An apical K+-dependent HCO3− transport pathway opposes transepithelial HCO3− absorption in rat medullary thick ascending limb

2004 ◽  
Vol 287 (1) ◽  
pp. F57-F63 ◽  
Author(s):  
Bruns A. Watts ◽  
David W. Good
Keyword(s):  
Initial Rate ◽  
Apical Membrane ◽  
Channel Blockers ◽  
Thick Ascending Limb ◽  
Acid Base ◽  
Transport Pathway ◽  
Transport Pathways ◽  
Medullary Thick Ascending Limb ◽  
And Function

Absorption of HCO3− in the medullary thick ascending limb (MTAL) is mediated by apical membrane Na+/H+ exchange. The identity and function of other apical acid-base transporters in this segment have not been defined. The present study was designed to examine apical membrane HCO3−/OH−/H+ transport pathways in the rat MTAL and to determine their role in transepithelial HCO3− absorption. MTALs were perfused in vitro in Na+- and Cl−-free solutions containing 25 mM HCO3−, 5% CO2. Lumen addition of either 120 mM Cl− or 50 mM Na+ (50 μM EIPA present) had no effect on intracellular pH (pHi). Lumen Cl− addition also had no effect on pHi in the presence of 145 mM Na+ or in the nominal absence of HCO3−/CO2. Thus there was no evidence for apical Cl−/HCO3− (OH−) exchange, Na+-dependent Cl−/HCO3− exchange, or Na+-HCO3− cotransport. In contrast, in tubules studied in Na+- and Cl−-free solutions containing 25 mM HCO3−, 5% CO2 and 120 mM K+, removal of luminal K+ induced a rapid and pronounced decrease in pHi (ΔpHi = 0.56 ± 0.06 pH U). pHi recovered following lumen K+ readdition. The initial rate of net base efflux induced by lumen K+ removal was decreased 85% at the same pHi in the nominal absence of HCO3−/CO2, indicating a dependence on HCO3−/CO2 and arguing against apical K+/H+ exchange. A combination of the apical K+ channel blockers quinidine (0.1 mM) and glybenclamide (0.25 mM) had no effect on the lumen K+-induced pHi changes, arguing against electrically coupled K+ and HCO3− conductances. The effect of lumen K+ on pHi was inhibited by 1 mM H2DIDS. In addition, lumen addition of DIDS increased transepithelial HCO3− absorption from 10.7 ± 0.7 to 14.9 ± 0.7 pmol·min−1·mm−1 ( P < 0.001) and increased pHi slightly in MTAL studied in physiological solutions (25 mM HCO3− and 4 mM K+). Lumen DIDS stimulated HCO3− absorption in the absence and presence of furosemide. These results are consistent with an apical membrane K+-dependent HCO3− transport pathway that mediates coupled transfer of K+ and HCO3− from cell to lumen in the MTAL. This mechanism, possibly an apical K+-HCO3− cotransporter, functions in parallel with apical Na+/H+ exchange and opposes transepithelial HCO3− absorption.

scholarly journals Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH4+ transport.

1994 ◽  
Vol 103 (5) ◽  
pp. 917-936 ◽  
Author(s):  
B A Watts ◽  
D W Good
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Apical Membrane ◽  
Ammonium Transport ◽  
Thick Ascending Limb ◽  
Transport Pathway ◽  
Critical Determinant ◽  
Medullary Thick Ascending Limb ◽  
Intracellular Alkalinization

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium-sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)-NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.

scholarly journals Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption.

1986 ◽  
Vol 87 (4) ◽  
pp. 567-590 ◽  
Author(s):  
S C Hebert ◽  
T E Andreoli
Keyword(s):  
Apical Membrane ◽  
Electrical Conductance ◽  
Transport Processes ◽  
Diffusion Resistance ◽  
Osmotic Gradient ◽  
Thick Ascending Limb ◽  
Nacl Absorption ◽  
Medullary Thick Ascending Limb ◽  
Paired Measurement

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.

Functional roles of apical membrane Na+/H+ exchange in rat medullary thick ascending limb

1996 ◽  
Vol 270 (4) ◽  
pp. F691-F699 ◽  
Author(s):  
D. W. Good ◽  
B. A. Watts
Keyword(s):  
Steady State ◽  
Apical Membrane ◽  
Important Determinant ◽  
Basolateral Membrane ◽  
Thick Ascending Limb ◽  
Functional Roles ◽  
Medullary Thick Ascending Limb ◽  
Apical Membranes ◽  
Exchange Activity

The medullary thick ascending limb (MTAL) of the rat actively absorbs both HCO3- and ammonium. The roles of apical membranes Na+/H+ exchange in these processes and in determining steady-state intracellular pH (pHi) were examined in MTAL perfused in vitro with solutions containing 146 mM Na+ and 25 mM HCO3- (pH 7.4). Addition of 1 mM amiloride or 50 microM ethylisopropylamiloride (EIPA) to the lumen decreased HCO3- absorption (JHCO3) from 10.6 +/- 0.5 to 2.3 +/- 0.3 pmol.min-1.mm-1 (P < 0.001) and pHi from 7.10 +/- 0.02 to 6.86 +/- 0.03 (P < 0.001). The combination of lumen Na+ replacement plus amiloride abolished JHCO3. Chronic metabolic acidosis (CMA) caused a 32% increase in JHCO3 that was inhibited by luminal amiloride. Addition of 4 mM NH4Cl to perfusate and bath markedly decreased pHi (from 7.10 to 6.70) but did not stimulate luminal H+ secretion as assessed by HCO3- absorption. With 4 mM NH4Cl in perfusate and bath, luminal addition of amiloride decreased pHi from 6.70 +/- 0.06 to 6.50 +/- 0.05 (P < 0.005) but had no effect on net ammonium absorption. These results demonstrate that 1) apical membrane Na+/H+ exchange mediates virtually all of HCO3- absorption and is an important determinant of steady-state pHi in the MTAL; 2) the adaptive increase in HCO3- absorption in CMA is mediated by an increase in apical membrane Na+/H+ exchange; 3) ammonium markedly acidifies the cells but does not stimulate luminal acidification, suggesting that pHi is not a predominant influence on apical Na+/H+ exchange activity and that H+ generated in the cells as the result of transcellular ammonium absorption is extruded across the basolateral membrane; and 4) apical membrane Na+/H+ exchange is not important for ammonium absorption.

scholarly journals High sodium intake increases HCO3− absorption in medullary thick ascending limb through adaptations in basolateral and apical Na+/H+ exchangers

2011 ◽  
Vol 301 (2) ◽  
pp. F334-F343 ◽  
Author(s):  
David W. Good ◽  
Thampi George ◽  
Bruns A. Watts
Keyword(s):  
Absorptive Capacity ◽  
Sodium Intake ◽  
Thick Ascending Limb ◽  
Acid Base Balance ◽  
High Sodium ◽  
High Sodium Intake ◽  
Medullary Thick Ascending Limb ◽  
Nhe1 Activity ◽  
Exchange Activity

A high sodium intake increases the capacity of the medullary thick ascending limb (MTAL) to absorb HCO3−. Here, we examined the role of the apical NHE3 and basolateral NHE1 Na+/H+ exchangers in this adaptation. MTALs from rats drinking H2O or 0.28 M NaCl for 5–7 days were perfused in vitro. High sodium intake increased HCO3− absorption rate by 60%. The increased HCO3− absorptive capacity was mediated by an increase in apical NHE3 activity. Inhibiting basolateral NHE1 with bath amiloride eliminated 60% of the adaptive increase in HCO3− absorption. Thus the majority of the increase in NHE3 activity was dependent on NHE1. A high sodium intake increased basolateral Na+/H+ exchange activity by 89% in association with an increase in NHE1 expression. High sodium intake increased apical Na+/H+ exchange activity by 30% under conditions in which basolateral Na+/H+ exchange was inhibited but did not change NHE3 abundance. These results suggest that high sodium intake increases HCO3− absorptive capacity in the MTAL through 1) an adaptive increase in basolateral NHE1 activity that results secondarily in an increase in apical NHE3 activity; and 2) an adaptive increase in NHE3 activity, independent of NHE1 activity. These studies support a role for NHE1 in the long-term regulation of renal tubule function and suggest that the regulatory interaction whereby NHE1 enhances the activity of NHE3 in the MTAL plays a role in the chronic regulation of HCO3− absorption. The adaptive increases in Na+/H+ exchange activity and HCO3− absorption in the MTAL may play a role in enabling the kidneys to regulate acid-base balance during changes in sodium and volume balance.

scholarly journals Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through a TLR4-TRIF-PI3K signaling pathway

2017 ◽  
Vol 313 (1) ◽  
pp. F103-F115 ◽  
Author(s):  
Bruns A. Watts ◽  
Thampi George ◽  
Edward R. Sherwood ◽  
David W. Good
Keyword(s):  
Bacterial Infections ◽  
Lipid A ◽  
Thick Ascending Limb ◽  
Akt Inhibitor ◽  
Erk Activation ◽  
Akt Phosphorylation ◽  
Medullary Thick Ascending Limb ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid

Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4−/−or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF)−/−mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.

scholarly journals Angiotensin II inhibits NaCl absorption in the rat medullary thick ascending limb

2004 ◽  
Vol 287 (3) ◽  
pp. F404-F410 ◽  
Author(s):  
Nicolas Lerolle ◽  
Soline Bourgeois ◽  
Françoise Leviel ◽  
Gaëtan Lebrun ◽  
Michel Paillard ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Plasma Membranes ◽  
Inhibitory Effect ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Experimental Conditions ◽  
Nacl Absorption ◽  
Medullary Thick Ascending Limb

NaCl reabsorption in the medullary thick ascending limb of Henle (MTALH) contributes to NaCl balance and is also responsible for the creation of medullary interstitial hypertonicity. Despite the presence of angiotensin II subtype 1 (AT1) receptors in both the luminal and the basolateral plasma membranes of MTALH cells, no information is available on the effect of angiotensin II on NaCl reabsorption in MTALH and, furthermore, on angiotensin II-dependent medullary interstitial osmolality. MTALHs from male Sprague-Dawley rats were isolated and microperfused in vitro; transepithelial net chloride absorption ( JCl) as well as transepithelial voltage ( Vte) were measured. Luminal or peritubular 10−11 and 10−10 M angiotensin II had no effect on JCl or Vte. However, 10−8 M luminal or peritubular angiotensin II reversibly decreased both JCl and Vte. The effect of both luminal and peritubular angiotensin II was prevented by the presence of losartan (10−6 M). By contrast, PD-23319, an AT2-receptor antagonist, did not alter the inhibitory effect of 10−8 M angiotensin II. Finally, no additive effect of luminal and peritubular angiotensin II was observed. We conclude that both luminal and peritubular angiotensin II inhibit NaCl absorption in the MTALH via AT1 receptors. Because of intrarenal angiotensin II synthesis, angiotensin II concentration in medullary tubular and interstitial fluids may be similar in vivo to the concentration that displays an inhibitory effect on NaCl reabsorption under the present experimental conditions.

Aldosterone inhibits apical NHE3 and HCO3− absorption via a nongenomic ERK-dependent pathway in medullary thick ascending limb

2006 ◽  
Vol 291 (5) ◽  
pp. F1005-F1013 ◽  
Author(s):  
Bruns A. Watts ◽  
Thampi George ◽  
David W. Good
Keyword(s):  
Signaling Pathway ◽  
Intracellular Signaling ◽  
Thick Ascending Limb ◽  
Erk Activation ◽  
Intracellular Signaling Pathway ◽  
Cellular Processes ◽  
Medullary Thick Ascending Limb ◽  
Erk Activity ◽  
Rapid Activation

Although aldosterone influences a variety of cellular processes through nongenomic mechanisms, the significance of nongenomic pathways for aldosterone-induced regulation of epithelial function is not understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO3− absorption in the medullary thick ascending limb (MTAL) through a nongenomic pathway. This inhibition is mediated through a direct cellular action of aldosterone to inhibit the apical membrane NHE3 Na+/H+ exchanger. The present study was designed to identify the intracellular signaling pathway(s) responsible for this aldosterone-induced transport regulation. In rat MTALs perfused in vitro, addition of 1 nM aldosterone to the bath decreased HCO3− absorption by 30%. This inhibition was not mediated by cAMP/PKA and was not prevented by inhibitors of PKC or PI3-K, pertussis toxin, or rapamycin. The inhibition of HCO3− absorption by aldosterone was largely eliminated by the MEK/ERK inhibitors U-0126 and PD-98059. Aldosterone increased ERK activity 1.8-fold in microdissected MTALs. This ERK activation is rapid (≤5 min) and is blocked by U-0126 or PD-98059 but is unaffected by spironolactone or actinomycin D. Pretreatment with U-0126 to block ERK activation prevented the effect of aldosterone to inhibit apical NHE3. These data demonstrate that aldosterone inhibits NHE3 and HCO3− absorption in the MTAL through rapid activation of the ERK signaling pathway. The results identify NHE3 as a target for nongenomic regulation by aldosterone and establish a role for ERK in the acute regulation of NHE3 and its epithelial absorptive functions.

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