Prostaglandin E2 is crucial in the response of podocytes to fluid flow shear stress

Article (PDF Available)inJournal of Cell Communication and Signaling 4(2):79-90 · June 2010with39 Reads
DOI: 10.1007/s12079-010-0088-9 · Source: PubMed
Abstract
Podocytes play a key role in maintaining and modulating the filtration barrier of the glomerulus. Because of their location, podocytes are exposed to mechanical strain in the form of fluid flow shear stress (FFSS). Several human diseases are characterized by glomerular hyperfiltration, such as diabetes mellitus and hypertension. The response of podocytes to FFSS at physiological or pathological levels is not known. We exposed cultured podocytes to FFSS, and studied changes in actin cytoskeleton, prostaglandin E(2) (PGE(2)) production and expression of cyclooxygenase-1 and-2 (COX-1, COX-2). FFSS caused a reduction in transversal F-actin stress filaments and the appearance of cortical actin network in the early recovery period. Cells exhibited a pattern similar to control state by 24 h following FFSS without significant loss of podocytes or apoptosis. FFSS caused increased levels of PGE(2) as early as 30 min after onset of shear stress, levels that increased over time. PGE(2) production by podocytes at post-2 h and post-24 h was also significantly increased compared to control cells (p < 0.039 and 0.012, respectively). Intracellular PGE(2) synthesis and expression of COX-2 was increased at post-2 h following FFSS. The expression of COX-1 mRNA was unchanged. We conclude that podocytes are sensitive and responsive to FFSS, exhibiting morphological and physiological changes. We believe that PGE(2) plays an important role in mechanoperception in podocytes.
RESEARCH ARTICLE
Prostaglandin E
2
is crucial in the response of podocytes
to fluid flow shear stress
Tarak Srivastava & Ellen T. McCarthy & Ram Sharma &
Patricia A. Cudmore & Mukut Sharma &
Mark L. Johnson & Lynda F. Bonewald
Received: 17 September 2009 / Accepted: 5 March 2010 / Published online: 8 April 2010
#
US Government 2010
Abstract Podocytes play a key role in maintaining and
modulating the filtration barrier of the glomerulus. Because
of their location, podocytes are exposed to mechanical
strain in the form of fluid flow shear stress (FFSS). Several
human diseases are charact erized by glomerular hyper-
filtration, such as diabetes mellitus and hypertension. The
response of podocytes to FFSS at physiological or
pathological levels is not known. We exposed cultured
podocytes to FFSS, and studied changes in actin cytoskel-
eton, prostaglandin E
2
(PGE
2
) production and expression of
cyclooxygenase-1 and2 (COX-1, COX-2). FFSS caused a
reduction in transversal F-actin stress filaments and the
appearance of cortical actin network in the early recover y
period. Cells exhibited a pattern similar to control state by
24 h following FFSS without significant loss of podocytes
or apoptosis. FFSS caused increased levels of PGE
2
as
early as 30 min after onset of shear stress, level s that
increased over time. PGE
2
production by podocytes at post-
2 h and post-24 h was also significantly increased
compared to control cells (p<0.039 and 0.012, respective-
ly). Intracellular PGE
2
synthesis and expression of COX-2
was increased at post-2 h following FFSS. The expression
of COX-1 mRNA was unchanged. We conclude that
podocytes are sensitive and responsive to FFSS, exhibiting
morphological and physiological changes. We believe that
PGE
2
plays an important role in mechanoperception in
podocytes.
Keywords Prostaglandin E
2
.
Cyclooxygenase
.
Actin
.
Mechanical strain
.
Shear stress
Introduction
The glomerular filtration barrier is made up of fenestrated
endothelial cells, glomerular basement membrane (GBM),
and podocytes through which ultrafiltrate passes from
capillary into the urinary space (Rodewald and Karnovsky
1974). Podocytes are believed to play an important role in
modulating ultrafiltration, stabilizing glomerular capillaries
and in preventing loss of proteins into the urine. Podocytes
under normal conditions are exposed to mechanical forces
arising from both glomerular capillary pressure and
glomerular ultrafiltration (Endlich and Endlich, 2 006 ).
Podocytes possess a dynamic actin cytoskeleton and are
thus uniquely situated to provide immediate response to
mechanical strain in the glomerulus. Podocytes have been
shown to respond to mechanical strain (Durvasula et al.
2004; Endlich et al. 2001; Friedrich et al. 2006). Response
T. Srivastava
:
P. A. Cudmore
Section of Nephrology, Childrens Mercy Hospital and University
of Missouri at Kansas City,
Kansas City, MO, USA
E. T. McCarthy
Kidney Institute, University of Kansas Medical Center,
Kansas City, KS, USA
R. Sharma (*)
Renal Research Laboratory, Research and Development,
Kansas City VA Medical Center,
Room F1-130, Building 15, 4801 Linwood Boulevard,
Kansas City, MO 64128, USA
e-mail: Ram.Sharma2@va.gov
M. Sharma
Kidney Disease Center, Medical College of Wisconsin,
Milwaukee, WI, USA
M. L. Johnson
:
L. F. Bonewald
Department of Oral Biology, University of Missouri at Kansas
CitySchool of Dentistry,
Kansas, MO, USA
J. Cell Commun. Signal. (2010) 4:7990
DOI 10.1007/s12079-010-0088-9
of podocytes to mechanical forces arising from capillary
distention due to glomerular capillary hydrostatic pressure
has been studied using the in vitro model of substrate
stretch. Podocytes respond to substrate stretch with an
increase in radial stress filaments and actin-rich centers, as
well as narrowing of the cell bodies and elongation of
cytoplasmic processes (Durvasula et al. 2004 ; Endlich et al.
2001). In addition to mechanical strain resulting from
intracapillary hydrostatic pressure, podocytes are exposed
to fluid flow shear stress (FFSS) resulting from flow of
ultrafiltrate through the filtration slits and over the apical
surface of podocytes. Podocytes in culture respond to FFSS
with a reduction in transversal F-actin stress filaments,
formation of a cortical actin network and disruption of the
cell monolayer (Friedrich et al. 2006).
Several pathways have been shown to be involved in
sensing and responding to mecha nical strain in other cell
types. Examples of such mediators implicated in early
response to mechanical strain include nitric oxide (NO)
(Klein-Nulend et al. 1995; Johnson et al. 1996; Zaman et al.
1999; McAllister et al. 2000), Ca
2+
(Jorgensen et al. 1997;
You et al. 2002; Jorgensen et al. 2002; Jorgensen et al.
2003; You et al. 2001 ) and PGE
2
(Wadhwa et al. 2002; Jee
et al. 1985; Klein-Nulend et al. 1997; Forwood 1996; Ajubi
et al. 1996, 1997). The signaling events invo lved in
mechanoperception and respon se to FFSS in podocytes
are not known.
Eicosanoids of the cyclooxygenase (COX) pathway such
as PGE
2
are synthesized by the glomerulus and act in an
autocrine fashion to modulate glomerular function (Breyer
et al. 1996). PGE
2
regul ates glomerular filtration rate
(GFR) and can modify the permselectivity of the glomer-
ular filtration barrier (Breyer and Breyer 2000 ; Schlondorff
1986; Kömhoff et al. 1997; McCarthy and Sharma 2002;
Sharma et al. 2006). Podocytes express both COX-1 and
COX-2 (Kö mhoff et al. 1997; Cheng et al. 2007). We
hypothesize that PGE
2
plays a role in the response of
cultured podocytes to mechanical strain in the form of
FFSS. In our current study, we confirmed that podocytes
are sensitive to F FSS by demonstrating changes in
podocyte cell morphology and F-actin cytoskeleton and
showed that PGE
2
plays a role in early response to FFSS.
Material and methods
Podocyte culture
The well-ch ar acteriz ed conditionally immort alized mouse
podocyte line (a kind gift from Dr. Peter Mundel) with
thermosensitive tsA58 mutant T-antigen was used in
these studies (Mundel et al. 1997; Shankland et al. 2007).
We followed the culture protocol as outlined in literature
and did not study cultured cells beyond 15 passages
(Shankland et al. 2007). In b ri ef , po do cyte s w er e m ai n-
tained in RPMI 1640 with L-glutam ine suppl emented with
10% fetal bovine serum, 100 units/ml p enicillin and
0.1 mg/ml streptomyc in (Invitro gen, Carl sbad , CA). To
propagate podocytes, cells were grown on collagen coated
(BD Biosciences, San Diego, CA) tis sue culture flas ks or
glass slides under permissive conditions, i.e. at 33°C wi th
10 units/ml of mouse γ-interferon (Cell Sciences , Nor-
wood, MA). To induce differentiation, cells were grown
under non-permissive conditions, i .e. at 37°C without
mouse γ-interferon. Podocytes were grown under non-
permissive conditions on 25×75×1 mm glass slides
(Fischer Scientific, Pitt sburg, PA) with 3 glass slides in
each culture dish containing 12 ml of culture media.
Differentiated podocytes on day 14 were used for FFSS
experiments.
Application of fluid flow shear stress
FFSS was applied to differentiated podocytes using a
FlexCell Streamer Gold apparatus (Flexcell International,
Hillsborough, NC). The apparatus was sterilized with
300 ml of 70% ethanol for 30 min, and checked for leaks.
This was followed by two wash steps with 300400 ml of
sterilized PBS for 5 min each. The PBS was then replaced
by 350 ml of media. The flow device chamber was then
moved into a sterile hood and using sterilized forceps, glass
slides with podocytes were placed in each one of the slots
of the flow device chamber (Fig. 1). All six slots were filled
to ensure consistent flow. The chamber was then replaced
in the incubator at 37°C with 5% CO
2
. The computer was
programmed to apply FFSS at a pre-determ ined level
(dynes/cm
2
) for a pre-determined period of time according
to experimental design. We applied FFSS (116 dynes/cm
2
)
for 30120 min to podocytes on a FlexCell Streamer Gold
in our pilot studies, and chose to use 2 dynes/cm
2
(or 75 ml/
min) for 2 h as our FFSS model based on these preliminary
studies. A 2 h of FFSS was selected based on our earlier
published experience with osteocyte cell line (Zhang et al.
2006; Cheng et al. 2001). In some experiments a 3-way
stop-cock was attached to collect 11.5 ml of media during
FFSS. Control podocytes were grow n on glass slides and
placed in the same hood and incubator as experimental cells
but were not exposed to FFSS. At the end of FFSS
treatment, the fluid device chamber was moved to the
sterile hood and was opened to remove the slides for
processing. The glass slides were then placed back in the
original medium after completion of FFSS, and podocytes
were allowed to recover for 24 h (Fig. 1). The study
samples obtained prior to onset of FFFSS was termed pre-
FFSS, 2 h and 24 h following cessation of FFSS as post-2 h
and post-24 h respectively.
80 T. Srivastava et al.
Assessment of morphology and actin cytoskeleton
Podocytes were washed twice with PBS, fixed with 2%
glutaraldehyde for 10 min and then stained with 0.1%
crystal violet for 20 min for assessment of cell morphology.
In separate experiments, immunostaining for F-acti n was
performed after washing with PBS and fixing with 4%
paraformaldehyde, using phalloidin tagged to Alexa Fluor
568 dye (Invitrogen, Carlsbad, CA), which binds to F-actin
filaments but not to monomeric G-actin . The images were
taken on fluorescence microscope Olympus BX60 (Ham-
burg, Germany). Finally, podocytes were incubated with
PGE
2
(020 µM for 2 and 24 h) and stained with crystal
violet for cell morphology and phalloidin for F-actin. The
images were obtained from 1020 random fields by an
unbiased obse rver. We als o in cuba ted podocytes w ith
indomethacin prior to (2.5 µM×1 h) and during (2.5 µM)
FFSS, and stained for F-actin with phalloidin and cell
morphology with crystal violet.
TUNEL assay for apoptosis
Podocytes exposed to FFSS and control podocytes were
assessed for apoptosis by TUNEL assay using a commer-
cial kit (Cell Deat h Detection Kit, Roche, Tucson, AZ).
Cells were studied at post-2 h and post-24 h FFSS along
with time-paired controls. The TUNEL assay was per-
formed directly on podocytes on glass slides and on cells
following cytospin preparation.
Media collection and preparation of cell lysate
We have previously used a dye diffusion assay to determine
that the equilibration time, i.e. the time taken for dye placed
at the inlet side of the slide chamber to become fully mixed
within the circulating media is approximately 2 min at a
flow rate of 16 dynes/cm
2
and 56 min at a flow rate of
2dynes/cm
2
. We collected 1.01.5 ml aliquots of medium
from the continuously circulating 350 ml medium during
application of FFSS at 0, 30 and 120 min using a 3-way
stop-cock (Fig. 1a). We also collected an aliquot of 1 ml
media from 12 ml of culture media immediately before
application of FFSS (pre-FFSS that had been changed 24 h
prior to application of FFSS. Once the glass slides were
returned to their original culture dish after FFSS, we
collected an aliquot of 1 ml 2 h after cessation of FFSS
(post-2 h FFSS) and the remaining 10 ml 24 h after cessation
of FFSS (post-24 h FFSS, Fig. 1b). Culture media were
collected from control podocytes at same time points. Because
PGE
2
concentration was very low given the large volume
(350 ml) of medium used during FFSS, it was concentrated
using a PGE
2
affinity column (#414018, Cayman Chemical,
Ann Arbor, MI). We used a 7-fold concentration, i.e. 840 μl
of medium was run over the affinity column and PGE
2
was
Pre FFSS Post 2 hr FFSS Post 24 hr FFSS
Control Podocytes - Media Collection
3 way stop cock
Pre FFSS Post 2 hr FFSS Post 24 hr FFSS
Flow device chamber
for media during
Experimental Podocytes - Media Collection
FFSS
AB
Fig. 1 The figure on the left shows the arrangement of apparatus used
to apply fluid flow shear stress (FFSS) to podocytes. It shows the
fluid device chamber in which six glass slides containing podocytes
are placed for FFSS studies, and the 3-way stop cock attachment to
collect media during application of FFSS. The figure on the right
provides the experiment design for collection of culture media at 3
different time points
Prostaglandin E
2
is crucial in the response of podocytes to fluid flow shear stress 81
eluted out in 120 μl in our column extraction method. Thus
we could express the measured PGE
2
as an absolute amount
of PGE
2
and as change from baseline/control as we had kept
the media volume fixed at 12 m l for culture dishes
containing 3 slides, 350 ml for FlexCell Streamer Gold
containing 6 slides, and PGE
2
affinity column extraction
fixed at a 7-fold concentration change.
Podocytes were washed with PBS following application
of FFSS and lysed with 100 μl of 1% Triton in PBS for
measurement of intracell ular PGE
2
. Measured PGE
2
was
corrected for DNA content of lysate, and intracellular PGE
2
was expressed as pg PGE
2
/μg DNA.
Measurement of PGE
2
PGE
2
was measured using PGE
2
EIA kit (#514010,
Cayman Chemical) in (a) medium obtained from the
FlexCell apparatus during FFSS, (b) culture medium after
application of FFSS, and (c) cell lysate. This kit measures
free PGE
2
in media and cell lysate and has a very low
cross-reactivity (0.02%) with the major PGE
2
metabolite
(13,14-dihydro-15-keto PGE
2
). PGE
2
accumulates in cul-
ture media and undergoes non-enzymatic oxidation while
intracellular PGE
2
undergoes enzymatic degradation as per
manufacturer. To further confirm this we analyzed the
media for 13,14-dihydro-15-keto PGE
2
using PGE
2
Metab-
olite EIA kit (#514531, Cayman Chemical) that in turn has
very low cross-reactivity (<0.01%) with PGE
2.
The detec-
tion limit of the PGE
2
and PGE
2
Metabolite EIA kits are
15 pg/ml and 2 pg/ml respectively. PGE
2
and PGE
2
metabolite levels were measured for each condition from
5 separate experiments.
mRNA extraction and RT-PCR
Total RNA was isolated from podocytes by homogenization
in 1 ml Trizol Reagent (Life Technologies, Gaithersburg,
MD) following the manufacturers protocol. Isolated total
RNA was subjected to DNase digestion and cleanup using
RNeasy Mini Kit (Qiagen, Valencia, CA) per manufac-
turers instructions. RNA 100 ng was reverse transcribed
with 50 µM Oligo (dT)20 using SuperScript III First-Strand
Synthesis SuperMix (Invitrogen, C arlsbad, CA). PCR
reactions were carried out by standard technique using
2 µl of the RT reaction (20 ng cDNA), and 22 U/ml
complexed recombinant Taq DNA Polymerase, Pyrococcus
species GB-D thermostable polymerase, and Platinum Taq
Antibody; 66 mM Tris-SO4 (pH 8.9); 19.8 mM (NH4)
2SO4; 2.4 mM MgSO4; 220 µM dNTPs; and stabilizers in
Platinum PCR SuperM ix High Fidelity (Invitrogen) under
the foll owing conditions: denaturation at 94°C for 30 s,
annealing at 52°C, and extension at 72°C for 30 s (35
cycles) after the initial denaturation at 94°C for 2 min and
with a final extension at 72°C. The amplification products
of 10 µl of each PCR reaction were separated on a 2%
NuSeive 3:1 agarose gel, stained with SYBR safe DNA gel
stain, and visualized by ultraviolet irradiation. The primer
sequences used were: COX-1forward 5-GGT CCT GCT
CGC AGA TCC TG-3, reverse 5-AGG ACC CAT CTT
TCC AGA GG-3, product size 580 bp; COX-2forward
5-CTG TAC AAG CAG TGG CAA-3, reverse 5-TTA
CAG CTC AGT TGA ACG CCT-3, product size 530 bp;
β-Actinforward 5'-ACC AAC TGG GAC GAC ATG
GAG-3', reverse 5' -GTC AGG ATC TTC ATG AGG TAG
TC-3', product size 380 bp.
Statistics
Data were analyzed with repeated measu res ANOVA
analyses using the SPSS 16.0 statistical software for group
and time comparison. A p value <0.05 was considered
significant.
Results
Fluid flow shear stress alters actin cytoskeleton
of podocytes
Podocytes were exposed to FFSS at 2 dynes/cm
2
for 2 h,
and F-actin filaments stained at mid-point (60 min), at end
of FFSS (120 min) and in the recovery period of post-2 h
and post-24 h following cessation of mechanical strain.
FFSS caused a reduction in t ransversal F-actin stress
filaments and the appearance of a cortical actin network at
mid-point (60 min), at end of FFSS (120 min) and in the
early recovery perio d (post-2 h) following FFSS (Fig. 2).
The F-actin filaments resumed a pattern that resembles
control state by 24 h following FFSS (post-24 h). In order
to further examine morphological changes that occur due to
FFSS, podocytes were stained using crystal violet. Podo-
cytes exhibited indistinct cellu lar margins and blunted
cytoplasmic processes at 2 h post-FFSS compared to
control cells (Fig. 3). The cells regained distinct cellular
margins and elongated, elaborate processes by 24 h
following cessation of FFSS, an appearance that closely
resembled that of control cells. Our results indicate that
FFSS causes alteration in podocyte morphology charact er-
ized by reorganization of F-actin filaments, and the
observed changes were largely reversed by 24 h post-
FFSS. We further incubated podocytes with indomethacin
prior to (2.5 µM×1 h) and during (2.5 µM) application of
FFSS, and stained for F-actin and cell morphology. As done
with FFSS without indomethacin, the glass slides were
placed back in the original medium containing indometh-
acin after completion of FFSS, and podocytes were allowed
82 T. Srivastava et al.
to recover for 24 h (Fig. 1 ). We found inhibition of COX
prevented changes in both actin cytoskeleton and cellular
morphology caused by FFSS, Figs. 2 and 3.
Fluid flow shear stress is not associated with cell loss
or apoptosis
Application of FFSS of 2 dynes/cm
2
for 2 h did not result in
significant cell loss (post-24 h FFSS 2.31±0.45×10
5
cells/
slide vs control 2.51±0.55×10
5
cells/slide, p=0.41). We
conclude that cell loss following application of FFSS in
these studies was minimal (8%). We then measured total
DNA in cell lysate as an indirect estimate of cell number
and again found no difference between experimental and
control (FFSS 39.0±5.4 µg DNA/slide post-2 h and 39.1±
8.3 µg DNA/slide post-24 h vs control 43.8±6.2 µg DNA/
slide, p=0.08 and p=0.18, respectively), FFSS did not
affect apoptosis as measured by the TUNEL assay at either
time point studied. When we studied cells grown on glass
slides we found no difference in percentage of apoptotic
podocytes in experimental and paired control groups (FFSS
post-2 h 6.2±5.3 vs contr ol 3.7±2.8, p=0.10, and FFSS
post-24 h 7.6±3.8 vs control 6.3±4.1, p=0.38). We repeat
the TUNEL assay on cytospin preparations of podocytes
and again did not find significant difference in percentage
of apoptotic podocytes (FFSS po st-2 h 3.7±2.1 vs control
2.9±2.2, p=0.31, and FFSS post-24 h 3.4±1.9 vs control
3.8±3.2, p=0.70). We conclude that FFSS at 2 dynes/cm
2
for 2 h is not associated with significant cell loss or
apoptosis.
Fluid flow shear stress increases PGE
2
in podocytes
We determined the effect of ongoing FFSS on podocyte
secretion of PGE
2
by studying aliquots of medium obtained
before and during FFSS. FFSS caused significantly in-
creased PGE
2
levels in the bathing medium by 30 min after
onset of mechanical strain and PGE
2
levels progressively
increased over time. PGE
2
level at onset of FFSS was
below the detection limit of the EIA kit. There was a
significant change over time for measured PGE
2
from
baseline at 120 min (p<0.05, Fig. 4). We conclude that
FFSS leads to increased synthesis of PGE
2
by cultured
podocytes.
Podocytes continue to produce PGE
2
during recovery
following fluid flow shear stress
In order to determine the pattern of PGE
2
secretion
following cessation of FFSS, aliquots of medium were
analyzed 2 and 24 h following treatment. Control consisted
of aliquots obtained from culture medium of untreated
podocytes (i.e. no FFSS) at 2 and 24 h. There was no
difference in the amount of PGE
2
in the culture media in
the pre-FFSS samples between control podocytes (3,911±
1,567 pg) and podocytes that were subjected to FFSS
(4,847±2,361 pg, p=0.48). PGE
2
levels in the medium at
both time points was significantly increased compared to
control (post-2 h FFSS 6,340±2,078 pg vs control 3,767±
1,056 pg, p=0.039, and post-24 h FFSS 6,980±2,606 pg vs
control 2,756±820 pg, p=0.012, respectively). There was a
trend for effect of group (p=0.059) and time (p=0.088),
and there was significant interaction for group by time
(p<0.001).
When analyzed separately, PGE
2
levels in the control
group fell over time probably due to ongoing nonenzymatic
degradation, though the levels were not significantly
different from those at baseline (post-2 h p=0.87 and
post-24 h p=0.10). Conversely, PGE
2
levels following
Control Control
End FFSS for 120 min
End FFSS for 120 min with
Indomethacin
Post-2hr FFSS
Post-2hr FFSS with
Indomethacin
Post-24hr FFSS with
Indomethacin
Post-24hr FFSS
Indomethacin
Fig. 2 The figure shows immunostaining for F-actin in control
podocytes, and in podocytes exposed to FFSS at end of FFSS
(120 min), during early recovery period following FFSS at 2 h (post-
2 h FFSS) and 24 h (post-24 h FFSS) in the left column (400X). The
right column shows podocytes pre-treated with indomethacin (2.5 μM
for 1 h) prior to application of FFSS during early recovery period at
2 h (post-2 h FFSS) and 24 h (post-24 h FFSS) in the presence of
indomethacin (200X). Indomethacin prevented FFSS-induced changes
in F-actin
Prostaglandin E
2
is crucial in the response of podocytes to fluid flow shear stress 83
FFSS increased over time, and were significantly different
at both post-2 h (p =0.01) and post-24 h (p =0.002)
compared to baseline. The pattern of change was similar
in all experiments studied (Fig. 5).
The rate of PGE
2
synthesis was increased following
FFSS. The rate of PGE
2
production between cessations of
FFSS to post-2 h sample was significantly different from
the rate of PGE
2
production seen during the same time
period for control podocyt es (0 h to post-2 h FFSS 746.0±
281.3 pg/hr vs control72.0±305.4 pg/hr, (p=0.03). Like-
wise, the rate of PGE
2
synthesis from cessation of FFSS to
post-24 h sample was significantly increased compared to
the same period for control (0 h to post-24 h FFSS 88.9±
36.2 pg/hr vs control 48.1±32.9 pg/hr, p=0.007).
Secreted PGE
2
does not undergo enzymatic degradation
We analyzed the media for PGE
2
metabolite 13,14-dihydro-
15-keto PGE
2
. There was no difference in the amount of
PGE
2
metabolite in the culture media between podocytes
Control
Control
Post-2hr FFSS Post-24hr FFSS
FFSS without Indomethacin
Post-2hr FFSS Post-24hr FFSS
FFSS with Indomethacin
Fig. 3 The figure shows control podocytes, and podocytes exposed to
FFSS (upper panel) stained with crystal violet for cellular morphology
during early recovery period following FFSS at 2 h (post-2 h FFSS) and
24 h (post-24 h FFSS). Podocytes show an indistinct cellular margin
and blunted cytoplasmic processes compared to control cells at post-2 h
following application of FFSS, changes that are resolving by post-24 h
FFSS. The lower panel shows podocytes pre-treated with indomethacin
(2.5 μM for 1 h) prior to application of FFSS during early recovery
period at 2 h (post-2 h FFSS) and 24 h (post-24 h FFSS). Indomethacin
prevented FFSS-induced changes in cell morphology in podocytes
Change in PGE2 in media during application of FFSS
*
40000
*
35000
30000
25000
20000
15000
10000
Protaglandins E2 (pg)
5000
0
Time
0 min 30 min 120 min
Fig. 4 Prostaglandin E
2
in media collected at time 0 min, 30 min and
120 min during application of fluid flow shear stress (FFSS) at
2 dynes/cm
2
are shown as bar figure with ±1 SE and the absolute
difference in prostaglandin E
2
from 5 separate experiments are shown
as a line figure (* denotes p<0.05)
84 T. Srivastava et al.
subjected to FFSS and control at any time point studied
(pre-FFSS 531.2±151.9 pg vs control 632.7±210.4 pg, p=
0.41; post-2 h FFSS 559.9±346.7 pg vs control 555.3±
171.1 pg, p=0.98; post-24 h FFSS 646.9±183.4 pg vs
control 578.3±152.2 pg, p=0.54). Additionally, there was
no effect of group (p=0.93), time (p=0.68) or group by
time (p=0.42). We conclude that secreted PGE
2
found in
culture medium does not undergo enzymatic degradation.
Intracellular levels of PGE
2
increases in podocytes
We measured intracellular PGE
2
in cell lysates and
normalized to DNA levels. Intracellular PGE
2
levels were
significantly elevated following FFSS compared to control
(i.e. pre-FFSS levels, 0.57±0.16 pgPGE
2
/μgDNA) at post-
2 h (1.56±0.71 pgPGE
2
/μgDNA, p=0.02), Fig. 6. Intracel-
lular PGE
2
levels were near baseline by post-24 h of FFSS
(0.81±0.11 pgPGE
2
/μgDNA, p=0.69 compared to control).
Unlike extracellular PGE
2
intracellular PGE
2
is subject to
enzymatic degradation.
Fluid flow shear stress increases expression of COX-2
Increased intraand extracellular levels of PGE
2
may be
due to increased expression of cyclooxygenase. RNA was
extracted from podocytes at post-2 h or post-24 h following
cessation of FFSS and expression of COX-1 and COX-2
were examined using reverse transcriptase PCR. COX-2
expression (n=6) was significantly increased at post-2 h
(16.84±9.99, p<0.001) and returned to near control levels
303.0
Change in Intracellular PGE2/DNA following
li ti f FFSS
2.5
)
app
li
ca
ti
on o
f
FFSS
*
g/µg)
2.0
NA (pg
15
2/ DN
1.5
din E2
10
gland
1.0
rostag
0.5
Pr
0.0
Pre FFSS Post 2 hr FFSS Post 24 hr FFSSPre-FFSS Post-2 hr FFSS Post-24 hr FFSS
*
Fig. 6 The measured intracellular PGE
2
/DNA and the change in PGE
2
/DNA from control podocytes following FFSS from 5 separate experiments
are shown as a bar figure with ±1 SE and as a line figure respectively (* denotes p<0.05)
Change in media PGE2 following application of FFSS
Change
in
media
PGE2
following
application
of
FFSS
*
4000.0
*
3000.0
*
2000.0
1000 01000.0
0.0
Pr e -FFSS Po s t-2h r FFSS Po s t-24h r FFSS
-1000.0
Prostglandin E2 (pg)
-2000 0-2000.0
-3000.0
Time
*
*
Fig. 5 Prostaglandin E
2
in culture media prior to FFSS, and following
recovery at 2 and 24 h post application of FFSS from control and
experimental podocytes are shown as bar figure with ±1 SE, and the
absolute difference in prostaglandin E
2
from 5 separate control and
FFSS experiments are shown as line figures (* denotes p<0.05)
Prostaglandin E
2
is crucial in the response of podocytes to fluid flow shear stress 85
by post-24 h (3.01±1.87, p=0.47) following FFSS, Fig. 7.
FFSS did not affect COX-1 expression (n=3). COX-1
expression was 0.76±0.29 (p=0.21) at post-2 h and 0.75±
0.21 (p=0.18) at post-24 h following FFSS.
Exogenous PGE
2
causes reorganization of actin
cytoskeleton
It is possible that morphological changes seen in podocytes
exposed to FFSS are related to some event other than the
increase in PGE
2
synthesis. To better understand if this is
the case, we incubated podocytes with PGE
2
(020 µM/ml)
for 2 and 24 h. PGE
2
(5 μM for 2 h) showed changes in F-
actin stress filament in F-actin compared to control, Fig. 8.
Changes in F-actin were marked at 10 μM and 20 μM, and
persisted at 24 h. We then examined effect of PGE
2
on cell
morphology of experimental and control cells using crystal
violet staining. PGE
2
(5 μM for 2 h) caused blunting of
cytoplasmic processes compared to control, Fig. 8.As
before, morphological changes were marked at 10 μM and
20 μM, and persisted at 24 h. We could reproduce part of
changes observed in podocytes with FFSS with direct
exposure to PGE
2
. We conclude that PGE
2
plays a role in
the cytoskeletal changes in FFSS.
Discussion
We have demonstrated that podocytes in culture are
responsive to mechanical strain in the form of FFSS. We
have shown that podocytes respond by alterations in actin
structure, in gene expression and eicosanoid synthesis. The
location of podocytes in the glomerulus is such that under
normal conditions it is exposed to mechanical forces arising
from both intracapillary pressure and flow of filtrate over
the cell surface (Endlich and Endlich 2006). Micropuncture
studies in animals indicate that the transmural hydrostatic
pressure gradient of the glomerular capillary is 40
50 mmHg (Arendshorst and Navare 1993). To ensure
structural stability, t hese outwardly directed expansile
forces from glomerular capillary pressure must be balanced
by inwardly directed forces on the wall. The podocytes are
believed to provide this counteracting tone by virtue of their
contractile actin cytoskeleton. The effect of substrate stretch
Fig. 7 The figure shows the gene expression for COX-1 and COX-2
in podocytes following application of FFSS. a The gel figure shows
an increase in COX-2 gene e xpression at post-2 h following
application of FFSS that begins to resolve at post-24 h FFSS. COX-
1 expression is unchanged with FFSS. b The bar graph denotes the
change in gene expression for COX-1 (n=3) and COX-2 (n=6) on RT-
PCR at post-2 h and post-24 h FFSS (* denotes p<0.05)
86 T. Srivastava et al.
on podocytes and other cell types has been studied using
instruments that cause biaxial elongation of cultured cells.
Podocytes are mechanosensitive to substrate stretch, and
exhibit an increase in radial stre ss filaments and actin-rich
centers, and narrowing of the cell bodies with sharp and
elongated cytopl asmic processes (Durvasula et al. 2004;
Endlich et al. 2001; Martineau et al. 2004).
Flow of ultrafiltrate through the filtration slits and over
the apical surface of podocytes exerts FFSS on podocytes.
The shear stress to which podocytes are exposed in vivo has
not been directly measured. The effect of FFSS on several
non-podocyte cell types, most notably endothelial cells and
osteocytes, has been studied using instruments that modu-
late flow of medium over the surface of cultured cells
(Klein-Nulend et al. 1995; Johnson et al. 1996; Zaman et al.
1999; McAllister et al. 2000; Jorgensen et al. 1997; You et
al. 2002; Jorgensen et al. 2002, 2003; You et al. 2001;
Wadhwa et al. 2002; Jee et al. 1985; Klein-Nulend et al.
1997; Forwood 1996; Ajubi et al. 1996, 1997). In designing
the present studies we turned to reports of the effect of
FFSS on osteocytes as a starting point. Osteocytes share
similarities wi th podocytes in that both cells types are
terminally differentiated and possess elaborate cytoplasmic
extensions. We have been among those investigators who
propose that osteocytes sense and respond to mechanical
strain in their environment (Cherian et al. 2005; Zhang et
al. 2006; Cheng et al. 2001). There are numerous reports
describing the effect of FFSS on osteocytes (Klein-Nulend
et al. 1995). We and others have commonly used FFSS
ranging from 4 to 16 dynes/cm
2
for 12hinin vitro studies
to examine responsiveness of osteocytes to mechanical
strain (Ajubi et al. 1997; Cherian et al. 2005; Zhang et al.
2006; Cheng et al. 2001; Bakker et al. 2006). We used
FFSS at 2 dynes/cm
2
for 2 h, and studied post-2 h and post-
24 h FFSS time points based on results from our pilot
studies with podocyte s and our previous studi es wit h
osteocytes (Zhang et al. 2006; Cheng et al. 2001).
There are few previous reports of the effect of FFSS on
podocytes. Investigators have used FFSS ranging from 0.015
to 649 dynes/cm
2
(Friedrich et al. 2006; Dandapani et al.
2007; Huang and Miller 2007). Friedrich et al (2006)useda
mathematical model to estimate that mouse podocytes are
exposed to in vivo FFSS of 0.3 dyne/cm
2
.Theydemonstrat-
ed a redu ction in transversal F-actin stress filaments,
formation of a cortical actin network and disruption of the
cell monolayer. We corroborated these results, and demon-
strated a reduction in transversal F-actin stress filaments and
development of a cortical actin network after 60 and 120 min
0 µM 1.25 µM 2.5 µM5 µM 10 µM 20 µM
F-Actin stain of podocytes exposed to PGE
2
for 2 hours
0 µM 1.25 µM 2.5 µM5 µM 10 µM 20 µM
Crystal violet stain of podocytes exposed to PGE
2
for 2 hours
0 µM 1.25 µM
2.5 µM5 µM 10 µM 20 µM
F-Actin stain of podocytes exposed to PGE
2
for 24 hours
0 µM 1.25 µM 2.5 µM5 µM 10 µM 20 µM
Crystal violet stain of podocytes exposed to PGE
2
for 24 hours
Fig. 8 The figure shows changes in F-actin on phalloidin staining and
cell morphology on crystal violet staining in podocytes following
continuous treatment with PGE
2
from 01.252.551020 µM (from
left to right) without FFSS for 2 h (upper two panels) and 24 h (lower
two panels). The early discernable changes start to appear at 5 μM and
are marked at 10 μM and 20 μM at both 2 h and 24 h
Prostaglandin E
2
is crucial in the response of podocytes to fluid flow shear stress 87
of FFSS (data not shown). In contrast to the findings of these
investigators, we did not see disruption of the cell monolayer
or significant loss of cells with FFSS at 2 dynes/cm
2
.
Explanations for these differences may include the shorter
length of time that we exposed cells to FFSS (2 h vs 20 h).
In order to substantiate our theory that PGE
2
was responsible
for changes seen following FFSS, we exposed normal
podocytes to exogenous PGE
2
. We were able to reproduce
the actin cytoskeleton and morphological changes that we
had seen with FFSS.
We found the FFSS-induced changes in actin cytoskel-
eton persisted for at least 2 h following termination of
mechanical strain, and actin had nearly resumed its baseline
organization by 24 h following FFSS. In other words, the
change in the podocyte actin cytoskeleton caused by FFSS
was at least partially reversible. Our findings of morpho-
logical changes in cell shape seen with crystal violet
staining though qualitative in nature support our resul ts of
F-actin staining. It is possible that the changes described
above could be due to cell injury rather than response to
FFSS. We do not believe that cell injury played a role in our
reported findings for the following reasons: (a) we did not
find a significant difference in apoptosis between experi-
mental and control podocytes, (b) the severity of mechan-
ical stra in was much lower than those used in osteocytes,
and (c) only living cells can resume near normal morphol-
ogy by 24 h after FFSS. These observations lead us to
conclude that podocyt e injury is not significant in our FFSS
model.
Eicosanoids are biologically active fatty acids derived
from oxygenation of arachidonic acid. Prostaglandins are
eicosanoids synthesized by cyclooxygenase (COX) and
function as autocoids (Creminon et al. 1995; FitzGerald
2002). Constitutive COX -1 and inducible COX-2 metabo-
lize arachidonic acid to PGG
2
and PGH
2
.PGH
2
is
subsequently metaboli zed to PGE
2
, PGI
2
, PGD
2
, PGF
2α
or thromboxane A
2
(TXA
2
) (Creminon et al. 1995;
FitzGerald 2002). PGE
2
is the major product of renal
arachidonic acid metabolism under physiological condi-
tions. PGE
2
interacts with E-prostanoid (EP) recept ors, four
of which have been cloned and characterized and are
designated EP
1
to EP
4
(Narumiya et al. 1999). EP
1
is
coupled to Ca
2+
mobilization, EP
2
and EP
4
are linked to G
s
protein and EP
3
is coupled to G
i
protein (Narumiya et al.
1999; Sugimoto et al. 1994). Mouse podocytes ex press EP
1
and EP
4
, and EP
4
has been postulated to be functionally
significant (Bek et al. 1999).
We have demonstrated for the first time the role of PGE
2
in podocytes following application of FFSS. We find the
response of podocytes to FFSS is similar to that described
by us and others in osteocytes [20,32]. Osteocytes exhibit
increased PGE
2
synthesis as an early response to FFSS
(Ajubi et al. 1997; Cherian et al. 2005 ; Cheng et al. 2001;
Bakker et al. 2006; Bakker et al. 2005). This increase is
associated with increased COX-2 expression and translo-
cation of connexin 43 hemichannels (Ajubi et al. 1997;
Cherian et al. 2005; Cheng et al. 2001; Bakker et al. 2005,
2006). These studies demons trate t hat PGE
2
plays an
important role in mechanoperception, though the signaling
pathways involved in mechanotransduction have yet to be
described fully. Possible candidate pathways in osteocytes
include the non-canonical Wnt/β-catenin pathway (Robin-
son et al. 2006).
Increased PGE
2
synthesis appears to be an early
response to FFSS, detectable by 30 min of FFSS and
continuing as long as 120 min of exposure. In addit ion, we
found that podocytes continued to secrete PGE
2
at 2 h
following discontinuation of FFSS, and that synthesis
declined by 24 h after cessation of FFSS. We observed an
increase in intracellular PGE
2
as well as an increase in
COX-2 gene expression during the same time period.
Interestingly, our results are very similar to those seen in
osteocytes (Cherian et al. 2005; Cheng et al. 2001).
Increased PGE
2
synthesis does not appear to be a general
response to mechanical strain, as neither we nor other
investigators found significantly increased levels following
subst rate stretch in podocytes (Martin eau et al. 2004;
Srivastava et al. 2008). We conclude that increased PGE
2
synthesis is a specific early response of podocytes to FFSS.
There is little data on dose response for PGE
2
on actin
cytoskeleton in podocytes; hence we performed a dose
response evaluation for PGE
2
from 1.25 to 20 μM for 2 h
and 24 h without addition of another stimulus. We have
found 5 μM PGE
2
without FFSS to be an opti mal dose in
our studies with osteocytes (unpublished observation). One
group found no change with 1 μM PGE
2
for 90 min until
the cells were primed with a stretch stimulus (not FFSS)
(Martineau et al. 2004). Thus we are not completely
surprised with the discrepancy between PGE
2
levels
observed in our FFSS experiments versus exogenous
concentration of PGE
2
without FFSS needed to show
similar changes in podocyte.
Endogenous PGE
2
plays a role in regulating renal blood
flow and glomerular filtration rate directly by its effect on
vascular smooth musc le, causing arteriolar vasodilatation of
the afferent arteriole (Arendshorst and Navare 1993;
Edwards 1985; Chatziantoniou and Arendshorst 1992). It
is postulated that PGE
2
attenuates vasoconstriction resulting
from activation of the renin-angiotensin-aldosterone system
and sympathetic nervous system, and the response to PGE
2
is dependent on baseline renal vascular tone ( De Forrest et
al. 1980; Conte et al. 1992). Additionally, we have shown
that PGE
2
acts on the glomerular permeability barrier
independently of any effect on vascular tone (McCarthy
and Sharma 2002). We hypothesize that podocytes respond
to alterations in FFSS with an increase in PGE
2
, permitting
88 T. Srivastava et al.
ultrafiltration to be more tightly regulated through changes
in both hemodynamic tone and barrier properties.
Increased PGE
2
synthesis occurs in models of hyper-
filtration such as the 5/6 nephrectomy (Stahl et al. 1986;
Griffin et al. 1992; Nath et al. 1987; Wang et al. 1998).
Additionally, we have shown that PGE
2
increases albumin
permeability in isolated rat glomeruli in an in vitro assay, an
effect that can be abrogated by COX inhibition (McCarthy
and Sharma 2002; Sharma et al. 2006). Thus PGE
2
appears
to be important in modifying permselectivity of the
glomerular filtration barrier. In clinical practice, use of
nonsteroidal anti-inflammatory agents (NSAIDs) can re-
duce proteinuria (Vriesendorp et al. 1986; Golbetz et al.
1989; Vriesendorp et al. 1985). Vriesendorp et al. (1986)
found that NSAIDs that reduce renal PGE
2
excretion also
decrease proteinuria, whereas sulindac which does not
influence PGE
2
synthesis had no impact on protein
excretion. Several studies have shown that use of chronic
inhibition of COX slows progression of renal injury in
animal models and human disease (Velosa et al. 1985;
Wang et al. 2000; Nakayama et al. 2007 ). There are
compelling data to suggest that PGE
2
plays a role in
progression of renal dysfunction in diseases characterized
by hyperfiltration.
In summary, we provide compelling evidence that
podocytes are sensitive and responsive to FFSS. FFSS
causes disruption of the actin cytoskeleton which is
reversible following discontinuation of mechanical strain.
FFSS causes increased expression of COX-2 and produc-
tion of PGE
2
in cultured podocytes. We hypothesize that
PGE
2
plays a role in mechanoperception in podocytes.
Further study is required to define the events of mechano-
transduction that result in altered podocyte structure. We
believe that examination of cellular events caused by FFSS
is crucial to understanding changes that occur in diseases
characterized by glomerular hyperfiltration, and may allow
us to design effective therapeutic strategies in future.
Acknowledgement This work was supported in part by The Sam
and Helen Kaplan Research Fund in Pediatric Nephrology, Marion
Merrell Dow Foundation Clinical Scholar Award and The Norman S.
Coplon Extramural Research Grant to TS and AR046798 to MLJ and
LFB. We are indebted to Ashley Sherman, MA for statistical
assistance.
Disclosure None of the authors of this work have financial interests
to disclose.
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90 T. Srivastava et al.
    • "However, by analogy with other EnC in the vasculature which communicate with underlying smooth muscle cells via nitric oxide (NO), it seems likely such mediators will be released by GEnC to have actions on other glomerular cells. Under physiological conditions, mean glomerular shear stress is estimated at 10–20 dyn/cm 2 (Ballermann et al., 1998 ), however , there is little data detailing the effects of LSS on GEnC (Eng and Ballermann, 2003) (Bevan et al., 2011), podocytes (Srivastava et al., 2010; Friedrich et al., 2006 ) or the glomerulus as a whole, and how this impacts on glomerular permeability and cell cross-talk. Here we hypothesise that chronic exposure of GEnC to LSS will modulate expression of KLF2 and its downstream targets and modify communication with podocytes via soluble mediators. "
    [Show abstract] [Hide abstract] ABSTRACT: Laminar shear stress (LSS), induced by flowing blood, plays a key role in determining vascular health by modulating endothelial behaviour and vascular tone. In systemic endothelium many of the beneficial effects of chronic LSS are mediated through the transcription factor Kruppel-like factor 2 (KLF2), but little is known regarding the role of chronic LSS in the renal glomerulus. We demonstrate that exposure of glomerular endothelial cells to chronic (>24h) LSS of 10 dyn/cm(2) increases phosphorylation of extra-cellular signal-related kinase 5 (ERK5) and increases expression of KLF2, leading to increased expression of the downstream molecules endothelial nitric oxide synthase (eNOS), thrombomodulin, endothelin-1 and nitric oxide. However, the proportion of eNOS which was phosphorylated at serine 1117 and threonine 495 residues was decreased. We demonstrated dependence of these effects on the ERK5 pathway by using the inhibitor UO126. We found high levels of KLF2 expression in human glomeruli confirming the relevance of our in vitro observations and, as KLF2 is specifically induced by chronic LSS, suggesting the physiological importance of shear stress in the glomerulus. Conditioned medium from glomerular endothelial cells under chronic LSS decreased podocyte monolayer resistance and increased phosphorylation of vasodilator-stimulated phosphoprotein. The latter effect was more pronounced using a novel insert-based direct co-culture system in which endothelial cells were exposed to chronic LSS. These data provide the first direct evidence of glomerular endothelial cell to podocyte cross-talk.
    Full-text · Article · Jun 2012
  • [Show abstract] [Hide abstract] ABSTRACT: Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of CD Na(+) reabsorption. Salt loading is thought to increase CD ET-1 production; however, definitive evidence of this, as well as understanding of the mechanisms transducing this effect, is lacking. Tubule fluid flow increases in response to Na(+) loading; hence, we studied flow modulation of CD ET-1 production. Three days of a high-salt diet increased mouse and rat inner medullary CD (IMCD) ET-1 mRNA expression. Acute furosemide infusion increased urinary ET-1 excretion in anesthetized rats. Primary cultures of mouse or rat IMCD detached in response to flow using a closed perfusion chamber, consequently a CD cell line (mpkCCDcl4) was examined. Flow increased ET-1 mRNA at shear stress rates exceeding 1 dyne/cm(2), with the maximal effect seen between 2 and 10 dyne/cm(2). Induction of ET-1 mRNA was first evident after 1 h, and most apparent after 2 h, of flow. Inhibition of calmodulin or dihydropyridine-sensitive Ca(2+) channels did not alter the flow response; however, chelation of intracellular Ca(2+) or removal of extracellular Ca(2+) largely prevented flow-stimulated ET-1 mRNA accumulation. Downregulation of protein kinase C (PKC) using phorbol 12-myristate 13-acetate, or PKC inhibition with calphostin C, markedly reduced flow-stimulated ET-1 mRNA levels. Flow-stimulated ET-1 mRNA accumulation was abolished by inhibition of phospholipase C (PLC). Taken together, these data indicate that flow increases CD ET-1 production and this is dependent on extracellular and intracellular Ca(2+), PKC, and PLC. These studies suggest a novel pathway for coupling alterations in extracellular fluid volume to CD ET-1 production and ultimately control of CD Na(+) reabsorption.
    Article · Dec 2010
  • [Show abstract] [Hide abstract] ABSTRACT: Cyclooxygenase-2 (COX-2) plays a critical role in modulating deleterious actions of angiotensin II (Ang II) where there is an inappropriate activation of the renin-angiotensin system (RAS). This review discusses the recent developments regarding the complex interactions by which COX-2 modulates the impact of an activated RAS on kidney function and blood pressure. Normal rats with increased COX-2 activity but with different intrarenal Ang II activity because of sodium restriction or chronic treatment with angiotensin-converting enzyme (ACE) inhibitors showed similar renal hemodynamic responses to COX-2-selective inhibition (nimesulide) indicating independence from the intrarenal Ang II activity. COX-2-dependent maintenance of medullary blood flow was consistent and not dependent on dietary salt or ACE inhibition. In contrast, COX-2 influences on sodium excretion were contingent on the prevailing RAS activity. In chronic hypertensive models, COX-2 inhibition elicited similar reductions in kidney function, but COX-2 metabolites contribute to rather than ameliorate the hypertension. The maintenance of renal hemodynamics reflects direct and opposing effects of Ang II and COX-2 metabolites. The antagonism in water and electrolyte reabsorption is dependent on the prevailing intrarenal Ang II activity. The recent functional experiments demonstrate a beneficial modulation of Ang II by COX-2 except in the presence of inflammation promoted by hypertension, hyperglycemia, and oxidative stress.
    Full-text · Article · Nov 2011
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