Protein kinase C-dependent phosphorylation of Na(+)-K(+)-ATPase alpha-subunit in rat kidney cortical tubules

Aldosterone binds to the mineralocorticoid receptor (MR) and increases renal Na+ reabsorption via up-regulation of the epithelial Na+ channel (ENaC) and the Na+-K+-ATPase in the collecting system (CS) and possibly also via the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations remains controversial. We used mice with deletion of MR in 20% of renal tubule cells (MR/X mice), in which MR-positive (MRwt) and -negative (MRko) cells can be studied sideby-side in the same physiological context. Adult MR/X mice showed similar mRNA and protein levels of renal ion transport proteins to control mice. In MR/X mice, no differences in NCC abundance and phosphorylation was seen between MRwt and MRko cells and dietary Na+ restriction up-regulated NCC to similar extent in both groups of cells. In contrast, MRko cells in the CS did not show any detectable alpha-ENaC abundance or apical targeting of ENaC neither on control diet nor in response to dietary Na+ restriction. Furthermore, Na+-K+-ATPase expression was unaffected in MRko cells of the DCT, while it was lost in MRko cells of the CS. In conclusion, MR is crucial for ENaC and Na+-K+-ATPase regulation in the CS, but is dispensable for NCC and Na+-K+-ATPase regulation in the DCT. PAEJ-D-16-00005 -NCC regulation is MR independent 2 ACKNOWLEDGEMENTSThe authors would like to thank Günter Schütz and Stefan Berger, Burkhard Becher, Celso E. Gomez-Sanchez, and Eric Feraille for kindly providing us with the MRlox/lox mouse line, the cmv-cre mouse line, the anti-MR antibodies, and the anti-Na + -K + -ATPase antibody, respectively. The expert technical assistance by Monique Carrel and Michèle Heidemeyer is gratefully acknowledged. GRANTS DISCLOSURESThe authors declare no conflict of interest, financial or otherwise. PAEJ-D-16-00005 -NCC regulation is MR independent 3 ABSTRACTAldosterone binds to the mineralocorticoid receptor (MR) and increases renal Na + reabsorption via up-regulation of the epithelial Na + channel (ENaC) and the Na + -K + -ATPase in the collecting system (CS) and possibly also via the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations remains controversial. We used mice with deletion of MR in ~20% of renal tubule cells (MR/X mice), in which MR-positive (MR wt ) and -negative (MR ko ) cells can be studied sideby-side in the same physiological context. Adult MR/X mice showed similar mRNA and protein levels of renal ion transport proteins to control mice. In MR/X mice, no differences in NCC abundance and phosphorylation was seen between MR wt and MR ko cells and dietary Na + restriction up-regulated NCC to similar extent in both groups of cells. In contrast, MR ko cells in the CS did not show any detectable alphaENaC abundance or apical targeting of ENaC neither on control diet nor in response to dietary Na + restriction. Furtherm...

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“…The anti-MR antibody was kindly provided by Dr. GomezSanchez [9]. The anti-Na + -K + -ATPase antibody was a kind gift from Dr. Feraille [7].…”

Section: Gacaggatgcagaaggagattactgmentioning

confidence: 99%

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

Czogalla

Vohra

Pentón

et al. 2016

Pflugers Arch - Eur J Physiol

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“…Proteins were electro-transfered to polyvinylidine difluoride membranes (Polyscreen, Dupont de Nemours). After blocking, blots were successively incubated with anti-Na ϩ ,K ϩ -ATPase antibody (dilution 1/10,000) raised against the holoenzyme purified from rat kidney 44 and goat anti-rabbit IgG antibody (dilution 1/20,000) coupled to horseradish peroxidase (Promega), and revealed by chemiluminescence with SuperSignal West Pico Substrate (Pierce Biotechnology, France). Densitometry was quantitated by digital image analysis (Image Scan, Molecular Dynamics), expressed as a function of the tubular length, and thenafter as percent of the mean densitometry of the corresponding control.…”

Section: Rt-real Time Pcrmentioning

confidence: 99%

Sodium retention and ascites formation in a cholestatic mice model

et al. 2007

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Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenalintact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodiumand potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium-and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum-and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium-and potassiumdependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. Conclusion: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium-and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium-and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids. (HEPATOLOGY 2007;46:173-179.) See Editorial on Page 9 P atients with liver cirrhosis and portal hypertension develop renal sodium retention which promotes formation of ascites and peripheral edema. The "underfill theory" initially attributed sodium retention to secondary hyperaldosteronism accounted for by renal hypoperfusion brought about by intraabdominal fluid sequestration. 1 However, the underfill theory cannot explain the early phase of cirrhosis as sodium retention may precedes formation of ascites. 2-7 Thus, the "overflow theory" postulated that edema and ascites formation resulted from primary renal sodium retention promoting plasma volume expansion. 8 This theory is supported by the fact that abolishing portal hypertension in cirrhotic dogs by sideto-side portocaval shunt prevented ascites formation but not sodium retention. 5,6 Later, the "revised underfill theory" proposed that peripheral arterial vasodilatation leading to decreased effec...

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“…Proteins were blotted onto a polyvinylidine difluoride membrane (Immobilon-P, Millipore), incubated overnight with a polyclonal antibody (dilution, 1:2500) raised against the ␣-subunit of Na,K-ATPase (27), and then incubated with a second anti-rabbit IgG antibody (dilution, 1:10000) coupled to horseradish peroxidase (Transduction Laboratories, Lexington, KY). Detection was performed by chemiluminescence using the Super Signal substrate method (Pierce).…”

Section: Measurement Of Total and Cell Surfacementioning

confidence: 99%

Short Term Effect of Aldosterone on Na,K-ATPase Cell Surface Expression in Kidney Collecting Duct Cells

Summa¹,

Mordasini²,

Roger³

et al. 2001

Journal of Biological Chemistry

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Aldosterone controls extracellular volume and blood pressure by regulating Na ؉ reabsorption, in particular by epithelia of the distal nephron. A main regulatory site of this transcellular transport is the epithelial sodium channel (ENaC) that mediates luminal Na ؉ influx. The Na,K-ATPase (Na ؉ pump) that coordinately extrudes Na ؉ across the basolateral membrane is known to be regulated by short term aldosterone as well. We now show that in the cortical collecting duct (CCD) from adrenalectomized rats, the increase in Na,K-ATPase activity (approximately 3-fold in 3 h), induced by a single aldosterone injection, can be fully accounted by the increase in Na,K-ATPase cell surface expression (؉ 497 ؎ 35%). The short term aldosterone action was further investigated in cultured mouse collecting duct principal cells mpkCCD cl4 . Within 2 h, maximal Na,K-ATPase function assessed by Na ؉ pump current (I p ) measurements and Na,K-ATPase cell surface expression were increased by 20 -50%. Aldosterone did not modify the Na ؉ dependence of the Na ؉ pumps and induced transcription-and translation-dependent actions on pump surface expression and current independently of ENaC-mediated Na ؉ influx. In summary, short term aldosterone directly increases the cell surface expression of preexisting Na ؉ pumps in kidney CCD target cells. Thus, aldosterone controls Na ؉ reabsorption in the short term not only by regulating the apical cell surface expression of ENaC (Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B. C., Firestone, G. L., Pearce, D., and Verrey, F. (2001) Am. J. Physiol. 280, F675-F682) but also by coordinately acting on the basolateral cell surface expression of the Na,K-ATPase.

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Protein kinase C-dependent phosphorylation of Na(+)-K(+)-ATPase alpha-subunit in rat kidney cortical tubules

Cited by 74 publications

References 16 publications

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

Sodium retention and ascites formation in a cholestatic mice model

Short Term Effect of Aldosterone on Na,K-ATPase Cell Surface Expression in Kidney Collecting Duct Cells

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