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Hydrogen sulfide ameliorates hyperhomocysteinemia-associated chronic
renal failure
Utpal Sen, Poulami Basu, Oluwasegun A. Abe, Srikanth Givvimani, Neetu Tyagi, Naira Metreveli,
Karan S. Shah, John C. Passmore, and Suresh C. Tyagi
Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky
Submitted 11 March 2009; accepted in final form 19 May 2009
Sen U, Basu P, Abe OA, Givvimani S, Tyagi N, Metreveli N,
Shah KS, Passmore JC, Tyagi SC. Hydrogen sulfide ameliorates
hyperhomocysteinemia-associated chronic renal failure. Am J Physiol
Renal Physiol 297: F410 –F419, 2009. First published May 27, 2009;
doi:10.1152/ajprenal.00145.2009.—Elevated level of homocysteine
(Hcy), known as hyperhomocysteinemia (HHcy), is associated with
end-stage renal diseases. Hcy metabolizes in the body to produce
hydrogen sulfide (H
2
S), and studies have demonstrated a protective
role of H
2
S in end-stage organ failure. However, the role of H
2
Sin
HHcy-associated renal diseases is unclear. The present study was
aimed to determine the role of H
2
S in HHcy-associated renal damage.
Cystathionine--synthase heterozygous (CBS⫹/⫺) and wild-type
(WT, C57BL/6J) mice with two kidney (2-K) were used in this study
and supplemented with or without NaHS (30 mol/l, H
2
S donor) in
the drinking water. To expedite the HHcy-associated glomerular
damage, uninephrectomized (1-K) CBS(⫹/⫺) and 1-K WT mice were
also used with or without NaHS supplementation. Plasma Hcy levels
were elevated in CBS(⫹/⫺) 2-K and 1-K and WT 1-K mice along
with increased proteinuria, whereas, plasma levels of H
2
S were
attenuated in these groups compared with WT 2-K mice. Interestingly,
H
2
S supplementation increased plasma H
2
S level and normalized the
urinary protein secretion in the similar groups of animals as above.
Increased activity of matrix metalloproteinase (MMP)-2 and -9 and
apoptotic cells were observed in the renal cortical tissues of
CBS(⫹/⫺) 2-K and 1-K and WT 1-K mice; however, H
2
S prevented
apoptotic cell death and normalized increased MMP activities. In-
creased expression of desmin and downregulation of nephrin in the
cortical tissue of CBS(⫹/⫺) 2-K and 1-K and WT 1-K mice were
ameliorated with H
2
S supplementation. Additionally, in the kidney
tissues of CBS(⫹/⫺) 2-K and 1-K and WT 1-K mice, increased
superoxide (O
2•⫺
) production and reduced glutathione (GSH)-to-
oxidized glutathione (GSSG) ratio were normalized with exogenous
H
2
S supplementation. These results demonstrate that HHcy-associ-
ated renal damage is related to decreased endogenous H
2
S generation
in the body. Additionally, here we demonstrate with evidence that
H
2
S supplementation prevents HHcy-associated renal damage, in part,
through its antioxidant properties.
cystathionine-synthase; cystathionine-␥lyase; homocysteine; ma-
trix metalloproteinase
HYDROGEN SULFIDE (H
2
S) is known as a toxic gas with a very
strong repulsive odor. Despite its toxicity, many prokaryotic
and eukaryotic organisms thrive in sulfidic habitats (11). Re-
cently, the presence of tissue H
2
S from sulfidic organisms to
the animals who live in a sulfur-free environment has been
confirmed by several independent investigations (10, 49).
These findings unambiguously suggest that H
2
S is a constituent
of cellular milieu. However, to date, very little is known about
the physiological role of H
2
S in normal animal and during
pathological stages of renal diseases.
Endogenously, H
2
S is generated in the mammalian tissue
from a nonprotein amino acid, homocysteine (Hcy). This is a
sulfur-containing amino acid, which is an intermediate product
of methionine metabolism. In the body, two enzymes, cysta-
thionine -synthase (CBS) and cystathionine ␥-lyase (CSE), of
the transulfuration pathway of methionine metabolism catalyze
conversion of H
2
S from Hcy. Studies from independent labo-
ratories reported that, at low level, H
2
S defends organs from
several pathophysiological conditions, such as oxidative stress,
ischemia-reperfusion, and hypertension (20, 52, 59). Recently,
Yan et al. (51) postulated that, being a reducing molecule, H
2
S
may modulate redox tone and redox cell signaling. These
authors, however, reported that at high levels H
2
S induces
reactive oxygen species (ROS) and reactive nitrogen species
(RNS) formation but at low levels decreases hydrogen perox-
ide (H
2
O
2
), peroxinitrite (ONOO
•⫺
), and superoxide anion
(O
2•⫺
) generation induced by Hcy in a cell culture model (51).
These antioxidant properties were partly mediated by increas-
ing the antioxidant properties of cellular antioxidant molecules.
Nevertheless, the endogenous status of H
2
S and its role in
hyperhomocysteinemia (HHcy)-associated renal failure remain
unknown.
Previously, we have reported that reduction in renal function
leads to an increased plasma Hcy level (36, 38). The elevated
plasma Hcy, in turn, causes renal insufficiency, which leads to
a vicious cycle (22). Accumulated evidences from independent
laboratories, including our own, suggested that, at an elevated
level, Hcy is an independent and graded risk factor for car-
diorenovascular diseases (7, 27, 38). Reports are also available
that it may contribute to the pathogenesis of atherosclerosis (2,
26), including glomerulosclerosis, cellular apoptosis, podocyte
injury, and proteinuria (22, 56). These pathophysiological
effects of Hcy are mediated through generation of ROS,
including O
2•⫺
and H
2
O
2
, and reduction in endothelial nitric
oxide (NO) bioavailability (41, 60).
It is well known that ROS plays a major role in the extra-
cellular matrix (ECM) remodeling during various pathophysi-
ological conditions. One of the early events of the ECM
remodeling process is the activation of matrix metalloprotein-
ases (MMPs) (8, 39). Among MMPs, elastinases and collag-
enases are particularly important of matrix degradation in
fibrotic occlusive diseases including renal fibrosis (5, 21). We
have previously reported that MMP-2 and MMP-9 play an
important role in matrix accumulation associated with diabetic
nephropathy and HHcy (39). However, the role of H
2
S, if any,
in the regulation of these two MMPs in HHcy-associated renal
diseases was not defined. Also, the physiological status of
glomerular podocytes, injured podocyte marker, desmin, as
Address for reprint requests and other correspondence: Utpal Sen, Dept. of
Physiology & Biophysics, Univ. of Louisville School of Medicine, 500 S.
Preston St., Louisville, KY 40202 (e-mail: u0sen001@louisville.edu).
Am J Physiol Renal Physiol 297: F410–F419, 2009.
First published May 27, 2009; doi:10.1152/ajprenal.00145.2009.
0363-6127/09 $8.00 Copyright ©2009 the American Physiological Society http://www.ajprenal.orgF410
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well as normal slit diaphragm component nephrin during HHcy
is not clear. Therefore, the present study aimed to determine
whether HHcy was associated with decreased plasma H
2
S level
and increased oxidative stress during chronic renal failure by
modulating MMP-2 and -9 activities, glomerular cell apopto-
sis, and increased urinary protein secretion. Additionally, the
possible preventive role of H
2
S, if any, has been explored to
these deleterious effects in the failing kidney.
MATERIALS AND METHODS
Animals. Wild-type (WT, C57BL/6J) male mice aged 8 wk were
obtained from Jackson Laboratories (Bar Harbor, ME) and housed in
the animal care facility at University of Louisville. Mice were
acclimatized for 2 wk before the start of experiments. WT (C57BL/
6J) and heterozygous CBS(⫹/⫺), a model for hyperhomocysteine-
mic mice, were used for this study. Mice were divided into two
sets, the first set of mice had two kidneys (2-K), and the groups were
as follows: 1) WT, 2) CBS(⫹/⫺), 3)WT⫹NaHS (30 mol/l, H
2
S
donor), and 4) CBS(⫹/⫺)⫹NaHS (30 mol/l). To speed up the renal
damaging effects, the second set of mice was uninephrectomized
(1-K) with all above four groups. NaHS were supplied for 8 wk to the
appropriate groups. At the end of the experiments, mice were deeply
anesthetized, blood was collected, and the animals were killed to
harvest the tissues. All animal procedures were in accordance with the
National Institute of Health Guidelines for animal research and were
approved by the Institutional Animal Care and Use Committee of the
University Of Louisville School Of Medicine.
Rationale for NaHS (H
2
S donor) dose. The physiological concen-
tration of H
2
S ranges from 10 –100 mol/l (59). NaHS in the aqueous
phase produces exactly equal concentration of H
2
S gas in the solution.
Therefore, we used 30 mol/l NaHS in the drinking water to supple-
ment animals with 30 mol/l of H
2
S.
Antibodies and reagents. Rabbit polyclonal antibodies to desmin
and nephrin were purchased from Abcam (Cambridge, MA). Anti--
actin antibody, NaHS, and other analytical reagents were from Sigma-
Aldrich (St. Louis, MO). Horseradish peroxidase-linked anti-rabbit
IgG antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).
PVDF membrane was from Bio-Rad (Hercules, CA).
Measurement of plasma Hcy. The high-performance liquid chro-
matography (HPLC) apparatus, chromatographic conditioning, and
sample preparation for plasma Hcy measurement were adopted from
a previously reported procedure (25) with modifications as described
earlier (39).
Measurement of plasma H
2
S. To measure the H
2
S concentration in
the plasma of each of the experimental groups, 100 l of aliquots were
mixed with 50 l of water in microcentrifuge tubes containing 300 l
of zinc acetate (1% wt/vol) to trap H
2
S. The reaction was stopped after
5 min by adding 200 lofN,N-dimethyl-p-phenylenediamine sulfate
(20 mM in 7.2 M HCl), immediately followed by addition of 200 l
of FeCl
3
(30 mM in 1.2 M HCl). The mixture was kept in the dark for
20 min. To precipitate protein from the plasma, 150 l of trichloro-
acetic acid (10% wt/vol) was added. The mixture was then centrifuged
at 10,000 gfor 10 min, and the absorbance of the resulting supernatant
was determined at 670 nm (53) in a 96-well plate using a spectro-
photometer (Spectramax M2; Molecular Devices, Sunnyvale, CA).
All samples were assayed in duplicate, and H
2
S concentration in the
plasma was calculated against a calibration curve of NaHS (3.125–
100 M).
Urinary protein measurement. Mice were housed in metabolic
cages to collect 24-h urine samples for quantitative determination of
protein content in the urine. Quantitative urinary protein concentration
was determined using the Bio-Rad protein assay reagent on the basis
of the Bradford dye-binding procedure (4). Protein concentrations
were calculated using the calibration curve prepared from a standard
solution (0 –2 mg/ml) of BSA (Sigma Chemical).
In vitro MMP-2, -9 activities assay. Gelatin zymography was
performed using 1.5% in gel gelatin as described elsewhere (28). In
brief, glomerular tissues were minced into small pieces in ice-cold
extraction buffer (1:3 wt/vol) containing (in mmol/l) 10 cacodylic
acid, 20 ZnCl, 1.5 NaN
3
, and 0.01% Triton X-100 (pH 5.0) and
incubated overnight at 4°C with gentle shaking. The homogenate was
then centrifuged for 10 min at 800 g, and supernatant was collected.
Protein concentration in the sample was measured using Bradford
method, and 100 g of the protein was electrophoretically resolved
for each sample in 8% SDS-PAGE containing 1.5% gelatin as MMP
substrate. Gels were washed in 2.5% Triton X-100 for 30 min to
remove SDS, rinsed in water, and incubated for at least 24 h in
activation buffer (50 mmol/l Tris䡠HCl, 5 mmol/l CaCl
2
, and 0.02%
NaN
3
, pH 7.5) at 37°C in a water bath with gentle shaking. Gels were
then transferred to staining solution (acetic acid:methanol:water, 10:
50:40) containing 0.5% Coomassie blue for1hatroom temperature.
MMP activity in the gel was detected in a dark blue background with
white bands.
Cryosectioning. The kidneys were excised, and appropriate por-
tions of the kidney were cryopreserved in Peel-A-Way disposable
plastic tissue embedding molds (Polysciences, Warrington, PA) con-
taining tissue freezing media (Triangle Biomedical Sciences, Durham,
NC). These molds were kept frozen (⫺70°C) until serial 5-m tissue
sections were made in Cryocut 1800 (Reichert-Jung). Cryosections
were placed on Superfrost/plus microscope slides and air dried.
TUNEL staining. Apoptotic cells were detected and quantified
using an in situ Apoptosis Detection Kit [TACS Terminal deoxynucleo-
tidyl Transferase (TdT) kit; R&D Systems, Minneapolis, MN] following
manufacturer instructions. Briefly, 5-m kidney tissue cryosections were
fixed with 3.7% formaldehyde solution and the permeabilized with
proteinase K. Endogenous peroxidase was quenched using hydrogen
peroxide (H
2
O
2
). Next, biotinylated nucleotides were incorporated into
3⬘-OH ends of the DNA fragments by TdT. Positive controls were
generated by treating samples with TACS-Nuclease before TdT labeling.
The biotinylated nucleotides were detected using streptavidin-horseradish
peroxidase conjugate followed by the substrate, TACS Blue Label.
Apoptotic cells were detected by those that exhibit blue nuclear staining.
Western blots. The kidney tissue homogenates were prepared using
protein extraction buffer (0.01 M cacodylic acid pH 5.0, 0.15 M NaCl,
1M ZnCl
2
, 0.02 M CaCl
2
, 0.0015 M NaN
3
, and 0.01% vol/vol
Triton X-100). The extracted proteins were collected, and pH rose to
7.5 by adding 0.1 M Tris. Equal amounts of protein were analyzed on
10% SDS-PAGE, transferred to PVDF membrane, and probed with
appropriate antibodies following the earlier adopted method (39).
Detection of ROS. The method to detect ROS, specifically super-
oxide, was adopted from Dayal et al. (9). The oxidative fluorescent
dye, dihydroethidium (DHE; Invitrogen, Carlsbad, CA), was used in
frozen kidney sections, and the intensity of the fluorescent was
measured by laser scanning confocal microscopy (Fluo View 1000,
Olympus). Control sections were preincubated for 30 min with 250
U/ml polyethylene glycol-superoxide dismutase (PEG-SOD; Sigma-
Aldrich) before incubation with DHE. Fluorescent images were ana-
lyzed with ImagePro software (Media Cybernetics, Bethesda, MD).
Glutathione assay. Kidney tissue levels of reduced glutathione
(GSH) and oxidized glutathione (GSSG) were measured using a
commercially available kit (Cayman Chemicals, Ann Arbor, MI). The
GSH-to-GSSG ratio was calculated for each sample according to
manufacturer’s instructions.
Statistical analysis. Values are given as means ⫾SE from n
number of animals in each group as mentioned in each of the figure
legends. Differences between groups were tested with the use of
two-way ANOVA for repeated measures. Comparisons between
groups were made with the use of Student’s independent t-test.
Significance was accepted at P⬍0.05.
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RESULTS
Total plasma Hcy significantly increased in CBS(
⫹
/
⫺
)
mice. HPLC was performed to measure plasma levels of Hcy
from the experimental and control samples. Hcy from the
samples were identified according to the retention times and
cochromatography with standards. In CBS(⫹/⫺) 2-K mice, the
plasma Hcy level was found to be significantly higher com-
pared with WT 2-K mice (Fig. 1). This increase of Hcy level
in 8-wk-postsurgery mice was even higher and more dramatic
in CBS(⫹/⫺) 1-K mice compared with age-matched WT 1-K
mice. Although there was a tendency of higher plasma Hcy
level in WT 1-K mice compared with WT 2-K mice, the
difference was not significant. Additionally, there were no
further changes of plasma Hcy levels in the similar groups of
mice supplemented with NaHS (Fig. 1). These results clearly
suggest that, although 2-K CBS(⫹/⫺) mice develop high Hcy,
this effect was more acute in CBS(⫹/⫺) 1-K mice, and H
2
S
supplementation does not have a role in the plasma Hcy level.
NaHS supplementation increased total plasma H
2
S level.
Plasma total H
2
S levels were decreased ⬃12% in WT 1-K
mice compared with WT 2-K mice, whereas there was virtually
no difference between WT 2-K mice supplemented with or
without NaHS (Fig. 2). However, a significant increase in
plasma H
2
S was observed in WT 1-K mice supplemented with
NaHS, which was comparable to WT 2-K mice. H
2
S level in
CBS(⫹/⫺) 2-K mice was found significantly lower than in WT
2-K littermates. This difference was even higher in CBS(⫹/⫺)
1-K mice. Interestingly, NaHS treatment in CBS(⫹/⫺) 2-K
mice showed a significant increase (⬃32%, compared with its
2-K littermates without NaHS supplementation) in plasma H
2
S
level, which was comparable to WT 2-K mice. Similar results
were observed (⬃29% increase of plasma H
2
S level, compared
with its 1-K littermates without NaHS supplementation) in
CBS(⫹/⫺) 1-K mice treated with NaHS (Fig. 2). These results
suggest that HHcy is associated with decreased plasma H
2
S
level and that exogenous supplementation of NaHS increases
plasma total H
2
S level.
Hydrogen sulfide prevented proteinuria in 1-K mice. Urinary
protein concentration was higher in CBS(⫹/⫺) 2-K mice
compared with WT 2-K mice (Fig. 3). In these animals,
however, NaHS did not affect total urinary protein excretion.
Compared with WT 2-K mice, WT 1-K mice showed signifi-
cant hyperproteinuria. This effect was even more acute in
CBS(⫹/⫺) 1-K mice. Interestingly, in response to NaHS, the
increased proteinuria in both the WT 1-K and CBS 1-K mice
was normalized. This result suggests that increased protein-
uria was attributable to HHcy-associated glomerular dam-
age, and this damage can be partially prevented by H
2
S
supplementation.
MMP activities attenuated by H
2
S. MMPs are involved in
the remodeling process in the normal and diseased vascular
beds. Hence, we examined the MMP-2 and -9 activities in the
kidney because these two MMPs are critically involved in the
glomerular matrix remodeling process. As shown in the Fig. 4,
Fig. 1. Effect of uninephrectomy (1-K) and H
2
S supplementation on plasma
homocysteine (Hcy) level. Total homocysteine was extracted from plasma and
analyzed by high-performance liquid chromatography as described in MATE-
RIALS AND METHODS using our previously adopted procedure. Data represent
the means ⫾SE, n⫽4; NaHS, 30 mol/l. *Significant difference (P⬍0.05)
compared with respective wild-type (WT) two kidney mice (2-K). **Signifi-
cant difference (P⬍0.05) compared with respective WT 1-K. CBS, cystathi-
onine--synthase.
Fig. 2. Effect of NaHS supplementation on plasma H
2
S level. Reduced H
2
S
production was observed in CBS(⫹/⫺) 2-K, as well as CBS(⫹/⫺) 1-K and
WT 1-K mice. Interestingly, NaHS supplementation increased total plasma
H
2
S level in these mice. *Significant differences (P⬍0.05) compared with
WT 2-K mice. **P⬍0.05 vs. CBS(⫹/⫺) 2-K mice; ⌽⌽P⬍0.05 vs.
CBS(⫹/⫺) 1-K. Data represent the means ⫾SE, n⫽6 animals in each group.
Fig. 3. Role of H
2
S in homocysteine-associated proteinuria. Urinary protein
was measured using Bradford method. *Significant difference (P⬍0.05)
compared with WT 2-K. **P⬍0.05 vs. WT 1-K mice; ⌽P⬍0.05 vs.
CBS(⫹/⫺) 2-K; ⌽⌽P⬍0.05 vs. CBS(⫹/⫺) 1-K. Data represent the means ⫾
SE, n⫽8 animals per group.
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WT 2-K mice showed basal level of the proform and active
form of MMP-9 activity as well as active form of MMP-2
activity in the cortical tissue extract. The activities of these
MMPs were elevated in WT 1-K mice. Contrary to WT 2-K
mice, CBS(⫹/⫺) 2-K mice showed a very high level of these
MMP activities, which were further elevated in CBS(⫹/⫺)
1-K mice (Fig. 4). Interestingly, H
2
S supplementation almost
normalized these MMP activities in WT 1-K and both in
CBS(⫹/⫺) 2-K and 1-K mice. These results suggest that both
the MMP-2 and -9 play a major role in high Hcy-associated
renopathy. Importantly, modulation of these two MMPs by
H
2
S suggests a possible trigger of both these of MMPs during
HHcy in the renal cortex.
H
2
S prevented HHcy-associated glomerular cell death. We
determined the glomerular cell apoptosis by TUNEL staining.
Representative images of TUNEL staining showed that, in the
glomerulus of WT 2-K kidney, there were very few apoptotic
podocytic cells (Fig. 5). Similarly, WT 2-K mice treated with
NaHS showed very few dead cells. WT 1-K mice showed a
relatively high number of apoptotic cells (Fig. 5). These in-
creases in apoptotic cells were absent when WT 1-K mice were
supplemented with NaHS.
In the CBS(⫹/⫺) 2-K mice, however, although a very
minimal number of apoptotic cells was identified, the number
of apoptotic cells was higher than in WT 2-K littermates (Fig.
5). This number was dramatically increased in CBS(⫹/⫺) 1-K
mice, which was almost completely prevented in the mice
treated with NaHS. Likewise, in the kidney of CBS(⫹/⫺) 2-K
mice treated with NaHS, a minimal number of apoptotic cells
was observed.
Expression of desmin and nephrin during HHcy. A basal
level of desmin expression was observed in CBS(⫹/⫺) 2-K
mice. This expression was almost comparable with WT 2-K
mice; however, the expression was increased significantly in
both WT 1-K and CBS(⫹/⫺) 1-K mice although the expres-
sion was higher in CBS(⫹/⫺) 1-K mice (Fig. 6). WT 1-K and
CBS(⫹/⫺)1-K mice supplemented with NaHS prevented the
increased expression of desmin, and the levels were compara-
ble to their respective 2-K littermates. Contrary to the desmin
expression, the expression of nephrin in the kidney was found
to be opposite in the respective experimental groups. NaHS
treatment, however, prevented the changes of nephrin expres-
sions in WT 1-K and both the CBS(⫹/⫺) 2-K and CBS(⫹/
⫺)1-K mice (Fig. 6).
Increased ROS production was associated with HHcy. Pro-
duction of ROS in the glomerulus was measured using oxida-
tive fluorescent dye, DHE (Fig. 7). The DHE fluorescence
indicates ROS production. When CBS(⫹/⫺) mice were sup-
plemented with exogenous H
2
S, a significant decrease in DHE
fluorescence was observed. The fluorescence intensity in
CBS(⫹/⫺) 2-K mice was higher compared with WT 2-K
age-matched littermates. Interestingly, in CBS(⫹/⫺) 1-K
mice, a robust increase of DHE fluorescence was observed.
PEG, which is covalently linked to SOD (PEG-SOD) and a
potent superoxide (O
2•⫺
) anion scavenger, dramatically dimin-
ished DHE fluorescence, suggesting that the increased DHE
fluorescence observed in CBS(⫹/⫺)1-K was attributable to
increased production of O
2•⫺
.
H
2
S increased cortical tissue GSH-to-GSSG ratio. GSH is a
potent intracellular antioxidant, and GSH-to-GSSG ratio cor-
responds to the capacity of a cell to attenuate oxidative stress
(13). To assess the oxidative redox state in our experimental
groups, we measured the cortical-tissue reduced GSH and
GSH-to-GSSG ratio, and the calculated data are shown in
Fig. 8. There was a significant decrease of GSH level in WT
1-K and both in CBS(⫹/⫺) 2-K and CBS(⫹/⫺)1-K mice
compared with WT 2-K control groups. Also, a significant
decrease was observed in GSH/GSSG ratio in these groups.
Interestingly, H
2
S supplementation normalized the GSH and
GSH/GSSG ratio, suggesting that H
2
S maintained the intracel-
lular antioxidant capacity.
Fig. 4. H
2
S regulates matrix metalloproteinase (MMP)-2
and -9 activities during hyperhomocysteinemia. In gel, gelatin
zymography was performed to measure MMP-2 and -9 activi-
ties in the kidney cortex tissue-extracted protein. Both proforms
and active forms of MMP-9 and active form of MMP-2 in WT
1-K, CBS(⫹/⫺) 2-K, and 1-K mice were found to be signifi-
cantly higher than WT 2-K littermates. These MMP activities
were attenuated by H
2
S supplementation (NaHS, 30 mol/l) for
the period of 8 wk postsurgery; data represent means ⫾SE, n⫽
5 for WT mice per group, and n⫽6 for CBS(⫹/⫺) mice per
group; *P⬍0.01 vs. WT 2-K.
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DISCUSSION
The present study clearly demonstrates that plasma Hcy was
increased in CBS(⫹/⫺) mice, and this increase was further
elevated when renal function was reduced by the removal of
one kidney. The plasma Hcy was also correlated with the
extent of renal damage as detected by proteinuria. Plasma H
2
S
levels were found to be the opposite to that of the Hcy levels,
and this decrease in H
2
S level was elevated back toward
normal with exogenous supplementation of NaHS, a donor of
H
2
S. The increased MMP-2 and -9 activities and the occur-
Fig. 5. Effect of H
2
S on glomerular cell death. Histological kidney sections were analyzed for in situ apoptosis as described in MATERIALS AND METHODS. Numbers
of dead cells were counted under the microscope from 15 randomized fields in each group, quantitated, and plotted as bar diagram as shown; n⫽7 in each group;
⫻200 magnification.
2K WT
2K WT
Fig. 6. H
2
S normalized tissue expression of desmin and
nephrin. Protein was extracted from the kidney tissue and
was analyzed by Western blot. Equal amount of protein
was loaded in each well, and the expression of each of
the proteins was normalized with -actin. *Significant
difference (P⬍0.05) compared with WT 2-K. **P⬍
0.05 vs. WT 1-K mice; ⌽P⬍0.05 vs. CBS(⫹/⫺) 2-K;
⌽⌽P⬍0.05 vs. CBS(⫹/⫺) 1-K. Data represent the
means ⫾SE, n⫽6 per group.
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rence of glomerular cell death were associated with the in-
creased oxidative stress, which has been shown to be associ-
ated with HHcy. In addition to that, the increased proteinuria
was corroborated with the renal damage, as evidenced by
increased expression of podocyte injury marker, desmin, and
decreased expression of glomerular slit diaphragm protein,
nephrin. Interestingly, the extent of HHcy-associated renal
damage was ameliorated by H
2
S supplementation. Although it
is not completely understood, it appears that the defense
mechanism of H
2
S was exhibited, in part, through reducing
oxidative stress and maintaining intracellular GSH-to-GSSG
ratio. This mechanism, in addition to normalizing the expres-
sion of desmin and nephrin, prevented glomerular cell apopto-
sis and restored basal MMP activities. These resulted in rever-
sals of proteinuria in our hyperhomocysteinemic experimental
animal model treated with H
2
S.
HHcy is a risk factor for chronic kidney disease and is
associated with end-stage renal disease. In disease condition,
such as in diabetic nephropathy, Hcy disposal and clearance
are impaired and therefore accumulate in the body, resulting in
increase of plasma and tissue levels of Hcy (43). This, in turn,
causes renal microvascular impairment and vasoconstriction
(37), which lead to renal volume retention and further accu-
mulation of Hcy (38). This is a vicious cycle and is often
associated with chronic renal failure. The purpose of unine-
phrectomy (1-K), in the present study, was to impair the renal
function to subnormal level, which further accumulates Hcy
through increased renal volume retention. The results from our
study suggest that 1-K mice have more Hcy in the plasma than
their respective 2-K control groups. Therefore, this model was
created and used to investigate Hcy-associated renal damage
and remodeling process in the kidney.
In the body, Hcy is metabolized by two enzymes, CBS and
CSE, and produces a gaseous substance, H
2
S (16, 42). CBS is
a predominant H
2
S-generating enzyme in the brain and ner-
vous tissues (1, 12), and CSE is mainly expressed in the liver,
kidney, and vascular smooth muscle cells (3, 61). Although
Hcy has been shown to promote CSE activity at its lower
Fig. 7. H
2
S mitigated reactive oxygen species (ROS) production. Production of ROS in the frozen kidney sections was measured using oxidative fluorescent dye,
dihydroethidium (DHE), as described in MATERIALS AND METHODS. DHE fluorescence was detected at a higher level in the glomerulus of CBS(⫹/⫺) 1-K mice.
Interestingly, CBS(⫹/⫺) 1-K mice supplemented with exogenous H
2
S showed a significant decrease in DHE fluorescence. In addition, preincubation of
CBS(⫹/⫺) 1-K kidney sections with polyethylene glycol-superoxide dismutase (PEG-SOD) dramatically diminished DHE fluorescence, suggesting that the
major portions of ROS are superoxide (O
2•⫺
). *Significant difference (P⬍0.05) vs. CBS(⫹/⫺) 1-K (means ⫾SE, n⫽5– 6 animals in each group).
Magnification, ⫻200.
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concentrations, inhibition of CSE activity was reported at its
higher concentration in the rat liver (54). At pathological
conditions, elevated levels of Hcy alter the transulfuration
pathway by inhibiting CSE enzyme activity (6), thereby reduc-
ing endogenous production of H
2
S in the body. In the present
study, we investigated the effect of an increased level of
plasma Hcy, not the glomerular tissue level of Hcy, on the
glomerulus and its consequences in the renal remodeling pro-
cess. CBS(⫹/⫺) mice show higher levels of plasma Hcy and a
very reliable model to test HHcy-related disease process.
Additionally, it is also reported that HHcy inhibits CSE (6);
therefore, the use of CBS(⫹/⫺) mice is advantageous over
CSE(⫹/⫺). Hence, we used CBS(⫹/⫺) mice for the present
study to investigate the HHcy-related renal-damaging effect.
The growing body of evidence suggests that, despite its past
reputation as a noxious gas, H
2
S is rapidly emerging as a third
gaseous transmitter, in addition to nitric oxide and carbon
monoxide (16, 17, 46); the physiological function of endoge-
nous H
2
S, however, is not clear. It is reported that H
2
Sis
involved in the regulation of vascular tone (24) and hyperten-
sion (52) and protects neuronal cells from oxidative stress by
increasing the intracellular concentration of antioxidant and
GSH (19, 48). Recently, Tripatara et al. (44) demonstrated that
generation of endogenous H
2
S limits renal ischemia/reperfu-
sion injury and dysfunction. In the present study, to test any
protective effects of H
2
S in HHcy-associated renal damage, we
used CBS(⫹/⫺) mutant mice. These mice have an ⬃50%
reduction in CBS mRNA and enzyme activity in the liver and
have twice the normal plasma Hcy levels than WT littermates
(35). Thus the CBS(⫹/⫺) mice develop mild HHcy and are a
very good model to study HHcy-related disease processes,
including cardiovascular-renal diseases. Reports are available
that CBS inhibitors hydroxylamine and amino-oxyacetate sup-
press the production of H
2
S and that a CBS activator, S-
adenosyl-L-methionine, enhances H
2
S production in the brain
tissue (1). A similar trend was observed by Xia et al. (50)
where inhibition of CBS reduced endogenous generation of
H
2
S in the kidney tissues. These reports indicated the regula-
tory role of CBS enzyme in the production of endogenous H
2
S.
Our present result (Fig. 1) suggested that HHcy was associated
with CBS deficiency, where CBS(⫹/⫺) mutant mice exhibited
a higher level of plasma Hcy. This increase of plasma Hcy
level was even higher in CBS(⫹/⫺) mice after uninephrec-
tomy (1-K). Thus the results clearly indicated a strong rela-
tionship between renal insufficiency and plasma Hcy level.
Because Hcy is one of the precursors of endogenous H
2
S
generation, it was expected that increase of plasma Hcy level
would eventually be elevated in the plasma H
2
S level. Contrary
to this mechanism, the data presented in Fig. 2 showed that the
plasma H
2
S level was, in fact, inversely regulated by plasma
Hcy level, where increased Hcy repressed H
2
S level in the
plasma. This may be due to a negative feedback mechanism,
where increased plasma Hcy inhibited its metabolizing en-
zyme, CSE and/or CBS, resulting in low production of H
2
S.
We previously reported that stress, such as volume overload,
decreased CSE expression and subsequent H
2
S generation in
the cardiac tissue (40). Whether this mechanism is still appli-
cable to HHcy-associated oxidative stress and regulates the
plasma H
2
S level needs to be verified. Nevertheless, the results
from our present study indicated the possibility of such mech-
anisms that may be involved in part, if not the only mechanism,
which triggered endogenous generation of H
2
S and subsequent
plasma level of this gaseous substance.
Along the same line, Wei et al. (48) showed that severe
oxidative stress was present in a model of hypoxic pulmonary
hypertension and was accompanied by a decrease in the en-
dogenous production of H
2
S in the lung tissue. H
2
S, however,
acted as an antioxidant during this oxidative stress, which was
a result of the attenuated GSSG content. More recently, per-
fusion of H
2
S in ischemia-reperfusion-injured lung has been
shown to reduce malondialdehyde production, potentiated
SOD, and catalase (CAT) activities and restrain superoxide
(O
2•⫺
) production in the lung, resulting in an attenuated oxi-
dative lung injury (15). This emerging evidence suggests the
potential antioxidant properties of H
2
S in normal and patho-
physiological conditions. To determine the antioxidant role of
H
2
S, we measured the reduced GSH content and reduced
GSH-to-GSSG ratio in our study. Our results suggested that
H
2
S supplementation normalized the reduced GSH and re-
duced-GSH-to-GSSG ratio associated with HHcy. In our ex-
periments, although we have found that H
2
S supplementation
increased GSH/GSSG ratio in the kidney tissues, the exact
mechanisms, however, were not elucidated. Recently, Liu et al.
(23) reported that H
2
S protected intestinal ischemia-reperfu-
sion injury by increasing serum and intestinal level of SOD and
GSH peroxidase (23). Our laboratory has previously shown
that, in vitro, H
2
S enhanced the inhibitory effects of CAT and
SOD in methionine-loaded oxidative stress in mouse brain
endothelial cells (45). This mechanism has clearly indicated
Fig. 8. Decreased glutathione (GSH)/oxidized glutathione (GSSG) ratio was
normalized by H
2
S. Cortical tissue reduced GSH, and GSH-to-GSSG ratio was
measured and calculated as described in MATERIALS AND METHODS.H
2
S therapy
significantly improved GSH level in WT 1-K and in CBS(⫹/⫺)2-K and
CBS(⫹/⫺)1-K mice. Additionally, H
2
S preserved the GSH/GSSG ratio com-
pared with respective nontreated groups. Data represent means ⫾SE, n⫽7
per group. *P⬍0.05 vs. 2-K WT.
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the antioxidant property of H
2
S and was partly mediated by
increasing intracellular CAT and SOD. The present study did
not aim to elucidate this mechanism; however, whether or not
the same mechanism applies to HHcy-associated renal remod-
eling needs to be explored comprehensively. Interestingly, in
the present study, increased superoxide (O
2•⫺
) production was
attenuated with H
2
S supplementation in the mice that exhibited
high Hcy. These results suggest that H
2
S protects glomerular
tissue, at least in part, through its antioxidant properties.
H
2
S had been reported to be a general protective mechanism
for degenerative organ damages. Herein, we have focused our
study to ameliorate HHcy-associated kidney damages, if any,
through exogenous H
2
S supplementation. It is a need of future
investigation to elucidate whether H
2
S could be such a protec-
tive factor in the hypertensive kidney damages. For example,
does H
2
S protect DOCA salt-induced kidney damage? It is
reported that DOCA salt-induced hypertension decreased SOD
in the rat aorta and that antioxidant therapy increased SOD
activity (31). Given the fact that both DOCA salt and Hcy
induce oxidative stress through inhibition of SOD, and H
2
Sis
an enhancer of intracellular SOD, it is possible that H
2
S may
also play a role to increase SOD activity in the kidney of
DOCA salt-induced hypertensive rat. Although this is a very
interesting area to study antioxidant properties of H
2
S, the
scope of the present investigation, however, was limited to
investigate the protective role of H
2
S in HHcy-associated renal
damages.
Clinical studies have implicated proteinuria as a key prog-
nostic factor for renal complications in hypertension; however,
the pathogenesis causing proteinuria is poorly understood (30).
It is reported that dysfunction of podocytes, the final filtration
barrier in the glomerulus, plays a pivotal role in proteinuria
(29, 32, 47). Yi et al. (55) have shown that urinary albumin
excretion increased at the second week of methionine, the
precursor of Hcy, treatment. These reports established a strong
correlation between high Hcy and kidney disease. In our
present study, we have found that proteinuria was strongly
correlated with high Hcy and attenuated H
2
S in the plasma.
Increased Hcy levels were associated with apoptosis of podo-
cytes, in part attributable to reduction of H
2
S and increased
oxidative stress, and therefore damaged the final filtration
barrier of the kidney. These changes in the glomerulus allowed
excessive protein excretion in the urine. On the contrary to this
mechanism, H
2
S supplementation prevented podocyte apopto-
sis, and this provided protection to the kidney against HHcy-
associated renal damage and proteinuria. It is important to men-
tion that, although the level of protein excretion was high in CBS
(⫹/⫺) 2-K mice compared with WT 2-K mice, the difference was
not significant even though the reduction of plasma H
2
S levels
was significant in CBS(⫹/⫺) 2-K mice compared with WT 2-K
mice. Herein, it is possible that other antioxidants in the tissue,
such as CAT, SOD, lipid peroxidase, etc., may have played a
similar role as of H
2
S, to repress the oxidative stress, and therefore
ameliorated proteinuria in CBS(⫹/⫺) 2-K mice. Thus the ob-
served proteinuria in CBS(⫹/⫺) mice was not parallel with the
reduction of H
2
S levels. Further understanding of the regulatory
mechanisms of proteinuria by H
2
S in HHcy-associated renal
failure may provide a new insight into the molecular mechanisms
of this disease process.
Proteinuria is a hallmark of renal complication and a major
deteriorating factor for the progression to end-stage renal
diseases (34). The outer aspect of glomerular basement mem-
brane is lined up with very specialized visceral epithelial cells,
named podocytes, and these podocytes serve as the final
defense against urinary protein loss in the normal glomerulus
(30). Any damage to the podocytes and their slit diaphragm is
intimately associated with proteinuria (32). Biochemical as-
sessment of normal slit diaphragm component, such as nephrin
(18), and injured podocyte marker desmin (14) are now there-
fore considered as two major sensitive markers of podocyte
injury and subsequently glomerulopathy in renal diseases.
Therefore, we measured desmin and nephrin in the kidney
tissue extract to asses whether the kidney injury is associated
with a high level of plasma Hcy and whether H
2
S supplemen-
tation can ameliorate this change. In our present study, we have
found a conspicuous increase in the expression of desmin,
whereas expression of nephrin was decreased in the mice
showing high Hcy levels and decreased plasma H
2
S level. H
2
S
supplementation reversed the effect on these two protein ex-
pressions associated with high Hcy, suggesting the regulatory
role of H
2
S on these two proteins during HHcy.
High Hcy has been reported to produce a sustained and
abnormal elevation of glomerular arterial wall stress through
generation of ROS (57, 58). This stress initiates a complex and
progressive glomerular remodeling, including activation of
MMPs, collagen degradation, glomerular hypertrophy, and
dysfunction (22, 37). One of the major causes of glomerular
sclerosis and renal dysfunction is the increase of glomerular
ECM. Glomerular ECM, which is composed of mesangial
matrix and basement membrane, plays an important role in
physical, mechanical, and structural functions of the glomeru-
lus. MMPs degrade both the collagenous and noncollagenous
components of the ECM and are thereby actively involved in
matrix turnover. In pathological conditions, collagenases initi-
ate the degradation process of ECM and denature collagen into
nonhelical gelatin derivatives. Gelatinases, which are a mem-
ber of the family of MMPs, digest these products into smaller
peptides. Of particular interest are gelatinases MMP-2 and
MMP-9, which have potential capability to disrupt the kidney
architecture by virtue of their specificity for various compo-
nents of basement membrane (33). Therefore, the measurement
of MMP-2 and -9 activities allows for an estimation of the
remodeling process during HHcy-associated glomerulopathies.
Data from our present report suggested that high Hcy induced
elevation of superoxide (O
2•⫺
) production in the CBS(⫹/⫺)
kidney, and this production of O
2•⫺
was even greater in 1-K
mice. The level of MMP-2 and -9 activities followed the
increasing trend of O
2•⫺
production in both 2-K and 1-K
CBS(⫹/⫺) mice. H
2
S supplementation normalized both MMP
activity level and the increased level of O
2•⫺
production in
these mice. This result suggests that the activities of MMP-2
and -9 are associated with increased O
2•⫺
production and that
H
2
S scavenges O
2•⫺
production, thereby regulating MMP
activities in our experimental condition. This regulatory mech-
anism of O
2•⫺
by H
2
S prevented renal damage and subsequent
renal failure associated with HHcy.
In summary, we have shown that elevation of plasma Hcy
level causes a decrease in the level of plasma H
2
S and is
associated with renal impairment. This increase of plasma Hcy
level induced glomerular oxidative stress, resulting in aug-
mented MMP activities and induction in glomerular cell apop-
tosis. Additionally, HHcy altered expression of desmin and the
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final filtration barrier regulatory protein, nephrin. These
changes in cellular and protein level indicated damage in the
kidney, which was exhibited by proteinuria, a marker of renal
failure. H
2
S supplementation, however, showed the reversal of
these deleterious changes associated with HHcy and is there-
fore protective to the kidney.
ACKNOWLEDGMENTS
This study was supported, in part, by NIH grants HL-71010, HL-88012,
HL-74185, and NS-51568.
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