1.Association of age-related hearing loss with mice which expressed only one copy of gene encoding NKCC1 co-transporter.
Han-Qi CHU ; Hao XIONG ; Fang HAN ; Zhen-Gong WU ; Xiao-Wen HUANG ; Yong-Hua CUI
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2006;41(7):537-541
OBJECTIVETo generate transgenic mice of NKCC1 +/- (heterozygous) and NKCC1 +/+ (wild-type) that have a targeted disruption in the NKCC1 gene in order to investigate the relationship of one copy of NKCC1 gene (NKCC1 +/-) and age-related hearing loss (AHL) and to study the possible pathogenesis of AHL METHODS: Auditory function of NKCC1 +/- mice was detected regularly by auditory brain response (ABR) and endocochlear potential (EP). Morphology of cochlea was observed by scanning electron microscope and content of NKCC1 protein was detected by Western blot.
RESULTSThe mean value for ABR thresholds was elevated in NKCC1 +/- mice more than that of NKCC1 +/+ mice (P < 0.01). A progression of age-related hearing loss was found in NKCC1 +/- mice. Compared with younger NKCC1 +/- mice, the mean value for ABR thresholds in aged NKCC1 +/- mice was significantly increased (P < 0.05). The EP of NKCC1 +/- aged mice was also significantly decreased more than that of the younger NKCC1 +/+ mice (P < 0.05). And content of NKCC1 protein were reduced with the growth of the age. The scanning electron microscope showed a kind of scattered punctiform absence of outer hair cells in elder NKCC1 +/- mice cochlea.
CONCLUSIONSNKCC1 gene maybe takes part in the pathogenesis of AHL. Mice that expressed only one copy of NKCC1 could lead to AHL. AHL may be correlative with the amounts of NKCC1 protein and its function and also with the loss of outer hair cells perhaps.
Age Factors ; Aging ; genetics ; physiology ; Animals ; Hearing Disorders ; etiology ; genetics ; Heterozygote ; Mice ; Mice, Knockout ; Mice, Transgenic ; Sodium-Potassium-Chloride Symporters ; genetics ; Solute Carrier Family 12, Member 2
2.Role of ion channel Na-K-2Cl and alpha2 Na, K-ATPase in cochlear potassium cycling and auditory function.
Han-qi CHU ; Xiao-wen HUANG ; Hao XIONG ; Fang HAN ; Zhen-gong WU ; Liang-qiang ZHOU ; Yong-hua CUI ; Chun-fang WANG ; Ping ZHANG
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2006;41(9):692-697
OBJECTIVETo investigate the auditory function and the role of NKCC1 and alpha2 Na, K-ATPase in the potassium recycling of cochlea.
METHODSNKCC1(+/-) / alpha2 Na, K-ATPase(+/-) mice model was established from NKCC1(+/-) and alpha2 Na, K-ATPase(+/-) mice. The auditory function of all strain mice were detected by auditory brainstem response (ABR) and endocochlear potential (EP) to investigate the role of NKCC1 and alpha2 Na, K-ATPase in the potassium recycling of cochlea. Furosemide and ouabain were applied to block the two channels in Castel mice line which can long-time maintain normal auditory function and then their auditory function was detected by ABR to authenticate the mode of potassium recycling in vivo and the relationship between cochlear potassium recycling and NKCC1(+/-) and alpha2 Na, K-ATPase.
RESULTSThe mean value for ABR thresholds in response to stimulus was elevated in NKCC1(+/-) and alpha2 Na, K-ATPase (+/-) mice [(38.49 +/- 12.29) dB and (53.32 +/- 7.62) dB) ] respectively, which was significantly increased compared with that observed in wild type mice [(23.13 +/- 3.78) dB, P < 0.05) ]; The EP value of NKCC1(+/-) [(78 +/- 7) mV] and alpha2 Na, K-ATPase(+/-) mice [(71 +/- 14) mV] was decreased compared with that of NKCC1(+/-) / alpha2 Na, K-ATPase(+/-) mice [( 86 +/- 11) mV]. The auditory function of NKCC1(+/-) / alpha2 Na, K-ATPase(+/-) mice could simulate the model of cochlear potassium recycling well. NKCC1 and Na, K-ATPase were great of importance in the potassium recycling, while the two ion channels were in restrict dynamic equilibrium. Castel mice line after administration with furosemide developed significant ABR threshold shifts (P < 0.05) compared with control group. Castel mice line after administration with ouabain also developed greatly significant ABR threshold shifts (P < 0.05) compared with control group. ABR threshold shifts in mice after administration both furosemide and ouabain was attenuated compared with only administration with furosemide (P < 0.01).
CONCLUSIONSIon channel NKCC1 and alpha2 Na, K-ATPase played important roles in the inner ear potassium recycling. Dysfunction of either of them could influence potassium concentration in the endolymph and lead to hearing loss subsequently. The role of NKCC1 and alpha2 Na, K-ATPase in cochlear potassium recycling was authenticated in vivo. The two ion channels contribute the key role for dynamic equilibrium in cochlear potassium recycling and are of great importance for the metabolism of potassium in the inner ear to maintain the normal auditory function.
Animals ; Auditory Threshold ; Cochlea ; metabolism ; physiology ; Evoked Potentials, Auditory, Brain Stem ; Genotype ; Mice ; Mice, Knockout ; Potassium ; metabolism ; Sodium-Potassium-Chloride Symporters ; metabolism ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Solute Carrier Family 12, Member 2
3.Association of age-related hearing loss with ion transporter KCNQ1 and NKCC1 in cochlea of C57BL/6J mice.
Jian-Ling LI ; Han-Qi CHU ; Liang-Qiang ZHOU ; Hao XIONG ; Yan WANG ; Qing-Guo CHEN ; Jin CHEN ; Zhi-Yong LI ; Yun LIU ; Yong-Hua CUI
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2011;46(2):139-143
OBJECTIVETo investigate the age-related expression of KCNQ1 and NKCC1 ion transporters in the stria vascularis in the cochlea of C57BL/6J mice, and to analyze the relationship between the two ion transporters and age-related hearing loss.
METHODSAuditory function of C57BL/6J mice was measured by auditory brainstem response (ABR) at the ages of 4, 8, 14, 24, 40 weeks old respectively. The location of KCNQ1 and NKCC1 ion transporters in the cochlea of C57BL/6J mice was detected by immunohistochemistry staining. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the levels of KCNQ1 and NKCC1 mRNA in the cochlea of C57BL/6J mice at different ages.
RESULTSThe mean values for ABR thresholds in response to click, 4 kHz and 8 kHz sound stimulus of C57BL/6J mice gradually increased with age. The ABR thresholds of the mice of over 14 weeks age were significantly elevated in comparison with lower ages (P < 0.05). In the lateral wall of C57BL/6J mice cochlea, the KCNQ1 protein was mainly expressed at the apical membrane of the strial marginal cells. The localization of NKCC1 protein was mainly present at the basolateral membrane of the stria marginal cells, spiral ligament and the fibrocytes in the inferior portion of spiral limbus. Expression of KCNQ1 and NKCC1 protein in cochlea of C57BL/6J mice showed age-related decreasing. The level of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J also showed a age-related decreasing trend. There was a significant reducing of KCNQ1 mRNA level between C57BL/6J mice of 40 and 4 weeks old (P < 0.05). In comparison with the C57BL/6J mice of 4 weeks old, the NKCC1 mRNA levels of 24 and 40 weeks old also showed significant reducing (P < 0.05).
CONCLUSIONSThe mean value for ABR thresholds of C57BL/6J mice gradually increased with age. Expression of KCNQ1 and NKCC1 protein in the stria vascularis of C57BL/6J mice decreases with age. The levels of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J showed a age-related reducing trend. Regulating after post-translation may also participate in the adjusting of the age-related decreasing of KCNQ1 and NKCC1 protein in the cochlea of C57BL/6J mice. KCNQ1 and NKCC1 ion transporters may play a critical role in maintaining normal hearing function of inner ear.
Age Factors ; Animals ; Cochlea ; metabolism ; physiology ; Evoked Potentials, Auditory, Brain Stem ; KCNQ1 Potassium Channel ; metabolism ; Mice ; Mice, Inbred C57BL ; Sodium-Potassium-Chloride Symporters ; metabolism ; Solute Carrier Family 12, Member 2
4.Kanamycin induced ototoxicity in three kinds of mouse strains and its effects on the expression of na-K-2Cl co-transporter-1 in stria vascularis.
Hao XIONG ; Han-Qi CHU ; Fang HAN ; Zhen-Gong WU ; Ping ZHANG ; Chun-Fang WANG ; Yong-Huo CUI
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2006;41(1):43-47
OBJECTIVETo establish a mice model of aminoglycoside antibiotics (AmAn) induced ototoxicity. Then to investigate the sensitivity of AmAn induced ototoxicity in three mouse strains and effect of kanamycin on the expression of Na-K-2Cl co-transporter-1 (NKCC1) in stria vascularis.
METHODSC57BL/ 6J, CBA/CaJ, NKCC1 +/- mice (each of twenty-four) were randomly divided into four experimental groups A, B, C and D (A kanamycin alone, B kanamycin plus 2, 3-dihydroxybenzoate, C 2, 3-dihydroxybenzoate alone, D control group). Mice were injected with kanamycin or/and 2, 3-dihydroxybenzoate for 14 days. Auditory function was measured by auditory brainstem response (ABR) and morphology of cochlea was observed by succinate dehydrogenase staining. Expression of NKCC1 was detected by immunohistochemistry.
RESULTSMice in group A developed significant ABR threshold shifts (P < 0.01), which were accompanied by out hair cells loss. Mice in group B significantly attenuated ABR threshold shifts with out hair cells loss (P <0.01). The immunostaining of NKCC1 in stria vascularis was attenuated significantly in group A compared with group D (P < 0.01) while the immunostaining in group B was enhanced than which in group A (P < 0.01). CBA/CaJ mice has the highest sensitivity to AmAn in three mouse strains.
CONCLUSIONSAn mouse model of AmAn induced ototoxicity could be established by administration of kanamycin. Kanamycin could inhibit the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate could attenuate AmAn induced ototoxicity maybe by enhancing the expression of NKCC1. Mice that had the characteristic of presbycusis didn't show additional sensitivity of AmAn induced ototoxicity.
Animals ; Anti-Bacterial Agents ; toxicity ; Blood Vessels ; drug effects ; metabolism ; Cochlea ; blood supply ; drug effects ; metabolism ; Kanamycin ; toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Parabens ; toxicity ; Sodium-Potassium-Chloride Symporters ; metabolism ; Solute Carrier Family 12, Member 2
5.Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line.
Soonhong PARK ; Sang Kyun KU ; Hye Won JI ; Jong Hoon CHOI ; Dong Min SHIN
The Korean Journal of Physiology and Pharmacology 2015;19(3):249-255
Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca2+ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca2+ concentration ([Ca2+]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca2+]i increase with nonspecific Ca2+ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca2+]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca2+ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca2+ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca2+ regulation of the WNK-OSR1 pathway in intact cells.
Cell Line*
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Cell Size
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Gadolinium
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Humans
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Lanthanum
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Oxidative Stress
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Phosphorylation
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Phosphotransferases
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Salivary Glands*
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Sodium Chloride Symporters
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Sodium-Potassium-Chloride Symporters
6.Altered expression of renal bumetanide-sensitive sodium-potassium-2 chloride cotransporter and Cl- channel -K2 gene in angiotensin II-infused hypertensive rats.
Tao YE ; Zhi-quan LIU ; Chao-feng SUN ; Yong ZHENG ; Ai-qun MA ; Yuan FANG
Chinese Medical Journal 2005;118(23):1945-1951
BACKGROUNDLittle information is available regarding the effect of angiotensin II (Ang II) on the bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2), the thiazide-sensitive sodium-chloride cotransporter (NCC), and the Cl- channel (CLC)-K2 at both mRNA and protein expression level in Ang II-induced hypertensive rats. This study was conducted to investigate the influence of Ang II with chronic subpressor infusion on nephron-specific gene expression of NKCC2, NCC and CLC-K2.
METHODSSprague Dawleys rats were treated subcutaneously with either Ang II (100 ng.kg-1.min-1) or vehicle for 14 days. Expression of NKCC2, NCC and CLC-K2 mRNA in kidneys was determined by real time polymerase chain reaction (PCR). Western blotting analysis was used to measure NKCC2 and NCC protein expression.
RESULTSAng II significantly increased blood pressure and up-regulated NKCC2 mRNA and protein expression in the kidney. Expression of CLC-K2 mRNA in the kidney increased 1.6 fold (P < 0.05). There were no changes in NCC mRNA or protein expression in AngII-treated rats versus control.
CONCLUSIONSChronic subpressor Ang II infusion can significantly alter NKCC2 and CLC-K2 mRNA expression in the kidney, and protein abundance of NKCC2 in kidney is positively regulated by Ang II. These effects may contribute to enhanced renal Na+ and Cl- reabsorption in response to Ang II.
Angiotensin II ; pharmacology ; Animals ; Blood Pressure ; drug effects ; Gene Expression Regulation ; drug effects ; Hypertension ; chemically induced ; metabolism ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Chloride Symporters ; genetics ; Solute Carrier Family 12, Member 1
7.Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis.
Han-qi CHU ; Hao XIONG ; Xiao-qin ZHOU ; Fang HAN ; Zhen-gong WU ; Ping ZHANG ; Xiao-wen HUANG ; Yong-hua CUI
Chinese Medical Journal 2006;119(12):980-985
BACKGROUNDAfter establishing a murine model of aminoglycoside antibiotic (AmAn) induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2Cl cotransporter-1 (NKCC1) in stria vascularis were investigated.
METHODSC57BL/6J, CBA/CaJ, NKCC1(+/-) mice (24 of each strain) were randomly divided into four experimental groups: A: kanamycin alone; B: kanamycin plus 2, 3-dihydroxybenzoate; C: 2, 3-dihydroxybenzoate alone; and D: control group. Mice were injected with kanamycin or/and 2, 3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response (ABR) was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically.
RESULTSAll three strains in groups A and B developed significant ABR threshold shifts (P < 0.01), which were accompanied by outer hair cell loss. NKCC1 expression in stria vascularis was the weakest in group A (A cf D, P < 0.01) and the strongest in groups C and D (P < 0.05). CBA/CaJ mice had the highest sensitivity to AmAn.
CONCLUSIONSAdministration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicity-possibly by enhancing the expression of NKCC1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.
Animals ; Anti-Bacterial Agents ; toxicity ; Auditory Threshold ; drug effects ; Hair Cells, Vestibular ; drug effects ; Kanamycin ; toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Sodium-Potassium-Chloride Symporters ; analysis ; drug effects ; Solute Carrier Family 12, Member 2 ; Stria Vascularis ; chemistry ; drug effects
8.Altered Regulation of Renal Sodium Transporters in Salt-Sensitive Hypertensive Rats Induced by Uninephrectomy.
Ji Yong JUNG ; Jay Wook LEE ; Sejoong KIM ; Eun Sook JUNG ; Hye Ryoun JANG ; Jin Suk HAN ; Kwon Wook JOO
Electrolytes & Blood Pressure 2009;7(2):58-66
Uninephrectomy (uNx) in young rats causes salt-sensitive hypertension (SSH). Alterations of sodium handling in residual nephrons may play a role in the pathogenesis. Therefore, we evaluated the adaptive alterations of renal sodium transporters according to salt intake in uNx-SSH rats. uNx or sham operations were performed in male Sprague-Dawley rats, and normal-salt diet was fed for 4 weeks. Four experimental groups were used: sham-operated rats raised on a high-salt diet for 2 weeks (CHH) or on a low-salt diet for 1 week after 1 week's high-salt diet (CHL) and uNx rats fed on the same diet (NHH, NHL) as the sham-operated rats were fed. Expression of major renal sodium transporters were determined by semiquantitative immunoblotting. Systolic blood pressure was increased in NHH and NHL groups, compared with CHH and CHL, respectively. Protein abundances of Na+/K+/2Cl- cotransporter (NKCC2) and Na+/Cl- cotransporter (NCC) in the CHH group were lower than the CHL group. Expression of epithelial sodium channel (ENaC)-gamma increased in the CHH group. In contrast, expressions of NKCC2 and NCC in the NHH group didn't show any significant alterations, compared to the NHL group. Expressions of ENaC-alpha and ENaC-beta in the NHH group were higher than the CHH group. Adaptive alterations of NKCC2 and NCC to changes of salt intake were different in the uNx group, and changes in ENaC-alpha and ENaC-beta were also different. These altered regulations of sodium transporters may be involved in the pathogenesis of SSH in the uNx rat model.
Animals
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Blood Pressure
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Diet
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Diet, Sodium-Restricted
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Epithelial Sodium Channels
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Handling (Psychology)
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Humans
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Hypertension
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Immunoblotting
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Male
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Nephrectomy
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Nephrons
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Rats
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Rats, Sprague-Dawley
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Salicylamides
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Social Control, Formal
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Sodium
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Sodium Chloride Symporters
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Sodium-Potassium-Chloride Symporters
9.Water and Sodium Regulation in Heart Failure.
Electrolytes & Blood Pressure 2009;7(2):38-41
Heart failure is the pathophysiological state characterized by ventricular dysfunction and associated clinical symptoms. Decreased cardiac output or peripheral vascular resistance lead to arterial underfilling. That is an important signal which triggers multiple neurohormonal systems to maintain adequate arterial pressure and peripheral perfusion of the vital organs. The kidney is the principal organ affected when cardiac output declines. Alterations of hemodynamics and neurohormonal systems in heart failure result in renal sodium and water retention. Activation of sympathetic nervous system, renin-angiotensin-aldosterone system and non-osmotic vasopressin release stimulate the renal tubular reabsorption of sodium and water. Dysregulation of aquaporin-2 and sodium transporters also play an important role in the pathogenesis of renal sodium and water retention.
Aquaporin 2
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Aquaporins
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Arterial Pressure
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Cardiac Output
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Epithelial Sodium Channels
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Heart
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Heart Failure
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Hemodynamics
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Kidney
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Perfusion
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Renin-Angiotensin System
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Retention (Psychology)
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Sodium
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Sodium Chloride Symporters
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Sodium-Potassium-Chloride Symporters
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Sympathetic Nervous System
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Vascular Resistance
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Vasopressins
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Ventricular Dysfunction
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Water-Electrolyte Imbalance
10.Gitelman syndrome combined with complete growth hormone deficiency.
Se Ra MIN ; Hyun Seok CHO ; Jeana HONG ; Hae Il CHEONG ; Sung Yeon AHN
Annals of Pediatric Endocrinology & Metabolism 2013;18(1):36-39
Gitelman syndrome is a rare autosomal recessive hereditary salt-losing tubulopathy, that manifests as hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. It is caused by mutations in the solute carrier family 12(sodium/chloride transporters), member 3 (SLC12A3) gene encoding the thiazide-sensitive sodium chloride cotransporter channel (NCCT) in the distal convoluted tubule of the kidney. It is associated with muscle weakness, cramps, tetany, vomiting, diarrhea, abdominal pain, and growth retardation. The incidence of growth retardation, the exact cause of which is unknown, is lower than that of Bartter syndrome. Herein, we discuss the case of an overweight 12.9-year-old girl of short stature presenting with hypokalemic metabolic alkalosis. The patient, on the basis of detection of a heterozygous mutation in the SLC12A3 gene and poor growth hormone (GH) responses in two provocative tests, was diagnosed with Gitelman syndrome combined with complete GH deficiency. GH treatment accompanied by magnesium oxide and potassium replacement was associated with a good clinical response.
Abdominal Pain
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Alkalosis
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Bartter Syndrome
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Diarrhea
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Gitelman Syndrome
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Growth Hormone
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Humans
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Incidence
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Kidney
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Magnesium Oxide
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Muscle Cramp
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Muscle Weakness
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Overweight
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Potassium
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Sodium Chloride Symporters
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Tetany
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Vomiting