Antidiuretic hormone and/or cAMP increase Na+ transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na+ transport could be explained quantitatively by an increased density of ENaC Na+ channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat α-, β-, and γENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. Proc. Natl. Acad. Sci. USA. 93:15370–15375). The density of ENaC subunits was quantified by specific binding of 125I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4–8 nM. Transepithelial Na+ transport was measured as the amiloride-sensitive short-circuit current (AS-I sc) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-I sc measured in the same experiments. Stimulation with cAMP (20 μM 8-p-chlorothio-cAMP plus 200 μM IBMX) significantly increased AS-I sc from 11.2 ± 1.3 to 18.1 ± 1.3 μA/cm2. M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I sc from 0.62 ± 0.13 to 1.16 ± 0.18 fmol/cm2. Based on the concentration dependence of M2 binding, the quantity of Na+ channels per unit of AS-I sc was calculated to be the same in the presence and absence of cAMP, 0.23 ± 0.04 and 0.21 ±0.05 fmol/μA, respectively. These values would be consistent with a single channel conductance of ∼5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-I sc to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I sc increase produced by cAMP.

OREBP (osmotic response element-binding protein), also called TonEBP or NFAT5, is thought to induce the expression of genes that increase the accumulation of organic osmolytes to protect cells against a hypertonic environment. To investigate the consequences of lacking OREBP activity, transgenic (Tg) mice that overexpress OREBPdn (dominant negative form of OREBP) specifically in the epithelial cells of the renal collecting tubules were generated. These mice showed impairment in their urine concentrating mechanism, most likely due to reduced expression of the aquaporin AQP2 and the urea transporter UT-A1 and UT-A2 mRNAs. When deprived of water or after the administration of a vasopressin analogue, urine osmolality of the Tg mice was significantly increased but not to the same extent as that of the wild type mice. The expression of AQP2 and UT-A1, but not UT-A2 mRNAs, was increased to the same level as that of the wild type mice in the water deprivation state, indicating that the vasopressin regulatory mechanism was not affected by OREBPdn. These data indicate that in addition to vasopressin, OREBP is another essential regulator of the urine concentrating mechanism. Furthermore, the OREBPdn Tg mice developed progressive hydronephrosis soon after weaning, confirming the osmoprotective function of OREBP implicated by the in vitro experiments.The mammalian kidney plays an important role in maintaining the homeostasis of osmolality and the electrolyte concentrations of the circulating fluid. The osmolality of the kidney inner medulla is highly hypertonic to facilitate the reabsorption of water from the urine. The cells in the collecting tubules guard against hypertonic stress by increasing the synthesis or import of several organic "compatible" osmolytes. These include sorbitol, which is synthesized by the enzyme aldose reductase, and betaine, myo-inositol, and taurine, which are imported by the betaine/␥-aminobutyric acid transporter, the Na ϩ -dependent myo-inositol transporter, and taurine transporter, respectively (1). The transcription of these genes is induced by a hypertonic medium and regulated by a protein called the osmotic response element-binding protein (OREBP) 1 (2) or tonicity element-binding protein (TonEBP) (3). This protein is also called NFAT5 because of its homology to the NFAT family of transcription factors (4).OREBP consists of a nuclear localization signal near the N terminus followed by a DNA binding domain, a dimerization domain, and a transactivation domain at the carboxyl end. Upon stimulation by hypertonicity, it is rapidly translocated into the nucleus and binds to the osmotic response elements (OREs) (5) in the promoter region of the osmoprotective genes (2) to stimulate transcription. OREBP is highly expressed in the inner medulla as well as in the inner stripe of the outer medulla in the rat kidney (6). In response to water loading, OREBP in the initial portion of the inner medullary collecting ducts (IMCDs) is primarily located in the cytoplasm of the epithelial cells. In dehydrated a...

Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl increases GPC in renal [Madin-Darby canine kidney (MDCK)] cells. The GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. The purpose of the present study was to test whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells. We find that in mouse inner medullary collecting duct cells, high NaCl increases NTE mRNA within 8 h and NTE protein within 16 h. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl. TonEBP͞OREBP is a transcription factor that is activated by high NaCl. Knockdown of TonEBP͞OREBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1 ؊/؊ mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. We conclude that high NaCl increases transcription of NTE, likely mediated by TonEBP͞OREBP, and that the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo.diisopropyl fluorophosphate ͉ phospholipase B

Hypokalemia is a prominent feature of Gitelman syndrome and a common side effect of thiazide use in the treatment of hypertension. It is widely recognized that genetic or pharmacological inhibition of the renal thiazide-sensitive sodium-chloride cotransporter (NCC) initiates the potentially severe renal potassium loss observed in these settings. Surprisingly, hypokalemia has not been detected in NCC (−/−) mice maintained on normal rodent diets (Schultheis PJ, Lorenz JN, Meneton P, Nieman ML, Riddle TM, Flagella M, Duffy JJ, Doetschman T, Miller ML, and Shull GE. J Biol Chem 273: 29150–29155, 1998). We show that modest reduction of dietary potassium induced a marked reduction in plasma potassium and elevated renal potassium excretion in NCC (−/−) mice that was associated with a pronounced polydipsia and polyuria of central origin. These findings are consistent with the development of potassium depletion in NCC (−/−) mice and were not seen in wild-type mice maintained on the same low-potassium diet. In addition, plasma aldosterone levels were significantly elevated in NCC (−/−) mice even in the presence of a low-potassium diet. Collectively, these findings suggest an early central component to the polyuria of Gitelman syndrome and show that both elevated aldosterone and dietary potassium content contribute to the development of hypokalemia in Gitelman syndrome. Therefore, NCC (−/−) mice are more sensitive to reductions in dietary potassium than wild-type mice and become hypokalemic, thus more faithfully representing the Gitelman phenotype seen in humans.