NKCC2 surface expression in mammalian cells: down-regulation by novel interaction with aldolase B.

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

Gated blood pool tomoscintigraphy with 4-dimensional optical flow motion analysis quantifies left ventricular mechanical activation and synchronization.

BACKGROUND: Gated blood pool tomoscintigraphy has the unique capacity to accurately assess myocardial motion in paced patients. Our goal was to develop a precise radionuclide angiography analysis of cardiac dynamics to evaluate ventricular synchronization in patients undergoing biventricular pacing. METHODS AND RESULTS: On the basis of a 4-dimensional deformable motion estimation algorithm, we developed a protocol allowing estimation of motion fields after gated blood pool tomoscintigraphy. We measured the mechanical activation times for 17 left ventricular (LV) segments and determined the main types of contraction pattern in 10 normal subjects, 17 patients with dilated cardiomyopathy, and 12 resynchronized patients. We analyzed intra-LV dyssynchrony: apex to base, septum to lateral wall, and anterior wall to inferior wall. Three-dimensional measurements of intra-LV activation time (r > .80, P < .001) and LV ejection fraction (r > 0.90, P < .0001) are linearly correlated to 2-dimensional values. LV contraction follows the electrical activation pattern. In normal subjects the anteroseptal and anterior segments are first activated, followed by the apex and inferolateral segments. In resynchronized patients contraction begins with the lateral and apicoseptal segments in correspondence to the LV and right ventricular lead implantation. CONCLUSIONS: By measuring mechanical activation times, this technique allows for the analysis of the regional synchronous contraction. This may help to assess the variation of the activation pattern according to the cardiomyopathy type and the role of septal resynchronization in ventricular functional recovery.

Bone status in primary hyperparathyroidism assessed by regional bone mineral density from the whole body scan and QUS imaging at calcaneus.

To assess the bone mineral density status in primary hyperparathyroidism (PHPT), we studied 64 females with PHPT and 17 healthy women. Regional BMD (arms, trunk, legs) from the whole body scan and conventional sites (lumbar spine, femur, radius) were assessed by DXA. Quantitative ultrasound (QUS) imaging measurements were performed at calcaneus. Sixteen women had history of renal lithiasis, 11 had low impact fracture and 37 women had neither renal lithiasis nor fracture. In the entire group, the mean Z-scores were significantly decreased at all sites (lumbar spine, femur, radius). In all clinical subgroups, the mean Z-scores were significantly decreased at radius. The mean Z-scores in premenopausal women were significantly decreased comparatively to postmenopausal women at lumbar spine and femur. In a group of PHPT females matched to controls for age and BMI, only BMD values at radius were lower in PHPT patients than in control (P < 0.03). However, from the whole body scan data, all sites but no trunk were significantly involved in PHPT patients (P < 0.04). Using QUS measurements at calcaneus, the BUA but not SOS in PHPT females was significantly lower (P = 0.03) than in controls. Our results suggest that low BMD at lumbar spine and femur is encountered preferentially in premenopausal women. The BMD decrease predominates at limbs in PHPT with presumably a gradient from proximal to distal part of the limbs. Indeed, the distal part of the limbs are the most affected areas in PHPT whatever the amount of cortical or trabecular bone.

Tissue kallikrein-deficient mice display a defect in renal tubular calcium absorption.

Renal tubular calcium (RTCa) transport is one of the main factors that determine serum Ca concentration and urinary Ca excretion. The distal convoluted and connecting tubules reabsorb a significant fraction (10%) of filtered Ca. These tubule segments also synthesize in large abundance tissue kallikrein (TK), a major kinin-forming enzyme. Tested was the hypothesis that TK and kinins are involved in controlling RTCa transport by studying TK (TK-/-) or kinin B2 receptor (B2-/-)-deficient mice on different Ca diets. On a 0.9% wt/wt Ca diet, 129Sv or C57Bl/6 TK-/- mice excreted significantly more Ca in urine than their wild-type (WT) littermates. There was no difference between TK-/- and WT mice for plasma concentrations of Ca, Mg, creatinine, parathyroid hormone, or 1,25-dihydroxyvitamin D. On a low Ca (LCa) diet (0.01% wt/wt), urinary Ca excretion decreased in both TK-/- and WT mice but still remained higher in TK-/- mice compared with WT. The plasma Ca concentration was unchanged in C57Bl/6 TK-/- mice but decreased significantly in 129Sv TK-/- mice. Taken together, these data demonstrate that TK deficiency led to impaired RTCa absorption. On the LCa diet, renal TK gene expression doubled in WT mice. No change in urinary Ca excretion was observed in B2-/- mice, even after treatment with a kinin B1-receptor antagonist, and these mice adapted normally to the LCa diet. TK deficiency had no effect on the renal abundance of distal Ca transporter mRNA. These data suggest that TK may be a physiologic regulator of RTCa transport, acting through a non-kinin-mediated mechanism.

Genetic ablation of Rhbg in the mouse does not impair renal ammonium excretion.

NH(4)(+) transport by the distal nephron and NH(4)(+) detoxification by the liver are critical for achieving regulation of acid-base balance and to avoid hyperammonemic hepatic encephalopathy, respectively. Therefore, it has been proposed that rhesus type B glycoprotein (Rhbg), a member of the Mep/Amt/Rh NH(3) channel superfamily, may be involved in some forms of distal tubular acidosis and congenital hyperammonemia. We have tested this hypothesis by inactivating the RHbg gene in the mouse by insertional mutagenesis. Histochemical studies analyses confirmed that RHbg knockout (KO) mice did not express Rhbg protein. Under basal conditions, the KO mice did not exhibit encephalopathy and survived well. They did not exhibit hallmarks of distal tubular acidosis because neither acid-base status, serum potassium concentration, nor bone mineral density was altered by RHbg disruption. They did not have hyperammonemia or disturbed hepatic NH(3) metabolism. Moreover, the KO mice adapted to a chronic acid-loading challenge by increasing urinary NH(4)(+) excretion as well as their wild-type controls. Finally, transepithelial NH(3) diffusive permeability, or NH(3) and NH(4)(+) entry across the basolateral membrane of cortical collecting duct cells, measured by in vitro microperfusion of collecting duct from KO and wild-type mice, was identical with no apparent effect of the absence of Rhbg protein. We conclude that Rhbg is not a critical determinant of NH(4)(+) excretion by the kidney and of NH(4)(+) detoxification by the liver in vivo.

Inactivation of the Na-Cl co-transporter (NCC) gene is associated with high BMD through both renal and bone mechanisms: analysis of patients with Gitelman syndrome and Ncc null mice.

UNLABELLED: Chronic thiazide treatment is associated with high BMD. We report that patients and mice with null mutations in the thiazide-sensitive NaCl cotransporter (NCC) have higher renal tubular Ca reabsorption, higher BMD, and lower bone remodeling than controls, as well as abnormalities in Ca metabolism, mainly caused by Mg depletion. INTRODUCTION: Chronic thiazide treatment decreases urinary Ca excretion (UVCa) and increases BMD. To understand the underlying mechanisms, Ca and bone metabolism were studied in two models of genetic inactivation of the thiazide-sensitive NaCl cotransporter (NCC): patients with Gitelman syndrome (GS) and Ncc knockout (Ncc(-/-)) mice. MATERIALS AND METHODS: Ca metabolism was analyzed in GS patients and Ncc(-/-) mice under conditions of low dietary Ca. BMD was measured by DXA in patients and mice, and bone histomorphometry was analyzed in mice. RESULTS: GS patients had low plasma Mg. They exhibited reduced UVCa, but similar serum Ca and GFR as control subjects, suggesting increased renal Ca reabsorption. Blood PTH was lower despite lower serum ionized Ca, and Mg repletion almost corrected both relative hypoparathyroidism and low UVCa. BMD was significantly increased in GS patients at both lumbar (+7%) and femoral (+16%) sites, and osteocalcin was reduced. In Ncc(-/-) mice, serum Ca and GFR were unchanged, but UVCa was reduced and PTH was elevated; Mg repletion largely corrected both abnormalities. Trabecular and cortical BMD were higher than in Ncc(+/+) mice (+4% and +5%, respectively), and despite elevated PTH, were associated with higher cortical thickness and lower endosteal osteoclastic surface. CONCLUSIONS: Higher BMD is observed in GS patients and Ncc(-/-) mice. Relative hypoparathyroidism (human) and bone resistance to PTH (mice), mainly caused by Mg depletion, can explain the low bone remodeling and normal/low serum Ca despite increased renal Ca reabsorption.

Predictive performance of the modification of diet in renal disease and Cockcroft-Gault equations for estimating renal function.

Recent recommendations emphasize the need to assess kidney function using creatinine-based predictive equations to optimize the care of patients with chronic kidney disease. The most widely used equations are the Cockcroft-Gault (CG) and the simplified Modification of Diet in Renal Disease (MDRD) formulas. However, they still need to be validated in large samples of subjects, including large non-U.S. cohorts. Renal clearance of (51)Cr-EDTA was compared with GFR estimated using either the CG equation or the MDRD formula in a cohort of 2095 adult Europeans (863 female and 1232 male; median age, 53.2 yr; median measured GFR, 59.8 ml/min per 1.73 m(2)). When the entire study population was considered, the CG and MDRD equations showed very limited bias. They overestimated measured GFR by 1.94 ml/min per 1.73 m(2) and underestimated it by 0.99 ml/min per 1.73 m(2), respectively. However, analysis of subgroups defined by age, gender, body mass index, and GFR level showed that the biases of the two formulas could be much larger in selected populations. Furthermore, analysis of the SD of the mean difference between estimated and measured GFR showed that both formulas lacked precision; the CG formula was less precise than the MDRD one in most cases. In the whole study population, the SD was 15.1 and 13.5 ml/min per 1.73 m(2) for the CG and MDRD formulas, respectively. Finally, 29.2 and 32.4% of subjects were misclassified when the CG and MDRD formulas were used to categorize subjects according to the Kidney Disease Outcomes Quality Initiative chronic kidney disease classification, respectively.

Regulation of the Cl-/HCO3- exchanger AE2 in rat thick ascending limb of Henle's loop in response to changes in acid-base and sodium balance.

The Cl(-)/HCO(3)(-) exchanger AE2 is believed to be involved in transcellular bicarbonate reabsorption that occurs in the thick ascending limb of Henle's loop (TAL). The purpose of this study was to test whether chronic changes in acid-base status and sodium intake regulate AE2 polypeptide abundance in the TAL of the rat. Rats were subjected to 6 d of loading with NaCl, NH(4)Cl, NaHCO(3), KCl, or KHCO(3). AE2 protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex and the outer medulla of treated and control rats. In the renal cortex, AE2 abundance was markedly increased in response to oral loading with NH(4)Cl or with NaCl. In contrast, AE2 abundance was unchanged in response to loading with KCl or with NaHCO(3) and was decreased by loading with KHCO(3). The response of AE2 in the outer medulla differed from that in the cortex in that HCO(3)(-) loading increased AE2 abundance when administered with Na(+) but had no effect when administered with K(+). Immunohistochemistry revealed that NaCl loading increased AE2 abundance in the basolateral membrane of both the cortical and the medullary TAL. In contrast, NH(4)Cl loading increased AE2 abundance only in the cortical TAL but not in the medullary TAL. These results suggest that regulation of the basolateral Cl(-)/HCO(3)(-) exchanger AE2 plays an important role in the adaptation of bicarbonate absorption in the TAL during chronic acid-base disturbances and high sodium intake. The present study also emphasizes the contribution of cortical TAL adaptation in the renal regulation of acid-base status.

The Cl-/HCO3- exchanger pendrin in the rat kidney is regulated in response to chronic alterations in chloride balance.

Pendrin (Pds; Slc26A4) is a new anion exchanger that is believed to mediate apical Cl(-)/HCO(3)(-) exchange in type B and non-A-non-B intercalated cells of the connecting tubule and cortical collecting duct. Recently, it has been proposed that this transporter may be involved in NaCl balance and blood pressure regulation in addition to its participation in the regulation of acid-base status. The purpose of our study was to determine the regulation of Pds protein abundance during chronic changes in chloride balance. Rats were subjected to either NaCl, NH(4)Cl, NaHCO(3), KCl, or KHCO(3) loading for 6 days or to a low-NaCl diet or chronic furosemide administration. Pds protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex of treated and control rats. We observed a consistent inverse relationship between Pds expression and diet-induced changes in chloride excretion independent of the administered cation. Conversely, NaCl depletion induced by furosemide was associated with increased Pds expression. We conclude that Pds expression is specifically regulated in response to changes in chloride balance.

Angiotensin II inhibits NaCl absorption in the rat 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 (AT(1)) 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 (J(Cl)) as well as transepithelial voltage (V(te)) were measured. Luminal or peritubular 10(-11) and 10(-10) M angiotensin II had no effect on J(Cl) or V(te). However, 10(-8) M luminal or peritubular angiotensin II reversibly decreased both J(Cl) and V(te). The effect of both luminal and peritubular angiotensin II was prevented by the presence of losartan (10(-6) M). By contrast, PD-23319, an AT(2)-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 AT(1) 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.

What keeps serum calcium levels stable?

Many body functions require that serum calcium levels remain stable over time. This stability is provided by cooperation among three organs: two effectors, the bone and the kidney, which control calcium movements into and out of the extracellular compartment, and the parathyroid glands, which produce and release parathyroid hormone (PTH). PTH acts on the bone and renal tubule. Provided the amount released is appropriate, this keeps extracellular calcium levels stable.

Normocalcemic primary hyperparathyroidism: evidence for a generalized target-tissue resistance to parathyroid hormone.

Primary hyperparathyroidism (PHPT) is usually characterized by fasting hypercalcemia associated with inappropriately high PTH concentration. Nevertheless, cases of proven PHPT have been reported in normocalcemic patients. The purpose of the study was to investigate the mechanism(s) of persistent normocalcemia in PHPT. One hundred seventy-eight patients with PHPT were studied after exclusion of any evident cause of masked hypercalcemia. Patients were separated into normocalcemic (n = 34) and hypercalcemic (n = 144) subgroups on the basis of their fasting serum ionized calcium value. Patients with normocalcemic PHPT had, on average, a milder excess in PTH secretion assessed by a lower serum PTH concentration. Because of a clear overlap in PTH values between the two groups, normocalcemic and hypercalcemic patients were matched on the basis of serum PTH concentration, age, and sex. Patients with normocalcemic PHPT had lower fasting urine calcium excretion and renal tubular calcium reabsorption. In addition, normocalcemic patients differed from hypercalcemic patients by lower values of markers of bone turnover and plasma 1,25 dihydroxyvitamin D and higher values of renal phosphate threshold. In conclusion, a significant proportion of patients with PHPT are truly normocalcemic, and in addition to a milder increase in PTH secretion, the normocalcemic patients appear to display resistance to PTH action on bone and kidney.

Urinary measurement of Na+/H+ exchanger isoform 3 (NHE3) protein as new marker of tubule injury in critically ill patients with ARF.

BACKGROUND: It has been shown that apical sodium transporters of the renal tubule can be detected by immunoblotting of urine membrane fraction from rats. We raised the hypothesis that protein levels of the Na+/H+ exchanger isoform 3 (NHE3), the most abundant apical sodium transporter in renal tubule, should be increased in urine of patients presenting with acute renal failure (ARF) with severe tubular cell damage and thus might be a noninvasive marker of acute tubular necrosis (ATN). METHODS: Sixty-eight patients admitted to the intensive care unit were studied prospectively (54 patients with ARF, 14 controls without renal dysfunction). Patients with ARF were divided into 3 subgroups as follows: prerenal azotemia, ATN, and intrinsic ARF other than ATN. Urinary NHE3 protein abundance was estimated from semiquantitative immunoblots of urine membrane fraction samples collected from patients. The amount of urinary NHE3 was compared with the fractional excretion of sodium (FeNa) and urinary retinol-binding protein (RBP). RESULTS: NHE3 was not detected in urine from controls. Levels of urinary NHE3 normalized to urinary creatinine level were increased in patients with prerenal azotemia and 6 times as much in patients with ATN, without overlap (ATN, 0.78 +/- 0.36; prerenal azotemia, 0.12 +/- 0.08; P < 0.001). Conversely, urinary NHE3 protein was not detected in patients with intrinsic ARF other than ATN. Normalized NHE3 level correlated positively with serum creatinine level in patients with tubular injury (R2 = 0.305; P = 0.0003). Values for FeNa and normalized urinary RBP did not discriminate ATN from intrinsic ARF other than ATN and prerenal azotemia, respectively. CONCLUSION: In patients with ARF, urinary NHE3 abundance might be a novel noninvasive marker of renal tubule damage, helping to differentiate prerenal azotemia, ATN, and intrinsic ARF other than ATN.

Basal asynchrony and resynchronization with biventricular pacing predict long-term improvement of LV function in heart failure patients.

Biventricular pacing (BiV) is emerging for patients with dilated cardiomyopathy (DCM) and asynchrony. We measured basal asynchrony and early resynchronization by radionuclide angioscintigraphy (RNA) in order to predict long-term evolution of ventricular function after BiV. Thirty-four patients (NYHA Class III-IV,65.4 +/- 11 years) with large QRS(179 +/- 18 ms)were implanted with BiV and studied by RNA before (D0), at day 8 (D8), and during follow-up(20 +/- 7 months). We calculated left and right ejection fractions, the interventricular dyssynchrony (TRVLV), and the apicobasal dyssynchrony (Tab). LVEF improved from 20.2 +/- 8.1%(D0) to27.1%+/- 12.6%(follow-up,P < 0.003 vs D0) and RVEF from 28.6%+/- 13%(D0) to 34.3 +/- 11.5%(follow-up,P < 0.03 vs D0). Inter- (DeltaTRVLV) and intraventricular resynchronization was immediate and remained stable: TRVLV decreased from 68.3 +/- 38 ms(D0) to 13.4 +/- 48.5 ms(D8) and1.8 +/- 39.2 ms(follow-up,P < 0.0001 vs D0); and Tab from 45.8 +/- 64.1 msto-18 +/- 68(D8) and-28.3 +/- 53.6 ms(follow-up,P < 0.0001 vs D0). Early inter- and intraventricular resynchronization (DeltaTab) at D8 were related to late LVEF and RVEF improvement. Together, an LVEF > 15% and a significant interventricular dyssynchrony (TRVLV > 60 ms) at D0 have a sensitivity of 79% and a positive predictive value of 83% to predict an improvement of LVEF superior to 5% at follow-up. In DCM patients, BiV resynchronizes ventricles early and in the long-term, while RVEF and LVEF improve progressively. Patients with large electromechanical dyssynchrony benefit most from BiV.

Angiotensin II stimulates NHE3 activity by exocytic insertion of the transporter: role of PI 3-kinase.

BACKGROUND: Low-concentration angiotensin II (Ang II) stimulates Na+/H+ exchanger 3 (NHE3) activity in renal proximal tubule mainly via angiotensin II type 1 (AT1) receptors. The mechanisms that mediate the increase in NHE3 activity elicited by Ang II remain incompletely settled. METHODS: To assess a potential role of NHE3 trafficking in the Ang II effect, NHE3 activity was measured by H+-driven initial rate of 22Na uptake resistant to 50 micromol/L of the Na+/H+ exchange inhibitor cariporide (HOE642), and sensitive to 300 micromol/L ethyl isopropyl amiloride (EIPA), in a model of cultured proximal tubular cells (MKCC), in which functional apical NHE3 and AT receptors are normally present. Apical expression of NHE3 protein was determined by cell surface biotinylation and immunoblotting. RESULTS: Ang II (10-10 mol/L, 43 minutes) increased NHE3 activity and biotinylated NHE3 protein without any change in total amount of NHE3 protein. Both effects were suppressed by specific AT1 receptor antagonists. When 2-mercaptoethanesulphonic acid (MESNA) was used to cleave biotin from all apical proteins, intracellular biotinylated NHE3 protein remained unchanged after Ang II incubation compared to control. When sulfo-N-hydrosuccinimide (NHS)-acetate was used first to block all apical reactive sites, an increase in biotinylated NHE3 protein was observed following Ang II incubation. To evaluate the role of phosphatidylinositol 3-kinase (PI 3-kinase), the specific inhibitor wortmannin was used. It suppressed Ang II-induced increase in NHE3 activity and trafficking. Furthermore, latrunculin B, inhibitor of actin filament polymerization, prevented both Ang II stimulatory effects. CONCLUSION: Ang II stimulates NHE3 activity, at least in part, by exocytic insertion of the protein into the apical membrane. This effect is mediated by PI 3-kinase and required integrity of actin cytoskeleton.

Differentiated thick ascending limb (TAL) cultured cells derived from SV40 transgenic mice express functional apical NHE2 isoform: effect of nitric oxide.

Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV(40) (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV(40) T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.

Circulating vasopressin levels in septic shock.

OBJECTIVE: To assess the frequency of vasopressin deficiency in septic shock. DESIGN: Prospective cohort study. SETTING: Intensive care unit at Raymond Poincaré University Hospital. PATIENTS: A cohort of 44 patients who met the usual criteria for septic shock for < 7 days. A second cohort of 18 septic shock patients were enrolled within the first 8 hrs of disease onset. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: General demographics, severity scores, vital signs, standard biochemical data, and circulating vasopressin levels were systematically obtained at baseline in the two cohorts. Vasopressin deficiency was defined by a normal plasma vasopressin level in the presence of a systolic blood pressure of <100 mm Hg or in the presence of hypernatremia. Baroreflex sensitivity was systematically evaluated in patients of the first cohort when vasopressin deficiency was noted. In the second cohort of patients, plasma levels of vasopressin were obtained at baseline, 6, 24, 48, and 96 hrs after shock onset. In the first population, plasma vasopressin levels were inversely correlated to the delay from shock onset. Fourteen patients had relative vasopressin deficiency: 12 patients had systolic blood pressure <100 mm Hg, with impaired baroreflex sensitivity in four, and three patients had hypernatremia. In the second population, only two patients had relative vasopressin deficiency. The plasma levels of vasopressin significantly decreased over time (p < 10-3). CONCLUSIONS: Plasma vasopressin levels are almost always increased at the initial phase of septic shock and decrease afterward. Relative vasopressin deficiency is seen in approximately one-third of late septic shock patients.

RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.

Two nonerythroid homologs of the blood group Rh proteins, RhCG and RhBG, which share homologies with specific ammonia transporters in primitive organisms and plants, could represent members of a new family of proteins involved in ammonia transport in the mammalian kidney. Consistent with this hypothesis, the expression of RhCG was recently reported at the apical pole of all connecting tubule (CNT) cells as well as in intercalated cells of collecting duct (CD). To assess the localization along the nephron of RhBG, polyclonal antibodies against the Rh type B glycoprotein were generated. In immunoblot experiments, a specific polypeptide of Mr approximately 50 kD was detected in rat kidney cortex and in outer and inner medulla membrane fractions. Immunocytochemical studies revealed RhBG expression in distal nephron segments within the cortical labyrinth, medullary rays, and outer and inner medulla. RhBG expression was restricted to the basolateral membrane of epithelial cells. The same localization was observed in rat and mouse kidney. RT-PCR analysis on microdissected rat nephron segments confirmed that RhBG mRNAs were chiefly expressed in CNT and cortical and outer medullary CD. Double immunostaining with RhCG demonstrated that RhBG and RhCG were coexpressed in the same cells, but with a basolateral and apical localization, respectively. In conclusion, RhBG and RhCG are present in a major site of ammonia secretion in the kidney, i.e., the CNT and CD, in agreement with their putative role in ammonium transport.

pH dependence of Na+/myo-inositol cotransporters in rat thick limb cells.

BACKGROUND: To balance medullary interstitium hypertonicity generated by transepithelial NaCl absorption, medullary thick ascending limb (MTAL) cells accumulate myo-inositol (MI). Expression of Na+-MI cotransporter (SMIT) mRNA in TAL is correlated with the NaCl absorption rate. Our present study aimed to determine the plasma membrane location and functional properties of the Na+-MI cotransporter in MTAL cells. METHODS: Preparation of basolateral (BLMV) and luminal (LMV) membrane vesicles were simultaneously isolated from purified rat MTAL suspension, and uptake of [3H]myo-inositol ([3H]MI) was used to assess Na+-MI cotransport activity. RESULTS: In the presence of an inside-negative membrane potential, imposing an inwardly-directed Na+-gradient versus tetramethylammonium (TMA) stimulated the initial [3H]MI uptake in BLMV and LMV. Phlorizin inhibited Na+ gradient-dependent initial [3H]MI uptake in both preparations, with IC50 values of 565 and 29 micromol/L in BLMV and LMV, respectively. 2-0,C-methylene myo-inositol (MMI), a competitive inhibitor of MI transport, only inhibited the BLMV Na+-MI cotransporter. Phlorizin-sensitive Na+ gradient-dependent initial [3H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar Vmax but different Km values of 51 and 107 micromol/L in BLMV and LMV, respectively. Finally, BLMV but not LMV Na+-MI cotransporter exhibited a marked pH dependence with sigmoidal patterns of activation, as intravesicular pH (pHi) was decreased from 8.0 to 6.0 at extravesicular pH (pHe) 8.0, and as pHe was increased from 6.0 to 8.0 at pHi 6.0. Maximal activation was observed at pHi 6.5 and pHe 7.5. CONCLUSIONS: In rat MTAL cells, Na+-MI cotransporter activity is present in both BLM and LM, and has markedly different functional properties, indicating the presence of distinct transporters. Basolateral Na+-MI cotransporter activity is maximal at physiological pH values of MTAL cells and interstitium, and a powerful modulation of the transporter activity may be exerted by pHe and pHi.