scholarly journals Differential regulation of NFAT5 by NKCC2 isoforms in medullary thick ascending limb (mTAL) cells

2011 ◽  
Vol 300 (4) ◽  
pp. F966-F975 ◽  
Author(s):  
Shoujin Hao ◽  
Hong Zhao ◽  
Zbigniew Darzynkiewicz ◽  
Sailaja Battula ◽  
Nicholas R. Ferreri
Keyword(s):  
Protein Expression ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
Primary Cultures ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Activated T Cells ◽  
Mrna Accumulation ◽  
Medullary Thick Ascending Limb ◽  
Nacl Concentration

The effects of Na+-K+-2Cl− cotransporter type 2 (NKCC2) isoforms on the regulation of nuclear factor of activated T cells isoform 5 (NFAT5) were determined in mouse medullary thick ascending limb (mTAL) cells exposed to high NaCl concentration. Primary cultures of mTAL cells and freshly isolated mTAL tubules, both derived from the outer medulla (outer stripe>inner stripe), express NKCC2 isoforms A and F. The relative expression of NKCC2A mRNA was approximately twofold greater than NKCC2F in these preparations. The abundance of NKCC2A mRNA, but not NKCC2F mRNA, increased approximately twofold when mTAL cells were exposed for 2 h to a change in osmolality from 300 to 500 mosmol/kgH2O, produced with NaCl. Total NKCC2 protein expression also increased. Moreover, a 2.5-fold increase in NFAT5 mRNA accumulation was observed after cells were exposed to 500 mosmol/kgH2O for 4 h. Laser-scanning cytometry detected a twofold increase in endogenous NFAT5 protein expression in response to high NaCl concentration. Pretreatment with the loop diuretic bumetanide dramatically reduced transcriptional activity of the NFAT5-specific reporter construct TonE-Luc in mTAL cells exposed to high NaCl. Transient transfection of mTAL cells with shRNA vectors targeting NKCC2A prevented increases in NFAT5 mRNA abundance and protein expression and inhibited NFAT5 transcriptional activity in response to hypertonic stress. Silencing of NKCC2F mRNA did not affect NFAT5 mRNA accumulation but partially inhibited NFAT5 transcriptional activity. These findings suggest that NKCC2A and NKCC2F exhibit differential effects on NFAT5 expression and transcriptional activity in response to hypertonicity produced by high NaCl concentration.

scholarly journals NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake

2013 ◽  
Vol 304 (5) ◽  
pp. F533-F542 ◽  
Author(s):  
Shoujin Hao ◽  
Lars Bellner ◽  
Nicholas R. Ferreri
Keyword(s):  
Tap Water ◽  
Primary Cultures ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Activated T Cells ◽  
Mrna Accumulation ◽  
Nacl Concentration ◽  
Promoter Construct ◽  
Mrna And Protein Expression

Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving the Na+-K+-2Cl− cotransporter (NKCC2). A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in nuclear factor of activated T cells 5 (NFAT5) mRNA and protein expression was observed in the outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in the outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately sixfold in response to an increase in osmolality to 400 mosmol/kgH2O produced with NaCl and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake.

scholarly journals Expression and function of NFAT5 in medullary thick ascending limb (mTAL) cells

2009 ◽  
Vol 296 (6) ◽  
pp. F1494-F1503 ◽  
Author(s):  
Shoujin Hao ◽  
Hong Zhao ◽  
Zbigniew Darzynkiewicz ◽  
Sailaja Battula ◽  
Nicholas R. Ferreri
Keyword(s):  
Primary Cultures ◽  
Constitutive Expression ◽  
Transient Transfection ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Activated T Cells ◽  
Mrna Accumulation ◽  
Culture Cells ◽  
Medullary Thick Ascending Limb

The contribution of nuclear factor of activated T cells 5 (NFAT5) to the regulation of tumor necrosis factor-α (TNF) production in medullary thick ascending limb (mTAL) cells is unclear. RT-PCR analysis was performed on primary cultures of mouse mTAL cells and freshly isolated mTAL tubules to determine which NFAT isoforms are present in this nephron segment. Primer pairs were designed, based on published sequences for mouse NFAT1-5, to produce fragments of ∼200 bp. Analysis of PCR products by gel electrophoresis and subsequent DNA sequencing indicated that cells and tubules contained mRNA for all five NFAT isoforms. The relative expression of NFAT isoforms was then determined using quantitative real-time RT-PCR. The data indicate that NFAT isoforms 5 ≥ 1 are the predominant isoforms present in mTAL cells and tubules. Western blot analysis demonstrated constitutive expression of NFAT5 in nuclear extracts from mTAL tubules and primary culture cells; expression in mTAL cells also was detected by immunofluorescence. Expression of NFAT5 was increased in mTAL cells transiently transfected with an NFAT5 overexpression vector (pcDNA3.1-NFAT5), resulting in increased basal and calcium-sensing receptor (CaR)-mediated TNF production. Transient transfection of mTAL cells with a small hairpin RNA vector that targeted exon 8 of NFAT5 (U6-N5 ex8) significantly inhibited TNF promoter activity. Transient transfection with U6-N5 ex8 also reduced nuclear expression of NFAT5, TNF mRNA accumulation, and attenuated CaR-mediated activation of Cl−entry into polarized mTAL cells. Collectively, these data suggest that activation of NFAT5 is part of a TNF-dependent pathway that inhibits apical Cl−influx in the mTAL after activation of CaR.

Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb

2003 ◽  
Vol 284 (5) ◽  
pp. F977-F986 ◽  
Author(s):  
Morgan Gallazzini ◽  
Amel Attmane-Elakeb ◽  
David B. Mount ◽  
Steven C. Hebert ◽  
Maurice Bichara
Keyword(s):  
Protein Expression ◽  
In Vivo Studies ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Immunoblotting Analysis ◽  
Medullary Thick Ascending Limb ◽  
Nacl Concentration ◽  
Mrna And Protein Expression

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH2O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA ( P < 0.04) and protein ( P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA ( P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium ( P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats ( P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.

CaR-mediated COX-2 expression in primary cultured mTAL cells

2001 ◽  
Vol 281 (4) ◽  
pp. F658-F664 ◽  
Author(s):  
Dairong Wang ◽  
Shao-Jian An ◽  
Wen-Hui Wang ◽  
John C. McGiff ◽  
Nicholas R. Ferreri
Keyword(s):  
Protein Expression ◽  
Intracellular Signaling ◽  
Primary Cultures ◽  
Extracellular Fluid ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Mrna Accumulation ◽  
Beneficial Effects ◽  
Cox 2 ◽  
Salt And Water Balance

Primary cultures of medullary thick ascending limb (mTAL) cells retain the capacity to express calcium-sensing receptor (CaR) mRNA and protein. Increases in cyclooxygenase-2 (COX-2) mRNA accumulation, protein expression, and PGE2 synthesis were observed in a dose- and time-dependent manner after exposure of these cells to extracellular calcium (Ca[Formula: see text]). Moreover, transfection of mTAL cells with a CaR overexpression vector significantly enhanced COX-2 expression and PGE2 production in response to calcium compared with cells transfected with an empty vector. Challenge with the CaR-selective agonist poly-l-arginine (PLA) also increased COX-2 mRNA accumulation, protein expression, and PGE2 synthesis. Furthermore, Ca[Formula: see text]- and PLA-mediated PGE2production was abolished in the presence of NS-398 or nimesulide, two different COX-2-selective inhibitors. These data suggest that intracellular signaling mechanisms initiated via activation of CaR contribute to COX-2-dependent PGE2 synthesis in the mTAL. Because Ca[Formula: see text] concentration varies along Henle's loop, calcium may contribute to salt and water balance via a COX-2- and CaR-dependent mechanism. Thus novel calcimimetics might be useful in conditions such as hypertension in which manipulation of extracellular fluid volume provides beneficial effects.

scholarly journals Prostaglandin E2 EP3 receptor regulates cyclooxygenase-2 expression in the kidney

2012 ◽  
Vol 303 (3) ◽  
pp. F449-F457 ◽  
Author(s):  
Carlos P. Vio ◽  
Mariana Quiroz-Munoz ◽  
Catherina A. Cuevas ◽  
Carlos Cespedes ◽  
Nicholas R. Ferreri
Keyword(s):  
Protein Expression ◽  
Negative Feedback ◽  
Fold Increase ◽  
Treated Group ◽  
Cyclooxygenase 2 ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Mrna Accumulation ◽  
Cox 2 ◽  
Ep3 Receptor

Cyclooxygenase-2 (COX-2) is constitutively expressed and highly regulated in the thick ascending limb (TAL). As COX-2 inhibitors (Coxibs) increase COX-2 expression, we tested the hypothesis that a negative feedback mechanism involving PGE2 EP3 receptors regulates COX-2 expression in the TAL. Sprague-Dawley rats were treated with a Coxib [celecoxib (20 mg·kg−1·day−1) or rofecoxib (10 mg·kg−1·day−1)], with or without sulprostone (20 μg·kg−1·day−1). Sulprostone was given using two protocols, namely, previous to Coxib treatment (prevention effect; Sulp7-Coxib5 group) and 5 days after initiation of Coxib treatment (regression effect; Coxib10-Sulp5 group). Immunohistochemical and morphometric analysis revealed that the stained area for COX-2-positive TAL cells (μm2/field) increased in Coxib-treated rats (Sham: 412 ± 56.3, Coxib: 794 ± 153.3). The Coxib effect was inhibited when sulprostone was used in either the prevention (285 ± 56.9) or regression (345 ± 51.1) protocols. Western blot analysis revealed a 2.1 ± 0.3-fold increase in COX-2 protein expression in the Coxib-treated group, an effect abolished by sulprostone using either the prevention (1.2 ± 0.3-fold) or regression (0.6 ± 0.4-fold vs. control, P < 0.05) protocols. Similarly, the 6.4 ± 0.6-fold increase in COX-2 mRNA abundance induced by Coxibs ( P < 0.05) was inhibited by sulprostone; prevention: 0.9 ± 0.3-fold ( P < 0.05) and regression: 0.6 ± 0.1 ( P < 0.05). Administration of a selective EP3 receptor antagonist, L-798106, also increased the area for COX-2-stained cells, COX-2 mRNA accumulation, and protein expression in the TAL. Collectively, the data suggest that COX-2 levels are regulated by a novel negative feedback loop mediated by PGE2 acting on its EP3 receptor in the TAL.

Dynamics of nucleotides in distal nephron of mice with nephrogenic diabetes insipidus

1986 ◽  
Vol 250 (1) ◽  
pp. F151-F158 ◽  
Author(s):  
E. Kusano ◽  
A. N. Yusufi ◽  
N. Murayama ◽  
J. Braun-Werness ◽  
T. P. Dousa
Keyword(s):  
Diabetes Insipidus ◽  
Nephrogenic Diabetes Insipidus ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Camp Phosphodiesterase ◽  
Camp Content ◽  
Medullary Thick Ascending Limb ◽  
Atp Levels ◽  
Collecting Tubules ◽  
Camp Levels

In mice with hereditary nephrogenic diabetes insipidus (NDI), the high activity of cAMP-phosphodiesterase (cAMP-PDIE) in medullary collecting tubules (MCT) prevents the increase in cAMP content in response to vasopressin [Arg8]vasopressin (AVP). Even when the cAMP response to AVP is partly corrected by cAMP-PDIE inhibitor 1-methyl-3-isobutylxanthine (MIX), under all tested conditions the cAMP levels in MCT of NDI mice remained much lower than in controls (B. A. Jackson, R. M. Edwards, H. Valtin, and T. P. Dousa, J. Clin. Invest. 66: 110-122, 1980). In the present study, we explored which factors may account for this defect. We determined contents of ATP, nicotinamide adenine dinucleotide (NAD), and the levels of cAMP in MCT and in medullary thick ascending limb of Henle's loop (MAL) microdissected from control and NDI mice. In the presence of 1 microM AVP and 0.05 mM MIX, the cAMP levels accumulated in MCT of NDI mice were four times lower compared with controls, but the levels of ATP and NAD were not different. ATP levels in MAL of NDI mice were slightly (delta -23%) lower than in MAL from controls, and in distal convoluted tubules (DCT) of NDI mice the ATP levels were also decreased (delta -49%). Although AVP alone had little effect on cAMP levels in mouse MAL in the presence of 0.1 mM forskolin, the AVP elicited a 20-fold increase of cAMP of both the control and NDI mice. Addition of 0.1 mM forskolin further increased the cAMP accumulation in MCT incubated with AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

NBCn1 is a basolateral \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}\mathrm{-HCO}_{3}^{-}\) \end{document} cotransporter in rat kidney inner medullary collecting ducts

2004 ◽  
Vol 286 (5) ◽  
pp. F903-F912 ◽  
Author(s):  
Jeppe Praetorius ◽  
Young-Hee Kim ◽  
Elena V. Bouzinova ◽  
Sebastian Frische ◽  
Aleksandra Rojek ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Thick Ascending Limb ◽  
Medullary Thick Ascending Limb ◽  
Collecting Ducts ◽  
Basolateral Plasma Membrane ◽  
Medullary Collecting Duct ◽  
Plasma Membrane Domain

Primary cultures of rat inner medullary collecting duct (IMCD) cells Na+ dependently import [Formula: see text] across the basolateral membrane through an undefined transport protein. We used RT-PCR, immunoblotting, and immunohistochemistry to identify candidate proteins for this basolateral [Formula: see text] cotransport. The mRNA encoding the electroneutral [Formula: see text] cotransporter NBCn1 was detected as the only [Formula: see text] cotransporter in the rat inner medulla (IM) among the five characterized Na+-dependent [Formula: see text] transporters. The mRNA of a yet uncharacterized transporter-like protein, BTR1, was also present in the IM, but its expression in microdissected tubules seemed restricted to the thin limbs of Henle's loop. Immunoblotting confirmed the presence of NBCn1 as an ∼180-kDa protein of the rat IM. Anti-NBCn1 immunolabeling was confined to the basolateral plasma membrane domain of IMCD cells in the papillary two-thirds of the IM. Consistent with the presence of NBCn1, IMCD cells possessed stilbene-insensitive, Na+- and [Formula: see text]-dependent pH recovery after acidification, as assessed by fluorescence microscopy using a pH-sensitive intracellular dye. In furosemide-induced alkalotic rats, NBCn1 protein abundance was decreased in both the IM and inner stripe of outer medulla (ISOM) as determined by immunoblotting and immunohistochemistry. In contrast, NBCn1 abundance in the IM and ISOM was unchanged in NaHCO3-loaded animals, and the NBCn1 abundance increased only in the ISOM after NH4Cl loading. In conclusion, NBCn1 is a basolateral [Formula: see text] cotransporter of IMCD cells and is differentially regulated in IMCD and medullary thick ascending limb.

Abstract 553: Prostaglandin E2 EP3 Receptor Downregulates COX2 Expression in the Medullary Thick Ascending Limb (mTAL) Induced by Hypertonic NaCl Intake

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Shoujin Hao ◽  
Carlos P Vio ◽  
Carlos Cespedes ◽  
Mariana Quiroz-Munoz ◽  
Nicholas R Ferreri
Keyword(s):  
Protein Expression ◽  
Tap Water ◽  
Receptor Expression ◽  
Free Access ◽  
Thick Ascending Limb ◽  
Mrna Levels ◽  
Mrna Accumulation ◽  
Cox 2 ◽  
Ep3 Receptor ◽  
In Cells

We recently showed that a novel negative feedback mechanism involving PGE 2 acting on EP3 receptors regulates cyclooxygenase-2 (COX-2) expression in the thick ascending limb (TAL) induced by the selective COX-2 inhibitors, celecoxib and rofecoxib. In the present study we tested the hypothesis that inhibition of EP3 facilitates COX-2 expression in the TAL induced by ingestion of hypertonic NaCl or exposure of mTAL cells to hypertonic media. COX-2 protein expression in the outer medulla (OM) increased 2.2±0.3 fold in mice given free access to 1% NaCl in the drinking water for 3 days compared with tap water; p<0.05. The increase was associated with a 4-fold elevation in COX-2 mRNA accumulation (tap water: 0.5±0.1 vs 1% NaCl: 1.9±0.4; p<0.05), and higher PGE 2 production by freshly isolated mTAL tubules (tap water: 87.6±9.4 vs 1% NaCl: 203.3±19.3 pg/mg protein; p<0.05). EP3 mRNA levels also increased approximately 2-fold in OM of mice ingesting 1% NaCl (tap water: 0.7±0.2 vs 1% NaCl: 1.3±0.2). Administration of a selective EP3 receptor antagonist (L-798106: 20μg/kg/day) for 2 days increased COX-2 mRNA accumulation in mTAL tubules (L-798106: 2.1±0.1 fold change vs control; p<0.05), and the elevation in COX-2 protein expression induced by 1% NaCl was increased an additional 50% in mice given L-798106. COX-2 mRNA accumulation in primary cultures of mTAL cells increased 2-fold in response to media made hypertonic by addition of NaCl (400 mosmol/kg H 2 O), compared with cells incubated in isotonic media. L-798106 increased COX-2 mRNA 2-fold in isotonic media and 4-fold in cells exposed to 400 mosmol/kg H 2 O. PGE 2 production by mTAL cells increased approximately 4-fold in response to challenge with 400 mosmol/kg H 2 O for 9 hr, and was inhibited in cells transiently transfected with a lentivirus shRNA construct (EGFP-C2-ex5) to silence COX-2 (286.1±14.8 to 64.3±5.2 pg/mg protein; p<0.05). Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP3 receptor expression, which attenuates COX-2 activity in this segment of the nephron. Moreover, PGE 2 signaling via EP3 receptors in the TAL may be part of a mechanism that regulates mTAL COX-2 expression and function in response to diverse stimuli.

Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells

2002 ◽  
Vol 283 (5) ◽  
pp. F963-F970 ◽  
Author(s):  
Dairong Wang ◽  
Paulina L. Pedraza ◽  
Huda Ismail Abdullah ◽  
John C. McGiff ◽  
Nicholas R. Ferreri
Keyword(s):  
Protein Expression ◽  
Intracellular Signaling ◽  
Significant Inhibition ◽  
Thick Ascending Limb ◽  
Water Excretion ◽  
Medullary Thick Ascending Limb ◽  
Promoter Construct ◽  
Intracellular Ca ◽  
Cox 2 ◽  
Tnf Gene

Medullary thick ascending limb (mTAL) cells in primary culture express the Ca2+-sensing receptor (CaR), a G protein-coupled receptor that senses changes in extracellular Ca2+(Ca[Formula: see text]) concentration, resulting in increases of intracellular Ca2+concentration and PKC activity. Exposure of mTAL cells to either Ca[Formula: see text] or the CaR-selective agonist poly-l-arginine increased TNF-α synthesis. Moreover, the response to Ca[Formula: see text] was enhanced in mTAL cells transfected with a CaR overexpression vector. Transfection of mTAL cells with a TNF promoter construct revealed an increase in reporter gene activity after exposure of the cells to Ca[Formula: see text], suggesting that intracellular signaling pathways initiated by means of activation of a CaR contribute to TNF synthesis by a mechanism that involves transcription of the TNF gene. Neutralization of TNF activity with an anti-TNF antibody attenuated Ca2+-mediated increases in cyclooxygenase-2 (COX-2) protein expression and PGE2synthesis, suggesting that TNF exerts an autocrine effect in the mTAL, which contributes to COX-2-mediated PGE2production. Preincubation with the PKC inhibitor bisindolylmaleimide I inhibited Ca2+-mediated TNF production. Significant inhibition of COX-2 protein expression and PGE2synthesis also was observed when cells were challenged with Ca[Formula: see text] in the presence of bisindolylmaleimide I. The data suggest that increases in TNF production subsequent to activation of the CaR may be the basis of an important renal mechanism that regulates salt and water excretion.

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