scholarly journals Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line

2013 ◽  
Vol 33 (5) ◽  
Author(s):  
Zhi-Yuan Yu ◽  
Qun Kong ◽  
Bruce C. Kone
Keyword(s):  
Glutathione Transferase ◽  
Collecting Duct ◽  
Chromosome 9 ◽  
Na Channel ◽  
Central Component ◽  
Mrna Levels ◽  
Functional Interaction ◽  
Α Subunit ◽  
Duct Cells ◽  
Collecting Duct Cells

The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in mIMCD3 CD (collecting duct) cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST (glutathione transferase)–Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2–Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.

Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells

1996 ◽  
Vol 271 (1) ◽  
pp. C423-C428 ◽  
Author(s):  
D. L. Denault ◽  
G. Fejes-Toth ◽  
A. Naray-Fejes-Toth
Keyword(s):  
Nested Pcr ◽  
Collecting Duct ◽  
Na Channel ◽  
Mrna Levels ◽  
Cortical Collecting Duct ◽  
Gamma Subunit ◽  
Subunit Mrna ◽  
Duct Cells ◽  
Pcr Product ◽  
Collecting Duct Cells

Specific regulatory mechanisms of aldosterone-stimulated Na+ reabsorption through the apical amiloride-sensitive channel are unknown. In this study, we examined the effects of aldosterone on Na+ channel gamma-subunit mRNA levels in cultured rabbit cortical collecting duct cells. With the use of reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from aldosterone-treated cells and degenerate primers, a 446-base pair (bp) PCR product was amplified and further characterized by nested PCR and sequencing. The nested PCR yielded a predicted 164-bp product. Sequencing of the 446-bp PCR product revealed 83% nucleotide and 91% amino acid identity to the rat colonic Na+ channel gamma-subunit. The relative abundance of Na+ channel mRNA was determined by quantitative PCR after a 24-h aldosterone treatment. The results demonstrate that Na+ channel gamma-subunit mRNA levels were significantly higher (2.6 +/- 0.42) in aldosterone-treated cultures vs. the controls. This increase, however, is less than the aldosterone-induced increase (3.2 +/- 2.0) in the amiloride-sensitive short-circuit current. These results indicate that Na+ channel gamma-subunit mRNA levels are increased by aldosterone and that this increase is likely to be responsible, at least in part, for the aldosterone-induced Na+ current in the kidney.

Whole cell and unitary amiloride-sensitive sodium currents in M-1 mouse cortical collecting duct cells

1996 ◽  
Vol 270 (4) ◽  
pp. C998-C1010 ◽  
Author(s):  
M. L. Chalfant ◽  
T. G. O'Brien ◽  
M. M. Civan
Keyword(s):  
Collecting Duct ◽  
Cell Permeability ◽  
Na Channel ◽  
Na Channels ◽  
Cortical Collecting Duct ◽  
Ionic Selectivity ◽  
Whole Cell ◽  
Duct Cells ◽  
Excised Patches ◽  
Collecting Duct Cells

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

1997 ◽  
Vol 273 (4) ◽  
pp. C1194-C1205 ◽  
Author(s):  
Hassane Amlal ◽  
Zhaohui Wang ◽  
Manoocher Soleimani
Keyword(s):  
Collecting Duct ◽  
Acid Stress ◽  
Northern Hybridization ◽  
Mrna Levels ◽  
Inner Medullary Collecting Duct ◽  
Duct Cells ◽  
Acid Load ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Mutant Cells

The response of H+-ATPase to lethal acid stress is unknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cycles of lethal acid stress. Cells were grown to confluence on coverslips, loaded with 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein, and monitored for intracellular pH (pHi) recovery from an acid load. The rate of Na+-independent pHi recovery from an acid load in mutant cells was approximately fourfold higher than in parent cells ( P < 0.001). The Na+-independent H+ extrusion was ATP dependent and K+ independent and was completely inhibited in the presence of diethylstilbestrol, N, N′-dicyclohexylcarbodiimide, or N-ethylmaleimide. These results indicate that the Na+-independent H+ extrusion in cultured medullary cells is mediated via H+-ATPase and is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remained unchanged in mutant cells compared with parent cells. We propose that lethal acid stress results in increased H+-ATPase activity in inner medullary collecting duct cells. Upregulation of H+-ATPase could play a protective role against cell death in severe intracellular acidosis.

scholarly journals Overexpression of the Epithelial Na+Channel γ Subunit in Collecting Duct Cells

2005 ◽  
Vol 280 (18) ◽  
pp. 18348-18354 ◽  
Author(s):  
Kenneth A. Volk ◽  
Russell F. Husted ◽  
Rita D. Sigmund ◽  
John B. Stokes
Keyword(s):  
Collecting Duct ◽  
Na Channel ◽  
Γ Subunit ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Epithelial Na Channel

scholarly journals Soluble (pro)renin receptor regulation of ENaC involved in aldosterone signaling in cultured collecting duct cells

2020 ◽  
Vol 318 (3) ◽  
pp. F817-F825 ◽  
Author(s):  
Fei Wang ◽  
Renfei Luo ◽  
Kexin Peng ◽  
Xiyang Liu ◽  
Chuanming Xu ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Α Subunit ◽  
Duct Cells ◽  
Nadph Oxidase 1 ◽  
Collecting Duct Cells ◽  
Two Phases

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na+ channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), the cleavage product of PRR in ENaC regulation, and further tested its relevance to aldosterone signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM within minutes induced a significant and transient increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique. The acute ENaC activation was blocked by the NADPH oxidase 1/4 inhibitor GKT137892 and siRNA against Nox4 but not the β-catenin inhibitor ICG-001. In primary rat inner medullary collecting duct cells, administration of sPRR-His at 10 nM for 24 h induced protein expression of the α-subunit but not β- or γ-subunits of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The transcriptional activation of α-ENaC was dependent on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Ussing chamber determination of short-circuit current showed that aldosterone-induced transepithelial Na+ transport was inhibited by the PRR decoy inhibitor PRO20 and PF-429242, an inhibitor of sPRR-generating enzyme site-1 protease, and the response was restored by the addition of sPRR-His. Medium sPRR was elevated by aldosterone and inhibited by PF-429242. Taken together, these results demonstrate that sPRR induces two phases of ENaC activation via distinct mechanisms and functions as a mediator of the natriferic action of aldosterone.

Norepinephrine stimulates the epithelial Na+ channel in cortical collecting duct cells via α2-adrenoceptors

2015 ◽  
Vol 308 (5) ◽  
pp. F450-F458 ◽  
Author(s):  
Morag K. Mansley ◽  
Winfried Neuhuber ◽  
Christoph Korbmacher ◽  
Marko Bertog
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Nerve Fibers ◽  
Good Evidence ◽  
Fine Tuning ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na+ absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na+ excretion takes place. Here, the appropriate regulation of transepithelial Na+ transport, mediated by the amiloride-sensitive epithelial Na+ channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na+ transport, we performed short-circuit current ( Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na + absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

scholarly journals Novel Regulation of the Epithelial Na + Channel by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

2008 ◽  
Vol 22 (S1) ◽  
Author(s):  
Kenneth R. Hallows ◽  
Nuria M. Pastor‐Soler ◽  
Robert S. Edinger ◽  
Nicholas M. Oyster ◽  
Huamin Wang ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Collecting Duct ◽  
Na Channel ◽  
Soluble Adenylyl Cyclase ◽  
Kidney Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Epithelial Na Channel

Epidermal growth factor inhibits amiloride-sensitive sodium absorption in renal collecting duct cells

2003 ◽  
Vol 284 (1) ◽  
pp. F57-F64 ◽  
Author(s):  
Jie-Pan Shen ◽  
Calvin U. Cotton
Keyword(s):  
Growth Factor ◽  
Chronic Exposure ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Mrna Levels ◽  
Erk Pathway ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Renal Collecting Duct

The effects of the ERK pathway on electrogenic transepithelial Na+ absorption by renal collecting duct cells were determined. Approximately 90% of the unstimulated short-circuit current (15 ± 1 μA/cm2, n = 10) across conditionally immortalized murine collecting duct epithelial cells (mCT1) is amiloride sensitive and is likely mediated by apical epithelial Na+ channels. Chronic exposure (24 h) of the epithelial monolayers to either EGF (50 ng/ml) or transforming growth factor-α (TGF-α; 20 ng/ml) reduced amiloride-sensitive short-circuit current by >60%. The inhibitory effect of EGF on Na+ absorption was not due to inhibition of basolateral Na+-K+-ATPase, because the pump current elicited by permeabilization of apical membrane with nystatin was not reduced by EGF. Chronic exposure of the mCT1 cells to EGF (20 ng/ml, 24 h) elicited a 70–85% decrease in epithelial Na+ channel subunit mRNA levels. Exposure of mCT1 cells to either EGF (20 ng/ml) or PMA (150 nM) induced rapid phosphorylation of p42/p44 (ERK1/2) and pretreatment of the monolayers with PD-98059 (an ERK kinase inhibitor; 30 μM) prevented phosphorylation of p42/p44. Similarly, pretreatment of mCT1 monolayers with PD-98059 prevented the EGF- and PMA-induced inhibition of amiloride-sensitive Na+ absorption. The results of these studies demonstrate that amiloride-sensitive Na+ absorption by renal collecting duct cells is regulated by the ERK pathway. This pathway may play a role in alterations in ion transport that occur in polycystic kidney disease.

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