Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) depletes the ovary of primordial and primary follicles through an increase in the natural process of atresia. Additionally, in vitro exposure of Postnatal Day 4 (PND 4) rat ovaries to VCD causes similar follicular depletion. This study was designed to investigate survival signaling pathways that may be associated with VCD-induced ovotoxicity in small preantral follicles. Female Fischer 344 rats (PND 28) were dosed daily (80 mg/kg/day VCD i.p.; 12 days in vivo), and PND 4 ovaries were cultured (VCD 20 or 30 microM; 8 days in vitro). Microarray analysis identified a subset of 14 genes whose expression was increased or decreased by VCD in both experiments (i.e., via both exposure routes). Particularly, the analysis showed that relative to controls, VCD did not affect mRNA expression of growth and differentiation factor 9 (Gdf9), whereas there were decreases in mRNA encoding bone morphogenic protein receptor 1a (Bmpr1a) and Kit. To confirm findings from microarray, the genes Gdf9, Bmpr1a, and Kit were further examined. When growth factors associated with these pathways were added to ovarian cultures during VCD exposure, GDF9 and BMP4 had no effect on VCD-induced ovotoxicity; however, KITL attenuated this follicle loss. Additionally, there was a decrease in Kit and an increase in Kitl expression (mRNA and protein) following VCD exposure, relative to control. These results support that VCD compromises KIT/KITL signaling, which is critical for follicular survival in primordial and primary follicles.
The kidneys "escape" from the Na-retaining effects of aldosterone when circulating levels of aldosterone are inappropriately elevated in the setting of normal or expanded extracellular fluid volume, e.g., in primary aldosteronism. Using a targeted proteomics approach, we screened renal protein extracts with rabbit polyclonal antibodies directed to each of the major Na transporters expressed along the nephron to determine whether escape from aldosterone-mediated Na retention is associated with decreased abundance of one or more of renal Na transporters. The analysis revealed that the renal abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) was profoundly and selectively decreased. None of the other apical solute-coupled Na transporters displayed decreases in abundance, nor were the total abundances of the three ENaC subunits significantly altered. Immunocytochemistry showed a strong decrease in NCC labeling in distal convoluted tubules of aldosterone-escape rats with no change in the cellular distribution of NCC. Ribonuclease protection assays (RPAs) revealed that the decrease in NCC protein abundance was not associated with altered NCC mRNA abundance. Thus, the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule appears to be the chief molecular target for regulatory processes responsible for mineralocorticoid escape, decreasing in abundance via a posttranscriptional mechanism.
2012 Young Investigator Award for Excellence in Renal Physiology.
The mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) inhibits monocyte chemotactic protein-1- and stromal-derived factor-1alpha (SDF-1alpha)-mediated chemotaxis and diminishes chemokinesis in macrophage-like RAW264.7 and U937 cells in a manner that may involve attenuation of the intracellular calcium signal. STC1 is strongly induced in the kidney following obstructive injury. We hypothesized that STC1 may serve to attenuate the influx of inflammatory cells to the site of tissue injury. In this study, we examined the effect of STC1 on the migration of freshly isolated human macrophages, neutrophils, and T and B lymphocytes through quiescent or IL-1beta-treated human umbilical vein endothelial cell (HUVEC) monolayers. STC1 inhibited transmigration of macrophages and T lymphocytes through quiescent or IL-1beta-activated HUVECs but did not attenuate the transmigration of neutrophils and B lymphocytes. STC1 regulates gene expression in cultured endothelial cells and is detected on the apical surface of endothelial cells in vivo. The data suggest that STC1 plays a critical role in transendothelial migration of inflammatory cells and is involved in the regulation of numerous aspects of endothelial function.
We have used peptide-directed antibodies to each major renal Na transporter and channel proteins to screen renal homogenates for changes in Na transporter protein expression after initiation of dietary NaCl restriction. After equilibration on a NaCl-replete diet (2.0 meq. 200 g body wt(-1). day(-1)), rats were switched to a NaCl-deficient diet (0.02 meq. 200 g body wt(-1). day(-1)). Na excretion fell to 25% of baseline levels on day 1, followed by a further decrease 4% of baseline levels on day 3, of NaCl restriction. The decreased Na excretion at day 1 occurred despite the absence of a significant increase in plasma aldosterone level or in the abundance of any of the major renal Na transporters. However, after a 1-day lag, plasma aldosterone levels increased in association with increases in abundances of three aldosterone-regulated Na transporter proteins: the thiazide-sensitive Na-Cl cotransporter (NCC), the alpha-subunit of the amiloride-sensitive epithelial Na channel (alpha-ENaC), and the 70-kDa form of gamma-ENaC. RNase protection assays of transporter mRNA levels revealed an increase in renal alpha-ENaC mRNA coincident with the increase in alpha-ENaC protein abundance. However, there was no change in NCC mRNA abundance, suggesting that the increase in NCC protein in response to dietary NaCl restriction was not a result of altered gene transcription. These results point to early regulatory processes that decrease renal Na excretion without an increase in the abundance of any Na transporter, followed by a late aldosterone-dependent response associated with upregulation of NCC and ENaC.
