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Inhibition of erythrocyte phosphatidylserine exposure by urea and Cl(-)

Article  in  American journal of physiology. Renal physiology · July 2004

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Am J Physiol Renal Physiol 286: F1046 –F1053, 2004;
10.1152/ajprenal.00263.2003.
Inhibition of erythrocyte phosphatidylserine exposure by urea and Cl⫺
Karl S. Lang,1 Swetlana Myssina,1 Philipp A. Lang,1 Valerie Tanneur,1 Daniela S. Kempe,1
Andreas F. Mack,2 Stephan M. Huber,1 Thomas Wieder,1 Florian Lang,1 and Christophe Duranton1
Departments of 1Physiology and 2Anatomy, University of Tübingen, D-72076 Tübingen, Germany
Submitted 19 December 2003; accepted in final form 12 January 2004
Lang, Karl S., Swetlana Myssina, Philipp A. Lang, Valerie
Tanneur, Daniela S. Kempe, Andreas F. Mack, Stephan M.
Huber, Thomas Wieder, Florian Lang, and Christophe Duranton.
Inhibition of erythrocyte phosphatidylserine exposure by urea and
Cl⫺. Am J Physiol Renal Physiol 286: F1046 –F1053, 2004. 10.1152/
ajprenal.00263.2003.—Osmotic shock by addition of sucrose to the
medium stimulates erythrocyte sphingomyelinase with subsequent
ceramide formation and triggers Ca2⫹ entry through stimulation of
cation channels. Both ceramide and Ca2⫹ activate an erythrocyte
scramblase, leading to breakdown of phosphatidylserine asymmetry, a
typical feature of apoptosis. Because erythrocytes are regularly ex-
posed to osmotic shock during passage of kidney medulla, the present
study explored the influence of NaCl and urea on erythrocyte phos-
phatidylserine exposure as determined by annexin binding. The per-
centage of annexin-binding erythrocytes increased from ⬍5 to 80 ⫾
4% (n ⫽ 4) upon addition of 650 mM sucrose, an effect paralleled by
activation of the cation channel and stimulation of ceramide forma-
tion. The number of annexin-binding erythrocytes increased only to
18% after addition of 325 mM NaCl and was not increased by
addition of 650 mM urea. According to whole cell patch-clamp
experiments, the cation conductance was activated by replacement of
extracellular Cl⫺ with gluconate at isotonic conditions or by addition
of hypertonic sucrose or urea. Although stimulating the cation con-
ductance, urea abrogated the annexin binding and concomitant in-
crease of ceramide levels induced by osmotic cell shrinkage. In vitro
sphingomyelinase assays demonstrated a direct inhibitory effect of
urea on sphingomyelinase activity. Urea did not significantly interfere
with annexin binding after addition of ceramide. In conclusion, both
Cl⫺ and urea blunt erythrocyte phosphatidylserine exposure after
osmotic shock. Whereas Cl⫺ is effective through inhibition of the
cation conductance, urea exerts its effect through inhibition of sphin-
gomyelinase, thus blunting formation of ceramide.
cation channels; sphingomyelinase; ceramide; apoptosis; cell volume;
osmolarity; phosphatidylserine
ERYTHROCYTES LACK MITOCHONDRIA and nuclei, intracellular or-
ganelles involved in the induction of apoptosis (22, 24). Nev-
ertheless, treatment of erythrocytes with the Ca2⫹ ionophore
ionomycin has recently been shown to induce erythrocyte
shrinkage, membrane blebbing, and breakdown of cell mem-
brane phosphatidylserine asymmetry, all typical features of
apoptosis in other cell types (5, 10, 15, 33). Cells exposing
phosphatidylserine at their surface are recognized by phospha-
tidylserine receptor-carrying macrophages (20), which bind,
phagocytose, and thus clear those cells from circulating blood.
The breakdown of phosphatidylserine asymmetry results
from the stimulation of a scramblase (63), which is activated
by an increase of cytosolic Ca2⫹ activity (16, 58, 59). Cyto-
solic Ca2⫹ activity is increased during exposure of erythrocytes
to osmotic shock by an increase of extracellular osmolarity,
Address for reprint requests and other correspondence: F. Lang, Physiolo-
gisches Institut, der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen
(E-mail: florian.lang@uni-tuebingen.de).
F1046 0363-6127/04 $5.00 Copyright © 2004 the American Physiological Society
which opens Ca2⫹-permeable cation conductance (33). More-
over, osmotic shock stimulates a sphingomyelinase (Smase)
with subsequent formation of ceramide (36), which potentiates
the effect of cytosolic Ca2⫹ activity on erythrocyte scramblase
(33, 36).
Because osmotic shock may trigger Ca2⫹ entry and cer-
amide formation, the question arises whether those events
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occur during passage of erythrocytes through kidney medulla
with the extraordinary NaCl and urea required to concentrate
the urine (49). The present study has been performed to test
whether hypertonic NaCl and urea similarly induce annexin
binding as hypertonic sucrose and to elucidate the related
mechanisms.
METHODS
Solutions. Erythrocytes were drawn from healthy volunteer donors
who gave informed consent, and the study was approved by the
Ethical Committee of the Medical Faculty. Experiments were per-
formed at 37°C in a standard Ringer solution containing (in mM): 125
NaCl, 5 KCl, 1 MgSO4, 32 HEPES/NaOH, 5 glucose, and 1 CaCl2
(pH ⫽ 7.4). Where indicated, osmolarity was increased by adding
sucrose, NaCl, or urea on top of isotonic Ringer. In some experiments,
extracellular Cl⫺ was removed by replacing NaCl, KCl, and CaCl2
from the standard Ringer solution with equimolar amounts of sodium-
D-gluconate (125 mM), potassium-D-gluconate (5 mM), and calcium-
(D-gluconate)2 (1 mM) or by NaNO3 (125 mM), KNO3 (5 mM), and
Ca(NO3)2 (1 mM).
Smase from Staphylococcus aureus was purchased from Biomol
and used at a final concentration of 0.005 U/ml by serial dilution from
a 100 U/ml stock solution. Externally added Smase has been shown to
be capable of degrading cell membrane sphingomyelin with the
formation of ceramide (27, 43, 46, 51). D-Erythro-N-hexanoylsphin-
gosine (C6-ceramide) was purchased from Biomol (Hamburg, Ger-
many). D-Erythro-N-hexanoylsphingosine was dissolved in DMSO to
give a 50 mM stock solution and further diluted to a final concentra-
tion of 50 ␮M in standard Ringer solution containing 0.1% BSA. The
maximum concentration of DMSO was in all cases 0.1%, a concen-
tration that did not induce annexin binding (data not shown).
A monoclonal anti-ceramide antibody (clone MID 15B4; isotype
IgM) was purchased from Alexis (Grünberg, Germany).
FACS analysis. FACS analysis was performed essentially as de-
scribed earlier (2, 31). After incubation, cells were washed in annexin-
binding buffer containing 125 mM NaCl, 10 mM HEPES/NaOH
(pH ⫽ 7.4), and 5 mM CaCl2. Erythrocytes were stained with
annexin-FLUOS (Böhringer Mannheim, Mannheim, Germany) at a
1:100 dilution. After 15 min, samples were diluted 1:5 and measured
by flow cytometric analysis on a FACS-Calibur (Becton-Dickinson,
Heidelberg, Germany). Cells were analyzed by forward and side
scatter, and annexin-fluorescence intensity was measured in FL-1.
Control experiments revealed that urea (600 mM) did not interfere
with the annexin-binding assay of hypertonically stressed erythrocytes
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
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 INHIBITION OF ERYTHROCYTE “APOPTOSIS” BY UREA AND CL⫺
(Ringer ⫹ 650 mM sucrose for 8 h) when added 5 min before the end
of incubation. In those experiments, the percentage of annexin-
binding cells was 63 ⫾ 6% (n ⫽ 6) in the absence and 62 ⫾ 5%
(n ⫽ 6) in the presence of urea.
For determination of ceramide formation, cells were stained for 1 h
at 4°C with 1 ␮g/ml anti-ceramide antibody in PBS containing 1%
FCS at a dilution of 1:5, as described recently (36). After three washes
with PBS/1% FCS, cells were stained with polyclonal FITC-conju-
gated goat anti-mouse Ig-specific antibody (Pharmingen, Hamburg,
Germany) in PBS/1% FCS at a dilution of 1:50 for 30 min. Unbound
secondary antibody was removed by washing the cells two times
with PBS/1% FCS, and samples were analyzed by flow cytometric
analysis on a FACS-Calibur. FITC-fluorescence intensity was mea-
sured in FL-1.
Immunofluorescence. Control or ischemic (30 min) kidneys were
quickly frozen in liquid nitrogen and transferred to a cryostat. Exper-
iments were performed according to the German Animal Protection
Law and were approved by the local authorities. Sections were taken
at 20 ␮m and stained unfixed with annexin-V-FLUOS (Roche) diluted
1:100 in buffer containing (in mM) 140 NaCl, 10 HEPES/NaOH, and
5 CaCl2 (pH 7.4). After being rinsed two times with the same buffer,
sections were covered with a coverslip and analyzed immediately.
Sections were examined on a Zeiss LSM510 confocal microscope
using an Argon laser with excitation at 488 nm, with appropriate
emission filters, and a ⫻40 oil immersion lens (numerical aperture
1.3). Images or small z-stacks of images from large blood vessels in
the outer medulla were scanned, and the number of annexin-V-
FLUOS-positive cells was counted. The confocal microscopy soft-
ware supplied by the manufacturer was used to measure the cross-
sectioned area of the blood vessels. Together with the optical section
thickness or z-dimension of the stack (ranging from 3 to 6 ␮m), we
calculated the number of stained cells per volume.
Measurement of intracellular ATP concentrations. The intracellu-
lar ATP concentration of human erythrocytes was quantified by a
luciferin-luciferase assay kit (Roche Diagnostics, Mannheim, Ger-
many) using a luminometer (Berthold Biolumat LB9500, Bad Wild-
bad, Germany). Briefly, fresh erythrocytes were washed (3 ⫻ 5 min)
in PBS Ca2⫹-free medium and centrifuged, and 100 ␮l of blood pellet
(hematocrit 100%) were incubated for 8 h at 37°C with standard
Ringer solution or Ringer supplemented with 650 mM urea or 650
mM sucrose (final hematocrit ⬃5–7%). After incubation, cells were
lysed in distillated water, and proteins were precipitated by HClO4
(5%). After centrifugation, an aliquot of the supernatant (400 ␮l) was
adjusted to pH 7.7 by addition of saturated KHCO3 solution. All of the
manipulations were performed at 4°C to avoid ATP degradation.
After dilution of the supernatant, the ATP concentration of the
aliquots was determined by luciferin/luciferase reaction according to
the manufacturer’s protocol. The given values are representative of
the average ATP concentration measured for 100 ␮l pelleted blood
(hematocrit 100%).
In vitro Smase assay. In vitro measurements of Smase activity were
performed essentially as described earlier (36) using total lipid ex-
tracts from erythrocytes supplemented with [choline-methyl-
3
H]sphingomyelin (with a specific radioactivity of 2.96 TBq/mmol;
purchased from American Radiolabeled Chemicals, St. Louis, MO) as
substrate. Erythrocytes (2 ⫻ 109) were washed two times with 1 ml
standard Ringer solution. Next, total lipids were extracted by resus-
pending the cell pellets in 500 ␮l methanol, 250 ␮l chloroform, and
200 ␮l water. Samples were stirred for 10 min on a vortex mixer and
centrifuged at 13,000 g for 2 min. Phase separation was accomplished
by the addition of 250 ␮l chloroform and 250 ␮l water. The suspen-
sion and centrifugation steps were repeated. [choline-methyl-
3
H]sphingomyelin (55.5 kBq) was added to 150 ␮l of the chloroform
phase and stirred for 1 min. The radioactive substrate solution (10 ␮l)
was dried under nitrogen and used for the in vitro Smase assay. The
radioactive substrate was resuspended in 100 ␮l of assay buffer (100
mM Tris·HCl, pH 7.4, 6 mM MgCl2, and 0.1% Triton X-100 in the
AJP-Renal Physiol • VOL
F1047
presence or absence of 600 mM urea). Samples were sonicated for 5
min, and 0.2 U/ml Smase from Staphylococcus aureus (Biomol) or
Streptomyces species (Sigma, Taufkirchen, Germany) were added.
Reaction mixtures were incubated for different times at 37°C. After 5,
10, and 30 min, 20 ␮l of the samples were removed, and the reactions
were stopped by addition of 133 ␮l chloroform and 67 ␮l methanol.
Phase separation was completed by addition of 20 ␮l water. Smase-
catalyzed release of [3H]phosphocholine was quantitated by counting
20 ␮l of the upper, aqueous phase. Blank reactions contained no
Smase, and values were subtracted from the sample values.
Patch clamp. Patch-clamp experiments have been performed as
described earlier (26, 36). Erythrocytes were recorded at 21°C. A
continuous superfusion was applied through a flow system inserted in
the dish. The bath was grounded via an NaCl bridge filled with bath
NaCl solution (see below). Borosilicate glass pipettes (8 –14 M⍀ tip
resistance; GC 150 TF-10; Clark Medical Instruments, Pangbourne,
UK) manufactured by a microprocessor-driven DMZ puller (Zeitz,
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.3 on May 11, 2017
Augsburg, Germany) were used in combination with an MS314
electrical micromanipulator (MW; Märzhäuser, Wetzlar, Germany).
The currents were recorded in voltage-clamp mode in fast whole cell
configuration by an EPC-9 amplifier (Heka, Lambrecht, Germany)
using Pulse software (Heka) and an ITC-16 Interface (Instrutech, Port
Washington, NY). The whole cell currents were evoked by a pulse
protocol, clamping the voltage in 11 successive 400-ms square pulses
from the ⫺10 mV holding potential to potentials between ⫺100 and
⫹100 mV.
The potentials were corrected for liquid junction potentials (3). The
original whole cell current traces are depicted after 500 Hz low-pass
filtering, and currents of the individual voltage square pulses are
superimposed. The applied voltages refer to the cytoplasmic face of
the cell membrane with respect to the extracellular space. The inward
currents, defined as flow of positive charge from the extracellular to
the cytoplasmic membrane face, are negative currents and depicted as
downward deflections of the original current traces.
Whole cell currents were recorded in standard isotonic bath solu-
tion containing (in mM) 115 NaCl, 10 HEPES, 5 CaCl2, and 10
MgCl2, titrated with NaOH to pH 7.4, in combination with a sodium-
D-gluconate or potassium-D-gluconate pipette solution containing (in
mM) 115 sodium-D-gluconate or potassium-D-gluconate, 10 NaCl, 1
EGTA, 1 MgCl2, 1 Mg-ATP, and 5 HEPES, titrated to pH 7.35 with
NaOH. A residual Cl⫺ concentration of 10 mM was used in these
pipette solutions to avoid shifts in offset potential.
Where indicated, NaCl bath solution was replaced by sodium-D-
gluconate solution containing (in mM): 140 sodium gluconate, 5
HEPES, 1 CaCl2, and 1 MgCl2. The removal of Cl⫺ eliminates the
outward currents generated by Cl⫺ influx and upregulates the cation
conductance (18). For some experiments, the osmolarity of the so-
dium-D-gluconate bath solution was increased by addition of 250 mM
sucrose or 250 mM urea.
Statistics. Data are expressed as arithmetic means ⫾ SE. Statistical
analysis was made by paired or unpaired t-test, where appropriate.
RESULTS
Effects of hypertonic sucrose, NaCl, and urea on erythrocyte
annexin binding. In isotonic extracellular fluid, only a small
fraction of human erythrocytes (4 ⫾ 1%, n ⫽ 12) showed
detectable annexin binding, reflecting the virtual absence of
phosphatidylserine at the extracellular face of the erythrocyte
cell membrane (Fig. 1A). Increase of osmolarity by addition of
sucrose, however, led to a sharp increase in annexin binding,
reflecting breakdown of the phosphatidylserine asymmetry
(Fig. 1A); after addition of 650 mM sucrose, 80 ⫾ 4% (n ⫽ 4)
of the human erythrocytes bound annexin within 8 h (Fig. 1, A
and B). The same increase in osmolarity by addition of 325
mM NaCl (650 mosmol/l) induced annexin binding only in
286 • JUNE 2004 • www.ajprenal.org
F1048 INHIBITION OF ERYTHROCYTE “APOPTOSIS” BY UREA AND CL⫺
Fig. 1. Effect of hypertonic sucrose, NaCl, and urea on erythrocyte annexin
binding and intracellular ATP. A: histograms showing the percentage of
annexin-binding erythrocytes after incubation for 8 h in isotonic Ringer
solution (top left), Ringer solution ⫹ 650 mM sucrose (top right), Ringer
solution ⫹ 325 mM NaCl (bottom left), or Ringer solution ⫹ 650 mM urea
(bottom right). Nos. refer to the calculated percentage of one single experi-
ment. B: percentage of annexin-binding erythrocytes after 8 h of treatment with
urea, NaCl, and sucrose as a function of osmolarity (arithmetic means ⫾ SE,
n ⫽ 4). The cells were suspended in Ringer NaCl solution (300 mosmol/l) with
additional increasing concentrations of sucrose (from 300 to 650 mM), NaCl
(from 150 to 325 mM), or urea (from 300 to 650 mM). C: histograms showing
the concentrations of intracellular ATP after incubation for 8 h in isotonic
Ringer solution, Ringer solution ⫹ 650 mM sucrose, or Ringer solution ⫹ 650
mM urea (arithmetic means ⫾ SE, n ⫽ 15). Data are expressed as mM
intracellular ATP/l blood (hematocrit ⬃100%, n ⫽ 10).
18 ⫾ 3% (n ⫽ 4) of the cells (Fig. 1, A and B). In sharp
contrast, addition of 650 mM urea did not significantly enhance
the percentage of annexin-binding cells (4 ⫾ 1%, n ⫽ 4; Fig.
1, A and B). As shown in Fig. 1B, a correlation is observed
between the number of annexin-binding cells and the hyper-
osmolarity by increasing concentrations of sucrose (varying
from 300 to 650 mM) or NaCl (varying from 150 to 325 mM)
but not urea (varying from 300 to 650 mM). The annexin
binding after osmotic shock was not significantly blunted in the
presence of the Na⫹-K⫹-2Cl⫺ cotransport inhibitor bumet-
anide. In this series of experiments, after 8 h of osmotic shock
(Ringer ⫹ 650 mM sucrose), the percentage of annexin-
binding cells was 45 ⫾ 2 (n ⫽ 4) in the absence and 46 ⫾ 2
(n ⫽ 4) in the presence of bumetanide (100 ␮M).
ATP measurements using luciferin-luciferase assay revealed
that an 8-h incubation in Ringer supplemented with 650 mM
urea or 650 mM sucrose induced a moderate decrease (8 and
32%, respectively, n ⫽ 15) of the intracellular ATP concen-
AJP-Renal Physiol • VOL 286 • JUNE 2004 •
tration compared with the control condition (isosmotic Ringer,
n ⫽ 15, Fig. 1C).
Inhibitory effect of urea on annexin binding in erythrocytes
exposed to osmotic shock. The inability of hypertonic urea to
induce annexin binding prompted us to explore whether urea
could inhibit the annexin binding of erythrocytes exposed to
hypertonic sucrose concentrations. In the absence of urea, the
addition of sucrose (Ringer ⫹ 650 mM) led within 8 h to
annexin binding in 73 ⫾ 5% of the cells (n ⫽ 4; Fig. 2A). The
addition of urea (600 mM) blunted the sucrose-induced an-
nexin binding despite a further increase of osmolarity (Fig.
2A). In contrast, an increase to the same osmolarity by addition
of sucrose (1,250 mM) induced annexin binding in 80 ⫾ 3%
(n ⫽ 6) of the cells. Figure 2B shows the dose-response curve
for urea-dependent inhibition of erythrocyte annexin binding at
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a constant concentration of sucrose (650 mM on top of isotonic
Ringer) and increasing concentrations of urea (added on top of
isotonic Ringer and sucrose). The additional presence of 600
mM urea decreased the percentage of annexin-binding cells to
20 ⫾ 5% (n ⫽ 4, Fig. 2, A and B) despite the presence of 650
mM sucrose on top of isotonic Ringer. Although the addition
of up to 300 mM sucrose on top of isotonic Ringer does not
significantly increase the number of annexin-binding cells (Fig.
1B), the addition of 250 mM sucrose and replacement of NaCl
with sodium gluconate significantly enhanced the number of
annexin-binding cells, an effect partially reversed by further
addition of urea (600 mM, Fig. 2C).
Because urea, unlike sucrose, rapidly enters cells (32), it
does not create an osmotic gradient across the cell membrane.
We thus explored whether phloretin, a nonspecific inhibitor of
urea transport (41), modifies the effect of urea on annexin
binding. Phloretin (700 ␮M) led to a slight increase of annexin
binding in isotonic Ringer (9 ⫾ 2%, n ⫽ 9) but did not
significantly interfere with the inhibitory effect of urea. Urea
(600 mM) reduced the annexin binding induced by hyperos-
motic shock (Ringer plus 650 mM sucrose for 8 h) by 44 ⫾ 8%
(n ⫽ 9) and 52 ⫾ 8% (n ⫽ 9) in the presence and absence of
phloretin, respectively.
In the absence of extracellular Ca2⫹, the effect of exposure
to hypertonic shock (Ringer ⫹ 650 mM sucrose) on annexin
binding was significantly blunted by 17 ⫾ 6% (n ⫽ 5), but the
inhibitory effect of urea was preserved. The percentage of
inhibition was 57 ⫾ 5% (n ⫽ 5) in the absence of Ca2⫹
compared with 58 ⫾ 5% (n ⫽ 5) in the presence of Ca2⫹.
Activation of cation conductance by Cl⫺ removal and hy-
perosmolarity. Patch-clamp experiments have been performed
to elucidate the role of osmolarity, Cl⫺, and urea in the
regulation of the nonselective cation conductance. As shown in
Fig. 3A, the cation conductance was activated by replacement
of NaCl with sodium gluconate, even in isotonic extracellular
fluid. Upon removal of external Cl⫺, the conductance (G; as
calculated between ⫹40 and ⫹100 mV) was 223 ⫾ 41 pS
(n ⫽ 17) and 251 ⫾ 51 pS (n ⫽ 8) when recorded with sodium
gluconate and potassium gluconate pipette solution, respec-
tively.
Addition of either sucrose (250 mM) or urea (250 mM) on
top of isotonic sodium gluconate further stimulated the chan-
nel. Figure 3B depicts the corresponding current-voltage rela-
tionships recorded with isosmotic sodium gluconate (n ⫽ 16),
sodium gluconate ⫹ 250 mM sucrose (n ⫽ 4), or sodium
gluconate ⫹ 250 mM urea (n ⫽ 4). Figure 3C shows the mean
www.ajprenal.org
 INHIBITION OF ERYTHROCYTE “APOPTOSIS” BY UREA AND CL⫺
slope conductance (calculated between ⫹40 and ⫹100 mV)
recorded with isosmotic sodium gluconate bath solution (G ⫽
0.31 ⫾ 0.06 nS, n ⫽ 16) and after increasing the osmolarity of
the sodium gluconate bath solution by addition of 250 mM
sucrose (G ⫽ 0.52 ⫾ 0.04 nS, n ⫽ 4) or 250 mM urea (G ⫽
0.47 ⫾ 0.06 nS, n ⫽ 4). Thus, similar to hypertonic sucrose,
hypertonic urea activates the cation conductance.
Fig. 3. Activation of cation channels by Cl⫺ removal and hyperosmolarity. A:
original whole cell current traces (sodium gluconate pipette solution) recorded
with isotonic bath solution (NaCl), after removal of Cl⫺ (sodium gluconate),
addition of 250 mM sucrose (sodium gluconate ⫹ sucrose), replacement of
sucrose by 250 mM urea (sodium gluconate ⫹ urea), and washout of urea by
isotonic bath solution (NaCl). B: current (I)-voltage (V) relationships recorded
as in A with isotonic (‚) and hypertonic sodium gluconate bath solution
(sodium gluconate ⫹ 250 mM sucrose; {) and after isosmotic replacement of
sucrose by urea (sodium gluconate ⫹ 250 mM urea, }). Data are means ⫾ SE
(n ⫽ 4). C: comparison of the mean conductance ⫾ SE (n ⫽ 4) calculated from
the outward current (⫹40 to ⫹100 mV) in the presence of isotonic sodium
gluconate bath solution or under hypertonic conditions after addition of 250
mM sucrose or 250 mM urea to the sodium gluconate bath solution. D: annexin
binding of erythrocytes after a 20-h treatment in isotonic extracellular fluid
without and with isosmotic replacement of NaCl by sodium gluconate (arith-
metic means ⫾ SE, n ⫽ 4).
AJP-Renal Physiol • VOL
F1049
Fig. 2. Inhibition of sucrose-induced annexin binding by
urea. A: histograms of annexin binding in a representa-
tive experiment of erythrocytes incubated in isotonic
Ringer solution (left), in Ringer solution ⫹ 650 mM
sucrose (suc; middle), and in Ringer solution ⫹ 650 mM
sucrose ⫹ 600 mM urea (right) for 8 h. B: percentage of
annexin-binding erythrocytes after 8 h of incubation
with a Ringer solution ⫹ 650 mM sucrose as a function
of the concentration of urea added on top of 650 mM
sucrose (arithmetic means ⫾ SE, n ⫽ 4). C: percentage
of annexin-binding erythrocytes after 8 h of incubation
with Cl⫺-free Ringer solution (NaCl replaced by so-
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dium gluconate) ⫹ 250 mM sucrose as a function of the
concentration of urea added on top of 250 mM sucrose
(arithmetic means ⫾ SE, n ⫽ 4).
Because removal of extracellular Cl⫺ activates the cation
conductance even without an increase of osmolarity, the effect
of Cl⫺ removal on annexin binding was tested. As shown in
Fig. 3D, replacement of isotonic Cl⫺ by isotonic gluconate
Fig. 4. Inhibition of sphingomyelinase (Smase)-induced annexin binding by
urea. A: representative histograms of erythrocytes incubated for 8 h in isotonic
Ringer solution with purified Smase (0.005 U/ml, top left), Smase (0.005
U/ml) ⫹ urea (600 mM, top right), ceramide (50 ␮M, bottom left), or ceramide
(50 ␮M) ⫹ urea (600 mM, bottom right). B: arithmetic means ⫾ SE (n ⫽ 4)
of the percentage of annexin-binding cells stimulated with Smase (0.005 U/ml)
or ceramide (50 ␮M) for 8 h in the presence of different urea concentrations.
Control refers to cells stimulated with Smase (0.005 U/ml) or ceramide (50
␮M) in the absence of urea.
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F1050 INHIBITION OF ERYTHROCYTE “APOPTOSIS” BY UREA AND CL⫺

within 20 h significantly increased the number of annexin-

binding cells. Similarly, replacement of extracellular Cl⫺
by isotonic NO⫺ 3 significantly increased the number of
annexin-binding cells from 5 ⫾ 1% (n ⫽ 10) to 11 ⫾ 2%
(n ⫽ 10).
Inhibitory effect of urea on Smase-induced annexin binding.
Because urea inhibited annexin binding despite the activation
of the nonselective cation conductance, experiments were per-
formed to test for a putative effect of urea on Smase-mediated
annexin binding. As shown in Fig. 4A, exposure of erythro-
cytes to purified Smase (0.005 U/ml) led within 8 h to annexin
binding in 68 ⫾ 2% (n ⫽ 4) of the cells. A similar increase of
the percentage of annexin-binding cells (62 ⫾ 11%, n ⫽ 4) was
observed after C6-ceramide exposure (50 ␮M). Figure 4B
shows the concentration-dependent effect of urea on Smase-

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induced annexin binding. In these experiments, addition of 600
mM urea virtually abolished (3 ⫾ 2%, n ⫽ 4) the breakdown
of phosphatidylserine asymmetry after addition of Smase. In
contrast, ceramide-induced annexin binding was further en-
hanced in the presence of urea, approaching 95 ⫾ 3% (n ⫽ 4)
of the cells for 600 mM urea (Fig. 4B).
Inhibition of ceramide formation and Smase activity by urea.
The observation that urea inhibits annexin binding after addi-
tion of Smase and hypertonicity, but not after addition of
ceramide, suggested that urea directly influences Smase. Fur-
ther experiments have been performed to test for an effect of
urea on Smase activity. As shown in Fig. 5, osmotic shock
induced by addition of sucrose (650 mM sucrose) stimulated

Fig. 6. Inhibition of in vitro Smase activity by urea. A: time-dependent release

of [3H]phosphocholine from [choline-methyl-3H]sphingomyelin by Smase was
measured in control assay buffer as described in METHODS. Arithmetic means ⫾
SE (n ⫽ 3) of liberated [3H]phosphocholine are given in dpm. B: arithmetic
means ⫾ SE (n ⫽ 3) of liberated [3H]phosphocholine/min after incubation in
assay buffer in the absence (control) or in the presence of 600 mM urea (urea).
Smase either from Staphylococcus aureus or Streptomyces species was used as
an enzyme source. *Significant difference from control assay buffer (2-tailed
t-test, P ⱕ 0.05).

ceramide formation in erythrocytes (the levels of ceramide

Fig. 5. Inhibition of ceramide formation by urea. A: representative histograms
of ceramide detection in control cells (isotonic Ringer, black, all histograms)
and cells exposed for 4 h to hypertonic sucrose solution (Ringer ⫹ 650 mM
sucrose) in the absence (gray, left) or in the presence (gray, middle) of 600 mM
urea. Cells exposed for 5 min to purified Smase in isotonic Ringer (1 U/ml)
served as a positive control for ceramide production (gray, right). B: arithmetic
means ⫾ SE (n ⫽ 4) of the mean ceramide-related fluorescence after 4 h of
incubation with an isotonic Ringer NaCl solution (control), after 4 h in
hypertonic sucrose solution (suc; Ringer ⫹ 650 mM sucrose), after 4 h in
hypertonic sucrose solution in the presence of urea (suc ⫹ urea, Ringer ⫹ 650
mM sucrose ⫹ 600 mM urea), and after 5 min in the presence of 1 U/ml
purified Smase in isotonic Ringer (Smase) as a positive control. *Significant
difference from control cells and #significant difference from cells in hyper-
tonic sucrose solution in the absence of urea (2-tailed t-test, P ⱕ 0.05).
AJP-Renal Physiol • VOL 286 • JUNE 2004 •
formation start to increase after 30 min and reach a plateau
after 4 h; data not shown). After 4 h, ceramide levels reached
181 ⫾ 30% of control (n ⫽ 4), an effect completely reversed
to 106 ⫾ 15% of control (n ⫽ 4) in the presence of urea (650
mM sucrose ⫹ 600 mM urea). As a positive control for
ceramide detection, erythrocytes were treated with purified
Smase (1 U/ml during 5 min). As expected, this maneuver led
to increased ceramide formation, approaching 303 ⫾ 48% (n ⫽
4) of control (Fig. 5B).
In vitro assays were performed in addition to test for the
influence of urea on Smase activity. For this, total erythrocyte
lipid extract supplemented with [choline-methyl-3H]sphingo-
myelin was used as substrate. As shown in Fig. 6A, production
of [3H]phosphocholine increased within 10 min of incubation
with Smase and could thus serve as a direct measure of Smase
activity. Urea significantly reduced the activity of Smase from
Staphylococcus aureus and Streptomyces species by 44 ⫾ 4%
(n ⫽ 3) and 35 ⫾ 2% (n ⫽ 3), respectively (Fig. 6B).
www.ajprenal.org
 INHIBITION OF ERYTHROCYTE “APOPTOSIS” BY UREA AND CL⫺
Induction of erythrocyte phosphatidylserine exposure in
ischemic mouse kidney. To test for a possible role in acute renal
failure, erythrocyte phosphatidylserine exposure and vessel
wall adherence were estimated in control and ischemic (30
min) mouse kidney by fluorescence microscopy. As shown in
Fig. 7, A and B, ischemia induced a moderate but significant
(P ⱕ 0.05) increase in the percentage of phosphatidylserine-
exposing erythrocytes (from 100 ⫾ 23 to 231 ⫾ 45%; n ⫽
6 – 8; Fig. 7C). Specifically, phosphatidylserine-exposing
erythrocytes were found in the large veins at the cortex-
medulla interface. In particular, a fraction of phosphatidyl-
serine-exposing erythrocytes was adjacent to the vessel wall,
suggesting cytoadherence to the endothelium.
DISCUSSION
The present study confirms the previous observations that
hyperosmotic shock stimulates breakdown of the phosphati-
dylserine asymmetry in erythrocytes (33, 35). In vivo, nucle-
ated cells (20) and erythrocytes (6, 19) exposing phosphatidyl-
serine will be eliminated by macrophages. As shown previ-
ously, hyperosmotic shock and oxidative stress open a
nonselective cation conductance allowing the passage of Ca2⫹
(18, 26). Volume-regulatory cation conductances are not only
expressed in erythrocytes but also in a wide variety of nucle-
ated cells (11, 12, 21, 30, 32, 54 –56). The cation conductance
in erythrocytes has been characterized previously (18, 26). In
those cells, the cation conductance probably does not serve cell
volume regulation. In contrast, Ca2⫹ entry through the cation
conductance (33) activates the Gardos channel (35), leading to
hyperpolarization, loss of cellular KCl, and cell shrinkage.
Increase of extracellular K⫹ rather blunts cell shrinkage and
erythrocyte annexin binding after an increase of cytosolic
Ca2⫹ (34).
Increase of cytosolic Ca2⫹ in the erythrocytes may result not
only from enhanced entry but also from impaired extrusion.
Osmotic shock leads to a decrease of ATP concentrations,
AJP-Renal Physiol • VOL
F1051
which may at least in part result from enhanced ATP hydro-
lysis (60). A decrease of ATP concentration may interfere with
Ca2⫹ extrusion by the Ca2⫹-ATPase and thus increase cyto-
solic Ca2⫹ concentration.
Beyond the activation of nonspecific cation conductance,
hyperosmotic cell shrinkage activates in erythrocytes an endo-
geneous Smase, leading within 30 min to the formation of
ceramide and with a slight delay to stimulation of annexin
binding (36). The formation of ceramide is paralleled by the
decrease of sphingomyelin (36). External application of cer-
amide triggers annexin binding in erythrocytes (36) and nucle-
ated cells (17, 38). Ceramide sensitizes erythrocytes for the
action of Ca2⫹ (36). Along those lines, the increase of annexin
binding after osmotic shock was blunted in the presence of the
putative Smase inhibitor dichloroisocoumarin (36). In nucle-
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.3 on May 11, 2017
ated cells (27, 43) and erythrocytes, addition of exogenous
Smase similarly stimulates ceramide formation and subsequent
increase of annexin binding. Involvement of ceramide in sig-
naling of apoptosis has been shown for nucleated cells, such as
CD95-induced lymphocyte death (23, 25) and treatment of
cells with various chemotherapeutic agents, e.g., hexade-
cylphosphocholine (57) or daunorubicin (9). The ability of
C6-ceramide to induce erythrocyte phosphatidylserine expo-
sure is surprising, since erythrocytes lack mitochondria, crucial
elements in the signaling cascade triggered by C6-ceramide in
nucleated cells. C6-ceramide must utilize a pathway distinct
from that described in nucleated cells. Thus the capacity of
C6-ceramide to trigger phosphatidylserine exposure in eryth-
rocytes discloses the presence of an alternative pathway that
may exist in nucleated cells as well. C6-ceramide triggers
erythrocyte phosphatidylserine exposure even though it does
not enhance Ca2⫹ uptake (36).
Excessive osmolarity, as it prevails in renal medulla, may
trigger apoptosis of nucleated cells (7, 8, 32, 35, 40, 45, 47,
48). Moreover, it compromises the function of lymphocytes,
which inhibits defense in kidney medulla (37). In cultured
Fig. 7. Annexin-binding erythrocytes in is-
chemic kidney. A and B: confocal laser scan
images from sections of control (A) and is-
chemic (30 min; B) mouse kidneys stained
with annexin-V-FLUOS to detect phosphati-
dylserine-exposing erythrocytes. Shown are
fluorescence images (left) and superimposed
fluorescence and transmission light images
(right) from veins at the cortex medulla bor-
der (as indicated by scheme on top right).
Arrowheads indicate phosphatidylserine-ex-
posing erythrocytes adhering at the vessel
wall. C: relative number of phosphatidyl-
serine-exposing erythrocytes in sections of
control (open bar) and ischemic (closed bar)
mouse kidneys. Data are means ⫾ SE of 6 – 8
z-stacks of vessel images from 2 kidneys for
each condition. OM, outer medulla; IM, in-
ner medulla.
286 • JUNE 2004 • www.ajprenal.org
F1052 INHIBITION OF ERYTHROCYTE “APOPTOSIS” BY UREA AND CL⫺
renal tubular epithelial cells, the apoptosis induced by NaCl
could be reversed by urea (53, 61, 62). On the other hand, urea
has been shown to sensitize cells to apoptosis (13) and lead to
cell cycle delay (45). In the present study, we clearly show that
urea (600 mM) inhibits hyperosmotic shock-induced erythro-
cyte annexin binding. Notably, erythrocytes exposed to 300
mM NaCl plus 600 mM urea for 8 h (which approximates the
hyperosmotic condition in the medullary tip) only show mar-
ginal annexin binding of 11.4 ⫾ 4.5% (n ⫽ 8) compared with
erythrocytes exposed to isotonic Ringer solution (2.9 ⫾ 1.4%,
n ⫽ 8). Thus the high Cl⫺ and urea concentrations prevailing
in renal medulla prevent erythrocyte phosphatidylserine expo-
sure despite increased osmolarity. Under physiological condi-
tions, the contact time of erythrocytes with renal medulla is too
short to trigger significant phosphatidylserine exposure. How-
ever, the contact time may be considerably increased in acute
renal failure, which typically leads to trapping of erythrocytes
in the transition from medulla to cortex (42). In the present
study, experimental renal ischemia, indeed, induced phospha-
tidylserine exposure of erythrocytes. Moreover, at least in
theory, urea could similarly interfere with ceramide formation
in renal medullary cells.
The mechanisms by which urea interferes with apoptotic
signaling are poorly understood. Urea may activate ras (52)
and increase phosphatidylinositol 3-kinase activity (62). In
erythrocytes, urea has been shown to modify several transport
processes, including the Na⫹-K⫹-ATPase, KCl cotransport,
and Na⫹-K⫹-2Cl⫺ cotransport (28, 29, 39, 50). Interestingly,
hydroxyurea protects erythrocytes against oxidative damage
(1) and prevents sickling (4, 14, 44). In this respect, it may be
of interest that sickle cells are especially sensitive to phospha-
tidylserine exposure induced by osmotic shock, oxidative
stress, and energy depletion (33). The direct ability of urea to
inhibit Smase and ceramide formation may well participate in
the regulation of phosphatidylserine exposure not only in
erythrocytes but also in nucleated cells.
In summary, similar to nucleated cells, erythrocytes loose
phosphatidylserine asymmetry, leading to annexin binding af-
ter exposure to hyperosmotic shock. The phosphatidylserine
exposure is mediated by both activation of cation conductance
and by Smase-related ceramide formation. Cl⫺ directly inhibits
the cation conductance, and urea decreases the Smase activity.
Thus, at least in erythrocytes, both extraordinary Cl⫺ and urea
concentrations, as they prevail in renal medulla, partially ab-
rogate the proapoptotic effects of hyperosmotic shock. The
present observations in erythrocytes may thus shed light on the
complex interplay of NaCl and urea in the regulation of
medullary kidney cell apoptosis during osmotic shock.
ACKNOWLEDGMENTS
We acknowledge the technical assistance of E. Faber and the meticulous
preparation of the manuscript by L. Subasic and T. Loch.
GRANTS
This study was supported by Deutsche Forschungsgemeinschaft Grant Nos.
La 315/4-3, La 315/6-1, and La 315/11-1, Bundesministerium für Bildung,
Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clin-
ical Research) Grant 01 KS 9602, and the Biomed program of the European
Union (BMH4-CT96 – 0602).
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