Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts.

Moe, Orson W.; Miller, R. Tyler; Horie, Shigeo; Cano, Adriana; Preisig, Patricia A.; Alpern, Robert J.

doi:10.1172/jci115487

Cited by 115 publications

(101 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Hence, we anticipated that we could successfully express NKCC2 protein by tagging its N-terminal domain with Myc. Therefore, full-length mouse NKCC2 cDNA was cloned into pCMV-expression vector downstream of the c-Myc tag and used for transient transfection in either OKP or MKTAL cells, two models of previously characterized renal epithelial cell lines (28,41,42). The expression of NKCC2 protein was determined by Western blot following immunoprecipitation using anti-Myc antibody.…”

Section: Nkcc2 Expression In Mammalianmentioning

confidence: 99%

“…As mentioned above, aldolase B protein expression in renal proximal tubule cells is unequivocally demonstrated (44 -46). Given that OKP cells are an excellent model of proximal tubule cells (41,42) and that transient transfection is very efficient in these cells (49,50), we assumed that they are an appropriate model for testing the ability of NKCC2 to interact with endogenous aldolase protein. To address this, a goat antibody raised against native aldolase protein was used.…”

Section: Endogenous Aldolase Protein Interacts and Co-localizes With mentioning

confidence: 99%

See 1 more Smart Citation

NKCC2 Surface Expression in Mammalian Cells

Benziane

Demaretz

Defontaine

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

Apical bumetanide-sensitive Na؉ -K ؉ -2Cl ؊ co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

show abstract

Section: Nkcc2 Expression In Mammalianmentioning

confidence: 99%

Section: Endogenous Aldolase Protein Interacts and Co-localizes With mentioning

confidence: 99%

NKCC2 Surface Expression in Mammalian Cells

Benziane

Demaretz

Defontaine

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…NHE1 has been shown to be stimulated by acid in both animals and cell culture but this is unlikely to be related to transepithelial transport of H + equivalents. [44][45][46] Chronic adaptation of NHE3 has been described in both the proximal tubule (increased NHE3 activity and protein) and TAL (increased NHE3 protein and transcript) in animals given a sufficient acid load to chronically suppress serum [HCO 3 − ]. 36,[47][48][49][50][51][52][53] Presently it is unclear whether the signals and mechanisms responsible for increased NHE3 are the same for the proximal tubule and TAL.…”

Section: Adaptation To Metabolic Acidosismentioning

confidence: 99%

Na+/H+ Exchangers in Renal Regulation of Acid-Base Balance

Bobulescu

Moe

2006

Seminars in Nephrology

View full text Add to dashboard Cite

The kidney plays key roles in extracellular fluid pH homeostasis by reclaiming bicarbonate (HCO 3 − ) filtered at the glomerulus and generating the consumed HCO 3 − by secreting protons (H + ) into the urine (renal acidification). Sodium-proton exchangers (NHEs) are ubiquitous transmembrane proteins mediating the countertransport of Na + and H + across lipid bilayers. In mammals, NHEs participate in the regulation of cell pH, volume, and intracellular sodium concentration, as well as in transepithelial ion transport. Five of the 10 isoforms (NHE1-4 and NHE8) are expressed at the plasma membrane of renal epithelial cells. The best-studied isoform for acid-base homeostasis is NHE3, which mediates both HCO 3 − absorption and H + excretion in the renal tubule. This article reviews some important aspects of NHEs in the kidney, with special emphasis on the role of renal NHE3 in the maintenance of acid-base balance.Keywords sodium/hydrogen exchange; renal acidification; bicarbonate absorption The free hydrogen ion (H + ) concentration in body fluids is regulated exquisitely around 40 nmol/L (pH 7.40) whereas H + flux through the body greatly exceeds this magnitude. In a 70-kg human being at a basal state, normal metabolic and dietary acid production rate is about 50 to 70 mmoles/d and respiratory volatile acid production at the basal state is around 15,000 mmoles/d, with peak production at maximal exercise reaching 200 mmoles/min. The flux of H + through the organism over 24 hours is 8 orders of magnitude greater than the total pool of free H + in total body water (<2 μmoles). This remarkable homeostatic feat is accomplished by concerted efforts of extracellular and intracellular buffers, highly efficient ventilatory responses, metabolic functions of the liver, and renal ammoniagenic and solute transport mechanisms. For excretion of nonvolatile acid and base loads, the kidney assumes the pivotal role.A filtration-reabsorption nephron bears an exorbitant burden of having to reclaim a vast amount of valuable solutes indiscriminately dispensed in the filtrate; one of which of course is the approximately 4,000 mmoles of bicarbonate (HCO 3 − ) per day. The luminal acid disequilibrium pH implies that the predominant mode of HCO 3 − absorption involves H + secretion, although a small concomitant degree of direct HCO 3 − reabsorption cannot be excluded. 1 Two points are noteworthy. First, complete reclamation of the approximately 4,000 mmoles of filtered HCO 3 − forestalls a physiologic disaster but does not lead to net acid secretion. Further elaboration of H + into the urine is necessary. Second, whether the organism is catering to the The era of phenomenologic analysis was supplemented by gene-and protein-specific reagents when the first mammalian NHE was cloned by Sardet et al. 7 The relationship between mammalian NHE genetic sequence and those from a multitude of organisms is shown in Fig 2. Na + /H + exchange across lipid bilayers and proteins that sustain this function are universal in prokaryotic, animal, and...

show abstract

mentioning

confidence: 99%

“…The effect on the Na/H antiporter can be simulated by incubation of a rabbit primary proximal tubule cell culture, or a number of renal cell lines, in acid medium for 24-48 h (6)(7)(8). This effect of acid incubation is dependent on protein synthesis and is associated with an increase in Na/H antiporter mRNA (mRNANa/H) abundance at 24 h (6)(7)(8). In rabbit proximal tubule primary cultures, chronic activation of protein kinase C (phorbol esters for 2 h) causes an increase in Na/H antiporter activity that persists 24 h after removal of phorbol ester, is dependent upon transcription and translation, and is associated with a 2-fold increase in mRNANa/H (9).…”

mentioning

confidence: 99%

Role of protein kinase C and transcription factor AP-1 in the acid-induced increase in Na/H antiporter activity.

Horie

Moe

Yamaji

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Chronic incubation of cultured renal tubular epithelial cells in acid medium causes an increase in Na/H antiporter activity that persists after removal from acid, is dependent on protein synthesis, and is associated with an increase in Na/H antiporter mRNA. Chronic activation of protein kinase C has similar effects in these cells. The present studies examined the role of protein kinase C in the effect of acid incubation. Incubation of MCT cells in acid for 24 h caused a 50% increase in Na/H antiporter activity. This was prevented by inhibition of protein kinase C, either with sphingosine or by protein kinase C downregulation. Pertussis toxin pretreatment did not prevent the increase in antiporter activity. Acid incubation caused an increase in transcription factor AP-1 activity, as shown by an increase in expression from a reporter gene containing six tandem AP-1 binding sites. This was associated with transient increases in c-fos and c-jun mRNAs. This response is typical of that for gene activation by protein kinase C. These studies demonstrate that acid activation of the Na/H antiporter requires protein kinase C and is associated with c-fos and c-jun expression and increased AP-1 activity.

show abstract

Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts.

Cited by 115 publications

References 32 publications

NKCC2 Surface Expression in Mammalian Cells

NKCC2 Surface Expression in Mammalian Cells

Na+/H+ Exchangers in Renal Regulation of Acid-Base Balance

Role of protein kinase C and transcription factor AP-1 in the acid-induced increase in Na/H antiporter activity.

Contact Info

Product

Resources

About