Potassium dependent H+/HCO3- transport mechanisms in cells of medullary thick ascending limb of rat kidney

Chloride/base exchange activity has been detected in every mammalian nephron segment in which it has been sought. However, in contrast to the Cl-/HCO3- exchanger AE1 in type A intercalated cells, localization of AE2 within the kidney has not been reported. We therefore studied AE2 expression in rat kidney. AE2 mRNA was present in cortex, outer medulla, and inner medulla. Semiquantitative polymerase chain reaction of cDNA from microdissected tubules revealed AE2 cDNA levels as follows [copies of cDNA derived per mm tubule (+/- SE)]: proximal convoluted tubule, 688 +/- 161; proximal straight tubule, 652 +/- 189; medullary thick ascending limb, 1,378 +/- 226; cortical thick ascending limb, 741 +/- 24; cortical collecting duct, 909 +/- 71; and outer medullary collecting duct, 579 +/- 132. AE2 cDNA was also amplified in thin limbs and in inner medullary collecting duct. AE2 polypeptide was detected in all kidney regions. AE2 mRNA and protein were also detected in several renal cell lines. The data are compatible with the postulated roles of AE2 in maintenance of intracellular pH and chloride concentration and with its possible participation in transepithelial transport.

Download Full-text

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.6.f1224 ◽

1995 ◽

Vol 268 (6) ◽

pp. F1224-F1228 ◽

Cited By ~ 10

Author(s):

P. Borensztein ◽

M. Froissart ◽

K. Laghmani ◽

M. Bichara ◽

M. Paillard

Keyword(s):

Rat Kidney ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Pcr Analysis ◽

Thick Ascending Limb ◽

Rt Pcr ◽

Medullary Thick Ascending Limb ◽

Pcr Products ◽

Polymerase Chain ◽

Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Download Full-text

PKC-dependent superoxide production by the renal medullary thick ascending limb from diabetic rats

AJP Renal Physiology ◽

10.1152/ajprenal.00314.2009 ◽

2009 ◽

Vol 297 (5) ◽

pp. F1220-F1228 ◽

Cited By ~ 18

Author(s):

Jing Yang ◽

Pascale H. Lane ◽

Jennifer S. Pollock ◽

Pamela K Carmines

Keyword(s):

Rat Kidney ◽

Diabetic Rats ◽

Thick Ascending Limb ◽

Protein Levels ◽

Medullary Thick Ascending Limb ◽

Pkc Isoforms ◽

Normal Rat ◽

Membrane Bound ◽

Pkc Activation

Type 1 diabetes (T1D) is a state of oxidative stress accompanied by PKC activation in many tissues. The primary site of O2•− production by the normal rat kidney is the medullary thick ascending limb (mTAL). We hypothesized that T1D increases O2•− production by the mTAL through a PKC-dependent mechanism involving increased expression and translocation of one or more PKC isoforms. mTAL suspensions were prepared from rats with streptozotocin-induced T1D (STZ mTALs) and from normal or sham rats (normal/sham mTALs). O2•− production by STZ mTALs was fivefold higher than normal/sham mTALs ( P < 0.05). PMA (30 min) mimicked the effect of T1D on O2•− production. Exposure to calphostin C or chelerythrine (PKC inhibitors), Gö6976 (PKCα/β inhibitor), or rottlerin (PKCδ inhibitor) decreased O2•− production to <20% of untreated baseline in both normal/sham and STZ mTALs. PKCβ inhibitors had no effect. PKC activity was increased in STZ mTALs ( P < 0.05 vs. normal/sham mTALs) and was unaltered by antioxidant exposure (tempol). PKCα protein levels were increased by 70% in STZ mTALs, with a ∼30% increase in the fraction associated with the membrane (both P < 0.05 vs. sham). PKCβ protein levels were elevated by 29% in STZ mTALs ( P < 0.05 vs. sham) with no change in the membrane-bound fraction. Neither PKCδ protein levels nor its membrane-bound fraction differed between groups. Thus STZ mTALs display PKC activation, upregulation of PKCα and PKCβ protein levels, increased PKCα translocation to the membrane, and accelerated O2•− production that is eradicated by inhibition of PKCα or PKCδ (but not PKCβ). We conclude that increased PKCα expression and activity are primarily responsible for PKC-dependent O2•− production by the mTAL during T1D.

Download Full-text

Cl(-)-dependent NH4+ transport mechanisms in medullary thick ascending limb cells

AJP Cell Physiology ◽

10.1152/ajpcell.1994.267.6.c1607 ◽

1994 ◽

Vol 267 (6) ◽

pp. C1607-C1615 ◽

Cited By ~ 43

Author(s):

H. Amlal ◽

M. Paillard ◽

M. Bichara

Keyword(s):

Membrane Potential ◽

Intracellular Ph ◽

Media Exposure ◽

Thick Ascending Limb ◽

Transport Mechanisms ◽

K Channels ◽

Initial Cell ◽

Medullary Thick Ascending Limb ◽

Free Media ◽

Cl Conductance

To characterize Cl(-)-dependent NH4+ transport mechanisms in renal medullary thick ascending limb (MTAL), intracellular pH (pHi) and membrane potential (PD) were monitored with use of 2',7'-bis(carboxyethyl)- 5(6)-carboxyfluorescein and 3,3'-dipropylthiadicarbocyanine, respectively, in suspensions of rat MTAL tubules in CO2-free media. Exposure of MTAL cells to 4 mM NH4Br caused, after an initial cell alkalinization due to NH3 entry, an NH4(+)-induced fall in pHi that was approximately 67% less pronounced in Cl(-)-free than in Cl(-)-containing media. The following experiments were performed in the presence of 1 microM amiloride to block the MTAL NH4+ conductance. When cells were preincubated in a Cl(-)-free gluconate medium in which K+ and Cl- conductances are greatly reduced, abrupt addition of 100 mN N-methyl-D-glucamine (NMDG)-Cl had no effect on cell PD and pHi in the absence of ammonia, but acutely acidified the cells by approximately 0.2 pH units in the presence of 4 mM NH4Br, which thus indicated nonelectrogenic (NMDG-Cl)-dependent NH4+ influx. The latter also occurred in a Cl(-)-free thiocyanate medium in which the Cl- conductance was blocked by 0.1 mM diphenylamine-2-carboxylate (DPC). An NMDG-Cl- dependent NH4(+)-induced fall in pHi was reduced approximately 33% by 10 mM Ba+, approximately 84% by 0.1 mM bumetanide, and 100% by 1.5 mM furosemide, whereas 1 mM hydrochlorothiazide had no effect; inhibition by Ba+ was observed even in the presence of 0.1 mM verapamil added to block both K+ channels and K+/NH4+ antiport.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

Basolateral membrane Cl−-, Na+-, and K+-coupled base transport mechanisms in rat MTALH

AJP Renal Physiology ◽

10.1152/ajprenal.00220.2000 ◽

2002 ◽

Vol 282 (4) ◽

pp. F655-F668 ◽

Cited By ~ 23

Author(s):

Soline Bourgeois ◽

Sandrine Massé ◽

Michel Paillard ◽

Pascal Houillier

Keyword(s):

Basolateral Membrane ◽

Disulfonic Acid ◽

Thick Ascending Limb ◽

Transport Mechanisms ◽

Medullary Thick Ascending Limb ◽

Bilateral Absence ◽

Transepithelial Voltage ◽

Intracellular Alkalinization ◽

Cellular Acidification

Mechanisms involved in basolateral HCO[Formula: see text] transport were examined in the in vitro microperfused rat medullary thick ascending limb of Henle (MTALH) by microfluorometric monitoring of cell pH. Removing peritubular Cl− induced a cellular alkalinization that was inhibited in the presence of peritubular 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and blunted in the absence of external CO2/HCO[Formula: see text]. The alkalinization elicited by removing peritubular Cl−persisted in the bilateral absence of Na+, together with a voltage clamp. When studied in Cl−-free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external CO2/HCO[Formula: see text]. Removing peritubular Na+ elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na+-HCO[Formula: see text] cotransport and inhibition of a basolateral Na+/H+exchange. Increasing bath K+ induced an intracellular alkalinization that was inhibited in the absence of external CO2/HCO[Formula: see text]. At 2 mM, peritubular Ba2+, which inhibits the K+-Cl−cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited K+-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral Ba2+-sensitive K+-Cl− cotransport that may operate as a K+-HCO[Formula: see text] cotransport. This cotransport was inhibited in the peritubular presence of furosemide, [(dihydroindenyl)oxy]alkanoic acid, 5-nitro-2-(3-phenylpropylamino)benzoate, or DIDS. At least three distinct basolateral HCO[Formula: see text] transport mechanisms are functional under physiological conditions: electroneutral Cl−/HCO[Formula: see text] exchange, DIDS- and EIPA-insensitive Na+-HCO[Formula: see text] cotransport, and Ba2+-sensitive electroneutral K+-Cl−(HCO[Formula: see text]) cotransport.

Download Full-text

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

AJP Renal Physiology ◽

10.1152/ajprenal.00437.2002 ◽

2004 ◽

Vol 286 (5) ◽

pp. F903-F912 ◽

Cited By ~ 44

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.

Download Full-text

Cloning, renal distribution, and regulation of the rat Na+- HCO 3 − cotransporter

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.6.f1119 ◽

1998 ◽

Vol 274 (6) ◽

pp. F1119-F1126 ◽

Cited By ~ 16

Author(s):

Charles E. Burnham ◽

Michael Flagella ◽

Zhaohui Wang ◽

Hassane Amlal ◽

Gary E. Shull ◽

...

Keyword(s):

Metabolic Acidosis ◽

Rat Kidney ◽

Human Kidney ◽

Cloning And Expression ◽

Thick Ascending Limb ◽

Mrna Levels ◽

Reading Frame ◽

Medullary Thick Ascending Limb ◽

Nephron Segment

We recently reported the cloning and expression of a human kidney Na+-[Formula: see text]cotransporter (NBC-1) (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111–19114, 1997). To expedite in vivo experimentation, we now report the cDNA sequence of rat kidney NBC-1. In addition, we describe both the organ and nephron segment distributions and the regulation of NBC-1 mRNA under three models of pH stress: chloride-depletion alkalosis (CDA), metabolic acidosis, and bicarbonate loading. Rat NBC-1 cDNA encodes an open reading frame of 1,035 amino acids, with 96 and 87% identity to human and salamander NBC-1, respectively. Rat NBC-1 mRNA is expressed at high levels in kidney and brain, with lower levels in colon, stomach, and heart. None appears in liver. In the kidney, NBC-1 is expressed mainly in the proximal tubule, with traces found in medullary thick ascending limb and papilla. [Formula: see text] loading decreased NBC-1 mRNA levels, which were unchanged either by metabolic acidosis or by CDA.

Download Full-text