Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line

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.

Expression of the Na(+)-K(+)-2Cl(-)-Cotransporter 2 in the Normal and Pressure-Induced Ischemic Rat Retina

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.

Association of age-related hearing loss with ion transporter KCNQ1 and NKCC1 in cochlea of C57BL/6J mice / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Auditory 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>The 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.</p>

High Dose Vitamin D3 Attenuates the Hypocalciuric Effect of Thiazide in Hypercalciuric Rats

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.

Water and Sodium Regulation in Heart Failure

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.

Altered Regulation of Renal Sodium Transporters in Salt-Sensitive Hypertensive Rats Induced by Uninephrectomy

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.

Effects of Thiazide on the Expression of TRPV5, Calbindin-D28K, and Sodium Transporters in Hypercalciuric Rats

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.

Altered Renal Sodium Transporter Expression in an Animal Model of Type 2 Diabetes Mellitus

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.

Association of age-related hearing loss with mice which expressed only one copy of gene encoding NKCC1 co-transporter / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>NKCC1 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.</p>

Role of ion channel Na-K-2Cl and alpha2 Na, K-ATPase in cochlear potassium cycling and auditory function / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the auditory function and the role of NKCC1 and alpha2 Na, K-ATPase in the potassium recycling of cochlea.</p><p><b>METHODS</b>NKCC1(+/-) / 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>Ion 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.</p>

Increased Expression of Sodium Transporters in Rats Chronically Inhibited of Nitric Oxide Synthesis

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.

Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>After 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.</p><p><b>METHODS</b>C57BL/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.</p><p><b>RESULTS</b>All 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.</p><p><b>CONCLUSIONS</b>Administration 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.</p>

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 / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>C57BL/ 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.</p><p><b>RESULTS</b>Mice 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.</p><p><b>CONCLUSIONS</b>An 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.</p>

Altered expression of renal bumetanide-sensitive sodium-potassium-2 chloride cotransporter and Cl- channel -K2 gene in angiotensin II-infused hypertensive rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Little 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.</p><p><b>METHODS</b>Sprague 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.</p><p><b>RESULTS</b>Ang 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.</p><p><b>CONCLUSIONS</b>Chronic 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.</p>

Angiotensin II AT1 Receptor Blockade Changes Expression of Renal Sodium Transporters in Rats with Chronic Renal Failure

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.