The Synthesized Plant Metabolite 3,4,5-Tri-O-Galloylquinic Acid Methyl Ester Inhibits Calcium Oxalate Crystal Growth in a Drosophila Model, Downregulates Renal Cell Surface Annexin A1 Expression, and Decreases Crystal Adhesion to Cells

Article abstract of DOI:10.1021/acs.jmedchem.7b01566

The plant metabolite 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME, compound 6) was synthesized, and its potential effect on calcium oxalate monohydrate (COM) crystal binding to the surface of Madin-Darby canine kidney cells type I (MDCKI) and crystal growth in a Drosophila melanogaster Malpighian tubule (MT) model were investigated. Membrane, cytosolic, and total annexin A1 (AxA1), α-enolase, and heat shock protein 90 (HSP90) amounts were examined by Western blot analysis after subcellular fractionation, then confirmed by immunofluorescence staining of cultured cells. Pretreatment of MDCKI cells with TGAME for up to 6 h significantly diminished COM crystal binding in a concentration-dependent manner. TGAME significantly inhibited AxA1 surface expression by immunofluorescence microscopy, whereas intracellular AxA1 increased. Western blot analysis confirmed AxA1 expression changes in the membrane and cytosolic fractions of compound-treated cells, whereas whole cell AxA1 remained unchanged. TGAME also significantly decreased the size, number, and growth of calcium oxalate (CaOx) crystals induced in a Drosophila melanogaster MT model and possessed a potent antioxidant activity in a DPPH assay.

Full text of DOI:10.1021/acs.jmedchem.7b01566

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Article
The Synthesized Plant Metabolite 3,4,5-Tri-O-Galloylquinic
Acid Methyl Ester Inhibits Calcium Oxalate Crystal Growth in a
Drosophila Model, Downregulates Renal Cell Surface Annexin
A1 Expression, and Decreases Crystal Adhesion to Cells
Mohamed Abd El-Salam, Jairo Kenupp Bastos, Jing Jing Han, Daniel Previdi,
Eduardo B. Coelho, Paulo M Donate, Michael F. Romero, and John Lieske
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01566 • Publication Date (Web): 06 Feb 2018
Downloaded from http://pubs.acs.org on February 7, 2018
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The Synthesized Plant Metabolite 3,4,5-Tri-O-Galloylquinic Acid Methyl
Ester Inhibits Calcium Oxalate Crystal Growth in a Drosophila Model,
Downregulates Renal Cell Surface Annexin A1 Expression, and Decreases
Crystal Adhesion to Cells
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Mohamed Abd El-Salam,† ‡ Jairo Kenupp Bastos, Jing Jing Han, Daniel Previdi, Eduardo B.
†
‡
§
⊥
§
‡,*
∥
⊥
Coelho, Paulo M. Donate, Michael F. Romero and John Lieske
†Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences, University of São Paulo, Ribeirão
Preto, SP 14040-903, Brazil
‡Department of Internal Medicine, Division of Nephrology and Hypertension, Mayo Clinic College of Medicine,
Rochester, MN 55905, USA
§Department of Chemistry, Faculty of Philosophy, Arts and Sciences, University of Sao Paulo, Ribeirão Preto, SP
1
4040-901, Brazil
∥Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto,
SP 14040-900, Brazil.
⊥Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905,
USA
ABSTRACT
The plant metabolite 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME, compound 6)
was synthesized and its potential effect on calcium oxalate monohydrate (COM) crystal binding
to the surface of Madin-Darby Canine Kidney Cells type I (MDCKI) and crystal growth in a
Drosophila melanogaster Malpighian tubule (MT) model was investigated. Membrane, cytosolic
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and total Annexin A1 (AxA1), α-enolase and heat shock protein 90 (HSP90) amounts were
examined by Western blot analysis after subcellular fractionation, then confirmed by
immunofluorescence staining of cultured cells. Pretreatment of MDCKI cells with TGAME for
up to 6 h significantly diminished COM crystal-binding in a concentration-dependent manner.
TGAME significantly inhibited AxA1 surface expression by immunofluorescence microscopy,
whereas intracellular AxA1 increased. Western blot analysis confirmed AxA1 expression
changes in the membrane and cytosolic fractions of compound-treated cells, whereas whole cell
AxA1 remained unchanged. TGAME also significantly decreased the size, number, and growth
of calcium oxalate (CaOx) crystals induced in a Drosophila melanogaster MT model and
possessed a potent antioxidant activity in a DPPH assay.
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INTRODUCTION
Renal stone disease, also known as urolithiasis, is common with a recent overall
estimated prevalence rate of 14.8% that appears to be rising, with a 5 year recurrence rate of up
to 50%. 1, 2 The first stages of stone formation include nucleation , crystallization and retention
of these crystals within the kidney. Crystal nucleation is the first step in crystallization and can
occur homogeneously or heterogeneously. Homogeneous nucleation demands a high degree of
supersaturation with respect to the mineral concerned. By contrast, heterogeneous nucleation is
3
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the much more likely mechanism through which crystal initiation occurs in the urine and this
process can occur in the presence of particulate matters consisting of cell debris, proteins or
crystals of another mineral, and is contained within receptacles lined with chemically active cell
4
surfaces. Once a crystal nucleus is formed inside the kidneys , exposure to the urine makes the
stone able to grow by encrustation. 5 There are two basic pathways (free-particle and fixed-
particle mechanisms) for the establishment of a stone nucleus, both of which can be active in any
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stone former. Crystallization in turn requires supersaturation of tubular and/or interstitial fluid
with respect to ionic compounds such as calcium oxalate (CaOx) and calcium phosphate (CaP).
If these processes occur in tubular fluid, the retention of crystals in the kidney may involve
aggregation of smaller crystals and/or adhesion to the tubular cell surface. 3, 6
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Calcium oxalate monohydrate (COM) is found in up to 70-80 % of stones. Many studies
have demonstrated that COM crystal binding to renal tubular cells triggers diverse cellular
responses including proliferation, mitochondrial dysfunction, oxidative stress, cytotoxicity and
cell death. 8, 9 One possible strategy to prevent stone formation is to arrest these early intrarenal
events after COM crystal nucleation including crystal growth, crystal aggregation, and crystal
adherence to renal epithelial cells. 10, 11 Previous studies have suggested some possible COM
crystal binding proteins on the apical surface of renal tubular cells (e.g. α-enolase, annexin A1
and heat shock protein 90 (HSP90)). 11-14
Several studies have reported the role of natural products in the prevention of renal stones
by inhibiting calcification and hyperoxaluria-induced oxidative stress in animal models of
nephrolithiasis. We previously demonstrated that Copaifera langsdorffii Desf. (Fabaceae) leaf
extract increased urinary magnesium and decreased urinary uric acid excretion in rats. 15
Moreover, treated rats had lower urinary oxalate and higher urinary citrate excretion, reduced
intratubular calcification, and decreased osteopontin (OPN) expression. 16 Galloylquinic acid
compounds are the major secondary metabolites found in C. langsdorffii leaf that might
significantly contribute to its antiurolithic activity. It is interesting to note that the compounds
have an affinity for the kidney. A high concentration (Cmax = 15.84 µg/g tissue) was measured in
renal tissue after an intravenous pharmacokinetic study performed in an animal model by our
group. 17 This relatively high distribution of the compounds within the kidney might make them
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particularly effective for preventing kidney stone formation. Recently, we isolated 16
galloylquinic acid compounds from the aqueous fraction of the leaves, which displayed potential
gastroprotective activity in a mouse model of gastric ulcer and cytotoxicity for gastric
adenocarcinoma cells. 18 The methyl and ethyl 3,4,5-tri-O-galloylquinic acid were previously
isolated from Lepidobotrys staudtii Egnl. stem bark and the leaves of Guiera senegalensis J.F.
Gmel, respectively. 1
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Previous studies also reported chemically related compounds to galloyquinic acids, such
as gallotannin, epigallocatechin-3-gallate and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose in
suppressing oxalate-induced oxidative stress and COM crystal binding to renal epithelial cells. 7
,
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1, 21
The present study involved the total synthesis of 3,4,5-tri-O-galloylquinic acid methyl ester
(TGAME, 6) (Figure 1) and its evaluation on renal cells and COM crystal adhesion, as well as
investigating its effect on CaOx crystallization in a Drosophila melanogaster Malpighian tubule
in vivo and ex vivo models. We also determined the antioxidant activity of the compound using
DPPH assay.
Figure 1. Chemical structure of 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME, 6).
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RESULTS AND DISCUSSION
Chemistry. The promising diverse bioactivities of plant extracts18, 20, 22 rich in galloylquinic acids
prompted our interest to synthesize the tri-substituted 3,4,5-tri-O-galloylquinic acid methyl ester,
with the goal of developing lead compounds for kidney stone prevention. The total synthesis
included six steps starting from commercially available quinic and Gallic acids. The key step in
the synthetic pathway was the affordable Steglich esterification 23 of methyl quinate with 3,4,5-
tribenzyloxybenzoic acid using dicyclohexylcarbodiimide (DCC) and N,N-(dimethylamino)
pyridine (DMAP) as the coupling reagents. The chemical structures of the final compound and
its synthetic intermediates were elucidated by all possible spectroscopic, spectrometric and
spectrophotometric methods of analyses.
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The designed synthetic route of the target TGAME is summarized in Scheme 1. The type
of esterification reaction used is a mild one, which allowed conversion of the sterically
demanding and acid labile methyl quinate, a compound with complex stereochemistry, into the
triester form. A protection protocol using benzyl chloride was applied for the hydroxyl groups of
Gallic acid to mask their reactivity and formation of undesired side products.
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Scheme 1. Synthesis of 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME, 6). Reagents and
conditions: (a) & (b) H
2
SO
4
, methanol, reflux/18 h for (a) and 24 h for (b); (c) KI, K
2
CO , benzyl
3
chloride, acetone, reflux/21 h; (d) NaOH, ethanol, reflux/2 h then HCl; (e) DCC, DMAP,
CH
CHCl
Pd/C for 6 h.
2
Cl
2
, reflux/72 h followed by purification using Silica gel column chromatography,
2
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/methanol (100:1); (f) H
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, pressure 80 p.s.i equivalent to 6 kgf/cm , ethyl acetate, 10%
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The proposed mechanism of the reaction is illustrated in Scheme 2. The chemical
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structure of TGAME and its intermediates were elucidated and confirmed by IR, H NMR, C
NMR, and mass spectrometric data, which were in full agreement with their structures. 18
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D
TGAME was obtained as a light yellow amorphous solid with a specific rotation [α] of - 42.6,
2
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which is levorotatory similar to the naturally occurring one in plants. The HPLC-UV analysis
of the compound at 280 nm and 254 nm revealed one single peak at 33.57 min. The UV
spectrum of the peak showed two characteristic bands at 225 and 275 nm, indicating that the
compound belongs to galloylquinic acids class. 18 The IR spectrum of TGAME showed a broad
-1
band at 3387 cm referring to the presence of H-bonded hydroxyl groups. A characteristic
-1
carbonyl group of the ester appeared at 1707 cm . The bands at 1216 and 1034 indicated the
presence of a C−O functional group. Two bending bands of C−H appeared at 1475 and 1338 cm-
1
reflecting an aliphatic methyl group. Other characteristic IR bands of the compound are
presented in the Supporting Information. The ESI-MS/MS analysis of the important synthetic
intermediate 5 (C92
80
H O18), a precursor of the final compound, manifested the following
+
+
+
molecular ions: m/z [M+H] 1473.5239, MS [M+Na] 1496.5005, MS [M+K] 1511.4664.
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Scheme 2. The proposed mechanism of the coupling reaction between compound 1
and 4 for the synthesis of 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME, 6).
The molecular weight of TGAME was determined by ESI-MS/MS in both the positive
and negative ion modes. The experimental and calculated m/z of the protonated molecular ion
+
[
M+H] were 663.1190 and 663.1200, respectively. The MS analysis in the negative ion mode
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-
revealed a molecular ion of m/z [M-H] at 661.0878. The base peak at m/z 169.0089 was formed
through McLafferty rearrangement pathway. Other fragments were formed through the loss of
water and CO
2
as shown in the proposed fragmentation pathways in Supporting Information. The
+
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sodiated molecular [M+Na] ion also was observed in the positive ion mode at m/z 685.1012
with an error of precision +1.75 ppm. The proposed fragmentation patterns of the protonated and
sodiated molecular ion are further illustrated in Supporting Information. The NMR and 2D NMR
(
HMBC and HSQC) spectral data of compound 5 and 6 agreed with our previously published
data. 18
MDCKI cell cytotoxicity of TGAME and its hydrolytic products. To determine the safe in vitro
concentration, cytotoxicity was assessed by the MTT assay after exposing Madin Darby Canine
Kidney type I (MDCKI) cells to increasing concentrations of TGAME (0, 5, 10, 25, 50 µM) for
2
4 h. Gallic acid and methyl quinate, which are products of TGAME hydrolysis, were also
tested. TGAME did not exert any apparent MDCKI cell cytotoxicity in a concentration up to
155+/-4 μM (Figure 2). The inhibitory concentration 50% (IC50) of the hydrolytic products,
methyl quinate and Gallic acid, were 120+/-7 μM and 34+/-6 μM, respectively. Therefore, the
nontoxic concentrations below this IC50 were used in subsequent experiments for the compounds.
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Conc (μM)
Compound 6
Gallic acid
Methyl quinate
Figure 2. Cytotoxic effects of TGAME (6) and its hydrolytic products in MDCKI cells. The
assay of cytotoxicity was evaluated by MTT assay. Cells were plated onto 96-well microplates
4
(
1×10 cells/well) and treated with various concentrations of each tested compound (0, 5, 10, 25,
5
0 µM in DMSO/PBS) for 24 h. Data were expressed as means ± S.D. of three independent
experiments.
COM crystal-adherence assay. MDCKI cells (originally derived from the distal nephron
of a female dog), were used as an in vitro model since the initial site of nephrolithiasis has been
2
5, 26
proposed to be in the distal nephron as supported by histopathological analysis.
cells were incubated with COM crystals (100 μg/mL of culture medium) after pretreatment with
0 μM TGAME for up to 6 h. The concentrations of TGAME used in this study were based upon
MDCKI
5
concentration-response and cytotoxicity studies together with results from the crystal binding
assay (concentration-dependence study) that demonstrated TGAME significantly decreased the
number of crystals bound to the cell surface at both 25 M and 50 M versus the control (Figure
3
), while the concentration of 50 M was more inhibitory compared to 25 M (p <0.05). Results
from the crystal-binding assay (time-course study) demonstrated that pretreatment with TGAME
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(
50 μM) significantly (p <0.05) decreased the number of cell surface associated-COM crystals
by approximately 50% at 3 h and 6 h compared to control (Figure 4). Assays were done at early
time points (not exceeding 6 h) to focus on the crystal-binding stage and largely avoiding
subsequent COM crystal phagocytosis. 11, 27
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(A)
Control
1 µM
10 µM
400 X
400 X
400 X
25 µM
50 µM
400 X
400 X
(B)
6
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4
3
2
1
0
0
0
0
0
0
0
*
**
Control
1 µM
10 µM
Compound 6
25 µM
50 µM
Figure 3. Effect of TGAME (6) on COM crystal-binding capability of the cells was dose
dependent. MDCKI cells were pretreated with various doses of compound 6 (0, 1, 10, 25, and 50
μM) for 3 h followed by incubation with COM crystals (100 μg/mL of culture medium) for 30
min. A | After washing with plain DMEM culture media, images of the remaining crystals were
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captured under a phase-contrast microscope for 15 high-power fields (HPF) in each well. The
original magnification power was ×400. B | The remaining crystals that adhered on the cell
surface were then counted. *p < 0.05 vs. control and **p <0.05 for the effect of the concentration
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(A)
Control
3 hr
6 hr
1
hr
_____
_____
_____
_____
20 μM
20 μM
20 μM
20 μM
(B)
45
4
3
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5
Control
20
2
5
5 µM
0 µM
*
1
1
5
0
5
0
*
*
1
hr
3 hr
Compound 6
6 hr
Figure 4. Pretreatment of TGAME (6) reduced COM crystal binding onto the cell surface.
MDCKI cells were treated with 0, 25 or 50 μM compound 6 for 1, 3 or 6 h prior to incubation
with COM crystals (100 μg/mL of culture medium) for 30 min. A | After washing with plain
DMEM culture media, images of the remaining crystals were captured under a phase-contrast
microscope for at least 15 high-power fields (HPF) in each well. B | Crystals remaining on the cell
surface were then counted. *p < 0.05 vs. control.
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A 3 h pretreatment time was selected to determine concentration-dependent effects (0-50
M), since effects were measureable and a longer time of 6 h did not increase the magnitude.

TGAME decreased crystal-binding in a concentration-dependent manner across this range with
maximal effects at 50 μM. Thus, further experiments used exposure to 50 μM of TGAME for 3
h. Of note, Gallic acid (one of the compound hydrolytic products) significantly (p <0.05)
decreased the crystal binding at a lower concentration (10 μM) after 3 h versus the control
1
1
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(
Figure 5). This indicates that TGAME can still generate active metabolites, if it undergoes in
vivo metabolism by body esterases.
60
50
40
*
30
20
10
0
1 µM
0 µM
1
Figure 5. Effects of TGAME (6) and its hydrolytic products on COM crystal binding to MDCKI
cells. The cells were treated with 0, 1 or 10 μM of each compound for 3 h prior to incubation with
COM crystals (100 μg/mL of culture medium) for 30 min. After washing with plain DMEM culture
media, images of the remaining crystals were captured under a phase-contrast microscope for 15 high-
power fields (HPF) in each well. Crystals remaining on the cell surface were then counted. *p < 0.05
vs. control.
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Analysis of COM-binding proteins (Annexin A1, α-enolase and HSP90) by Western blot. We
reported previously that when COM crystals were pre-coated with gallotannin (in green tea),
7
their subsequent adhesion to renal tubular cells was reduced. A similar study demonstrated that
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aluminum citrate decreased COM crystal binding to the same cells, when the crystals were pre-
coated with it. 28 Moreover, we recently showed that an aqueous extract of Costus arabicus, a
plant used in Brazilian folk medicine to treat urolithiasis, inhibited the growth of COM crystals
and their adhesion to renal epithelial cells. 29 The inhibitory effect on the binding of crystals to
the cell was only significant when pre-coated COM crystals were investigated. Pretreatment of
cells with this extract had no effect on crystal adhesion. Unlike the previous studies, we found
that TGAME exerted effects when added directly to renal cells followed by treatment with
uncoated COM crystals. We propose that the observed biological activities might be related to
cellular effects of TGAME and reduction of COM crystal-binding protein expression on the
surface of renal cells. One of these proteins is AxA1 , 14 in addition to α-enolase and heat shock
protein 90 (HSP90). 6, 30
A previous publication documented that epigallocatechin gallate strongly interacts with
phospholipid bilayers due to hydrogen bond formation, and it inhibits COM crystal adhesion to
renal tubular cells by decreasing α-enolase surface expression. 11 It is noteworthy that AxA1, α-
enolase, and HSP90 are all in the database of the lipid raft proteome (http://lipid-raft-
database.di.uq.edu.au/). AxA1 is present in the basolateral cell membrane, cytoplasm, cell
projection and nucleus. Thus TGAME could decrease cell surface expression of these proteins
through transcriptional suppression, altered molecular shuttling, or trafficking from the
cytoplasm to the cell surface, since the compound is polyphenolic and could interact with lipid
bilayers through hydrogen bond formation. We studied surface expression of these three putative
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COM crystal receptors on MDCKI cells pretreated with TGAME by immunofluorescence
microscopy and confirmed results by Western blot analysis of whole cell lysate and subcellular
fractions. The analysis revealed that TGAME significantly reduced the presence of AxA1 and
the other two proteins in the membrane fractions (P <0.05), whereas their concentrations in
cytosolic fractions was greater (Figure 6). Overall, the total amount of protein in the whole cell
lysate was the same. Interestingly, the inhibitory effect of TGAME on AxA1 in the membrane
fraction was more significant in comparison with its effect on α-enolase and HSP90 proteins (p
1
1
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<
0.05).
(A)
Whole Cell
Membrane
Cytosolic
5
0 μM
50 μM
Cpd 6
50 μM
Cpd 6
Control
Cpd 6
Control
Control
Annexin A1
(37 kDa)
HSP90
(90 kDa)
Enolase-1
(47 kDa)
β-Actin
(42 kDa)
(B)
0.25
.2
0.4
.35
0.6
*
0
0
0
0
.5
.4
0
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.3
.25
.2
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.15
0.1
.05
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0.3
*
*
.15
0.1
0
.2
.1
0
0
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0.05
0
Control
50μM Cpd 6
Control
50μM Cpd 6
Control 50μM Cpd 6
1.4
1.2
1
1.4
1.2
1
1.2
1
*
0.8
0.6
0.4
0.2
0
*
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
Control 50 µM Cpd 6
Control
50 µM Cpd 6
Control
50 µM Cpd 6
1
1.2
1
*
0.6
.5
0.4
.3
0
.9
0.8
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.7
.6
.5
.4
.3
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0.6
0
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.4
.2
0
0.2
0.1
0
0.2
0
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.1
0
Control
50 μM cpd 6
Control
50 µM cpd 6
Control
50 µM cpd 6
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Figure 6. A | Western blot analysis of COM crystal-binding proteins. Proteins obtained from whole
cell lysate, cytosolic fraction and membrane fraction of MDCKI with or without 50 µM TGAME (6)
treatment, were subjected to Western blotting for annexin A1, α-enolase, HSP90. β-actin served as the
loading control. B | Bands intensity was quantitated using AlphaEase FC software (Mayo Clinic).
Each bar represents mean ± SEM from 3 independent experiments. *p < 0.05 vs. control. **p < 0.05
for the effect of TGAME on Annexin A1 vs. α-enolase and HSP90.
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These data suggested that TGAME decreased AxA1 cell surface expression by inhibiting
2
+
or re-localizing its shuttling from cytoplasm to the cell membrane. Annexins are Ca -regulated
membrane binding proteins found throughout the animal and plant kingdoms. The Annexin core
2+
(
Ca /membrane binding unit) is thought to be one essential element that facilitates membrane
interactions. Thus Annexins can organize and assemble phospholipids into specific membrane
domains. 31 AxA1 protein also plays important roles by controlling cellular Ca storage and thus
2+
2
+
Ca -dependent signal transduction. Overall, AxA1 localizes to many cellular structures
including the plasma, cytoplasm and organelle membranes. 31
Alpha-enolase is another COM crystal binding protein on the surface of MDCKI cells,
and it was reported that expression of this protein on the MDCKI cell surface increased after
exposure to oxalate. 11 Another study confirmed a role of α-enolase in COM crystal binding by
using a specific antibody to block α-enolase and demonstrating decreased adhesion to the cell
surface. 30
HSP90 is also found on the surface of renal cells as a putative crystal binding molecule.
Downregulating HSP90 expression using specific anti-HSP90 antibodies or small interfering
RNA (siRNA) decreased COM crystal adhesion by 50% and 75%, respectively. 13
To further support our hypothesis that the COM crystal adhesion inhibition by TGAME
is mediated in part by surface AxA1, we used a specific anti-AxA1 antibody. As expected, COM
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crystal adhesion to the surface of renal cells was significantly reduced (p <0.05) in comparison
with the control and control isotypic-IgG treated cells (Figure 7).
Blank control
Isotype-controlled IgG
Anti-Annexin A1
(A)
1
1
1
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(B)
2
0
5
1
*
10
5
0
Blank Control
Isotype-controlled IgG
Anti-Annexin A1
Figure 7. Cell-COM crystal adhesion assay and neutralization by a specific anti-Annexin A1. The
confluent polarized MDCKI cell monolayer was incubated with 0.2 μg/mL anti-Annexin A1 antibody
or 0.2 μg/mL rabbit isotype-controlled IgG prior to cell-crystals adhesion assay, whereas the cells
without antibody treatment served as the blank control. A | After removal of unbound crystals, crystals
adherent on the cell surface were imaged by phase-contrast microscopy. B | The adherent crystals were
counted from 15 random high power fields (HPF). Data were expressed as means ± SEM of three
independent experiments. *p < 0.05 vs. controls.
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Examining the expression of surface and intracellular Annexin A1 by immunofluorescence
assay and laser-scanning confocal microscopy. Laser-scanning confocal microscopy
demonstrated that AxA1 cell surface expression was dramatically inhibited by pretreatment with
0
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0 μM of TGAME to approximately 50% of control (Figure 8). However, Annexin A1
expression inside cells increased after TGAME treatment.
(A)
Permeabilized MDCKI cells
Nucleus
(B)
Non-permeabilized MDCKI cells
Annexin A1
Nucleus Merged
Annexin A1
Merged
Z-stack
Sagittal
Z-stack
Sagittal
Z-stack
Sagittal
Z-stack
Sagittal
C) 300
(
250
200
150
100
50
0
Control
Treatment
0
10
20
30
Distance (μm)
Figure 8. Confocal and laser scanning microscopy. TGAME (6) reduced the surface expression of
Annexin A1, but increased its intracellular level. A | Intracellular expression of Annexin A1 (with cell
permeabilization). B | Surface expression of Annexin A1 (without cell permeabilization). MDCKI cells
were pretreated with 50 μM compound 6 for 3 h and processed for immunofluorescence study using
anti-Annexin A1 as a primary antibody and secondary antibody conjugated with Alexfluor 488 Donkey
anti-rabbit (shown in green), whereas nuclei were counterstained with TOT-3 dye (shown in blue).
Original magnification power was ×400. C | The graph peaks represent the fluorescence intensities of
the staining at the given distances (in m) along the red bar.
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In vivo feeding and ex vivo oxalate ± TGAME birefringence experiment in Drosophila
melanogaster Malpighian tubule model. Our group previously studied the effect of thiosulfate
and sulfate on Malpighian tubule (MT) CaOx crystallization in a Drosophila melanogaster (fruit
fly) model. 32 Chen et al. fed fruit flies lithogenic agents such as sodium oxalate (NaOx). Their
analysis used polarized light microscopy of dissected tubules to show that NaOx feeding resulted
in the formation of crystals within the Malpighian tubule lumen in a dose-dependent manner.
These crystals were identified as CaOx using scanning electron microscopy and energy-
dispersive X-ray spectroscopy. The study also demonstrated that crystal formation decreased
lifespan of the flies and that the use of potassium citrate (KCit) as an antiurolithic agent, reduced
crystallization inside MT and decreased mortality. Thus, Drosophila appears to be a useful
model to study CaOx renal stones, before embarking on the use of higher order experimental
animals. 33 Therefore, we investigated the effect of TGAME on the formation of CaOx crystals
within MT in feeding experiment and after ex vivo exposure of the tubules to oxalate.
1
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The results of the ex vivo experiment (Figure 9) showed that TGAME (100 µM)
significantly (p <0.05) decreased CaOx crystallization, size and total crystal area within
Malpighian tubules. Flies fed oxalate (10 µM) + TGAME (50 µM) also manifested a decrease in
CaOx crystal number, size and total crystal area within MT after 24 h exposure in comparison to
those fed oxalate alone, but these findings were not significant (p >0.05) (Figure 10). However,
in the factors such as pharmacokinetic parameters affecting TGAME absorption to reach the
blood stream, or the need for doses greater than 50 µM in vivo to deliver sufficient amounts to
MT in order to alter CaOx crystallization.
Annexin b11 (Axb11) is also found in Drosophila 34 as a Ca -dependent phospholipids
2+
binding protein (http://flybase.org/reports/FBgn0030749.html) that is known to play important
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role in cell adhesion, involved in several functions, among them differentiation, cell signaling
and membrane fusions. In this case, we speculate that the effect of our compound TGAME in the
fly model might be of its action on Axb11, where the decrease in cell surface Axb11 might
subsequently decrease nucleation on the cell surface.
0
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(A)
(B)
_
_______
0 x
________
10 x
1
(
C)
(D)
(
E) 45
6
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50
00
40
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2
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*
150
2
5
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*
100
20
*
15
10
5
50
1
0
0
0
0
Oxalate
Oxalate+3,4,5-tri-O-galloyl quinic
acid methyl ester
Oxalate
Oxalate+3,4,5-tri-O-galloyl quinic
acid methyl ester
Oxalate
Oxalate+3,4,5-tri-O-galloyl quinic
acid methyl ester
Figure 9. Malpighian tubule (MT) images from wild type female fruit flies incubated ex vivo with A |
oxalate (10 mM) or B | oxalate (10 mM) and 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME) (100
μM) for 1 h. The arrows indicate CaOx crystals within the lumen of the MT. C | CaOx crystal number, D |
total crystal area, and E | average area/crystal per 600 μm of tubule were determined using ImageJ. TGAME
significantly decreased crystal number, total crystal area and average area/crystal. Values are means ± SE (n
=
12 for both oxalate control group and compound 6-treated group). *p < 0.05 vs. control.
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0
_
_______
0 x
________
10 x
1
(D)
(C)
600
500
(E) 20
3
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18
16
400
14
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300
10
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00
00
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4
1
2
0
Oxalate
Oxalate+3,4,5-tri-O-galloyl quinic
acid methyl ester
Oxalate
Oxalate+3,4,5-tri-O-galloyl quinic
acid methyl ester
Oxalate
Oxalate+3,4,5-tri-O-galloyl quinic
acid methyl ester
Figure 10. Malpighian tubule (MT) images from wild type female fruit flies fed A | oxalate (10 mM) or
B | oxalate (10 mM) and 3,4,5-tri-O-galloylquinic acid methyl ester (TGAME) (50 μM) for 24 h. The
arrows indicate CaOx crystals within the lumen of the MT. C | CaOx crystal number, D | total crystal
area, and E | average area/crystal per 600 μm of tubule were determined using ImageJ. Values are means
±
SE (n = 11 for both oxalate control group and compound 6-treated group). p >0.05.
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Antioxidant activity and DPPH assay. Studies suggest that renal epithelial cell exposure to
CaOx crystals, CaP crystals, and oxalate ions can lead to the generation of reactive oxygen
species (ROS), potential cause of injury and/or promote inflammation. Further, the urine of stone
formers and hyperoxaluric rats is enriched with markers of inflammation and oxidative stress
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(
OS). 35 Stone formers also have been reported to have reduced peripheral concentrations of
antioxidants including α- and β-carotenes. There are many reported mechanisms whereby ROS
can increase stone risk including decreased function of macromolecular inhibitors and/or cellular
injury that promotes increased crystal adhesion. 36 Antioxidant treatments have also been shown
to reduce pathological injury of crystal deposition in hyperoxaluria animal models.
In this study, the DPPH assay used to gauge OS, revealed that TGAME and its metabolite
Gallic acid can scavenge free radicals to a similar extent as the standard antioxidant vitamin C
with an EC50 11+/-0.3, 3 and 45+/-2 (Figure 11), respectively. Therefore, the compound is
considered to be a potent antioxidant. Methyl quinate did not show any radical scavenging
activity as expected, since it is a sugar with no phenolic properties.
In total, our study supports the hypothesis of Verkoelen and Verhulst that crystal binding
is preceded by pathologic alterations in cell surface binding molecules. Nonetheless, TGAME-
treatment could not entirely prevent COM adhesion to cells, suggesting that these interactions
involve multiple processes and many molecules, perhaps including annexin II, osteopontin and
hyaluronan. 6, 37 Therefore, other unknown cell surface molecules might mediate the inhibitory
effect of TGAME on COM crystal-binding to MDCKI cells.
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Gallic acid
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Compound 6
Vitamin C
Methyl quinate
20
1
0
0
0
2
4
6
8
10
12
Conc (µM)
Figure 11. Antiradical efficiency (AE) of TGAME (6) and its hydrolytic products (Gallic acid and
methyl quinate) in DPPH assay using vitamin C as a standard. Different volumes (2 - 20μl) of the
compounds (2-20 µM in DMSO) were made up to 40μL with DMSO and 1.96mL DPPH (0.1mM)
solution was added. The reaction mixture was incubated in dark condition at room temperature for 20
min. Then, the absorbance of the mixture was read at 517 nm. DPPH solution was taken as control and
the % radical scavenging activity of each compound was calculated.
CONCLUSIONS
We report that TGAME inhibited COM crystal adhesion to renal cells and this effect is
mediated by decreased expression of AxA1 on cell surface. Thus, cell surface expression of
AxA1 might be a key pathogenic factor in crystal retention and urinary stone formation in vivo.
Moreover, the compound significantly decreased CaOx crystal number, size and total crystal area
within MT of Drosophila models, as well as it showed a potential antioxidant activity by free
radicals scavenging capability. Our findings may also be relevant for the observed decrease of
crystal deposition when hyperoxaluric animals treated with C. langsdorffii leaf extract. Finally,
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our findings support a promising role for TGAME in the prevention and modulation of new or
recurrent renal stone formation. Further pre-clinical and clinical studies should be performed for
the use of this compound in urolithiasis.
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EXPERIMENTAL SECTION
Materials and methods
The purities of the tested compound were determined by HPLC analysis and MS error of
precision data as being ≥ 98%. See the Supporting Information for full details. Biological assays
were conducted at Department of Internal Medicine, Division of Nephrology and Hypertension,
Mayo Clinic Medical School, USA.
All methods were performed using anhydrous solvents purchased from commercial
vendors and used without further purification. Quinic and Gallic acids were purchased from
Sigma Adrich, Germany in addition to all other reagents. Hydrogenolysis was performed using a
hydrogen reactor system (FAMABRAS, IND. BRAS. 1/8, 255046, Brazil) supported with
°
pressure controller (CLASSE B ABNT). Melting points ( C, uncorrected) were determined in
25
capillary tubes using Fisatom™ melting point device (model 431, Brazil). Optical rotation [α]D
was done using polarimeter (Jasco P-2000, Japan) at 25°C and a wavelength of 589 nm.
25
Methanol and chloroform were used as solvents. Three readings of [α] were recorded and the
D
average was taken. Chromatographic purification was performed based on flash chromatography
using silica gel (pore size 60 A°, 40-63 µm, Sigma, batch #MKBC6227) and the solvent systems
are expressed as v/v percent ratios as indicated. All reactions were monitored by TLC using
fluorescent pre-coated silica gel plates (Merck, Germany) at short wavelength. Molybdate/H
2
SO
4
spray solution was used as the raveling reagent. The IR spectra were recorded using FT-IR
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-1
spectrophotometer (Perkin Elmer Spectrum Two) in the range of 400-4000 cm by KBr pellet
technique. The analytical HPLC analysis was performed on a Shimadzu LC-10ADvp (Japan)
operated with multisolvent delivery system, equipped with a Shimadzu SPD-MICAvp
photodiode array detector (PDA). Analyses were performed using analytical reversed phase
column (Polar-RP C 80 A°) with dimensions of 150 mm × 4.6 mm (Shimadzu) and a particle
diameter of 4 μm; the mobile phase consisted of acidified water (A) (0.1% formic acid in water)
and methanol (B) in gradient conditions as follows: 15-50% of B (45 min), 50-90% of B (45-65
min) and 90-15% of B up to 75 min; flow rate, 1 mL/min, with injection volume of 20 µL. All
samples were dissolved in HPLC grade methanol (2 mg/mL) 0.45 µm filtered prior to automatic
injection. Mass spectra were acquired on a high resolution MicroTOF II-Q mass Spectrometer
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(
Bruker Daltonics, Billerica, MA, US) fitted with an electrospray ionization (ESI) operating
system in the positive and negative ion modes. Accurate masses were obtained using TFA-Na+
(sodiated trifluoroacetic acid, 10 mg/mL) as the internal standard; end plate: -500 volts;
capillary: 3500 volts; dry gas temperature: 180°C with flow rate 4L/min; nebulizer gas pressure:
1
13
0
.4 bar of N gas; infusion bomb model: Cole Parmer with a flow rate 300μL/ h. H NMR, C
2
NMR, and two-dimensional (2D) spectroscopic techniques were recorded on a Bruker-Avance
DRX500 spectrometer operating at a frequency of 500 MHz. Samples were dissolved in Aldrich
deuterated dimethyl sulfoxide (DMSO-d6) for compounds 1-4, 6 and chloroform (CHCl -d) for
3
1
compound 5. Data for H NMR are reported as follows: chemical shift (δ, ppm), multiplicity (s =
singlet, d = doublet, t = triplet, m = multiplet, coupling constant (s) in Hz, integration). Data for
13C NMR are recorded in terms of chemical shift (δ, ppm).
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Synthetic steps of TGAME
Synthesis of quinic acid methyl ester. Methyl-(1S,3R,4S,5R)-1,3,4,5
tetrahydroxycyclohexanecarboxylate (1)
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To a stirred solution of methanol (160 mL) and sulfuric acid (2 mL), quinic acid (4g) was
°
added. The mixture was refluxed at 79-80 C for 18 h. TLC analysis demonstrated a whole
conversion of quinic acid into a single product spot (R
f
= 0.56; ethyl acetate
/methanol/water/formic acid = 6:3:3:0.2). The reaction mixture then was poured over solid
NaHCO until no effervescence (neutralization was detected by litmus paper), and was filtered.
3
The filtrate was evaporated to give a solid crude hygroscopic residue (8.67 g) that was
recrystallized in methanol to give rosette crystals (4.60 g, 91%) with undefined melting point;
molecular formula C
8
H
14
6
O .
Synthesis of methyl 3,4,5-trihydroxybenzoate (2)
Sulfuric acid (1 mL) was added drop-wise to a stirred solution of Gallic acid in methanol
(
10 g dissolved in 100 mL). The resulting reaction mixture was refluxed for 24 h. The solvent
was evaporated and an off-white precipitate was obtained which was dissolved in ethyl acetate
150 mL). The organic phase was washed with distilled water (3 X 20 mL) and sodium
bicarbonate solution (3 X 20 mL), successively. The organic phase was dried over anhydrous
sodium sulphate, then evaporated to furnish 8 g of a yellowish-white precipitate (R = 6.3; ethyl
(
f
acetate/formic acid = 8:2). The product was purified by recrystallization in methanol and clusters
°
of plate-like crystals were formed (6 g, 60%); mp 200-202 C; molecular formula C
8
H
8
5
O .
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Synthesis of methyl 3,4,5-tribenzyloxybenzoic acid (3)
Compound 2 (5 g (27.15 mmol)), 2 g (12 mmol) of KI and 22 g (159 mmol) of dry
K
2
CO
11 g (87 mmol) of benzyl chloride in 50 mL of acetone was added portion-wise. The suspension
was refluxed for 21 h. TLC screening revealed a single spot of the formed product (R = 0.30;
hexane/ethyl acetate = 8:2). The reaction mixture was filtered, then the filtrate was concentrated
under a vacuum. The resulting residue was re-suspended in 200 mL of CH Cl , then filtered
through Celite and was concentrated under a vacuum. The product was obtained as off-white
solid (12 g, 97%); molecular formula C29
3
were mixed in 250 mL of acetone and stirred for 20 min at room temperature (RT). Next,
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0
f
2
2
H
26
5
O .
Synthesis of 3,4,5-tribenzyloxybenzoic acid (4)
Compound 3 (10 g) was mixed with a solution of 1.77 g (44.25 mmol) sodium hydroxide
in 250 mL of 95% ethanol. The resulting mixture was heated up for 2 h with reflux, followed by
immediate pouring into 260 mL of 0.6 M HCl and stirred for 10 min, and finally filtered under
vacuum. The resulting product was washed with a 1:1 mixture of 95% ethanol and water (50
mL), followed by pure water (50 mL), 95% ethanol (50 mL), methanol (2 X 25 mL), and di-ethyl
ether (25 mL). The yield was dried under vacuum overnight to give a white solid (8.9 g, 94 %);
ο
mp 192-195 C; C28
H
24
5
O .
Synthesis of (1S,3R,4S,5R)-5-hydroxy-5-(methoxycarbonyl)cyclohexane-1,2,3-triyl tris
(3,4,5-tris (benzyloxy) benzoate) (5)
A suspension of 7 g (16 mmol) of 3,4,5-tribenzyloxybenzoic acid, 0.82 g (4 mmol) of
methyl quinate, 4 g (20 mmol) of dicyclohexylcarbodiimide (DCC), and 12.24 g (18.4 mmol) of
N,N-(dimethylamino) pyridine (DMAP) in 500 mL dry dichloromethane, was refluxed for 72 h.
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After cooling to RT, the mixture was filtered, then concentrated using a rotary evaporator. The
resulting crude residue (14.5 g) was purified by flash chromatography (two times, 97 fractions of
20 mL each were collected/per one time) using a mixture of chloroform/methanol (100:1). Both
the reaction and the column fractions were monitored by TLC (dichloromethane/toluene/ethyl
acetate = 7.5:2.5:0.1) and the desired product fractions (f 71 to 81 and f 82 to 91) were
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combined and concentrated to give (1.74 g, 29%) of a faint yellowish-white glassy material (R =
f
2
D
5
0
.2); mp 129-130°C; molecular formula C92
H
80
O
18; [α] : - 49.2 (c 0.15; CHCl
); m/z [M+H]+
3
+
+
1
473.5239, MS [M+Na] 1496.5005, MS [M+K] 1511.4664.
Synthesis of TGAME, (1S,3R,4S,5R)-1-hydroxy-3,4,5-tris ((3,4,5-trihydroxybenzoyl) oxy)
cyclohexanecarboxylic acid (6)
A suspension of 44 mg of palladium (10 wt % on activated carbon) and 200 mg of
compound 5 in 15 mL of dry ethyl acetate was stirred at RT for 6 h inside a cell of a hydrogen
reactor system at RT under a hydrogen gas atmosphere with a pressure of 80 p.s.i, equivalent to 6
2
kgf/cm . Next, the reaction mixture was filtered through Celite filter-aid using a mixture of ethyl
acetate and methanol. The filtrate was concentrated under vacuum and the obtained residue was
lyophilized, recrystallized in ethyl acetate to give a light yellow amorphous solid (93 mg, 99%);
°
mp 137-140 C. UV λmax (methanol): 280 and 254 nm. Molecular formula C29
26
H O18; MS: m/z
+
−
+
25
D
6
63.1190 [M+H] , 661.0878 [M-H] , 685.1012 [M+Na] . [α] : − 42.6 (c 0.50; CH
3
OH). IR
-1
-1
-1
υ
max (KBr): (3387 cm , O−H “H-bonded”), (2953 cm , C−H aliphatic “stretch”), (1707 cm ,
-1
-1
C═O), (1616 cm , C−C aromatics), (1475 and 1338 cm , C−H (─ CH
3
) “bend”), (1216 and
-1
-1
1
1
034 cm , C−O), (764 cm , C−H aromatics “out-of-plane bend”). HNMR (DMSO/TMS): δ
(ppm) 2.30 (m, 2H), 2.40 (m, 2H), 3.43 (s, 1H), 3.49 (s, 3H), 3.75 (s, 9H), 4.90 (m, 1H), 5.40
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(
dd, 1H), 5.60 (m, 1H), 6.89 (s, 2H), 6.92 (s, 2H), 6.94 (s, 2H). C NMR (DMSO/TMS): δ
(
ppm) 29.2, 35.5, 52.4 (-CH ), 63.3, 68.4, 72.8, 109.2, 119.2, 119.8, 128.1, 128.2, 128.4, 128.7,
3
139.0, 139.3, 145.8, 145.9, 165.0, 165.2, 165.6, 173.9.
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1
13
For more details on H, C NMR, 2D NMR, DEPT and FT-IR spectral data of all synthetic
compounds, see Supporting Information.
Cytotoxicity by MTT assay
The cytotoxicity of TGAME, Gallic acid and methyl quinate was evaluated by MTT
colorimetric assay (MTT Kit ThermoFisher Scientific, USA). The cells of MDCKI (ATCC®
CRL-2935™, USA, provided by Dr John Lieske, Mayo Clinic Medical School, MN, USA) were
4
seeded onto 96-well microplates at a density of 1×10 cells per well and treated with different
concentrations (0, 5, 10, 25, 50 µM in DMSO/ phosphate buffered saline (PBS)) of the
compounds for 24 h. The MTT (15 µL) working solution (5 mg/mL in PBS) was added to each
well and incubated at 37°C for 3 h. After the addition of the stop solution (100 µL) the optical
density (OD) was measured at 570 nm using a microplate reader (SpectraMax 190 Microplate
Reader, USA). Cell viability was calculated as a percentage of viable cells in compound-treated
group versus untreated control by the following equation:
Cell viability (%) = [OD (compound) − OD (blank)] / [OD (control) − OD (blank)]×100
Cell-COM crystal binding assay and neutralization by a specific anti-Annexin A1 antibody
Preparation of COM crystals: NaOx (1mM) in Tris buffer saline (50 mL) containing 90 mM
Sodium chloride (pH 7.4) was added to a gently stirred solution of 10 mM Calcium chloride (50
mL). The mixture was incubated at RT overnight and then centrifuged at 3,000 rpm for 5 min.
Crystals were re-suspended in methanol (1 mL) and centrifuged for 5 min at 3,000 rpm. The
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