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J Am Soc Nephrol 13:604-610, 2002
© 2002 American Society of Nephrology

Posttranscriptional Compensation for Heterozygous Disruption of the Kidney-Specific NaK2Cl Cotransporter Gene

Nobuyuki Takahashi*, Heddwen L. Brooks{dagger}, James B. Wade{ddagger}, Wen Liu§, Yoshiaki Kondo§, Sadayoshi Ito|, Mark A. Knepper{dagger} and Oliver Smithies*

*Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; {dagger}National Heart Lung and Blood Institute, National Institute of Health, Bethesda, Maryland; {ddagger}Department of Physiology, University of Maryland, Baltimore, Maryland; §Department of Pediatrics and |Department of Nephrology, Hypertension and Endocrinology, Tohoku University Graduate School of Medicine, Sendai, Japan.

Correspondence to: Dr. Oliver Smithies, Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, 701 Brinkhous-Bullitt Building, Chapel Hill, NC 27599-7525. Phone: 919-966-6912; Fax: 919-966-8800; E-mail: ntakaha{at}med.unc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Mice homozygous for a loss of function mutation of the kidney-specific NaK2Cl cotransporter, BSC1/NKCC2, do not survive. Here the effects of loss of one copy of the gene are studied. NKCC2 mRNA of NKCC2 +/- kidney was 55 ± 6% of +/+, yet no differences were found between NKCC2 +/+ and +/- mice in BP, blood gas, electrolytes, creatinine, plasma renin concentration, urine volume and osmolality, ability to concentrate and dilute urine, and response to furosemide. When mice were challenged with 180 mM NH4Cl, plasma ammonia and urinary ammonia excretion were increased twofold and fivefold, respectively, but there was still no difference between the two genotypes. NKCC2 +/- mice had a near-normal level of NKCC2 protein and no clear change in the distribution of NKCC2 in the thick ascending limb (TAL) cells. In vitro microperfusion of isolated TAL showed no significant difference between the two genotypes in the basal and vasopressin-stimulated capacity to reabsorb NaCl. There was no difference in the mRNA expressions of thiazide-sensitive NaCl cotransporter, epithelial Na channel (ENaC), aquaporin-2, ROMK, and NaKATPase. Halving the mRNA expression of NKCC2 does not affect BP or fluid balance because of compensatory factors that restore the protein level to near normal. One possible factor is a regulated increase in the movement of cytoplasmic protein to the luminal membrane leading to a restoration of functional transporter to an essentially wild type level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The kidney-specific NaK2Cl cotransporter, NKCC2/BSC1, is expressed in the luminal membrane of the thick ascending limb (TAL) of Henle’s loop and the macula densa (1–4). In the TAL, NaCl is actively reabsorbed from the luminal fluid (5). The reabsorbed NaCl accumulates in the renal medulla, where the countercurrent mechanism generates a hypertonic state. In turn, the hypertonicity of the renal medulla stimulates passive water reabsorption in the collecting duct and the thin descending limb, leading to the production of concentrated urine. The macula densa comprises a specialized form of TAL cells that are capable of sensing Cl- concentration in the adjacent luminal fluid and of controlling renin release and GFR (tubuloglomerular feedback) (6).

The loss of function mutation of NKCC2 is responsible for Bartter syndrome (7), characterized by polyuria, hypokalemic metabolic alkalosis, high plasma renin and aldosterone, and hypotension (8). We recently generated a mouse model of this syndrome by disrupting the NKCC2 gene (9). Mice homozygous for this loss of function mutation of NKCC2 do not survive (9). NKCC2 inhibitors, furosemide and bumetanide, are used to treat patients with hypertension. We therefore expected that NKCC2 heterozygotes would have half the amount of the gene product, and consequently have a less than normal BP. Current data from the families of Bartter syndrome patients are inconclusive in this respect, because of the heterogeneous genetic background and environment of human populations. Mouse models generated by gene targeting, on the other hand, allow tests of the effects of mild quantitative changes in the expression of genes in a constant environment and without the problems of genetic heterogeneity. The aim of this study is to clarify whether the loss of one copy of the NKCC2 gene would affect their phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Mice lacking NKCC2 were maintained on a 129SvEv inbred background (9). Male mice 3 mo of age were used except for in vitro microperfusion experiments. Mice were housed in standard cages and allowed free access to 0.4% NaCl chow and water except where indicated, and handled in accordance with the National Institute of Health guidelines for the use and care of experimental animals.

Analyses of Blood, Plasma Renin Concentration, and BP Measurement
Blood and plasma renin concentration were analyzed as described previously (9). BP was measured by using a computerized tail cuff method (10).

Ability to Concentrate and Dilute Urine
The ability to concentrate and dilute urine was analyzed as described previously (11,12). Briefly, the mice received an acute water load equivalent to 4% body wt into the stomach by gavage. Urine osmolality was measured before and 1 and 2 h after water challenge. Additional food and water were withheld after administration of water until the end of the experiment. Minimum osmolality of urine was used as a measure of diluting ability. The ability to concentrate urine was assessed by measuring osmolality of urine after 14 h of water deprivation.

Furosemide Protocol
Mice were given drinking water containing 0, 80, 160, and 320 µg/ml furosemide for 2 wk and then housed in metabolic cages for 3 consecutive days. Furosemide intake was calculated from water intake and plotted against urine volume.

Ammonium Chloride Protocol
The protocol is based with minor modifications on previous experiments with rats (13). We first gave our mice 50 mM NH4Cl as drinking water. This is the standard dose for rats and increases their ammonia excretion fourfold (13). We found that 50 mM NH4Cl increased urinary excretion of NH4Cl only about 1.5-fold in mice. We therefore gave the mice drinking water containing 180 mM NH4Cl for 2 wk and again housed them in metabolic cages for 3 consecutive days. Urine volume and ammonia excretion were normalized to 20 g body wt.

Quantification of mRNA Expression
Gene expression was quantified with the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The primers are 5'-AGG CTC TGT CCT ATG TGA GT-3' and 5'-CAT GGG TCC GCC TGT TAA G-3' for Slc12a1 (NKCC2), 5'-GGC TGG CTC CTA CAA TCT G-3' and 5'-GGG GCG GTA GTT CTT GAT G-3' for Slc12a3 (NaCl cotransporter, NCC), 5'-AGC GCG TCT TCC AGT GTA C-3' and 5'-GAT TTG TTC TGG TTG CAC AGT-3' for Scnn1a ({alpha}subunit of epithelial Na channel, {alpha}ENaC), 5'-CAA CAG CAG CAA CCC GGC-3' and 5'-CTG GTG AAG TTC CGC AAG G-3' for Scnn1b (ßENaC), 5'-CAC TGG TCG GAA GCG GAA A-3' and 5'-GCA CAG TCA GAG GTG TCA TT-3' for Scnn1g ({gamma}ENaC), 5'-TAC GTG GCT GCC CAG CTG-3' and 5'-GGC TGT TGC ATT GTT GTG GA-3' for Aqp2 (aquaporin-2), 5'-GCA CAG TAG AAT CCA CCA GT-3' and 5'-AAA GCA CCT CTT CTG GGA TG-3' for Kcnj1 (ROMK K channel), 5'-GAG ATG AGG GAG AAA TAG AG-3' and 5'-CTC AGA TGC ATT TGG GTT CT-3' for Atp1a1 (NaKATPase {alpha}1 subunit), and 5'-GGA CGA CAT GAT TTT CGA GG-3' and 5'-CTC TCC TCG TTC GTG ATT GA-3' for Atp1a2 (NaKATPase ß1 subunit). Probes are 5'-FAM-TAG ACA ACG CTC TGG AAT TAA CCA CAG-TAMRA-3' for Slc12a1, 5'-FAM-TCG TTG AGG CCC ACG GAG TAG CTC A-TAMRA-3' for Slc12a3, 5'-FAM-CAA CAA TCC CCA AGT GGA CAG GAA GG-TAMRA-3' for Scnn1a, 5'-FAM-AGT TCC ATT GGC ACT GCA CAG CCT C-TAMRA-3' for Scnn1b, 5'-FAM-ACA AGG CTT CTA ATG TCA TGC ACG TTC-TAMRA-3' for Scnn1g, 5'-FAM-CAT GAG ATT ACC CCT GTA GAA ATC CGC-TAMRA-3' for Aqp2, 5'-FAM-CAA CCT GCC AAG TCC GCA CAT CA-TAMRA-3' for Kcnj1, 5'-FAM-AGA TTC CCT TCA ACT CCA CCA ACA AG-TAMRA-3'for Atp1a1, and 5'-FAM-TGT GGC AAT GTT CCC AGT GAA CCC A-TAMRA-3'for Atp1a2. The amount of kidney total RNA for each reaction was adjusted within the range 0.05 to 0.2 µg depending on the gene to ensure that gene expression was within the range of linear correlation between the log (amount of total RNA) and threshold cycle number. Relative levels of gene expression, expressed as a percentage of wild type, were determined by the dCt method using ß-actin as an internal standard. The primers and probe for ß-actin were previously described (14).

Antibodies
Rabbit polyclonal antibodies to the following renal transporters and channels were used: NKCC2 (15), NCC (16), all three subunits of ENaC (17), ROMK (18), the type 2 Na-phosphate cotransporter (NaPi-2, NPT2) (19), the type 3 Na-H exchanger (NHE3) (20), AQP1 (21), and the vasopressin-regulated water channels, AQP2 (22) and AQP3 (23). The antisera were affinity-purified against the immunizing peptides as described previously (15,16). We also used a mouse monoclonal antibody against the NaKATPase {alpha}1 subunit (product number 05–369, Upstate Biotechnology, Lake Placid, NY).

Semiquantitative Immunoblotting
Semiquantitative immunoblotting was used to compare protein abundance as described previously (15,21,24). The left kidneys were homogenized intact. For each set of samples (NKCC2 +/- versus +/+), after solubilization in Laemli sample buffer, an initial gel was stained with Coomassie blue as described previously (25) to confirm equal loading among samples. For immunoblotting, sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed on 7.5%, 10%, or 12% polyacrylamide gels (Ready Gels, Bio-Rad, Hercules, CA), and the proteins were transferred from the gel electrophoretically to nitrocellulose membranes. Membranes were probed overnight at 4°C with the respective primary antibodies and then exposed to secondary antibody (goat anti-rabbit IgG conjugated with horseradish peroxidase, Pierce no. 31463, diluted to 1:5000) for 1 hr at room temperature. Sites of antibody-antigen reaction were visualized by using a luminol-based enhanced chemiluminescence substrate (LumiGLO, Kirkegaard and Perry Laboratories, Gaithersburg, MD) before exposure to x-ray film (165–1579; Eastman Kodak, Rochester, NY). The band densities were quantified by laser densitometry (PDS1-P90; Amersham Biosciences, Sunnyvale, CA).

Fixation of Tissue and Immunocytochemistry
Kidneys were fixed by perfusion with 2% paraformaldehyde, and each protein was immunolocalized on frozen sections as described previously (26). Sections were incubated overnight at 4°C with primary antibodies diluted to 10 µg/ml. Secondary antibodies were donkey anti-rabbit antibodies (Jackson Immunoresearch Labs, West Grove, PA) coupled with Alexa 488 (Molecular Probes, Eugene, OR). Sections were examined with a Zeiss LSM410 confocal microscope (Carl Zeiss, Thornwood, NY).

In Vitro Microperfusion
In vitro microperfusion experiments using isolated tubules were executed as described previously (27). Medullary thick ascending limbs were isolated from the inner stripe of outer medulla of male NKCC2 +/- and +/+ mice 5 to 6 wk of age.

Statistical Analyses
All values are expressed as mean ± SEM. t test was used for statistical evaluations. Analysis of covariance was used for the test of furosemide response.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BP, Analyses of Blood, and Urine
We first measured BP, plasma renin concentration (PRC), daily urine volume, and osmolality in the NKCC2 +/- mice and found them indistinguishable from wild type (Table 1). Blood data (Na+,K+,Cl-, creatinine, urea nitrogen, protein, hemoglobin, hematocrit, and blood cell counts) of the two genotypes were also indistinguishable, as were the concentrations and daily excretion of Na+,K+,Cl-, creatinine, protein, Ca2+, and Mg2+ (data not shown).


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  		Table 1. Blood pressure, renin, and urine Dataa
 
Ability to Concentrate and Dilute Urine
To further investigate the NKCC2 +/- phenotype, we exposed animals to stressed conditions that affect kidney function. NKCC2 is indispensable for removing NaCl from the luminal fluid of the thick ascending limb (5). Consequently, we can test the ability of the kidney to dilute urine, if animals are challenged with water. Likewise, water deprivation will test the ability of the kidney to concentrate urine. Neither of these tests revealed any difference between the NKCC2 +/- and +/+ mice (Table 1).

Response to Furosemide
If the expression level of NKCC2 in heterozygous mice is lower than wild type, they would be expected to be more sensitive to furosemide. However, again there was no significant difference (P = 0.66) between the two genotypes (Figure 1).



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  		Figure 1. Response to furosemide of the NKCC2 +/- and +/+ mice. Daily furosemide intake and urine volume were normalized to 20 g of body wt. Plain and dashed lines represent fitted lines for NKCC2 +/+ and +/- .

 
Ammonium Chloride Challenge
Acid load by NH4Cl enhances ammonium ion accumulation in the renal medulla by counter current multiplication and enhances the ammonium ion gradient along the corticomedullary axis (28). NKCC2 is indispensable for creating this gradient because NH4+ substitutes for K+ in the cotransporter. If NKCC2 +/- mice have an impaired ability to create an ammonium ion gradient, then NH4Cl loading should reveal a difference between NKCC2 +/- and +/+. To test this, we gave the animals 180 mM NH4Cl as drinking water, which increased their ammonia excretion about fivefold. This still did not reveal any significant differences in blood and urine parameters between the two genotypes (Table 2).


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  		Table 2. Blood and urine data from ammonium chloride challengea
 
Quantification of mRNA, Protein, and Immunohistochemistry of NKCC2
We detected no phenotypic differences between the +/- and +/+ mice; we, therefore, investigated the possibility that some unexpected compensation had increased the mRNA of NKCC2 in the heterozygotes. To do this, we assayed NKCC2 mRNA from kidneys of NKCC2 +/- mice by quantitative reverse transcriptase–PCR and found it to be 55 ± 6% of wild type, which is not significantly different from the expected 50% (Figure 2A). By immunoblot, we then tested the hypothesis that kidneys from NKCC2 +/- mice express half of the wild type amount of NKCC2 protein (Figure 2B). Because of the limited linearity between amount of protein and band density, we loaded twice as much protein from the NKCC2 +/- mice as from the controls, allowing the null hypothesis to be tested by direct comparison of band densities. The band densities for the double-loaded NKCC2 +/- mice were significantly greater than those for single-loaded NKCC +/+ mice (P < 0.05) but were not significantly different from twice the +/+ level. Thus, NKCC2 +/- mice do not differ significantly from wild type in their expression of the NKCC2 protein. Moreover, immunohistochemistry shows no detectable difference in the cellular distribution and amount of NKCC2 and ROMK between +/- and +/+ mice (Figure 2C). Note that the immunoreactive NKCC2 is located on the luminal side of the cells not in the cytoplasm. These observations were reproduced in three mice of each genotype. Thus, in the heterozygous mice, some compensation of NKCC2 occurs at the protein level.



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  		Figure 2. Expression of NKCC2 in NKCC2 +/- and +/+ mice. (A) NKCC2 mRNA expression in the kidney of NKCC2 +/- compared with +/+ mice. (n = 7 each). {square}: NKCC2 +/+; {blacksquare}: +/-. *P < 0.0005. (B) NKCC2 protein level. Twice as much protein was loaded for the NKCC2 +/- mice than for the +/+ mice. The relative amount of NKCC2 protein in double-loaded NKCC2 +/- is expressed as a percentage of single-loaded protein of wild type. P < 0.05. (C) Immunohistochemical localization of NKCC2 (top panels) and ROMK (bottom panels) in serial sections from the inner stripe of outer medulla of NKCC2 +/+ and +/- mice. Scale bar, 10 µm.

 
In Vitro Microperfusion
The absence of phenotype combined with the essentially normal expression of NKCC2 protein in the kidney of NKCC2 +/- mice suggests that the compensation could be entirely within the TAL. If this is the case, there should be no difference in the NaCl reabsorption of TAL in vitro. This can be tested by measuring the response of transepithelial voltage (Vt) of the medullary TAL to vasopressin and to various inhibitors of the TAL transport, such as bumetanide or barium (a K channel inhibitor) applied to the lumen of the TAL, as well as the Vt response to ouabain (a NaKATPase inhibitor) or 5-nitro-2-(3-phenylpropylamino)-benzoic acid and NPPB (a Cl channel inhibitor) applied to the basolateral side. We therefore isolated fragments of medullary TAL of the NKCC2 +/- and +/+ mice and studied their function by microperfusing them in vitro at a physiologic rate of 2 nl/min inside the lumen. There was no significant difference in basal and vasopressin-stimulated Vt between the nephrons from two genotypes (Figure 3A). Likewise, the nephrons of the NKCC2 +/+ and +/- mice showed no difference in response to various inhibitors (Figure 3B). These results provide strong evidence that compensation is achieved within the TAL and, by inference, that the induction of changes in the expression of other transporters and channels involved in NaCl transport elsewhere in the nephron is not involved.



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  		Figure 3. In vitro microperfusion of thick ascending limb (TAL) of Henle’s loop. (A) Transepithelial voltages (Vt) of isolated microperfused medullary TAL from the +/+ (n = 22) and NKCC2 +/- (n = 21) mice. (B) Changes in Vt after addition of 100 µM bumetanide, 1 mM barium, 1 mM ouabain, 100 µM 5-nitro-2-(3-phenylpropylamino)-benzoic acid, NPPB (n = 8 to 9, each), or 1 nM vasopressin (n = 12 for +/+ and 13 for +/-). {square}: NKCC2 +/+; {blacksquare}: NKCC2 +/-.

 
Quantification of Other Kidney Genes Involved in Salt and Water Transport
To test the validity of this inference, we looked for any differences in the expression of genes involved in water and NaCl handling in the other parts of the kidney, including thiazide-sensitive NaCl cotransporter (NCC), epithelial Na channel (ENaC), AQP2, ROMK potassium channel, and NaKATPase. As shown in Figure 4, the mRNA expression of none of these genes differed between the +/+ and +/- mice. Moreover, there was no significant change in the protein expression of other kidney genes tested, except for a decrease in ß and {gamma} ENaC expression (Figure 5).



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  		Figure 4. mRNA expression of the genes responsible for salt and water reabsorption in the kidney. Relative mRNA amount of the NKCC2 +/- mice is expressed as a percentage of wild type (n = 7 each). {square}: NKCC2 +/+; {blacksquare}: NKCC2 +/-.

 


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  		Figure 5. Immunoblots of proteins responsible for salt and water reabsorption in the kidney. +/+, wild type; +/-, NKCC2 +/-. The expression of ß and {gamma} ENaC of NKCC2 +/- mice was significantly lower than that of wild type (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that the disruption of one copy of the NKCC2 gene has no direct effect on the physiologic phenotype of the heterozygous mice, in that it does not affect BP, plasma renin concentration, and water and electrolyte handling by the kidney, even though the expression of NKCC2 mRNA in the heterozygotes is only 50% of wild type. Nor do the heterozygotes differ from wild type in their responses to several stressed conditions, such as water challenge, water deprivation, furosemide, or ammonium chloride challenge.

An absence of detectable phenotype in mice heterozygous for gene disruption (+/-) has been reported for several other channels and transporters involved in water and salt transport in the kidney, although as with NKCC2, homozygous loss of function (-/-) causes a severe phenotype. Examples include ENaC, NHE3, AQP1, AQP2, and CLCNK-1 (29–33). Consequently, clarifying the mechanism behind the absence of phenotype in these heterozygous gene disruptions is of general biologic importance, not limited to the NKCC2 gene. A clue to understanding how this can be achieved mechanistically is provided by our observations that the amount of NKCC2 protein expressed in the kidney of NKCC2 +/- mice is not distinguishable from normal, despite the 50% level of NKCC2 mRNA, and that the amount and cellular distribution of NKCC2 protein in NKCC2 +/- mice is likewise indistinguishable from wild type. Consistent with these results is our finding that there were no differences in the basal and vasopressin-stimulated Vt and in the response of Vt to a variety of agents in isolated microperfused TAL fragments from the NKCC2 +/- and +/+ mice. The compensation could conceivably be the result of a change in RNA splicing that produces a shorter protein that functions as NaCl cotransporter and has a dominant negative effect on NaK2Cl cotransporter, the majority of NKCC2 product (34,35). However, this is not likely because (1) we barely detected the short spliced variant by reverse transcriptase–PCR in kidneys of both wild type and NKCC2 +/- mice (our unpublished observation) and (2) there was no difference in the magnitude of Vt response to vasopressin between the NKCC2 +/- and +/+ mice.

Observations by other investigators on NKCC1 are relevant to the development of an understanding of our findings. NKCC1 is another NaK2Cl cotransporter in the same gene family as NKCC2; it is expressed in many tissues, although not in the TAL, and it is thought to be involved in cell volume regulation (36). The experiment using squid giant axons showed that decreases in either intracellular Na+ or Cl- concentrations ([Na+]i or [Cl-]i) increase NaCl uptake via NKCC1 (36,37). We hypothesize a similar situation for NKCC2; namely, that NKCC2 is also regulated by [Na+]i and [Cl-]i in such a way that the number of active NKCC2 cotransporters on the luminal membrane of NKCC2 +/- mice is effectively normal, as is indicated by their normal kidney physiology and immunohistochemistry (Figure 2). This hypothesis coupled with the observations of an essentially normal amount of membrane bound protein in the +/- heterozygotes predicts that the effective half-life of NKCC2 protein in these mice should be close to twice normal. It should be possible to test this prediction by using TAL cell lines from NKCC2 +/- and +/+ mice.

We conclude that mild quantitative changes in the expression of NKCC2 at the mRNA level, which occur in the heterozygous parents of patients with Bartter syndrome and are likely to be observed in human populations as polymorphisms, do not affect the phenotype, probably because of a regulated increase in the movement of NKCC2 protein molecules from the cytoplasm to the plasma membrane.


   Acknowledgments
 
We thank Drs. Thomas M. Coffman, Nobuyo Maeda and Hyung-Suk Kim for discussions and critical reading of the manuscript. The authors gratefully acknowledge the expert technical assistance of Mr. John H. Hagaman, Mr. David Carraway, Ms. Melissa A. Taylor, and Ms. Jie Liu. Our work was supported by the National Institute of Health HL49277 (OS), GM20069 (OS), W. M. Keck Foundation (OS), DK32839 (JBW), Burroughs Wellcome Fund (OS), The Ministry of Education, Science and Culture of Japan (YK and SI), and by The Salt Science Research Foundation in Japan (YK).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication October 1, 2001. Accepted for publication November 14, 2001.




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