Corticosteroid-Dependent Sodium Transport in a Novel Immortalized Mouse Collecting Duct Principal Cell Line

Abstract

Abstract. The final control of sodium balance takes place in the cortical collecting duct (CCD) of the nephron, where corticosteroid hormones regulate sodium reabsorption by acting through mineralocorticoid (MR) and/or glucocorticoid (GR) receptors. A clone of principal CCD cells (mpkCCDc14) has been established that is derived from a transgenic mouse (SV40 large T antigen under the control of the SV40 enhancer/L-type pyruvate kinase promoter). Cells grown on filters form polarized monolayers with high electrical transepithelial resistance (RT approximately 4700 Ω × cm2) and potential difference (PD approximately -50 mV) and have an amiloride-sensitive electrogenic sodium transport, as assessed by the short-circuit current method (Isc approximately 11 μA/cm2). Reverse transcription-PCR experiments using rat MR primers, [3H]aldosterone, and [3H]dexamethasone binding and competition studies indicated that the mpkCCDc14 cells exhibit specific MR and GR. Aldosterone increased Isc in a dose- (10-10 to 10-6 M) and time-dependent (2 to 72 h) manner, whereas corticosterone only transiently increased Isc (2 to 6 h). Consistent with the expression of 11β-hydroxysteroid dehydrogenase type 2, which metabolizes glucocorticoids to inactive 11-dehydroderivates, carbenoxolone potentiated the corticosterone-stimulated Isc. Aldosterone (5 × 10-7 M)-induced Isc (fourfold) was associated with a three- to fivefold increase in α-ENaC mRNA (but not in those for β- or γ-ENaC) and three- to 10-fold increases in α-ENaC protein synthesis. In conclusion, this new immortalized mammalian CCD clonal cell line has retained a high level of epithelial differentiation and sodium transport stimulated by aldosterone and therefore represents a useful mammalian cell system for identifying the genes controlled by aldosterone.

Epac1–/– and Epac2–/– mice exhibit deficient epithelial Na+ channel regulation and impaired urinary Na+ conservation

“…Using a high-sensitivity chemiluminescence kit on the same nitrocellulose membrane, 3 bands of a similar size were detected by antibodies against αENaC ( Figure 4A ). However, only the lowest molecular weight band (shown with arrow) was present in whole cell lysates from mpkCCD c14 cells ( Supplemental Figure 3 ), a generally accepted model of principal cell of the collecting duct ( 31 ). Thus, we considered that this is the cleaved form of αENaC and the upper 2 bands are not specific.…”

Statistical distortion of supervised learning predictions in optical microscopy induced by image compression

“…Microspheres were imaged with a 20 × objective and used for segmentation tests, as well as for the measurement of the microscope’s point spread function (PSF). Two cell lines with variable confluence have been cultured: human neural stem cells (HIP) (A3890101, ThermoFisher) 27 and mpkCCDC14 mouse kidney collecting duct cells (MPK) 28 , 29 . Both cell lines were cultured and grown on a cell treated plastic surface at a temperature of 37 °C with an air atmosphere enriched with 5% CO 2 .…”

A Vasopressin-Induced Change in Prostaglandin Receptor Subtype Expression Explains the Differential Effect of PGE2 on AQP2 Expression

“…However, a problem with in vivo studies investigating the effect of prostaglandins on the collecting duct is that these studies are complicated by the effect of prostaglandins on AVP release and on medullary osmolality, both of which will influence the AQP2 expression (Yamamoto et al, 1976;Stoff et al, 1981;Hasler et al, 2005). To study the effect of prostaglandins directly on principal cells, experiments were performed in mpkCCD cells, shown to display the essential functionalities characteristic of principal cells like the AVPregulated AQP2 expression and aldosterone-mediated sodium transport via the epithelial sodium channel (Bens et al, 1999;Hasler et al, 2002).…”