null mice are hypotensive, in part, from the absence of NKCC1-mediated vasoconstriction. Whether these mice have renal defects in NaCl and water handling which contribute to the hypotension is unexplored. Therefore, we asked 1) whether NKCC1 (Ϫ/Ϫ) mice have a defect in the regulation of NaCl and water balance, which might contribute to the observed hypotension and 2) whether the hypotension observed in these mice is accompanied by endocrine abnormalities and/or downregulation of renal Na ϩ transporter expression. Thus we performed balance studies, semiquantitative immunoblotting, and immunohistochemistry of kidney tissue from NKCC1 (ϩ/ϩ) and NKCC1 (Ϫ/Ϫ) mice which consumed either a high (2.8% NaCl)-or a low-NaCl (0.01% NaCl) diet for 7 days. Blood pressure was lower in NKCC1 (Ϫ/Ϫ) than NKCC1 (ϩ/ϩ) mice following either high or low dietary NaCl intake. Relative to wild-type mice, NKCC1 null mice had a lower plasma ANP concentration, a higher plasma renin and a higher serum K ϩ concentration with inappropriately low urinary K ϩ excretion, although serum aldosterone was either the same or only slightly increased in the mutant mice. Expression of NHE3, the ␣-subunit of the Na-K-ATPase, NCC, and NKCC2 were higher in NKCC1 null than in wild-type mice, although differences were generally greater during NaCl restriction. NKCC1 null mice had a reduced capacity to excrete free water than wild-type mice, which resulted in hypochloremia following the NaCl-deficient diet. Hypochloremia did not occur from increased aquaporin-1 (AQP1) or 2 protein expression or from redistribution of AQP2 to the apical regions of principal cells. Instead, NKCC1 null mice had a blunted increase in urinary osmolality following vasopressin administration, which should increase free water excretion and attenuate the hypochloremia. In conclusion, aldosterone release is inappropriately low in NKCC1 null mice. Moreover, the action of aldosterone and vasopressin is altered within kidneys of NKCC1 null mice, which likely contributes to their hypotension. Increased Na ϩ transporter expression, increased plasma renin, and reduced plasma ANP, as observed in NKCC1 null mice, should increase vascular volume and blood pressure, thus minimizing hypotension.

In rat kidney the "secretory" isoform of the Na+-K+-2Cl- cotransporter (NKCC1) localizes to the basolateral membrane of the alpha-intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes Cl- and whether transepithelial Cl- transport occurs, in part, through Cl- uptake across the basolateral membrane mediated by NKCC1 in series with Cl- efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical HCO/CO2-buffered solutions. Cl- secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 microM), when added to the bath, reduced Cl- secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive Cl- secretion was dependent on extracellular Na+ and either K+ or NH, consistent with the ion dependency of NKCC1-mediated Cl- transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete Cl- into the luminal fluid through NKCC1-mediated Cl- uptake across the basolateral membrane in series with Cl- efflux across the apical membrane. The physiological role of NKCC1-mediated Cl- uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated.

In rat outer medullary collecting duct (OMCD), the mechanism(s) and regulation of H+ secretion are not understood fully. The effect of changes in acid-base balance and the renin-angiotensin system on net H+ secretion was explored. Rats received NaCl, NaHCO3, NH4Cl, or nothing in their drinking water for 7 days. Total ammonia and total CO2 (JtCO2) fluxes were measured in OMCD tubules perfused in vitro from rats in each treatment group. JtCO2 was reduced in tubules from rats drinking NH4Cl relative to those drinking NaHCO3. Because NH4Cl intake increases plasma renin and aldosterone, we asked if upregulation of the renin-angiotensin system reduces net H+ secretion. Deoxycorticosterone pivalate administered in vivo did not affect JtCO2. However, ANG II given in vivo at 0.1 ng/min reduced JtCO2 by 35%. To determine if ANG II has a direct effect on acid secretion, JtCO2 was measured with ANG II applied in vitro. ANG II (10-8 M) present in the bath solution reduced JtCO2 by 35%. This ANG II effect was not observed in the presence of the AT1 receptor blocker candesartan. In conclusion, in rat OMCD, JtCO2 is paradoxically reduced with NH4Cl ingestion. Increased circulating ANG II, as occurs during metabolic acidosis, reduces JtCO2.

In rat terminal inner medullary collecting duct (tIMCD), the Na,K-ATPase mediates NH[Formula: see text] uptake, which increases secretion of net H+ equivalents. K+ and NH[Formula: see text]compete for a common binding site on the Na,K-ATPase. Therefore, NH[Formula: see text] uptake should increase during hypokalemia because interstitial K+ concentration is reduced. We asked whether upregulation of the Na,K-ATPase during hypokalemia also increases basolateral NH[Formula: see text] uptake. To induce hypokalemia, rats ate a diet with a low K+ content. In tIMCD tubules from rats given 3 days of dietary K+restriction, Na,K-ATPase β1-subunit (NK-β1) protein expression increased although NK-α1 protein expression and Na,K-ATPase activity were unchanged relative to K+-replete controls. However, after 7 days of K+ restriction, both NK-α1 and NK-β1 subunit protein expression and Na,K-ATPase activity increased. The magnitude of Na,K-ATPase-mediated NH[Formula: see text]uptake across the basolateral membrane ( J [Formula: see text]) was determined in tIMCD tubules perfused in vitro from rats after 3 days of a normal or a K+-restricted diet. J [Formula: see text] was the same in tubules from rats on either diet when measured at the same extracellular K+ concentration. However, in either treatment group, increasing K+ concentration from 10 to 30 mM reduced J [Formula: see text] >60%. In conclusion, with 3 days of K+ restriction, NH[Formula: see text] uptake by Na,K-ATPase is increased in the tIMCD primarily from the reduced interstitial K+ concentration.

ABSTRACT. In rat kidney, the “secretory” isoform of the Na-K-Cl cotransporter, NKCC1 (BSC-2), localizes to the basolateral membrane of the α intercalated cell, the acid secreting cell of the outer medullary collecting duct (OMCD). This laboratory has reported that NKCC1 mediates Cl− uptake across the basolateral membrane in series with Cl− secretion across the apical membrane in rat OMCD. NKCC1 transports NH4+, K+, and Na+ as well as Cl−; therefore, a role for the cotransporter in the process of HCl, NH4Cl, KCl, and NaCl secretion has been suggested. Thus, it was determined if bumetanide, an inhibitor of NKCC1, alters transepithelial cation transport in rat OMCD. OMCD tubules from deoxycorticosterone pivalate (DOCP)–treated rats were perfused in vitro. Hydration of CO2, rather than NH4+, provides the principle source of H+ for net acid secretion. In HCO3−/CO2-buffered solutions, no effect of bumetanide on net K+ flux was detected. Under some conditions, bumetanide addition resulted in a small reduction in secretion of net H+ equivalents. Transepithelial Na+ flux, JNa, was −1.5 ± 1.7 pmol/mm per min, values not different from zero. However, with the application of bumetanide to the bath, JNa was +5.2 ± 1.3 pmol/mm per min (P < 0.05), which indicates net Na+ absorption. In conclusion, inhibition of NKCC1 in rat OMCD changes transepithelial movement of Na+ and Cl−. The role of NKCC1 in the secretion of net H+ equivalents is small.