Isolation and characterization of luminal and basolateral plasma membrane vesicles from the medullary thick ascending loop of Henle

Attmane-Elakeb, Amel; Chambrey, Régine; Tsimaratos, Michel; Leviel, F.; Blanchard, Anne; Warnock, David G.; Paillard, Michel; Podevin, René-Alexandre

doi:10.1038/ki.1996.408

Cited by 36 publications

(47 citation statements)

References 14 publications

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“…At the same time, in our view it is unsurprising that there was no diminution of Na¤-H¤ exchange when using the low-sodium perfusate. Immunohistochemical studies and Western blot analyses using polyclonal antibodies have demonstrated intense staining for NHE_3 in the apical membrane of rat medullary and cortical TALH (Amemiya et al 1995;Attmane-Elakeb et al 1996), whereas staining in the pars recta was relatively meagre in the Sµ segment and completely absent from the S× segment (Amemiya et al 1995). Furthermore, apical carbonic anhydrase (type IV) is present in the TALH and the Sµ segment of the pars recta, but not in the S× segment (Brown et al 1990).…”

Section: Discussionmentioning

confidence: 99%

Contribution of Na⁺‐H⁺ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

Shirley

Walter

Unwin

et al. 1998

The Journal of Physiology

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1. The contribution of apical Na¤-H¤ exchange to sodium reabsorption in the thick ascending limb of the loop of Henle (TALH) in vivo was examined in anaesthetized rats by perfusing loops of Henle of superficial nephrons with solutions containing the Na¤-H¤ exchange inhibitor, ethyl isopropyl amiloride (EIPA). 2. Using a standard perfusate, no statistically significant effect of EIPA on net sodium reabsorption (JNa) was detected. However, when sodium reabsorption in the pars recta of the proximal tubule was minimized by using a low-sodium perfusate, EIPA reduced JNa from 828 ± 41 to 726 ± 37 pmol min¢ (P < 0·05), indicating that apical Na¤-H¤ exchange can make a small contribution to net sodium reabsorption in the TALH in vivo. This contribution appears to be dependent on the bicarbonate load, since an increase in the latter led to an enhancement of EIPA-sensitive sodium transport. 3. Addition of the Na¤-K¤-2Cl¦ cotransport inhibitor, bumetanide, to the low-sodium perfusate reduced baseline JNa to 86 ± 27 pmol min¢. In this setting, EIPA reduced JNa further, to −24 ± 18 pmol min¢ (P < 0·05), an effect similar to that seen in the absence of bumetanide. This finding argues against previous suggestions (based on in vitro evidence) that inhibition of the Na¤-K¤-2Cl¦ cotransporter leads to an increase in apical Na¤-H¤ exchange in the TALH.

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Section: Discussionmentioning

confidence: 99%

Contribution of Na⁺‐H⁺ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

Shirley

Walter

Unwin

et al. 1998

The Journal of Physiology

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“…We have shown previously that, in the rat MTAL, NHE-1 and NHE-3 are expressed, but not NHE-2 and NHE-4 (18). Since NHE-3 is the apical Na ϩ /H ϩ exchanger of the rat MTAL (9,20), the present experiments were designed to determine if CMA is associated with intrinsic adaptation of NHE-3, and which mechanisms are involved. Our results show that NHE-3 mRNA, protein abundance, and transport activity are increased in MTAL cells freshly isolated from chronically acidotic rats.…”

Section: Discussionmentioning

confidence: 99%

“…We have shown recently, in microdissected rat MTAL, that NHE-3 mRNA is strongly expressed and NHE-1 mRNA is also detected, whereas NHE-2 and NHE-4 mRNAs are not detected (18). Furthermore, recent data have demonstrated that NHE-1 and NHE-3 are localized to the basolateral and apical plasma membrane of MTAL cells, respectively (9,(19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Laghmani¹,

Borensztein²,

Ambühl³

et al. 1997

J. Clin. Invest.

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Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025 Ϯ 0.003 to 0.042 Ϯ 0.009 amol/ng total RNA ( P Ͻ 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Nadepleted acidified MTAL cells in the presence of 50 M HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity ( P Ͻ 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance ( P Ͻ 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity. ( J. Clin. Invest. 1997. 99:24-30.)

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“…The method used for isolating MTAL tubules was the same as that previously described (11,12). Briefly, under stereomicroscopic control, the inner stripe of the outer medulla was carefully excised.…”

Section: Preparation Of Mtal Tubulesmentioning

confidence: 99%

“…LMVs and BLMVs were prepared from purified rat MTAL tubules as described previously in detail (11,12).…”

Section: Isolation Of Plasma Membranesmentioning

confidence: 99%

Polarized Expression of Different Monocarboxylate Transporters in Rat Medullary Thick Limbs of Henle

Eladari

Chambrey

Irinopoulou

et al. 1999

Journal of Biological Chemistry

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Extracellular lactic acid is a major fuel for the mammalian medullary thick ascending limb (MTAL), whereas under anoxic conditions, this nephron segment generates a large amount of lactic acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs. Furthermore, this L-lactate self-exchange was markedly inhibited by ␣-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a stereospecific carrier-mediated lactate transport system in LMVs. Using immunofluorescence confocal microscopy and immunoblotting, the monocarboxylate transporter (MCT)-2 isoform was shown to be specifically expressed on the basolateral domain of the rat MTAL, whereas the MCT1 isoform could not be detected in this nephron segment. This study thus demonstrates the presence of different monocarboxylate transporters in rat MTALs; the basolateral H ؉ /L-lactate cotransporter (MCT2) and the luminal H ؉ -independent organic anion exchanger are adapted to play distinct roles in the transport of monocarboxylates in MTALs. The medullary thick ascending limb (MTAL)1 is significantly engaged in the active absorption of NaCl, NH 4 ϩ , and HCO 3 Ϫ .Extracellular lactate has been shown to be a major fuel to support this large energy demand (for review, see Ref. 1), indicating that lactate uptake is a crucial process in the regulation of energy metabolism in this nephron segment. Conversely, under anoxic conditions, the increase in lactic acid production in the MTAL greatly exceeds the increase seen in other nephron segments (2). There is thus a need for facilitating efflux of glycolytically derived lactic acid from the MTAL. In most cells, transport of lactic acid occurs via monocarboxylate transporters (MCTs) exhibiting the following properties: 1) cotransport of H ϩ with a monocarboxylate (or its equivalent, OH Ϫ /monocarboxylate exchange); 2) broad specificity for short chain monocarboxylates, including lactate and pyruvate; 3) inhibition by anion transport blockers such as ␣-cyano-4-hydroxycinnamate (CHC), DIDS, and phloretin; and 4) transstimulation of L-lactate uptake by pyruvate, L-lactate (but not by D-lactate), and other various substituted monocarboxylates. Seven different cDNAs have been identified that encode for MCTs in mammalians, designated MCT1 to MCT7 (3-6). MCT1 and MCT2 show a broad tissue distribution and are present in hamster (3, 4) and rat (7) kidneys. On the other hand, the expression of rat MCT3 and MCT4 is restricted to the retinal pigment epithelium (5) and muscle fibers (8), respectively. Three additional MCT isoforms, MCT5, MCT6, and MCT7, have been identified in human tissues (6).Although the mechanisms of lactate export and import have been extensively investigated in a variety of tissues (for review, see Ref. 9), little information has been available concerning the mechanisms of lactate...

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Isolation and characterization of luminal and basolateral plasma membrane vesicles from the medullary thick ascending loop of Henle

Cited by 36 publications

References 14 publications

Contribution of Na⁺‐H⁺ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

Contribution of Na⁺‐H⁺ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Polarized Expression of Different Monocarboxylate Transporters in Rat Medullary Thick Limbs of Henle

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Isolation and characterization of luminal and basolateral plasma membrane vesicles from the medullary thick ascending loop of Henle

Cited by 36 publications

References 14 publications

Contribution of Na+‐H+ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

Contribution of Na+‐H+ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Polarized Expression of Different Monocarboxylate Transporters in Rat Medullary Thick Limbs of Henle

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Contribution of Na⁺‐H⁺ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

Contribution of Na⁺‐H⁺ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats