Upregulation of Renal Sodium Transporters in D₅ Dopamine Receptor Deficient Mice

Abstract

D₅ dopamine receptor (D₅R) deficient (D₅-/-) mice have hypertension that is aggravated by an increase in sodium intake. The current experiments were designed to test the hypothesis that a dysregulation of renal sodium transporters is related to the salt sensitivity in D₅-/- mice. D₅R was expressed in the renal proximal tubule, thick ascending limb, distal convoluted tubule, and cortical and outer medullary collecting ducts in D₅ +/+ mice. On a control Na⁺ diet, renal protein expressions of sodium-potassium-2 chloride cotransporter (NKCC2), sodium chloride cotransporter (NCC), α and γ subunits of the epithelial sodium channel (ENaC) were greater in D₅-/- than in D₅+/+ mice. Renal renin abundance and urine aldosterone levels were similar but renal angiotensin II receptor (AT₁R) protein expression was increased in D₅-/- mice. An elevated Na⁺ diet increased further the elevated blood pressure of D₅-/- mice but did not affect the normal blood pressure of D₅+/+ mice. The increased levels of NKCC2, NCC, α and γ subunits of ENaC persisted with the elevated Na⁺ diet and unaffected by chronic AT₁R blockade (losartan) in D₅-/- mice. The expressions of proximal sodium transporters, sodium hydrogen exchanger type 3 (NHE3) and sodium phosphate cotransporter type 2 (NaPi2) were increased by the elevated Na⁺ diet in D₅-/- mice; the increased expression of NHE3 but not NaPi2 was abolished by AT₁R blockade. Our findings suggest that the increased protein expression of sodium transporters/channels in distal nephron segments may be the direct consequence of the disruption of D₅R, independent of the renin-angiotensin aldosterone system.

Introduction

Dopamine receptors include the D₁-like (D₁R and D₅R) and D₂-like (D₂R, D₃R, and D₄R) receptor subfamilies. All dopamine receptor subtypes are expressed in the kidney (1, 2). Lack or dysfunction of one dopamine receptor gene may affect renal tubular sodium transport, which may result in sodium retention and contribute to the development of hypertension. However, the differential nephron segment expression of dopamine receptor subtypes suggests that they may regulate sodium transport and blood pressure via different mechanisms (3).

The locus of the human D₅R gene, 4p15.1–16.1 and its pseudogenes, 1q21.1 and 2p11.1-p11.2 (4), have been linked to human essential hypertension (5-8). Several single nucleotide polymorphisms of the human D₅R gene are associated with diminished function (9). Disruption of the D₅R gene in mice increases blood pressure that is aggravated by an increase in sodium intake (10,11). It is possible that the salt sensitivity in D₅-/- mice is caused by increased sodium transport in the kidney but the nephron segment(s) involved has not been established.

Active sodium transport along the nephron occurs mainly through transport proteins at the apical membrane: sodium-hydrogen exchanger isoform 3 (NHE3) and the type 2 sodium phosphate cotransporter (NaPi2) in the proximal tubule, the bumetanide-sensitive sodium-potassium-2 chloride cotransporter (BSC1 or NKCC2) and NHE3 in the thick ascending limb of loop of Henle, the thiazide-sensitive sodium-chloride cotransporter (TSC or NCC) in the distal convoluted tubule and the amiloride-sensitive epithelial sodium channel (ENaC, subunits α, β and γ) in the connecting tubule and collecting duct, as well as Na⁺K⁺ ATPase at the basolateral membrane in almost all nephron segments. However, it is not known if the D₅R can regulate the expression of these proteins, in part, because the nephron segments in which the D₅R is expressed in mice have not been determined (10-12). It is also not clear if the knockout of the D₅R gene alters the renal expression of the different dopamine receptor subtypes. Therefore, the current experiments were designed to determine a novel renal mechanism of salt-sensitive hypertension by testing the hypothesis that a dysregulation of renal sodium transporters is related to the salt sensitivity of the blood pressure of D₅-/- mice. We measured blood pressure, urinary sodium and aldosterone excretions, and renal protein expressions of dopamine receptors, the type 1 angiotensin II receptor (AT₁R), renin and sodium transporters/channels in D₅-/- mice and D₅+/+ littermates on a control and an elevated sodium diet. In order to determine if the increased expression and activity of AT₁R is responsible for any alteration in renal sodium transporters in D₅-/- mice (13), we also measured renal sodium transporters/channels in mice chronically given the AT₁R blocker losartan.

Methods

Generation of D₅-/- mice

Heterozygous mice (D₅ +/-) were generated as reported (10,11). The D₅-/- and D₅+/+ (6^th generation in C57BL/6/Taconic) mice were identified by DNA genotyping. C57BL/6/Taconic mice were used instead of C57BL/6 Jackson mice because C57BL/6/Taconic mice, unlike C57BL/6 Jackson mice, do not develop elevated blood pressure with an increase in sodium intake (11, 14). The studies were conducted in accordance with NIH guidelines for the ethical treatment and handling of animals in research, and approved by the Institutional Animal Care and Use Committee, initially at Georgetown University, and subsequently at the Children's National Medical Center.

Sodium balance study in ration-fed mice

D₅-/- and D₅+/+ mice (4-6 months old, mixed gender, n=5) were housed in metabolic cages. The mice, for each 25 g of body weight, were ration-fed for 10 days with gelled control sodium diet (0.2-0.3% Na⁺) consisting of ground rodent chow (5 g) and melted agar (0.04 g) in 10 ml distilled water (15-17). Ration feeding of gelled food eliminates the possibility of food crumbs contaminating the urine collections. Blood pressures were measured in the aortae, via the femoral arteries of mice under anesthesia (pentobarbital, 50 mg/kg intravenously) (10,11,15). Urine and blood samples were analyzed for electrolytes (E4A Electrolyte system, Beckman, Fullerton, CA), creatinine (Creatinine Analyzer 2, Beckman), and aldosterone (radioimmunoassay, Diagnostic Products Corp., Los Angeles, CA).

Semi-quantitative immunoblotting

The kidneys, removed after euthanasia, were homogenized (15-17). Renal plasma membrane-enriched fractions were obtained by centrifugation of kidney homogenates at 17,000 × g, 30 min at 4°C (18); the 17,000 × g fraction resulted in the enrichment of Na⁺K⁺ ATPase α subunit by more than 60%, relative to whole kidney homogenates (data not shown). Equal amounts of protein were separated by 7.5 or 10% SDS PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were probed with the indicated primary antibodies, and then exposed to horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL). The primary antibodies were rabbit polyclonal antibodies against NHE3, NaPi2, NKCC2, NCC, ENaC (gifts from Dr. Mark A. Knepper, LKEM, NHLBI, NIH), D_1-5R, actin (Sigma), and mouse monoclonal antibody to Na⁺K⁺ ATPase α subunit (Upstate Biotechnology, Lake Placid, NY). The specificities of these antibodies have been reported (16-26). The specificity of the polyclonal D₅R antibody was initially determined by the ability to the antibody to detect human D₅R protein heterologously expressed in HEK293 cells (positive control) without and with peptide-pre-adsorption (negative control) (12). In this report we show that the D₅R antibody recognizes D₅R in kidneys from mice with D₅R gene (D₅R +/+) but not in mice without the D₅R gene (D₅R -/-) (Figure 1). The chemiluminescent signals were quantified by the NIH Image J program and normalized against the corresponding actin bands.

Figure 1.

Renal D₅R expression

Figure 1A: Immunoblots of D₅R and actin in whole kidney homogenates from D₅-/- (n=5) and D₅+/+ mice (n=5, upper panel). A 55 kDa band is found in D₅+/+ but not in D₅-/- mice, confirming the disruption of the D₅R gene and the specificity of the D₅R antibody. Actin is present in both mouse strains. Renal immunocytochemistry of D₅R (lower panel: 40×). Positive staining (purple) is observed in specific nephron segments in the D₅+/+ but not in the D₅-/- mice.

Figure 1B. Renal D₅R expression in D₅+/+ mice. The D₅R staining (purple, 400×) is mainly located in the proximal convoluted tubule (PCT), distal convoluted tubule (DCT), and cortical collecting duct (CCD). There is also D₅R immunostaining in the proximal straight tubule (PST), thick ascending limb of Henle (TAL), and outer medullary collecting duct (OMCD). Little or negative staining is found in the juxtaglomerular cell (JGC), afferent arteriole (AA), macula densa, glomerulus (G), and thin descending limb (TDL) of Henle.

Renal immunocytochemistry

In separate studies, kidneys from anesthetized D₅+/+ and D₅-/- mice were perfused with PBS (pH 7.4) and fixed with Histochoice (Amresco, Solon, OK). Three μm sections were cut from paraffin-embedded tissues for H&E staining and indirect immunoperoxidase labeling (12). Immunoreactivity was visualized with the VIP kit (Vector, Burlingame, CA) and bright-field microscopy, under the same conditions and magnifications (Eclipse E600, Nikon, Tokyo, Japan). The experiments were performed at least 3 times.

Chronic salt loading

In additional studies, D₅+/+ and D₅-/- mice were fed an elevated sodium (2-3% Na⁺) diet for 3 weeks. After euthanasia, the kidneys were harvested for immunoblotting, as above.

Telemetry

D₅+/+ and D₅-/- mice were fed a control sodium diet (0.2-0.3 %Na⁺) for 3-4 weeks, then switched to an elevated sodium (2-3% Na⁺)(27) diet for 2 weeks. The mice had free access to food and water. Blood pressures were monitored by telemetry, via pre-implanted transmitters in conscious mice (11, 15). Mice were acclimated to the apparatus and the environment for a total of 7 days before measurements were taken.

Losartan treatment

D₅+/+ and D₅-/- mice were fed with an elevated sodium (2-3% Na⁺) diet for 3 weeks. In the last 5-7 days of the study, the AT₁R blocker, losartan, was injected intraperitoneally (20 mg (100 μl) /kg/day, × 5-7 days) (26). After euthanasia, the kidneys were harvested for immunoblotting, as above.

Statistical analysis

Data are expressed as mean ± SE and as percent of D₅+/+ mice for immunoblotting. Unpaired Student's t-test was used for a two-group comparison and ANOVA, Holm-Sidak test for multi-group comparison. P<0.05 was considered significant.

Results

Distribution of D₅R along the nephron

Whole kidney homogenates immunoblotted with the polyclonal antibody to D₅R showed a 55 kDa band in D₅+/+ but not in D₅-/- mice. Strong immunostaining was observed in kidneys from D₅+/+ mice but not in D₅-/- mice (Figure 1A). No immunostaining was observed when the antibody was blocked by pre-adsorption with the immunizing peptide (not shown). These studies confirmed the specificity of the D₅R antibody and the disruption of the D₅R gene in D₅-/- mice.

D₅R was expressed in the proximal convoluted and straight tubules (including brush border membranes), thick ascending limb of Henle, distal convoluted tubule, and cortical and outer medullary collecting ducts. Minimal staining was observed in the juxtaglomerular cell, glomerulus, macula densa, afferent arteriole, and thin descending limb of Henle (Figure 1B).

Effect of the disruption of D₅R gene (D₅-/-) on some physiological parameters in mice fed a control sodium diet

All physiological data are summarized in Table1. We have reported that the blood pressures are higher in D₅-/- mice than D₅+/+ littermates when measured by telemetry in the conscious state or when measured from the aorta via the femoral artery in the anesthetized state (10,11). The systolic and diastolic blood pressures were measured under anesthesia in this group of mice in the current studies. The higher blood pressure in D₅-/- compared to D₅+/+ mice was confirmed (Table 1). There were no differences in heart rate, body weight, and food/water intake between the two groups. Serum Na⁺, K⁺, Cl^- and creatinine concentrations and creatinine clearance were also similar in the two mouse strains. No differences were found in daily urinary sodium and water excretion between the two mouse strains (only the data on the last day of the balance study are shown in Table 1). The higher blood pressure in D₅-/- mice than in D₅+/+ littermates was not associated with a greater urinary sodium, suggesting that a state of sodium retention may have already been achieved by the time the variables were measured. Mice retain sodium for several hours and then achieve sodium balance by the first or second day after a high salt load (28, 29).

Table 1.

Physiological data of D₅-/- and D₅+/+ mice ration-fed a control sodium diet

Mouse group	D5+/+	D5-/-
SBP(mmHg)	93± 2	122± 5*
DBP(mmHg)	78 ± 3	101± 2*
Heart rate (beats/min)	347 ± 8	328 ± 14
Body weight (g)	26± 0.9	25 ± 0.2
Serum Na+ (mmol/l)	152± 1	148 ± 2
Serum K+ (mmol/l)	4.3±0.4	4.5 ±0.8
Serum Cl- (mmol/l)	124±0.4	126 ± 2.9
Serum creatinine (μmol/l)	26.4±3.5	31.7±6.2
Gelled-food intake (g/day)	14.7± 0.2	13.2 ±0.7
Water intake (ml/g/day)	0.39±0.01	0.35±0.02
Urine output (ml/day)	2.9± 0.3	3.1 ± 0.9
Na+ excretion (mmol/day)	0.31 ± 0.03	0.29± 0.07
Creatinine clearance (ml/day)	159± 33	120 ± 32

Data are M ± SE.

^*P<0.05, vs. D₅+/+ mice (4-6 months old, mixed gender, n=5/group), Student's t-test,. SBP: systolic blood pressure, DBP: Diastolic blood pressure, MAP: Mean arterial blood pressure. BPs were measured under pentobarbital anesthesia.

Effect of the disruption of D₅R gene (D₅-/-) on the expression of renal sodium transporter, channels, and pump in mice fed a control sodium diet

The protein expressions of renal sodium transporters, channels, and pump in D₅-/- and D₅+/+ mice are shown in Figures 2A, B &C. NHE3 expression did not differ between D₅-/- and D₅+/+ mice while NaPi2 protein was decreased in D₅-/- relative to D₅+/+ mice. Expressions of NKCC2, a sodium transporter in the thick ascending limb of Henle, and NCC, a sodium transporter in the distal convoluted tubule, were increased in D₅-/- mice relative to D₅+/+ mice (Figure 2), although the nephron segment distribution of NCC (Figure 3) was not altered in D₅-/- mice. The α and γ subunits of ENaC were also increased in D₅-/- mice but no differences were found in the expression of the β subunit of ENaC, and the α subunit of Na⁺K⁺ ATPase (Figures 2 B & C) between the two groups.

Figure 2.

Immunoblotting of whole kidney homogenates for sodium transporters, channels, and pump in D₅-/- and D₅+/+ mice fed a control sodium diet.

Figure 2A. Immunoblots of NHE3, NaPi2, NKCC2 and NCC in D₅-/- (n=5) and D₅+/+ mice (n=5).

Figure 2B. Immunoblots of the α, β, and γ subunits of ENaC and α subunit of Na⁺K⁺ ATPase (αNKA) in D₅-/- (n=5) and D₅+/+ mice (n=5).

Figure 2C. Densitometric analysis of the immunoblots. The protein expressions of NKCC2, NCC, αENaC and γENaC are increased and the protein expression of NaPi2 is decreased, while the protein expression of NHE3 and Na⁺K⁺ ATPase α subunit (αNKA) is not altered in D₅-/- mice relative to D₅R+/+ mice. *P<0.05, Student's t-test.

Figure 3.

Renal immunostaining of NCC in D₅-/- and D₅+/+ mice. NCC protein (purple) is expressed in the luminal membrane of the distal convoluted tubule; the expression is greater in D₅-/- than in D₅+/+ mice (upper panel, 1000×). Nephron segment distribution of NCC (lower panel 200×) is similar in D₅-/- and D₅+/+ mice.

Effect of the disruption of D₅R gene (D₅-/-) on renal dopamine receptors, AT₁R, and renin in mice fed a control sodium diet

The renal protein expression of all D₂-like dopamine receptors was similar in D₅-/- and D₅+/+ mice, but the renal protein expression of D₁R was lower in D₅-/- than in D₅+/+ mice (Figure 4A), indicating that there was no compensatory upregulation of the other dopamine receptor subtypes in the kidney as a consequence of the deletion of the D₅R gene.

Figure 4.

Immunoblotting of dopamine receptor subtypes, renin, and AT₁R in D₅+/+ and D₅-/- mice fed a control sodium diet.

Figure 4A. Immunoblots in whole kidney homogenates from D₅+/+ (n=5) and D₅-/- mice (n=4). The protein expression of D₁R is decreased but not the expression of D₂R, D₃R, and D₄R in D₅-/- mice. *P<0.05, Student's t-test.

Figure 4B. Immunoblotting in whole kidney homogenates for AT₁R, and renin in D₅-/- and D₅+/+ mice (n=4). Relative to D₅+/+ mice, the protein expression of AT₁R is increased in D₅-/- mice, but the renin protein abundance is similar in the two mouse strains. *P<0.05, Student's t-test.

Renal AT₁R protein was increased in D₅-/- mice, in agreement with our previous report (13, 26) but renal renin protein was similar in D₅-/- and D₅+/+ mice (Figure 4B). The specificity of the antibodies against AT₁R (AT₁R, N-10, Santa Cruz, CA) and renin (Innovative Research, Southfield, MI) were verified (data not shown) prior to these experiments. Urinary aldosterone excretion was also similar in the two mouse strains (D₅-/-: 2.9±1.1, D₅+/+: 2.5±0.7, ng/mg of creatinine, n=6/group), close to the values reported in the literature (30, 31). We have also reported that the levels of renal and plasma catecholamines are similar in D₅-/- and D₅+/+ mice (10, 26).

Effect of the disruption of D₅R gene (D₅-/-) in mice fed an elevated sodium diet

Figure 5 shows the mean arterial blood pressure in D₅-/- and D₅+/+ mice. The data included some of the mice reported in our previous publication (11). On the control sodium diet, blood pressure was higher in D₅-/- mice than in D₅+/+ littermates. The elevated sodium diet increased blood pressure further in D₅-/- mice but did not affect the normal blood pressure of D₅+/+ mice, confirming the salt sensitivity of blood pressure of D₅-/- mice (11).

Figure 5.

Effect of an elevated sodium diet on blood pressure in D₅+/+ (n=8) and D₅-/- mice (n=6). Blood pressure (average shown for each diet) was measured by telemetry in conscious mice. The mean arterial blood pressure (MAP) was higher in D₅-/- than D₅+/+ mice fed a control sodium diet (2 weeks). MAP was increased further by an elevated sodium diet (2 weeks) in D₅-/- mice. P<0.05, * vs. D₅+/+; # vs. others, ANOVA, Holm-Sidak test.

The increased protein levels of renal distal sodium transporters, channels, and pump in the D₅-/- mice fed the control sodium diet, including NKCC2, NCC and α and γ subunits of ENaC persisted in the D₅-/- mice fed the elevated sodium diet. In addition, NHE3, NaPi2 and the α subunit Na⁺K⁺ ATPase were also increased in D₅-/- mice, relative to D₅+/+ mice. The β subunit of ENaC was similar in the two mouse strains (Table 2).

Table 2.

Results of the immunoblotting of Na⁺ transporters/channels in mice fed an elevated sodium diet

Mouse group	D5+/+ (n=3)	D5-/-(n=3)
NHE3	100 ± 6	287 ± 62*
NaPi2	100 ± 9	177 ± 19*
NKCC2	100 ± 3	174 ± 15*
NCC	100 ±39	705 ± 141*
αENaC	100 ± 7	227 ±13*
βENaC	100 ± 27	94 ± 5
γENaC	100 ± 5	454 ± 110*
αNKA	100 ±7	322 ± 27*

Data are M ± SE, % of D₅+/+ mice, corrected by actin.

^*P<0.05, vs. D₅+/+, Student's t-test αNKA= Na⁺K⁺ ATPase, α subunit

Effects of losartan on renal sodium transporters in D₅-/- and D₅+/+ mice

To determine if the increased AT₁R expression in the kidney contributed to the alterations in sodium transporters, channels, and pump in D₅-/- mice, we studied mice treated with the AT₁R blocker losartan, and fed the elevated sodium diet (Figure 6A, B&C). We have reported that renal AT₁R is increased (13, 26) and losartan treatment normalized the high blood pressure of D₅-/- mice (26).

Figure 6.

Renal sodium transporters, channels, and pump in D₅+/+ (n=4) and D₅-/- mice (n=5) fed an elevated sodium (HS) diet and treated with an AT₁R blocker (Losartan)

Figure 6A. Immunoblots of the proximal sodium transporters and Na⁺K⁺ ATPase α subunit (αNKA).

Figure 6B. Immunoblots of the renal distal sodium transporters. Arrow points to 90 kDa γENaC which is probably an inactive form (21).

Figure 6C. Densitometric analysis of the immunoblots. NaPi2 and Na⁺K⁺ ATPase α subunit (αNKA) are increased in D₅-/- relative to D₅+/+ littermates. D₅-/- mice continued to have increased renal expression of NKCC2, NCC and α and γ subunits of ENaC, despite losartan treatment. *P<0.05, Student's t-test.

The increased protein expression of the distal sodium transporters (NKCC2, NCC, α and γ subunits of ENaC) (Figure 6B and 6C) in D₅-/- mice, on the control and elevated sodium diets, respectively, persisted in the losartan-treated D₅-/- mice. There were 2 bands for γENaC, a higher band (90kDa), and a lower band (75kDa), which is believed to be the active form of γENaC, following the proteolysis or deglycosylation of the larger, inactive form (90kDa) (21). The higher band was absent and the density of the lower band was increased in D₅-/- mice, relative to their D₅+/+ littermates. We do not know the reason for the absence of the higher band in the D₅-/- mice but it is possible that all the “inactive” forms are converted to the “active” forms in these mice. Losartan abrogated the increased expression of NHE3 but not the increased expression of NaPi2 and Na⁺K⁺ ATPase α subunit observed in D₅-/- mice (Figure 6, Table 2); losartan also decreased the expression of the β subunit of ENaC.

Discussion

Under normal circumstances, an increase in blood pressure activates renal mechanisms to increase sodium excretion to maintain a normal blood pressure (pressure-natriuresis phenomenon) (32). In the current report, the sodium intake and urinary sodium excretion on the control sodium diet are similar in D_5-/- mice and D₅+/+ littermates, indicating the achievement of sodium balance. However, this was achieved at the expense of higher blood pressure in the D₅-/- mice, indicating a blunting of the pressure-natriuresis relationship with the disruption of the D₅R gene.

The natriuresis associated with increased sodium intake and blood pressure, thought to be an example of aldosterone escape, has been reported to caused by a decrease in the protein abundance/activity of NCC in the renal distal convoluted tubule (25). It is possible that the impaired pressure-natriuresis mechanism in D₅-/- mice may be due to increased renal expression of NCC. In the current study, D₅-/- mice not only have increased protein expression of NCC, but also one of the sodium transporters in the thick ascending limb (NKCC2) and the α and γ subunits of ENaC in the collecting duct. The increase in renal sodium transporters with the elevated sodium diet in D₅-/- mice is not in agreement with the effect of high salt intake in normotensive rats (27,33). There may be species and strain differences because an elevated sodium diet for one week does not affect NHE3 or NKCC2 expression in SJL mice (unpublished data), similar to that reported in normotensive rats; these transporters are decreased by the elevated sodium diet in wild-type C57BL/6 mice, the genetic background of D₅-/- mice. NCC expression, which is decreased by an elevated sodium diet in the rat in one study (27) but not in another (33), is also decreased by the elevated sodium diet in SJL mice but not in C57BL/6 wild-type mice (unpublished data). In contrast, the elevated sodium diet in C57BL/6 mice with deleted D₅R increases NCC expression (Table 2). The disruption of the D₅R gene in mice results in the up-regulation of renal sodium transporters/channels in all of the distal nephron segments. The increased expression of these distal tubule sodium transporters/channels in D₅-/- mice may have attenuated the magnitude of the natriuresis that would have normally accompanied the increase in systemic blood pressure.

Using a specific antibody against D₅R, we find D₅R expression in the thick ascending limb, distal convoluted tubule, as well as the cortical and outer medullary collecting ducts, the nephron segments that express the sodium transporter/channels that are increased in D₅-/- mice fed either control or elevated sodium diet. These data suggest that the D₅R may have a direct inhibitory effect on the expression of renal sodium transporters and channels in the distal nephron segments. An effect of D₅R on distal sodium transporter and channel expression could also be indirect, as a result of increased AT₁R expression. D₅R promotes the degradation of the AT₁R (26) and renal AT₁R expression is increased in D₅-/- mice (13, 26), confirmed in the current report. Both NCC and αENaC are regulated by AT₁R (34, 35). The protein abundance of NCC and αENaC is decreased in AT_1A null mice (34) and in rats treated with AT₁R antagonists but increased in rats treated with angiotensin II (35).

Renal renin abundance and urinary aldosterone levels are not increased in D₅-/- mice. That the D₅R may not regulate renin and indirectly, aldosterone secretion, is in keeping with the absence of D₅R immunostaining in the juxtaglomerular apparatus, including the juxtaglomerular cell, and macula densa. These data suggest that the D₅R, unlike the D₁R (1-3, 36), may not regulate the secretion/release of renin. High salt intake suppresses renin secretion in kidney. Renin secretion is probably suppressed by salt intake to a similar extent in both D₅+/+ and D₅-/- mice since there is no difference in renal renin level between the mouse strains placed on high salt diet (26). It is, therefore, unlikely that alterations in angiotensin II levels are responsible for the differential expression of distal sodium transporters in D₅-/- and D₅+/+ mice. Nevertheless, we determined whether the increased expression and activity of the AT₁R is responsible for the increased expression of distal tubule sodium transporters/channel subunits in D₅-/- mice by blocking AT₁R function with losartan. In spite of AT₁R blockade, the increase in renal distal sodium transporters/channel subunits observed in D₅-/- mice fed the elevated sodium diet persisted, suggesting that the increased expression of distal renal sodium transporters/channel subunits in D₅-/- mice is independent of the AT₁R. The increase in NHE3, but not NaPi2, is abolished by AT₁R blockade, suggesting an additional effect of AT₁R on NHE3 expression in D₅-/- mice. The absence of D₅R seems to enhance the renal tubular effects of AT₁R when sodium intake is increased because losartan treatment abolishes not only the increase in NHE3 expression, but also the salt-sensitivity in D₅-/- mice (26). Endothelin is probably not involved in the hypertension of D₅-/- mice because endothelin A and B receptor antagonists, BQ610 (37) and BQ788 (38), respectively, do not affect the arterial blood pressure of D₅-/- or D₅+/+ mice (10). The role of increased sympathetic tone (10) and reactive oxygen species production (11) in the increased expression of renal distal tubule transporters in D₅-/- mice was not evaluated (3).

In summary, relative to D₅+/+ littermates, D₅-/- mice have higher blood pressure and greater renal protein expressions of NKCC2, NCC, and α and γ subunits of ENaC on control and elevated sodium diet which is not abrogated by AT₁R blockade. The expression of proximal sodium transporters, NHE3 and NaPi2, is increased only on elevated sodium diet and the increase in NHE3 but not NaPi2 is abolished by AT₁R blockade in D₅-/- mice. We suggest that the increased renal protein expression of these transporters/channels may play an important role in the pathogenesis of salt-sensitive hypertension that occurs with the disruption of the D₅R gene in mice.

Perspectives

Several single nucleotide polymorphisms of the human D₅R gene are associated with diminished function of the receptor (9). The increased renal distal sodium transporters and channels (NKCC2, NCC and ENaC) suggest that inhibitors of distal sodium transport may have a greater anti-hypertensive effect in humans with impaired function of the D₅R