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The Effects of

Intermittent
Hypobaric Hypoxia
Exposure on
Glutathione Level
in Renal Tissue of
Rats

Diandra Safirina

1206222521 – FMUI 2012


Background

– Hypoxia in high altitudes  due to decreasing level of oxygen  can cause oxidative
stress
– An increased reactive oxygen species (ROS) can cause cell membrane damages and
impaired ATP production1,2
– Intermittent hypobaric hypoxia (IHH) will cause adaptation response in order to
protect tissue cells from ROS  antioxidant play role, such as glutathione reductase
(GSH)
– Intermittent hypobaric hypoxia (IHH) induces higher levels of lipid peroxidation and
reduces GSH activity and increase of oxidative damage in kidney2
– Oxidative stress level is important as study model
Background

– Kidney  an organ that mostly exposed into hypoxia due to anatomical


structure (blood vessels)
– There is a significant difference in blood supply and oxygenation
– Oxygen tension found comparatively low with high rated blood flow and oxygen
delivery  renal medulla
– The phenomenon caused by parallel arrangement of arterial and venous pre-
glomerular and post-glomerular vessels  allows oxygen diffuse from arterioles into
post-capillary venous system (via shunt diffusion)
– Due to limitation in renal oxygen supply, kidney is susceptible to hypoxia 
important factor in pathogenesis of acute renal injury and chronic renal disease3
Problem Identification

– Since hypoxia triggered reactive oxidative species (ROS) proved by elevating


carbonyl level in previous study, the effect of intermittent hypobaric hypoxia
(IHH) treatment on glutathione level as endogenous antioxidant tissue,
however, is still unknown.
Research Question

– How is the effect of hypobaric hypoxia on glutathione level of renal tissue found
in rats?
Hypothesis

– Glutathione level is increased in intermittent hypobaric hypoxia (IHH)


treatment
Objectives

– To increase public awareness by providing information about oxidative agent


such as glutathione and its relation with hypobaric hypoxia condition
– To obtain information and knowledge regarding reactive oxidative species (ROS)
and how it connects with hypobaric hypoxia condition on renal tissue samples
– As a prominence effort in conducting and contributing research primarily in
biochemistry subject
Literature Review

– Hypoxia is defined as a state of reduced supply of oxygen into cellular network


below its physiological levels even when the blood perfusion is still adequate3
– Environmental condition that commonly correlated to hypoxia showed several
characteristics  decreased temperature, lower humidity, increased UV
penetration, declined sufficient oxygen pressure
– Renal is usually affected by hypobaric hypoxia  considered as an acute
physiological stress that leads to oxidative stress
– Hypoxia can occur in high altitudes with low atmospheric pressure
– Typically contemplated by groups that are exposed  military aviators
Literature Review

– In normal state, ROS produced are fully inactivated by a series of cellular and extracellular
antioxidant defense system
– Any changes in renal function at high altitude arise from direct effects of renal hypoxia and
different compensatory adapting mechanisms
– Urine output and sodium excretion in renal are vary and associated with partial pressure of
oxygen4
– Renal blood flow is usually increases from 8 to 20% during hypoxia and then return to its
normal baseline after several days
– Renal increases bicarbonate excretion in order to compensate with oxygen-lacking
environment due to hypoxia
– Body fluid homeostasis involves a complex interaction with physiological system
– In high altitude, plasma vasopressin was reduced or remain unchanged (due to increase plasma
cortisol concentration)
Literature Review

– Hypoxia can induced inhibition of renin aldosterone system thus leads to reduction
in creatinine clearance and low natriuretic response
– Hypobaric hypoxia induces elevated BP, hypovolemia and hyper osmolality due to
the suppression of vasopressin release or reduced renal sensitivity to vasopressin
– Renal produces ROS from normal metabolism
– Contributed by glomerular cells
– Can reduce tubular ion transport and increase medullary perfusion
– Glutathione normally present in high amounts of tubular cells and neutralize ROS
– Cellular glutathione level falls with hypoxia
– Reduced cellular glutathione level sensitizes cells to oxidative stress
Research Design and Research
Duration
– Experimental methods in rat
subjects (kidney) as the most
appropriate method
– Induction of hypoxia has previously
held by doctoral students as
researchers in faculty of medicine
University Indonesia
– Conducted in laboratory of faculty
of medicine University Indonesia
from August 2015 to March 2016
Data Sources

– Obtained from primary sources, directly acquired from protein measurement


and glutathione assay under hypobaric hypoxia exposure
– Using previous sample, rats renal tissue were divided into 2 main groups:
control and ‘treatment’  ‘treatment’ – groups that were exposed to
intermittent hypobaric hypoxia (IHH)
Data Sources

– Treatment groups consist of four groups which classified according to how many
exposure towards hypobaric hypoxia listed below:
– H1 group treated under 1 (one) time hypobaric hypoxia condition (type I chamber
flight profile (max. altitude of 35.000 feet, hypoxia demonstration at 35.000 feet))
– H2 group treated under 2 (two) times hypobaric hypoxia (type II chamber flight
profile (max. altitude of 43.000 feet, hypoxia demonstration at 25.000 feet)
– H3 group treated under 3 (three) times hypobaric hypoxia (just like H2 group with
addition of 1 (one) time type II chamber flight profile)
– H4 group treated under 4 (four) times hypobaric hypoxia (just like H3 group with
addition of 1 (one) time type II chamber flight profile)
Techniques of Sample Selection

– Rats renal sample taken from deep-freeze, placed into dry ice for several
minutes, being removed, and placed in an ice box at -20ºC
– Sample then were cut into smaller piece and weighed using proper scale
– 1 mL PBS 0.1M (pH 7.4) were added into each sample for homogenization
preparation using a vorter; after that each sample were centrifuged at 3500
rpm for 10 minutes
– Supernatant was obtained for experimental results, yet the homogenate used in
this research was diluted for 100 times
Sample Size

– Samples were obtained from the kidney of male Sprague-Dawley rats weighing
150-200 grams
– Minimum sample size of the research is determined according to Mead’s
Formula: (E = N – T) with T as ‘treatment’ component, E as 10-20 or degrees of
freedom, and N as number of rats in each group(s) times number of group(s) 
total = 15 rats
– The treatment (T) in this experiment is reflecting the 5 groups that has been
divided for each days criteria and the control group which was exposed to
normal environment
– The sampling method used in this research was simple random sampling
Data Analysis

– Glutathione assay  using Ellman’s method  presenting GSH level per total
protein renal tissue (ng/mg protein)
– Protein measurement  using Christian Warburg’s method
– The plotted data were tested whether they were normally distributed or not
(using Shapiro Wilk test) and homogenous before being statistically analyzed
using ANOVA in SPSS version 21
– The result were estimated to be significant if the P value < 0.05
Results

– GSH standard curve was determined


using different concentration of GSH Standard Curve
0.45
standard GSH in order to calculate

Mean of Absorbance (412 nm)


0.4 y = 0.0416x + 0.0038
0.35
R² = 0.99965
total content of GSH level in renal 0.3

samples 0.25
0.2
0.15
– Absorbance of different GSH 0.1
0.05
concentration (0, 1, 2, 4, 5, 10 μg/mL) 0
0 2 4 6 8 10 12
was measured at 412 nm Standard GSH Concentration μg/mL
Results

– Total GSH content of renal samples (μg/mL)


was measured using GSH standard curve and Total GSH Content of Renal
calculated using the formula X = [(Y-a)/b] thus Samples
the value represented in mean ± SD 0.09

– GSH concentration was calculated by dividing 0.08


0.07
GSH content with protein concentration 0.06
(μg/mg protein) 0.05
0.04
– GSH level firstly increased in line after first 0.03
exposure until the second hypoxia, then a 0.02
decreased trend in third hypoxia are seen 0.01
0
before it shown almost stabilized level on the kontrol h1 h2 h3 h4
fourth hypoxia (Figure 4.2.) Figure 4.2. Renal GSH Level of Rats with Different Duration
of Exposure in Intermittent Systemic Hypoxia. (kontrol = no
– Statistically, the data were not significant exposure to hypoxia; h1 = 1x exposure to hypoxia; h2 = 2x exposure
according to ANOVA and Post Hoc LSD to hypoxia; h3 = 3x exposure to hypoxia; h4 = 4x exposure to
Analysis (p = 0.268). hypoxia).
Discussions

– Glutathione level is affected with hypobaric hypoxia  seen on its trend 


matches with carbonyl level trend according to previous study5
– Abraham study analysis4  significant elevation in carbonyl level (oxidative stress
marker) on group that exposed for 2x hypobaric hypoxia  due to an increase in ROS
level
– Reduced glutathione (GSH) is the parameter  present abundantly in renal
tubular cells as one of the major non-enzymatic antioxidant defenses in renal
– There is an increase of GSH level on the first and second hypoxia exposure until
certain amount of time, here represented by third hypoxia exposure and then
decreased, before continued to slightly increased again (almost stabilized)
Discussions

– Hypoxia is associated to lowered cellular GSH level in different cell types  the
longer the exposure to systemic hypoxia, the lower renal GSH would be6, although
the research result was slightly performed different matter
– The renal GSH level within hypoxia exposure has increased until certain amount of
time  peak was found on first and second times of exposure testing (0.053 ±
0.024)  represented maximum GSH concentration that can be produced by renal
under hypoxia to overcome ROS level
– The renal GSH level decreased on third times of hypoxia exposure (0.02 ± 0.02) 
due to adjustment mechanism of hypoxia, renal GSH level was eventually lowered
– The renal GSH level has slightly risen on the fourth times of hypoxia exposure (0.028
± 0.007)  almost stabilized
Discussions

– Increasing amount to hypoxia exposure leads to elevated mitochondrial ROS


production since GSH becoming to oxidized faster  function to scavenge
excessive ROS to keep it within normal state
– Renal antioxidant capacity is maintained by GSH, glutathione peroxidase and
reductase  to counter balance increased ROS formation during IHH
– Clinically, sufficient hypobaric hypoxia treatment in renal tissue of rats might
have renoprotective effects (such as inhibit renal structural function)
Discussions

– Mean difference of GSH level found to be highest in comparison of second and third times
hypoxia exposures
– Data found to be insignificant  perhaps due to limited research sample number of rats renal
tissue  until it reached its maximum time period, GSH level will be lowered due to hypoxia
exposure
– Analogues with Nakanishi et al. (1995) who found that GSH levels were not significantly changed in
hypobaric hypoxia rats rather than in control rats by 21 days of hypoxia exposure
– Nakanishi study also shown that MnSOD, one of enzymatic defense system play role in mitochondria
has no significant difference  proving that duration of hypoxia exposure is very influential against
oxidative stress
– MDA (lipid peroxidation product) has increased under systemic hypoxia treatment in day 37
– Decreasing level in the research (third times exposure) was due to maximum adaptation to hypoxia
Conclusions

– The level of renal GSH in rats which exposed to intermittent hypobaric hypoxia,
after certain adjustment, create variation of GSH level due to renal adaptive
responses system
– One of the main factor that influenced GSH level in renal tissue was the
exposure time to intermittent hypobaric hypoxia
– As the hypoxia get higher, glutathione gave protective level on renal rats tissue
Recommendation

– Further studies such as ROS detection analysis should be attempted to


overcome the research limitation as well as adding more samples in the
research in order to reach more definitive conclusion
References

1. Berra E. The magic of the hypoxia-signaling cascade. Cell. Mol. Life Sci. 65 (7-8): 1133–49,
2008.
2. Brezis M, Rosen S. Hypoxia of the renal medulla: Its implications for disease. N Engl J Med
332 :647– 655,1995.
3. Dorland’s illustrated medical dictionary. Philadelphia: WB Saunders, 28th edition; 1994; pp
89:812.
4. Yakub, Abraham. The Effect of Hypobaric Hypoxia Exposure Towards Carbonyl Concentration
as Oxidative Stress Marker in Rats Kidney [Thesis]. Indonesia. Universitas Indonesia: 2014.
5. Gonchar, O. Effect of moderate hypoxia/reozygenation on mitochondrial adaptation to acute
severe hypoxia. Acta Biol. Hung., 60: 185-194; 2009.
6. Syarifin, Andi. W.A. Jusman, Sri. Sadikin, Mohamad. Gene Expression and Enzyme Activities
of Carbonic Anhydrase and Glutaminase in Rat Kidneys Induced by Chronic Systemic
Hypoxia; 2014.
7. Dosek, A. H. Ohno. Z. Acs, A. Taylor and Z. Radak. High altitude and oxidative stress. Resp.
Physiol. Neurobiol, 158: 128-131; 2007