[Role of the Na(+)-K+(NH4+)-2Cl cotransporter of the medullary ascending limb in the regulation of renal acid-base equilibrium]. / Rôle du cotransporteur Na(+)-K+(NH4+)-2Cl- de la branche large ascendante dans la régulation rénale de l'équilibre acido-basique.

NH4+ absorption by the medullary thick ascending limb (MTAL) of Henle's loop, which causes the accumulation of NH4+/NH3 in the medullary interstitium, is a key step in the renal handling of ammonia. Accumulation of NH4+/NH3 in the medullary interstitium is necessary to the secretion of ammonia in the medullary collecting ducts and then to NH4+ excretion in the urine. The MTAL apical Na(+)-K+(NH4+)-2Cl- cotransporter BSC1/NKCC2 is responsible for the majority of the MTAL luminal NH4+ uptake. Stimulation of BSC1 expression by metabolic acidosis accounts for the increase of the MTAL ability to absorb NH4+ during this condition. Metabolic acidosis increases the mRNA and protein abudance and the transport activity of BSC1. Two factors have been demonstrated to mediate the effects of acidosis, an acid pH and glucocorticoids whose production augments during metabolic acidosis. These two factors thus control in a coordinated manner ammoniagenesis in the proximal tubule and MTAL NH4+ transport to ensure urinary acid excretion rates appropriate to the acid-base status.

Ammonium carriers in medullary thick ascending limb.

Absorption of NH(4)(+) by the medullary thick ascending limb (MTAL) is a key event in the renal handling of NH(4)(+), leading to accumulation of NH(4)(+)/NH(3) in the renal medulla, which favors NH(4)(+) secretion in medullary collecting ducts and excretion in urine. The Na(+)-K(+)(NH(4)(+))-2Cl(-) cotransporter (BSC1/NKCC2) ensures approximately 50-65% of MTAL active luminal NH(4)(+) uptake under basal conditions. Apical barium- and verapamil-sensitive K(+)/NH(4)(+) antiport and amiloride-sensitive NH(4)(+) conductance account for the rest of active luminal NH(4)(+) transport. The presence of a K(+)/NH(4)(+) antiport besides BSC1 allows NH(4)(+) and NaCl absorption by MTAL to be independently regulated by vasopressin. At the basolateral step, the roles of NH(3) diffusion coupled to Na(+)/H(+) exchange or Na(+)/NH(4)(+) exchange, which favors NH(4)(+) absorption, and of Na(+)/K(+)(NH(4)(+))-ATPase, NH(4)(+)-Cl(-) cotransport, and NH(4)(+) conductance, which oppose NH(4)(+) absorption, have not been quantitatively defined. The increased ability of the MTAL to absorb NH(4)(+) during chronic metabolic acidosis involves an increase in BSC1 expression, but fine regulation of MTAL NH(4)(+) transport probably requires coordinated effects on various apical and basolateral MTAL carriers.

Regulation by glucocorticoids of expression and activity of rBSC1, the Na+-K+(NH4+)-2Cl- cotransporter of medullary thick ascending limb.

To assess whether glucocorticoids regulate rBSC1, the apical Na(+)-K(+)(NH(4)(+))-2Cl(-) cotransporter of kidney medullary thick ascending limb (MTAL), studies were performed in normal rats, adrenalectomized (ADX) rats, and ADX rats infused with dexamethasone for 6 days. The effects of dexamethasone on rBSC1 were also studied in vitro using isolated rat MTAL segments. Cotransport activity was estimated by intracellular pH measurements; rBSC1 protein was quantified in MTAL crude membranes by immunoblotting analysis, and mRNA was quantified by quantitative reverse transcription-polymerase chain reaction. The abundance of rBSC1 protein and mRNA increased in ADX rats infused with dexamethasone compared with ADX rats (p < 0. 04). In addition, application of dexamethasone for 1-3 h to MTALs caused rBSC1 protein and mRNA abundance and cotransport activity to significantly increase in a hyperosmotic medium (450 mosmol/kg of H(2)O) containing 0.7 nm arginine vasopressin, which is an in vitro experimental condition that resembles the in vivo MTAL environment. Results obtained in various media and with 8-bromo-cAMP indicated that stimulation of rBSC1 expression by glucocorticoids required interactions between glucocorticoid receptor- and cAMP-dependent factors. Up to 100 nm d-aldosterone had no effect on cotransport activity in vitro. Thus glucocorticoids directly stimulate MTAL rBSC1 expression and activity, which contributes to glucocorticoid-dependent effects on the renal regulation of acid-base balance and urinary concentrating ability.

Stimulation by in vivo and in vitro metabolic acidosis of expression of rBSC-1, the Na+-K+(NH4+)-2Cl- cotransporter of the rat medullary thick ascending limb.

To assess whether metabolic acidosis per se regulates rBSC-1, the rat medullary thick ascending limb (MTAL) apical Na+-K+(NH4+)-2Cl- cotransporter, rat MTALs were incubated for 16 h in an acid 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's medium. Cotransport activity was estimated in intact cells and membrane vesicles by intracellular pH and 22Na+ uptake measurements, respectively; rBSC-1 protein was quantified by immunoblotting analysis and mRNA by quantitative reverse transcription-polymerase chain reaction. As compared with incubation at pH approximately 7.35, acid incubation (pH approximately 7.10) up-regulated by 35-100% rBSC-1 transport activity in cells and membrane vesicles, and rBSC-1 protein and mRNA abundance. In contrast, acid incubation did not alter alkaline phosphatase and Na+/K+-ATPase enzyme activities or beta-actin protein abundance. After 3 h of in vivo chronic metabolic acidosis (CMA) rBSC-1 mRNA abundance increased in freshly harvested MTALs, which was accompanied after 1-6 days of CMA with enhanced rBSC-1 protein abundance. These results demonstrate that both in vivo and in vitro CMA stimulate rBSC-1 expression, which would contribute to the adaptive increase in MTAL absorption and urinary excretion of NH4+ in response to CMA.

Long-term shake suspension and membrane vesicles of medullary thick ascending limb.

Cultured medullary thick ascending limb (MTAL) cells may lack some of the main carriers of fresh MTAL cells, such as apical Na+-K+(NH4+)-2Cl- cotransporter (BSC-1) and Na+/H+ exchanger (NHE-3). We have developed a technique to maintain rat MTALs several hours in suspension and in a good state of viability. Medullary thick ascending limbs were suspended in a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium (HDMEM) supplemented with 25 mM HCO3- and gassed with 95% O2/5% CO2; the resulting mixture was placed in a rotary shaking water bath at 37 degrees C for 16 hours. As seen by electron microscopy, MTALs from the HDMEM-suspension retained a virtually normal tubular organization. Na+-K+(NH4+)-2Cl- cotransport activity and NHE consistent with both apical NHE-3 and basolateral NHE-1 activities were underscored both in intact cells by intracellular pH measurements and in a membrane fraction enriched in apical and basolateral membranes by 22Na+ uptake experiments. These results demonstrate that freshly harvested MTALs can be maintained in a well differentiated state for at least 16 hours; this preparation should make long-term in vitro studies of MTAL transport regulations possible.

Apical location and inhibition by arginine vasopressin of K+/H+ antiport of the medullary thick ascending limb of rat kidney.

To characterize and localize a K+/H+ antiport mechanism in the renal medullary thick ascending limb (MTAL), membrane vesicles were isolated from a rat MTAL homogenate. K+/H+ antiport (in > out H+ gradient-stimulated 86Rb+ uptake) was abolished by barium and verapamil (apparent Ki of 55 microM) but unaffected by other K+ channel blockers such as quinidine and high amiloride concentrations. SCH 28080, a H+/K+-ATPase blocker, did not affect K+/H+ antiport. K+/H+ antiport activity was correlated positively with the enrichment factor of the membranes in the apical marker enzyme alkaline phosphatase (r = 0.875, p < 0.01) and negatively correlated with the enrichment factor in basolateral Na+/K+-ATPase (r = -0.665, p < 0.05). Moreover, a functional interaction occurred with Na+/H+ exchange (NHE) consistent with colocation of K+/H+ antiport and apical NHE-3, not basolateral NHE-1. K+/H+ antiport was shown by intracellular pH measurements to be inhibited by arginine vasopressin and 8-bromo-cAMP through cAMP-dependent protein kinase (protein kinase A) activation. These results demonstrate the presence of a K+/H+ antiport mechanism, which is inhibited by arginine vasopressin via protein kinase A, in the apical membrane of the MTAL.