPR39 is apical or basolateral, we cloned murine Gpr39 with a C-terminal EGFP tag and transiently transfected this construct into either polarized M-1 cells (murine collecting duct cell line) or polarized MDCK cells (Madin-Darby canine kidney cells); in both cases, cells were grown to confluence on permeable supports. In both cell lines, heterologous GPR39 trafficked to the basolateral membrane. Based on the localization of GPR39 to principal cells, we hypothesized that GPR39 activation may alter AQP2 expression or trafficking. To test this hypothesis in vitro, we first validated mpkCCD (mouse principal cell kidney cortical collecting duct) cells as a model. We cultured mpkCCD on transwells until polarized (TER >5kΩ·cm2). In the absence of dDAVP, mpkCCD do not express AQP2 by western or IF. Basolateral treatment with 1nM dDAVP for 4 days induced AQP2 expression (western blot) and apical trafficking (confocal IF). We also confirmed that withdrawal of dDAVP (4h) resulted in a cytosolic accumulation of AQP2, while the re-introduction of dDAVP (1h) returned AQP2 to the apical membrane by IF. Moreover, we found that Gpr39 expression was unaffected by dDAVP by qPCR (n=3, ΔCt ± SEM: control = 7.9±0.3, dDAVP = 7.2±0.1) and western blot. In a preliminary study, we treated mpkCCD with 13nM of a GPR39 selective agonist, cpd1324, or with a vehicle control, and probed for changes in AQP2 by IF. Both groups were treated with 1nM dDAVP and with 10μM ZnCl2 (a GPR39 cofactor). We found that cpd1324 treatment decreased AQP2 by IF (mean integrated fluorescence density ± SEM (A.U.): cpd1324 = 233.5 ± 20.8, control = 373.8 ± 13.0). Additionally, cpd1324 treatment reduced the apical AQP2 abundance (mean integrated fluorescence density ± SEM (A.U.) of the most apical Z-slice: cpd1324 = 6.05e+05 ± 3.9e+04, control = 2.6e+06 ± 4.3e+05). These results suggest that GPR39 agonism may antagonize vasopressin-induced AQP2 trafficking and/or expression. NIH T32HL007534 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.