Urol Res (2005) 33: 340–348
DOI 10.1007/s00240-005-0491-5
O R I GI N A L P A P E R
Eddie L. Greene Æ Gerard Farell Æ Shihui Yu
Tori Matthews Æ Vivek Kumar Æ John C. Lieske
Renal cell adaptation to oxalate
Received: 6 May 2005 / Accepted: 8 June 2005 / Published online: 13 November 2005
Springer-Verlag 2005
Abstract Renal manifestations of chronic hyperoxaluria
include nephrolithiasis and, when extreme, interstitial
scarring and progressive loss of function. Exposure of
cultured renal cells to oxalate has been reported to cause
cell death, as well as proliferation. The current study was
performed to assess the time course and cell-type specificity of these responses. Proximal (LLC-PK1) and distal
[cIMCD and primary human renal (HRC1)] renal epithelial cells, as well as interstitial KNRK cells, were
exposed to oxalate (0.5–2.0 mM) for 24–72 h. The generation of reactive oxygen species (ROS) was measured
using the fluorescent probe DCF, and cell number was
determined with CyQuant reagent. HSP-70 expression
was assessed via real time PCR and quantitative Western
blot. In response to all oxalate concentrations (0.5–
2.0 mM) and lengths of exposure (15 min–2 h), cultured
proximal and distal renal epithelial cells and renal fibroblasts generated ROS. After 24 h, cells demonstrated
initial cell death and decrease in cell numbers, but by 48–
72 h adapted and grew, despite the continued presence
of oxalate. This response was associated with increased
expression of HSP-70 mRNA and protein. Renal cells in
vivo may possess adaptive mechanisms to withstand
chronic hyperoxaluria, including increased expression of
chaperone molecules such as HSP-70.
Keywords cIMCD Æ HSP-70 Æ KNRK Æ LLC-PK1 Æ
Primary cultures
E. L. Greene Æ G. Farell Æ S. Yu Æ T. Matthews Æ V. Kumar
J. C. Lieske (&)
Division of Nephrology, Mayo Clinic College of Medicine,
200 First Street SW, Rochester, MN 55905 USA
E-mail: Lieske.John@mayo.edu
Tel.: +1-507-2667960
Fax: +1-507-266-9315
Introduction
Renal manifestations of chronic hyperoxaluria include
nephrolithiasis and, when extreme, interstitial scarring
and progressive loss of function. The clinical outcome in
many patients with primary hyperoxaluria, a genetic
disorder characterized by hepatic overproduction of
oxalate, is dismal because multiple complications of the
disease develop including nephrolithiasis, nephrocalcinosis and renal failure often resulting in death at a
young age [1, 2]. Although primary hyperoxaluria is
relatively rare, hyperoxaluria secondary to gastrointestinal malabsorption is not [3], and in these circumstances
oxalosis is also sometimes observed [4]. Furthermore,
the formation of kidney stones is extremely common [5],
and evidence suggests that minimal, perhaps transient
elevations in urinary oxalate concentration may be an
important factor in at least a subgroup of patients with
idiopathic calcium oxalate urolithiasis [6].
In the cases of primary and secondary hyperoxaluria,
oxalate appears to be a key mediator of renal manifestations that include tubulointerstitial scarring as well as
nephrolithiasis [4, 7]. For patients with idiopathic calcium oxalate stone disease and mild hyperoxaluria, it
has been postulated that stimulation of these same
pathways can result in a local nidus for stone formation
[8]. Since different regions of the kidney are likely to be
exposed to different oxalate concentrations [9], in the
current study we defined cell-type responses to oxalate,
including reactive oxygen species (ROS) generation, cell
growth, and induction of a representative stress response
protein.
Materials and methods
Cell culture
Rat continuous inner medullary collecting duct (cIMCD)
cells were a generous gift of Dr Jack Kleinman, Medical
341
College of Wisconsin, Milwaukee, WI, and porcine
proximal tubular LLC-PK1 cells a gift of Dr. Thomas
Dousa, Mayo Clinic, Rochester, MN. Mouse renal
fibroblastic KNRK cells were obtained from the American Tissue Culture Collection (CL-101).
Human renal cells were isolated from the urine of a
healthy, non-stone forming male using the method of
Dörrenhaus [10]. Briefly, a fresh urine sample (50 ml)
was centrifuged 100 g for 5 min), and rinsed twice with
Hams F-12 culture medium containing penicillin
(100 U/ml), streptomycin (100 lg/ml), amphotericin
(1.25 lg/ml) and 20% calf serum. The resulting pellet
was resuspended in 50 ll of medium and seeded in a
single well of a 24-well collagen-coated plate (Falcon) to
which an additional 50 ll of medium was placed. Wells
were fed with an additional 100 ll of medium the following day, and the medium replaced every 2–3 days
thereafter. Once confluent, cells were trypsinized and
replated onto other collagen-coated wells. Resulting cell
lines were then propagated on standard Falcon plates in
DMEM with 10% calf serum. One line (HRC1) was
fully characterized for these studies. HRC1 cells formed
a uniform tight monolayer, stained with lectins from
Jacalin, Concanavalin A, but not Vicia villosa (VVL),
Erythrina crystagalli (ECA) or Griffonia simplficolia
(GSII). Alkaline phosphatase activity was also detected.
Together, the evidence is most consistent that the cells
are a primary culture of distal renal cells [11].
Stock plates were grown in Dulbecco-Vogt modified
Eagle’s medium containing 25 mM glucose (DMEM)
and 10% fetal calf serum at 37C in a CO2 incubator as
previously described [12]. For assays in 96 well plates,
cells were trypsinized and resuspended in DMEM containing 1.6 lM biotin and 5% (cIMCD, KNRK, HRC1)
or 10% (LLC-PK1) fetal calf serum at a density designed
to achieve an initial confluence of 50%. Initial plating
densities were approximately 25·104 cells/ml (HRC1),
30·104 cells/ml (cIMCD and KNRK), and 50·104 cells/
ml (LLC-PK1). After 8 h, the media was changed to
DMEM containing 0.05% fatty acid free bovine serum
albumin (BSA, Sigma) and 1.6 lM biotin. The media
was changed daily for three more days with DMEM
0.05% BSA and 1.6 lM biotin. On day 4, when cells
had reached 75–85% confluence, they were exposed to
vehicle or potassium oxalate (0.1–2.0 mM) prepared in
phenol red free DMEM (BioWhittaker) plus 0.05% BSA
and 1.6 lM biotin. For exposures longer than 24 h,
media was changed daily with fresh medium containing
the same oxalate concentrations.
Earle’s balanced salt solution containing 10 lM DCFDA (Molecular Probes) and 1% bovine serum albumin
for 30 min at 37̊C. The medium was then removed and
replaced with fresh phenol red free DMEM containing
0.05% BSA and 1.6 lM biotin to which vehicle or
potassium oxalate (0.1–2.0 mM) was added. Relative
fluorescence intensity measurements were obtained over
time (15–120 min) by fluorometry using a Fusion-a
Fluorometer containing a 96 well plate reader (Packard
Instruments Downers Grove, Ill., USA). Fluormetric
readings were obtained using an excitation wavelength
of 485 nm and emission wavelength of 530 nm.
Quantification of cell proliferation
To quantitate cell proliferation, cells were plated into 96
well plates in phenol red-free DMEM containing 2%
fetal calf serum at the densities indicated above. After
1 day, media was changed to phenol red free DMEM
containing 0.05% BSA. The following day (day 2), and
each subsequent day the media was changed to phenolred free DMEM that contained potassium oxalate (0.5–
2.0 mM) or vehicle. On days 3, 4, and 5, media was
aspirated and plates were frozen to 80C to lyse cells.
To quantitate cell number, plates were thawed and 50 ll
of CyQuant reagent (Molecular Probes) was added to
each well. Plates were incubated at room temperature for
5 min prior to measuring relative fluorescence (480 nm
excitation, 530 nm emission).
Live/dead staining
For live/dead staining, cells were plated on glass coverslips in 24-well dishes at the same densities and conditions
as above. After exposure to potassium oxalate (0.5–
2.0 mM) for 4 or 24 h, media was aspirated and replaced
with 200 ll of Live/Dead mix and placed in the dark for
40 min at room temperature. Excess stain was then blotted off, and coverslips mounted on glass slides using
fluorescent mounting medium. One hundred cells per slide
were counted for live/dead staining using a fluorescence
microscope. For live staining (calcein), absorbance was
set at 494 nm and emission at 517 nm; for dead staining
(ethidium homodimer-1) absorbance was at 528 nm and
emission at 617 nm.
HSP-70 expression
Detection of reactive oxygen species
The peroxide sensitive fluorescent probe, 2¢,7¢-dichlorofluorescin diacetate (DCF-DA) was used to assess the
generation of reactive oxygen species (ROS) [13]. Briefly,
cells were plated into 96 well plates containing phenol
red-free DMEM and 1% fetal calf serum. After equilibration and attachment, the medium was changed to
HRC1 cells were grown as above in 100-mm dishes and
exposed to two concentrations of oxalate chosen to
model mild and extreme hyperoxaluria (0.5 and 2.0 mM),
for 4, 24 and 48 h each. Cells were then harvested for total
RNA using the Trizol reagent (Invitrogen), and cDNA
was prepared using the SuperScript II kit (Invitrogen).
Primer pairs were constructed using the human sequences
in the database for 18sRNA and HSP-70 (Table 1). PCR
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Fig. 1 Generation of ROS by
cultured renal cells. Cells were
exposed to oxalate (0.1–
2.0 mM) for times ranging
between 15 and 120 min, and
ROS generation was assessed
using DCF. All cells
demonstrated increased ROS
levels in response to oxalate
(0.5 mM) for 1 h (A), although
KNRK cells generated a
smaller percentage increase.
ROS were detected as early as
15 min after 0.5 mM oxalate
exposure, and there was no
clear trend for further increase
up to 2 h later (B). After 1 h
exposure, cells generated ROS
in response to oxalate
concentrations of between 0.5
and 2.0 mM, without any clear
trend for increased levels
in response to higher oxalate
levels (C)
conditions were optimized for each so that a single PCR
product was obtained on gel electrophoresis, as verified
using cDNA prepared from control cells as a template.
Real-time quantitative PCR (RT-PCR) was performed
using the SYBR Green JumpStart Taq ReadyMix (Sigma) and the Opticon DNA engine (MJ Research). Gene
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Table 1 Real time PCR primers
Gene
Forward primer
Reverse primer
PCR product size
HSP-70
18sRNA
5¢-aggccgacaagaagaaggtgct-3¢
5¢-cggctaccacatccaaggaa-3¢
5¢-tggtacagtccgctgatgatgg-3¢
5¢-gctggaattaccgcggct-3¢
139 bp
186 bp
expression under each condition was normalized for
18sRNA, and expressed relative to the matched controls.
Quantitative Western blots for HSP-70
Cells were grown in 24-well plates and exposed to oxalate as above. The medium was aspirated and the
monolayer rinsed once with ice-cold phosphate-buffered
saline (pH 7.4) prior to the addition of 1· cell culture
lysis reagent (200 ll, Promega, Madison, Wis.) after
which cells were scraped and vortexed for lysis. The
resulting cell lysate was centrifuged at 10,000 g for 2 min
in a refrigerated centrifuge, and the supernatant collected and stored at 80C. At the time of assay this
lysate was thawed, mixed with two parts of SDS-PAGE
Fig. 2 Growth of LLCPK1 cells in the presence of oxalate. Cells
were exposed to oxalate (0.5–2.0 mM) from time 0. At 24, 48, and
72 h cell counts were assessed fluorimetrically using the CyQuant
reagent. Cell numbers were reduced in the presence of the higher
sample buffer, boiled in a water-bath for 3 min, and the
proteins were resolved on 4–12% Bis-Tris gradient gels
(NuPage, Invitrogen). Gels were electroblotted onto an
Immobilon-P membrane (Millipore, Bedford, Mass.),
stained with Coommasie blue, and probed for HSP-70
using a monoclonal anti-HSP70 antibody (Sigma, St.
Louis, Mo.). Signals of the appropriate size (70 kDa)
were densitometrically quantified using a Kodak Gel
Logic 100 imaging system (Eastman Kodak, Rochester,
N.Y.). Each sample was run in triplicate and averaged.
Materials
Reagents were purchased from Sigma unless otherwise
indicated.
concentrations of oxalate at 24 and 48 h, but by 72 h cell numbers
were similar to control wells never exposed to oxalate. An asterisk
indicates P<0.05 vs control
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Fig. 3 Growth of cIMCD cells in the presence of oxalate. Cells
were exposed to oxalate (0.5–2.0 mM) from time 0. At 24, 48, and
72 h cell counts were assessed fluorometrically using the CyQuant
reagent. Cell numbers were reduced in the presence of the higher
concentrations of oxalate at 24 but not 48 or 72 . An asterisk
indicates P<0.05 vs control
Statistics
the quantity of ROS generation and oxalate concentration, although the highest percent increases were in
general at the highest oxalate level studied (2.0 mM).
Data were compared by Student’s t-test; P values <0.05
were accepted as significant. Values presented are
means±SEM. Points on graphs are each the mean of 16
or more values (n=8 for each experiment performed at
least twice). For real time PCR, results are the mean of
two separate experiments (oxalate exposure, RNA isolation and real time PCR).
Results
Renal cells generate ROS in response to oxalate
exposure
As assessed using DCF, all four renal cell lines generated
ROS within 1 h in response to a moderate and acute
oxalate exposure (0.5 mM·1 h, Fig. 1A). ROS could be
detected within 15 min, and persisted for the full 2 h
studied. In general, at all time points ROS generation
was greater for the three epithelial cell lines than KNRK
cells, and greatest for the HRC1 line (Fig. 1B). When
measured at 1 h, there was no clear relationship between
Cell growth in the presence of oxalate
Apoptosis, cell death and DNA synthesis have all been
reported in response to acute oxalate exposure. Therefore, we determined the effect of a prolonged oxalate
exposure on these disparate cell lines. For all four cell
types, cell numbers were lower after 24 h exposure to the
higher oxalate concentrations studied (1.0–2.0 mM,
Figs. 2, 3, 4, 5). By 48–72 h, however, numbers were
similar to the control for cells exposed to all levels of
oxalate. Live/dead staining confirmed that there were an
increased number of dead cells at 24 h in those exposed
to the higher concentrations of oxalate (that also had
reduced cell numbers), however, the number of dead
cells returned to baseline at 48 and 72 h, and in all instances was the same as control values at these later
times (not shown). There was no clear cell-type difference in sensitivity to the effects of oxalate, expect for the
suggestion that KNRK cells might exhibit a proliferative
345
Fig. 4 Growth of HRC1 cells in the presence of oxalate. Cells were
exposed to oxalate (0.5–2.0 mM) from time 0. At 24, 48, and 72 h
cell counts were assessed fluorometrically using the CyQuant
reagent. Cell numbers were reduced in the presence of the higher
concentrations of oxalate at 24 and 48, but not 72 h. An asterisk
indicates P<0.05 vs control
response to moderate oxalate levels (0.5 mM), with
some persistent toxicity at higher levels and prolonged
time lines (2.0 mM for 72 h, Fig. 5).
motion a series of events that can result in kidney stone
formation, or promote renal scarring when hyperoxaluria exposure is extreme and/or prolonged. [8]. However, many of the toxic effects of oxalate on renal cells
were observed in cell culture systems, and the most
dramatic adverse effects occurred when relatively high
oxalate concentrations were employed [14, 15]. Furthermore, greater toxicity was reported in proximal
compared to distal tubular-derived cell lines [16]. In
vivo, most observations have been made in rats made
hyperoxaluric by administration of ethylene glycol in the
drinking water [17], under conditions of high urinary
oxalate concentration which could not be precisely
controlled, and where metabolites of ethylene glycol
other than oxalate could have contributed to the cellular
toxicity that was observed.
With these caveats in mind, numerous studies suggest
that acute exposure to high concentrations of oxalate,
similar to those seen in the urine of primary or secondary hyperoxaluric patients, can injure cultured renal
tubular cells in the absence of crystal formation [14, 15,
16]. In lower concentrations oxalate ions induce cellular
proliferation [18]. Prior evidence also suggests that
oxalate ions damage cells via oxidative pathways [19,
20], and a recent study indicates that diphenyleneiodium
(DPI) can ameliorate oxalate ion, calcium oxalate
HSP-70
Given the ability of cells to recover and proliferate in the
presence of even high concentrations of oxalate, we next
looked at HSP-70 expression as an index of stress
response. As assessed by real time PCR, HSP-70 gene
expression was induced after 24 h exposure to 0.5 mM
(nine-fold) and 2.0 mM (27-fold) oxalate, then returned
close to baseline by 48 h (Fig. 6A). HSP-70 protein
levels were also increased in whole cell lysates both 24
and 48 h after oxalate exposure (10–30%; Fig. 6B),
although the difference reached statistical significance
only for 2.0 mM oxalate at 24 h. Therefore, HSP-70
induction could be part of a protective response induced
by prolonged oxalate exposure.
Discussion
Other investigators have hypothesized that tubular cell
injury occurs following oxalate exposure, setting into
346
Fig. 5 Growth of KNRK cells in the presence of oxalate. Cells
were exposed to oxalate (0.5–2.0 mM) from time 0. At 24, 48, and
72 h cell counts were assessed fluorometrically using the CyQuant
reagent. Cell numbers were reduced in the presence of the higher
concentrations of oxalate at 24 h, and increased at moderate
oxalate exposure (0.5 mM) at 72 h. An asterisk indicates P<0.05
vs control
monohydrate crystal and brushite crystal induced upregulation of the inflammatory cytokine MCP-1 in NRK
52E cells [21]. Since NADPH oxidase subunits are
known to be present in renal epithelial cells, this pathway is implicated as a source of ROS in cells exposed to
oxalate [22]. Consistent with other studies using cultured
renal cells, our investigation confirmed that diverse renal
cell types can generate ROS in response to acute oxalate
exposure. Marked cell-type differences were not
observed, although interstitial KNRK cells generated
less ROS at all oxalate concentrations and times of
exposure (Fig. 1). Perhaps these fibroblastic cells lack
important cell membrane transporters or channels to
allow efficient oxalate entry into the cytoplasm.
Overall, after an acute oxalate exposure, our most
striking finding was that surviving cells went on to
proliferate normally, without further toxicity. These
events correlated with enhanced expression of the stress
response chaperone HSP-70. Cell death is not a feature
of renal tissue derived from patients with nephrolithiasis, or even primary or secondary hyperoxaluria [23].
Although urinary oxalate excretion can vary in relation
to the timing of meals [6], it is likely that renal cells in
vivo are continuously exposed to an ambient baseline
level of oxalate. Our experiments suggest that cells are
able to adapt and even proliferate under these circumstances. In some ways, this situation could be analogous
to adaptive responses of inner medullary cells to markedly hyperosmolar conditions [24, 25].
It is possible that crystals may be initiating some of
the pathologic changes observed under hyperoxaluric
conditions in vivo. Calcium oxalate crystals can induce
renal epithelial cellular proliferation both in culture
models [12] and in vivo [7], but may also cause renal cell
injury when present at high concentrations [16]. Once a
calcium oxalate crystal is attached, endocytosis [26] appears to be followed by transport of the crystal to the
tubulo-interstitium where an inflammatory response is
initiated with recruitment of lymphocytes, macrophages,
and fibroblasts [27], perhaps leading to renal scarring.
Importantly, interstitial inflammation and fibrosis
associated with crystal deposition have been observed in
renal tissue of hyperoxaluric patients who developed
renal insufficiency [4, 7].
In conclusion, both proximal and distal renal cells, as
well as fibroblasts, generate ROS in response to oxalate
exposure. Cells demonstrate initial cell death and decrease in cell numbers, but by 48–72 h adapt and grow,
despite the continued presence of oxalate. This adaptive
response may involve chaperones such as HSP-70. Renal
347
Fig. 6 HSP-70 gene and protein
expression after oxalate
exposure. A Expression of the
gene encoding HSP-70 was
upregulated at 24 h after
exposure to oxalate (0.5 or
2.0 mM). HSP-70 protein
expression, measured by
quantitative Western blots of
whole cell lysates, was also
increased by 10–30% 1 or
2 days after oxalate exposure,
but was significant only for
2.0 mM oxalate at 24 hours
(B). An asterisk indicates
P<0.05
348
cells in vivo may have similar mechanisms to withstand
chronic hyperoxaluria.
Acknowledgments This work was supported by grants to J.C.L.
and E.L.G. from the National Institutes of Health (DK 53399, DK
60707), the Oxalosis and Hyperoxaluria Foundation (J.C.L.), the
Robert Wood Johnson Foundation (E.L.G.) and the Mayo
Foundation (J.C.L. and E.L.G.).
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