500
Kidney llj3-HSD2 Is Inhibited by Glycyrrhetinic
Acid-Like Factors in Human Urine
Ying H Lo, Michael F Sheff, Syed A. Latif, Carla Rtbeno, Helene Silver,
Andrew S Brem, David J Morns
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Abstract We have previously shown that human urme contams substances that, like glycyrrhetmlc acid, mhtblt 1 lfi-HSDI
We have named these substances “glycyrrhetmlc acid-like factors” or GALFs We now have found that human urme contams
measurable quanttttes of both llP(HSDl)and lIb(HSD2)GALF mhlbltory substances Both are markedly elevated m pregnancy Their chemical and high-performance hqmd chromatography (HPLC) charactensttcs suggest that several of the GALFs
are steroidal Large quantities of neutral llP(HSDl)and
ll@(HSD2)-GALFs
can be extracted directly from urine mto
ethyl acetate, yteldmg fractton EA, Hydrolyses of the GALFs
remaining m the aqueous phase by P-glucuromdase markedly
mcreases the total amounts of GALFs, with the maJonty now
bemg ethyl acetate extractable (fraction EAJ These EA2 posthydrolysis GALFs can be separated by HPLC resulting m at least
w
e have previously
suggested
that endogenous
substances might exist in humans and other
mammalian species that, like the hcortce derivative GA, inhibit the steroid mactivatmg enzymes 1loHSD and 5P-reductase 1 This followed from the tmportant work of Uhck, New, Monder, and coworker+4 who
demonstrated that hypertensive children with the syndrome of AME lack both 1l&HSD and S@reductase enzyme activity. Diminished activity of these enzymatic steroid metabohc pathways results m changes m the
peripheral metabolism of the glucocorticoid cortisol It
has been postulated that the resulting higher mtrarenal
concentrations of cortisol may then interact with mmeralocorticoid receptors and promote Na+ reabsorptton 3-5
Edwards et a16 and Funder et a17 proposed that m vtvo
renal mmeralocortmoid receptors remain aldosteronespecific because the enzyme lip-HSD
metabolizes
corttsol and corttcosterone (B) to cortisone and ll-dehydro-B, respectively These 1 l-dehydro products, which
have low bmding affinities for MR,7 do not elicit MClike effects and are considered mactive
Our mttial experiments 1demonstrated that partially purified extracts of urine from men and nonpregnant women
dtd indeed contain substances that exhrbtted GA-hke activity, mhibitmg both lip-HSD and SD-reductase We
termed these mhibttory substances “GA-like factors”
(GALFs) The level of GALF mhtbttory acttvtty was elevated m urine of pregnant women and shown to increase
From the Department of Pathology and Laboratory MedIcme, The
Mmam Hospital, Woman and Infants Hospital (H S ), and Rhode
Island Hospital (AS B.), Brown Umverslty School of Medune,
Providence,
RI
Correspondence
and Laboratory
to Dr David J Moms,
Department
Medlcme,
The Mmam
HospltalEkown
164 Summit Ave, Providence, RI 02906
0 1997 American
Heart
Assoclahon,
Inc
of Pathology
Unlverslty,
SIX components with mhlbltory actlvlty agamst each lsoenzyme
Only two GALF peaks are active against both 1 lp-HSD 1 and
1 I&HSD2 The others are peaks with specific 1 lP(HSD l)- and
1 lP(HSD2)-GALF
mhlbltory actlvlty The GALFs m the same
posthydrolysts EA1 extract are also mhlbltory toward the 1 IDHSD 1 that IS present m vascular smooth muscle where they may
play a role m the mechanisms controllmg blood pressure We
have also found that 1 I&HSD2 IS selectively mhlbtted by 5a(but not by 5@-) reduced steroids GC-MS analysts of the
llP(HSD2)-GALFs
m EA? IS now bemg performed to determme
whether this group includes 3cy,5a-nng A-tetrahydr+reduced denvatlves of steroids (Hypertenswn. 1997;29[part
2]:500-505.)
Key Words l hcorlce l
glycyrrhetmlc acid l urine
several-fold
dunng
l
hydroxysterold dehydrogenase
GALF l pregnancy
the second
and third
tnmesters
l
of
pregnancy GALF mhibitory activity is also markedly elevated m patients with congestive heart fatlure.8 These
earher experiments used mtcrosomal preparations of rat
liver or ktdney that predommantly contain 1 ID-HSD isoform 1 This tsoform, 11/I-HSDl, has a high K,,, for B
(~2 ,umol/L), utilizes NADP+, and ts bt-dtrecttonal9 Immediately thereafter, 1l@HSD isoform 2 (1 l,&HSD2)
was discovered m MR contammg cortical-collecting duct
cells of kidney rO.rrThis was followed by the finding that
AME patients exhibit mutations of this umdtrectional
NAD+-dependent, low K,,, for B (~10 to 20 nmol/L)
1 lp-HSD tsoform 2.~13 These experiments focused attention on renal 1 lfl-HSD2 as the “guardian” enzyme
that most likely confers MC specificity on MR-mediated
mechanisms on Na+ ll,&HSD2 has been shown to be
the major tsoform of this enzyme m sheep and human
kidney and has now been cloned from those tissues. 14~5
We have continued our efforts to isolate, further punfy,
and characterize the GALF substances m pregnant human
urine. We now report our experiments utihzmg sheep ktdney mtcrosomal 1l/I-HSD2 m addmon to rat liver mtcrosomal 1I/?-HSDl for the quantttation and chemtcal charactertzation of these 1l/?-HSD mhtbnory substances
present m pregnant human urine
Preparation
Methods
of Urine Extracts
Urme samples were collected from five pregnant
months) and five age-matched nonpregnant women and
-20°C until assayed They were desalted and partially
by the Sep-Pak Ct8 solid-phase extractlon method, which
previously described 1 Bnefly, Sep-Pak Cl8 cartrldges
Chromatography
Dlvlslon,
Mdhpore
Co)
were
prlmed
(6 to 8
stored at
purified
we have
(Waters
with
suc-
cesslve washes of 5 mL methanol and 5 mL water Ahquots of
5 to 20 mL of urme were then passed through the cartrldges
Followmg elutlon of unbound solutes with 5 mL of water, the
LO et al
Selected Abbreviations and Acronyms
1l/3-HSD = 1lp-hydroxysteroid dehydrogenase
l l-dehydro-B = 11-dehydrocorticosterone
AME = apparent mmeralocorticoid excess
CBX = carbenoxolone sochum
DOC = deoxycorticosterone
EA = ethyl acetate
GA = glycyrrhetmlc acid
GALF = glycyrrhetmic acid-hke factor
HPLC = high-performance liquid chromatography
MC = mmeralocorticolds
MR = mmeralocortlcoid receptor
VSM = vascular smooth muscle
compounds of interest were eluted with 3 mL of 100% methanol
These methanohc eluates were dried under reduced pressure m a
Savant Speed-Vat system (Savant Instruments Inc) and then redissolved m water, to one fifth of the original volume These
aqueous preparations were designated LJ, and were stored at 4°C
until used
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Fractionation
of U,, Extracts
The U0 preparations were extracted three times with equal volumes of EA, and the EA extracts (EA,) were dried under Nz and
reconstituted with water to the volume of U0 used for the extraction The remammg aqueous-phase extracts (AqJ were incubated
overmght at 37’C with P-glucuromdase (2 U/mL) m 0 1 mol/L
Na acetate buffer (pH 6 8) and were then passed through SepPaks as described above to remove the enzyme and buffer (see
flowsheet m Fig 1) Small ahquots of these samples, of known
volume, were removed and stored at 4°C for later assay of
1lP(HSDl)and 1lP(HSD2)-GALF
mhlbltory activity The remamder of the P-glucuromdase-treated
aqueous extracts (Aq,G)
were each extracted three times with equal volumes of EA to
yield the second EA extracts (EA,) and the second aqueous
extracts (Aqz) As before, ahquots of the extracts from each
step were taken and stored at 4°C before measurement of their
llP(HSDl)and 1 l,&HSD2)-GALF
mlubitory activity
The abilities of these extracts to inhibit 1 I/?-HSD activities m
mlcrosomal preparations from rat liver (1 lb-HSDl)
and sheep
kidney (1 lp-HSD2) were measured m duplicate as previously
described 16Briefly, for the assay of I l/I-HSDl enzymatic activIty, rat liver nncrosomes (50 to 100 pg protein) were incubated
at 37°C for 10 minutes with 5 ymol/L corticosterone (B) and
3H-corticosteione (1 &I) as tracer m 50 mmol/L Tns-HCL buffer
(pH 8 5) containing 3 4 mmol/L, NADP+ m a total volume of
0 25 mL For the assay of 1 lp-HSD2 enzymatic actlvlty, sheep
kidney tmcrosomes (4 to 30 pg protein) were incubated at 37°C
for 10 minutes with 50 nmoln 3H-corhcosterone (1 &I) as tracer
and substrate m 50 mmol/L Tns-HCl buffer (pH 8 5), contammg
200 pmol/L NAD* m a total volume of 0.25 mL 11 P-HSDl
from homogenates of rat aorta VSM cell preparations was measured as previously described17 and also used for studies of mhibition For all assays, an ahquot of either water (controls), urine
extracts, or aqueous solutions of known quantities of CBX were
added The CBX was used for the construction of standard curves
of mlnbltory activity against which the mhibltory activities of the
urine extracts could be compaied The reactions were terminated
by addition of 0 75 mL of 100% methanol
HPLC Assay of Enzyme Activity
The conversion of B to I I-dehydrocortlcosterone
(A) was
measured by separating the compounds by HPLC and detecting
and quantifying them with an on-line system Ahquots of the
methanol extracts from the incubation media were diluted with
water to 45% methanol and chromatographed on Du Pont Zorbax
C8 reverse-phase columns at 44°C usmg lsocratlc 62% aqueous
methanol This system allowed the adequate separation of radtoactive peaks (A at 6 8 mmutes and B at 8 8 minutes) The quantities of A and B were measured from the radioactivity detected
Kidney
ll&HSDZ
Inhibitors
in Human
Urine
501
and integrated by the on-line P-detection system (Rachomatlc
model FLO-ONE\Beta,
rachochromatography detector, Packard
Instrument Co) The percentage converSion was calculated and
used as a measure of enzyme activity Inhibitory activity was
expressed as the percentage reduction m enzyme activity resultmg from the presence of either known amounts of CBX or known
volumes of urine extract m the mcubation mixture The percent
of mhibition produced by varying quantities of CBX was plotted
against Its concentrations as a reference curve, and the mhlbltory
actlvlty of the urine samples was expressed as the nanograms or
micrograms of CBX that produced the same amount of mhibition
under the standardized conditions Since the ICs0 for CBX agamst
rat-hver 1l,&HSDI IS two orders of magnitude smaller than that
against sheep-kidney 1 lb-HSD2, independent GALF CBX units
for the two lsoenzymes are required These differ greatly The
range used for the CBX standard curve for rat-liver 1 lb-HSDl
1s 2 ng/mL to 32 ng/mL, while that used for sheep-kidney 1 lpHSD2 1s 200 ng/mL to 3200 ng/mL These separate GALF units
represent only the relative mlnbitory potencies of the GALFs m
the samples compared with CBX They are not an absolute measure of the quantities of the mhtbitory substances.
It should be noted that smnlar patterns of mhlbitory activity
were found when ‘H-cortisol was used as a substrate to determine
1 lp-HSD2 activity
Generation of HPLC Inhibitor
Graphs
In our mitral expenments to separate the GALF substances, we
found that there was more than one 1 l&HSD mhibitor m each
of the urines Therefore, we have chromatographed the mdivldual
EAI and EA2 extracts on a Du Pont Zorbax C8 reverse-phase
column, elutmg the mdlvidual fractions with a methanol gradient
beginning with 52% aqueous methanol that increased concavely
to 100% methanol by 35 minutes Each mdhhter of the eluant
was collected mto a borosdicate test tube usmg an automated
fraction collector and was later tested for its effects on rat-hver
(1 l&HSDl)
and sheep-kidney (1 l&HSD2) enzyme acuvity Radtolabeled cortisol (F), corticosterone (B), DOC, progesterone
(P), and unlabelled 3aJ&tetrahydro-progesterone
(detected at
191 nm absorbance) were added to the chromatographlc samples
as internal standards and were used for comparisons of the retention times of the GALFs
The results are plotted as a graph of mhibitory activity against
fraction number This 1s termed the “mhibltor graph” and shows
the mlnbitory profile for each EA, and EA2 sample
Chemicals
[1,2-3H] was obtained
Methanol (HPLC-grade)
NADPH, Tns-HCI buffer,
were obtained from Sigma
from Du Pont New England Nuclear
was obtained from Fisher Sclentlfic
NADP+, NAD+, B, and 11 dehydroB
Chemical Co
Results
Measurement of lip-HSD2 GALF in UOExtracts
from Pregnant and Nonpregnant (Control)
Human Urine
When U,, extracts (Fig 1) from pregnant and control human
urines were assayed for their mhlbltory achvltles agamst
sheep-ladney 1l/3-HSD2, the amounts of 1l,&HSD2)-GALF
mtibltory activity found m the pregnant urine extracts were
approximately four times higher than those m control extracts
(Table).
EA Extraction
Aq, and EA, extracts (prepared as described above)
from both pregnant and control urmes were also assayed
for inhibitory activity The amount of 1 I@(HSD2)-GALF
inhibitory activity was again approximately four times
higher m both fractions (Aq, and EA,) from pregnant urine
extracts (Table). However, the majority (>70%) of the
Hypertension
502
Vol29, No 1, Part 2 January 1997
Urine
pregnant urme contained approximately 6 to 7 times more
inhibitory activity than that m the corresponding extracts
from controls (Table) As with 11/3(HSD2)-GALF, the
bulk of the 1 l/?(HSDl)-GALF inhrbrtory actrvity m both
pregnant and control urine extracts was found m the EA,
fraction
b
Sep pak
uoEA extraetmnJx
\1
A, -
HPLC
Hydrolysis by j!LGlucuronidase
0 glueuron~dase Hydrolysm
Aqtc
EA extractmn, 3x
\1
t
3/
42
FIG 1
EA2 -HPLC
Procedure
for fractionation
of urine
samples
1l,@HSD2)-GALF mhrbrtory actrvrty was recovered m
the EA, extracts of both groups
Hydrolysis by /SGlucuronidase
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
There was a considerable increase m mhlbrtory activity
when the Aq, extracts from both pregnant and control
urines were hydrolyzed wrth P-glucuronidase.
Compared wrth Aq,, mhrbrtory actlvrty of the posthydrolysrs Aq,G was increased 2 to 3 times m pregnant and
4 to 5 times in control urines Followmg EA extractron,
the maJonty (>75%) of the increased mhrbrtory actrvrty
m Aq,G was recovered m the EA2 fractions m both pregnant and control urines and IS comparable m amount to
that m the EA, extracts Therefore, the majority of the
originally water-soluble polar GALF substances m the Aq,
fractrons must be glucuromdes that, after P-glucuromdase
hydrolyses became aglycones, which partmoned into the
EA phase (EA2) (Table). The llP(HSD2)-GALF mhrbrtory actrvrty present m EAZ fractrons of pregnant urine was
approximately 2.5 to 3 times higher than that m control
EAa fractions
Measurement of ll@(HSDl)-GALF
in Pregnant and
Nonpregnant (Control) Human Urine
Comparison of llP(HSD2)-GALF
Inhibitor
From Pregnant and Control Urine
EA Extraction
Aq, and EA1 extracts were also tested for 1lP(HSDl)GALF inhibitory actrvrty Both of the extracts from the
Graphs From EAI Extracts
The 1l@(HSD2)-GALF inhibitor graphs from EA, extracts of both pregnant and control urmes (Fig 2) have
three common peaks of mhrbrtory activity m the more polar region (fraction numbers 11, 13, and 18) and three common peaks m the less polar region (fraction numbers 32,
35, and 39) An addrtronal highly polar peak of
1lP(HSD2)-GALF inhibitory activity (fraction number 5)
was present m the control urine but not m the pregnant
urine EA, extracts The areas under the peaks of fraction
and llP(HSDl)-GALF
Inhibitory Actiwty in Aqueous
From Pregnant and Control Human Urine
GALF
lnhlbltory
ActWyl24
11/3(HSD2)*
Fractions
Pregnant
h
1 lfi(HSDl)t
Controls
Pregnant
Controls
uo
26
6
63
8
EAT
Aql
Ip-Glucuronidase
22
9
5
2
43
23
6
4
24
19
8
9
7
2
135
109
12
41
38
5
hydrolysis
Aq,G
EA,
Aqz
*Expressed
tExpressed
as mg of CBX producing
as pg of CBX producing
Graphs
The llB(HSD2)-GALF
substances m EA, and EA,
fractions were further separated mto a series of “polar”
and “less polar” mhrbrtory fractions by HPLC reversephase chromatography as described m “Methods ” This
solvent system reproducibly separates the steroid standards cortrsol, cortrcosterone, DOC, progesterone, and
3a5/3-tetrahydroprogesterone with retention times of 11 8,
17.4, 24.5, 31 3, and 34 2 minutes, respectively EA, and
EA2 extracts, obtained from comparable volumes of U0
from pregnant and control urines, were chromatographed
and each 1 mL fraction assayed for GALF mhtbrtory activity as previously described The results are plotted as
the mhtbrtor graphs m Ags 2 through 5.
Inhibitor
When U0 extracts (Ftg 1) from pregnant and control
urines were tested against rat-liver 1l/?-HSDl, the
1 l,f?(HSDl)-GALF mhrbrtory activity was approximately
8 times higher m the pregnant urme extracts than in the
controls (Table).
11/3(HSD2)-GALF
and EA Fractions
After the Aq, fractions were hydrolyzed by P-glucuronrdase, there was an increase of approximately 6 times m
the 1lP(HSDl)-GALF mhrbrtory activity m the pregnant
urme AqrG extracts and approximately 10 trmes m the
equivalent control extracts (Table). As m the case of
1 lP(HSD2)-GALF substances, the maJorrty (>7.5%) of
the originally polar 1lP(HSDl)-GALF substances then
partitioned mto the EA phase (EA,) after P-glucuromdase
hydrolyses 1lP(HSDl)-GALF mhrbrtory activity became
srgmficantly hrgher m the EA2 extracts when compared
wrth the EA, extracts m both pregnant and control groups.
The final llP(HSDl)-GALF
mhrbrtory actrvtty m pregnant urine EA1 fractions was approxrmately 3 times larger
than those of the EA2 extracts from the control urmes
same degree
same degree
of tnhibitory
of mhlbitory
activity
actiwty
Lo et al
Kidney
ll&HSD2
90
--ir
-
Control
Pregnant
Inhibitors
’
’
in Human
‘fC
80
’
-*
-c
70
5
'5
g
=
's
*
0
503
Urine
Rat hepatlc
Ill3 HSDl
Sheep renal
HI3 HSDZ
60
50
40
30
20
10
0
0
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
HPLC
Fraction
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
numbers 11, 13, and 18 were higher m pregnant urine extracts, whereas those of fraction numbers 32, 35, and 39
were reduced compared with controls.
Graphs From EAz Extracts
The profile of 1I,f?(HSD2)-GALF mhtbttory actwlty
m
the EA2 extracts from pregnant urine (Ftg 3) showed the
presence of 6 principal peaks together with at least 6 secondary peaks of mhtbttory actrvtty By contrast, HPLC of
control urine EA2 extracts showed the presence of two
principal peaks together with 6 secondary peaks of
1 IP(HSD2)-GALF inhibitory acttvrty Although the major
peaks eluted in the same region of the solvent gradient as
cortrsol, corttcosterone, and DOC, they dtd not exhibit the
UV charactenstrcs of these steroid standards Large drfferences were observed m the areas under these peaks
when the inhibitor graph of the pregnant urine EA2 extracts
was compared wtth those of the controls Smce the total
quantity of inhibitory activity IS less m the EA2 sample
from the control urine than m the EA1 extracts from the
pregnant urmes, all of the control peaks were smaller than
those from the pregnant urine. However, the peaks elutmg
at 16, 18, 25, and 28 minutes were drsproportronately
markedly reduced (Fig 3) Fraction numbers 23 and 28 m
EA2, whtch contam both ll/?(HSDl)- and ll@(HSD2)GALF mhtbttory activmes (Fig 4), are not present m EAl
extracts of either control or pregnant urines
f
90
Q
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
HPLC
FIG 2
A representative
1 lP(HSD2)
inhlbttor
graph from EA, extracts of pooled urine from pregnant
and control humans
Arrows
lndlcate
the fraction
numbers
where cot-hsol (F), corkosterone
(B), deoxycortlcosterone
(DOC) and progesterone
(P) elute
Inhibitor
4
Number
Dr
80
$
Number
of 1 lP(HSDl)and 1 l@(HSD2)-GALF
FIG 4
Comparison
rtor graphs
of EA* extracts
from pregnant
human urine
Inhlb-
Comparison of 11/3(HSDl)- and llP(HSD2)-GALF
Inhibitor Graphs of EA2 Extracts From
Pregnant Urine
Fractions of the EA2 extracts from pregnant urines separated by HPLC were also tested for both 1IP(HSDI)GALF and 1 lP(HSD2)-GALF mhtbttory activity. Constderable differences m the resulting mhtbttor graphs were
observed and are shown m Rg 4. With the exception of
the two common peaks of mhtbttory material m fraction
numbers 23 and 27/28, the major peaks of 11/3(HSD2)GALF inhibitory activity appeared m the polar region (left
of the steroid standard DOC), whereas those of
1l@(HSDl)-GALF appeared m the nonpolar region (rtght
of DOC) of the chromatograph However, both sets of
GALFs elute in the same region of the HPLC solvent gradient that encompasses the steroid standards. As indicated
by the differences m the mhtbttory acttvtty of the HPLC
separated peaks of GALF substances against 1lp-HSDl
and lip-HSD2, two peaks mhrbtt both ll&HSDl
and
1l&HSD2 acttvtty, while the others dlfferenttally inhibit
either 1lp-HSDl or 1lp-HSD2.
We also used rat VSM cell preparations as a second
source of 1I&HSDl. Other experiments m our laboratortes17 have shown that rat aorta preparations aud VSM
cells cultured from rat aorta contain a bi-directional
NADP+-dependent I I@HSDI, whrch has a smaller K,,,for
B (100 nmol/L) than that for rat-liver mtcrosomal preparations. When U0 extracts (Fig 1) from pregnant and control urines were assayed against homogenates of VSM
00
-- Control
-
Fraction
i
1
Q
DOC
J,
80
B -- VSMC
-c
Pregnant
70
Ill3 HSDl
Sheep renal
Illl HSDZ
z 60
:E
2 50
c
= 40
%
$ 3c
2c
i
IC
y
C
4
6
HPLC
FIG
tracts
3
4
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
A representative
of pooled urine
Fraction
1 lP(HSD2)
from pregnant
InhIbItor
graph from EA, exand control
humans
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
HPLC
Number
5
inhibitor
FIG
Fraction
Number
Comparison
of VSM 1 lp(HSDl)with
graphs
of EA, extracts
from pregnant
1 lP(HSD2)-GALF
human urine
504
Hypertension
Vol29,
No 1, Part 2
January
1997
cells,‘7 both types of urine showed measurable quantities
of 1 lP(HSD1) vsM-GALF mhibnory acttvtty
HPLC fractions of the EA2 extracts from pregnant
urines were also tested for 1l@(HSD1)vsM-GALF inhibitory actrvity for comparison with their mhtbitory activity
against lip-HSD2. The profile of the inhibitor graph is
similar to that obtained by when rat liver microsomal preparations of 1 l@-HSDl were used (Fig 5)
Discussion
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
The discovery that mgestion of licorice by humans
mimtcs the clinical features and changes m corns01 metabolism observed m patients with AME’s led to our hypothesis that GA (m licorice) may mimic the physiological
acttvity of endogenous factor(s), GALFs It was hypothesized that these GALF substances would serve to cause
glucocorttcotds, and possibly other steroids, to ehcit Naf
retention by MR-mediated mechamsms and so augment,
either naturally or in dtsease states, the Na+ retammg actions of aldosterone.
We had earlier shown m viva that the succmate derivative of GA CBX conferred renal MR-medtated Naf retention on cortisol and corticosterone (B) m adrenalectomized rats. 19.20We have shown that endogenous substances
such as the bile acid, chenodeoxychohc acid, and several
progesterone derivatives likewise conferred Na+ retention
on B 21.22However, the onginal 1 l@-HSD “guardian” hypothesis first proposed by Edwards et al,6 Funder et al,7
and others2 was later refined by Rusvai and Naray-FeJesToth,” who proposed that the 1 l@HSD tsoform 2 (1 lpHSD2) in the distal portions of the kidney acts as the
“protective” mechanism that normally prevents glucocorticoids from accessing renal MR. The more recent finding
that AME patients exhibit mutations of the NAD+-dependent 1lp-HSD2Q13 has offered considerable evidence
supporting the “guardian” role of 1 lo-HSD2 The role of
1l@-HSDl, which IS present predominantly m liver but
also in several other tissues such as VSM,i7 IS less clear
at this time.
In this report, we demonstrate that human urine contains, in addition to 1l/?(HSDl)-GALF, measurable quantities of 1l@(HSD2)-GALF Inhibitory substances and that
both are markedly elevated in pregnancy. Using HPLC,
we have now separated several peaks of GALF mhtbttory
substances, some of which possess specific 1l@(HSD2)GALF, while others possess only 1 lP(HSDl)-GALF mhibitory activity. Two HPLC peaks, which elute in fraction
numbers 23 and 28 (Fig 4), possess both ll/?(HSDl)GALF and 1l&HSD2)-GALF
inhibitory activity Each
tsolated GALF-contammg peak from urine of pregnant humans was markedly elevated when compared wtth those
isolated from nonpregnant females of similar age
Sigmficant quanttnes of both 1lP(HSDl)- and 1lP(HSD2)GALF mhibttory substances were extractable from the
partially purified urine extracts with EA (EA,, see flow
sheet, Ftg 1). Interestingly, when the GALF mhtbitory substances then remammg m the aqueous phase were subjected to hydrolysis by a P-glucuromdase preparation (devoid of any sulfatase activity), not only did the measurable
quantities of 1 l/?(HSDl)- and 1 lP(HSD2)-GALF mhibttory substances markedly increase by at least 2- to lo-fold,
but the maJonty then became EA (EA,) extractable The
HPLC Inhibitor graphs indicated that the GALFs released
after hydrolysts (and EA2 soluble) did not possessthe same
elution times as those isolated and separated by HPLC of
the EA, fraction. Thus, significant quantities of neutral
(EA soluble) GALF substances (aglycones) are released,
which are more active as 1lP(HSDl)- and 1lP(HSD2)GALFs than their glucuromde comugate forms. Monder
and White9 had previously shown reduced potency of glycyrrhtzm (the glycostde) compared with the 1l&HSD mhibitory activity of GA itself The GALF mhrbitory substances remaining m the aqueous phase (Aq,G) are now
being studied to determine the proportion and chemical
tdentmes of those that may be present as sulfates and be
susceptible to hydrolysis by pure sulfatase enzyme
preparations
Several of the 1lP(HSDl)- and 1 lD(HSD2)-GALF mhibttory substances m the EA, extractable fraction, and m
the EAZ extractable fraction (followmg ,&glucuromdase
hydrolyses), yielded separated peaks of acttvtty after
HPLC, with retention times m the vicmity of known steroid hormone standards (eg, cornsol, corticosterone, DOC,
and progesterone) However, they did not exhibit the UV
characteristics of these steroid standards
The majority of steroid hormones are excreted by humans as glucuromde and sulfate conJugates of then rmg
A-3o,SaTH- and 3o,SPTH-reduced derivattves We have
recently shownI that sheep-kidney mtcrosomal preparations of llfi-HSD2 are selectively mhtbited by these
3a,%+tetrahydro dertvatives of steroids but not by their
3cr,5/3-tetrahydro derivatives Several of the posthydrolysis 1 l/J(HSD2)-GALF mhtbttory substances m the
EAZ fractton, chromatograph where known standards of
3a,5a-rmg A reduced steroids, such as 3a,Sa-tetrahydro-B
and 3a,Scr-tetrahydro-l l-dehydro-B and 1lp-hydroxyand l l-keto-3a,5o-tetrahydro derivatives of progesterone
elute. Thus, if the 1 lP(HSD2)-GALFs present m EA2 extracts are of steroidal ongm, they would most likely be
3a,5a-TH steroid derivatives
We have recently demonstrated So-derivatives of the
glucocorticotd B (eg, So-DHB and 3a,Sa-THB) and 1I/?OH-progesterone23 as well as its 3a,5a-tetrahydro derivative, which all potently inhibit lip-HSD2, can m vivo
confer Na+-retammg acttvny on B m adrenalectomized
rats and can also cause increases m blood pressure when
infused mto usually normotensive Sprague-Dawley rats
(private communication, G.W Souness and D J. Morris,
1996) We plan m future experiments to test whether these
GALF substances m the EA2 extracts have similar btological activities.
Each of the HPLC-separated peaks of 1 l,&HSDl)- and
1 l/?(HSD2)-GALF substances is currently being analyzed
using GC-MS analysis These studies will also determine
whether the llP(HSD2)-GALFs m EA2 mclude 3a,5crring A-tetrahydro-reduced dertvattves of steroids It is tmportant to point out that the chemical structure and tdentity
of the mdtvidual GALF substances have not been ascertamed at this time Several GALF substances possesscharactertstics similar to those of neutral steroids and steroid
glucoromdes, and several of the EA extractable substances
chromatographed wtth retention times close to (but not
identical with) those of known steroid standards Nevertheless, the complexity and the number of substances excreted m urme of human pregnant mdtvtduals will make
the identification of the mdtvidual llP(HSDl)and
1lP(HSD2)-GALF substances difficult
Considerable addttional expertmentatton is now necessary to conclusively provide evidence as to the ongm and
physiological role(s) of the GALF substances The present
experiments clearly provide evidence for the existence of
both endogenous 1lP(HSDl)- and 11/3(HSD2)-GALF
Lo et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
substances and that one class IS most hkely steroidal m
ongm. 1 lp-HSDl has been clearly shown to be present in
VSM2425 and to play a functional rolel7.26in the regulation
of blood pressure The finding that the isolated GALF material(s) also inhibits rat VSM preparations of 1l&HSDI
possibly m&cates an additional role for GALFs
The recent experiments of Takeda et a127showing elevated levels of 1 lP(HSD%)-GALF mhibltory substances
m the urme of patients with low renm essential hypertension, and that they can be regulated by dietary Na+, are
indeed extremely interesting Similarly, the recent findings
of Soro et al*8 clearly demonstrate clmunlshed 1 l/3-HSD
and SP-reductase activity in patients with essential hypertension Their findmgs confirm the earhel work of Komel
et alz9J0that the %-ring A-reduced pathway 1sfavored m
these patients, with increases m the excretion of both 5achhydro- and 3a~,.%(allo)-tetrahydro denvatlves of several
steroid hormones These are very interesting findings, even
though initial studies using measurements of 1 lP(HSDl)GALF have not yet found consistently elevated levels of
total GALF m patients with essential hypertension 8.31
Further studies are now most important to determine
not only the chemical identities of the various farnihes
of substances that can function as GALFs but to reveal
the source and regulation of their synthesis and their
physlologlcal role(s).
II
Acknowledgments
20
This work was supportedby Natlonal Institutes of Health National Heart, Lung, and Blood Institute grant HL-52972 and by
the Miriam Hospital ResearchFoundation We thank Elizabeth
Glfford for excellent secretarlalassistance
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Hypertension. 1997;29:500-505
doi: 10.1161/01.HYP.29.1.500
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