Na+-Cl- cotransporter-mediated chloride uptake contributes to hypertension and renal damage in aldosterone-infused rats.

Recently, in addition to epithelial sodium channel alpha-subunit (αENaC), the thiazide-sensitive sodium-chloride cotransporter (NCC) and pendrin, also known as sodium-independent chloride/iodide transporter, were reported to be activated by aldosterone. Here, we investigated whether chloride (Cl-) is responsible for hypertension, inflammation, and renal damage in aldosterone-infused rats. Following left nephrectomy, 8-wk-old male Sprague-Dawley rats were allocated into four groups: 1) drinking 1.0% sodium chloride solution with aldosterone infusion (Aldo/NaCl rats); 2) drinking 1.44% sodium bicarbonate solution with aldosterone infusion (Aldo/NaHCO3 rats); 3) drinking distilled water with aldosterone infusion (Aldo/water rats); and 4) drinking distilled water without aldosterone infusion (sham rats). Additionally, heminephrectomized rats with aldosterone infusion were fed a 0.26% NaCl diet (control); 8.0% NaCl diet (high-Na/high-Cl); or a 4.0% NaCl 6.67% sodium citrate diet (high-Na/half-Cl). Last, Aldo/NaCl rats were treated with or without hydrochlorothiazide. Blood pressure in the Aldo/NaCl rats was significantly higher than in the Aldo/NaHCO3 rats, which was associated with the increased expression of NCC. Expression of markers of inflammation (CD3, CD68, interleukin-17A) and fibrosis (α-smooth muscle actin, collagen 1) were also increased in Aldo/NaCl rats. Similarly, aldosterone-infused rats fed a high-Na/half-Cl diet had lower blood pressure than those fed a high-Na/high-Cl diet, with a reduction of phosphorylated NCC, but not αENaC and pendrin. NCC inhibition with hydrochlorothiazide attenuated interleukin-17A protein expression along with the phosphorylation of NCC in Aldo/NaCl rats. These findings suggest that NCC-mediated Cl- uptake plays important roles in the development of aldosterone-induced hypertension and renal injury.


INTRODUCTION
The kidneys retain the balance between salt and water within body fluid, playing an important role in maintaining blood pressure. Excessive dietary salt intake is a well-known major factor for the induction of hypertension. Guyton et al. (15) first proposed that a decrease in renal excretion of salt leads to fluid retention, resulting in hypertension through an increase in cardiac output. Several studies have demonstrated that salt susceptibility to hypertension, also called "salt sensitivity," is different in each person (8), and that salt-sensitive hypertension is caused by several factors, including activation of the sympathetic nervous system, the renin-angiotensin-aldosterone system, and hyperinsulinemia (3,17,28). However, the precise mechanisms behind salt sensitivity remain unclear.
A recent study suggested that dietary salt per se accelerates sodium (Na ϩ ) reabsorption through RAS-related C3 botulinum toxin substrate 1 activation in the salt-sensitive phenotype (31). Dietary salt comprises both Na ϩ and chloride (Cl Ϫ ) ions, and their excretion is regulated at distal nephrons, which consist of a distal tubule and a collecting duct. Although Na ϩ is considered to be a major contributor to hypertension, previous studies suggest that Cl Ϫ , rather than Na ϩ , is essential for maintaining blood pressure and volume retention (6,42). Studies also report that selective Cl Ϫ loading causes vasocontraction and microangiopathy in spontaneously hypertensive rats (30,35). In contrast, oral intake of sodium bicarbonate (NaHCO 3 ) did not elevate blood pressure in rats treated with deoxycorticosterone acetate (DOCA) (45). These findings raise the possibility that the dual roles of NaCl in the pathogenesis of salt-sensitive hypertension may be explained by Cl Ϫ reabsorption at the distal nephron.
Among the various models of salt-sensitive hypertension, aldosterone/salt-treated animals exhibit infiltration of the kidneys by numerous immune cells, including lymphocytes and macrophages (4). A previous study found that administration of mycophenolate mofetil (MMF), an immunosuppressant, ameliorated aldosterone/salt-induced hypertension (5). We also reported that another immunosuppressive agent, mizoribine, attenuated not only renal inflammation but also hypertension in a rat aldosterone/NaCl model (10). These findings suggest that aldosterone/NaCl-induced inflammation may be implicated in the development of hypertension (13). Notably, recent studies have demonstrated that excess intake of NaCl causes inflammation through upregulation of interleukin-17A (IL-17A) derived from Th17 cells (1,23). However, the role of Cl Ϫ in renal inflammation and hypertension in aldosterone-infused rats remains unclear.
In this study, we investigated whether Cl Ϫ is responsible for the observed hypertension, inflammation, and renal damage in aldosterone-infused rats. We found that administration of NaHCO 3 solution resulted in less renal inflammation, fibrosis, and hypertension in aldosterone-infused rats compared with administration of NaCl. Next, we show that a 4.0% NaCl 6.67% sodium citrate (high-Na/half-Cl) diet had less hypertension compared with an 8.0% NaCl (high-Na/high-Cl) diet in aldosterone-infused rats. Last, Na ϩ -Cl Ϫ cotransporter (NCC) inhibition with hydrochlorothiazide (HCTZ) attenuated IL-17A protein expression in aldosterone/NaCl-infused rats. Both Cl Ϫ restriction and HCTZ administration attenuated protein expression of functional phosphorylated NCC in the membrane fraction. These results suggest that NCC-mediated Cl Ϫ uptake plays important roles in the development of aldosterone-induced hypertension and renal injury.

Ethical Considerations
All experiments were carried out in accordance with recommendations for the Care and Use of Laboratory Animals in the National Institutes of Health Guidelines. The Institutional Animal Care and Use Committee of Hiroshima University (Hiroshima, Japan) approved the experimental protocols (Permit Number A10 -52). All efforts were taken to minimize pain and distress to animals.

Surgical Procedures
Eight-week-old male Sprague-Dawley rats (290 -320 g) were purchased from Charles River Laboratories Japan (Yokohama, Japan). Rats underwent left nephrectomy under anesthesia with an intraperitoneal injection of sodium pentobarbital or a medetomidine-midazolam-butorphanol combination. Aldosterone (Sigma-Aldrich, St. Louis, MO) was dissolved in distilled water containing dimethyl sulfoxide, and the solution was administered to rats using Alzet osmotic pumps (Durect, Cupertino, CA) (10). Pumps were subcutaneously implanted in rats, ensuring constant aldosterone infusion throughout the 6-wk study period.

Measurement of Biological Parameters
Blood pressure was measured weekly (experiments 1 and 3) or biweekly (experiment 2) using the tail cuff method (Softron, Tokyo). Twenty-four-hour urine samples were collected using metabolic cages (Natsume, Tokyo). Rats were kept in the metabolic cages for 1 day of acclimatization before starting urine collection. At the end of the fifth (experiments 1 and 3) or the sixth (experiment 2) week, blood samples were taken by cardiac puncture. Measurement of blood and urine samples was outsourced to SRL (Tokyo). Blood pH was analyzed by I-STAT (chem 8ϩ cartridge; Abbott Point of Care, Princeton, NJ).

Immunoblot Assays
To extract protein from whole tissue, frozen renal tissue samples were lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA) and homogenized for 40 s using an ultrasonic homogenizer (VP-050; Taitec, Saitama, Japan) at 20% power. Soluble protein was additionally sonicated for 20 s. Protein from the membrane fraction was extracted using a Minute plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN). The concentration of the protein solution was measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL), and then the concentration was adjusted uniformly. An equal amount of each sample was analyzed by immunoblot analysis, as previously described (10,38).

Statistical Analysis
Results for parametric data are expressed as means Ϯ SD for each group of rats. Statistical analysis was performed using SPSS (version 22.0; IBM, Armonk, NY). Comparisons between two groups were tested using Student's t-test, whereas those among groups were tested by analysis of variance (ANOVA) followed by Tukey's post hoc test. The glomerulosclerosis score was analyzed by Kruskal-Wallis analysis followed by the Steel-Dwass test. The general linear model was applied for comparisons of blood pressure values and urinary samples at each time point and each group. Values of P Ͻ 0.05 were considered to be statistically significant.

NaCl, but not NaHCO 3 , Induced Hypertension in Aldosterone-Infused Rats
We first evaluated the effect of NaCl and NaHCO 3 on the physical characteristics of aldosterone-infused rats. At the fifth week, Aldo/NaCl rats demonstrated lower body weights compared with Aldo/NaHCO 3 rats. Moreover, severe renal hypertrophy was observed in Aldo/NaCl rats. Although the serum Na ϩ concentration did not show any significant differences among the groups, serum levels of potassium (K ϩ ) and Cl Ϫ were lower in Aldo/NaCl and Aldo/NaHCO 3 rats than in sham and Aldo/water rats ( Table 2).
Systolic blood pressure values in Aldo/NaCl rats gradually rose during the observation period. From week 2, blood pressure values showed a significant increase in the Aldo/NaCl rats compared with the Aldo/NaHCO 3 rats (Fig. 1A). The urinary excretion of Na ϩ and Cl Ϫ was increased in Aldo/NaCl rats; however, K ϩ did not differ. Urinary protein excretion was also significantly increased in Aldo/NaCl rats (Fig. 1B).

Expression of NCC and ␣ENaC, but not Pendrin, Was Increased in Aldo/NaCl Rats Compared with Aldo/NaHCO 3 Rats
Although aldosterone is known to upregulate the expression of NCC, ␣ENaC, and pendrin, the effect of NaHCO 3 on expression of these factors in aldosterone-infused rats remains unclear. We extracted proteins located in the membrane fraction from renal samples and adjusted the protein concentration of each sample. Expression levels of transporter proteins from the membrane lysates were examined using immunoblot analysis. Total NCC expression in membrane lysates was significantly increased in Aldo/NaCl rats compared with Aldo/NaHCO 3 rats, whereas ␣ENaC and pendrin expression did not differ significantly between the two groups (Fig. 2, A and B). To evaluate the activated form of NCC, we examined expression levels of phosphorylated-NCC proteins, specifically T53-NCC, T58-NCC, and P71-NCC. The expression of phosphorylated-NCC proteins was higher in Aldo/NaCl rats than in Aldo/NaHCO 3 and Aldo/ water rats (Fig. 2, C and D).

Number of Cell Death and Expression of Fibrotic Markers Were Increased in the Kidneys of Aldo/NaCl Rats Compared with Aldo/NaHCO 3 Rats
To evaluate renal injury, formalin-fixed, paraffin-embedded tissues were processed for histological analysis. By H-E staining, Aldo/NaCl rats displayed an increase in renal tubular showing protein expression levels of ␣SMA in sham rats, Aldo/water rats, Aldo/NaCl rats, and Aldo/NaHCO3 rats. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. F: graph shows relative protein expression of ␣SMA. Sham rats (sham): rats given water after left nephrectomy. Aldo/water rats (Aldo/water): heminephrectomized rats drinking distilled water with aldosterone infusion. Aldo/NaHCO3 rats (Aldo/NaHCO3): heminephrectomized rats drinking 1.44% NaHCO3 with aldosterone infusion. Aldo/NaCl rats (Aldo/NaCl): heminephrectomized rats drinking 1.0% NaCl with aldosterone infusion. Values are means Ϯ SD. Parametric data were analyzed with ANOVA followed by Tukey's test. Glomerulosclerosis score was analyzed with Kruskal-Wallis analysis followed by the Steel-Dwass test. Statistical differences are indicated as *P Ͻ 0.05, **P Ͻ 0.01.

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CHLORIDE AND ALDOSTERONE NEPHROPATHY dilation and immune cell infiltration into the tubulointerstitium compared with Aldo/NaHCO 3 and Aldo/water rats (Fig. 3A). Kidneys from Aldo/NaCl rats showed large areas stained with aniline blue after M-T staining, which indicated fibrotic tissue (Fig. 3A). Some glomeruli from Aldo/NaCl rats demonstrated marked glomerulosclerosis and adhesions to the Bowman's capsule along with focal tubulointerstitial injury, such as tubular dilation and protein casts (Fig. 3A). Conversely, Aldo/ NaHCO 3 and Aldo/water rats showed few sclerotic changes (Fig. 3B).
Immunohistochemical staining was performed to investigate renal injury more closely. TUNEL staining was performed to determine cell death. TUNEL-positive cells were observed mainly in the tubulointerstitium (Fig. 3C). Kidneys from Aldo/ NaCl rats had more TUNEL-positive cells than Aldo/NaHCO 3 rats (Fig. 3D). To assess the effects of NaCl and NaHCO 3 on fibrosis in aldosterone-infused rats, the expressions of ␣SMA and collagen 1 were used as markers of myofibroblasts and extracellular matrix protein, respectively. Whereas increased areas of fibrosis were observed in the renal tubulointerstitium of Aldo/NaCl rats, there were few fibrotic changes in Aldo/ NaHCO 3 rats (Fig. 3, C and D). Protein expression of ␣SMA was also examined by immunoblot assay, and the findings were compatible with the results from immunohistochemical staining (Fig. 3, E and F). showing inflammatory cell infiltration of kidneys of sham rats, Aldo/water rats, Aldo/NaCl rats, and Aldo/NaHCO3 rats. B: quantification of CD3-and CD68positive cells. C: immunoblot assays showing protein expression of interleukin 17A (IL-17A). D: quantification of protein expression of IL-17A. Sham rats (sham): rats given water after left nephrectomy. Aldo/water rats (Aldo/ water): heminephrectomized rats drinking distilled water with aldosterone infusion. Aldo/NaHCO3 rats (Aldo/ NaHCO3): heminephrectomized rats drinking 1.44% NaHCO3 with aldosterone infusion. Aldo/NaCl rats (Aldo/ NaCl): heminephrectomized rats drinking 1.0% NaCl with aldosterone infusion. Values are means Ϯ SD. Data were analyzed with ANOVA followed with Tukey's test. Statistical differences are indicated as *P Ͻ 0.05, **P Ͻ 0.01. cently been reported that differentiation of naive T cells into T helper 17 (Th17) cells is central to inflammation in the salt-sensitive state (18). Therefore, we performed immunohistochemical staining for CD3, a maker of T lymphocytes, and CD68, a marker of macrophages and monocytes. Aldo/ NaCl rats demonstrated numerous CD3-and CD68-positive cells infiltrating the tubulointerstitium of the kidneys (Fig.  4, A and B). Expression of IL-17A was also upregulated in Aldo/NaCl rats; however, IL-17A expression in the Aldo/ NaHCO 3 rats did not differ from the Aldo/water rats (Fig. 4,  C and D).

Dietary Cl Ϫ Restriction Suppressed Hypertension and Inhibited NCC Phosphorylation in Aldosterone-Infused Rats with Na ϩ Overload
To confirm the effect of Cl Ϫ on salt-sensitive hypertension under conditions of equalized Na ϩ overload, we measured blood pressure and examined the expression of NCC, ␣ENaC, and pendrin in rats on control, high-Na/high-Cl, and high-Na/ half-Cl diets. During the observation period, dietary consumption and body weight were not significantly different between the high-Na/high-Cl and high-Na/half-Cl groups (Fig. 5A and Reduction of Cl Ϫ intake prevented hypertension and inhibited protein expression of the Na ϩ -Cl Ϫ transporter. A: line graph indicates weekly dietary intake of control, high-Na/high-Cl, and high-Na/half-Cl groups during the observation period (n ϭ 8 for each group). B: line graph showing sequential blood pressure values measured biweekly. C: immunoblot assay showing membrane expression of ␣ENaC, pendrin, total NCC, T53, T58, and P71-NCC among control rats, high-Na/high-Cl rats, and high-Na/ half-Cl rats. D: quantification of protein expression of ␣ENaC, pendrin, total NCC, T53, T58, and P71-NCC in the membrane fraction isolated from the kidney lysate. ␣ENaC, epithelial sodium channel alphasubunit; NCC, thiazide-sensitive Na ϩ -Cl Ϫ cotransporter. Control rats (control): rats given a 0.26% NaCl (0.10% Na/0.16% Cl) diet. High-Na/high-Cl rats (high-Na/high-Cl): rats given an 8.0% NaCl (3.14% Na/ 4.85% Cl) diet. High-Na/half-Cl rats (high-Na/half-Cl): rats given a 4.0% NaCl 6.67% sodium citrate (3.14% Na/2.43% Cl) diet. All rats were subjected to heminephrectomy and aldosterone infusion. Values are means Ϯ SD. Dietary intake and blood pressure data at each time point were analyzed using the general linear model, and statistical differences compared with high-Na/half-Cl rats are indicated as §P Ͻ 0.05. Immunoblot data were analyzed with ANOVA followed by Tukey's test. Statistical differences are indicated as *P Ͻ 0.05, **P Ͻ 0.01. Table 3). At the third week, urinary excretion of Na ϩ did not differ between the high-Na/high-Cl and high-Na/half-Cl rats, whereas urinary Cl Ϫ was increased in high-Na/high-Cl rats compared with high-Na/half-Cl rats (Table 3). Similar to Aldo/ NaCl rats, blood pressure of high-Na/high-Cl rats was higher than that of high-Na/half-Cl rats (Fig. 5B). By immunoblot analysis, phosphorylated-NCC expression in membrane lysates was significantly upregulated in high-Na/high-Cl rats compared with high-Na/half-Cl rats (Fig. 5, C and D).

HCTZ Alleviated Hypertension and Attenuated ␣SMA and IL-17A Expression
To determine the role of NCC in Aldo/NaCl rats, we investigated the effect of HCTZ on blood pressure and renal inflammation and damage. Oral administration of HCTZ, a pharmacological inhibitor of NCC, at a dose of 25 mg·kg Ϫ1 ·day Ϫ1 for 4 wk, suppressed the rise in blood pressure in Aldo/NaCl rats, along with reduced urinary protein excretion (Fig. 6, A and B). Immunoblot assays also revealed that, in addition to ␣SMA, IL-17A decreased in HCTZ-treated rats compared with vehicle-treated rats (Fig. 6C).

HCTZ Suppressed Phosphorylated-NCC Expression in the Membrane Fraction
To identify the inhibitory effect of NCC on transporters at the distal nephron in Aldo/NaCl rats, we investigated the protein expression of NCC, ␣ENaC, and pendrin between vehicle-treated and HCTZ-treated rats. Although expression levels of total and phosphorylated NCC in membrane lysates were significantly decreased in HCTZ-treated rats, those of ␣ENaC and pendrin did not change, irrespective of HCTZ treatment (Fig. 7, A and B).

DISCUSSION
In this study, we found that aldosterone-induced salt-sensitive hypertension increased in rats administered NaCl, but not NaHCO 3 , in their drinking water which was accompanied by significantly increased expression of NCC. Administration of NaCl, but not NaHCO 3 , increased not only renal fibrosis but also infiltration of inflammatory cells along with upregulated expression of IL-17A in aldosterone-infused rats. Similarly, even with the equimolar Na ϩ overloading, a high-Na/high-Cl diet showed higher blood pressure than the high-Na/half-Cl diet in aldosterone-infused rats. High-Na/high-Cl diet upregulated expression of phosphorylated NCC, whereas the high-Na/ half-Cl diet did not. Last, NCC inhibition with HCTZ improved hypertension, renal fibrosis, and inflammation in Aldo/ NaCl rats. These findings suggest that NCC-mediated Cl Ϫ reabsorption plays a pivotal role in the development of hypertension and renal damage in this rat model of aldosteroneinduced salt-sensitive hypertension.
We found that Aldo/NaCl treatment induced elevated blood pressure and increased the expression of NCC and pendrin compared with Aldo/water treatment. Among several inducers of salt-sensitive hypertension, aldosterone is well known to increase renal NaCl reabsorption through activating ␣ENaC and NCC (7,9,20). Thus exogenous aldosterone infusion is regularly used for animal models of salt-sensitive hypertension. Recently, DOCA was reported to increase expression levels of pendrin in mice (27,41). Previous studies have demonstrated that pendrin enhances ␥ENaC function, possibly through its ability to keep channels open and alter subcellular protein distribution (25), and that elevated blood pressure is not observed in pendrin-null mice with DOCA/NaCl treatment (41). In contrast, other studies have reported that specific deletion of the pendrin gene did not attenuate salt-sensitive hypertension (32,44). Therefore, the role of pendrin on the progression of hypertension remains controversial. In the current study, we found that in rats that drank NaHCO 3 , or had the high-Na/half-Cl diet or received HCTZ treatment, salt-sensitive hypertension improved without changes in pendrin expression. Taken together, the results suggest that pendrin is not likely to be responsible for Aldo/NaCl-induced hypertension.
Aldo/NaHCO 3 and high-Na/half-Cl rats did not show significant elevations in blood pressure nor did they show upregulation of NCC, indicating that, in addition to aldosterone, Cl Ϫ is essential for increased salt-sensitivity through activation of NCC. We also observed that inhibition of NCC with oral administration of HCTZ attenuated not only hypertension and renal damage, including proteinuria, interstitial fibrosis and inflammation, in Aldo/NaCl rats but also reduced total and phosphorylated-NCC expression. NCC phosphorylation is induced by various stimuli, including hypokalemia, aldosterone, angiotensin, hyperinsulinemia, and sympathetic stimulation, which are all known to contribute to salt sensitivity (22,34,36,37,39). Our data indicate that NCC-mediated Cl Ϫ reabsorption plays an important role in the development of salt-sensitive hypertension.
Previous research has shown that NCC knockout mice upregulate not only ␥ENaC expression but also pendrin expression (14). Additionally, aldosterone-induced alkalosis leads to enhanced expression of pendrin (29,41). These findings suggest that either ␥ENaC or pendrin is complementarily overex- Values are means Ϯ SD. Control rats: rats fed a 0.26% NaCl (0.10% Na/0.16% Cl) diet. High-Na/high-Cl rats: rats fed an 8.0% NaCl (3.14% Na/4.85% Cl) diet. High-Na/half-Cl rats: rats fed a 4.0% NaCl 6.67% sodium citrate (3.14% Na/2.43% Cl) diet. All rats were subject to heminephrectomy followed by aldosterone infusion. Data were analyzed with ANOVA followed by Tukey's test. Statistical differences are indicated as *P Ͻ 0.05 (vs. control rats), †P Ͻ 0.05 (vs. High-Na/half-Cl rats). pressed to retain NaCl as well as to maintain acid-base homeostasis during blocking of the NCC function (32). However, in the current study, NCC inhibition with HCTZ was not compensated for by increased expression of ␥ENaC (data not shown) and pendrin. One possible explanation for this finding is that the biological half-life of oral HCTZ is only 1.7 and 13.1 h for the ␣and ␤-phases, respectively, indicating that HCTZ administration only has a temporary inhibitory effect on NCC, unlike that observed in knockout animals (2). Another possible mechanism is that ␥ENaC and pendrin might have already been upregulated in Aldo/NaCl rats. In either case, our data suggest that ␥ENaC and pendrin do not confer the ability to compensate for NCC.
Previous studies found that administration of DOCA or angiotensin II could not induce hypertension in rodents lacking T and B lymphocytes (16,33). It is also reported that immunosuppressants, such as TNF-␣ inhibitors, MMF, or mizoribine, prevent hypertension in salt-sensitive animal models (5,10,11). Importantly, recent studies have clarified that NaCl promotes autoimmune disease through Th17 (18), and that deletion of IL-17A ameliorates hypertension and renal injury in DOCA/NaCl rats (1). These findings suggest that T cell-driven inflammation is involved in aldosterone-induced hypertension.
In the current study, the protein expression level of IL-17A was suppressed in Aldo/NaHCO 3 rats and Aldo/NaCl rats with HCTZ treatment. Taken together, it appears that Cl Ϫ plays an essential role in the development of salt-induced inflammation.
Dysregulated Th17 cells are regarded as a therapeutic target not only for salt-sensitive hypertension but also inflammatory renal diseases, including lupus nephritis, IgA nephropathy, crescentic glomerulonephritis, and posttransplant rejection (12,19,26,40). Chronic inflammation is recognized to cause subsequent fibrosis through production of transforming growth factor-␤1 (21). A recent study reported that T cells are required for both collagen deposition and hypertension (43). Antiinflammatory therapies suppress renal fibrosis, suggesting that inhibition of T cells, at least in part, has antifibrotic effects. These findings raise the possibility that Cl Ϫ -mediated inflammation contributes to renal fibrosis as well as salt-sensitive hypertension.
In conclusion, aldosterone-infused rats with NaCl in their drinking water had increased hypertension and renal inflammation compared with NaHCO 3 -treated controls. Reduction of dietary Cl Ϫ also decreased the development of hypertension as well as NCC activation, even with equimolar Na ϩ overloading. As activation of T lymphocytes is known to participate in the Aldo/NaCl rats given distilled water. Values are means Ϯ SD. Blood pressure data at each time point were analyzed using the general linear model, and statistical differences compared with vehicle-treated rats are indicated as §P Ͻ 0.05. The other data were analyzed with Student's t-test for comparisons between two groups. Statistical differences are indicated as *P Ͻ 0.05, **P Ͻ 0.01. development of both hypertension and renal damage, Cl Ϫ overload may also be involved in this critical process. HCTZ treatment was found to ameliorate blood pressure elevation and renal damage through the reduction of NCC expression. These results suggest that NCC-mediated Cl Ϫ reabsorption plays an important role in the pathogenesis of salt-sensitive hypertension.

GRANTS
This work was supported by the Tsuchiya Memorial Medical Foundation.

DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors. Fig. 7. Hydrochlorothiazide attenuated the protein expression level of Na ϩ -Cl Ϫ transporter and its phosphorylated forms in the membrane fraction. A: immunoblot assay showing membrane expression of Na ϩ -Cl Ϫ transporters in Aldo/NaCl rats with or without hydrochlorothiazide (HCTZ) treatment. B: quantification of protein expression in the membrane fraction isolated from the kidney of Aldo/NaCl rats with or without HCTZ treatment. HCTZ-treated rats (HCTZ): Aldo/NaCl rats given 25 mg·kg Ϫ1 ·day Ϫ1 hydrochlorothiazide by oral gavage. Vehicle-treated rats (vehicle): Aldo/NaCl rats given distilled water. Values are means Ϯ SD. Data were analyzed using Student's t-test for comparisons between two groups. Statistical differences are indicated as *P Ͻ 0.05.