Hypotension in NKCC1 null mice: role of the kidneys
http://www.100md.com
《美国生理学杂志》
1Renal Division, Emory University School of Medicine, Atlanta, Georgia
2Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung Blood Institute, National Institutes of Health, Bethesda, Maryland
3Renal Division, University of Texas Medical School, Houston, Texas
4Departments of Molecular Genetics, Biochemistry and Microbiology
5Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
6Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit Medical Campus of Case Western Reserve University School of Medicine, Detroit, Michigan
7Department of Physiology and Biophysics, College of Medicine, University of South Florida, Tampa, Florida
ABSTRACT
NKCC1 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 (–/–) 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+ transporter expression. Thus we performed balance studies, semiquantitative immunoblotting, and immunohistochemistry of kidney tissue from NKCC1 (+/+) and NKCC1 (–/–) 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 (–/–) than NKCC1 (+/+) 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+ concentration with inappropriately low urinary K+ 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+ 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.
Na+ transporters; chloride; water; renin; aldosterone
NKCC1 (BSC-2) is a ubiquitous Na+-K+-2Cl– cotransporter (9), which serves a number of cellular functions such as cell volume regulation, while in polarized epithelia it participates in Cl– secretion (8, 9, 25, 27). NKCC1 is highly expressed in the salivary gland and inner ear where it contributes to the production of saliva (8) and to the formation of endolymphatic fluid (5).
In the kidney, NKCC1 localizes to the juxtaglomerular afferent arteriole as well as the glomerular and extraglomerular mesangium (10, 13), where it is thought to participate in the process of tubuloglomerular feedback and the regulation of blood pressure (13). In the mouse, NKCC1 is most highly expressed along the basolateral membrane of the terminal inner medullary collecting duct (13). In rat NKCC1 localizes to the basolateral regions of the type A intercalated cell of the outer medullary collecting duct (OMCD), a H+-secreting cell type (10). However, in the rat OMCD NKCC1 does not participate in net H+ secretion (25) but rather contributes to transepithelial transport of Na+ and Cl– (25, 27). NKCC1 may also participate in the process of fluid secretion along the collecting duct during physiological and pathophysiological states, such as with cyst formation in polycystic kidney disease (15, 30). Thus genetic disruption of NKCC1 would be predicted to reduce Cl– secretion along the collecting duct, leading to volume expansion and possibly hypertension. However, hypotension rather than hypertension is observed in NKCC1 null mice (9, 19), which suggests a renal defect in NaCl and water handling.
The purpose of the present study was to determine whether NKCC1 (–/–) mice have a defect in the regulation of NaCl and water excretion that might contribute to the observed hypotension.
METHODS
Animals
Series 1. NKCC1 null mice developed by Flagella et al. (9) were studied. Heterozygous offspring were used to establish breeding colonies in the UTHSC and Emory vivaria. Age- and sex-matched NKCC1 (–/–) and (+/+) littermates, weighing 20–40 g, were pair fed a balanced, NaCl-deficient diet (1.78 meq Na+/kg food, <1.13 meq Cl–/kg food, no. 53140000, Zeigler Brothers, Gardners, PA) for 7 days before death, prepared as a gel (0.01% NaCl, low-NaCl diet). Thus mice received 9 ml H2O/day and <0.01 meq/day NaCl. In some experiments, the gelled diet described above was supplemented with NaCl. In this protocol, mice received 9 ml H2O/day and 1.40 meq NaCl/day (2.8% NaCl, high-NaCl diet). Thus the high- and low-NaCl diets differed only in NaCl intake. Mice had no access to water other than that given in the gel. Mice were placed in metabolic cages, and urine was collected at 4°C under oil for 24 h before death under anesthesia with isofluorane (24). When measuring blood pressure through an arterial catheter, mice were anesthetized with ketamine (50 mg/kg) and Inactin (100 mg/kg) as separate intraperitoneal injections. Changes in urinary osmolality following vasopressin administration were measured using the protocol of Takahashi et al. (22). Mice ate rodent chow (Harlan Teklad LM-485) containing 139 meq Na+/kg food (0.8% NaCl) and water containing 5% dextrose ad libitum for 3 days. On day 4, urinary osmolality was measured before and 2 h after administration of 1 ng/g body wt intraperitoneal vasopressin (desmopressin, dDAVP) (22). Mice invariably voided their urine when handled. Thus mice were handled at 1 and 2 h after the administration of dDAVP to induce voiding of urine that had accumulated during the first and second hours following administration of the drug. Urine was voided onto a piece of parafilm and then immediately collected with a pipette tip for measurement of osmolality. The Institutional Animal Care and Use Committee at Emory, the University of Cincinnati, and UTHSC approved all animal treatment protocols.
Measurement of Blood Pressure, Serum and Urine Chemistries, Arterial Blood Gases, and Glomerular Filtration Rate
Systolic blood pressure in conscious mice was measured by tail cuff or femoral artery catheter as described previously (16, 24). Blood was collected for serum chemistries through the abdominal aorta under isofluorane anesthesia (24). Unless noted, urine and serum chemistries were measured at IDEXX Laboratories (West Sacramento, CA) (24). Arterial blood gases, serum osmolality, urine osmolality, and urinary total ammonia concentration were measured as described previously (24, 29). Urine Na+ and Cl– concentration from mice on a low-salt diet as well as plasma renin concentration and serum aldosterone concentration were measured as described previously (17, 29). Plasma atrial natriuretic peptide (ANP) concentration was measured as reported previously with one measurement per mouse (6). Osmolar clearance (Cosm) was calculated as Uosm/Sosm x V, where Uosm and Sosm are urine and serum osmolality, respectively, and where V is urinary flow rate (in ml/min). CH2O was calculated as Cosm – V. Glomerular filtration rate (GFR) was measured using FITC-inulin, as described previously (16), after 7 days of the NaCl-deficient diet. Urinary NO was measured using a kit (Nitrate/Nitrite Colorimetric Assay Kit, Cayman Chemical, Ann Arbor, MI).
Mouse Kidney Lysates and Immunoblotting
Preparation of kidney lysates and immunoblotting were performed as reported previously (26). Equal total protein in each lane was confirmed with gels run in parallel stained with Coomassie blue dye. Blots were probed with affinity-purified, peptide-directed rabbit polyclonal antibodies that specifically recognize the major renal Na+ and water transporters (NHE3, NKCC2, the -, -, and -subunits of ENaC and the thiazide-sensitive cotransporter, TSC/NCC, AQP1, AQP2) (1, 7, 23) and a mouse monoclonal anti-Na-K-ATPase, -subunit antibody (Upstate Biotechnology, Lake Placid, NY) (26). Donkey anti-rabbit IgG conjugated to horseradish peroxidase (31458, Pierce, Rockford, IL) or sheep anti-mouse IgG conjugated with horseradish peroxidase (Amersham-Pharmacia Biotech, Piscataway, NJ) was used as secondary antibodies. Antibody-antigen reactions were visualized using enhanced chemiluminescence before exposure of X-ray film (26). Band densities were quantified (26) and normalized to the mean band density of lysates from wild-type mice measured in parallel in the same gel.
Immunohistochemistry
Mice were anesthetized with isofluorane and the kidneys were fixed in situ as reported previously (28). Immunoperoxidase labeling of 2-μm sections of paraffin-embedded tissue was performed using a postembedding method described previously (14). AQP2 abundance and distribution were determined using a rabbit polyclonal antibody described previously (7, 23).
Statistical Analysis
For data without a normal distribution or equal variance, a Mann-Whitney rank sum test was used. In all other studies, comparisons were made between two groups using an unpaired Student's t-test. P < 0.05 indicates statistical significance. Data are means ± SE.
RESULTS
NKCC1 (–/–) Mice Are Constitutively Hypotensive
After 7 days of a high-NaCl diet, blood pressure was lower in NKCC1 (–/–) than NKCC1 (+/+) mice (P < 0.05), when measured either in conscious mice by tail cuff (Fig. 1) or in anesthetized mice by femoral artery catheter (Table 1). Hypotension persisted unchanged after 7 days of either a NaCl-deficient or a high-NaCl diet (Fig. 1). We conclude that NKCC1 (–/–) mice are hypotensive independent of NaCl intake, consistent with previous observations (20).
NKCC1 (–/–) Mice Have a Higher Plasma Renin Concentration Than Wild-Type Mice, Although Their Circulating Aldosterone Is Inappropriately Low During Some Treatment Conditions
Normal kidneys respond to hypotension, and thus provide a corrective response, by increasing the release of renin, ANG II, and aldosterone which reduce NaCl excretion. Therefore, if NKCC1 null mice have low blood pressure and normal renal function, renal NaCl excretion should decrease at least transiently, thereby increasing vascular volume, which restores blood pressure to normal. Thus we asked whether the hypotension observed in NKCC1 null mice occurs from inappropriately reduced circulating levels of renin and/or aldosterone. On a high-salt diet, plasma renin concentration was fourfold higher in NKCC1 knockout mice than in wild-type mice, although differences between wild-type and knockout mice were not detected with a low-NaCl diet (Table 2). Thus plasma renin concentration is higher in NKCC1 null than wild-type mice, as expected given the hypotension observed in these mutant mice.
Because plasma renin was higher in NKCC1 null than in wild-type mice, we asked whether NKCC1 null mice excrete more Cl– than wild-type mice, which makes their Cl– balance more negative thus increasing plasma renin concentration. Therefore, cumulative urinary Cl– excretion was measured over the first 72 h following the transition from a low- to a high-NaCl diet (Fig. 2). As shown, NKCC1 null mice did not excrete more Cl– over this time period than pair-fed wild-type mice. Thus the elevated plasma renin concentrations observed in NKCC1 null mice do not occur because they excrete more Cl– than pair-fed wild-type mice.
High serum K+ and plasma renin concentrations, as observed in NKCC1 null mice on a high-salt diet, should markedly increase circulating aldosterone. However, on a high-NaCl diet, serum aldosterone was not higher in NKCC1 null than in wild-type mice (Table 2). Thus serum aldosterone is inappropriately low in NKCC1 null mice ingesting a high-NaCl diet.
After ingestion of the low-NaCl diet, the increased serum K+ and aldosterone concentration observed in NKCC1 null mice should produce a kaliuresis. Because NKCC1 null mice did not excrete more K+ than wild-type mice, renal K+ excretion is inappropriately low in these mutant mice (Table 2). These data demonstrate that NKCC1 null mice have defects in renal Na+ and K+ handling.
Hypotension in NKCC1 Null Mice Does Not Occur from an Increase in Nitric Oxide or Atrial Natriuretic Peptide
Nitric oxide (NO) and ANP relax vascular smooth muscle and promote diuresis and natriuresis (21, 32). Thus increased synthesis and/or release of NO or ANP should reduce blood pressure. However, the hypotension observed in NKCC1 null mice does not occur from increased NO synthesis, as NO excretion was the same as in wild-type mice (Table 2). Moreover, on either a high- or a low-NaCl diet, plasma ANP concentration was lower in NKCC1 null than in wild-type mice (Table 2). Thus the hypotension observed in NKCC1 null mice does not occur due to increased synthesis and/or release of NO or ANP. Furthermore, the inappropriately low serum aldosterone concentration observed in NKCC1 null mice eating a high-NaCl diet does not occur from increased ANP release.
Na+ Transporter Expression Is Upregulated in NKCC1 (–/–) Mice
We asked whether the hypotension observed in NKCC1 null mice occurs in tandem with downregulation of one or more of the renal Na+ transporters or water channels that micropuncture and isolated, perfused tubule experiments have shown mediate Na+ and water absorption in specific nephron segments of the kidney (1). On a high-NaCl diet, expression of the thiazide-sensitive cotransporter of the DCT (NCC) was greater in NKCC1 (–/–) than NKCC1 (+/+) mice, whereas expression of the other Na+ transporters was the same in kidneys from NKCC1 null and wild-type mice (Fig. 3). However, on a low-NaCl diet, expression of the proximal Na+/H+ exchanger (NHE3), the thick ascending limb Na+-K+-2Cl– cotransporter (NKCC2), the -subunit of the Na-K-ATPase and NCC were all increased in NKCC1 null relative to wild-type mice, although expression of the three subunits of ENaC was unchanged (Fig. 4). Thus the hypotension observed in NKCC1 null mice does not occur from inappropriately reduced expression of a major transport mechanism of renal Na+ absorption. Instead, increased renal Na+ transporter expression is likely a compensatory response to the low blood pressure and high plasma renin concentration observed in these mutant mice.
NKCC1 (–/–) Mice Are Hypochloremic and Vasopressin Resistant
Table 2 shows that NKCC1 (–/–) mice consuming a low-NaCl diet develop hypochloremia. This hypochloremia was accompanied by reduced serum osmolality and was not explained by increased serum HCO3–. Despite the hypochloremia, urinary volume was lower in NKCC1 null than in (+/+) mice, although excretion of chloride and total osmoles was the same. Thus free water excretion did not respond appropriately to the hypochloremia observed in NKCC1 null mice.
We asked whether the hypochloremia and reduced urinary volume are due to increased water absorption by the kidney or reduced GFR. Because NKCC1 null mice have reduced urinary volume and are hypochloremic, we asked whether AQP1 or 2 is upregulated in kidney from NKCC1 (–/–) mice either through increased protein expression or through selective redistribution to the apical plasma membrane, which might lead to increased water absorption. As shown in Figs. 3 and 4, AQP1 and 2 protein expression was the same in wild-type and NKCC1 null mice. However, we observed that AQP2 is redistributed to the basal regions of principal cells from NKCC1 null mice (Fig. 5).
Because AQP2 protein is redistributed within principal cells of NKCC1 null mice, we asked whether these mice are vasopressin resistant. Thus changes in urinary osmolality following the administration of vasopressin were measured in NKCC1 null and wild-type mice. Baseline urinary osmolality was the same in NKCC1 null and wild-type mice (696 ± 187 mosmol/kgH2O in wild-type mice, n = 9, vs. 392 ± 81 mosmol/kgH2O in NKCC1 null mice, n = 8, P = not significant). Two hours following the administration of dDAVP, urinary osmolality was lower in NKCC1 null than wild-type mice (1,866 ± 203 mosmol/kgH2O in wild-type mice, n = 9, vs. 1,103 ± 152 mosmol/kgH2O in NKCC1 null mice, n = 8, P < 0.05). Thus NKCC1 null mice have a blunted increase in urinary osmolality following the administration of vasopressin, which may reflect the subcellular redistribution of AQP2 observed within principal cells. This vasopressin resistance should increase free water excretion, thus limiting the hypochloremia observed in NKCC1 null mice.
Because kidney size is smaller in NKCC1 null than in wild-type mice (Figs. 2 and 3), reduced GFR is expected in NKCC1 knockout mice, although this was not demonstrated statistically (Table 1). Thus we cannot exclude the possibility that the lower renal mass of NKCC1 (–/–) mice attenuates their ability to excrete free water.
DISCUSSION
Whether NKCC1 modulates blood pressure through vascular and/or renal effects is unknown. NKCC1 is expressed in vascular smooth muscle where it modulates vascular contractility, likely through changes in intracellular calcium generated by NKCC1-mediated Cl– uptake (3). In vascular smooth muscle, NKCC1 is upregulated by aldosterone, which contributes to the heightened vascular tone and hypertension observed in this treatment model (12). Because vascular tone is reduced in NKCC1 knockout mice (19), NKCC1-dependent changes in blood pressure are thought to be primarily vascular rather than renal events (19).
The role of the kidney in NKCC1-mediated changes in blood pressure has been unexplored. This study demonstrates that in addition to vascular changes, NKCC1 null mice have diminished aldosterone release from the adrenal cortex as well as defects within the kidney which blunt the renal response to vasopressin and aldosterone. The result is abnormal regulation of Na+, K+, and water excretion.
Within the kidney, NKCC1 is expressed in the afferent arteriole and glomerular and extraglomerular mesangium and may be involved in the sensing of luminal Cl– concentration, which ultimately leads to changes in afferent arteriolar tone (TGF) (13). Because NKCC1 is present in cells responsible for the synthesis of renin within the afferent arteriole, the hypotension observed in NKCC1 knockout mice might result from failure of the kidney to release renin on demand. However, the present study shows increased plasma renin concentration in NKCC1 null mice on a NaCl-replete diet and demonstrates increased expression of ANG II-sensitive Na+ transporters such as the TSC/NCC. Thus the hypotension observed in NKCC1 knockout mice does not occur from a defect in the release of renin or from an inability of renal Na+ transporter expression to respond to the expected increase in ANG II. Moreover, these results are consistent with the recent observation that NKCC1 suppresses the release of renin (2), which therefore explains the marked elevation in plasma renin observed in NKCC1 null mice. Instead, the hypotension observed may occur from an altered TGF set point (13), which increases NaCl delivery to the distal convoluted tubule and transiently increases GFR. NaCl loss would ensue, which lowers blood pressure and returns GFR and distal delivery of NaCl to normal. However, in the transition from a NaCl-deficient to a high-NaCl diet, NKCC1 null mice did not excrete more Cl– than wild-type mice. Alternatively, the hypotension observed likely reflects, at least in part, the lack of NKCC1-mediated vasoconstriction. Increased Na+ transporter expression may therefore be a compensatory mechanism for reduced vasoconstriction. Whether NKCC1 null mice have reduced renal vascular resistance remains to be determined.
NKCC1 null mice are hyperkalemic independent of NaCl intake, which might occur through a renal or a renal-independent mechanism. During K+ stress, a rise in serum K+ is buffered through K+ uptake in skeletal muscle mediated by the Na-K-ATPase (18) and to some extent by NKCC1 (31). Thus in the absence of NKCC1-mediated K+ uptake in skeletal muscle, the kidney should respond by increasing renal K+ excretion. However, renal excretion of K+ was not increased in NKCC1 null mice. Thus in NKCC1 null mice, renal K+ excretion does not respond appropriately to the increase in serum aldosterone and K+ concentration. The absence of NKCC1-mediated Cl– secretion may attenuate secretion of K+ along the collecting duct. Why NKCC1 null mice have inappropriately low renal K+ excretion is not apparent and will require further studies in native tissue.
NKCC1 null mice have reduced water excretion by both the kidney and the salivary gland (8). NKCC1 participates in secretion of isotonic fluid by the salivary acinar cells (8). Downstream, NaCl absorption occurs within the salivary duct cells, which results in the production of hypotonic saliva (8). Thus NKCC1 null mice have a reduced ability to produce hypotonic saliva (8). When consuming a NaCl-deficient diet with high water intake, NKCC1 null mice retain free water in excess of NaCl, which results in hypochloremia. If NKCC1 functions in the kidney through a process analogous to the salivary gland, then it should participate in the generation of hypotonic urine and the renal excretion of free water. Testing this hypothesis will require further studies in isolated tubules perfused in vitro.
Vasopressin resistance is likely a compensatory mechanism which attenuates the hypochloremia observed in NKCC1 null mice ingesting the low-NaCl diet. Whether vasopressin resistance results from the AQP2 redistribution observed within cortical collecting duct (CCD) principal cells of NKCC1 null mice is unclear. Christensen et al. (4) reported that acute administration of V2 receptor antagonists leads to redistribution of AQP2 to the basolateral membrane of rat principal cells within the CCD in tandem with increased water excretion. However, redistribution of AQP2 to the basolateral membrane of principal cells has also been reported in rat models associated with reduced free water excretion (4, 11). Whether targeting of AQP2 to the basolateral membrane, as observed in NKCC1 null mice, promotes or inhibits the absorption of water requires further study.
In conclusion, the hypotension observed in NKCC1 null mice persists unchanged when NaCl intake is varied. NKCC1 is critical to the release and/or action of vasopressin, renin, and aldosterone and the regulation of renal Na+, K+, Cl– and water excretion. The action of vasopressin as well as the action and release of aldosterone are attenuated in kidneys from NKCC1 null mice, which likely contributes to their hypotension. Increased Na+ transporter expression, increased plasma renin and reduced plasma ANP, as observed in NKCC1 null mice, should increase vascular volume and blood pressure, thereby limiting the hypotension.
GRANTS
This study was supported by National Institutes of Health Grants DK-46493 (to S. M. Wall), DK-50594 (to G. E. Shull), DK-57552 (to J. N. Lorenz), and Z01-HL-01282-KE (to M. A. Knepper).
ACKNOWLEDGMENTS
We thank Dr. James E. Melvin (University of Rochester) for generously supplying NKCC1 (+/–) mice following Tropical Storm Allison.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Brooks HL, Allred AJ, Beutler KT, Coffman TM, and Knepper MA. Targeted proteomic profiling of renal Na+ transporter and channel abundances in angiotensin II type 1a receptor knockout mice. Hypertension 39: 470–473, 2002.
Castrop H, Lorenz JN, Hansen P, Friis U, Mizel D, Oppermann M, Jensen B, Briggs JP, Skott O, and Schnermann J. Contribution of the basolateral isoform of the Na-K,2Cl-cotransporter (NKCC1/BSC2) to renin secretion. Am J Physiol Renal Physiol 289: F1185–F1192, 2005.
Chipperfield AR and Harper AA. Chloride in smooth muscle. Prog Biophys Mol Biol 74: 175–221, 2000.
Christensen BM, Wang W, Frokiaer J, and Nielsen S. Axial hetergeneity in basolateral AQP2 localization in rat kidney: effect of vasopressin. Am J Physiol Renal Physiol 284: F701–F717, 2003.
Delpire E, Lu J, England R, Dull C, and Thorne T. Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl cotransporter. Nat Genet 22: 192–195, 1999.
Dietz JR and Nazian SJ. Release of atrial natriuretic factor in hypophysectomized rats. Am J Physiol Regul Integr Comp Physiol 255: R534–R538, 1988.
DiGiovanni SR, Nielsen S, Christensen EI, and Knepper MA. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc Natl Acad Sci USA 91: 8984–8988, 1994.
Evans RL, Park K, Turner RJ, Watson GE, Nguyen HV, Dennett MR, Hand AR, Flagella M, Shull GE, and Melvin JE. Severe impairment of salivation in Na+-K+-2Cl– cotransporter (NKCC1)-deficient mice. J Biol Chem 275: 26720–26726, 2000.
Flagella M, Clarke LL, Miller ML, Erway LC, Giannella RA, Andringa A, Gawenis LR, Kramer J, Duffy JJ, Doetschman T, Lorenz JN, Yamoah EN, Cardell EL, and Shull GE. Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf. J Biol Chem 274: 26946–26955, 1999.
Ginns SM, Knepper MA, Ecelbarger CA, Terris J, He X, Coleman RA, and Wade JB. Immunolocalization of the secretory isoform of the Na-K-Cl cotransporter in rat renal intercalated cells. J Am Soc Nephrol 7: 2533–2542, 1996.
Jeon US, Joo KW, Na KY, Kim YS, Lee JS, Kim J, Kim GH, Nielsen S, Knepper MA, and Han JS. Oxytocin induces apical and basolateral redistribution of aquaporin-2 in rat kidney. Nephron Exp Nephrol 93: e36–e45, 2003.
Jiang G, Cobbs S, Klein JD, and O'Neill WC. Aldosterone regulates the Na-K-2Cl cotransporter in vascular smooth muscle. Hypertension 41: 1131–1135, 2003.
Kaplan MR, Plotkin MD, Brown D, Hebert SC, and Delpire E. Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium and the glomerular afferent arteriole. J Clin Invest 98: 723–730, 1996.
Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frokiaer J, and Nielsen S. Altered expression of renal acid-base transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol 285: F1244–F1257, 2003.
Lebeau C, Hanaoka K, Moore-Hoon ML, Guggino WB, Beauwens R, and Devuyst O. Basolateral chloride transporters in autosomal dominant polycystic kidney disease. Pflügers Arch 444: 722–731, 2002.
Lorenz JN and Gruenstein E. A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin. Am J Physiol Renal Physiol 276: F172–F177, 1999.
Lum C, Shesely EG, Potter DL, and Beierwaltes WH. Cardiovascular and renal phenotype in mice with one or two renin genes. Hypertension 43: 79–86, 2004.
McDonough AA, Thompson CB, and Youn JH. Skeletal muscle regulates extracellular potassium. Am J Physiol Renal Physiol 282: F967–F974, 2002.
Meyer JW, Flagella M, Sutliff RL, Lorenz JN, Nieman ML, Weber CS, Paul RJ, and Shull GE. Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na+-K+-2Cl– cotransporter. Am J Physiol Heart Circ Physiol 283: H1846–H1855, 2002.
Pace AJ, Lee E, Athirakul K, Coffman TM, O'Brien DA, and Koller BH. Failure of spermatogenesis in mouse lines deficient in the Na+-K+-2Cl– cotransporter. J Clin Invest 105: 441–450, 2000.
Sabrane K, Kruse MN, Fabritz L, Zetsche B, Mitko D, Skryabin BV, Zwiener M, Baba HA, Yanagisawa M, and Kuhn M. Vascular endothelium is critically involved in the hypotensive and hypovolemic actions of atrial natriuretic peptide. J Clin Invest 115: 1666–1674, 2005.
Takahashi N, Chernavvsky DR, Gomez RA, Igarashi P, Gitelman HJ, and Smithies O. Uncompensated polyuria in a mouse model of Bartter's syndrome. Proc Natl Acad Sci USA 97: 5434–5439, 2000.
Terris J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414–F422, 1996.
Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett LA, Green ED, and Wall SM. Deoxycorticosterone upregulates Pds (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003.
Wall SM and Fischer MP. Contribution of the Na+-K+-2Cl– cotransporter (NKCC1) to transepithelial transport of H+, NH4+, K+ and Na+ in rat outer medullary collecting duct. J Am Soc Nephrol 13: 827–835, 2002.
Wall SM, Fischer MP, Kim GH, Nguyen BM, and Hassell KA. In rat inner medullary collecting duct, NH4+ uptake by the Na-K-ATPase is increased during hypokalemia. Am J Physiol Renal Physiol 282: F91–F102, 2002.
Wall SM, Fischer MP, Mehta P, Hassell KA, and Park SJ. Contribution of the Na+-K+-2Cl– cotransporter (NKCC1) to Cl– secretion in rat outer medullary collecting duct. Am J Physiol Renal Physiol 280: F913–F921, 2001.
Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, and Verlander JW. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284: F229–F241, 2003.
Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, and Verlander JW. NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl– conservation. Hypertension 44: 1–6, 2004.
Wallace DP, Rome LA, Sullivan LP, and Grantham JJ. cAMP-dependent fluid secretion in rat inner medullary collecting ducts. Am J Physiol Renal Physiol 280: F1019–F1029, 2001.
Wong JA, Fu L, Schneider EG, and Thomason DB. Molecular and functional evidence for Na+-K+-2Cl– cotransporter expression in rat skeletal muscle. Am J Physiol Regul Integr Comp Physiol 277: R154–R161, 1999.
Zhou MS, Schulman IH, and Raij L. Nitric oxide, angiotensin II and hypertension. Semin Nephrol 24: 366–378, 2004.(Susan M. Wall, Mark A. Knepper, Kathryn )
2Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung Blood Institute, National Institutes of Health, Bethesda, Maryland
3Renal Division, University of Texas Medical School, Houston, Texas
4Departments of Molecular Genetics, Biochemistry and Microbiology
5Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
6Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit Medical Campus of Case Western Reserve University School of Medicine, Detroit, Michigan
7Department of Physiology and Biophysics, College of Medicine, University of South Florida, Tampa, Florida
ABSTRACT
NKCC1 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 (–/–) 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+ transporter expression. Thus we performed balance studies, semiquantitative immunoblotting, and immunohistochemistry of kidney tissue from NKCC1 (+/+) and NKCC1 (–/–) 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 (–/–) than NKCC1 (+/+) 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+ concentration with inappropriately low urinary K+ 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+ 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.
Na+ transporters; chloride; water; renin; aldosterone
NKCC1 (BSC-2) is a ubiquitous Na+-K+-2Cl– cotransporter (9), which serves a number of cellular functions such as cell volume regulation, while in polarized epithelia it participates in Cl– secretion (8, 9, 25, 27). NKCC1 is highly expressed in the salivary gland and inner ear where it contributes to the production of saliva (8) and to the formation of endolymphatic fluid (5).
In the kidney, NKCC1 localizes to the juxtaglomerular afferent arteriole as well as the glomerular and extraglomerular mesangium (10, 13), where it is thought to participate in the process of tubuloglomerular feedback and the regulation of blood pressure (13). In the mouse, NKCC1 is most highly expressed along the basolateral membrane of the terminal inner medullary collecting duct (13). In rat NKCC1 localizes to the basolateral regions of the type A intercalated cell of the outer medullary collecting duct (OMCD), a H+-secreting cell type (10). However, in the rat OMCD NKCC1 does not participate in net H+ secretion (25) but rather contributes to transepithelial transport of Na+ and Cl– (25, 27). NKCC1 may also participate in the process of fluid secretion along the collecting duct during physiological and pathophysiological states, such as with cyst formation in polycystic kidney disease (15, 30). Thus genetic disruption of NKCC1 would be predicted to reduce Cl– secretion along the collecting duct, leading to volume expansion and possibly hypertension. However, hypotension rather than hypertension is observed in NKCC1 null mice (9, 19), which suggests a renal defect in NaCl and water handling.
The purpose of the present study was to determine whether NKCC1 (–/–) mice have a defect in the regulation of NaCl and water excretion that might contribute to the observed hypotension.
METHODS
Animals
Series 1. NKCC1 null mice developed by Flagella et al. (9) were studied. Heterozygous offspring were used to establish breeding colonies in the UTHSC and Emory vivaria. Age- and sex-matched NKCC1 (–/–) and (+/+) littermates, weighing 20–40 g, were pair fed a balanced, NaCl-deficient diet (1.78 meq Na+/kg food, <1.13 meq Cl–/kg food, no. 53140000, Zeigler Brothers, Gardners, PA) for 7 days before death, prepared as a gel (0.01% NaCl, low-NaCl diet). Thus mice received 9 ml H2O/day and <0.01 meq/day NaCl. In some experiments, the gelled diet described above was supplemented with NaCl. In this protocol, mice received 9 ml H2O/day and 1.40 meq NaCl/day (2.8% NaCl, high-NaCl diet). Thus the high- and low-NaCl diets differed only in NaCl intake. Mice had no access to water other than that given in the gel. Mice were placed in metabolic cages, and urine was collected at 4°C under oil for 24 h before death under anesthesia with isofluorane (24). When measuring blood pressure through an arterial catheter, mice were anesthetized with ketamine (50 mg/kg) and Inactin (100 mg/kg) as separate intraperitoneal injections. Changes in urinary osmolality following vasopressin administration were measured using the protocol of Takahashi et al. (22). Mice ate rodent chow (Harlan Teklad LM-485) containing 139 meq Na+/kg food (0.8% NaCl) and water containing 5% dextrose ad libitum for 3 days. On day 4, urinary osmolality was measured before and 2 h after administration of 1 ng/g body wt intraperitoneal vasopressin (desmopressin, dDAVP) (22). Mice invariably voided their urine when handled. Thus mice were handled at 1 and 2 h after the administration of dDAVP to induce voiding of urine that had accumulated during the first and second hours following administration of the drug. Urine was voided onto a piece of parafilm and then immediately collected with a pipette tip for measurement of osmolality. The Institutional Animal Care and Use Committee at Emory, the University of Cincinnati, and UTHSC approved all animal treatment protocols.
Measurement of Blood Pressure, Serum and Urine Chemistries, Arterial Blood Gases, and Glomerular Filtration Rate
Systolic blood pressure in conscious mice was measured by tail cuff or femoral artery catheter as described previously (16, 24). Blood was collected for serum chemistries through the abdominal aorta under isofluorane anesthesia (24). Unless noted, urine and serum chemistries were measured at IDEXX Laboratories (West Sacramento, CA) (24). Arterial blood gases, serum osmolality, urine osmolality, and urinary total ammonia concentration were measured as described previously (24, 29). Urine Na+ and Cl– concentration from mice on a low-salt diet as well as plasma renin concentration and serum aldosterone concentration were measured as described previously (17, 29). Plasma atrial natriuretic peptide (ANP) concentration was measured as reported previously with one measurement per mouse (6). Osmolar clearance (Cosm) was calculated as Uosm/Sosm x V, where Uosm and Sosm are urine and serum osmolality, respectively, and where V is urinary flow rate (in ml/min). CH2O was calculated as Cosm – V. Glomerular filtration rate (GFR) was measured using FITC-inulin, as described previously (16), after 7 days of the NaCl-deficient diet. Urinary NO was measured using a kit (Nitrate/Nitrite Colorimetric Assay Kit, Cayman Chemical, Ann Arbor, MI).
Mouse Kidney Lysates and Immunoblotting
Preparation of kidney lysates and immunoblotting were performed as reported previously (26). Equal total protein in each lane was confirmed with gels run in parallel stained with Coomassie blue dye. Blots were probed with affinity-purified, peptide-directed rabbit polyclonal antibodies that specifically recognize the major renal Na+ and water transporters (NHE3, NKCC2, the -, -, and -subunits of ENaC and the thiazide-sensitive cotransporter, TSC/NCC, AQP1, AQP2) (1, 7, 23) and a mouse monoclonal anti-Na-K-ATPase, -subunit antibody (Upstate Biotechnology, Lake Placid, NY) (26). Donkey anti-rabbit IgG conjugated to horseradish peroxidase (31458, Pierce, Rockford, IL) or sheep anti-mouse IgG conjugated with horseradish peroxidase (Amersham-Pharmacia Biotech, Piscataway, NJ) was used as secondary antibodies. Antibody-antigen reactions were visualized using enhanced chemiluminescence before exposure of X-ray film (26). Band densities were quantified (26) and normalized to the mean band density of lysates from wild-type mice measured in parallel in the same gel.
Immunohistochemistry
Mice were anesthetized with isofluorane and the kidneys were fixed in situ as reported previously (28). Immunoperoxidase labeling of 2-μm sections of paraffin-embedded tissue was performed using a postembedding method described previously (14). AQP2 abundance and distribution were determined using a rabbit polyclonal antibody described previously (7, 23).
Statistical Analysis
For data without a normal distribution or equal variance, a Mann-Whitney rank sum test was used. In all other studies, comparisons were made between two groups using an unpaired Student's t-test. P < 0.05 indicates statistical significance. Data are means ± SE.
RESULTS
NKCC1 (–/–) Mice Are Constitutively Hypotensive
After 7 days of a high-NaCl diet, blood pressure was lower in NKCC1 (–/–) than NKCC1 (+/+) mice (P < 0.05), when measured either in conscious mice by tail cuff (Fig. 1) or in anesthetized mice by femoral artery catheter (Table 1). Hypotension persisted unchanged after 7 days of either a NaCl-deficient or a high-NaCl diet (Fig. 1). We conclude that NKCC1 (–/–) mice are hypotensive independent of NaCl intake, consistent with previous observations (20).
NKCC1 (–/–) Mice Have a Higher Plasma Renin Concentration Than Wild-Type Mice, Although Their Circulating Aldosterone Is Inappropriately Low During Some Treatment Conditions
Normal kidneys respond to hypotension, and thus provide a corrective response, by increasing the release of renin, ANG II, and aldosterone which reduce NaCl excretion. Therefore, if NKCC1 null mice have low blood pressure and normal renal function, renal NaCl excretion should decrease at least transiently, thereby increasing vascular volume, which restores blood pressure to normal. Thus we asked whether the hypotension observed in NKCC1 null mice occurs from inappropriately reduced circulating levels of renin and/or aldosterone. On a high-salt diet, plasma renin concentration was fourfold higher in NKCC1 knockout mice than in wild-type mice, although differences between wild-type and knockout mice were not detected with a low-NaCl diet (Table 2). Thus plasma renin concentration is higher in NKCC1 null than wild-type mice, as expected given the hypotension observed in these mutant mice.
Because plasma renin was higher in NKCC1 null than in wild-type mice, we asked whether NKCC1 null mice excrete more Cl– than wild-type mice, which makes their Cl– balance more negative thus increasing plasma renin concentration. Therefore, cumulative urinary Cl– excretion was measured over the first 72 h following the transition from a low- to a high-NaCl diet (Fig. 2). As shown, NKCC1 null mice did not excrete more Cl– over this time period than pair-fed wild-type mice. Thus the elevated plasma renin concentrations observed in NKCC1 null mice do not occur because they excrete more Cl– than pair-fed wild-type mice.
High serum K+ and plasma renin concentrations, as observed in NKCC1 null mice on a high-salt diet, should markedly increase circulating aldosterone. However, on a high-NaCl diet, serum aldosterone was not higher in NKCC1 null than in wild-type mice (Table 2). Thus serum aldosterone is inappropriately low in NKCC1 null mice ingesting a high-NaCl diet.
After ingestion of the low-NaCl diet, the increased serum K+ and aldosterone concentration observed in NKCC1 null mice should produce a kaliuresis. Because NKCC1 null mice did not excrete more K+ than wild-type mice, renal K+ excretion is inappropriately low in these mutant mice (Table 2). These data demonstrate that NKCC1 null mice have defects in renal Na+ and K+ handling.
Hypotension in NKCC1 Null Mice Does Not Occur from an Increase in Nitric Oxide or Atrial Natriuretic Peptide
Nitric oxide (NO) and ANP relax vascular smooth muscle and promote diuresis and natriuresis (21, 32). Thus increased synthesis and/or release of NO or ANP should reduce blood pressure. However, the hypotension observed in NKCC1 null mice does not occur from increased NO synthesis, as NO excretion was the same as in wild-type mice (Table 2). Moreover, on either a high- or a low-NaCl diet, plasma ANP concentration was lower in NKCC1 null than in wild-type mice (Table 2). Thus the hypotension observed in NKCC1 null mice does not occur due to increased synthesis and/or release of NO or ANP. Furthermore, the inappropriately low serum aldosterone concentration observed in NKCC1 null mice eating a high-NaCl diet does not occur from increased ANP release.
Na+ Transporter Expression Is Upregulated in NKCC1 (–/–) Mice
We asked whether the hypotension observed in NKCC1 null mice occurs in tandem with downregulation of one or more of the renal Na+ transporters or water channels that micropuncture and isolated, perfused tubule experiments have shown mediate Na+ and water absorption in specific nephron segments of the kidney (1). On a high-NaCl diet, expression of the thiazide-sensitive cotransporter of the DCT (NCC) was greater in NKCC1 (–/–) than NKCC1 (+/+) mice, whereas expression of the other Na+ transporters was the same in kidneys from NKCC1 null and wild-type mice (Fig. 3). However, on a low-NaCl diet, expression of the proximal Na+/H+ exchanger (NHE3), the thick ascending limb Na+-K+-2Cl– cotransporter (NKCC2), the -subunit of the Na-K-ATPase and NCC were all increased in NKCC1 null relative to wild-type mice, although expression of the three subunits of ENaC was unchanged (Fig. 4). Thus the hypotension observed in NKCC1 null mice does not occur from inappropriately reduced expression of a major transport mechanism of renal Na+ absorption. Instead, increased renal Na+ transporter expression is likely a compensatory response to the low blood pressure and high plasma renin concentration observed in these mutant mice.
NKCC1 (–/–) Mice Are Hypochloremic and Vasopressin Resistant
Table 2 shows that NKCC1 (–/–) mice consuming a low-NaCl diet develop hypochloremia. This hypochloremia was accompanied by reduced serum osmolality and was not explained by increased serum HCO3–. Despite the hypochloremia, urinary volume was lower in NKCC1 null than in (+/+) mice, although excretion of chloride and total osmoles was the same. Thus free water excretion did not respond appropriately to the hypochloremia observed in NKCC1 null mice.
We asked whether the hypochloremia and reduced urinary volume are due to increased water absorption by the kidney or reduced GFR. Because NKCC1 null mice have reduced urinary volume and are hypochloremic, we asked whether AQP1 or 2 is upregulated in kidney from NKCC1 (–/–) mice either through increased protein expression or through selective redistribution to the apical plasma membrane, which might lead to increased water absorption. As shown in Figs. 3 and 4, AQP1 and 2 protein expression was the same in wild-type and NKCC1 null mice. However, we observed that AQP2 is redistributed to the basal regions of principal cells from NKCC1 null mice (Fig. 5).
Because AQP2 protein is redistributed within principal cells of NKCC1 null mice, we asked whether these mice are vasopressin resistant. Thus changes in urinary osmolality following the administration of vasopressin were measured in NKCC1 null and wild-type mice. Baseline urinary osmolality was the same in NKCC1 null and wild-type mice (696 ± 187 mosmol/kgH2O in wild-type mice, n = 9, vs. 392 ± 81 mosmol/kgH2O in NKCC1 null mice, n = 8, P = not significant). Two hours following the administration of dDAVP, urinary osmolality was lower in NKCC1 null than wild-type mice (1,866 ± 203 mosmol/kgH2O in wild-type mice, n = 9, vs. 1,103 ± 152 mosmol/kgH2O in NKCC1 null mice, n = 8, P < 0.05). Thus NKCC1 null mice have a blunted increase in urinary osmolality following the administration of vasopressin, which may reflect the subcellular redistribution of AQP2 observed within principal cells. This vasopressin resistance should increase free water excretion, thus limiting the hypochloremia observed in NKCC1 null mice.
Because kidney size is smaller in NKCC1 null than in wild-type mice (Figs. 2 and 3), reduced GFR is expected in NKCC1 knockout mice, although this was not demonstrated statistically (Table 1). Thus we cannot exclude the possibility that the lower renal mass of NKCC1 (–/–) mice attenuates their ability to excrete free water.
DISCUSSION
Whether NKCC1 modulates blood pressure through vascular and/or renal effects is unknown. NKCC1 is expressed in vascular smooth muscle where it modulates vascular contractility, likely through changes in intracellular calcium generated by NKCC1-mediated Cl– uptake (3). In vascular smooth muscle, NKCC1 is upregulated by aldosterone, which contributes to the heightened vascular tone and hypertension observed in this treatment model (12). Because vascular tone is reduced in NKCC1 knockout mice (19), NKCC1-dependent changes in blood pressure are thought to be primarily vascular rather than renal events (19).
The role of the kidney in NKCC1-mediated changes in blood pressure has been unexplored. This study demonstrates that in addition to vascular changes, NKCC1 null mice have diminished aldosterone release from the adrenal cortex as well as defects within the kidney which blunt the renal response to vasopressin and aldosterone. The result is abnormal regulation of Na+, K+, and water excretion.
Within the kidney, NKCC1 is expressed in the afferent arteriole and glomerular and extraglomerular mesangium and may be involved in the sensing of luminal Cl– concentration, which ultimately leads to changes in afferent arteriolar tone (TGF) (13). Because NKCC1 is present in cells responsible for the synthesis of renin within the afferent arteriole, the hypotension observed in NKCC1 knockout mice might result from failure of the kidney to release renin on demand. However, the present study shows increased plasma renin concentration in NKCC1 null mice on a NaCl-replete diet and demonstrates increased expression of ANG II-sensitive Na+ transporters such as the TSC/NCC. Thus the hypotension observed in NKCC1 knockout mice does not occur from a defect in the release of renin or from an inability of renal Na+ transporter expression to respond to the expected increase in ANG II. Moreover, these results are consistent with the recent observation that NKCC1 suppresses the release of renin (2), which therefore explains the marked elevation in plasma renin observed in NKCC1 null mice. Instead, the hypotension observed may occur from an altered TGF set point (13), which increases NaCl delivery to the distal convoluted tubule and transiently increases GFR. NaCl loss would ensue, which lowers blood pressure and returns GFR and distal delivery of NaCl to normal. However, in the transition from a NaCl-deficient to a high-NaCl diet, NKCC1 null mice did not excrete more Cl– than wild-type mice. Alternatively, the hypotension observed likely reflects, at least in part, the lack of NKCC1-mediated vasoconstriction. Increased Na+ transporter expression may therefore be a compensatory mechanism for reduced vasoconstriction. Whether NKCC1 null mice have reduced renal vascular resistance remains to be determined.
NKCC1 null mice are hyperkalemic independent of NaCl intake, which might occur through a renal or a renal-independent mechanism. During K+ stress, a rise in serum K+ is buffered through K+ uptake in skeletal muscle mediated by the Na-K-ATPase (18) and to some extent by NKCC1 (31). Thus in the absence of NKCC1-mediated K+ uptake in skeletal muscle, the kidney should respond by increasing renal K+ excretion. However, renal excretion of K+ was not increased in NKCC1 null mice. Thus in NKCC1 null mice, renal K+ excretion does not respond appropriately to the increase in serum aldosterone and K+ concentration. The absence of NKCC1-mediated Cl– secretion may attenuate secretion of K+ along the collecting duct. Why NKCC1 null mice have inappropriately low renal K+ excretion is not apparent and will require further studies in native tissue.
NKCC1 null mice have reduced water excretion by both the kidney and the salivary gland (8). NKCC1 participates in secretion of isotonic fluid by the salivary acinar cells (8). Downstream, NaCl absorption occurs within the salivary duct cells, which results in the production of hypotonic saliva (8). Thus NKCC1 null mice have a reduced ability to produce hypotonic saliva (8). When consuming a NaCl-deficient diet with high water intake, NKCC1 null mice retain free water in excess of NaCl, which results in hypochloremia. If NKCC1 functions in the kidney through a process analogous to the salivary gland, then it should participate in the generation of hypotonic urine and the renal excretion of free water. Testing this hypothesis will require further studies in isolated tubules perfused in vitro.
Vasopressin resistance is likely a compensatory mechanism which attenuates the hypochloremia observed in NKCC1 null mice ingesting the low-NaCl diet. Whether vasopressin resistance results from the AQP2 redistribution observed within cortical collecting duct (CCD) principal cells of NKCC1 null mice is unclear. Christensen et al. (4) reported that acute administration of V2 receptor antagonists leads to redistribution of AQP2 to the basolateral membrane of rat principal cells within the CCD in tandem with increased water excretion. However, redistribution of AQP2 to the basolateral membrane of principal cells has also been reported in rat models associated with reduced free water excretion (4, 11). Whether targeting of AQP2 to the basolateral membrane, as observed in NKCC1 null mice, promotes or inhibits the absorption of water requires further study.
In conclusion, the hypotension observed in NKCC1 null mice persists unchanged when NaCl intake is varied. NKCC1 is critical to the release and/or action of vasopressin, renin, and aldosterone and the regulation of renal Na+, K+, Cl– and water excretion. The action of vasopressin as well as the action and release of aldosterone are attenuated in kidneys from NKCC1 null mice, which likely contributes to their hypotension. Increased Na+ transporter expression, increased plasma renin and reduced plasma ANP, as observed in NKCC1 null mice, should increase vascular volume and blood pressure, thereby limiting the hypotension.
GRANTS
This study was supported by National Institutes of Health Grants DK-46493 (to S. M. Wall), DK-50594 (to G. E. Shull), DK-57552 (to J. N. Lorenz), and Z01-HL-01282-KE (to M. A. Knepper).
ACKNOWLEDGMENTS
We thank Dr. James E. Melvin (University of Rochester) for generously supplying NKCC1 (+/–) mice following Tropical Storm Allison.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Brooks HL, Allred AJ, Beutler KT, Coffman TM, and Knepper MA. Targeted proteomic profiling of renal Na+ transporter and channel abundances in angiotensin II type 1a receptor knockout mice. Hypertension 39: 470–473, 2002.
Castrop H, Lorenz JN, Hansen P, Friis U, Mizel D, Oppermann M, Jensen B, Briggs JP, Skott O, and Schnermann J. Contribution of the basolateral isoform of the Na-K,2Cl-cotransporter (NKCC1/BSC2) to renin secretion. Am J Physiol Renal Physiol 289: F1185–F1192, 2005.
Chipperfield AR and Harper AA. Chloride in smooth muscle. Prog Biophys Mol Biol 74: 175–221, 2000.
Christensen BM, Wang W, Frokiaer J, and Nielsen S. Axial hetergeneity in basolateral AQP2 localization in rat kidney: effect of vasopressin. Am J Physiol Renal Physiol 284: F701–F717, 2003.
Delpire E, Lu J, England R, Dull C, and Thorne T. Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl cotransporter. Nat Genet 22: 192–195, 1999.
Dietz JR and Nazian SJ. Release of atrial natriuretic factor in hypophysectomized rats. Am J Physiol Regul Integr Comp Physiol 255: R534–R538, 1988.
DiGiovanni SR, Nielsen S, Christensen EI, and Knepper MA. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc Natl Acad Sci USA 91: 8984–8988, 1994.
Evans RL, Park K, Turner RJ, Watson GE, Nguyen HV, Dennett MR, Hand AR, Flagella M, Shull GE, and Melvin JE. Severe impairment of salivation in Na+-K+-2Cl– cotransporter (NKCC1)-deficient mice. J Biol Chem 275: 26720–26726, 2000.
Flagella M, Clarke LL, Miller ML, Erway LC, Giannella RA, Andringa A, Gawenis LR, Kramer J, Duffy JJ, Doetschman T, Lorenz JN, Yamoah EN, Cardell EL, and Shull GE. Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf. J Biol Chem 274: 26946–26955, 1999.
Ginns SM, Knepper MA, Ecelbarger CA, Terris J, He X, Coleman RA, and Wade JB. Immunolocalization of the secretory isoform of the Na-K-Cl cotransporter in rat renal intercalated cells. J Am Soc Nephrol 7: 2533–2542, 1996.
Jeon US, Joo KW, Na KY, Kim YS, Lee JS, Kim J, Kim GH, Nielsen S, Knepper MA, and Han JS. Oxytocin induces apical and basolateral redistribution of aquaporin-2 in rat kidney. Nephron Exp Nephrol 93: e36–e45, 2003.
Jiang G, Cobbs S, Klein JD, and O'Neill WC. Aldosterone regulates the Na-K-2Cl cotransporter in vascular smooth muscle. Hypertension 41: 1131–1135, 2003.
Kaplan MR, Plotkin MD, Brown D, Hebert SC, and Delpire E. Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium and the glomerular afferent arteriole. J Clin Invest 98: 723–730, 1996.
Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frokiaer J, and Nielsen S. Altered expression of renal acid-base transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol 285: F1244–F1257, 2003.
Lebeau C, Hanaoka K, Moore-Hoon ML, Guggino WB, Beauwens R, and Devuyst O. Basolateral chloride transporters in autosomal dominant polycystic kidney disease. Pflügers Arch 444: 722–731, 2002.
Lorenz JN and Gruenstein E. A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin. Am J Physiol Renal Physiol 276: F172–F177, 1999.
Lum C, Shesely EG, Potter DL, and Beierwaltes WH. Cardiovascular and renal phenotype in mice with one or two renin genes. Hypertension 43: 79–86, 2004.
McDonough AA, Thompson CB, and Youn JH. Skeletal muscle regulates extracellular potassium. Am J Physiol Renal Physiol 282: F967–F974, 2002.
Meyer JW, Flagella M, Sutliff RL, Lorenz JN, Nieman ML, Weber CS, Paul RJ, and Shull GE. Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na+-K+-2Cl– cotransporter. Am J Physiol Heart Circ Physiol 283: H1846–H1855, 2002.
Pace AJ, Lee E, Athirakul K, Coffman TM, O'Brien DA, and Koller BH. Failure of spermatogenesis in mouse lines deficient in the Na+-K+-2Cl– cotransporter. J Clin Invest 105: 441–450, 2000.
Sabrane K, Kruse MN, Fabritz L, Zetsche B, Mitko D, Skryabin BV, Zwiener M, Baba HA, Yanagisawa M, and Kuhn M. Vascular endothelium is critically involved in the hypotensive and hypovolemic actions of atrial natriuretic peptide. J Clin Invest 115: 1666–1674, 2005.
Takahashi N, Chernavvsky DR, Gomez RA, Igarashi P, Gitelman HJ, and Smithies O. Uncompensated polyuria in a mouse model of Bartter's syndrome. Proc Natl Acad Sci USA 97: 5434–5439, 2000.
Terris J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414–F422, 1996.
Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett LA, Green ED, and Wall SM. Deoxycorticosterone upregulates Pds (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003.
Wall SM and Fischer MP. Contribution of the Na+-K+-2Cl– cotransporter (NKCC1) to transepithelial transport of H+, NH4+, K+ and Na+ in rat outer medullary collecting duct. J Am Soc Nephrol 13: 827–835, 2002.
Wall SM, Fischer MP, Kim GH, Nguyen BM, and Hassell KA. In rat inner medullary collecting duct, NH4+ uptake by the Na-K-ATPase is increased during hypokalemia. Am J Physiol Renal Physiol 282: F91–F102, 2002.
Wall SM, Fischer MP, Mehta P, Hassell KA, and Park SJ. Contribution of the Na+-K+-2Cl– cotransporter (NKCC1) to Cl– secretion in rat outer medullary collecting duct. Am J Physiol Renal Physiol 280: F913–F921, 2001.
Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, and Verlander JW. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284: F229–F241, 2003.
Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, and Verlander JW. NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl– conservation. Hypertension 44: 1–6, 2004.
Wallace DP, Rome LA, Sullivan LP, and Grantham JJ. cAMP-dependent fluid secretion in rat inner medullary collecting ducts. Am J Physiol Renal Physiol 280: F1019–F1029, 2001.
Wong JA, Fu L, Schneider EG, and Thomason DB. Molecular and functional evidence for Na+-K+-2Cl– cotransporter expression in rat skeletal muscle. Am J Physiol Regul Integr Comp Physiol 277: R154–R161, 1999.
Zhou MS, Schulman IH, and Raij L. Nitric oxide, angiotensin II and hypertension. Semin Nephrol 24: 366–378, 2004.(Susan M. Wall, Mark A. Knepper, Kathryn )