OJHAS Vol. 9, Issue 2:
and Thiol Stress in patients with acute renal failure
Department of Biochemistry and Genetics, St Matthew’s University, School of Medicine, Grand Cayman, Cayman Islands, BWI,
Vivekananda Kedage, Manjunatha S Muttigi,
Department of Biochemistry,
Nataraj K, Waqas Wahid Baig, Ravindra Prabhu Attur,
Department of Nephrology,
Medical College, Manipal - 576104, India
Address For Correspondence
Associate Professor in biochemistry and genetics,
St Matthew’s University, School of Medicine,
P.O.BOX 30992, Regatta Office Park,
Leeward Three, Grand Cayman KY1-1204,
CAYMAN ISLANDS, BWI
Prakash M, Kedage V, Muttigi MS, Nataraj K, Baig WW, Attur RP. Glutathione-S-Transferase
and Thiol Stress in patients with acute renal failure. Online J Health Allied Scs.
Submitted: May 23,
2009; Accepted: Apr 25, 2010; Published: Jul 30, 2010
Tubular damage is common finding in acute renal failure
(ARF). Various etiologies have been put forth to explain the tubular
damage in ARF, one important mechanism among them is oxidative damage
to renal tubules. Several biomolecules including low-molecular weight
peptides and enzymes in urine have been proposed as early markers of
renal failure. Current study has been undertaken to study the thiol
stress and glutathione-S-transferase (GST) levels in ARF patients. Method:
58 ARF patients and 55 healthy controls were selected based on inclusion
and exclusion criteria. Serum thiols, GST, malanoldehyde (MDA) and urine
thiols were determined by spectrophotometer based methods. Results:
Serum thiols and urine thiols were significantly decreased (p<0.0001),
and serum GST and MDA levels were significantly increased (p<0.0001)
in ARF patients compared to healthy controls. Serum GST and MDA correlated
positively in ARF cases (r2 = 0.6938, p<0.0001). Conclusion:
There is significant thiol stress and increased lipid peroxidation in
ARF patients which leads to tubular cell membrane damage and release
of GST into blood stream and into urine. This may be possible mechanism
for the increased presence of GST in urine (enzymuria) found in other
Key Words: Glutathione-S-transferase; thiol stress; acute renal failure; urine
renal failure (ARF) is characterized by a sudden or gradual decline
in glomerular filtration rate (GFR), a slow and steady accumulation
of nitrogenous waste products, and an inability of the kidney to regulate
the balance of sodium, electrolytes, acid, and water.(1) The ischemic
damage in ARF is generally most severe in the early proximal tubule
(S3 segment) and the thick ascending limb of the loop of Henle.(2) Poor
oxygenation leads to a variety of secondary factors that promote the
development of tubular injury, including the intracellular accumulation
of calcium, the generation of reactive oxygen species, depletion of
adenosine triphosphate, and apoptosis.(2-4) Many tubular enzymes have
been studied as markers of the necrotic/apoptotic damage or dysfunction
of (proximal) tubular cells. Three major origins have been identified:
the lysosomes, the brush-border membrane, and the cytoplasm of the cells.(5,6)
studies have demonstrated that increased urinary amounts of enzymes
are useful to detect acute tubular damage at a very early stage, but
increased enzymuria may also be induced by a reversible mild dysfunction
of the cells not necessarily associated with irreversible damage. The
usefulness of enzymuria may be obscured by the low threshold for release
of tubular enzymes, even in response to injury that may not proceed
to ARF.(7) However, enzymes are also released during chronic glomerular
diseases, which might limit their use as a marker of tubular injury
only.(8-11) Some of the best-characterized tubular enzymes to detect
tubular injury are glutathione-S-transferases (GSTs), γ-glutamyl transferase
(γ-GT ), alkaline phosphatase (AP), lactate dehydrogenase (LDH), NAG,
fructose-1,6-biphosphatase, and Ala-(Leu-Gly)-aminopeptidase.(8,9) Increased
urinary excretion of these proteins implies tubular injury.
are important in intracellular binding and transport of numerous compounds,
and play a central role in human detoxification process. Human GSTs
mainly consists of class Pi (GST π), Alpha (GST α), Mu (GST μ) and
Theta (GST θ) enzymes, each subdivided into one or more isoenzymes.
They catalyze the conjugation of glutathione with wide variety of xenobiotics
such as carcinogens, pharmacologically active agents, as well as reactive
oxygen species (ROS). The conjugation may result in the formation of
more water soluble and less biologically toxic molecules that may be
easily excreted. In addition to detoxification, GSH is important in
storage and transport of amino acids. The characteristic feature of
the tripeptide GSH (γ-glutamylcysteinylglycine) is the presence of
reactive sulphydryl (–SH) group donated by cysteine in GSH is provided
by cysteine, and this dictates the chemistry of GSH.(12)
oxygen species (ROS) have been implicated in the renal cell injury that
occurs with reperfusion after ischemia. Products of lipid peroxidation
are generated on reperfusion, and these are presumed to derive from
ROS action on membrane lipids.(13,14) Scavengers such as superoxide
dismutase (SOD), glutathione, and vitamin E, as well as inhibitors of
ROS production, such as the iron chelator deferoxamine, have been reported
to protect against ischemic injury.(15,16) Exposure of kidney subcellular
organelles or microsomes to ROS-generating systems mimics some features
of ischemic injury.(14-17) Lipid peroxidation is frequently used as
an indicator of oxidative damage in the kidney.(13,18,19)
the current study we have determined the GST activity, thiols status
along with lipid peroxidation markers in ARF patients and compared them
with that of healthy individuals to know the difference and to understand
the biochemical basis for the change observed.
eight subjects with ARF were selected as cases. Fifty five healthy controls
were participated in this study. Inclusion criteria for ARV cases are:
age > 18 years, ARF of any etiology, defined by more than 30% rise
in serum creatinine from baseline, patients with renal failure presenting
to the hospital for the first time with short history (<3 months
duration), and ultrasound showing normal sized kidneys (>8.5cm).
Exclusion criteria: age <18 years, obstructive acute renal failure,
patients with preexisting history of renal failure (acute on chronic
renal failure), patients with history of diabetes mellitus or hypertension,
kidney size <8.5cm on ultrasound or evidence of hydronephrosis,
patients presenting as sepsis with acute renal failure. Healthy controls
aged more than 18 years with no past or present history of any medical
illness, not on any kind of medication, non-smokers; non-alcoholics
were included in the study.
aseptic conditions blood was drawn into plain vacutainers from ARF cases
and healthy controls, allowed to clot for 30 min, and then centrifuged
at 3000 rpm for 15 min for separation of serum. All assays were performed
immediately after serum was separated. Twenty four hour urine sample
from 58 ARF cases and 55 healthy controls was collected in a brown bottle
containing toluene as urine preservative, urine sample bottle was stored
at 4şC during the period of collection. Samples were centrifuged at
3000 rpm for 10 minutes and were analyzed immediately after the collection
period. Informed consent from the subjects
involved in the study and ethical clearance from institutional review
board was taken.
chemicals like reduced glutathione (GSH), 1-cholro 2,4-dinitrobenzene
(CDNB), 5’ 5’ dithio-bis (2-nitrobenzoic
acid) (DTNB), 1, 1, 3, 3-tetraethoxypropane and thiobarbituric acid
(TBA) were obtained from Sigma chemicals, St Louis, MO, USA. All other
reagents were of analytical grade.
GST and MDA, and serum and urine total thiols were measured using Genesys
10UV spectrophotomter. Urine creatinine levels were determined by automated
clinical chemistry analyzer Hitachi 912.
mL reaction mixture containing 850 µL of 0.1 M Phosphate buffer pH
6.5, 50 µL CDNB 20 mM, 50 µL 20 mM GSH, was preincubated at 37o
C for 10 min. Reaction was started by adding 50 µL serum or urine.
GST activities were assayed kinetically by noting changes in absorbance
at every 1 min interval for 5 min at 340 nm. Serum and urine GST activity
was determined by using molar extinction coefficient 9.6 mM-1
cm-1 (20-22) and was expressed in IU.
urine total thiol assay
µL serum or urine was added to reaction mixture containing 900 µL
2 mM Na2EDTA in 0.2 M Na2HPO4, 20 µL
10 mM DTNB in 0.2 M Na2HPO4, incubated at room
temperature for 5 min and absorbance was read at 412 nm. Similarly absorbance
of sample blank and reagent blank was subtracted from serum and urine
absorbance values to obtain corrected values. The calibration curve
was produced using GSH dissolved in phosphate buffered saline (PBS).
Total thiol levels were determined using molar extinction coefficient
have followed Satoh’s method (24), where 100 μl of
sample, 1000 μl of 0.67% TBA and 500 μl of 20% TCA were
ncubate at 100°C for 20 minutes; transfered the content to Eppendorf
tube and centrifuged at 12,000 rpm for 5 minutes. The absorbance of
the supernatant was read at 532 nm against water blank. 1, 1, 3, 3-tetraethoxypropane
(1 μmol/L) was used as a standard for MDA standard graph and to obtain
extinction coefficient (Є) for the malonaldehyde-TBA complex which
was 1.56×105 M-1.L.Cm-1.
statistical analysis was done using statistical package for social sciences
(SPSS) version 16. Independent sample t test and Mann Whitney U test
was done to compare mean values. A Pearson’s correlation was used
to correlate between the parameters. P value <0.05 was considered
significant. Microsoft office excel 2 was used to prepare correlation
depicted in Table 1, we have found significant decrease in the serum thiols in ARF patients compared to healthy controls (p<0.0001), however,
urine thiols were increased in ARF cases (p<0.0001). Serum GST activity
found to be increased in ARF cases compared to healthy controls (p<0.0001).
Membrane lipid peroxidation marker MDA levels were found to be higher
in ARF cases compared to healthy controls (p<0.0001). We have observed
significant skewed values in all the parameters that we have determined
(mentioned in Table 1 as minimum and maximum values).
Table 1: Independent
sample t test for all the determined biochemical parameters in both
healthy controls and acute renal failure cases (values expressed as
mean ± standard error of mean, both minimum and maximum value observed
(n = 55)
|Acute Renal Failure Cases (n = 58)|
Serum Thiols (μmoles/L)
Urine thiols (μmoles/L)
Min: 1.88, Max: 51.25
Min: 4.40, Max: 552.50
Serum GST (IU/L)
Serum MDA (nmoles/L)
||0.76±0.06Min:0.08, Max: 1.57
compared to healthy controls.
Because of wide
variation in the observed parameters, we have also analyzed the above
parameters by Manny Whitney rank sum test. As mentioned in Table 2,
there was significant decrease in the serum thiols (p<0.0001), and
significant increase in serum GST (p<0.0001), serum MDA (p<0.001)
and urine thiols (p<0.0001) in ARF patients compared to healthy controls.
On applying Pearson’s correlation, we have seen serum GST correlated
positively with serum MDA (r2 = 0.694, p<0.0001) (Figure
Mann Whitney Rank Sum test for the all the determined biochemical parameters
in both healthy controls and acute renal failure cases
||Serum Thiols (μmoles/L)
||Urine thiols (μmoles/L)
||Serum GST (IU/L)
||Serum MDA (nmoles/L)
||Urine Creatinine (gm/L)
Asymp. Sig. (2-tailed)
Correlation between serum GST and MDA in acute renal failure cases
stress is known to modify plasma proteins, and these modifications can
serve as excellent in vivo biomarkers of oxidative stress status.
The ready accessibility of plasma proteins for sampling, the relatively
long plasma half-lives of many proteins, and the well-characterized
biochemical pathways of protein and amino acid oxidation make plasma
protein oxidation an attractive in vivo
biomarker of oxidative reactions.(25-29) Thiols are organic sulfur derivatives
that are characterized by the presence of sulfhydryl residues at the
active site. Halliwell and others (30-32) have demonstrated that protein-associated
thiols, particularly in the albumin molecule, constitute a major defense
against oxidative stress in plasma. In our previous study we have shown
that there is protein thiol oxidation and lipid peroxidation in patients
with uremia.(33) Glutathione, normally present in high amounts in tubular
cells, can react with and neutralize ROS. Cellular glutathione levels
fall with ischemia (34), and reduced cellular glutathione levels sensitize
cells to oxidative stress.(35) Protective effects of glutathione have
been reported, although it remains controversial as to whether these
effects are due to the antioxidant characteristics of this compound
or due to the generation of glycine, its metabolic product, independent
of ROS scavenging. As with other ROS scavengers, glutathione administration
has yielded inconsistent results.(36,37)
our study, we have determined the total thiol status which includes
both glutathione and protein thiols, and found significant decrease
in serum thiols in ARF patients. We have also found increase in the
levels of lipid peroxidation marker MDA in ARF patients indicating the
increased presence of oxidative stress in these patients. The significant
decrease in thiol status in combination with increased presence of MDA
suggest that there is possibility of generation of enormous amount of
ROS species causing tubular membrane damage and loss of cytosol contents
into blood stream and urine. This may possibly explain the increased
presence of serum GST that we have found in ARF patients. Renal tubular
damage and possible leak of glutathione and protein bound thiols into
urine also explains increased presence of urine thiols in ARF patients
observed by us. In total our study, in line with similar previous study
with different experimental designs agrees with the fact that ARF causes
increased generation of ROS generation and depletion of antioxidants.
Furthermore, previous authors have observed inverse association between
plasma protein thiol content and the plasma levels of proinflammatory
cytokines IL-6, IL-8, and TNF- suggest that inflammation and oxidative
stress. Critically ill patients with ARF manifest a marked increase
in plasma protein oxidation, including plasma protein thiol group oxidation
and carbonyl formation.(38)
can damage tissue in a variety of ways. They can cause lipid peroxidation
by abstracting a hydrogen atom from a polyunsaturated fatty acid of
membrane phospholipids; a conjugated diene forms after molecular rearrangement
of the fatty acid. The diene then reacts with oxygen to form a peroxide
radical, which can remove hydrogen atoms from other lipids, generating
a chain reaction. Lipid peroxidation can increase plasma and subcellular
membranes' permeability (39), impair enzymatic processes and ion pumps
(14), and damage DNA.(40,41) In addition, direct oxidation of membrane
proteins occurs (42), affecting critical proteins such as the sodium-potassium ATPase and the Ca2 ATPase. The role of ROS in ischemic renal injury
remains controversial because investigators do not all agree that antioxidants
confer protection (43,44), nor do all agree on the presence of increased
lipid peroxidation or ROS generation in ischemia.(43,44)
conclusion, we have observed increased presence of oxidative stress
environment in patients with ARF as denoted by depletion of thiol status
and increased presence of MDA causing membrane damage and hence leakage
of GST and thiols into urine.
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