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OXIDATIVE IMBALANCE AND KIDNEY DAMAGE IN SPONTANEOUSLY

HYPERTENSIVE RATS: ACTIVATION OF EXTRINSIC APOPTOTIC PATHWAYS

D. La Russa§, E. Brunelli§, D. Pellegrino*

Dept. Biology, Ecology and Earth Sciences, Univ. Calabria, 87036, Cosenza, Italy
§
La Russa and Brunelli equally contributed to this research
*Corresponding author: Laboratory of Physiology, Department of Biology, Ecology and
Earth Sciences, University of Calabria, 87036, Arcavacata di Rende (CS), Italy
[email protected]

ABSTRACT

In both humans and animals, essential hypertension acts as a risk factor for subclinical
kidney damage and precedes renal dysfunction. Several lines of evidence indicate that
hypertension and oxidative stress are closely related. The increase in vascular oxidative stress

ACCEPTED MANUSCRIPT
plays a key role in the pathophysiological consequences of hypertension, including kidney
disease. Our study examined this issue in SHR, a reliable model of essential hypertension.
We used SHR 20 weeks-old when hypertension is stably developed, vascular
remodeling started, but kidney function is preserved. We examined plasmatic pro-oxidant
and antioxidant status showing a significant alteration in oxidative balance in SHR. As index of
oxidative damage, we evaluated lipid peroxidation in kidney, liver and skeletal muscle, detecting a
significant rise in lipid peroxidation levels in all SHR tissues, particularly relevant in
kidney. In addition, we analyzed the expression of cytoplasmic antioxidant enzymes, SOD1
and GSTP1. In SHR liver, SOD1 expression slight increased while we have not detected any
variation in other tissues. Concerning GSTP1, SHR renal tissues did not display variations in
enzyme expression, while, in the other tissues, we observed a significant increase of both
monomeric and pro-apoptotic dimeric form of the enzyme. By analyzing apoptotic signal, we
founded JNK activation in all SHR tissues, but only kidney presented extrinsic apoptotic
pathway activation. Our results suggest that, in hypertensive animals with preserved renal
function, despite the remarkable oxidative damage of renal tissues, only the extrinsic apoptotic
pathway is activated.

KEYWORDS Hypertension; oxidative stress; biological antioxidant potential; lipid peroxidation; SOD1; GSTP1;
apoptosis; JNK; PARP-1; caspase-8; caspase-9.

ABBREVIATIONS: SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rat; ROSs, reactive oxygen species; BP,
blood pressure; SOD1, superoxide dismutase 1; GSTP1, glutatione S-tranferasi P1; JNK, c-Jun N-terminal
kinase; pJNK, phosphorylated JNK; MAPK, mitogen-activated protein kinase; PARP-1, Poly [ADP-ribose] polymerase 1.

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INTRODUCTION

Renal diseases are a major public health concern and the main consequences include
loss in renal function and cardiovascular complications [1]. Moreover, hypertension has
been linked with the development of endothelial dysfunction, inflammation, and renal injury
[2,3,4]. In both humans and animals, essential hypertension acts as a risk factor for
subclinical kidney damage and precedes renal dysfunction [5] but mechanisms that
correlate hypertension and kidney disease have not been elucidated extensively. Although
elevated blood pressure is the major factor contributing to hypertensive organ damage,
complex biochemical, hormonal and hemodynamic mechanisms are also involved in tissue
damage [6].

Several experimental and clinical data prove that hypertension and oxidative stress are
closely related [7,8], although it is unclear whether oxidative stress is a cause or an effect
of hypertension [9,10]. The important pathophysiological role of ROSs in hypertension
development is due, in large part, to oxygen excess and decreased NO bioavailability in
vasculature and kidneys [11]. Increased oxidative stress has been revealed in genetic and
experimental models of hypertension although the effectiveness of antioxidant treatments
in reducing blood pressure has not been not fully verified [12]. Oxidative stress seems to
be a salient feature also in human hypertension, indeed hypertensive patients show both
increased oxidative stress and reduced antioxidant capacity [11,13]. Another important
finding is that the increase in vascular oxidative stress plays a key role in the
pathophysiological consequences of hypertension, including kidney disease [6]. Recent
evidence states that oxidative stress is a crucial molecular mechanism involved in the
pathogenesis of hypertensive renal damage [14] and that apoptosis occurs in critical
organs (heart, brain, or kidney) during hypertension [15].

In this work we used a genetic model of hypertension, the SHR, derived from the
normotensive WKY rat [16]. The SHR, besides being the most widely used model for
essential hypertension, is also an excellent model for studying the development of renal
damage in the context of human hypertension and hypertensive kidney disease, as
highlighted in several recent publications [17,18]. We utilized kidney as target organ and
liver and skeletal muscle as control tissues, since these organs do not appear particularly
susceptible to hypertensive damage [15].

We hypothesized that the increased oxidative stress in genetic models of hypertension

may contribute to organ damage through activation of apoptosis signaling pathways. In
order to test this assumption, we evaluated (i) plasmatic pro-oxidant/antioxidant status, (ii)
tissue lipid peroxidation as an index of oxidative damage, (iii) expression of cytoplasmic
antioxidant enzymes (SOD1 and GSTP1) and (iv) extrinsic and intrinsic apoptotic
pathways.
MATERIALS AND METHODS

Animals

Twenty-week-old male SHRs and WKY rats (Harlan Laboratories s.r.l. Udine, Italy) were
housed under controlled lighting and temperature conditions and fed ad libitum with a
standard diet and with free water access. BP measured before each experiment by tail-cuff
method, was: WKY: Systolic BP= 127.565.4 mmHg and Diastolic BP= 83.564.5 mmHg;
SHR: Systolic BP = 181.967.9 mmHg and Diastolic BP =124.366.2 mmHg. Ethics
Statement: the experiments were conducted in accordance with the Directive 2010/63/EU
of the European Parliament and Italian law (D.L. 116/92). All surgery was performed under
anesthesia and all efforts were made to minimize animal suffering.

Measurement of plasma and tissue oxidative status

Plasma and tissue oxidative status determinations were measured by using photometric
measurement kits and a free radical analyzer system with a spectrophotometric device
reader (FREE Carpe Diem, Diacron International, Grosseto, Italy), which are routinely
used in our laboratory [19]. Plasma oxidative stress was assayed using a Diacron-reactive
oxygen metabolite (d-ROM) test. The d-ROM test helps to determine the oxidant ability of
a plasma sample that measures the presence of Reactive Oxygen Metabolite derivatives,
in particular hydroperoxides. By means of an appropriate acidic buffer, transition metal
ions (essentially iron), originating by protein, are converted to alkoxy and peroxy radicals
that react with hydroperoxides thus forming new radicals; aromatic amine (N,N-
diethylparaphenylene-diamine) react with these new radicals originating a coloured cation
radical spectrophotometrically detectable at 505 nm. Results are expressed in Carratelli
Units (UC; 1UC=0.8 mg/L of hydrogen peroxide). Total plasma antioxidant capacity was
assayed using a biological antioxidant potential (BAP) test. Total plasma antioxidant
capacity was assayed using a biological antioxidant potential (BAP) test. The BAP test
provides an overall measure of the biological antioxidant potential measuring the blood
concentration of antioxidants (such as bilirubin, uric acid, vitamins C and E and proteins)
capable of reducing iron from the ferric to the ferrous form; in fact, when the plasma is
mixed with a colored solution (ferric chloride and thiocyanate) a decoloration occurs whose
intensity is related to the ability of the plasma to reduce iron ions. The intensity of
decoloration is spectrophotometrically detectable at 505 nm. Results are expressed in
µmol/L of the reduced ferric ions. Tissue lipid peroxidation was assayed using a LIPO
tissue test based on the ability of peroxides to induce the oxidation of Fe2+ to Fe3+. The
resulting Fe3+ binds to thiocyanate, causing a formation of a colored complex that can be
measured photometrically. The increase in absorbance is proportional to the concentration
of lipoperoxides present in the sample. Briefly, samples (200 mg) were homogenized in 1
ml distilled water, centrifuged (15 min at 15000g) and washed twice with distilled water.
After removing the supernatant, 2 ml of the indicator mixture (R1) was added, mixed (5
min) and centrifuged (5 min at 1400g). Then, 10 µl of supernatant or standard (4000 µEq/L
terbutilhydroperoxide) was added in 1 ml indicator mixture, followed by the addition of 10
µl Fe2+(R2 reagent). After incubation (5 min at 37 °C), the optical density was read at 505
nm and the concentration of lipoperoxides was calculated and expressed as
nanoequivalent hydroperoxydes/g tissue.

Western Blot and densitometric Analysis

Tissue samples (800 mg) were lysed in RIPA buffer (1.6 ml) with the protease inhibitor
cocktail (Sigma, St Louis, MO, USA) and centrifuged at 14,000 rpm for 20 min at 4°C.
Supernatant was collected and protein was quantified with a Bradford reagent kit (Sigma,
St Louis, MO, USA). Samples of supernatants containing 50 µg of proteins were heated for
five minutes in Laemmli buffer (Sigma, St Louis, MO, USA), separated by SDS-PAGE in a
Bio-Rad Mini Protean III, and then electroblotted onto nitrocellulose membrane (NitroBind,
Maine Manufacturing, Maine, USA) using a mini trans-blot (Bio-Rad Laboratories,
Hercules, CA, USA). Membrane was blocked with TBS-T buffer containing 5% non-fat dry
milk. For immunodetection, the blots were incubated overnight at 4°C with the following
antibodies diluted in TBS-T: SOD1 (polyclonal goat antibody, Santa Cruz Biotechnology,
inc.); GSTP1 (monoclonal mouse antibody, Santa Cruz Biotechnology, inc.); pJNK
(monoclonal mouse antibody, Santa Cruz Biotechnology, inc.); PARP-1 (polyclonal rabbit
antibody, Santa Cruz Biotechnology, inc.); Caspase 8 (monoclonal mouse antibody, Santa
Cruz Biotechnology, inc.); Caspase 9 (polyclonal rabbit antibody, Santa Cruz
Biotechnology, inc.). Peroxidase linked secondary antibodies (Santa Cruz Biotechnology,
inc.) were diluted 1:2000. Immunodetection was performed by using an enhanced
chemiluminescence kit (ECL Plus, Amersham). Autoradiographs were obtained by
exposure to X-ray Films (Hyperfilm ECL, Amersham). Immunoblots were digitalized and
the densitometric analysis of the bands obtained was carried out using WCIF Image J
based on 256 grey values (0 ¼ white; 256 ¼ black). Quantification of the bands was
obtained by measuring (5 times on each band) the mean optical density of a square area
after the background area had been subtracted. The results of absorbance measurements
and the grey values obtained from the densitometric analysis were expressed as means ±
SE (standard error) of five determinations for each sample.

Statistical Analysis

Data was analyzed using the GraphPad/Prism version 5.01 statistical software (SAS
Institute, Abacus Concept, Inc., Berkeley, CA, USA). Statistical differences were examined
using one-way analysis of variance (ANOVA) followed by the Bonferroni multiple
comparisons test. Data are expressed as the mean ± SE.
RESULTS

Plasma and tissue oxidative status

We analyzed the trend of both oxidative stress (dROMs) and antioxidant barrier efficiency
(BAP) in plasma of SHR and WKY rats. As shown in Table 1, we found a significant
increase in oxidative stress and a remarkable reduction in antioxidant barrier efficiency in
SHR compared to normotensive animals. We also evaluated tissue lipid peroxidation
(estimated by LIPO tissue test) in kidney, liver and skeletal muscle. Our results showed a
very significant increase in hydroperoxide level in SHR respect to WKY rats in all analyzed
tissues (Figure 1). The hydroperoxide level increase is particularly significant in kidney
tissue (nearly twofold).

Antioxidant enzymes' expression

We examined the expression of two important cytoplasmatic antioxidant enzymes, SOD1

and GSTP1, in both SHR and WKY rats. We noticed a slight increase in SOD1 expression
in SHR liver, while in other tissues (kidney and skeletal muscle) we did not find any
variation in hypertensive animals (Figure 2a). Noteworthy, SOD1 expression in liver was
significantly higher respect to other tissues in both WKY and SHR (Figure 2a). Our results
showed that in kidney tissue there is no variation in GSTP1 expression in SHR with
relation to WKY rats while in both liver and skeletal SHR muscle, we found a significant
increase in the expression of both the monomeric (23 kDa) and dimeric (46 kDa)
enzymatically active form (Figure 2b and 2c). The basal GSTP1 amount resulted
significantly different in all tissues examined and was clearly visible in kidney tissue in both
SHR and WKY rats (Figure 2b and 2c).

Apoptotic pathways

We analyzed apoptosis activation by evaluating the expression of pJNK and PARP-1,

apoptotic extrinsic pathways by evaluating the expression of caspase 8, and apoptotic
intrinsic pathways by evaluating the expression of caspase 9. Our results showed the
apoptosis activation in all SHR tissues examined (kidney, liver and skeletal muscle) as
highlighted by (i) significant upregulation of pJNK detected as double bands (pJNK1, 46
kDa and pJNK2, 54 kDa; Figure 3a) and (ii) significant upregulation of PARP-1 active
cleaved fragment (89 kDa; Figure 3b). We observed the apoptotic extrinsic pathway
activation only in SHR kidney as highlighted by an increased expression of two caspase 8
active fragments cleaved (10 kDa and 18 kDa) that were absent in other tissues (Figure
4a). We observed the apoptotic intrinsic pathway activation in SHR liver and skeletal
muscle as highlighted by an increased expression of caspase 9 active fragment cleaved
(10 kDa; Figure 4b).
DISCUSSION

The present study was designed to assess how the alteration of the oxidative balance in
the essential hypertension model can influence tissue alterations, in terms of both
oxidative damage (lipid peroxidation, altered expression antioxidant enzymes) and
apoptotic pathways (intrinsic/extrinsic) activation. Our results are presented in graphical
form in Figure 5. We showed significant alterations in plasmatic oxidative balance and
tissue oxidative damage in SHR. In particular, we found a particularly relevant lipid
peroxidation level in SHR kidney, whereas SOD1 and GSTP1 expression remained
unaltered. Concerning the apoptotic signal, we found pJNK and PARP-1 activation in all
SHR tissues tested, while we found intrinsic apoptotic pathway activation in liver and
skeletal muscle and extrinsic apoptotic pathway activation only in kidney tissue.

Hypertension acts as an important risk factor for renal diseases as highlighted by several
studies in animal models of hypertension, in particular in spontaneously hypertensive
animals [17, 18]. In our study, we used a genetic model of essential hypertension that
represents the most popular model of hypertension with over 20.000 articles in PubMed.
Furthermore, the SHRs are currently widely used as an important model in order to
analyze the intricate link between blood pressure (BP) and kidney function [17,18,20]. The
timeline of the development of hypertension and renal damage is well known in this
experimental model: hypertension develops within the first 10 weeks after birth and
remains stable or changes gradually as age increases; vascular remodeling begins very
early (4-5 weeks) and appears concurrent with increased renal autoregulatory efficiency;
renal function (renal blood flow and glomerular filtration rate) remains constant up to 20
weeks of age; hypertensive kidney damage is not morphologically evident before 30
weeks of age [17]. For our purpose we used 20 weeks-old SHR, when hypertension is fully
developed, vascular remodeling has started, but kidney function is preserved.

The first stage of this research examined the plasma and tissue oxidative status in SHR
showing significant alterations in plasmatic oxidative balance and tissue oxidative damage,
which was particularly relevant in kidney tissue [21,22]. To detect total plasma redox
balance, we used a simple and standardized methodology [19] that allowed us to analyze
both oxidative stress (dROM) and antioxidant capacity (BAP). The analysis of the overall
redox balance does not allow us to identify the alterations in the systems but it provides a
global vision that reflects the balance created after the perturbation of the individual
factors. Our results clearly show a significant increase in oxidative stress values and a
significant depletion in the efficiency of total plasma antioxidant barrier in SHR, while in
normotensive WKY we detected normal values of oxidative balance, similar to those in
humans [19,23]. In SHR, the plasmatic oxidative stress increase has been already
detected at 2 weeks of age, which is when the rats are still normotensive [24]. Our results
confirm literature reports in both animal models and humans [11,13]. To quantify the tissue
damage by oxidative stress, we detected lipid peroxidation. Lipids that contain unsaturated
fatty acids with more than one double bond are particularly susceptible to the deleterious
action of free radicals with consequent impairment of the biological membranes' structure
and function. In our study, we detected a significant increase in tissue LIPO levels (kidney,
liver and skeletal muscle), as markers of lipid peroxidation, in SHR in contrast with WKY
animals. This increase appears particularly relevant in kidney tissue, where the lipid
peroxidation level results twofold in the hypertensive animal compared to the normotensive
one. It is well known that the kidney is a target organ for hypertensive oxidative stress, and
several studies have confirmed that oxidative damage appears as a primary abnormality in
the kidney before development of hypertension [24]. Interestingly, in the liver, commonly
used as an indicator for systemic oxidative stress [25], we detected a lower degree of lipid
peroxidation compared to other tissues examined. Probably, being the main center of
detoxification, the liver detoxifies various ROS by efficient antioxidant defense
mechanisms.

In the second part of our work, we examined the expression of two important antioxidant
enzymes, SOD1 and GSTP1. The high ROS levels determine a depletion of non-
enzymatic antioxidants since the ROS species neutralization implies their consumption
[10]. Concerning enzymatic antioxidants, the issue is more complex: in the case of
low/medium oxidative stimulation, enzymatic antioxidant activity can increase, but if
oxidative stress is persisting, or its level is very high, the damage caused to proteins
becomes profound and a decreased expression/activity may occur via direct oxidative
damage of the molecules and/or oxidative-altered gene expression. Hypertensive patients
experience reduced activity and decreased content of antioxidant enzymes, including
SOD1, the most important preventive antioxidants which catalyze the dismutation reaction
of superoxide anion to the more stable hydrogen peroxide [11,26]. However, in
experimental models of hypertension, it has been reported either as a reduction in
antioxidant enzyme expression/activity or as an adaptive increase in antioxidant enzyme
activities [27,28,29]. In addition, also physiological post-translational oxidative modification
can influence the stability/activity of SOD1, as recently highlighted [30]. In our study, we
showed a slight increase in SOD1 expression only in liver tissue. Noteworthy, SOD1
expression in the liver was significantly higher when compared to other tissues in both
WKY and SHR. These results highlight the importance of SOD1 as the first line of cellular
defense against oxidative injury. Indeed, in liver tissue, we found an increase in SOD1
expression and a low rate of lipid peroxidation, thus confirming the important protective
role of SOD1 against oxidative damage. The GST family comprises dimeric isoenzymes
widely expressed in mammalian tissues that catalyze the conjugation of reduced
glutathione with a wide variety of electrophiles, including various oxidative stress products
such as oxidized DNA and lipid [31]. In addition, several lines of evidence suggested a
non-catalytic role for GSTP1 as an integral determinant for stress response cellular
pathways, proliferation and apoptosis in both humans and rodents [32,33]. In particular,
although GSTs are dimeric proteins, the monomeric form of GSTP1 acts as a JNK-
proliferative pathway activator [34]. Several studies have shown that GSTP1 acts as a
stress response protein which multimerizes through disulphide crosslinks, if affected by
oxidative stress, and loses its ability to bind JNK, causing an increase in JNK-apoptotic
pathways [32]. Our results showed that in kidney tissue, where GSTP1 resulted markedly
expressed, there is no variation in SHR respect to WKY while in both liver and skeletal
muscle, we found a significant increase in both monomeric and dimeric forms of the
enzyme in hypertensive animals. Despite the lack of remarkable difference in SOD1 and
GSTP1 expression, SHR show a significant increase in lipid peroxidation, therefore, future
studies should be directed at measuring the enzymatic activities of both SOD1 and GSTP1
in hypertension.

In the final stage of our research we examined the apoptotic signal showing a significant
increase in both pJNK and PARP-1 in all SHR tissues tested respect to WKY controls.
Apoptosis signaling has been widely classified into extrinsic (initiated by death receptors)
and intrinsic (initiated by mitochondrial events) pathways, and pJNK plays a central role in
both of these pathways [35,36]. JNK, an important member of the MAPK family, plays a
critical physiological and pathophysiological role in cells and a complex role in apoptosis.
Several lines of evidence suggest that JNK is an important mediator in oxidative stress-
induced apoptotic cell death, particularly significant in the proapoptotic state associated
with hypertension [37]. Gomes and coworkers [37] suggest that enhanced vulnerability to
oxidative stress-induced apoptosis in SHR renal cells may result from impaired catalase
activity and JNK hyper activation. Our result confirmed this finding, indeed in our SHR
tissues (including kidney) we detected a specific and robust pJNK2 activation, and we
support the hypothesis that enhanced JNK activity is a vital mechanism underlying the
apoptotic response to oxidant injury in hypertension. The activation of the extrinsic
pathway is mediated by caspase-8 [38] and the activation of the intrinsic pathway is
mediated by caspase-9 [39]. Caspase mediated apoptotic cell death is accomplished
through the cleavage of key proteins required for cellular functioning/survival, and PARP-1
is one of caspase cellular substrates. Cleavage of PARP-1 by all the caspases is
considered to be a hallmark for apoptosis [40]. Accordingly, all SHR tissues analyzed by
us showed the activation of the mechanisms leading to cell death. Apoptosis occurs in
critical organs (heart, brain, kidney, liver, skeletal muscle) during hypertension
[15,41,42,43] and emerging data confirms that excessive ROS induced by hypertension
can damage tissue components including lipids, proteins, and ultimately lead to cellular
loss via apoptosis or necrosis [41,44]. Indeed, adult SHRs present a significant increase in
renal interstitial fibrosis and apoptosis [45], while in newborn animals apoptosis was minor
in SHRs' kidneys compared with their normotensive controls [46]. In the current study, by
analyzing both the extrinsic and intrinsic apoptotic pathways, we found that only in kidney
tissue extrinsic apoptotic pathway activation was present while liver and skeletal muscle
showed intrinsic apoptotic pathway activation. In the rat model for kidney disease, the
apoptotic process is associated with both intrinsic and extrinsic pathways [47]. Also in the
ventricle of hypertensive rats caspase−8 and caspase−9 activity was substantial compared
to that in the control group [48]. In our model, in which hypertension is stably developed
whereas kidney function is preserved, the remarkable renal oxidative damage (i.e. lipid
peroxidation) in SHR does not lead to the activation of the mitochondrial-mediated intrinsic
apoptotic pathway. Furthermore, in SHR tissue we did not detect an increase in the
dimeric proapoptotic form of GSTP1, which was higher in SHR liver and skeletal muscle
where the intrinsic apoptotic pathway is activated. These results suggest that, in our
model, the increase in the dimeric form of GSTP1 can exert its proapoptotic action by
activating the intrinsic pathway.

In conclusion, data collected in the current study shows that, in addition to a direct effect
on BP, the redox disequilibrium in both plasma and tissue is extremely important in the
hypertensive tissue alteration in terms of both oxidative damage (lipid peroxidation, altered
expression antioxidant enzymes) and apoptotic pathways (intrinsic/extrinsic) activation.
Furthermore, our results highlight the strong causality link between oxidative damage in
renal tissue and hypertension and suggest the presence of additional (genetic and/or
acquired) factors intrinsic to the kidney involved in the susceptibility to BP alterations.

CLINICAL PERSPECTIVES

(I) Hypertension is complex systemic condition resulting from anatomical, physiological

and molecular events, which can cause an anomalous environment for cells and tissues.
The aim of our study was to assess the relationship between the oxidative balance
alteration and the tissue alterations, in terms of both oxidative damage (lipid peroxidation,
altered expression antioxidant enzymes) and apoptotic pathway (intrinsic/extrinsic)
activation. For our purpose we utilized SHR, a useful model of essential hypertension.

(II) Our results showed significant alterations in plasmatic oxidative balance and tissue
oxidative damage in SHR. In particular, despite the lack of remarkable difference in SOD1
and GSTP1 expression, SHR show a significant increase in lipid peroxidation, particularly
relevant in kidney. Concerning the apoptotic signal, we found pJNK and PARP-1 activation
in all SHR tissues tested, but only kidney tissue experienced extrinsic apoptotic pathway
activation.

(III) A deeper understanding of the mechanisms underlying the relationship between

hypertension and progression of renal tissue damage can provide new treatment
possibilities for both renal and cardiovascular diseases.

Author Contribution: D.P. developed and led the project. All of the authors designed,
performed the experiments and analyzed the results. D.P. wrote the paper with input from
E.B. and D.L.R.

Funding: This work was supported by University of Calabria.

Competing Interests: The Authors declare that there are no competing interests
associated with the manuscript.
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Figures and Tables captions

Table 1. The dROM and BAP values for WKY and SHR rat plasma (n= 5).

Figure 1. Evaluation of lipoperoxidation levels in SHR and WKY rat tissues. Data are
means ± SE of five determinations for each animal (n= 5). Statistical differences were
evaluated by one-way ANOVA followed by Bonferroni multiple comparisons test
(***p<0.001).

Figure 2. (a) Western blotting of SOD1 in the kidney, liver and skeletal muscle extracts of
SHR and WKY rats; (a1) shows the densitometric quantification of the blots. Protein
loading was verified by using the anti-β-actin antibody. (b,c) Western blotting of monomeric
GSTP1 form (b) and dimeric GSTP1 form (c) in the kidney, liver and skeletal muscle
extracts of SHR and WKY rats; (b1) and (c1) show the densitometric quantification of the
blots. Protein loading was verified by using the anti-β-actin antibody. Data are means ± SE
of five determinations for each animal (n= 3). Statistical differences were evaluated by
one-way ANOVA followed by Bonferroni multiple comparisons test (*p < 0.05; **p < 0.025;
***p < 0.001).

Figure 3. Western blotting of pJNK (a) and PARP-1 (b) in the kidney, liver and skeletal
muscle extracts of SHR and WKY rats; (a1) and (b1) show the densitometric quantification
of the blots: (a1) pJNK double bands (pJNK1, 46 kDa and pJNK2, 54 kDa); (b1) PARP-1
active cleaved fragment (89 kDa). Protein loading was verified by using the anti-GAPDH
antibody. Data are means ± SE of five determinations for each animal (n= 3). Statistical
differences were evaluated by one-way ANOVA followed by Bonferroni multiple
comparisons test (***p < 0.001).

Figure 4. Western blotting of Caspase 8 (a) and Caspase 9 (b) in the kidney, liver and
skeletal muscle extracts of SHR and WKY rats; (a1) and (b1) show the densitometric
quantification of the blots: (a1) caspase 8 active fragments cleaved (10 kDa and 18 kDa);
(b1) caspase 9 active fragment cleaved (10 kDa). Protein loading was verified by using the
anti-GAPDH antibody. Data are means ± SE of five determinations for each animal (n= 3).
Statistical differences were evaluated by one-way ANOVA followed by Bonferroni multiple
comparisons test (*p < 0.05; ***p < 0.001).

Figure 5. Key findings in graphical form.