1.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
2.Altered Renal Sodium Transporter Expression in an Animal Model of Type 2 Diabetes Mellitus.
Yun Kyu OH ; Kwon Wook JOO ; Jay Wook LEE ; Un Sil JEON ; Chun Soo LIM ; Jin Suk HAN ; Mark A KNEPPER ; Ki Young NA
Journal of Korean Medical Science 2007;22(6):1034-1041
Hemodynamic factors play an important role in the development and/or progression of diabetic nephropathy. We hypothesized that renal sodium transporter dysregulation might contribute to the hemodynamic alterations in diabetic nephropathy. Otsuka Long Evans Tokushima Fatty (OLETF) rats were used as an animal model for type 2 diabetes. Long Evans Tokushima (LETO) rats were used as controls. Renal sodium transporter regulation was investigated by semiquantitative immunoblotting and immunohistochemistry of the kidneys of 40-week-old animals. The mean serum glucose level in OLETF rats was increased to 235+/-25 mg/dL at 25 weeks, and the hyperglycemia continued up to the end of 40 weeks. Urine protein/ creatinine ratios were 10 times higher in OLETF rats than in LETO rats. At 40th week, the abundance of the epithelial sodium channel (ENaC) beta-subunit was increased in OLETF rats, but the abundance of the ENaC gamma-subunit was decreased. No significant differences were observed in the ENaC alpha-subunit or other major sodium transporters. Immunohistochemistry for the ENaC beta-subunit showed increased immunoreactivity in OLETF rats, whereas the ENaC gamma-subunit showed reduced immunoreactivity in these rats. In OLETF rats, ENaC beta-subunit upregulation and ENaC gamma-subunit downregulation after the development of diabetic nephropathy may reflect an abnormal sodium balance.
Animals
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Blood Glucose/analysis
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Diabetes Mellitus, Type 2/*metabolism
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*Disease Models, Animal
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Epithelial Sodium Channel/*analysis
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Hypertension/complications
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Immunoblotting
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Immunohistochemistry
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Kidney/*metabolism
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Male
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Rats
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Sodium/*metabolism
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Sodium-Hydrogen Antiporter/genetics
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Sodium-Potassium-Chloride Symporters/genetics
3.Increased Expression of Sodium Transporters in Rats Chronically Inhibited of Nitric Oxide Synthesis.
Joon Sik KIM ; Ki Chul CHOI ; Myung Ho JEONG ; Soo Wan KIM ; Yoon Wha OH ; Jong Un LEE
Journal of Korean Medical Science 2006;21(1):1-4
The present study was done to determine whether endogenous nitric oxide (NO) plays a role in the regulation of sodium transporters in the kidney. Male Sprague-Dawley rats were treated with NG-nitro-L-arginine methyl ester (L-NAME, 100 mg/L drinking water) for 4 weeks. Control rats were supplied with tap water without drugs. Expression of Na, K-ATPase, type 3 Na/H exchanger (NHE3), Na/K/2Cl cotransporter (BSC1), and thiazide-sensitive Na/Cl cotransporter (TSC) proteins was determined in the kidney by Western blot analysis. Catalytic activity of Na,K-ATPase was also determined. The treatment with L-NAME significantly and steadily increased the systemic blood pressure. Total and fractional excretion of urinary sodium decreased significantly, while creatinine clearance remained unaltered. Neither plasma renin activity nor aldosterone concentration was significantly altered. The alpha1 subunit expression and the catalytic activity of Na, K-ATPase were increased in the kidney. The expression of NHE3, BSC1 and TSC was also increased significantly. These results suggest that endogenously-derived NO exerts a tonic inhibitory effect on the expression of sodium transporters, including Na, K-ATPase, NHE3, BSC1, and TSC, in the kidney.
Animals
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Blotting, Western
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Carrier Proteins/*biosynthesis
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Enzyme Inhibitors/pharmacology
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Kidney/drug effects/metabolism
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Male
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NG-Nitroarginine Methyl Ester/*pharmacology
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Na(+)-K(+)-Exchanging ATPase/biosynthesis
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Nitric Oxide Synthase/*antagonists & inhibitors/metabolism
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Rats
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Rats, Sprague-Dawley
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Receptors, Drug/biosynthesis
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Sodium/*metabolism
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Sodium Chloride Symporters/biosynthesis
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Sodium-Hydrogen Antiporter/biosynthesis
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Sodium-Potassium-Chloride Symporters/biosynthesis
4.Expression of the Na(+)-K(+)-2Cl(-)-Cotransporter 2 in the Normal and Pressure-Induced Ischemic Rat Retina.
Korean Journal of Ophthalmology 2012;26(3):203-211
PURPOSE: To evaluate the expression of the Na(+)-K(+)-2Cl(-)-cotransporter 2 (NKCC2) in the ischemic rat retina. METHODS: Retinal ischemia was induced by pressures 90 to 120 mmHg, above systemic systolic pressure. Immunohistochemistry and western blot analysis were performed. RESULTS: NKCC2 is expressed in the normal retina and its expression is increased by ischemia caused by intraocular pressure elevation. NKCC2 immunoreactivity was observed mainly in axon bundles of ganglion cells and horizontal cell processes in the retina. NKCC2 expression continuously increased with a peak value 3 days (to 415% of normal levels) after ischemic injury, and then gradually decreased to 314% of controls until 2 weeks post injury. The mean density of NKCC2-labeled ganglion cells per mm2 changed from 1,255 +/- 109 in normal retinas to 391 +/- 49 and 185 +/- 37 at 3 days and 2 weeks after ischemia, respectively (p < 0.05), implying cell death of ganglion cells labeled with NKCC2. CONCLUSIONS: Taken together, these results suggest that NKCC2, which is expressed in retinal ganglion and horizontal cells, may contribute to cell death by ischemic injury in the retina, although the molecular mechanisms involved remain to be clarified.
Animals
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Blotting, Western
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Disease Models, Animal
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Immunohistochemistry
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Intraocular Pressure
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Ischemia/etiology/*metabolism
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Male
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Microscopy, Confocal
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Ocular Hypertension/*complications/metabolism/physiopathology
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Rats
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Rats, Sprague-Dawley
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Retinal Diseases/etiology/*metabolism
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Retinal Ganglion Cells/*metabolism/pathology
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Sodium-Potassium-Chloride Symporters/*biosynthesis
5.High Dose Vitamin D3 Attenuates the Hypocalciuric Effect of Thiazide in Hypercalciuric Rats.
Hye Ryoun JANG ; Jay Wook LEE ; Sejoong KIM ; Nam Ju HEO ; Jeong Hwan LEE ; Hyo Sang KIM ; Ji Yong JUNG ; Yun Kyu OH ; Ki Young NA ; Jin Suk HAN ; Kwon Wook JOO
Journal of Korean Medical Science 2010;25(9):1305-1312
Thiazide is known to decrease urinary calcium excretion. We hypothesized that thiazide shows different hypocalciuric effects depending on the stimuli causing hypercalciuria. The hypocalciuric effect of hydrochlorothiazide (HCTZ) and the expression of transient receptor potential vanilloid 5 (TRPV5), calbindin-D(28K), and several sodium transporters were assessed in hypercalciuric rats induced by high calcium diet and vitamin D3. Urine calcium excretion and the expression of transporters were measured from 4 groups of Sprague-Dawley rats; control, HCTZ, high calcium-vitamin D, and high calcium-vitamin D with HCTZ groups. HCTZ decreased urinary calcium excretion by 51.4% in the HCTZ group and only 15% in the high calcium-vitamin D with HCTZ group. TRPV5 protein abundance was not changed by HCTZ in the high calcium-vitamin D with HCTZ group compared to the high calcium-vitamin D group. Protein abundance of NHE3, SGLT1, and NKCC2 decreased in the hypercalciuric rats, and only SGLT1 protein abundance was increased by HCTZ in the hypercalciuric rats. The hypocalciuric effect of HCTZ is attenuated in high calcium and vitamin D-induced hypercalciuric rats. This attenuation seems to have resulted from the lack of HCTZ's effect on protein abundance of TRPV5 in severe hypercalciuric condition induced by high calcium and vitamin D.
Animals
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Calcium/therapeutic use/urine
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Calcium Channels/genetics/metabolism
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Cholecalciferol/*toxicity
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Hydrochlorothiazide/*therapeutic use
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Hypercalciuria/chemically induced/*drug therapy
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Rats
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Rats, Sprague-Dawley
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Sodium Chloride Symporter Inhibitors/*therapeutic use
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Sodium-Glucose Transporter 1/genetics/metabolism
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Sodium-Hydrogen Antiporter/genetics/metabolism
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Sodium-Potassium-Chloride Symporters/genetics/metabolism
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TRPV Cation Channels/genetics/metabolism
6.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
7.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
8.Angiotensin II AT1 Receptor Blockade Changes Expression of Renal Sodium Transporters in Rats with Chronic Renal Failure.
Eun Jung KIM ; Yong Wuk JUNG ; Tae Hwan KWON
Journal of Korean Medical Science 2005;20(2):248-255
We aimed to examine the effects of angiotensin II AT1 receptor blocker on the expression of major renal sodium transporters and aquaporin-2 (AQP2) in rats with chronic renal failure (CRF). During 2 wks after 5/6 nephrectomy or sham operation, both CRF rats (n=10) and sham-operated control rats (n=7) received a fixed amount of low sodium diet and had free access to water. CRF rats (n=10) were divided into two groups which were either candesartan-treated (CRF-C, n=4) or vehicletreated (CRF-V, n=6). Both CRF-C and CRF-V demonstrated azotemia, decreased GFR, polyuria, and decreased urine osmolality compared with sham-operated rats. When compared with CRF-V, CRF-C was associated with significantly higher BUN levels and lower remnant kidney weight. Semiquantitative immunoblotting demonstrated decreased AQP2 expression in both CRF-C (54% of control levels) and CRF-V (57%), whereas BSC-1 expression was increased in both CRF groups. Particularly, CRF-C was associated with higher BSC-1 expression (611%) compared with CRF-V (289%). In contrast, the expression of NHE3 (25%) and TSC (27%) was decreased in CRF-C, whereas no changes were observed in CRF-V. In conclusion, 1) candesartan treatment in an early phase of CRF is associated with decreased renal hypertrophy and increased BUN level; 2) decreased AQP2 level in CRF is likely to play a role in the decreased urine concentration, and the downregulation is not altered in response to candesartan treatment; 3) candesartan treatment decreases NHE3 and TSC expression; and 4) an increase of BSC-1 is prominent in candesartan-treated CRF rats, which could be associated with the increased delivery of sodium and water to the thick ascending limb.
Angiotensin II Type 1 Receptor Blockers
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Animals
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Aquaporins/genetics
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Benzimidazoles/*pharmacology
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Blood Urea Nitrogen
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Kidney Failure, Chronic/drug therapy/*metabolism
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Male
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Organ Size/drug effects
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Rats
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Rats, Sprague-Dawley
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Receptors, Drug/*genetics
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Research Support, Non-U.S. Gov't
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Sodium-Hydrogen Antiporter/*genetics
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Sodium-Potassium-Chloride Symporters/*genetics
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Symporters/*genetics
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Tetrazoles/*pharmacology
9.Effects of Thiazide on the Expression of TRPV5, Calbindin-D28K, and Sodium Transporters in Hypercalciuric Rats.
Hye Ryoun JANG ; Sejoong KIM ; Nam Ju HEO ; Jeong Hwan LEE ; Hyo Sang KIM ; Soren NIELSEN ; Un Sil JEON ; Yun Kyu OH ; Ki Young NA ; Kwon Wook JOO ; Jin Suk HAN
Journal of Korean Medical Science 2009;24(Suppl 1):S161-S169
TRPV5 is believed to play an important role in the regulation of urinary calcium excretion. We assessed the effects of hydrochlorothiazide (HCTZ) on the expression of TRPV5, calbindin-D28K, and several sodium transporters in hypercalciuric rats. Sprague- Dawley rats were divided into 4 groups; control, HCTZ, high salt, and high salt with HCTZ group in experiment 1; control, HCTZ, high calcium (Ca), and high Ca with HCTZ group in experiment 2. To quantitate the expression of TRPV5, calbindin- D28K, and sodium transporters, western blotting was performed. In both experiments, HCTZ significantly decreased urinary calcium excretion. TRPV5 protein abundance decreased in all hypercalciuric rats, and restored by HCTZ in both high salt with HCTZ and high Ca with HCTZ group. Calbindin-D28K protein abundance increased in the high salt and high salt with HCTZ groups, but did not differ among groups in experiment 2. Protein abundance of NHE3 and NKCC2 decreased in all hypercalciuric rats, and were restored by HCTZ in only high Ca-induced hypercalciuric rats. In summary, protein abundance of TRPV5, NHE3, and NKCC2 decreased in all hypercalciuric rats. The hypocalciuric effect of HCTZ is associated with increased protein abundance of TRPV5 in high salt or calcium diet-induced hypercalciuric rats.
Animals
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Biological Transport
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Calcium/urine
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Calcium Channels/chemistry
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Calcium-Binding Protein, Vitamin D-Dependent/*biosynthesis
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Hydrochlorothiazide/pharmacology
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Hypercalciuria/*therapy
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Male
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Models, Biological
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Rats
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Rats, Sprague-Dawley
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Sodium/*metabolism
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Sodium-Hydrogen Antiporter/chemistry
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Sodium-Potassium-Chloride Symporters/metabolism
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TRPV Cation Channels/*biosynthesis/chemistry
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Thiazides/*pharmacology