ARTICLES

J Am Soc Nephrol 13: 14251432, 2002

Secretory-Defect Distal Renal Tubular Acidosis Is Associated with Transporter Defect in H-ATPase and Anion Exchanger-1
JIN SUK HAN,* GHEUN-HO KIM, JIN KIM, UN SIL JEON, KWON WOOK JOO, KI YOUNG NA,* CURIE AHN,* SUHNGGWON KIM,* SANG EUN LEE, and JUNG SANG LEE*
*Department of Internal Medicine, Seoul National University, Clinical Research Institute of Seoul National University Hospital, Seoul, Korea; Department of Internal Medicine, Hallym University Hangang Sacred Heart Hospital, Seoul, Korea; Department of Anatomy, Catholic University, Seoul, Korea; Department of Internal Medicine, Gyeong-sang National University, Chinju, Korea; Department of Internal Medicine, Gachon Medical School, Incheon, Korea; Department of Urology, Seoul National University, Seoul, Korea.

Abstract. Recent progress in molecular physiology has permitted us to understand pathophysiology of various channelopathies at a molecular level. The secretion of H from -intercalated cells is mediated by apical plasma membrane HATPase and basolateral plasma membrane anion exchanger-1 (AE1). Studies have demonstrated the lack of H-ATPase immunostaining in the intercalated cells in a few patients with distal renal tubular acidosis (dRTA). Mutations in H-ATPase and AE1 gene have recently been reported to cause dRTA. This study extends the investigation of the role of transporter defect in dRTA by using immunohistochemical methods. Eleven patients with hyperchloremic metabolic acidosis were diagnosed functionally to have secretory-defect dRTA: urine pH 5.5 during acidemia, normokalemia or hypokalemia, and urine-to-blood pCO2 25 mmHg during bicarbonaturia. Renal

biopsy tissue was obtained from each patient, and immunohistochemistry was carried out using antibodies to H-ATPase and AE1. For comparison, renal tissues from the patients who had no evidences of distal acidification defect by functional studies were used: four with glomerulopathy or tubulointerstitial nephritis (disease controls) and three from nephrectomized kidneys for renal cell carcinoma (normal controls). The HATPase immunoreactivity in -intercalated cells was almost absent in all of the 11 patients with secretory-defect dRTA. In addition, 7 of 11 patients with secretory-defect dRTA were accompanied by negative AE1 immunoreactivity. In both disease controls and normal controls, the immunoreactivity of H-ATPase and AE1 was strong in -intercalated cells. In conclusion, significant defect in acid-base transporters is the major cause of secretory-defect dRTA. lished (2). There is evidence that -intercalated cells also have an apical H-K-ATPase (3) that may account for a significant fraction of HCO3 transport (4). Functionally, normal distal acidification requires an impermeant luminal membrane that is capable of sustaining large pH gradients, a lumen-negative potential difference in the cortical collecting duct supporting both H and K secretion in this segment, and a rate of H secretion by the -intercalated cells of the cortical and medullary collecting ducts (5). Therefore, distal renal tubular acidosis (dRTA) has been functionally classified into gradient (backleak), voltage-dependent, and secretory-defect on the basis of the deranged physiologic mechanisms of urinary acidification (6). In principle, the secretory-defect dRTA should be caused by an impairment of distal H secretion, which may arise from reduction in the number of intercalated cells, reduction in the quantity of H-ATPase and/or H-K-ATPase in the intercalated cells, or an abnormal distribution of proton pump(s) with a reduced quantity at the apical membrane (5). We and others have demonstrated the lack of H-ATPase immunostaining in the intercalated cells in a few patients with

Renal tubular acidosis comprises a diverse group of tubular disorders that are characterized by an impairment of urinary acidification. Urinary acidification can be divided into proximal and distal categories on the basis of which nephron segment is involved in H secretion and HCO3 reabsorption. The proximal tubule is the major site for reabsorption of filtered HCO3, and the distal nephron, primarily the collecting duct, is responsible for reabsorption of the remaining 10 to 20% of filtered HCO3 and for elimination of excess H (1). The importance of the apical H-ATPase and basolateral anion exchanger-1 (AE1) in distal acidification is well estabReceived January 17, 2002. Accepted February 2, 2002. Correspondence to: Dr. Jung Sang Lee, Department of Internal Medicine, Seoul National University, Research Institute of Seoul National University Hospital, 28, Yongun-dong, Chongno-gu, Seoul, 110-744, South Korea. Phone: 82-2-760-2265; Fax : 82-2-742-9031; E-mail : jsleemd@plaza.snu.ac.kr 1046-6673/1306-1425 Journal of the American Society of Nephrology Copyright 2002 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000013882.73122.2B

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secretory-defect dRTA (710). Mutations in H-ATPase (11) and AE1 gene (1216) have recently been reported to cause dRTA. Here, we extend our investigation on the role of acidbase transporter defect in the patients with secretory-defect dRTA by using immunohistochemical methods. This study suggests that reduction in the quantity of H-ATPase and AE1 may be the major cause of functionally diagnosed secretorydefect dRTA.

Materials and Methods

Patients
Diagnosis and classification of renal tubular acidosis were done by the functional tests described below. Eleven patients were enrolled in the group of secretory-defect dRTA. The causes of dRTA were idiopathic, tubulointerstitial nephropathies, and autoimmune disorders such as Sjogrens syndrome. Four patients with intrinsic renal diseases but without any defect in urinary acidification were enrolled in disease controls. Three patients who underwent surgical nephrectomy due to renal cell carcinoma served as normal controls. For immunohistochemistry, percutaneous renal biopsy specimens were obtained from the group of secretory-defect dRTA and disease controls. Normal tissue fractions of the nephrectomized kidneys were also examined from normal controls.

Triton X-100 in phosphate-buffered saline (PBS), and then blocked with normal goat serum diluted 1:10 in PBS for 15 min. Subsequently, the sections were incubated overnight at 4C with rabbit polyclonal antibodies to the 70-kd catalytic subunit of the vacuolar H-ATPase (kindly provided by Dr. Dennis Stone, University of Texas Southwestern, Dallas, TX) and erythrocyte Cl/HCO3 exchanger, AE1 (kindly provided by Dr. Philip S. Low, Purdue University, West Lafayette, IL), a mouse monoclonal antibody (7C6) raised against chicken carbonic anhydrase II (kindly provided by Dr. Paul Linser, Whitney Marine Laboratory, University of Florida, Gainesville, FL), and a mouse monoclonal antibody to Na-K-ATPase 1 subunit (Upstate Biotechnology, Lake Placid, NY). The sections were rinsed in PBS and incubated with the biotinylated secondary antibody for 60 min and subsequently with the Vectastain ABC reagent for 60 min. After being rinsed with 0.1 M Tris buffer, the sections were incubated with a mixture of 0.05% 3.3'-diaminobenzidine and 0.033% H2O2 for 1 to 2 min at room temperature. After being rinsed with Tris buffer again, the sections were counterstained with hematoxylin and examined with light microscopy.

Results
Table 1 shows the results of urinary acidification tests from the patients and controls. In normal controls, as expected, urine pH was low with NH4Cl loading and urine-to-blood pCO2 gradient was large with NaHCO3 loading. In disease controls, who had glomerulopathy or tubulointerstitial nephritis but without evidences of acidification defect, urine pH was below 5.3 during acidemia, which was induced by short-term NH4Cl loading, indicating intact distal acidification. Eleven patients with hyperchloremic metabolic acidosis were diagnosed to have secretory-defect dRTA on the basis of urine pH 5.5 during acidemia, normokalemia or hypokalemia, and urine-to-blood pCO2 25 mmHg during bicarbonaturia. Eight patients were hypokalemic, and the other three patients were normokalemic. The serum anion gap, calculated by Na (Cl HCO3), ranged from 4 to 10 mmol/L. The urine anion gap (Na K Cl) was positive in all of the patients, which is suggestive of impaired urinary NH4 excretion in the clinical setting of hyperchloremic metabolic acidosis (19,20). Even with NH4Cl loading, urinary NH4 excretion was not so high as in the controls (Table 1). As expected, urine pH was above 5.5 during acidemia that was spontaneous or induced by short-term NH4Cl loading. Additionally, urine pH did not decrease after an oral administration of furosemide, confirming a defect in H secretion (21). Urine pCO2 measured after intravenous NaHCO3 loading did not increase normally, and the urine-to-blood pCO2 gradient was 25 mmHg when it was calculated under the condition of urine pH 7.5. Therefore, we concluded that our 11 patients had secretorydefect dRTA. However, in three patients, fractional excretion of bicarbonate was 15% with intravenous NaHCO3 loading. These patients seemed to have combined proximal and distal acidification defect. Renal biopsy tissue was obtained from each patient, and immunohistochemistry was carried out using antibodies to H-ATPase, AE1, carbonic anhydrase II, and Na-K-ATPase 1 subunit. Renal biopsy specimens from the disease controls were also used for comparison. We also examined

Physiologic Tests for Urinary Acidification

To evaluate urinary acidification, we performed short-term ammonium chloride loading, furosemide test, and sodium bicarbonate loading (17), as described below. The ammonium chloride loading test was omitted in cases of overt acidemia (plasma HCO3 18 mmol/ L). Urine pH was measured using a pH meter (Beckman, Fullerton, CA), and urine and blood pCO2 were measured by a blood gas analyzer (Nova, Waltham, MA). Serum and urine electrolytes and creatinine were measured on an autoanalyzer (Beckman, Fullerton, CA). Urine ammonium was determined by the enzymatic method (18). In the short-term ammonium chloride loading test, urine was collected under mineral oil at 2-h intervals from 4 to 8 h after the administration of NH4Cl at a dose of 0.2 g/kg body wt. Blood samples were also taken at the end of each 2-h urine collection to ensure that plasma the bicarbonate level decreased to 20 mmol/L or less. For the furosemide test, urine was collected at 2-h intervals from 4 to 6 h after an oral dose (1 mg/kg body weight) of furosemide. To increase the sensitivity of the test by ensuring a state of sodium avidity, 1 mg of fludrocortisone was administered orally the evening before the testing. In the bicarbonate loading test, 2.75% NaHCO3 solution was infused intravenously at a rate of 4 ml/kg per h. Urine and blood samples were taken at 2-h intervals until plasma bicarbonate concentration reached 26 mmol/L. The values of urine-to-blood pCO2 gradient and fractional excretion of bicarbonate were calculated when urine pH was raised to 7.5.

Immunohistochemistry from Renal Tissues

The renal tissues from the dRTA patients, disease controls and normal controls were preserved in periodate-lysine-paraformaldehyde. The fixed tissues were dehydrated and embedded in polyester wax, and sections were cut to 4-m thickness and mounted on gelatin-coated glass slides. The sections were dewaxed with xylene and ethanol and were treated with methanolic H2O2 for 30 min after rinsing under tap water. Before incubation with primary antibodies, the sections were permeabilized by incubation for 15 min in 0.5%

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Table 1. Results of urinary acidification tests

Cases Urine NH4 (mmol/d)a Urine pHa Urine pHb (U-B)pCO2 (mmHg)c FEHCO3 (%)c

Normal control A 71.8 5.1 B 93.6 5.3 C 83.3 4.9 Disease control D 69.1 4.6 E 75.5 5.0 F 84.6 4.9 G 72.4 5.2 Secretory-defect distal renal tubular acidosis H 12.8 8.0 I 23.1 6.9 J 22.1 7.1 K 28.4 6.6 L 30.1 5.8 M 29.3 6.2 N 31.8 6.6 O 27.9 6.6 P 24.6 6.9 Q 11.8 6.5 R 20.6 5.9
a b

ND ND ND ND ND ND ND ND 7.9 6.6 6.5 7.0 7.3 6.0 ND ND ND 6.2

67.1 58.9 53.4 ND ND ND ND 20.7 3.0 8.8 3.2 8.0 8.0 4.5 20.3 1.9 17.4 21.0

2.1 3.2 1.9 ND ND ND ND 9.9 4.2 6.1 3.8 8.6 2.0 1.3 3.4 18.3 17.0 26.0

Measured after short-term NH4Cl loading. Measured after furosemide administration. c Measured after NaHCO3 loading; FEHCO3, fractional excretion of bicarbonate; ND, not done.

three different normal tissue sections from nephrectomized kidneys for renal cell carcinoma (normal controls). Figure 1 shows H-ATPase immunohistochemistry in connecting tubule and cortical collecting duct from the normal controls, disease controls, and dRTA patients. In both normal controls and disease controls, the immunostaining of HATPase was strongly positive on the apical membranes of -intercalated cells. However, the secretory-defect dRTA patients revealed absolute decrease in immunostaining of HATPase along the connecting tubule and collecting duct. The H-ATPase immunostaining was moderately positive in proximal tubule cells of disease controls and normal controls. Although most dRTA patients showed positive HATPase immunostaining in proximal tubule, three dRTA patients had negative or trace H-ATPase immunoreactivity in their proximal tubule cells (Figure 2). We could not find any significant correlation between the lack of proximal tubule H-ATPase immunoreactivity and the presence of combined proximal acidification defect. Figure 3 shows AE1 immunohistochemistry in collecting duct from the normal controls, disease controls, and dRTA patients. In both normal controls and disease controls, the immunostaining of AE1 was strongly positive on the basolateral membranes of -intercalated cells. However, most of the secretory-defect dRTA patients revealed absolute decrease in immunostaining of AE1 along the connecting tubule and collecting duct.

Carbonic anhydrase II (CA II) is present in the intercalated cells to facilitate acid-base transport in the distal nephron. The CA II immunoreactivity was also markedly different between controls and secretory-defect dRTA patients (Figure 4). In both normal controls and disease controls, the immunostaining for CA II was strong in the intercalated cells of the connecting tubule and collecting duct. In the secretory-defect dRTA patients, however, immunostaining for CA II in the connecting tubule and collecting duct was markedly decreased in intensity. We also tested Na-K-ATPase immunoreactivity in the kidney to verify whether the absence of H-ATPase and AE1 immunostaining were specific findings or not. In intercalated cells, the Na-K-ATPase 1 subunit immunoreactivity in our wax sections was too weak to compare between the patients and controls. However, the Na-K-ATPase 1 subunit immunostaining was equally strong on the basolateral membranes of distal convoluted tubules and thick ascending limbs in the normal controls, disease controls and secretory-defect dRTA patients as well (Figure 5). Therefore, the lack of H-ATPase and AE1 immunostaining was thought to be selective in secretory-defect dRTA. Table 2 summarizes relative immunoreactivities of H-ATPase, AE1, and Na-K-ATPase from the kidneys of normal controls, disease controls, and secretory-defect dRTA patients. The H-ATPase immunoreactivity in -intercalated cells was almost absent in all of the 11 patients with secretory-defect dRTA. Notably, more than half (7 of 11) of them were accom-

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Figure 2. DIC micrographs illustrating immunostaining for H-ATPase in the PT from normal control (A), disease control (B), and a secretory-defect dRTA patient (C). Strong positive immunoreactivity of H-ATPase in normal and disease controls is observed in the PT. In the secretory-defect dRTA patient, who has combined defect in proximal acidification, H-ATPase immunoreactivity is decreased in the PT compared with controls. Magnification, 400.

panied by negative AE1 immunoreactivity in -intercalated cells.

Discussion
We present here important structural-functional correlates through our immunohistochemical study of renal tissue using antibodies against H-ATPase and AE1 in renal biopsy specimens from secretory-defect dRTA patients in whom careful physiologic studies were performed. It was concluded that functionally diagnosed secretory-defect dRTA may be caused by H-ATPase (with or without AE1) defect in intercalated cells. Although there have been some case reports demonstrating the lack of H-ATPase and/or AE1 in the collecting ducts of the patients with dRTA, we confirmed the absolute reduction of these acid-base transporters in our collection of secretory-defect dRTA patients. In addition, we demonstrated coexistence of H-ATPase and AE1 defect in most of our secretory-defect dRTA patients. The existence of secretory-defect dRTA has been reasonably well characterized in patients with different diseases causing acquired dRTA (22). Derangements of collecting duct function determined genetically (e.g., hereditary dRTA), associated with structural abnormalities (e.g., medullary sponge kidney), or immunologically mediated (e.g., Sjogrens syndrome) could all interfere with the integrity of the proton pump secretory apparatus (22). Our dRTA patients were functionally diagnosed on the basis of the following results to have a secretory defect. First, urine pH was not lowered below 5.5, not only during acidemia, but also after stimulation of sodium-dependent distal acidification by the administration of furosemide. Second, the urine pCO2 measured after bicarbonate loading did not increase normally, reflecting that the rate of collecting duct H secretion is reduced. In the collecting duct, H-ATPase is likely responsible for much of the H secretion that takes place in this segment (23). Therefore, the secretory-defect dRTA could be due to any alterations causing an impairment of the collecting duct acid-

Figure 1. Differential interference contrast (DIC) micrographs of renal cortex from normal controls (A and B), disease controls (C and D), and secretory-defect distal renal tubular acidosis (dRTA) patients (E and F) illustrating immunostaining for H-ATPase. In normal controls, numerous intercalated cells with apical (arrows) and diffuse (open arrow) H-ATPase immunoreactivity are observed in the connecting tubule (CNT) and cortical collecting duct (CCD). In disease controls, intercalated cells with strong H-ATPase immunoreactivity (arrows) are seen in the CNT and CCD. In secretory-defect dRTA patients, H-ATPaselabeled intercalated cells are decreased both in number and in intensity of immunoreactivity for H-ATPase along the CNT and CCD. PT, proximal tubule. Magnification, 400.

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Figure 3. DIC micrographs of renal cortex from normal control (A), disease control (B), and secretory-defect dRTA patients (C and D) illustrating immunostaining for anion exchanger-1 (AE1). In normal and disease controls, a lot of intercalated cells (arrows) with basolateral immunostaining for AE1 are seen in the CNT and CCD. In secretory-defect dRTA patients, only a few intercalated cells weakly labeled with AE1 are seen in the connecting tubule and collecting ducts. Magnification, 400.

ification by primarily interfering with the H -ATPase. Cohen et al. (7) first demonstrated absence of H-ATPase staining in collecting duct cells from a renal biopsy specimen from a patient with secretory-defect dRTA and Sjogrens syndrome. They found the presence on electron microscopy of cells with typical ultrastructural features of intercalated cells, and they suggested that the distal acidification defect was due to the absence of intact H-ATPase rather than the selective loss of -intercalated cells. We have also reported that a patient with idiopathic hypokalemic dRTA (Albrights disease) had absence of H-ATPase staining in the intercalated cells (10).

Figure 4. DIC micrographs of renal cortex from normal control (A and B), disease control (C and D), and a secretory-defect dRTA patient (E and F) illustrating immunostaining for carbonic anhydrase II (CA II) in the CNT (A, C, and E) and CCD (B, D, and F). In normal and disease control, intercalated cells (arrows) in both the CNT and CCD exhibit strong immunoreactivity. In the secretory-defect dRTA patient, however, immunostaining for CA II both in CNT and CCD is markedly decreased in intensity. Magnification, 400.

Taken together with the results from our current study, it is strongly indicated that lack of H-ATPase in the intercalated cells of the collecting duct may be the chief cellular mechanism responsible for the secretory-defect dRTA.

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Fig. 5. DIC micrographs of renal cortex from normal control (A), disease control (B), and a secretory-defect dRTA patient (C) illustrating immunostaining for Na-K-ATPase 1 subunit. Positive immunoreactivity of Na-K-ATPase in normal and disease controls is observed in the distal convoluted tubules and thick ascending limbs (*). The secretory-defect dRTA patient also shows strong immunostaining for Na-K-ATPase in both nephron segments. Magnification, 400.

We found that not only H-ATPase but also AE1 immunostaining were absent in the -intercalated cells in the majority of our secretory-defect dRTA patients, suggesting failure of -intercalated cells to express both the H-ATPase and AE1 proteins. Although the reason is unclear, few -intercalated cells were noted in our biopsy tissue. Therefore, we could not describe any changes in the -intercalated cells. In addition, the immunoreactivity for both H-ATPase and AE1 was so

weak in the secretory-defect dRTA patients that we could not analyze further on subtype defects in the intercalated cells. Bastani and associates (8,9) previously examined kidney biopsies from two patients with Sjogrens syndrome, and neither patient showed any immunostaining of intercalated cells with the antibodies against H-ATPase and AE1. The ultrastructural presence of intercalated cells and the absence of discernible staining for H-ATPase and AE1 suggest that the defect in proton secretion may represent a defect involving the assembly of at least two of the ion transport pumps that are essential for the normal maintenance of acid-base homeostasis by the intercalated cells (8). Consistent with this view, we found that CA II immunostaining in the intercalated cells of the connecting tubule and cortical collecting duct was markedly decreased in the secretory-defect dRTA patients. The parallel decrease in H-ATPase and CA II expression might be explained by the fact that the H-secreting process is accelerated by cytosolic or membrane-associated carbonic anhydrase in most H-secreting cells (24). Deficiency of CA II is an autosomal recessive disorder producing a distinctive syndrome of renal tubular acidosis, osteopetrosis, cerebral calcification, and mental retardation (25), and most patients with CA II deficiency syndrome have both proximal and distal components to the renal tubular acidosis (26). Three patients from our current study were diagnosed on the basis of high fractional excretion of bicarbonate to have combined proximal and distal acidification defect.

Table 2. Relative immunoreactivity of H-ATPase, AE1, and Na-K-ATPase in the kidneysa

Cases H-ATPase (PT) H-ATPase (CD) AE1 (CD) Na-K-ATPase (DT)

Normal control A 2 B 2 C 2 Disease control D 1 E 2 F 2 G 2 Secretory defect distal renal tubular acidosis H I 2 J 1 K 2 L M 2 N 2 O 2 P 1 Q 2 R

4 4 4 2 4 3 2 1

4 4 4 2 4 4 3 4 1 2

4 4 4 4 4 4 4 4 4 4 4 4 3 4 4 4 4 4

a The techniques used were semiquantitative, and the scheme 0 to 4 is used to indicate relative expression level as detected by eye. AE1, anion exchanger 1; PT, proximal tubule; CD, collecting duct; DT, distal tubule.

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However, we could not find any clinical evidence for CA II deficiency syndrome in these patients. Our immunostaining for CA II in the proximal tubule was weakly positive in both the patients and controls. Although we found negative or trace H-ATPase immunostaining in proximal tubule cells in three dRTA patients, there was no clear association between the lack of proximal tubule H-ATPase immunoreactivity and the presence of combined proximal acidification defect. The Na-K-ATPase, located in the basolateral membranes of renal tubular epithelial cells, also has a role in pathophysiology of urinary acidification defects (27), because H-ATPase activity can be secondarily affected if Na-K-ATPase is altered (28). In this study, intercalated cells did not show enough Na-K-ATPase immunostaining to compare the patients with the controls. The distal tubules, on the other hand, showed strong Na-K-ATPase immunoreactivity in both the patients and the controls. These findings seemed compatible with the results of Na-K-ATPase activity measured in individual segments of the nephron (29). We believe that intrinsic failure of H-ATPase would be associated with features of dRTA. The pathogenic mechanisms causing the absence of immunostaining for H-ATPase and/or AE1 remain elusive. Although the nature of the defects is unknown, point mutations producing abnormal acid-base transporters may underlie primary dRTA in adults. A variety of tubulointerstitial disease may produce secondary dRTA. Conceivably, the inflammatory process prevents the normal assembly and/or insertion of HATPase into the apical plasma membrane. However, the renal tissue from our dRTA patients revealed only occasional findings of interstitial infiltration, and thus it appears that other mechanisms may play a role in the development of the acidification defect. We postulate existence of as-yet-unknown renal molecules that may affect synthesis, trafficking, or degradation of acid-base transporter protein. Finally, most of our dRTA patients were hypokalemic. Hypokalemia is presumably due to increased distal potassium secretion, possibly mediated by secondary hyperaldosteronism and increased distal delivery of sodium or a defect in H-KATPase (6,30). Unfortunately, the lack of antibodies prevented us from testing the possible role of a deficiency of H-KATPase as the mechanism underlying hypokalemic dRTA. Further studies are needed to elucidate a role for H-KATPase in the pathogenesis of hypokalemic dRTA.

Acknowledgments
This work is supported by the Grant 97-N1 02-04-A-05 from Ministry of Science and Technology of Korea and by the Brain Korea 21 Project from Korea Research Foundation. The authors are indebted to Young-Hee Kim and So-Young Kim for technical assistance.

References
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