Selective renal vasoconstriction, exaggerated natriuresis

and excretion rates of exosomic proteins in essential

hypertension

M. Damkjær,1,* P. H. Jensen,1,2,* V. Schw€ammle,2 R. R. Sprenger,2 I. A. Jacobsen,3

O. N. Jensen2 and P. Bie1

1 Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense,

Denmark

2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

3 Department of Endocrinology, Odense University Hospital, Odense, Denmark

Received 5 April 2014,

revision requested 21 April 2014,

revision received 7 July 2014,

accepted 8 July 2014

Correspondence: P. Bie, Depart-

ment of Cardiovascular and Renal

Research, Institute of Molecular

Medicine, University of Southern

Denmark, 21 Winslowparken,

5000 Odense C, Denmark.

E-mail: pbie@health.sdu.dk

*These authors contributed

equally to this manuscript.

Abstract

Aim: In essential hypertension (EH), the regulation of renal sodium excre-

tion is aberrant. We hypothesized that in mild EH, (i) abnormal dynamics

of plasma renin concentration (PRC) and atrial natriuretic peptide (ANP)

are responsible for the exaggerated natriuresis, and (ii) exosomic protein

patterns reflect the renal tubular abnormality involved in the dysregulation

of sodium excretion.Methods: After 2-week drug washout and 4-day diet, systemic and renal

hemodynamics, cardio-renal hormones, glomerular filtration and renal

excretion were studied in male patients during saline loading (SL). Excre-

tion rates of exosome-related urinary proteins including apical membrane

transporters were determined by proteomics-based methods.Results: In patients, baseline renal vascular conductance was reduced

(�44%, P < 0.001), but non-renal vascular conductances were normal

while PRC was reduced and ANP elevated (both P < 0.01). SL induced

exaggerated natriuresis and reduced PRC (P < 0.01), at normal suppres-

sion rate. SL increased arterial pressure in patients (+11 mmHg,

P < 0.001), but not in controls; however, during time control, patients

showed identical increases (+10 mmHg, P < 0.005) apparently dissociating

arterial pressure from natriuresis.

At baseline, excretion rates of 438 proteins ranged from 0.07 to

49.8 pmol (mmol creatinine)�1; 12 proteins were found in all subjects,

and 21 proteins were found in two or more patients, but not in controls.

In patients, the excretion rate of retinoic acid-induced gene 2 protein was

reduced, and excretion rates of other proteins showed increased variances

compatible with pathophysiological and clinical applicability.Conclusion: Essential hypertension patients exhibit selective renal vaso-

constriction and individually varying excretion rates of several exosome-

related proteins. Hormonal changes, rather than arterial pressure, seem to

cause exaggeration of natriuresis.

Keywords hormones, hypertension, kidney, proteomics, renin, vascular

resistance.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12345 1

Acta Physiol 2014

The link between acute and chronic renal sodium han-

dling and essential hypertension (EH) is unclear, but

so are even the mechanisms of normal sodium homeo-

stasis (Damkjaer et al. 2013). Although serious flaws

seem to exist in most trials, which have attempted to

establish the relation between sodium intakes to blood

pressure-related cardiovascular disease (CVD) (Cobb

et al. 2014), the concept of a positive relation between

sodium intake and arterial blood pressure driving

CVD remains widely accepted (Strom et al. 2013,

Cook et al. 2014) and referred to as ‘salt sensitivity’.

Surprisingly, a standardized test for presence of salt

sensitivity does not exist; multiple protocols including

intravenous SL or changes in dietary intake have been

applied with and without the use of diuretics. Possibly

related to this lack of standardization, a consistent

role of the renin angiotensin aldosterone system

(RAAS) in the development of EH has not been estab-

lished although the RAAS is a pivotal element of

sodium homeostasis. Recent experimental evidence

shows that levels of circulating AngII, which are not

immediately vasoactive, act on the CNS to increase

sympathetic drive to the kidney (Malpas 2010) possi-

bly in an organ selective manner. Organ-specific

changes in sympathetic nerve traffic have been demon-

strated convincingly (Ramchandra et al. 2009, 2013,

Yoshimoto et al. 2010a,b), recently also in response

to sodium loading (Frithiof et al. 2014). It has been

known since the 50s that in EH, the natriuretic

response to acute SL is exaggerated (Green et al.

1952, Solomon et al. 1960). In case increased renal

sympathetic tone is a primary element of the aetiology

of EH, the exaggeration of the natriuretic response to

saline is puzzling and suitable as basis for a working

hypothesis with regard to the dynamics of the hor-

monal response to sodium loading.

A certain degree of sodium retention often is assumed

to be involved in hypertension (Johnson et al. 2008,

Herrera & Coffman 2012), but precise measurements

of body fluid compartments are technically difficult and

not all studies of EH demonstrate (alterations compati-

ble with) increases in total body sodium. Normally,

hypertension per se with or without sodium retention

would be expected to suppress renin system activity and

elevate plasma natriuretic peptide concentrations. As

such changes are not constant findings, malfunctions of

the hormonal regulation of kidney function seem to be

a part of EH. However, standardization of patients

with regard to sex, degree of EH and target organ dam-

age as well as of circumstances (dietary regimen, body

position, SL) is essential for studies of hormonal regula-

tion; therefore, the study included only highly selected

patients without signs of organ damage.

Urinary exosomes are membrane-limited nanoparti-

cles derived from the apical membrane endocytotic

pathway (Raposo & Stoorvogel 2013). Released con-

tinuously from cells facing the lumen of the renal

tubules, the exosome-related proteins may reflect cel-

lular events crucial for NaCl and water transport and

may even play a role in intercellular signalling (van

Balkom et al. 2011). The patterns of excretion of

these may be valuable indicators of renal functions

because it can be assumed that all such proteins are of

renal origin. Methodological advances within proteo-

mics now allow label-free absolute quantification of

multiple proteins (Creskey et al. 2012) to be applied

to urine samples; the results include absolute rates of

excretion in picomoles per hour as well as per milli-

mole of excreted creatinine possibly reflecting patho-

physiological events in the kidney (Alvarez et al.

2013, Hogan et al. 2014, Wood et al. 2013). It

seemed attractive to associate urinary protein profiles

also with the dynamic operation of renal regulation

with the response to an acute salt load in EH.

We hypothesized that in EH, the homeostatic, neu-

rohumoral pathways to the kidney, for example,

AngII and aldosterone, are distorted generating a

baseline condition of relative antinatriuresis and that

augmented responses to SL [e.g. deactivation of RAAS

and secretion of atrial natriuretic peptide (ANP)] are

responsible for the exaggeration of the natriuretic

response. Furthermore, it was hypothesized that in

EH, the urinary rates of excretion of specific, mem-

brane-associated proteins are different from those of

control subjects reflecting the tubular molecular mech-

anisms involved.

Methods

The present setting is defined by selected, mildly

hypertensive, male patients without signs of organ

damage being exposed to slow, body mass-adjusted

volume expansion in the sitting position after meta-

bolic standardization including low normal sodium

intake. Full time control measurements in the patients

and parallel measurements in a matched control group

are included for comparisons.

Patients

Eleven patients with mild, monosymptomatic hyper-

tension were studied. Middle-aged (40–60 years),

male, non-smoking patients with EH grade I–II were

recruited from the local outpatient clinic. The diagno-

sis of EH was based on the criteria defined by the

European Society of Hypertension (Mancia et al.

2007). Exclusion criteria were body mass index (BMI)

>28, abdominal circumference >100 cm, any anam-

nestic information of co-morbidity, any sign of target

organ damage by echocardiography, elevated plasma

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.123452

Hemodynamics and proteomics in hypertension · M Damkjær et al. Acta Physiol 2014

creatinine (no value exceeded 110 lmol L�1) or

microalbuminuria. Twelve healthy age and BMI-

matched males were recruited as control group. None

of the control subjects had any past medical history,

and all were normotensive and had normal plasma

sodium, potassium and creatinine concentrations. For

details, see Table S1 in Data S1.

Experimental protocol

Prior to the investigation, all drugs were withheld for

18 days. In the period of drug withdrawal, the

patients were seen by a physician at the outpatient

clinic up to twice weekly for recordings of ambulatory

blood pressure. If systolic blood pressure exceeded

180 mmHg or diastolic pressure exceeded 110 mmHg,

the patient immediately resumed regular medication

and thereby dropped out of the study. During the last

4 days, the subjects received a low-salt diet providing

a daily intake of 50–60 mmol Na+; 24-h urinary

sodium excretion (07:00–07:00 hour) was measured

immediately prior to investigation to assess dietary

compliance. Patients were studied on two consecutive

days (time control and SL). Day 1 was the control

day. After instrumentation, followed by bolus injec-

tions of para-aminohippurate (PAH) and 51Cr-EDTA

(see below), cardiovascular, hormonal and renal vari-

ables were sampled for six consecutive hours. On day

2, sampling of baseline variables for 1 h was followed

by infusion of saline (135 mmol L�1, i.e. slightly

hypotonic) intravenously for 4 h as described previ-

ously (Damkjær et al. 2012). Cardiac output (CO)

was measured continuously by impedance cardiogra-

phy (PhysioFlow PF-03; Manatec Biomedical, Macher-

en, France), the mean difference between the CO

values obtained in normal subjects at rest by the direct

Fick method and PhysioFlow methods has been found

to be negligible (0.07 L min�1) (Charloux et al.

2000). Blood pressures was measured every 10 min by

automated oscillometry, glomerular filtration rate

(GFR) by the clearance of constantly infused51Cr-EDTA and effective renal plasma flow (ERPF) by

clearance of infused p-aminohippurate (PAH). Derived

variables include systemic vascular resistance, effective

renal blood flow (ERBF) renal vascular resistance

(RVR) as well as renal and non-renal vascular conduc-

tances. RVR was estimated as mean arterial blood

pressure (MABP)/ERBF. ERBF was estimated as the

ERPF divided by (1 � haematocrit) which was set at

0.45 for all subjects. Osmolality, electrolytes and hor-

mones were determined by routine methods. The sub-

jects were given 100 mL of glucose solution (10%)

orally every 30 min throughout the experiment to (i)

maintain the standardized degree of hydration, (ii)

generate a uniform diuresis facilitating the clearance

measurements and (iii) suppress the feeling of hunger,

which otherwise would have occurred during the

experiment. For other details, see Data S1.

Proteomic analysis of exosomal proteins

Urinary exosomes were isolated by centrifugation and

repeated ultracentrifugation steps (Pisitkun et al.

2004, Gonzales et al. 2009), resulting in microgram

quantities of exosomic protein per sample. After

depletion of Tamm–Horsfall protein and enzymatic

digestion, peptides were concentrated and desalted

prior to mass spectrometric (MS) analysis using cus-

tomized microcolumns (Gobom et al. 1999). One

microgram of each sample was analysed by liquid

chromatography tandem mass spectrometry in a high/

low collision energy switching mode [LC-MSE (Silva

et al. 2006)]. Each sample was spiked with 100 fmol

yeast enolase as internal standard to enable determina-

tion of protein concentrations and to address technical

variation (Silva et al. 2006). The data were processed

and searched with ProteinLynx Global Server (PLGS,

version 2.4; Waters, Milford, MA, USA). Protein iden-

tifications were obtained by searching the human

SwissProt entries of a UniProt database (release

2010_05) modified to include known N-terminal pro-

cessing of proteins using the protein maturation device

software tool (Martens et al. 2005, Reisinger & Mar-

tens 2009). The output from the PLGS Ion Accounting

algorithm (Li et al. 2009) was used for further analy-

sis, which was limited to proteins identified in at least

two of three triplicates. Peptide identifications were

performed with <10 ppm mass deviation. The ‘top3/

HI3’ method embedded in PLGS was used for label-

free absolute protein quantification as preferred

method providing direct proportionality to protein

abundance over the full quantification range with

good accuracy and precision (Ahrne et al. 2013). Pro-

tein excretion rates were calculated and normalized to

creatinine excretion (pmol protein (mmol creati-

nine)�1). For details, see Data S1.

Statistics

Data are presented as mean � SEM and were analy-

sed by GRAPHPAD PRISM (GraphPad Software, San

Diego, CA, USA). Comparisons between two groups

were performed by Student’s t-test. Differences

between variances were assessed by F-test, and if

appropriate, Welch’s correction for uneven variances

was applied. Time-dependent changes were analysed

by two-way ANOVA. Differences were considered signif-

icant at P < 0.05. A number of hypotheses were

tested, but obviously these were not independent. No

correction for multiple testing was performed.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12345 3

Acta Physiol 2014 M Damkjær et al. ·Hemodynamics and proteomics in hypertension

Results

Essential hypertension patients and control subjects

were indistinguishable with regard to age, body mass,

plasma sodium and potassium concentrations, as well

as to 24-h renal sodium excretion (Table S1).

Baseline cardio-renal function

Heart rate and cardiac output were very similar in the

two groups. In EH, MABP was elevated modestly due

to an 18% increase in total peripheral resistance

(Table 1). This was analysed further in terms of con-

ductances. At baseline, the numerically lower ERPF in

patients (509 � 92 mL min�1) was not statistically

different form controls (613 � 109 mL min�1). How-

ever, partly due to the contribution of the elevated

MABP, renal vascular conductances were markedly

different (8 � 2 and 15 � 3 ml (mmHg min)�1 in

patients and controls, respectively, P < 0.01, Table 1),

that is, reduced by 44% in the patients. The non-renal

vascular conductances in patients and controls were

similar (Table 1). Consequently, the increase in total

peripheral resistance in EH was due to renal vasocon-

striction. Over the course of the experiment, GFR in

patients was reduced (P < 0.05) and calculated filtered

load of sodium was 28% less than in controls

(P < 0.01); however, the baseline sodium excretion

rate was unaffected. The calculated fractional sodium

excretion (FENa) was 0.06 � 0.01% in patients and

0.05 � 0.01% in control subjects (P = ns). In patients

at baseline, plasma renin concentration (PRC) aver-

aged 61% of that of control subjects (P < 0.01), while

plasma ANP was elevated (+84%, P < 0.001).

Cardio-renal response to saline infusion

In control subjects (n = 12), the SL left MABP

unchanged (87 � 2 to 88 � 2 mmHg, Fig. 1). In

patients (n = 11), a significant increase in MABP

occurred (105 � 2 to 116 � 3 mmHg, P < 0.001,

Fig. 1). However, during the time control study of the

patients, MABP increased similarly (105 � 2 to

115 � 3 mmHg, P < 0.005, Figure S1) without any

change in sodium excretion. This does not indicate that

the rise in MABP occurring during SL was without any

effect on the rate of excretion of sodium, but it does

show that spontaneous changes in MABP are not driv-

ing sodium excretion under the present circumstances.

Based on the results obtained by two-way ANOVA,

renal plasma flow in patients was lower than in con-

trol subjects (Fig. 1). After SL, ERPF in patients were

markedly lower than in controls (final values, t-test,

P = 0.02). By two-way ANOVA, the trends towards

decrease and increase in renal conductances within the

EH patients and the control subjects, respectively, did

not reach statistical significance (Table 2).

As expected, the patients showed exaggerated

natriuresis (Fig. 1 and Figure S1): the 2.3-fold increase in

sodium excretion in the patients (63–141 lmol min�1,

P < 0.001) was significantly larger (P < 0.01) than

the 1.4-fold change in the control group

Table 1 Baseline values during low-sodium diet in control subjects (CON) and patients with essential hypertension (EH). Val-

ues are mean � SEM, n = 11 (EH) and 12 (CON)

Variable CON EH Diff. (%) P<

Mean arterial blood pressure (mmHg) 87 � 2 104 � 2 +20 0.001

Systolic blood pressure (mmHg) 123 � 3 140 � 2 +14 0.001

Diastolic blood pressure (mmHg) 69 � 2 86 � 2 +27 0.01

Heart rate (bpm) 71 � 2 70 � 3 – ns

Cardiac output (L min�1) 4.9 � 0.2 5.0 � 0.2 – ns

Total peripheral resistance (mmHg (L min�1)�1) 17.7 � 0.6 20.9 � 1.0 +18 0.01

Total peripheral conductance (mL (min 9 mmHg)�1) 57.5 � 2.1 48.7 � 2.1 �15 0.01

Renal vascular conductance (mL (min 9 mmHg)�1) 14.6 � 2.8 8.2 � 1.7 �44 0.01

Extrarenal vascular conductance (mL (min 9 mmHg)�1) 42.7 � 3.0 40.5 � 2.4 – ns

Effective renal plasma flow (mL min�1) 613 � 109 509 � 92 – ns

Glomerular filtration rate (mL min�1) 127 � 8 86 � 9 �32 0.05

Filtered load of sodium (mmol min�1) 17 � 1 12 � 1 �28 0.01

Plasma renin activity (mIU L�1) 75 � 5 44 � 7 �39 0.01

Plasma aldosterone concentration (pg mL�1) 186 � 22 138 � 13 – ns

Plasma ANP concentration (pg mL�1) 25 � 4 46 � 6 +84 0.001

Plasma noradrenaline concentration (nmol L�1) 2.49 � 0.25 3.79 � 0.51 +52 0.05

Plasma vasopressin concentration (pg mL�1) 2.9 � 0.5 2.1 � 0.5 – ns

ANP, atrial natriuretic peptide; Diff., Difference (EH – CON); ns, non-significant. Values are mean � SE. See text for details.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.123454

Hemodynamics and proteomics in hypertension · M Damkjær et al. Acta Physiol 2014

(49–71 lmol min�1, P < 0.01). During the time control

sessions, sodium excretion did not change (Figure S1).

During SL of patients and controls, the relative

decreases in PRC (Pt: �38%, Con: �48%) and plasma

aldosterone (Pt: �52%, Con: �49%) were similar

(Fig. 1). The time courses of the suppression of renin

secretion during saline infusion to EH and control sub-

jects were virtually identical (Fig. 1). During the time

0 1 2 3 4 5 63.5

4.0

4.5

5.0

5.5

h

Car

diac

out

put (

l/min

) Saline loading

0 1 2 3 4 5 660

80

100

120

140

h

GFR

(ml/m

in)

Saline loading

0 1 2 3 4 5 6200

400

600

800

1000

h

ERPF

(ml/m

in)

Saline loading

0 1 2 3 4 5 60

50

100

150

h

Perc

ent c

hang

e A

ldo

Saline loading

0 1 2 3 4 5 60

50

100

150

h

Perc

ent c

hang

e re

nin

Saline loading

0 1 2 3 4 5 680

100

120

140

160

h

Perc

ent c

hang

e N

A

Saline loading

0 1 2 3 4 5 6

85

105

125

h

MA

BP

(mm

Hg)

Saline loading(a) (b) (c)

(e)

***

0 1 2 3 4 5 60

50

100

150

200

h

NaE

x (µ

mol

/min

)

Saline loading(f)(d)

(g) (h) (i)

****

***

+++

****

*

0 1 2 3 4 5 655

60

65

70

75

h

Hea

rt ra

te (b

pm)

Saline loading

*

**

+

**

*

Figure 1 Cardio-renal responses to saline infusion in hypertensive patients (red) and control subjects (black). (a) mean arterial

blood pressure (MABP); dotted blue line: MABP in hypertensive patients during the time control day. (b) cardiac output (CO).

(c) heart rate (HR). (d) effective renal plasma flow (ERPF). (e) glomerular filtration rate (GFR). (f) renal sodium excretion

(NaEx). (g) per cent change from baseline value in plasma renin. (h) per cent change from baseline value in plasma aldosterone.

(i) per cent change from baseline value in plasma noradrenaline. Within series statistically significant change from baseline

value: *P < 0.05, **P < 0.01, ***P < 0.001. Statistically significant difference between experimental series: +P < 0.05,

++P < 0.01.

Table 2 Hemodynamics before and after saline loading

Variable Unit

Control subjects Hypertensive pts.

P-value after vs.

before

Before After Before After Controls Patients

Mean arterial blood pressure mmHg 87 � 2 88 � 2 105** � 2 116** � 3 ns <0.001

Total peripheral resistance mmHg min L�1 17.8 � 0.6 21.5 � 1.3 21.0* � 1.0 26.2* � 1.0 <0.01 <0.001

Total conductance ml (mmHg min)�1 57.5 � 2.1 47.5 � 2.5 48.7* � 2.1 38.2* � 1.5 <0.01 <0.001

Renal conductance ml (mmHg min)�1 14.6 � 2.8 17.4 � 2.1 8.2* � 1.7 7.5* � 1.7 ns ns

Extrarenal conductance ml (mmHg min)�1 42.7 � 3.0 30.1 � 2.8 40.5 � 2.4 30.7 � 3.1 <0.01 <0.01

Values are mean � SE, n = 9 or 10. Different from corresponding values in control subjects at *P < 0.01, **P < 0.001.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12345 5

Acta Physiol 2014 M Damkjær et al. ·Hemodynamics and proteomics in hypertension

control sessions, PRC did not change significantly (Fig-

ure S1). In patients, the saline infusion increased plasma

noradrenaline by 23 � 10% (Fig. 1) in contrast to the

control subjects (Fig. 1). In patients, SL did not change

plasma ANP (46 � 6 to 45 � 7 pg mL�1). In every

control subject, the saline caused a modest increase in

plasma ANP (average 25 � 4 to 30 � 6 pg mL�1,

P < 0.05).

Baseline exosomic proteins

Full data sets of excretion rates of exosomic proteins

before and/or after SL are available from seven

patients and five control subjects. The overall techni-

cal coefficient of variation for protein measurement

was 16 � 3%. At baseline, 438 proteins were quanti-

fied (Fig. 2) in amounts ranging from 0.07 to

49.8 pmol mmol�1 of creatinine (Table S2). In indi-

vidual urine samples, up to 274 individual proteins

were identified and quantified. To avoid ambiguity,

the description of proteins includes database accession

numbers (SwissProt, http://www.uniprot.org). The

data set was filtered using different criteria (see below)

to identify interesting subgroups of proteins poten-

tially useful in describing differences between the EH

patients and controls.

Twelve proteins were detected in all baseline sam-

ples irrespective of blood pressure (Tables 3 and S3).

Of these, b-actin (SwissProt #P60709) was the most

abundant. Several are involved in renal sodium han-

dling, such as aminopeptidase N (#P15144), neprilysin

(#P08473) and solute carrier family 12 member 1

(NKCC2, #Q13621). Most of these proteins were

excreted at rates which were similar in EH patients and

controls (Tables 3 and S3). However, the rates of excre-

tion of retinoic acid-induced gene 2 protein (RAIG-2,

(a) (b)

Figure 2 Excretion rates of proteins in baseline urine samples. (a) 276 proteins detected in control subjects (blue symbols)

ranked by excretion rates. Abscissa: protein with highest excretion rate has rank number 1; increase in rank number reflects

decrease in rate of excretion. Ordinate: rate of excretion in pmol per mmol creatinine (log scale). Dark blue: proteins common

to control subjects and patients (red squares). Light blue: proteins detected only in control subjects. (b) 162 proteins detected

only in EH patients ranked by excretion rates. The sigmoidal curves obtained are characteristic for the log normal distribution

expected from protein quantitation data. It also reflects the dynamic range of the samples analysed. The relatively large scatter

of patient data illustrates the biological variance between samples of a potentially diverse group. For protein identities, see Table

S2a,b. EH, essential hypertension.

Table 3 Exosomic proteins present in all baseline urine samples

Protein Acc.# Excretion rate CON Excretion rate EH

Actin, cytoplasmatic 1 P60709 50 � 21 28 � 9.7

G-protein coupled receptor family C, group 5, member C (RAIG-3) Q9NQ84 25 � 9.4 6.9 � 2.5

Aminopeptidase N P15144 24 � 10 14 � 8.4

Glyceraldehyde-3-P dehydrogenase P04406 11 � 6.4 11 � 4.7

Neprilysin P08473 11 � 4.5 6.2 � 3.5

Ezrin P15311 9.7 � 3.1 5.5 � 2.0

Keratin, type II cytoskeletal 1 P04264 9.4 � 4.7 46 � 40

Alpha-enolase P06733 6.5 � 2.1 7.6 � 3.1

L-lactate dehydrogenase B chain P07195 6.3 � 2.8 5.6 � 2.8

Solute carrier family 12 member 1 (NKCC2) Q13621 5.9 � 1.8 9.8 � 5.9

Heat-shock cognate 71 kDa protein P11142 3.2 � 0.8 1.9 � 0.7

Annexin A2 P07355 2.0 � 0.5 2.2 � 0.8

Acc.#, SwissProt accession number; Excretion rate, rate of excretion (pmol protein (mmol creatinine)�1), mean � SEM; CON,

control subjects; EH, patients with essential hypertension. See Table S3 for more details.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.123456

Hemodynamics and proteomics in hypertension · M Damkjær et al. Acta Physiol 2014

#Q9NZH0) and syntenin-1 (#O00560) were signifi-

cantly lower in patients than in controls (Table S4,

Fig. 3c,d). For other proteins, RAIG-3 (#Q9NQ84),

NKCC2 and TSC (#P55017), the pattern of excretion

at baseline in EH appeared different from the corre-

sponding pattern in the controls: the average rates of

excretion were 4–25 pmol (mmol creatinine)�1 and

statistically indistinguishable (Fig. 3, Tables 3, S3 and

S4), but the variances were markedly larger in EH (all

P < 0.03). Notably, it is the same four patients who

exhibit low rates of excretion of the proteins shown in

Fig. 3. See Data S1 for details on protein function.

Other proteins of interest were the 21 proteins

found in the baseline samples of two or three

patients, but not of any control subject (Tables 4

and S5) including S100-A9 (calgranulin B, #P06702)

and alcohol dehydrogenase class III (ADH5,

#P11766).

Exosomic response to saline infusion

In general, the urinary concentrations of the individual

proteins decreased during SL, while urine flow

increased substantially. Therefore, the rates of excretion

of individual proteins did not change markedly with SL.

Notably, the excretion rates of a number of V-H+AT-

Pase subunits did not follow this pattern (Figure S2,

Table S6). See Data S1 for further details.

A number of proteins were common, but not ubiq-

uitous; 17 proteins were found in all five control sub-

jects and in five or six of the seven EH patients (Table

S4), including dipeptidyl peptidase IV (DPP4,

#P27487), which was excreted at rates averaging 3.6–

7.6 pmol (mmol creatinine)�1 apparently independent

of blood pressure and SL.

Ninety-three proteins were identified once in the

baseline samples from patients, but not in controls,

and remain potentially interesting (Table S7).

Discussion

Our study had two objectives: firstly to assess under

carefully controlled conditions the mechanisms

responsible for the exaggerated natriuresis of EH and

secondly to provide proof of concept of proteomics-

based quantitative measurements of exosomic proteins

as a tool in hypertension research.

Circulatory, renal and endocrine dynamics and

exaggerated natriuresis

The novel hemodynamic and hormonal results are (i)

that the increase in RVR in EH is solely responsible

for the increase in total peripheral resistance, (ii) that

baseline renin system activity and plasma ANP reflect

an endocrine setting favouring natriuresis and likely to

(a) (b)

(c) (d)

Figure 3 Baseline excretion rates of

individual proteins in hypertensive

patients and normotensive controls. (a)

solute carrier family 12 member 1

(NKCC2, #Q13621). (b) solute carrier

family 12 member 3 (TSC, #P55017).

Note log scale of Y axes. (c) retinoic

acid-induced gene 2 protein (RAIG-2,

#Q9NZH0). (d) syntenin-1 protein

(#O00560). (a, b) diuretic-sensitive pro-

teins; (c, d) unknown renal function.

Mean values � SEM.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12345 7

Acta Physiol 2014 M Damkjær et al. ·Hemodynamics and proteomics in hypertension

provide at least part of the background for the exag-

geration of the natriuretic response to saline infusion,

(iii) that the time course of the deactivation of the

renin system during SL in patients was virtually identi-

cal to that of controls and (iv) that the increase in

blood pressure seen during SL unexpectedly occurred

also during the control day where renin system activ-

ity and sodium excretion did not change. The EH

patients showed increased total peripheral resistance

and decreases in GFR and filtered load of sodium as

expected (Goldring et al. 1941, Lowenstein et al.

1967, London et al. 1984).

The study focused on volume expansion in mild,

uncomplicated EH; patients were highly selected to

avoid the confounding effect of target organ damage,

including microalbuminuria. In addition, electrolyte

intake was standardized to low normal values

(Whelton et al. 2012) facilitating measurements of the

dynamic deactivation of the renin system, SL was per-

formed at individual rates of infusion adjusted to body

mass, and the concentration of sodium chloride in the

infusate was adjusted to provide sodium concentra-

tions slightly below those of plasma. Furthermore, the

subjects were investigated in the sitting position.

Together, this provides an unprecedented background

for disclosure of the volume-mediated reactivity of the

renin system and the associated renal excretory

responses.

Under less controlled conditions including more het-

erogeneous groups of patients, EH is assumed to be

associated with varying levels of renin system activity

(Laragh & Sealey 2011). In addition, early measure-

ments of strongly stimulated plasma renin levels (after

low-salt diet and standing) seemed to warrant subdivi-

sion of patients with EH (cf. Tuck et al., 1976). How-

ever, with the present standardization, PRC was

clearly reduced and plasma ANP concentrations

undoubtedly increased, thus providing a pronatriuretic

endocrine setting. It is well known that the natriuretic

effect of ANP is markedly augmented when renin sys-

tem activity is reduced (Bie et al. 1990). The notion of

normal function of the renin system in our patients is

supported by the finding that the acute deactivation of

the renin system during saline infusion was superim-

posable to the response of the normotensive controls.

Increased levels of plasma renin have been reported

before (Schalekamp et al. 1970, Birkenhager et al.

1972), and the differences between these and other

studies may be related to patient selection and stan-

dardization procedures. The present selection is very

restrictive, but the conditions of low normal salt

intake and seated position seem realistic for normal

life. It seems plausible, therefore, that the renal vaso-

constriction in the present hypertensive patients con-

stitutes a primary, renin-independent, inappropriate

antinatriuretic mechanism, which is counterbalanced

by homeostatically appropriate changes in the endo-

crine regulation of sodium excretion. This renal vaso-

constriction may well be neurogenic in consonance

with the original analysis of Hollenberg (Hollenberg

& Adams 1976) and the experimental studies of Esler

et al. (1989). The new twist is that the (inappropriate)

vasoconstriction apparently is occurring solely in the

kidney.

The reports on hemodynamic changes in EH are

numerous, and cardiac output and renal blood flow

have been measured simultaneously in a number of

studies (e.g. Sullivan et al. 1976, Schalekamp et al.

1977, London et al. 1984, van den Meiracker et al.

1989), but non-renal and renal vascular conductances

do not seem to have been assessed previously. Notably,

the extensive analysis of renal hemodynamics in EH

Table 4 Exosomic proteins occurring in baseline urine

samples from at least two patients, but not from any control

subjects

Protein name Acc.#

Excretion

rate

Protein S100-A9 P06702 6.3

Heat-shock protein b-1 P04792 3.7

Alcohol dehydrogenase class 3 P11766 2.6

Lysosome membrane protein 2 Q14108 2.1

Destrin P60981 2.0

Rho GDP-dissociation inhibitor 1 P52565 1.9

Aspartate aminotransferase,

cytoplasmatic

P17174 1.8

Multivesicular body subunit 12A Q96EY5 1.8

Rho-related GTP binding protein

RhoC

P08134 1.7

3-hydroxybutyrate dehydrogenase

type 2

Q9BUT1 1.4

Aldo-ketoreductase family 1

member C3

P42330 1.3

Elongation factor 2 P13639 1.3

Flavin reductase (NADPH) P30043 1.3

Protein NDGR1 Q92597 1.3

D-3-phosphoglycerate dehydrogenase O43175 1.1

Phenazine biosynthesis-like

domain containing protein

P30039 0.9

Unconventional myosin-Ib O43795 0.9

Myosin-9 P35579 0.8

Heat-shock-related 70 kDa protein 2 P54652 0.6

Epidermal growth factor receptor

kinase substrate 8-like protein 1

Q8TE68 0.5

V-type proton ATPase subunit d2 Q8N8Y2 0.4

Acc.#, SwissProt accession number; Excretion rate, rate of

excretion (pmol protein (mmol creatinine)�1), mean values

from two or three patients. No protein occurred in more

than three patients.

See Table S5 for more details.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.123458

Hemodynamics and proteomics in hypertension · M Damkjær et al. Acta Physiol 2014

performed by the group of Hollenberg did not include

renal conductance (cf. Williams et al. 1982, Shoback

et al. 1983, Rydstedt et al. 1986). Our finding that

renal vascular conductance was reduced to an extent

accounting for all the increase in total peripheral resis-

tance supports the notion that renal vasoconstriction is

an essential early element in the development of EH.

In EH, the SL increased blood pressure in contrast

to the control subjects seemingly indicating ‘salt sensi-

tivity’ in EH. However, this may be incorrect; during

the control day, blood pressure increased similarly,

but renal sodium excretion remained unchanged. This

demonstrates firstly that a peculiar diurnal blood pres-

sure rhythm was apparent in the EH patients and, sec-

ondly, that under the present conditions of small

changes (10–12 mmHg) in arterial blood pressure,

these do not necessarily contribute to drive renal

sodium excretion. Consequently, sodium excretion

seems to be regulated primarily by renovascular and

neurohumoral mechanisms.

Together, the hemodynamic and neurohumoral

results indicate that selective renal vasoconstriction

most likely driven by sympathetic nerve activity, or

renal vasomotor events independent hereof, is an early

– if not a primary – process of mild EH and that the

pro-natriuretic setting is an appropriate, compensatory

reaction. The concentration of noradrenaline in

plasma is not a reliable marker of ‘sympathetic drive’,

much less an index of renal nerve activity. Neverthe-

less, the modest increase in plasma noradrenaline in

hypertensive patients at least is compatible with

increased renal sympathetic tone.

Quantitative measurements of exosomic proteins

In recent years, proteomic methods have been applied

to the study of urinary proteins (Valente et al. 2012,

Carty et al. 2013) also of the proteins associated with

exosomes (Moon et al. 2011b, Raj et al. 2012, Rai-

mondo et al. 2013). The novel aspects of the present

study include (i) the measurement of the rates of

excretion for hundreds of individual exosome-related

proteins present in one human urine sample, (ii) the

different excretion patterns of proteins known to be

relevant to hypertension (bumetanide- and thiazide-

sensitive renal ion channel proteins) and (iii) the

excretion patterns of several members of the G-protein

coupled receptor family not previously associated with

EH.

A limited element of probability is involved in the

identification of proteins by mass spectrometry. We

applied conservative criteria for the identification of

individual proteins. Only proteins identified by a mini-

mum of three peptides in at least two of three techni-

cal replicates were analysed further. The MSE data

acquisition strategy enabled reproducible absolute

quantification of protein amounts, in turn making it

possible to calculate excretion rates per unit creatinine,

per unit b-actin and per hour. For the majority of the

proteins, rates of excretion were in the order of 1–

10 pmol mmol�1 of creatinine (Fig. 2). Remarkably,

the excretion rates of most proteins were unaffected by

the about threefold increase in urine flow occurring dur-

ing the SL, indicating that the appearance of exosomic

proteins in the urine is not flow dependent. So-called

household proteins [b-actin and glyceraldehyde-3-P

dehydrogenase (GAPDH)] were among the most abun-

dant proteins present in all baseline samples. GAPDH

may not be entirely ‘household’ (Nicholls et al. 2012),

as it may be part of the potassium channel (ROMK2)

supramolecular complex in the thick ascending loop of

Henle (Renigunta et al. 2011). The abundance of b-actin allows the use of this protein for normalization.

The proteomics approach has been applied to the

analysis of pooled normal human urine from eight

subjects by the group of Pisitkun and Knepper (Gonz-

ales et al. 2009); after elaborate fractionation, they

identified more than 1100 proteins, that is, several

times the number of proteins found in this study.

Using quantitative methods similar to this study,

Moon et al. (2011a)reported to have identified almost

1900 exosomic proteins; however, ambiguous proteins

seem to be included; after proper filtering, the result

was a total of 358 unique proteins, that is, slightly

lower than the present 438 proteins. Among these,

specific groups of proteins included the subset of pro-

teins found in this study in all baseline samples and

the group occurring only in the baseline samples from

patients. Twenty-seven of these 33 proteins (Tables 3

and 4) were also found by Gonzales et al. (2009); the

most notable exception is that the form of actin (cyto-

plasmatic 1, #P60709), found most abundantly in our

study and potentially useful for normalization, does

not occur in the lists of Gonzales et al. (2009). Nota-

bly, this particular protein was present in all four nor-

mal subjects from which individual data were

reported by Moon et al. (2011a). The reason for these

discrepancies is not known, but their existence calls for

elaborate standardization of protocols for sampling,

extraction and measurements. In the study of Gonzales

et al. (2009), a total of 177 proteins were assumed to

be associated with diseases (34 with renal diseases)

based on presence on the ‘Online Mendelian Inheri-

tance in Man’ database. Some of these were found in

this study; of the proteins common to all baseline sam-

ples, two of 12 (neprilysin and NKCC2, Table 3)

belonged to these disease-related proteins; however,

among the proteins occurring in baseline samples only

from patients, a similar fraction (four of 21) was ‘dis-

ease related’ (#O43175, #P04792, #P35579, #Q92597,

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12345 9

Acta Physiol 2014 M Damkjær et al. ·Hemodynamics and proteomics in hypertension

Table 4). Urine samples of hypertensive patients do not

appear to show predominance of individual disease-

related exosomic proteins. Studies of the abundance of

exosome proteins by immunological methods (McKee

et al. 2000, Esteva-Font et al. 2010, van der Lubbe

et al. 2012) have confirmed the presence of sodium

transporters (NKCC2 and TSC), but not provided the

sufficient basis for clinical applications.

In hypertensive patients, the level of excretion of

RAIG-2 protein (RAIG-2) was significantly (P < 0.05)

reduced. RAIG-2 is a G-protein coupled receptor

(family C, group 5, member B, GPRC5B), and its spe-

cific function is unknown, but it has recently been

shown to be elevated in acute renal injury (Kwon

et al. 2014). RAIG-3 (Tables 3 and S3) is another

related G-protein coupled receptor (family C, group 5,

member C) for which gene transcription is induced by

retinoic acid (Robbins et al. 2000); in normal subjects,

the rate of excretion averaged 25 pmol (mmol creati-

nine)�1, but the excretion rates of RAIG-3 were very

low in some patients. Retinoids, including retinoic

acid (all-trans-retinoic acid), are derivates of vitamin

A (retinol) exerting antiproliferative and anti-inflam-

matory actions via retinoid acid receptors (among oth-

ers); retinoid receptors are expressed in the kidney,

where they modify the expression of target genes via

modulation of gene transcription (Dechow et al.

2001). Treatment with retinoic acid has been found to

blunt glomerulosclerosis after 5/6 nephrectomy (Liu

et al. 2011) as well as cardiac remodelling after aortic

constriction (Choudhary et al. 2008) and to decrease

AngII synthesis and receptor expression in a nephritis

model (Dechow et al. 2001). Assuming that urinary

RAIG-2 reflects the activity of retinoic acid dependent

renal pathway(s), we may hypothesize that these path-

ways, in addition to their roles in organ differentiation

and pathophysiology (Gudas 2012), are associated

with the aetiology of EH. Irrespective of molecular

mechanics, RAIG proteins seem to be potential uri-

nary biomarkers of the disease.

Overall, the excretion rates of syntenin are signifi-

cantly lover (P < 0.05) in hypertension (Fig. 3d). Syn-

tenin is a PDZ domain containing adapter protein

involved in intracellular and cell surface targeting

(Sarkar et al. 2004) notably playing an important role

in exosome formation together with ALIX [apoptosis-

linked gene 2 (ALG-2) interacting protein X] (Baietti

et al. 2012, Hurley & Odorizzi 2012). The functional

significance of a possible reduction in the abundance

of syntenin-ALIX-dependent exosomes in the urine in

some of the patients remains unclear.

Other proteins were excreted in conspicuous patterns

in hypertensive patients, for example, the thiazide-sen-

sitive NaCl co-transporter (TSC) for which the variance

of the excretion data in the hypertensive patients was

significantly higher than in controls. The fact that the

same patients exhibited low excretion rates for several

proteins (Fig. 3) indicates a coupling between excretion

patterns. These results support the assumptions that the

excretion rates of individual proteins follow different

distributions in clinically similar EH patients and that

these proteins are potentially useful as means of identifi-

cation of patient subgroups.

This study has several limitations. Firstly, admixture

of proteins from the reproductive tract and the epithe-

lium of the urinary tract undoubtedly occurred, how-

ever, at present to an unknown extent. Secondly,

proteins may remain undetected by the present

method, but still be measured by sensitive antibody-

based techniques. Thirdly, our results demonstrate the

methodological feasibility of quantitative studies of

large ensembles of proteins, but do not prove the exis-

tence of exosomic proteins specific for EH. However,

162 exosome-associated proteins were found in the

urine of all seven hypertensive patients, but not in any

of the samples from any of five normotensive controls.

Although it is very likely that this number is substan-

tially higher than the real difference between the two

groups, it is equally likely that the list includes poten-

tial biomarkers valuable for diagnosis, subdivision and

treatment of patients with EH.

Perspectives on mild, uncomplicated EH

The present results are compatible with the notions (i)

that an early pathophysiological mechanism is selec-

tive renal vasoconstriction mediated by renal nerves,

(ii) that changes in renin system activity and natri-

uretic peptide concentrations contribute to the exag-

geration of natriuresis and (iii) that the excretion rates

of specific proteins of renal origin with or without

known functions may serve to identify subgroups of

patients.

Conflict of interest

None.

The technical assistance of Eva C. Østerlund, Bodil Kristen-

sen and Charlotte Nielsen is greatly appreciated. We thank

Dr. Alexei Bagrov, National Institute of Aging, NIH, Balti-

more, MD, for the analyses of marinobufagenin.

Funding

The work was supported by the Danish Cardiovascu-

lar Research Academy (DaCRA), the Faculty of

Health Sciences, University of Southern Denmark

(SDU), the SDU Center for Clinical Proteomics, Lisa

and Gudmund Jørgensens Foundation, and Annie and

Otto Detlefs’ Foundation.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.1234510

Hemodynamics and proteomics in hypertension · M Damkjær et al. Acta Physiol 2014

References

Ahrne, E., Molzahn, L., Glatter, T. & Schmidt, A. 2013.

Critical assessment of proteome-wide label-free absolute

abundance estimation strategies. Proteomics 13, 2567–

2578.

Alvarez, S., Suazo, C., Boltansky, A., Ursu, M., Carvajal, D.,

Innocenti, G., Vukusich, A., Hurtado, M., Villanueva, S.,

Carreno, J.E., Rogelio, A. & Irarrazabal, C.E. 2013. Urinary

exosomes as a source of kidney dysfunction biomarker in

renal transplantation. Transplant Proc 45, 3719–3723.

Baietti, M.F., Zhang, Z., Mortier, E., Melchior, A., Degeest,

G., Geeraerts, A., Ivarsson, Y., Depoortere, F., Coomans,

C., Vermeiren, E., Zimmermann, P. & David, G. 2012.

Syndecan-syntenin-ALIX regulates the biogenesis of exo-

somes. Nat Cell Biol 14, 677–685.

van Balkom, B.W.M., Pisitkun, T., Verhaar, M.C. & Knep-

per, M.A. 2011. Exosomes and the kidney: prospects for

diagnosis and therapy of renal diseases. Kidney Int 80,

1138–1145.

Bie, P., Wang, B.C., Leadley, R.J. & Goetz, K.L. 1990.

Enhanced atrial peptide natriuresis during angiotensin and

aldosterone blockade in dogs. Am J Physiol Reg Int Comp

Physiol 258, R1101–R1107.

Birkenhager, W.H., Schalekamp, M.A., Krauss, X.H., Kol-

sters, G., Schalekamp-Kuyken, M.P., Kroon, B.J. & Teu-

lings, F.A. 1972. Systemic and renal haemodynamics, body

fluids and renin in benign essential hypertension with special

reference to natural history. Eur J Clin Invest 2, 115–122.

Carty, D.M., Schiffer, E. & Delles, C. 2013. Proteomics in

hypertension. J Hum Hypertens 27, 211–216.

Charloux, A., Lonsdorfer-Wolf, E., Richard, R., Lampert, E.,

Oswald-Mammosser, M., Mettauer, B., Geny, B. & Lons-

dorfer, J. 2000. A new impedance cardiograph device for

the non-invasive evaluation of cardiac output at rest and

during exercise: comparison with the “direct” Fick

method. Eur J Appl Physiol 82, 313–320.

Choudhary, R., Palm-Leis, A., Scott, R.C., Guleria, R.S.,

Rachut, E., Baker, K.M. & Pan, J. 2008. All-trans retinoic

acid prevents development of cardiac remodeling in aortic

banded rats by inhibiting the renin-angiotensin system. Am

J Physiol Heart Circ Physiol 294, H633–H644.

Cobb, L.K., Anderson, C.A., Elliott, P., Hu, F.B., Liu, K.,

Neaton, J.D., Whelton, P.K., Woodward, M., Appel, L.J.

& American Heart Association Council on Lifestyle and

Metabolic Health. 2014. Methodological issues in cohort

studies that relate sodium intake to cardiovascular disease

outcomes: a science advisory from the American heart

association. Circulation 129, 1173–1186.

Cook, N.R., Appel, L.J. & Whelton, P.K. 2014. Lower levels

of sodium intake and reduced cardiovascular risk. Circula-

tion 129, 981–989.

Creskey, M.C., Li, C., Wang, J., Girard, M., Lorbetskie, B.,

Gravel, C., Farnsworth, A., Li, X., Smith, D.G.S. & Cyr,

T.D. 2012. Simultaneous quantification of the viral

antigens hemagglutinin and neuraminidase in influenza

vaccines by LC–MSE. Vaccine 30, 4762–4770.

Damkjær, M., Vafaee, M., Braad, P.E., Petersen, H., Høil-

und-Carlsen, P.F. & Bie, P. 2012. Renal cortical and

medullary blood flow during modest saline loading in

humans. Acta Physiol (Oxf) 205, 472–483.

Damkjaer, M., Isaksson, G.L., Stubbe, J., Jensen, B.L., Asser-

sen, K. & Bie, P. 2013. Renal renin secretion as regulator

of body fluid homeostasis. Pflugers Archiv 465, 153–165.

Dechow, C., Morath, C., Peters, J., Lehrke, I., Waldherr, R.,

Haxsen, V., Ritz, E. & Wagner, J. 2001. Effects of all-

trans retinoic acid on renin-angiotensin system in rats with

experimental nephritis. Am J Physiol Renal Physiol 281,

F909–F919.

Esler, M., Lambert, G. & Jennings, G. 1989. Regional nor-

epinephrine turnover in human hypertension. Clin Exp

Hypertens A 11(Suppl 1), 75–89.

Esteva-Font, C., Wang, X., Ars, E., Guillen-Gomez, E., Sans,

L., Gonzalez Saavedra, I., Torres, F., Torra, R., Masilama-

ni, S., Ballarin, J.A. & Fernandez-Llama, P. 2010. Are

sodium transporters in urinary exosomes reliable markers

of tubular sodium reabsorption in hypertensive patients?.

Nephron Physiol 114, 25–34.

Frithiof, R., Xing, T., McKinley, M.J., May, C.N. & Ramch-

andra, R. 2014. Intracarotid hypertonic sodium chloride

differentially modulates sympathetic nerve activity to the

heart and kidney. Am J Physiol Regul Integr Comp Physiol

306, R567–R575.

Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R. &

Roepstorff, P. 1999. Sample purification and preparation

technique based on nano-scale reversed-phase columns for

the sensitive analysis of complex peptide mixtures by

matrix-assisted laser desorption/ionization mass spectrome-

try. J Mass Spectrom 34, 105–116.

Goldring, W., Chasis, H., Ranges, H.A. & Smith, H.W.

1941. Effective renal blood flow in subjects with essential

hypertension. J Clin Invest 20, 637–653.

Gonzales, P.A., Pisitkun, T., Hoffert, J.D., Tchapyjnikov, D.,

Star, R.A., Kleta, R., Wang, N.S. & Knepper, M.A. 2009.

Large-scale proteomics and phosphoproteomics of urinary

exosomes. J Am Soc Nephrol 20, 363–379.

Green, D.M., Wedell, H.G., Wald, M.H. & Learned, B.

1952. The relation of water and sodium excretion in blood

pressure in human subjects. Circulation 6, 919–924.

Gudas, L.J. 2012. Emerging roles for retinoids in regenera-

tion and differentiation in normal and disease states. Bio-

chim Biophys Acta 1821, 213–221.

Herrera, M. & Coffman, T.M. 2012. The kidney and hyper-

tension: novel insights from transgenic models. Curr Opin

Nephrol Hypertens 21, 171–178.

Hogan, M.C., Johnson, K.L., Zenka, R.M., Cristine Charles-

worth, M., Madden, B.J., Mahoney, D.W., Oberg, A.L.,

Huang, B.Q., Leontovich, A.A., Nesbitt, L.L., Bakeberg,

J.L., McCormick, D.J., Robert Bergen, H. & Ward, C.J.

2014. Subfractionation, characterization, and in-depth pro-

teomic analysis of glomerular membrane vesicles in human

urine. Kidney Int 85, 1225–1237.

Hollenberg, N.K. & Adams, D.F. 1976. The renal circulation

in hypertensive disease. Am J Med 60, 773–784.

Hurley, J.H. & Odorizzi, G. 2012. Get on the exosome bus

with ALIX. Nat Cell Biol 14, 654–655.

Johnson, R.J., Feig, D.I., Nakagawa, T., Sanchez-Lozada,

L.G. & Rodriguez-Iturbe, B. 2008. Pathogenesis of

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12345 11

Acta Physiol 2014 M Damkjær et al. ·Hemodynamics and proteomics in hypertension

essential hypertension: historical paradigms and modern

insights. J Hypertens 26, 381–391.

Kwon, S.H., Liu, K.D. & Mostov, K.E. 2014. Intercellular

transfer of GPRC5B via exosomes drives HGF-mediated

outward growth. Curr Biol 24, 199–204.

Laragh, J.H. & Sealey, J.E. 2011. The plasma renin test

reveals the contribution of body sodium-volume content

(V) and renin-angiotensin (R) vasoconstriction to long-term

blood pressure. Am J Hypertens 24, 1164–1180.

Li, G.-Z., Vissers, J.P.C., Silva, J.C., Golick, D., Gorenstein,

M.V. & Geromanos, S.J. 2009. Database searching and

accounting of multiplexed precursor and product ion spec-

tra from the data independent analysis of simple and com-

plex peptide mixtures. Proteomics 9, 1696–1719.

Liu, X., Lu, L., Tao, B.-B., Zhou, A.-L. & Zhu, Y.-C. 2011.

Amelioration of glomerulosclerosis with all-trans retinoic

acid is linked to decreased plasminogen activator inhibitor-

1 and [alpha]-smooth muscle actin. Acta Pharmacol Sin

32, 70–78.

London, G.M., Safar, M.E., Sassard, J.E., Levenson, J.A. &

Simon, A.C. 1984. Renal and systemic hemodynamics in

sustained essential hypertension. Hypertension 6, 743–754.

Lowenstein, J., Steinmetz, P.R., Effros, R.M., Demeester, M.,

Chasis, H., Baldwin, D.S. & Gomez, D.M. 1967. The dis-

tribution of intrarenal blood flow in normal and hyperten-

sive man. Circulation 35, 250–259.

van der Lubbe, N., Jansen, P.M., Salih, M., Fenton, R.A.,

van den Meiracker, A.H., Danser, A.H.J., Zietse, R. &

Hoorn, E.J. 2012. The phosphorylated sodium chloride

cotransporter in urinary exosomes is superior to prostasin

as a marker for aldosteronism/novelty and significance.

Hypertension 60, 741–748.

Malpas, S.C. 2010. Sympathetic nervous system overactivity

and its role in the development of cardiovascular disease.

Physiol Rev 90, 513–557.

Mancia, G., De Backer, G., Dominiczak, A., Cifkova, R.,

Fagard, R., Germano, G., Grassi, G., Heagerty, A.M.,

Kjeldsen, S.E., Laurent, S. et al. 2007. 2007 Guidelines for

the management of arterial hypertension. Eur Heart J 28,

1462–1536.

Martens, L., Vandekerckhove, J. & Gevaert, K. 2005.

DBToolkit: processing protein databases for peptide-centric

proteomics. Bioinformatics 21, 3584–3585.

McKee, J.A., Kumar, S., Ecelbarger, C.A., Fernandez-Llama,

P., Terris, J. & Knepper, M.A. 2000. Detection of Na(+)

transporter proteins in urine. J Am Soc Nephrol 11, 2128–

2132.

van den Meiracker, A.H., Man in’t Veld, A.J., Boomsma, F.,

Fischberg, D.J., Molinoff, P.B. & Schalekamp, M.A. 1989.

Hemodynamic and beta-adrenergic receptor adaptations

during long-term beta-adrenoceptor blockade. Studies with

acebutolol, atenolol, pindolol, and propranolol in hyper-

tensive patients. Circulation 80, 903–914.

Moon, P.-G., Lee, J.-E., You, S., Kim, T.-K., Cho, J.-H.,

Kim, I.-S., Kwon, T.-H., Kim, C.-D., Park, S.-H., Hwang,

D., Kim, Y.-L. & Baek, M.-C. 2011a. Proteomic analysis

of urinary exosomes from patients of early IgA nephropa-

thy and thin basement membrane nephropathy. Proteomics

11, 2459–2475.

Moon, P.-G., You, S., Lee, J.-E., Hwang, D. & Baek, M.-C.

2011b. Urinary exosomes and proteomics. Mass Spectrom

Rev 30, 1185–1202.

Nicholls, C., Li, H. & Liu, J.-P. 2012. GAPDH: a common

enzyme with uncommon functions. Clin Exp Pharmacol

Physiol 39, 674–679.

Pisitkun, T., Shen, R.-F. & Knepper, M.A. 2004. Identifica-

tion and proteomic profiling of exosomes in human urine.

Proc Natl Acad Sci USA 101, 13368–13373.

Raimondo, F., Corbetta, S., Morosi, L., Chinello, C., Gianaz-

za, E., Castoldi, G., Di Gioia, C., Bombardi, C., Stella, A.,

Battaglia, C., Bianchi, C., Magni, F. & Pitto, M. 2013.

Urinary exosomes and diabetic nephropathy: a proteomic

approach. Mol BioSyst 9, 1139–1146.

Raj, D.A., Fiume, I., Capasso, G. & Pocsfalvi, G. 2012. A mul-

tiplex quantitative proteomics strategy for protein biomar-

ker studies in urinary exosomes. Kidney Int 81, 1263–1272.

Ramchandra, R., Hood, S.G., Frithiof, R. & May, C.N.

2009. Discharge properties of cardiac and renal sympa-

thetic nerves and their impaired responses to changes in

blood volume in heart failure. Am J Physiol Regul Integr

Comp Physiol 297, R665–R674.

Ramchandra, R., Hood, S.G., Frithiof, R., McKinley, M.J. &

May, C.N. 2013. The role of the paraventricular nucleus

of the hypothalamus in the regulation of cardiac and renal

sympathetic nerve activity in conscious normal and heart

failure sheep. J Physiol 591, 93–107.

Raposo, G. & Stoorvogel, W. 2013. Extracellular vesicles:

exosomes, microvesicles, and friends. J Cell Biol 200, 373–

383.

Reisinger, F. & Martens, L. 2009. Database on demand – an

online tool for the custom generation of FASTA-formatted

sequence databases. Proteomics 9, 4421–4424.

Renigunta, A., Mutig, K., Rottermann, K., Schlichth€orl, G.,

Preisig-M€uller, R., Daut, J., Waldegger, S. & Renigunta,

V. 2011. The glycolytic enzymes glyceraldehyde 3-phos-

phate dehydrogenase and enolase interact with the renal

epithelial K+ channel ROMK2 and regulate its function.

Cell Physiol Biochem 28, 663–672.

Robbins, M.J., Michalovich, D., Hill, J., Calver, A.R., Med-

hurst, A.D., Gloger, I., Sims, M., Middlemiss, D.N. & Panga-

los, M.N. 2000. Molecular cloning and characterization of

two novel retinoic acid-inducible orphan G-protein-coupled

receptors (GPRC5B and GPRC5C).Genomics 67, 8–18.

Rydstedt, L.L., Williams, G.H. & Hollenberg, N.K. 1986.

Renal and endocrine response to saline infusion in essential

hypertension. Hypertension 8, 217–222.

Sarkar, D., Boukerche, H., Su, Z.Z. & Fisher, P.B. 2004.

mda-9/syntenin: recent insights into a novel cell signaling

and metastasis-associated gene. Pharmacol Ther 104, 101–

115.

Schalekamp, M.A., Schalekamp-Kuyken, M.P. & Birkenhag-

er, W.H. 1970. Abnormal renal haemodynamics and renin

suppression in hypertensive patients. Clin Sci 38, 101–110.

Schalekamp, M., Birkenh€ager, W., Zaal, G. & Kolsters, G.

1977. Haemodynamic characteristics of low-renin hyper-

tension. Clin Sci Mol Med 52, 405–412.

Shoback, D.M., Williams, G.H., Moore, T.J., Dluhy, R.G.,

Podolsky, S. & Hollenberg, N.K. 1983. Defect in the

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.1234512

Hemodynamics and proteomics in hypertension · M Damkjær et al. Acta Physiol 2014

sodium-modulated tissue responsiveness to angiotensin II

in essential hypertension. J Clin Invest 72, 2115–2124.

Silva, J.C., Gorenstein, M.V., Li, G.-Z., Vissers, J.P.C. &

Geromanos, S.J. 2006. Absolute quantification of proteins

by LCMSE. Mol Cell Proteomics 5, 144–156.

Solomon, P., Belsky, J.L. & Bleifer, K.H. 1960. The response

to the administration of an isotonic sodium chloride-lactate

solution in patients with essential hypertension. J Clin

Invest 39, 876–884.

Strom, B.L., Yaktine, A.L. & Oria, M. 2013. Sodium Intake

in Populations: Assessment of Evidence. Institute of Medi-

cine, Washington, DC.

Sullivan, J.M., Adams, D.F. & Hollenberg, N.K. 1976. beta-

adrenergic blockade in essential hypertension: reduced

renin release despite renal vasoconstriction. Circ Res 39,

532–536.

Tuck, M.L., Williams, G.H., Dluhy, R.G., Greenfield, M. &

Moore, T.J. 1976. A delayed suppression of the renin-

aldosterone axis following saline infusion in human hyper-

tension. Circ Res 39, 711–717.

Valente, M.A.E., Damman, K., Dunselman, P.H.J.M., Hil-

lege, H.L. & Voors, A.A. 2012. Urinary proteins in heart

failure. Prog Cardiovasc Dis 55, 44–55.

Whelton, P.K., Appel, L.J., Sacco, R.L., Anderson, C.A.M.,

Antman, E.M., Campbell, N., Dunbar, S.B., Frohlich,

E.D., Hall, J.E., Jessup, M. et al. 2012. Sodium, blood

pressure, and cardiovascular disease. Circulation 126,

2880–2889.

Williams, G.H., Tuck, M.L., Sullivan, J.M., Dluhy, R.G. &

Hollenberg, N.K. 1982. Parallel adrenal and renal abnor-

malities in young patients with essential hypertension. Am

J Med 72, 907–914.

Wood, S.L., Knowles, M.A., Thompson, D., Selby, P.J. &

Banks, R.E. 2013. Proteomic studies of urinary biomarkers

for prostate, bladder and kidney cancers. Nat Rev Urol 10,

206–218.

Yoshimoto, M., Miki, K., Fink, G.D., King, A. & Osborn,

J.W. 2010a. Chronic angiotensin II infusion causes differ-

ential responses in regional sympathetic nerve activity in

rats. Hypertension 55, 644–651.

Yoshimoto, M., Nagata, K. & Miki, K. 2010b. Differential

control of renal and lumbar sympathetic nerve activity dur-

ing freezing behavior in conscious rats. Am J Physiol Regul

Integr Comp Physiol 299, R1114–R1120.

Supporting Information

Additional Supporting Information may be found in

the online version of this article:

Figure S1. Hemodynamics, plasma hormones and

renal sodium excretion in hypertensive patients during

time control (blue) and saline infusion (Na-load in red).

Figure S2. Changes in excretion rates of V-ATPase

subunits with saline loading in three control subjects

(panel a) and five hypertensive patients (panel b).

Data S1. Methods.

Table S1. Entry characteristics of hypertensive

patients (EH) and control subjects (CON).

Table S2. Exosomic proteins detected in baseline

samples and ranked according to abundance.

Table S3. Exosomic proteins present in all baseline

samples ranked according to abundance in control

subjects.

Table S4. Exosomic proteins present in all baseline

samples of normal subjects, but not all patients.

Table S5. Exosomic proteins occurring in baseline

urine samples from at least 2 patients, but not from

any control subjects.

Table S6. Excretion rates of exosomic V-ATPase

subunits in hypertensives and control subjects, at base-

line and after saline loading.

Table S7. Proteins identified in baseline sample from

one hypertensive patient, but not in any control sub-

ject.

Table S8. Raw peptide and protein data.

Table S9. Excretion rates (pmol mmol�1 creatinine)

with standard deviation and coefficient of variation

(CV) for controls (C) and patients (P) after n ≥ 2 fil-

tering of technical replicates and normalization of

determined absolute protein amounts by measured

creatine levels.

Table S10. Excretion rates (pmol mmol�1 creati-

nine) for proteins identified in ≥2 replicate runs for

individual controls (C) and patients (P), before and

after saline loading, as well as per group

(mean � SEM).

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12345 13

Acta Physiol 2014 M Damkjær et al. ·Hemodynamics and proteomics in hypertension