Am J Physiol Renal Physiol 309: F204–F215, 2015.
First published June 3, 2015; doi:10.1152/ajprenal.00150.2015.
Kidney glycosphingolipids are elevated early in diabetic nephropathy and
mediate hypertrophy of mesangial cells
Marimuthu Subathra,1 Midhun Korrapati,2 Lauren A. Howell,3 John M. Arthur,4,5 James A. Shayman,6
Rick G. Schnellmann,2,7 and Leah J. Siskind1
1
Department of Pharmacology and Toxicology, James Graham Brown Cancer Center, University of Louisville, Louisville,
Kentucky; 2Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South
Carolina; 3Department of Biomedical Sciences, Florida State University, Tallahassee, Florida; 4University of Arkansas for
Medical Sciences, Little Rock, Arkansas; 5Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; 6Nephrology
Division, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan; and 7Ralph H.
Johnson Veterans Administration Medical Center, Charleston, South Carolina
Subathra M, Korrapati M, Howell LA, Arthur JM, Shayman
JA, Schnellmann RG, Siskind LJ. Kidney glycosphingolipids are
elevated early in diabetic nephropathy and mediate hypertrophy of
mesangial cells. Am J Physiol Renal Physiol 309: F204 –F215, 2015.
First published June 3, 2015; doi:10.1152/ajprenal.00150.2015.—
Glycosphingolipids (GSLs) play a role in insulin resistance and
diabetes, but their role in diabetic nephropathy (DN) has received
limited attention. We used 9- and 17-wk-old nondiabetic db/m and
diabetic db/db mice to examine the role of GSLs in DN. Cerebrosides
or monoglycosylated GSLs [hexosylceramides (HexCers); glucosyland galactosylceramides] and lactosylceramide (LacCers) were elevated in db/db mouse kidney cortices, specifically in glomeruli, and
also in urine. In our recent paper (25), we observed that the kidneys
exhibited glomerular hypertrophy and proximal tubular vacuolization
and increased fibrosis markers at these time points. Mesangial cells
contribute to hyperglycemia-induced glomerular hypertrophy in DN.
Hyperglycemic culture conditions, similar to that present in diabetes,
were sufficient to elevate mesangial cell HexCers and increase markers of fibrosis, extracellular matrix proteins, and cellular hypertrophy.
Inhibition of glucosylceramide synthase or lowering glucose levels
decreased markers of fibrosis and extracellular matrix proteins and
reversed mesangial cell hypertrophy. Hyperglycemia increased phosphorylated (p)SMAD3 and pAkt levels and reduced phosphatase and
tensin homolog levels, which were reversed with glucosylceramide
synthase inhibition. These data suggest that inhibition of glucosylceramide synthase reversed mesangial cell hypertrophy through decreased pAkt and pSmad3 and increased pathways responsible for
protein degradation. Importantly, urinary GSL levels were higher in
patients with DN compared with healthy control subjects, implicating
a role for these lipids in human DN. Thus, hyperglycemia in type II
diabetes leads to renal dysfunction at least in part by inducing
accumulation of HexCers and LacCers in mesangial cells, resulting in
fibrosis, extracellular matrix production, and hypertrophy.
glycosphingolipids; kidney; diabetic nephropathy; hypertrophy; mesangial cells
(DN) is major complication of diabetes
mellitus and is the most prevalent cause of chronic renal failure
and end-stage renal disease in the Western world (13). DN is
clinically characterized by an initial rise in the glomerular
filtration rate followed later by an increase in urinary albumin
DIABETIC NEPHROPATHY
Address for reprint requests and other correspondence: L. J. Siskind, Dept.
of Pharmacology and Toxicology, Univ. of Louisville, 505 S. Hancock St.,
CTRB 203, Louisville, KY (e-mail: [email protected])
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excretion. Without intervention, the glomerular filtration rate
decreases over time.
DN is thought to arise from hemodynamic and metabolic
factors that act independently and in concert with one another
(14, 23). Hemodynamic factors include alterations in intraglomerular vascular pressure as well as vasoactive hormone pathways such as the renin-angiotensin aldosterone system and
endothelin (14, 23). Chronic hyperglycemia has been reported
to induce an initial phase of increased mesangial cell proliferation that is followed by mesangial matrix expansion and renal
hypertrophy (17, 23, 29, 62). In addition, there is an induction
of inflammatory signaling pathways within the glomerulus that
results in the activation of macrophages and infiltration of
immune cells (20). The mechanisms by which chronic hyperglycemia induces these changes in the glomeruli are not completely understood.
Glycosphingolipids (GSLs) are a heterogenous class of lipids in the sphingolipid family. One of the most simple subclass
of GSLs is glucosylceramide, which is generated from ceramides synthesized in the endoplasmic reticulum that are
transported to the Golgi, where UDP-Glc:ceramide glucosyltransferase [glucosylceramide synthase (GCS)] catalyzes the
addition of glucose (35). Once formed, a galactose (from
UDP-galactose) can be added to glucosylceramide to form
lactosylceramides (LacCer). LacCer is the central hub for the
synthesis of complex GSLs, including other neutral GSLs, such
as globotriaosylceramide (Gb3), and acidic GSLs, such as
ganglioside GM3.
GSLs regulate a number of cellular processes, including cell
proliferation and inflammation, and have been implicated in
metabolic syndrome and diabetes (7, 12, 26, 27, 35, 38, 55, 63).
Inhibition of GCS by D,L-threo-1-phenyl-2-decanoylamino-3morpholino-1-propanol hydrochloride (PDMP), D,L-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), deoxynojirimycin, and miglustat protect from various disease conditions
in rodent models; in particular, inhibition of GCS prevents and
reverses insulin resistance and hepatic steatosis in rodent models
of diabetes (1, 59, 65). GCS inhibitors, such as miglustat and
eliglustat, are beneficial in the treatment of Gaucher disease in
humans (9, 28, 36, 43, 52).
GSLs are highly abundant in the kidney, including podocytes, mesangial cells, and tubular epithelial cells (35). Defects
in GSL metabolism that either result in their accumulation or
enhanced degradation occur in Fabry’s kidney disease, polycystic kidney disease, and renal cell carcinoma (35). Renal
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Submitted 10 April 2015; accepted in final form 27 May 2015
GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
MATERIALS AND METHODS
Materials. DMEM (low and high glucose), trypsin-EDTA solution,
HEPES, FBS, and penicillin-streptomycin solution were from
GIBCO/Invitrogen. F-12 HAM’s supplement was purchased from
Hyclone. The BCA protein assay kit was from Pierce (catalog nos.
23223 and 23224). We used an inhibitor of glucosylceramide synthase, D-threo-1-ethylendioxyphenyl-2-decanoylamino-3-pyrrolidinopropanol, the 10-carbon analog of eliglustat (C10) (33). Concentration
determined by glucosylceramide synthase activity assays verified that
48 h after a single treatment of 0.15 ␮M C10, the activity of
glucosylceramide synthase was significantly reduced (decreased by
90%) and did not decrease viability (data not shown).
Mice. Female diabetic db/db mice (BKS.Cg-m ⫹/⫹ Leprdb/J;
related genotype: a/a⫹Leprdb/⫹ Leprdb) and female nondiabetic db/m
mice (BKS.Cg-m ⫹/⫹ Leprdb/J; related genotype: a/a⫹Dock7m ⫹/⫹
Leprdb) were purchased from Jackson Laboratory (stock no. 000642,
Bar Harbor, ME) and housed in temperature-controlled conditions
under a light-dark cycle with food and water supplied ad libitum. At
9 wk (n ⫽ 11 mice/group) and 17 wk (n ⫽ 6 mice/group) of age, db/m
and db/db mice were placed in metabolism cages for 24 h and then
euthanized as previously described (25), and body weight, serum
glucose, proinflammatory markers, fibrosis, creatinine clearance, and
urinary protein excretion were evaluated (25).
Isolation of glomeruli from db/db and db/m mice. Db/db and db/m
mice were euthanized by cervical dislocation, and kidneys were
dissected. The kidneys of three mice were pooled (6 total). The kidney
cortex was dissected, and the cortices were minced with a razor in
sterile ice-cold PBS. Kidney cortex homogenate was filtered through
three consecutive nylon sieves arranged largest pore size on top to
smallest pore size on bottom (pore sizes: 180, 106, and 53 ␮m). The
end of a plunger from a 10-ml syringe was used to press the
homogenate through the top sieve. The contents of the final 53-␮m
sieve were washed into a beaker using ice-cold PBS and centrifuged
for 5 min at 500 g at 4°C. The resulting pellet was resuspended in
collagenase solution that was prewarmed at 37°C (3 ml of 5 mg/ml
collagenase type II, catalog no. 17101-015, GIBCO) and incubated at
37°C for 30 min with gentle vortexing every 10 min. After the
collagenase digestion, 5 ml ice-cold PBS was added, and the homogenate was then centrifuged at 500 g for 5 min. The resulting pellet was
washed twice by resuspending in ice-cold PBS and centrifugation at
500 g for 5 min. The final pellet was snap frozen and stored at ⫺80°C
until analysis.
Mesangial cell culture and treatment. Mouse mesangial cells were
obtained from the American Type Culture Collection (CRL 1927).
Cells were cultured in a 3:1 (vol/vol) mixture of high-glucose DMEM,
Ham’s F-12 medium, and 14 mM HEPES and supplemented with 5%
FBS and 1% penicillin-streptomycin solution. The total glucose concentration of this media composition was 18.8 mM. Once we received
this cell line, one set of cells was switched to normal glucose
conditions (cells were grown for 25 days in normal glucose and split
when cells were confluent; this set was frozen and used for further
experiments, which used a total glucose concentration of 5.5 mM).
Glucose concentrations were controlled by altering the glucose level
of DMEM to achieve either a high (18.8 mM) or normal (5.5 mM)
total glucose concentration in the culture media. As an osmotic
control, mannitol was used at an equal concentration as glucose. In all
cases, media were filter sterilized before use. Cells were grown at
37°C in a humidified 5% CO2 incubator not exceeding seven passages.
Sphingolipidomic mass spectrometry. For kidney measurements,
the cortex of the kidney was homogenized using a Tissue-Tearor
(Biospec Products) in 20 mM Tris·HCl buffer. A Complete Protease
Inhibitor Cocktail Tablet was added to the buffer (Sigma, St. Louis,
MO). Protein was quantified using a BCA Protein Determination Kit.
From each kidney cortex homogenate, 1 mg of protein was sent for
sphingolipid analysis. For measurement of urine GSLs, 300 ␮l mouse
or 100 ␮l human urine was centrifuged, and the supernatant was
transferred to a glass tube. To obtain enough material for the quantification of hexosylceramides (HexCers), purified glomeruli from the
kidneys of three mice were combined into a single sample. For
cultured mesangial cells, ⬃3 ⫻ 106 cells were sent for sphingolipid
analysis. All samples were snap frozen and submitted for sphingolipid
analysis performed at the Medical University of South Carolina
(Lipidomics Core) on a Thermo Finnigan TSQ 7000, triple-stage
quadrupole mass spectrometer operating in a multiple reaction monitoring positive ionization mode, as previously described (3). Data
were normalized to total lipid phosphate, and urine sample data were
normalized to creatinine.
Mesangial cell proliferation by counting. Mouse mesangial cells
grown in normal or high-glucose medium were plated in six-well
plates at a density of 1.6 ⫻ 105 cells/well (in 2 ml of complete growth
medium). At 24 or 48 h, cells were washed with PBS, trypsinized, and
resuspended in growth media. Cell counts were determined using a
Bio-Rad TC20 automated cell counter.
Mesangial cell proliferation by Alamar blue assay. Mouse mesangial cells grown in normal or high-glucose medium were plated in
96-well plates at a density of 3 ⫻ 103 cells/well (in 200 ␮l of complete
growth medium) for 24, 48, and 72 h, at which point 10% of Alamar
blue reagent from Invitrogen (catalogue no. DAL1100) was added to
each well. Plates were then incubated, and the fluorescence of the
Alamar blue reduction was determined on a SPECTRAmax Gemini
EM plate reader every hour until untreated wells were midlinear
(⬃4,000 arbitrary units, 540-nm excitation and 594-nm emission).
Mesangial cell proliferation by bromodeoxyuridine/7-aminoactinomycin D flow cytometry. Mouse mesangial cells grown in normal or
high-glucose medium were plated in 10-cm2 dishes at a density of
0.5 ⫻ 106 cells/plate (in 2 ml of complete growth medium). At 24 or
48 h, cells were washed with PBS, trypsinized, and resuspended in
growth media. At 45 min before trypsinization, bromodeoxyuridine
(BrdU; catalog no. 552598, BD Pharmingen) was added at a final
concentration of 10 ␮M. Cells were fixed, permeabilized, and washed
according to the manufacturer’s instructions. BrdU and 7-aminoactinomycin D (7-AAD) were analyzed via flow cytometry (FACS scan,
DxP multicolor upgrades powered by Cytek Development).
Mesangial cell proliferation by BrdU ELISA. Mouse mesangial
cells grown in normal or high-glucose medium were plated in 96-well
plates at a density of 3 ⫻ 103 cells/well (in 200 ␮l of complete growth
medium). At the indicated time points, 20 ␮l of 1⫻ BrdU solution was
added and incubated for 24 h, and cells were then fixed. BrdU
measurements were performed according to the manufacturer’s instructions (catalog no. 2750, Millipore). The absorbance was then read
at 450 nm.
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GSLs have been shown to play a role in streptozotocin (STZ)induced type I DN in rats, and their inhibition with PDMP
reversed glomerular hypertrophy (61). However, their role in
nephropathy associated with type II diabetes has not been
determined.
We hypothesized that chronic hyperglycemia in type II
diabetes elevates levels of kidney GSLs, which lead to glomerular pathology. We tested this in vivo in the db/db mouse
model of type II diabetes and in vitro in cultured mesangial
cells. We observed elevation of GSLs in the kidney of diabetic
db/db mice and that inhibition of the synthesis of these lipids
reversed hyperglycemia-induced mesangial cell hypertrophy
through decreased phosphorylated (p)Smad3 and pAkt signaling and enhanced p-phosphatase and tensin homolog (pPTEN)mediated protein degradation pathways. The present work
indicates a novel role for GSLs in the induction of glomerular
hypertrophy in response to hyperglycemia in DN.
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GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
creatinine assay kit (BioAssay Systems, Hayward, CA) as per the
manufacturer’s instructions.
RESULTS
GSLs are elevated in the kidney cortex and urine of diabetic
db/db mice. We have previously reported that the db/db mouse
model of type II diabetes has elevated markers of inflammation
and fibrosis in the kidney as early 9 wk. A decline in kidney
function and pathological changes typical of DN, such as
mesangial matrix expansion, thickening of the glomerular
basement membrane, and vacuolization of the proximal tubular
cells, were observed at 17 wk of age (25). Thus, we defined 9
and 17 wk of age as early and late DN in these mice. Using the
same db/db diabetic and db/m nondiabetic control mice from
these two time points (25), we measured HexCers and LacCers
in kidney cortex homogenates. HexCers are synthesized from
ceramides, which vary in the chain length of the N-linked fatty
acyl chain. In the diabetic kidney, several major species of
HexCers and LacCers were elevated in the kidney cortex at
both 9 and 17 wk in db/db mice compared with age-matched
db/m control mice (Fig. 1, A and B). Very-long-chain species
of HexCers (C22- to C26-HexCers) were elevated to a larger
extent than long-chain species of C16- and C18-HexCers
(Fig. 1A). However, the pattern for LacCers was the opposite, with C16-, C18-, and C20-LacCers increased to a larger
extent than very-long-chain species (C24-, C24:1-, and C26LacCers; Fig. 1B).
Because GSLs are elevated in several kidney diseases and
can be secreted in the urine, we determined if GSLs were
higher in the urine of db/db diabetic mice. Total GSLs were
elevated in the urine of 9-wk-old db/db mice compared with
db/m control mice (Fig. 1C). These data demonstrate that renal
and urinary HexCers and LacCers are elevated early in DN and
represent multiple species with very-long-chain species of
HexCers and long-chain species of LacCers being elevated.
HexCer levels are higher in glomeruli obtained from db/db
mice, and hyperglycemia induces their generation in mesangial cells in vitro. Because DN is characterized in part by
glomerular changes, we determined if GSLs were elevated in
glomeruli. Glomeruli-enriched fractions were obtained from
kidney cortices of 9-wk-old db/db diabetic and db/m control
mice, and HexCers were quantified. HexCers were higher in
glomeruli obtained from db/db mice [long-chain (C16, C18, and
C20) HexCer but not-very-long chain (C22, C22:1, and C24:1)
HexCer; Fig. 2A]. Kidney GSLs were elevated after STZinduced type I DN in rats, and administration of insulin to these
rats reduced kidney GSLs, suggesting that they accumulate as
a result of hyperglycemia (61). Thus, we hypothesized that
hyperglycemia in diabetes is sufficient to induce the generation
of HexCers. To test this, we used mouse mesangial cells grown
in culture and determined if hyperglycemic conditions, similar
to that present in DN, increase HexCers. HexCers were measured in cells cultured in normal glucose (5.5 mM supplemented with 13.3 mM mannitol to control for changes in
osmolarity) or high-glucose (18.8 mM) media for 48 h. Highglucose media led to the accumulation of long-chain and
very-long-chain HexCers (C16-, C18-, C20-, and C24-HexCer),
with the largest changes being long-chain-species (C16- and
C20-HexCers; Fig. 2B). The pattern of accumulated HexCers in
mesangial cells cultured in hyperglycemic conditions was sim-
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Mesangial cell thickness by confocal microscopy. Mouse mesangial
cells grown in normal or high-glucose medium were plated in 35-mm
diameter confocal dishes at a density of 1.6 ⫻ 105 cells/plate (in 2 ml
of complete growth medium). Where indicated, cells were treated
with GCS inhibitor (0.15 ␮M C10) at the time of plating. After 48 h,
cells were fixed with 4% formaldehyde for 10 min at room temperature (24°C). After plates were washed twice with PBS for 5 min each,
cells were permeabilized with blocking buffer (2 ml, 1⫻ PBS, 5%
FBS, and 0.3% Triton X-100) for 10 min at room temperature.
Blocking buffer was aspirated, and cells were stained with the F-actin
stain phalloidin (diluted 1:1,000 in PBS, Alexa fluor568, catalog no.
12380, Life Technologies) at room temperature for 10 min. After cells
had been washed three times for 5 min each with PBS, they were
stained with the nuclear stain DRAQ (catalog no. 4084, Cell Signalling) at room temperature for 10 min. Cells were then washed three
times for 10 min each at room temperature. PBS remained on the cells
to avoid drying until they were imaged on a Nikon (NIS-Elements,
version 4.00) confocal microscope. The microscope was setup to take
z-stacks every 0.5 ␮m from several micrometers above the cells until
the bottom of the cells at the surface of the slide. The average number
of z-stacks required to transverse the cells (where we saw F-actin stain
from the top to bottom of the cells) was defined the average thickness
of the cells. z-Stacks were performed using the ⫻60 objective on
samples prepared from three independent experiments. Measurements
of at least 10 fields of view were taken per experiment.
Immunoblot analysis. Mouse mesangial cells grown in normal or
high-glucose medium were plated in 10-cm2 dishes at a density of
0.5 ⫻ 106 cells/plate (in 10 ml of complete medium). Where indicated, cells were treated with GCS inhibitor (0.15 ␮M C10) at the
time of plating. After 48 h, media were removed, and cells were
washed with ice-cold PBS and scraped with 1 ml PBS. Cells were
centrifuged, and the supernatant was removed. Cells were resuspended with Nonidet P-40 lysis buffer containing Pierce Halt protease
and phosphatase inhibitor. Processing of Western blot samples was
performed as previously described by Qureshi et al. (45). Protein (30
␮g) was used for Western blot analysis. Membranes were then
incubated with primary antibodies in 1:2,000 dilutions and incubated
overnight with continuous shaking at 4°C. The primary antibodies
were as follows: Akt, pAkt (Ser473), PTEN, pPTEN (Ser380/Thr382/383), and
Smad3 (catalog nos. 4691S, 4060S, 9552S, 9549S, and 9513S, respectively, Cell Signaling); pSmad3 (Ser423/Ser425, catalog no. 600401-919, Rockland); and fibronectin and ␤-actin (catalog nos. F3648
and A5441, respectively, Sigma). After extensive washes, membranes
were incubated with peroxidase-conjugated donkey anti-rabbit (1:
10,000, catalog no. SC-2313, Santa Cruz Biotechnology). Signals
were visualized using ECL or SuperSignal west Dura (Thermo Scientific).
Data and statistical analysis. Data are expressed as means ⫾ SD
for all experiments. Multiple comparisons of normally distributed data
were analyzed by one-way ANOVA, as appropriate, and group means
were compared using the Student-Newman-Keuls post hoc test. Single
comparisons were analyzed by Student’s t-test where appropriate.
Animal usage. All animal and treatment protocols were in compliance with the National Institutes of Health Guide for the Care and Use
of Laboratory Animals and were approved by the Institutional Animal
Care and Use Committee of the Medical University of South Carolina.
Human urine samples. Clean catch urine samples for the LacCer
analyses were obtained from frozen, centrifuged, and aliquoted samples that were collected for an unrelated urine biomarker study. All
volunteers gave documented informed consent to participate in Institutional Review Board-approved protocols. A 100-␮l aliquot of urine
was submitted to the Medical University of South Carolina Lipidomics Core facility for the quantification of LacCers. In human urine,
LacCers can be easily quantified, but HexCers are often below the
detectable limit (data not shown). This may reflect differences in their
stability in solution and/or their rates of shedding into the urine.
LacCers were normalized to milligram of urine creatinine using a
GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
F207
ilar to those that accumulated in vivo in glomerular-enriched
fractions (Fig. 2A). These data reveal that elevated glucose is
sufficient to increase GSLs in mesangial cells.
High glucose induces mesangial cell hypertrophy, which is
reversed by glucosylceramide synthase inhibition. Glomerular
hypertrophy occurs in DN, and in the db/db model of type II
DN, glomerular hypertrophy is observed at 17 wk of age (25).
Hyperglycemia-induced glomerular hypertrophy is thought to
occur in part by an initial transient phase of increased mesangial cell proliferation, which is followed by mesangial cell
expansion, extracellular matrix accumulation, podocyte hypertrophy, and thickening of the glomerular basement membrane;
hyperglycemic culture conditions have been shown to be sufficient to induce many of these changes (2, 30, 32, 34, 42, 49,
51, 57, 58, 62). We cultured mesangial cells in high-glucose
media, and cell size was measured by determining the average
cell thickness using confocal microscopy. Cells grown in
normal glucose media had an average cell thickness of 7.7 ⫾
0.3 ␮m (mean ⫾ SD; Fig. 3, A and D). In contrast, the cell
thickness of mesangial cells grown in high-glucose media
increased to 12.3 ⫾ 0.8 ␮m (mean ⫾ SD; Fig. 3, B and D). As
mesangial cell GSLs were elevated in high-glucose media (Fig.
2B), we determined if pharmacological inhibition of glucosylceramide synthase was sufficient to reduce mesangial cell
thickness in high-glucose media. Mesangial cells grown in
high-glucose media and treated with an inhibitor of glucosylceramide synthase, D-threo-1-ethylendioxyphenyl-2-decanoylamino-3-pyrrolidino-propanol, the 10-carbon analog of eliglustat (C10), for 48 h exhibited decreased cell thickness to 8.0 ⫾
0.7 ␮m (mean ⫾ SD; Fig. 3, C and D), similar to cells grown
in normal glucose medium.
Chronic high glucose does not increase mesangial cell
proliferation. Several studies have reported that glomerular
hypertrophy in DN is associated with an initial phase of
enhanced mesangial cell proliferation (23, 62). High-glucose
media in vitro has also been reported to increase cell proliferation of mesangial cells (5, 39, 60, 62). In addition, GSLs have
been reported to play a role in cell proliferation (6). To test if
cell proliferation played a role in this system, we compared
mesangial cells chronically cultured in high-glucose conditions
with those chronically cultured in normal glucose conditions.
Cell proliferation was examined using multiple approaches,
including counting cell numbers (Fig. 4A), Alamar blue fluorescence (Fig. 4B), and BrdU/7-AAD staining and flow cytom-
Fig. 2. HexCer levels are higher in glomerular cells obtained from db/db mice and mesangial cells grown in elevated glucose media in vitro. A: HexCers were
elevated in glomerular enriched fractions obtained from the kidneys of 9-wk-old diabetic db/db mice compared with age-matched nondiabetic db/m control mice.
Cells were purified as described in MATERIALS AND METHODS from the kidney cortex. Kidneys from two mice were combined to create a single sample of three
million cells, and three separate samples were analyzed. B: elevated glucose induces an accumulation of HexCers in cultured mesangial cells. Mouse mesangial
cells were cultured for 48 h in normal or high-glucose medium. Sample of three million cells and three separate samples were analyzed. HexCers were measured
by HPLC-MS/MS, normalized to total lipid phosphate, and expressed as fold changes over db/m control mice (A) and mannitol-treated control cells (B). Data
are means ⫾ SD; n ⫽ 3. *P ⱕ 0.01 (as determined by a Student’s t-test).
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Fig. 1. Glycosphingolipids (GSLs) are elevated in the kidney cortex and urine of diabetic db/db mice. GSLs [hexosylceramides (HexCers) and lactosylceramides
(LacCers)] were quantified by HPLC-tandem mass spectroscopy (HPLC-MS/MS). HexCers (A) and LacCers (B) were quantified in 1 mg of kidney cortex
homogenate obtained from either db/m or db/db mice at 9 or 17 wk of age. Lipids in A and B were normalized to total lipid phophate. C: total HexCers and
LacCers in a 24-h urine sample collected from nondiabetic db/m or diabetic db/db mice at 9 wk of age. Lipids were normalized to creatinine. Data in A–C are
means ⫾ SD; n ⫽ 5. *P ⱕ 0.01 compared with age-matched nondiabetic control mice.
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GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
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Fig. 3. High glucose induces mesangial cell hypertrophy, which is reversed by glucosylceramide synthase inhibition. Mesangial cells were grown in
normal glucose (NG; A), high glucose (HG; B), or
HG supplemented with 0.15 ␮M C10 (C). At 48 h,
cells were fixed, permeabilized, and stained with the
F-actin marker phalloidin and the nuclear stain
DRAQ. Serial 0.5-␮m-thick z-sections were obtained using confocal microscopy. D: average thickness of mesangial cells calculated by the distance
from the top to the bottom of the F-actin staining.
Data are means ⫾ SD; n ⱖ 5. ***P ⱕ 0.0001, HG
compared with NG; $$$P ⱕ 0.0001, HG ⫹ C10
compared with HG (as determined by one-way
ANOVA). Scale bars ⫽ 50 ␮m.
etry (Fig. 4C). These results show that chronic high-glucose
culture conditions do not alter mesangial cell proliferation. In
addition, we determined the effects of a switching normal
glucose to high-glucose media and high-glucose to normal
glucose complete growth media for 24 or 48 h on cell prolif-
eration by measuring BrdU incorporation (Fig. 4D). There
were no changes in cell proliferation under any condition (Fig.
4D). Therefore, the data shown in Fig. 3 provide evidence that
hyperglycemia-induced cell hypertrophy is not accompanied
by enhanced cellular proliferation.
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GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
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Chronic high glucose increases signaling pathways associated with enhanced cell growth, extracellular matrix production, and fibrosis, which are reduced with glucosylceramide
synthase inhibitors. To decipher the mechanism by which
inhibition of glucosylceramide synthase reverses hyperglycemia-induced mesangial cell hypertrophy, we measured the
expression of key molecules that regulate cell growth, protein
degradation, and extracellular matrix protein fibronectin. It has
been shown that growth stimulatory signals and inhibition of
protein degradation pathways can cause cell hypertrophy (10,
32, 64). Mesangial cells in high-glucose media had elevated
cell growth signals with increased pAkt (Ser473) with no
change in total Akt (Fig. 5, A and B) and decreased levels of the
protein degradation marker pPTEN (Fig. 5D) even though total
PTEN was increased (Fig. 5C). Our results indicate that hyperglycemia increases growth signals in association with decreased protein degradation by decreasing PTEN phosphorylation in mesangial cells.
Transforming growth factor (TGF)-␤ has been implicated in
renal hypertrophy and the production of extracellular matrix in
DN (22). Because TGF-␤ mediates phosphorylation of Smad3,
we analyzed Smad3 and pSmad3 in mesangial cells in the
presence of normal and high glucose. High-glucose culture
conditions increased levels of Smad3 and pSmad3 in mesangial
cells, suggesting enhanced TGF-␤ signaling (Fig. 5, E and F).
TGF-␤ production is upstream of and regulates extracellular
matrix production. It has been established that mesangial
extracellular matrix buildup is a characteristic feature of DN
(37). High-glucose media have also been reported to induce
expression of cell-associated fibronectin in mesangial cells
(37). High glucose enhanced levels of total fibronectin associated with mesangial cells (Fig. 5G). In conclusion, highglucose culture conditions enhanced pSmad3 and pAkt, decreased pPTEN, and increased production of the extracellular
matrix protein fibronectin via Smad3 and pAkt induced by
TGF-␤.
To determine the role of GSLs in high-glucose mesangial
cell signaling, we treated mesangial cells grown in hyperglycemic conditions with the C10 inhibitor of GCS and analyzed
the above signaling pathways. Cells were treated with a 0.15
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Fig. 3. Continued.
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GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
␮M concentration of C10 for 48 h. GCS inhibition did not alter
total Akt (Fig. 6A) but decreased Akt phosphorylation (Fig. 6B),
suggesting reduced Akt signaling. Similarly, C10 did not alter
total PTEN levels (Fig. 6C) but increased levels of pPTEN
(Fig. 6D), suggesting enhanced protein degradation pathways.
To determine if GCS inhibition alters signaling through
TGF-␤, we analyzed levels of pSmad3 and total Smad3. C10
reduced levels of total Smad3 and pSmad3 (Fig. 6, E and F)
in cells grown in high glucose, suggesting inhibition of
TGF-␤ signaling pathways. Likewise, C10 reduced levels of
fibronectin (Fig. 6G), suggesting reduced levels of extracellular matrix proteins. The data shown in Fig. 6 suggest that
inhibition of GCS reverses mesangial cell hypertrophy by
reducing Akt and Smad3 signaling and reducing extracellular matrix proteins by enhanced protein degradation pathways.
Urinary LacCers are elevated in humans with DN. The data
presented thus far indicate that in the db/db mouse model of
type II DN, GSLs are elevated in the kidney and urine and play
a role in mediating DN and the functional role of GSLs on
mesangial hypertrophy. We hypothesized that GSLs would
also be elevated in the urine of patients with DN. GSLs were
quantified in urine obtained from either patients diagnosed with
DN or from healthy control subjects. Total urine LacCers were
increased ⬃12-fold in patients with DN compared with levels
detected in healthy control subjects (2.9 ⫾ 4.1 vs. 35.1 ⫾ 14
Fig. 5. Chronic HG alters protein expression. Mesangial cells were grown in NG or HG. At 48 h, cells
were collected and immunobloted for cell growth
signaling pathways (A and B), protein degradation
markers (C and D), and fibrosis and extracellular
matrix (ECM) markers (E–G). p, Phosphorylated;
PTEN, phosphatase and tensin homolog. The blots
shown are from one experiment and were reprobed
for ␤-actin, which was used as an internal control.
Results are representative of at least 3 independent
experiments. Data are means ⫾ SD. *P ⱕ 0.05;
***P ⱕ 0.001; ****P ⱕ 0.0001 (as determined by
a Student’s t-test).
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Fig. 4. Chronic HG does not increase mesangial cell
proliferation. Cells were plated in NG or HG, and
cell proliferation determined by counting cells (A),
determining relative cell numbers with Alamar
blue fluorescence (B), measuring incorporation of
bromodeoxyuridine (BrdU) at the indicated time
points by flowcytometry (C), and measuring the
incorporation of BrdU at the indicated time points
by ELISA after plating (D). Data are means ⫾
SD; n ⫽ 3.
GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
F211
pmol LacCer/mg creatinine; Fig. 7). These data suggest a
possible role for GSLs in human DN.
DISCUSSION
GSLs have been documented to play a role in metabolic
syndrome, insulin resistance, and diabetes. In particular, inhibition of GCS prevents and reverses insulin resistance in a
variety of rodent models of diabetes (1). Furthermore, GSL
accumulation has been shown to play a role in nephropathy
resulting from STZ-induced type I diabetes in rats, and inhibition of their production using the glucosylceramide synthase
inhibitor PDMP reversed renal hypertrophy (61). In that study
(61), GSLs were measured by thin-layer chromatography,
which can separate glucosylceramide and galactosylceramide
Fig. 7. Urinary LacCers are elevated in humans with diabetic nephropathy
(DN). LacCers were quantified in the urine of control subjects without
nephropathy or from patients with confirmed DN via HPLC-MS/MS. Lipids
were normalized to urine creatinine. Data are means ⫾ SD; n ⫽ 10. *P ⱕ 0.01
compared with control subjects (as determined by a Student’s t-test).
using borate-impregnated plates but does not separate individual species of GSLs based on fatty acyl groups. The mass
spectroscopy methods used in the present study do not distinguish between glucosylceramide and galactosylceramide.
However, given that UDP-glucose and UDP-galactose are
interconvertable through a ubiquitously expressed epimerase, it
is likely that total HexCer content reflects changes in glucosylceramide. In addition, the location within the kidney in
which GSLs were generated and their role in nephropathy were
also not determined (61). Our data reveal that GSLs accumulated in the kidney cortex in glomeruli suggest that they are
mediators of type II DN via the induction of glomerular
hypertrophy. Our in vitro data indicate that GSLs play a role
in mesangial cell hypertrophy. There are many cell types in
addition to mesangial cells that are present and play a role
in glomerular hypertrophy in DN. Future work will be aimed at
determining the specific glomerular cell type in which GSLs
accumulate during DN.
Db/db mice become very obese and develop insulin resistance and hyperglycemia at a very young age. Changes in the
kidney such as inflammation, immune cell infiltration, and
fibrosis occur early (9 wk), whereas changes such as altered
creatinine clearance occur later (17 wk) (25). Mice in this
model develop glomerular hypertrophy and proximal tubule
vacuolization similar to human disease (25). Here, we documented renal cortical accumulation of the GSLs HexCers and
LacCers early in DN (9 wk) and continuing at later stages of
DN (17 wk; Fig. 1, A and B). We have recently reported (41)
increased GSL levels in the kidneys and urine of lupus-prone
mice. Similarly, urinary hexosyl and LacCers were increased in
db/db mice at 9 wk of age (Fig. 1C). As we previously reported
in lupus nephritis (41), GSLs in db/db mice were elevated at
least in part in the glomeruli. We have recently reported that
GSL levels in hematopoietic and leukemia cells are in part
determined by glucose availability (47). Similarly, culture of
mesangial cells in hyperglycemic conditions, similar to those
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Fig. 6. Inhibition of glucosylceramide synthase reduces mesangial hypertrophy by reducing pAkt and
pSmad3 and increasing pPTEN. Mesangial cells
were grown in HG, plated, and supplemented with
DMSO or 0.15 ␮M C10. At 48 h, cells were collected and immunobloted for cell growth signaling
pathways (A and B), protein degradation markers (C
and D), and fibrosis and ECM marker (E–G). The
blots shown are from one experiment and were
reprobed for ␤-actin, which was used as an internal
control. Results are representative of at least 3
independent experiments. Data are means ⫾ SD.
*P ⱕ 0.05; **P ⱕ 0.01; ***P ⱕ 0.005; ****P ⱕ
0.0001 (as determined by a Student’s t-test).
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GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
ited pharmacologically with C10, mesangial cell thickness was
reduced to the size of cells grown in normal glucose (Fig. 3, C
and D). Our data are consistent with those reported in the
STZ-induced model of type I DN in which PDMP administration to diabetic rats reversed glomerular hypertrophy (61). In
summary, these data indicate that GSLs mediate hyperglycemia-induced mesangial cell hypertrophy.
To explore the molecular mechanism by which GCS inhibition reverses hyperglycemia-induced mesangial cell hypertrophy, we evaluated the expression of key signaling molecules
that mediate this process. Chronic hyperglycemia induced
enhanced Akt activation and reduced PTEN expression (Fig. 5,
B and D). A recent report (64) has indicated that activated Akt
and a reduction in PTEN levels are crucially involved in vivo
in db/db mouse model DN and in vitro in hyperglycemiainduced hypertrophy. Overexpression of wild-type PTEN reduces Akt phosphorylation and prevents hyperglycemia-induced mesangial cell hypertrophy, whereas overexpression of
dominant negative PTEN is sufficient to induce hypertrophy in
mesangial cells even when grown in normal glucose conditions
(32). Our data suggest that GSLs and glucosylceramide syn-
Fig. 8. Schematic representation of the glucosylceramide-induced signaling
cascade of the DN process. Excessive glucose enhances GSL production by
increasing the availability of substrate ceramide and UDP-glucose. GSLs
activate signaling through Smad3 and downregulate pPTEN phosphorylation,
which increases Akt phosphorylation, leading to hypertrophy and ECM accumulation in diabetic mesangial cells. Inhibition of GSL by C10 improves the
pathology by reversing the signaling pathway.
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present in the diabetic kidney, were sufficient to induce the
accumulation of GSLs.
The mechanism by which elevated glucose leads to increased levels of HexCers is unproven. In vivo data provide
evidence that blood glucose levels regulate at least in part
kidney GSL concentrations; administration of insulin to rats
with insulin-dependent diabetes as a result of STZ treatment
significantly decreased blood glucose levels as well as kidney
GSLs (61). Shayman and coauthors (61) have suggested that
UDP-glucose/UDP-galactose are required substrates for the
synthesis of glucosylceramide and LacCer and that the levels
of UDP-glucose/UDP-galactose in the kidney are lower than
the Km of glucsosylceramide synthase and thus are rate limiting. These substrates are generated in the glycogen synthesis
pathway. In addition, NADPH produced in the pentose phosphate pathway is rate limiting for the synthesis of fatty acids,
the building blocks of all lipids, including sphingolipids. UTP
generated in the pentose phosphate pathway feeds into the
glycogen synthesis pathway for the generation of UDP-glucose
and UDP-galactose. It is possible that the enhanced flux of
glucose into these otherwise minor pathways in the kidney
during diabetes leads to an enhanced production of ratelimiting substrates required for a glucosylceramide synthesis.
Indeed, our recent data suggest that this is the case (47). Cells
made to be highly glycolytic have elevated HexCers, which are
depleted upon glucose withdrawal. In addition, inhibitors of
glycolysis or the pentose phosphate pathway significantly decreased HexCers (47). These changes in HexCers were likely
substrate driven, as they were independent of glucosylceramide
synthase expression or activity.
In addition to hyperglycemia, ROS generation in the diabetic
kidney could also play a role in GSL production. Grove et al.
(16) previously reported that the ROS inhibitor pyridoxamine,
when given to endothelial nitric oxide synthase-deficient
C57BLKS db/db DN mice upon the development of hyperglycemia (⬃6 wk of age), prevented the accumulation in glomeruli and/or tubules of specific phospho- and glycolipid species
from four different classes, such as gangliosides, sulfoglycosphingolipids, lysophospholipids, and phosphatidylethanolamines, and prevented nephropathy. Pyridoxamine is also
efficacious when used in several other animal models of
diabetes (11, 48, 50) and in clinical trials in patients with early
stages of the disease. Indeed, treatment of mesangial cells with
the oxidant H2O2 increases HexCer levels (data not shown).
Thus, in addition to hyperglycemia, increased ROS in DN
could further enhance synthesis of GSLs.
Hyperglycemia is a prerequisite for the development of DN
and mediates early glomerular pathological changes. Consistent with the literature (8, 18, 21, 32, 34, 44, 49), our data
indicate that high-glucose media induce hypertrophy of mesangial cells. Transgenic mice overexpressing glucose transporter (GLUT)1 in glomeruli develop glomerular hypertrophy,
glomerulosclerosis, and decreased renal function (54); these
data reveal that hyperglycemia induces many pathological
changes in glomeruli even in the absence of diabetes and
hypertension. Overexpression of GLUT1 in mesangial cells in
culture is sufficient to induce hypertrophy (19). We showed
that mesangial cells grown in high-glucose media are 1.6-fold
greater in size than those grown in normal glucose media (Fig.
3, B and D). High glucose induced an accumulation of GSLs in
mesangial cells (Fig. 2B), and when GSL synthesis was inhib-
GLYCOSPHINGOLIPIDS IN DIABETIC NEPHROPATHY
indeed direct mediators of DN. Pharmacological inhibitors of
glucosylceramide synthase, such as eliglustat, are currently
being used in humans for the treatment of Gaucher disease
and improve platelet and hemoglobin levels as well as
reduce spleen and liver size (28, 31). These inhibitors are
also well tolerated for long-term use. Thus, glucosylceramide synthase inhibitors may have therapeutic potential for
the treatment of DN.
GRANTS
This work was supported by National Institutes of Health (NIH) Grants
P20-RR-17677 (COBRE in Lipidomics and Pathobiology pilot project), R01DK-093462 (to L. J. Siskind), R01-DK-101034 (to J. M. Arthur), and R01GM-084147 (to R. G. Schnellmann), Veterans Administration Merit Review
Grant BX-000851 (to R. G. Schnellmann), Veterans Affairs Research Enhancement Award Program pilot project funding (to L. J. Siskind, J. M. Arthur,
and R. G. Schnellmann), and the Lipidomics Shared Resource of the Hollings
Cancer Center at the Medical University of South Carolina, supported by NIH
Grant P30-CA-138313.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
DISCLAIMER
The contents of this publication do not represent the views of the Department of Veterans Affairs or the United States Government or any of the other
funding entities.
AUTHOR CONTRIBUTIONS
Author contributions: M.S., M.K., J.M.A., J.A.S., R.G.S., and L.J.S. conception and design of research; M.S., M.K., L.A.H., and L.J.S. performed
experiments; M.S., L.A.H., and L.J.S. analyzed data; M.S., R.G.S., and L.J.S.
interpreted results of experiments; M.S. and L.J.S. prepared figures; M.S. and
L.J.S. drafted manuscript; M.S., J.A.S., R.G.S., and L.J.S. edited and revised
manuscript; M.S., R.G.S., and L.J.S. approved final version of manuscript.
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Inhibition of glucosylceramide synthase may be a viable
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