CALL FOR PAPERS Programming Normal Renal Development and Modeling
BIM deficiency differentially impacts the function of kidney endothelial
and epithelial cells through modulation of their local microenvironment
Nader Sheibani,1,2Margaret E. Morrison,3Zafer Gurel,1SunYoung Park,1and Christine M. Sorenson3
Departments of1Ophthalmology and Visual Sciences,2Pharmacology, and3Pediatrics, University of Wisconsin School
of Medicine and Public Health, Madison, Wisconsin
Submitted 31 August 2011; accepted in final form 11 December 2011
Sheibani N, Morrison ME, Gurel Z, Park S, Sorenson CM. BIM
deficiency differentially impacts the function of kidney endothelial
and epithelial cells through modulation of their local microenviron-
ment. Am J Physiol Renal Physiol 302: F809–F819, 2012. First
published December 14, 2011; doi:10.1152/ajprenal.00498.2011.—
The extracellular matrix (ECM) acts as a scaffold for kidney cellular
organization. Local secretion of the ECM allows kidney cells to
readily adapt to changes occurring within the kidney. In addition to
providing structural support for cells, the ECM also modulates cell
survival, migration, proliferation, and differentiation. Although aber-
rant regulation of ECM proteins can play a causative role in many
diseases, it is not known whether ECM production, cell adhesion, and
migration are regulated in a similar manner in kidney epithelial and
endothelial cells. Here, we demonstrate that lack of BIM expression
differentially impacts kidney endothelial and epithelial cell ECM
production, migration, and adhesion, further emphasizing the special-
ized role of these cell types in kidney function. Bim ?/? kidney
epithelial cells demonstrated decreased migration, increased adhesion,
and sustained expression of osteopontin and thrombospondin-1
(TSP1). In contrast, bim ?/? kidney endothelial cells demonstrated
increased cell migration, and decreased expression of osteopontin and
TSP1. We also observed a fivefold increase in VEGF expression in
bim ?/? kidney endothelial cells consistent with their increased
migration and capillary morphogenesis. These cells also had de-
creased endothelial nitric oxide synthase activity and nitric oxide
bioavailability. Thus kidney endothelial and epithelial cells make
unique contributions to the regulation of their ECM composition, with
specific impact on adhesive and migratory properties that are essential
for their proper function.
angiogenesis; apoptosis; capillary morphogenesis; extracellular ma-
trix proteins; organogenesis
BCL-2 IS THE FOUNDING MEMBER of a family of proteins that influence
apoptosis. Family members contain conserved regions denoted as
Bcl-2 homology (BH) domains. Proapoptotic members are di-
vided into those that only contain a BH3 domain and those that
contain multiple BH domains (14). BIM is a BH3 only-containing
matrix detachment. BIM is a critical mediator of anoikis in
epithelial cells, acting as a sensor of integrin and growth factor
signals to the Erk pathway (17). Although it is well established
that extracellular matrix (ECM) expression can impact cell sur-
vival, less is known as to whether modulation of proteins that
influence apoptosis can impact ECM production and tissue ho-
meostasis in a cell type-specific manner.
The mammalian kidney is a complex organ that contains over
25 different cell types. It is a highly vascularized organ in which
the various segments of the vascular tree accomplish specialized
regional functions (5). The microenvironment of the kidney con-
sists of epithelial, vascular, fibroblast, and smooth muscle cells
embedded in a complex network of ECM proteins, which en-
hances the complexity of this microenvironment. The ECM is
locally secreted and acts as a scaffold for tissue organization,
regulating growth factor and cytokine availability to aid tissue
homeostasis. ECM composition adapts to the changing conditions
within the organ, including injury. In addition to providing struc-
tural support for cells, the ECM also modulates several cell
functions including cell survival, migration, proliferation, and
differentiation (3). Thus changes in the ECM milieu can affect
kidney structure/function through aberrant modulation of cell
function such as cell survival.
Cell-cell and cell-matrix interactions impact cell proliferation,
migration, differentiation, and apoptosis. The ability of the cell to
ECM and neighboring cells is essential for tissue homeostasis.
Altered ECM expression can impact cell-adhesive mechanisms
influencing tissue architecture and function. Disruption of this
delicately balanced microenvironment can also lead to many
disease states. However, it is not well understood whether this
delicate balance is regulated similarly in all cell types housed
within this microenvironment or how they may vary in their
responses. Previous work from this laboratory demonstrated that
bcl-2 not only regulates apoptosis but also influences the ECM
milieu, with significant impact on adhesion and migration char-
acteristics of kidney epithelial cells. Loss of bcl-2 expression
resulted in precocious downregulation of thrombospondin-1
(TSP1) and osteopontin, increased cell migration, and decreased
cell adhesion (28).
To begin to address whether loss of a specific pro-apoptotic
protein, BIM, in kidney epithelial and endothelial cells would
have a similar impact on the microenvironment, we prepared and
characterized kidney cells from weanling wild-type and bim ?/?
mice. Kidney epithelial cells demonstrated sustained expression
of TSP1 and osteopontin, while kidney endothelial cells demon-
strated decreased expression. These changes corresponded with
decreased migration and increased adhesion to fibronectin, vitro-
Address for reprint requests and other correspondence: C. M. Sorenson, Univ.
of Wisconsin School of Medicine and Public Health, Dept. of Pediatrics, 600
Highland Ave., H4/444 CSC, Madison, WI 53792-4108 (e-mail: cmsorenson
Am J Physiol Renal Physiol 302: F809–F819, 2012.
First published December 14, 2011; doi:10.1152/ajprenal.00498.2011.
1931-857X/12 Copyright © 2012 the American Physiological Societyhttp://www.ajprenal.orgF809
nectin and collagen IV in bim ?/? kidney epithelial cells. In
contrast, bim ?/? kidney endothelial cells demonstrated in-
creased migration, enhanced capillary morphogenesis, decreased
phosphorylated endothelial nitric oxide synthase (p-eNOS) ex-
pression, a twofold decrease in nitric oxide (NO) production, and
a fivefold increase in VEGF expression. Thus loss of bim expres-
sion differentially impacts kidney endothelial and epithelial cell
function through modulation of their responses to their distinct
MATERIALS AND METHODS
Experimental animals and cell cultures. The mice used for these
studies were maintained and treated in accordance with our protocol
approved by the University of Wisconsin Animal Care and Use Com-
mittee. Immortomice expressing a temperature-sensitive SV40 large T
antigen were obtained from Charles River Laboratories (Wilmington,
MA). As previously described (7, 28), bim ?/? mice (Jackson Labora-
tory, Bar Harbor, ME) were crossed with the Immortomouse and
screened. To isolate kidney endothelial cells, kidneys from two to three
aseptically and placed in serum-free DMEM containing penicillin/strep-
tomycin (Sigma, St. Louis, MO). The kidneys were pooled, rinsed with
DMEM, minced into small pieces in a 60-mm tissue culture dish using
sterilized razor blades, and digested in 5 ml of collagenase type I (1
mg/ml in serum-free DMEM, Worthington, Lakewood, NJ) for 30–45
min at 37°C. Following digestion, DMEM with 10% FBS was added and
cells were pelleted. The cellular digests were then filtered through a
double layer of sterile 40-?m nylon mesh (Sefar America, Hanover Park,
IL), centrifuged at 400 g for 10 min to pellet cells, and the cells were then
washed twice with DMEM containing 10% FBS. The cells were resus-
pended in 1.5 ml medium (DMEM with 10% FBS) and incubated with
sheep anti-rat magnetic beads precoated with anti-platelet endothelial cell
adhesion molecule (PECAM)-1 antibody (MEC13.3, BD Biosciences,
Bedford, MA), as described previously (22). After affinity binding,
magnetic beads were washed six times with DMEM with 10% FBS, and
the bound cells were plated into a single well of a 24-well plate precoated
with 2 ?g/ml of human fibronectin (BD Biosciences) in endothelial
growth medium. Endothelial cells were grown in DMEM containing
10% FBS, 2 mM L-glutamine, 2 mM sodium pyruvate, 20 mM HEPES,
1% nonessential amino acids, 100 ?g/ml streptomycin, 100 U/ml peni-
cillin, 55 U/ml heparin (Sigma), 100 ?g/ml endothelial growth supple-
ment (Sigma), and murine recombinant interferon-? (R&D Systems,
Minneapolis, MN) at 44 U/ml. Cells were maintained at 33°C with 5%
CO2. Cells were progressively passed to larger plates, maintained, and
propagated in 1% gelatin-coated 60-mm dishes. Kidney endothelial cells
were positive for B4-lectin (a mouse endothelial cell-specific lectin) and
expressed PECAM-1 and vascular endothelial (VE)-cadherin as previ-
ously described (7, 10). The experiments described here were performed
with three separate isolations of cells with similar results.
To isolate collecting duct epithelial cells (referred subsequently to as
kidney epithelial cells), both kidneys from 4 wk-old wild-type and
bim ?/? Immortomice were minced into small pieces in a 60-mm tissue
culture dish using sterile razor blades and digested in 5 ml of collagenase
type I (1 mg/ml in serum-free DMEM, Worthington) for 30–45 min at
and the cells were pelleted and rinsed twice in DMEM containing 10%
FBS. The cells were resuspended in growth medium (DMEM:F12,
Invitrogen, Carlsbad, CA) supplemented with 1% FBS, 5? MITO (BD
Biosciences, Franklin Lakes, NJ), 44 U/ml ?-interferon (R&D Systems),
2 mM glutamine, 50 ?g/ml streptomycin/50 U/ml penicillin (Sigma), 50
?g/ml gentamicin (Invitrogen), and 50 U/ml nystatin (Sigma) and plated
on a 35-mm dish precoated with Matrigel (1:400 in serum-free DMEM:
F12). The cells were plated, grown to near confluence, and expanded in
60-mm dishes coated with Matrigel. Cells from two 60-mm dishes were
harvested by incubation with 2 mM EDTA in Tris-buffered saline
containing 0.05% BSA for 10 min and scraping. The cells were rinsed
with serum-free DMEM:F12 and incubated with magnetic beads pre-
coated with Dolichos biflorus agglutinin (DBA) (28). After binding, the
magnetic beads were washed six times with DMEM containing 10%
FBS, and the bound cells were plated into a single well of a 24-well plate
precoated with Matrigel (1:400) in growth medium. The cells were
maintained at 33°C with 5% CO2. Cells were progressively passed to
larger plates, maintained, and propagated on Matrigel (1:400)-coated
60-mm plates. The selection process was repeated twice. Collecting duct
epithelial cells expressed aquaporin 2 and calbindin and were DBA
positive (mouse collecting duct-specific lectin) as we previously de-
scribed (20, 28).
Cell apoptosis assays. As an apoptotic stimulus, cells were incubated
cells) or growth medium for 48 h at 37°C. The rate of apoptotic cells was
determined by in situ monitoring of caspase activity using the CaspACE
FITC-VAD-FMK in situ marker (Promega, Madison, WI) or Caspase 3/7
Glo (Promega), as recommended by the supplier (20).
Scratch-wound assay. Cells (4 ? 105) were plated in 60-mm tissue
culture dishes and allowed to reach confluence (2–3 days). After aspira-
tion of the medium, cell layers were wounded using a 1-ml micropipette
tip. Plates were then rinsed with PBS, fed with growth medium contain-
ing 100 ng/ml of 5-FU to rule out potential contribution of differences in
cell proliferation, and incubated at 37°C for the duration of the experi-
ment. The wounds were observed and photographed up to 72 h. The
distance migrated was determined as the percentage of total distance for
quantitative assessments as described previously (7). These experiments
were repeated at least twice with two different isolations with similar
Capillary morphogenesis in Matrigel. Matrigel (10 mg/ml; BD
Biosciences) was applied at 0.5 ml/35-mm tissue culture dish and incu-
bated at 37°C for at least 30 min to harden. Cells were removed using
trypsin-EDTA, washed with growth medium once, and resuspended at
1 ? 105cells/ml in serum-free growth medium. Cells (2 ml) were gently
added to the Matrigel-coated plates, incubated at 37°C, monitored for
6–24 h, and photographed using a Nikon microscope equipped with a
digital camera. For quantitative assessment of the data, the mean number
of branch points in 10 high-power fields (?100) was determined after 24
h. A longer incubation of the cells did not result in further branching
Cell adhesion assays. Cell adhesion to various matrix proteins was
performed as previously described (18). Briefly, varying concentrations
of fibronectin, vitronectin, collagen I, collagen IV, and laminin (BD
Biosciences) prepared in TBS with Ca2?and Mg2?(2 mM each; TBS
with Ca/Mg) were coated on 96-well plates (50 ?l/well; Nunc Maxisorbe
with 1% BSA. Plates were rinsed four times with 200 ?l of TBS with
Ca/Mg and blocked with 200 ?l of 1% BSA prepared in TBS with
Ca/Mg for at least 1 h at room temperature. Cells were removed by the
dissociation solution (Sigma), washed with TBS, and resuspended at 5 ?
108cells/ml in HBS (20 mM HEPES, 150 mM NaCl, pH 7.6, and 4
mg/ml BSA). After blocking, plates were rinsed with TBS with Ca/Mg
Table 1. Primer sequences
Forward 5= to 3=Reverse 5= to 3=
BIM-DEFICIENT KIDNEY ENDOTHELIAL AND EPITHELIAL CELLS
AJP-Renal Physiol • doi:10.1152/ajprenal.00498.2011 • www.ajprenal.org
leads to enhanced vascular density (7, 22, 25). Here, we also
show increased migration, capillary morphogenesis, and vas-
cular density in the absence of BIM. Unlike endothelial cells,
decreased cell migration of bim ?/? epithelial cells did not
adversely impact tubular morphogenesis in Matrigel. Previ-
ously, studies from our laboratories demonstrated that en-
hanced kidney epithelial migration led to an inability to un-
dergo tubular morphogenesis in Matrigel and branching mor-
phogenesis in vivo (20). The impact of the modulation of
migration has on capillary/tubular morphogenesis in Matrigel
appears to be regulated differently in endothelial and epithelial
cells. In summary, the studies presented here emphasize the
importance of considering all cell types within an organ in
designing treatment modalities with minimal off-target effects.
The authors thank Robert Gordon for assistance with graphics.
This work was supported by grants from the University of Wisconsin
Department of Pediatrics Research and Development Fund and University of
Wisconsin Medical School Research Committee (C. M. Sorenson). C. M.
Sorenson was funded, in part, by National Institutes of Health (NIH) Grant
DK067120 and American Heart Association Research Award 0950057G.
N. Sheibani is supported by NIH grants EY016995, EY018179, and
RC4EY021357, P30 CA014520 UW Paul P. Carbone Cancer Center support
grant, P30 EY016665, and an unrestricted departmental award from Research
to Prevent Blindness. N. Sheibani is the recipient of a Research Award from
the American Diabetes Association (1-10-BS-160) and the Retina Research
Foundation. M. E. Morrison is a recipient of a Senior Thesis Grant from the
College of Letters and Science at the University of Wisconsin-Madison. S. Y.
Park is a recipient of a predoctoral studentship from AstraZeneca.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: N.S. and C.M.S. provided conception and design of
research; N.S., M.E.M., Z.G., S.P., and C.M.S. performed experiments; N.S.,
M.E.M., Z.G., S.P., and C.M.S. analyzed data; N.S. and C.M.S. interpreted
results of experiments; N.S. and C.M.S. edited and revised manuscript; N.S.
and C.M.S. approved final version of manuscript; M.E.M., Z.G., S.P., and
C.M.S. prepared figures; C.M.S. drafted manuscript.
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AJP-Renal Physiol • doi:10.1152/ajprenal.00498.2011 • www.ajprenal.org