Altered expression of renal aquaporins and α-adducin polymorphisms may contribute to the establishment of salt-sensitive hypertension

822 JULY 2011 | VOLUME 24 NUMBER 7 | 822-828 | AMERICAN JOURNAL OF HYPERTENSION

nature publishing groupORIGINAL CONTRIBUTIONS

Essential hypertension is a complex syndrome determined by genetic as well as environmental factors. In ~50% of the patients, the hypertension is found to be salt-sensitive,1 a fea-ture that may depend on genetic factors.2

The kidney plays an important role in the genesis and main-tenance of salt-sensitive hypertension in animal models as well as in humans. In fact, transplantation of kidneys from hyper-tensive (HT) rats into normotensive (NT) rats can induce the HT phenotype.3

Animal models of genetic hypertension have provided an excellent tool to study the mechanisms that leads to the devel-opment and maintenance of hypertension, and the role of the kidney in these processes.4

In one of these models, the rat Milan HT strain (MHS), hypertension develops because of a primary increase in renal tubular Na+ reabsorption,5 induced by an increased activity and expression of Na+-K+-ATPase6 associated with mutation of the cytoskeletal protein adducin.7–10 Adducin mutations reduce Na+-K+-ATPase endocytosis, thereby increasing the ability of tubular cells to reabsorb Na+ ions.11,12

Interestingly, linkage studies have shown that adducin poly-morphisms are associated with a subset of patients affected by hypertension.13

Arterial blood pressure is largely dependent on regulation of the extracellular fluid volume which, in turn, depends on selective reabsorption of sodium and water occurring in spe-cialized nephron tubule segments. The sodium transporters and the water channel aquaporins (AQPs) expressed in kidney tubules are therefore critical for blood pressure regulation.

At least six AQPs (AQP1, 2, 3, 4, 6, and 7) are currently known to be expressed in the kidney.14,15 A physiologically relevant role in water reabsorption has been demonstrated

1Department of General and Environmental Physiology, University of Bari, Bari, Italy; 2Prassis Research Institute, Sigma Tau, Milan, Italy; 3Division of Nephrology, Dialysis, and Hypertension, University Vita-Salute San Raffaele, Milan, Italy. Correspondence: Giuseppe Procino ([email protected])

Received 17 August 2010; first decision 12 October 2010; accepted 15 January 2011.

© 2011 American Journal of Hypertension, Ltd.

Altered Expression of Renal Aquaporins and α-Adducin Polymorphisms May Contribute to the Establishment of Salt-Sensitive HypertensionGiuseppe Procino1, Francesca Romano1, Lucia Torielli2, Patrizia Ferrari2, Giuseppe Bianchi3, Maria Svelto1 and Giovanna Valenti1

BACKGROUNDSodium-sensitive hypertension is caused by renal tubular dysfunction, leading to increased retention of sodium and water. Previous findings have suggested that single-nucleotide polymorphisms of the cytoskeletal protein, α-adducin, are associated with increased membrane expression of the Na/K pump and abnormal renal sodium transport in Milan hypertensive strain (MHS) rats and in humans. However, the possible contribution of renal aquaporins (AQPs) to water retention remains undefined in MHS rats.

METHODSKidneys from MHS rats were analyzed and compared with those from age-matched Milan normotensive strain (MNS) animals by quantitative-PCR, immunoblotting, and immunoperoxidase. Endocytosis assay was performed on renal cells stably expressing AQP4 and co-transfected either with wild-type normotensive (NT) or with mutated hypertensive (HT) α-adducin.

RESULTSSemiquantitative immunoblotting revealed that AQP1 abundance was significantly decreased only in HT MHS whereas AQP2 was

reduced in both young pre-HT and adult-HT animals. On the other hand, AQP4 was dramatically upregulated in MHS regardless of the age. These results were confirmed by immunoperoxidase microscopy. Endocytosis assays clearly showed that the expression of mutated adducin strongly reduced the rate of constitutive AQP4 endocytosis, thereby increasing its abundance at the plasma membrane.

CONCLUSIONSWe provide here the first evidence that AQP1, AQP2, and AQP4 are dysregulated in the kidneys of MHS animals. In particular, we provide evidence that α-adducin mutations may be responsible for AQP4 upregulation. The downregulation of AQP1 and AQP2 and the upregulation of AQP4 may be relevant for the onset and maintenance of salt-sensitive hypertension.

Keywords: adducin; aquaporins; blood pressure; hypertension; MHS

American Journal of Hypertension, advance online publication 31 March 2011; doi:10.1038/ajh.2011.47

AMERICAN JOURNAL OF HYPERTENSION | VOLUME 24 NUMBER 7 | JULY 2011 823

ORIGINAL CONTRIBUTIONSAquaporins and Hypertension in MHS Rats

for AQP1–4. AQP1 is abundantly expressed in the proximal tubule and in the descending thin Henle’s limbs.16 AQP2 and AQP3/4 are expressed in the collecting duct that is the major site for vasopressin-regulated water reabsorption. Vasopressin increases water reabsorption in principal cells of the renal col-lecting duct by inducing an exocytotic fusion of AQP2 vesicles into the plasma membrane. In this manner, water reabsorption is hormonally regulated in the distal part of the nephron and is not secondary to sodium transport. Any alteration in this mechanism may be crucial in conditions such as hypertension that are characterized by modification of the extracellular fluid volume.

More generally, dysregulation of renal AQPs and sodium transporters could be important in the development and/or maintenance of hypertension.

The expression of renal AQPs has been studied in animal models of induced as well as spontaneous hypertension, such as spontaneously HT rats (SHR),17,18 the DOCA-salt HT rats,19 and the rat model of two-kidney, one clip hypertension.20 In all these animal models, an alteration in the expression of renal AQPs has been observed.

In this study, we investigated, for the first time, the expression of renal AQPs in MHS HT rats and their possible dysregulation, potentially leading to abnormal water reabsorption and either contributing to or counteracting the increase in blood pressure.

METHODSAnimals. All the experiments involving animals were per-formed on MHS and Milan NT strain (MNS) male rats, gen-erously provided by Prassis Research Institute (Milan, Italy). The studies were performed in accordance with animal wel-fare laws and in conformity with the Italian Guidelines for the use of laboratory animals, which conforms with the European Community Directive published in 1986 (86/609/EEC).

Tissue sampling. The kidneys were rapidly excised, immersed in RNAlater (Applied Biosystems Ambion, Austin, TX) and stored at −80 °C until total RNA isolation or Western blot experiments. The kidney cortex and inner medulla/papilla were dissected under a stereomicroscope.

Isolation of total RNA, reverse transcription, and real-time PCR. For quantitation of AQP1, AQP2, AQP3, and AQP4 mRNAs, total RNA was extracted from kidney papillae using the TRIzol method (Invitrogen, San Giuliano Milanese, Milan, Italy). RNA was reverse-transcribed, and real-time PCRs were performed in triplicate using the Applied Biosystems StepONE Fast Real-time PCR system and TaqMan GenExpression Assays (Applied Biosystems, Austin, TX) for rat AQP1 (Assay ID Rn00562834_m1), AQP2 (Assay ID Rn00563755_m1), AQP3 (Assay ID Rn00581754_m1), and AQP4 (Assay ID Rn00563196). Rat GAPD (GAPDH) (VIC/MGB Probe; Primer Limited, Applied Biosystems) was used as endogenous control. Each reaction was carried on as a singleplex reaction. The reactions consisted of 1 µl of complementary DNA (100 ng), 1 µl of TaqMan primer set, 10 µl Taq (TaqMan Fast Universal PCR master mix (2×),

No AmpErase UNG; no. 4366072), and 8 µl of H2O under the following PCR conditions: step 1, 95 °C for 20 s (enzyme acti-vation); step 2, 95 °C for 3 s (melting); and step 3, 60 °C for 30 s (annealing and extension); steps 2 and 3 were repeated 40 times.

Western blotting experiments. Kidney cortex and papilla were homogenized in RIPA buffer containing protease inhibitors (20 mmol/l pepstatin, 20 mmol/l leupeptin, and 1 mmol/l phe-nylmethylsulfonyl fluoride). Lysates were sonicated for 30 s and centrifuged at 13,000g for 15 min at 4 °C. The protein con-centration of the supernatants was determined.

In order to evaluate the abundance of AQP1, AQP2, AQP3, and AQP4, kidney lysates from cortex (AQP1) and papilla (AQP2–4), were separated on 4–12% NuPAGE Bis–Tris gels (Invitrogen) under reducing conditions. Protein bands were electrophoretically transferred to a polyvinylidene fluoride membrane. The blots were blocked with 3% bovine serum albu-min in Tris-buffered saline Tween-20 pH 7.5 (20 mmol/l Tris, 137 mmol/l NaCl, 0.1% Tween-20) for 1 h at room tempera-ture. The membranes were then incubated with the respective primary antibodies diluted in 3% bovine serum albumin/Tris-buffered saline Tween-20: AQP1 (Rabbit 1:500; Santa Cruz, Santa Cruz, CA), AQP2 (Rabbit 1:500; affinity-purified), AQP3 (Rabbit 1:500; Santa Cruz), and AQP4 (Goat 1:500; Santa Cruz).

Immunoreactive bands were detected with secondary anti-body conjugated to horseradish peroxidase and developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Densitometry analysis was performed using ImageJ (NIH) software. The results of AQPs bands densitom-etry were normalized for the tubulin signal and expressed as percentage of the values obtained from control. P values were calculated by Student’s t-test for unpaired data. Difference were considered to be significant P < 0.05.

Immunoperoxidase on kidney cryosections. Kidney samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C and infiltrated in 30% sucrose in PBS for 12 h. Tissues were frozen in dry ice in Cryomatrix (Thermo Shandon, Waltham, MA), and 5 µm sections were cut with a cryostat. The sections were pretreated with hydrogen peroxide (H2O2) 3% in PBS for 5–30 min and blocked with bovine serum albumin 1% in PBS for 1 h at room tempera-ture. The sections were then incubated with primary antibod-ies against AQP1–4 in blocking solution for 2 h. Next, the sec-tions were incubated with secondary antibodies conjugated to horseradish peroxidase diluted 1:20,000 in blocking solution. The slides were incubated with a solution containing the chro-mogenic substrate (3,3 diaminobenzidine 0.05% in 50 mmol/l Tris/HCl pH 7.4, 0.01% H2O2) for 5 min at room temperature. The reaction was stopped by washing the sections with distilled water. The tissues were finally dehydrated in graded ethanols (50–100%), cleared with Histolemon (Carlo Erba, Rodano, Italy), and mounted with a cover slip using a drop of mounting medium. Images were captured with a digital camera (Nikon DXM 1200-F; Nikon, Melville, NY) mounted on a Leica DM

824 JULY 2011 | VOLUME 24 NUMBER 7 | AMERICAN JOURNAL OF HYPERTENSION

ORIGINAL CONTRIBUTIONS Aquaporins and Hypertension in MHS Rats

RXA microscope (Leica Microsystems, Wetzlar, Germany) equipped with a ×100 oil immersion objective.

Cell culture and transfection. M1-AQP4 cells21 were transiently transfected with rat HT and NT variants of α-adducin using the following constructs (kind gift of Dr Tripodi):

Plasmid NM: green fluorescent protein (GFP)-Add1normo (F316) in EGFPN1 and plasmid HYP: GFP-Add1hyper (Y316) in EGFPN1 (hereinafter referred to as NT and HT adducin, respectively).

Transfection was performed using Lipofectamine 2000 Reagent (Invitrogen). The efficiency of the transfection (~80% of the cells were transfected) was verified by fluorescence visualization.

Biochemical internalization assay. Forty-eight hours after trans-fection with NT or HT adducin constructs, M1-AQP4 cells, grown on 0.4 μm cell culture inserts, were subjected to endo-cytosis assays as described previously.21 All procedures were performed on ice unless otherwise indicated. Briefly, filters were washed in Earle’s balanced salt solution buffer and the basolateral side was incubated with biotin (2.5 mg/ml in Earle’s balanced salt solution buffer) for 30 min. Unbound biotin was quenched by incubation for 10 min in quenching buffer. Basolateral pro-teins were permitted to become internalized by warming the cells to 37 °C for 0, 15, and 30 min. Next, the cells were treated 3 × 15 min with 100 mmol/l MesNa in 100 mmol/l NaCl, 1 mmol/l EDTA, 50 mmol/l Tris (pH 8.6), 0.2% bovine serum albumin. Excess MesNa was quenched with 120 mmol/l iodoacetic acid in PBS, and the cells were lysed. Lysates were sonicated for 30 s, unsolubilized material was pelletted at 13,000g for 10 min and biotinylated proteins were precipitated with streptavidin beads suspension at 4 °C overnight. Biotinylated proteins were extracted in NuPAGE LDS sample buffer (Invitrogen) with 100 mmol/l DTT at 95 °C for 10 min and resolved on NuPAGE gels. The abundance of AQP4 was revealed by Western blotting and semiquantitated as described earlier.

RESULTSExpression profile of renal AQPs in MHS ratsImmunoblotting. The abundance and the subcellular distri-bution of renal AQPs (AQP1–4) were investigated in MHS rats and compared with the findings in NT rats (MNS). In 3-week-old pre-HT MHS rats no significant change in AQP1 abundance in the kidney cortex was observed with respect to age-matched NT MNS animals (Figure 1a,b). In contrast, a strong downregulation of AQP2 (nearly 60%) was observed in the papilla of young MHS rats. Conversely, AQP4 protein was dramatically increased in young MHS rats as compared to MNS rats.

In HT adult rats (3-month-old MHS animals), AQP1 expres-sion was significantly lower in the kidney cortex as compared to MNS animals (Figure 1c,d). As seen in young pre-HT ani-mals, the adult HT animals also showed significantly down-regulation of AQP2 and dramatic upregulation of AQP4 when compared with adult MNS animals.

No significant differences were observed between MHS and MNS rats as regards the abundance of AQP3, either in the young rats or in the adult animals (data not shown).

Quantitative reverse transcriptase-PCR. In order to investigate whether the difference in abundance of renal AQPs observed in MHS rats relative to MNS rats reflects post-transcriptional or post-translational regulatory mechanisms, real-time reverse transcriptase PCR assays were performed. In young MHS rats, the abundance of both the AQP1 and AQP2 mRNAs was similar to those in MNS rats (Figure 2a,b). However, the abundance of AQP4 mRNA was two times higher in MHS animals relative to MNS animals (Figure 2c).

120

100

80

60

40

20

0

AQ

P1

dens

itom

etry

(per

cent

age

of M

NS

)

3 Weeks old 3 Months old

MNS MHS

120

100

80

60

40

20

0

AQ

P2

dens

itom

etry

(per

cent

age

of M

NS

)

MNS MHS

100200300400500600700800900

0

AQ

P4

dens

itom

etry

(per

cent

age

of M

NS

)

MNS MHS

*

50100150200250300350400450

0

AQ

P4

dens

itom

etry

(per

cent

age

of M

NS

)

MNS MHS

*

*

120

100

80

60

40

20

0

AQ

P2

dens

itom

etry

(per

cent

age

of M

NS

)

MNS MHS

*

120

100

80

60

40

20

0

AQ

P1

dens

itom

etry

(per

cent

age

of M

NS

)

MNS MHS

*

Tubulin

AQP2

AQP1

AQP4

3 Weeks old 3 Months old

MNS MHS MNS MHS

a c

b d

Figure 1 | Western blotting analysis of renal aquaporins (AQP) in Milan hypertensive strain (MHS) rats. Renal papillae from MHS and control Milan normotensive strain (MNS) rats were excised, lysed in RIPA buffer and lysates analyzed by Western blotting for the abundance of AQP1, AQP2, and AQP4. (a) Immunoblotting analysis of protein extracts from 3-week-old prehypertensive rats (MHS) compared to age-matched normotensive animals (MNS). (b) Densitometric analysis *P values <0.05 were considered significant. (c) Immunoblotting analysis of protein extracts from 3-month-old hypertensive rats (MHS) compared to age-matched normotensive animals (MNS). (d) Densitometric analysis *P < 0.05 were considered significant. N = 8 animals per group.

AMERICAN JOURNAL OF HYPERTENSION | VOLUME 24 NUMBER 7 | JULY 2011 825

ORIGINAL CONTRIBUTIONSAquaporins and Hypertension in MHS Rats

In adult MHS rats, the AQP1 mRNA was significantly lower relative to MNS animals (Figure 2d). A tendency toward AQP2 mRNA downregulation was observed in MHS rats, but this was not statistically significant (Figure 2e). AQP4 mRNA abundance in adult-HT MHS animals (Figure 2f) was similar to that in MNS animals.

Immunolocalization of renal AQPs in MHS rats. AQP1 stain-ing in proximal tubule epithelial cells from HT (3-month-old) MHS rats was proportionally lower, both at the apical and the basolateral membrane, consistent with the Western blot-ting data (data not shown). In contrast, an opposite pattern of AQP2 and AQP4 staining was observed in the principal cells of collecting ducts (Figure 3). Specifically, apical AQP2 staining appeared strongly reduced in MHS rats as compared to MNS animals (Figure 3; upper panel), whereas AQP4 immunoreactivity in the basolateral membrane was much higher in MHS rats. Therefore, two distinct AQPs expressed in the same cell type are regulated in opposite directions. It has to be underlined that, while AQP2 was downregulated in MHS animals, the expressed AQP2 in kidney tissues (cortex,

outer medulla, and inner medulla) of these animals displayed a distribution of the staining between intracellular vesicles and the apical membrane, comparable with the distribution pattern observed in MNS animals. This suggests that AQP2 trafficking was not impaired. No statistically significant differ-ences between MHS and MNS animals were seen as regards the number of AQP2-positive tubules. Moreover, no differ-ences were observed in the labeling intensity among the dif-ferent kidney zones cortex, outer medulla, and inner medulla (Figure 3).

No differences were observed between MHS and MNS animals as regards AQP3 immunoreactivity and localization; this finding was in agreement with the results of Western blotting tests (not shown). Taken together, these data indi-cate that, in the MHS rat model of hypertension, the abun-dance, but not the subcellular localization, of AQP1-2-4 is altered.

Mutant α-adducin reduces the constitutive endocytic rate of AQP4 in renal cellsIt has been shown that heterologous expressions of rat and human mutants of the α-adducin in renal cells results in a reduction of the constitutive endocytosis of Na+/K+-ATPase.11 Given that both Na+/K+-ATPase and AQP4 have basola-teral localization in renal cells, it is possible to speculate that α-adducin mutations might also affect the rate of AQP4 endocytosis.

Cor

tex

Out

er m

edul

laIn

ner

med

ulla

Hig

h m

agni

ficat

ion

Cor

tex

Out

er m

edul

laIn

ner

med

ulla

Hig

h m

agni

ficat

ion

MNS MHS MNS MHS

AQP2 AQP4

Figure 3 | Immunoperoxidase localization of AQP2 and AQP4 in the kidney of adult Milan hypertensive strain (MHS) and Milan normotensive strain (MNS) rats. The subcellular localization of AQP2 and AQP4 was analyzed in different kidney portions (cortex, outer medulla, and inner medulla) of adult hypertensive MHS rats and compared to that of control normotensive MNS rats.In both strains AQP2 displayed a comparable distribution in the subapical cytoplasm although the intensity of AQP2 staining was clearly reduced in MHS animals. AQP4 was associated with the basolateral membrane of collecting duct principal cells in both MHS and MHS animals and was dramatically upregulated in MHS animals. AQP, aquaporins.

1.5

1

0.5

0MNS MHS MNS MHS

MNS MHS

MNS MHS MNS MHS

MNS MHS

Rat

io A

QP

1/G

AP

DH

mR

NA

s

1.5

1

0.5

0Rat

io A

QP

2/G

AP

DH

mR

NA

sR

atio

AQ

P4/

GA

PD

H m

RN

As

Rat

io A

QP

4/G

AP

DH

mR

NA

s

1.5

1

0.5

0

Rat

io A

QP

2/G

AP

DH

mR

NA

s

1.5

1

0.5

0

Rat

io A

QP

1/G

AP

DH

mR

NA

s

3 Weeks old 3 Months old

*

*2.5

2

1.5

1

0.5

0

2.5

2

1.5

1

0.5

0

qRT-PCR

a d

b e

c f

Figure 2 | Abundance of renal aquaporins (AQP) mRNA in Milan hypertensive strain (MHS) compared to age-matched Milan normotensive strain (MNS) rats. Renal mRNA expression levels of (a,d) AQP1, (b,e) AQP2, and (c,f) AQP4 were determined by real-time quantitative (qRT) PCR analysis and are depicted as the ratio to GAPDH. Quantitative analysis was performed in the inner medulla of both 3-week-old prehypertensive MHS and 3-month-old hypertensive MHS animals. Age-matched normotensive MNS rats were used as controls. N = 5 animals per group. Data are presented as mean ± s.e.m. *P < 0.05 were considered significant.

826 JULY 2011 | VOLUME 24 NUMBER 7 | AMERICAN JOURNAL OF HYPERTENSION

ORIGINAL CONTRIBUTIONS Aquaporins and Hypertension in MHS Rats

The internalization of AQP4 and Na+/K+-ATPase after cell surface biotinylation was evaluated in adducin-transfected mouse M-1 renal cells.

M-1 cells stably expressing rat AQP421 were co-transfected with constructs encoding for wild-type NT adducin (NT) or mutated rat HT adducin. Both constructs were tagged with GFP.

Figure 4a shows the results of the immunoblotting analy-sis performed on M1-AQP4 cells transiently transfected with both adducin variants (NT and HT) and probed with antibod-ies against the GFP tag. Both constructs were expressed with comparable efficiency, and the overexpression of both adducin forms did not change the amount of total AQP4. After basola-teral labeling with cleavable biotin, membrane proteins were allowed to be internalized for different intervals of time (see Methods section for details).

Biotinylated AQP4 and Na+/K+-ATPase were visualized by Western blotting (Figure 4b and relative statistical analysis of densitometry in Figure 4c).

In M1-AQP4 cells, the overexpression of NT adducin affected neither the amount of membrane-expressed AQP4 nor the rate of AQP4 endocytosis at each time point indicated (0, 15, 30 min) as compared to untransfected cells. Interestingly, the ectopic expression of HT adducin significantly increased the amount of membrane-expressed AQP4 and dramatically reduced the amount of AQP4 that was internalized at 15 and 30 min. A reduction in endocytosis in the presence of HT adducin was also seen as regards the Na/K pump, consistent with previous reports from studies in Madin–Darby canine kidney epithelial cells.11

DISCUSSIONIn this article, we analyzed, for the first time, the expression of the major renal AQPs (AQP1–4) in MHS, a rat model of sodium-sensitive hypertension carrying mutations of the α-adducin protein. MHS rats have been previously charac-terized, showing that, at week 3 after weaning, blood pres-sure is 40–50 mm Hg higher as compared to NT rats.5 MHS rats showed significantly greater sodium retention, and con-sequently the fractional urinary sodium excretion reduced significantly starting at week 2 after weaning.5 Interestingly, previous studies showed that α-adducin polymorphisms are positively associated with hypertension in humans also.8,9,22 In vitro studies demonstrated that α-adducin polymorphisms induced upregulation of Na+K+-ATPase activity/expression and, consequently, Na+ reabsorption from the kidney tubules. Given that the sodium gradient provides the driving force for AQPs-mediated water reabsorption in the kidney, in this study we evaluated the possible contribution of renal AQPs to the establishment and/or maintenance of the HT phenotype in MHS animals.

Our study provides evidence that, during their HT phase, MHS rats show a heavy downregulation of both AQP1 and AQP2, whereas AQP4 is dramatically upregulated.

The dramatic downregulation of AQP1 in MHS animals could be interpreted as a compensatory mechanism to pre-vent excessive water reabsorption in the proximal tubule and consequent expansion in extracellular fluid volume. Alteration in the renin–angiotensin system is a possible explanation for the downregulation of AQP1 in MHS rats, given that these animals have been shown to have lower plasma levels of ren-nin.23 In fact, MHS rats have elevated plasma aldosterone24 that could negatively modulate renin release by the kidney. Renin, in turn, can modulate plasma concentrations of angiotensin II that may have direct as well as indirect roles in water and salt homeostasis.

Several studies have demonstrated the regulatory role of angiotensin II in modulating AQP1 expression in the kidney.25,26

Perturbation of the renin–angiotensin system might also explain the downregulation of AQP2 that we observed in MHS animals. In vitro studies have demonstrated that angiotensin II potentiates vasopressin-dependent cAMP accumulation in Chinese hamster ovary cells, and that there is crosstalk between the signal pathways of the two receptors.26,27

GFP

AQP4

Untransfected NT adducin HT adducina

UntransfectedNT adducinHT adducin

70

60

50

40

30

20

10

0

Den

sito

met

ry (

arbi

trar

y un

its)

t0 t0 t15 t30

* *

*c

AQP4

Untransfected NT adducin HT adducin

Na+K+ ATPase

MesNa:Min: 0 0 15 30 0 0 0 0 15 303015

b

Figure 4 | Internalization assay showing the effect of adducin polymorphisms on AQP4 endocytosis in renal cells. (a) M1-AQP4 cells, grown on permeable supports, were left untransfected or transfected either with green fluorescent protein (GFP)-tagged normotensive (NT) or with GFP-tagged hypertensive (HT) adducin. After 48 h, the efficiency of transfection was assessed by Western blotting using anti-GFP antibodies. (b) Cells were biotinylated from the basolateral side. Subsequently, cells were allowed to internalize biotinylated proteins for the indicated time (0, 15, and 30 min) at 37 °C. The remaining surface-accessible biotin was stripped with MesNa, where indicated (MesNa−/+). Biotinylated proteins were recovered and immunoblotted for AQP4. (c) Densitometric analysis of biotinylated AQP4 band. Values are expressed as means ± s.e. of three independent experiments. *P < 0.05 were considered significant. AQP, aquaporins.

AMERICAN JOURNAL OF HYPERTENSION | VOLUME 24 NUMBER 7 | JULY 2011 827

ORIGINAL CONTRIBUTIONSAquaporins and Hypertension in MHS Rats

The hypercalciuria that characterized MHS animals may also provide an alternative explanation for AQP2 downregu-lation.28 We have previously shown that kidney concentrating ability is impaired in patients with hypercalciuria.29 Moreover, more recent data from studies in cultured renal cells have shown that high concentrations of extracellular calcium atten-uate arginine vasopressin-induced AQP2 expression by acti-vating the calcium sensing receptor.30

In MHS rats, the high concentration of calcium in urine might induce AQP2 downregulation by activating the calcium sensing receptor.

Whether AQP2 in these animals is downregulated in response to the increased Na reabsorption (and consequent low plasma renin) or in response to high luminal calcium, both phenomena are likely to be adaptive responses counteracting, rather than promoting, water retention.

On the other hand, the expression of basolateral AQP4 in these animals is dramatically increased regardless of age. The upregulation of AQP4 during the onset of hypertension might suggest that increased water reabsorption in the collecting duct might contribute to the extracellular fluid volume expan-sion that is a typical characteristic of Na-sensitive hypertension. Interestingly, MHS rats show significantly high diuresis rates at the time of weaning but not during adulthood when they start to retain sodium and develop hypertension. At that time, an increase of AQP4-mediated water reabsorption in the kidney might both normalize the diuresis and promote water retention.

In analogy to Na+-K+-ATPase, AQP4 is also expressed at the basolateral membrane of the kidney tubule epithelial cells. As discussed earlier, the presence of single-nucleotide polymorphisms of α-adducin has been proposed to explain the increased expression and activity of the Na+-K+-ATPase through a mechanism of inhibition of the constitutive clathrin-mediated endocytosis of the pump.11 According to this hypothesis, the presence of mutated adducin induces hyperphosphorylation of the AP2 adaptor protein.12 The increased expression of AQP4 at the basolateral membrane of collecting duct suggests that the presence of adducin sin-gle-nucleotide polymorphisms might also contibute to this phenomenon. In analogy to Na+-K+-ATPase, AQP4 endocy-tosis is regulated by interaction with the AP2 adaptor com-plex.31 In our study, the in vitro endocytosis assay performed on AQP4-expressing renal cells clearly shows that the con-stitutive endocytosis of AQP4 is strongly inhibited by the presence of the adducin mutant. Once endocytosed, AQP4 is efficiently targeted to lysosomes for degradation;31 there-fore reduced endocytosis would explain the increase in total AQP4 in MHS rats.

A detailed analysis of the expression of renal AQPs has been performed earlier in a different rat model of hypertension (SHR rats). AQP1 was found reduced in the inner medulla of SHR rats as compared to control Wistar-Kyoto rats.32 AQP2 was found increased and more abundant at the apical mem-brane of the inner medulla of SHR animals.32,33 In contrast, Kim and co-workers found a small increase in AQP2 in the inner stripe of outer medulla of young pre-HT SHR rats but

no changes in adult SHR animals with severe hypertension, although a more prominent apical staining was observed in the samples from the latter animals.18

In SHR rats, AQP4 expression was found unchanged in the inner medulla whereas AQP3 showed an increase.32

It appears, therefore that different patterns of alteration in renal AQPs are observed in these two animal models of hyper-tension, thereby suggesting that the differing contributions and/or adaptation of renal AQPs to hypertension in these two different models of hypertension may depend on the differing etiologies of hypertension in these two strains.

The pathogenesis of hypertension in SHR rats appears to be heterogeneous; cellular, central nervous system, neurohu-moral, and renal abnormalities have been proposed. The SHR rat represents a “normal renin” model, and its blood pressure is relatively sodium-independent.

On the other hand, in the sodium-sensitive MHS animals, hypertension develops because of a primary increase in renal tubular Na+ reabsorption.5 However, a new finding in this work is the dramatic upregulation of AQP4 in the kidney inner medulla of MHS rats as compared to age-matched MNS animals.

We might speculate that, in MHS, AQP4 upregulation increases water reabsorption in the kidney and contributes toward generating and sustaining the HT phenotype in these animals.

AQP4 is constitutively expressed only at the basolateral plasma membrane in the collecting duct. This nephron seg-ment is characterized by a higher resistance with respect to the proximal tubule, and therefore water reabsorption is mostly transcellular.

During antidiuresis, vasopressin increases the water per-meability of the apical membrane through insertion of AQP2 channels into the membrane. However, a significant amount of AQP2 is expressed at the plasma membrane even under conditions of low vasopressin levels. Because it has been shown that AQP2 is much more abundant than AQP4 in con-trol mammalian kidneys34 it is possible that the amount of AQP4 expressed at the basolateral membrane represents the real limiting barrier for water reabsorption in the collecting duct. Considering the fact that, despite AQP2 being down-regulated in MHS, AQP4 is increased up to sevenfold even in young MHS, we can speculate that this condition, associ-ated with the presumably higher medulla osmolality (caused by the higher sodium reabsorption), might increase the net fluid absorption toward the interstitium and promote fluid retention.

This interpretation sheds new light on the possible contri-bution of AQP4 to the establishment and maintenance of sodium-dependent hypertension in animals and humans in the presence of adducin variants. Further investigations are needed to demonstrate that AQP4 is also upregulated in humans carrying adducin mutations. If this proves to be so, AQP4 inhibitors might be considered as a class of poten-tial pharmacological tools for the treatment of subsets of HT patients characterized by adducin polymorphisms.

828 JULY 2011 | VOLUME 24 NUMBER 7 | AMERICAN JOURNAL OF HYPERTENSION

ORIGINAL CONTRIBUTIONS Aquaporins and Hypertension in MHS Rats

Acknowledgments: We are grateful to Dr Lisa Mastrofrancesco and Domenica Lasorsa for their excellent assistance during the experiments. The research was supported by PRIN (grant code 2006065339_002 and 2008W5AZEC_005 to G.V.), PRIN (grant code 20078ZZMZW to M.S.), the Regional Strategic Grant (grant code CIP PS_144 to G.V.), FIRB (grant code RBIN04PHZ7 to M.S.), and by Centro di Eccellenza di Genomica in campo Biomedico ed Agrario (CEGBA).

Disclosure: The authors declared no conflict of interest.

1. Poch E, González D, Giner V, Bragulat E, Coca A, de La Sierra A. Molecular basis of salt sensitivity in human hypertension. Evaluation of renin-angiotensin-aldosterone system gene polymorphisms. Hypertension 2001; 38:1204–1209.

2. Luft FC, Miller JZ, Weinberger MH, Christian JC, Skrabal F. Genetic influences on the response to dietary salt reduction, acute salt loading, or salt depletion in humans. J Cardiovasc Pharmacol 1988; 12 Suppl 3:S49–S55.

3. Bianchi G, Fox U, Di Francesco GF, Giovanetti AM, Pagetti D. Blood pressure changes produced by kidney cross-transplantation between spontaneously hypertensive rats and normotensive rats. Clin Sci Mol Med 1974; 47:435–448.

4. Crowley SD, Gurley SB, Herrera MJ, Ruiz P, Griffiths R, Kumar AP, Kim HS, Smithies O, Le TH, Coffman TM. Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proc Natl Acad Sci USA 2006; 103:17985–17990.

5. Bianchi G, Baer PG, Fox U, Duzzi L, Pagetti D, Giovannetti AM. Changes in renin, water balance, and sodium balance during development of high blood pressure in genetically hypertensive rats. Circ Res 1975; 36:153–161.

6. Melzi ML, Syrén ML, Assael BM, Sereni F, Aperia A. Increased renal tubular Na-K-ATPase activity in Milan hypertensive rats in the prehypertensive period. Pediatr Nephrol 1991; 5:700–703.

7. Hughes CA, Bennett V. Adducin: a physical model with implications for function in assembly of spectrin-actin complexes. J Biol Chem 1995; 270:18990–18996.

8. Bianchi G, Manunta P, Glorioso N. Clinical impact of adducin polymorphism. J Hypertens 2009; 27:1325–1327; author reply 1327.

9. Citterio L, Lanzani C, Manunta P, Bianchi G. Genetics of primary hypertension: the clinical impact of adducin polymorphisms. Biochim Biophys Acta 2010; 1802:1285–1298.

10. Tripodi G, Valtorta F, Torielli L, Chieregatti E, Salardi S, Trusolino L, Menegon A, Ferrari P, Marchisio PC, Bianchi G. Hypertension-associated point mutations in the adducin α and β subunits affect actin cytoskeleton and ion transport. J Clin Invest 1996; 97:2815–2822.

11. Torielli L, Tivodar S, Montella RC, Iacone R, Padoani G, Tarsini P, Russo O, Sarnataro D, Strazzullo P, Ferrari P, Bianchi G, Zurzolo C. α-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption. Am J Physiol Renal Physiol 2008; 295:F478–F487.

12. Efendiev R, Krmar RT, Ogimoto G, Zwiller J, Tripodi G, Katz AI, Bianchi G, Pedemonte CH, Bertorello AM. Hypertension-linked mutation in the adducin α-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+,K+-ATPase trafficking in response to GPCR signals and intracellular sodium. Circ Res 2004; 95:1100–1108.

13. Bianchi G. Genetic variations of tubular sodium reabsorption leading to “primary” hypertension: from gene polymorphism to clinical symptoms. Am J Physiol Regul Integr Comp Physiol 2005; 289:R1536–R1549.

14. Knepper MA, Wade JB, Terris J, Ecelbarger CA, Marples D, Mandon B, Chou CL, Kishore BK, Nielsen S. Renal aquaporins. Kidney Int 1996; 49:1712–1717.

15. Nielsen S, Kwon TH, Frøkiaer J, Knepper MA. Key Roles of Renal Aquaporins in Water Balance and Water-Balance Disorders. News Physiol Sci 2000; 15:136–143.

16. Maunsbach AB, Marples D, Chin E, Ning G, Bondy C, Agre P, Nielsen S. Aquaporin-1 water channel expression in human kidney. J Am Soc Nephrol 1997; 8:1–14.

17. Buemi M, Nostro L, Di Pasquale G, Cavallaro E, Sturiale A, Floccari F, Aloisi C, Ruello A, Calapai G, Corica F, Frisina N. Aquaporin-2 water channels in spontaneously hypertensive rats. Am J Hypertens 2004; 17:1170–1178.

18. Kim SW, Wang W, Kwon TH, Knepper MA, Frøkiaer J, Nielsen S. Increased expression of ENaC subunits and increased apical targeting of AQP2 in the kidneys of spontaneously hypertensive rats. Am J Physiol Renal Physiol 2005; 289:F957–F968.

19. Lee J, Kang DG, Kim Y. Increased expression and shuttling of aquaporin-2 water channels in the kidney in DOCA-salt hypertensive rats. Clin Exp Hypertens 2000; 22:531–541.

20. Lee J, Oh Y, Kim SW. Altered renal expression of aquaporin-2 water channels in rats with experimental two-kidney, one clip hypertension. J Korean Med Sci 2001; 16:462–466.

21. Procino G, Barbieri C, Carmosino M, Rizzo F, Valenti G, Svelto M. Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells. Am J Physiol Renal Physiol 2010; 298: F266–F278.

22. Bianchi G, Ferrari P, Staessen JA. Adducin polymorphism: detection and impact on hypertension and related disorders. Hypertension 2005; 45:331–340.

23. Ferrari P, Barber BR, Torielli L, Ferrandi M, Salardi S, Bianchi G. The Milan hypertensive rat as a model for studying cation transport abnormality in genetic hypertension. Hypertension 1987; 10:I32–I36.

24. Lloyd-MacGilp SA, Torielli L, Bechtel S, Tripodi G, Gomez-Sanchez CE, Zagato L, Bernhardt R, Kenyon CJ. Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences. Am J Physiol Endocrinol Metab 2002; 282:E608–E617.

25. Bouley R, Palomino Z, Tang SS, Nunes P, Kobori H, Lu HA, Shum WW, Sabolic I, Brown D, Ingelfinger JR, Jung FF. Angiotensin II and hypertonicity modulate proximal tubular aquaporin 1 expression. Am J Physiol Renal Physiol 2009; 297:F1575–F1586.

26. Kwon TH, Nielsen J, Knepper MA, Frøkiaer J, Nielsen S. Angiotensin II AT1 receptor blockade decreases vasopressin-induced water reabsorption and AQP2 levels in NaCl-restricted rats. Am J Physiol Renal Physiol 2005; 288: F673–F684.

27. Klingler C, Ancellin N, Barrault MB, Morel A, Buhler JM, Elalouf JM, Clauser E, Lugnier C, Corman B. Angiotensin II potentiates vasopressin-dependent cAMP accumulation in CHO transfected cells. Mechanisms of cross-talk between AT1A and V2 receptors. Cell Signal 1998; 10:65–74.

28. Cirillo M, Galletti F, Strazzullo P, Torielli L, Melloni MC. On the pathogenetic mechanism of hypercalciuria in genetically hypertensive rats of the Milan strain. Am J Hypertens 1989; 2:741–746.

29. Valenti G, Laera A, Gouraud S, Pace G, Aceto G, Penza R, Selvaggi FP, Svelto M. Low-calcium diet in hypercalciuric enuretic children restores AQP2 excretion and improves clinical symptoms. Am J Physiol Renal Physiol 2002; 283:F895–F903.

30. Bustamante M, Hasler U, Leroy V, de Seigneux S, Dimitrov M, Mordasini D, Rousselot M, Martin PY, Féraille E. Calcium-sensing receptor attenuates AVP-induced aquaporin-2 expression via a calmodulin-dependent mechanism. J Am Soc Nephrol 2008; 19:109–116.

31. Madrid R, Le Maout S, Barrault MB, Janvier K, Benichou S, Mérot J. Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes. EMBO J 2001; 20:7008–7021.

32. Lee J, Kim S, Kim J, Jeong MH, Oh Y, Choi KC. Increased expression of renal aquaporin water channels in spontaneously hypertensive rats. Kidney Blood Press Res 2006; 29:18–23.

33. Sonalker PA, Tofovic SP, Jackson EK. Increased expression of the sodium transporter BSC-1 in spontaneously hypertensive rats. J Pharmacol Exp Ther 2004; 311:1052–1061.

34. Yamamoto T, Sasaki S. Aquaporins in the kidney: emerging new aspects. Kidney Int 1998; 54:1041–1051.