Role of renal heme oxygenase-1 in the response to ischemia/reperfusion in rats
preconditioned by hypoxia and/or remote ischemia
低氧預處理和/或遠端缺血預處理中第一型血基質氧化酶對於缺血再灌流
腎臟之保護角色
趙玉雯 1,3、楊芝青 2、黃菁英 6、張朝富 1,2,3、陳達隆 4、范俊雄 1、鄭劍廷 5、陳朝峰 6
台北市立聯合醫院和平院區 1,陽明大學醫學院醫學系 2、臨醫所 3,台北市立聯合醫院忠
孝院區 4,台大臨醫所 5,台大生理所 6
摘要
背景: 血基質氧化酶 (heme oxygenase, HO)，為血基質代謝分解過程中之速率決定步驟
酵素，其中第一型血基質氧化酶(HO-1)為誘發表現型。 HO-1 可受到缺氧的刺激而產
生，並可以對具傷害性之刺激產生適應性，降低組織的傷害，包括了腎臟缺血再灌流
(renal ischemia reperfusion, I/R) 之傷害。 因此在本研究探討遠端缺血預處理和/或低氧
預處理，對腎臟缺血再灌流傷害的影響，以及 HO-1 在其中所扮演的角色。
方法: 本實驗將所使用的雌性 Wistar 大鼠分成四組：分別為海平面組（sea level, SL;
101.3 千帕）；遠端缺血預處理組（remote ischemic preconditioning, SLR）；低氧預處理
組（hypoxia adapted, HA; 50.7 千帕之低氧艙四週）
；低氧預處理後進行遠端缺血預處理
組（HAR）
。各組動物再隨機分為對照亞組及接受腎臟缺血再灌流(I/R: 將腎動脈夾閉四
十五分鐘隨後再灌流四小時）亞組。
結果: 以西方墨點法觀察 HO-1 的蛋白質表現與酵素活性測定，在 SLR、HA 和 HAR
1
組都明顯的較 SL 組增加。在腎功能測定方面，則給予 HO 抑制劑 鋅前吡喀紫質
（zinc-protoporphyrin Ⅸ, ZnPP Ⅸ）後，觀察腎小球過濾率（glomerular filtration rate, GFR）
和腎血流量的變化。本實驗在接受腎臟缺血再灌流亞組中，SLR、HA 和 HAR 組在腎
小球過濾率和腎血流的表現皆優於 SL 組。
結論: 低氧預處理和/或遠端缺血預處理可誘發大鼠腎臟內 HO-1 的增加，對於腎臟缺血
再灌流所造成的傷害具有緩和的效果。
關鍵字: 血基質氧化酶，缺血預處理，低氧預處理，缺血再灌流，遠距缺血預處理
2
Role of renal heme oxygenase-1 in the response to ischemia/reperfusion in rats
preconditioned by hypoxia and/or remote ischemia
1,3
2
Yu-Wen Chao , Jing-Ying Huang6, Chih-Ching Yang , Chau-Fu Chang1,2,3, Dar-Long
4
1
5
Cheng , Jung-Siong Fan , Chiang-Ting Chien , Chau-Fong Chen
6
Taipei City Hospital-Heping Branch1, Taipei, Taiwan
2
Faculty of Medicine , College of Medicine and Institute of clinical medicine3, National
Yang-Ming University, Taipei, Taiwan
Taipei City Hospital, Zhongxiao Branch4, Taipei, Taiwan
Department of Medical Research5, National Taiwan University College of Medicine and
National Taiwan University Hospital, Taipei, Taiwan
6
Graduate Institute of Physiology , College of Medicine of National Taiwan University, Taipei,
Taiwan
Correspondence author: Prof. Chau-Fong Chen, Graduate Institute of Physiology, College of
Medicine of National Taiwan University, Taipei, Taiwan
Tel: +886-2-23889595 ext.2535
Fax: +886-2-23889622
E-mail: DAL31@tpech.gov.tw; ywchaoo@yahoo.com
3
Abstract
Background: Heme oxygenase (HO) is the rate-limiting enzyme for heme degradation, and
HO-1 is the inducible isoform. Induction of HO-1 is an adaptive response to injurious stimuli
such as renal ischemia reperfusion (I/R). We examined the effect of preconditioning on
response to renal I/R injury and the role of HO-1 in this response.
Methods: Female Wistar rats were divided into four groups: sea level (SL, 1 atm = 101.3
kPa ); hypoxic preconditioning (HA, 4 wks at 50.7 kPa); remote surgical ischemic
preconditioning (SLR); and preconditioning by hypoxia and remote surgical ischemia (HAR).
Each group was randomized into control and renal I/R subgroups.
Results: HO-1 protein expression and enzymatic activity was significantly higher in the HA,
SLR, and HAR groups than the SL group. ZnPP IX (HO inhibitor) was administered to assess
changes of renal function, as determined by glomerular filtration rate (GFR) and renal blood
flow. The renal function of rats undergoing I/R in the SLR, HA, and HAR groups were better
than that of rats in the SL group.
Conclusion: Renal preconditioning by hypoxia or a remote surgical procedure increased
renal HO-1 expression and enzymatic activity. This appears to have a protective effect on the
kidney in response to renal I/R injury.
Key Words: Heme oxygenase, hypoxic preconditioning, ischemic preconditioning, ischemic
reperfusion, remote ischemic preconditioning
4
Introduction
Preconditioning can be classified as pharmacological, ischemic, or hypoxic.
Preconditioning procedures generally protect against subsequent injury (Bonventre JV, 2002,
Kidney ischemic preconditioning. Cur Opin Nephrol Hypertens 11: 43-48.), but the
mechanisms underlying the protective effects of the different types of preconditioning are not
yet well established.
In 1986, Murry et al. (1) introduced the term “ischemic preconditioning” (IP) to describe
a treatment in which the induction of brief ischemia followed by a short period of reperfusion
produced a beneficial effect on subsequent long term ischemia. Initially, IP was reported to
affect the myocardium, but subsequent studies have identified beneficial effects in other
organs, including the kidney (2, 3). Remote ischemic preconditioning, or “preconditioning at
a distance”, is a method that protects the myocardium by applying single or repetitive remote
ischemia of the small intestine (4), kidney (5), or a hind limb (6). Recent research has shown
that brief liver ischemia can protect the kidney from ischemia reperfusion (I/R) injury by
reducing cellular oxidative stress (7).
Physiological adaptation to chronic hypoxia involves the induction of erythropoietin and
an increase in red blood cell mass, resulting in an increased oxygen-carrying capacity of
blood (8). Studies of cardiac preconditioning have demonstrated the cytoprotective effect of
hypoxic preconditioning. For example, exposure of rats to a period of hypoxia increases the
5
cardiac tolerance to subsequent ischemic insults (25). In the kidney, the glomerular filtration
rate (GFR) is usually well maintained (10), and elicits renal vasodilation due to the release of
vasodilators such as NO or CO (11).
Heme oxygenase (HO) is the rate limiting enzyme for the degradation of heme to
biliverdin, and this reaction is accompanied by the release of free iron and carbon monoxide
(CO). Biliverdin is further metabolized to bilirubin, a strong antioxidant. By means of soluble
quanylyl cyclase (sGC), CO acts as an intracellular messenger, similar to nitric oxide (NO).
There are three HO isoforms: HO-1, HO-2, and HO-3. HO-1, also known as known as heat
shock protein 32, is induced by numerous stimuli, including hypoxia, hyperoxia, angiotensin
II, and oxidative stress. The HO-1 promoter contains several regulatory sites, including
hypoxia-inducible factor-1 (HIF-1) (12). HO-1 protects against hepatic I/R injury in rats
following hypoxic preconditioning (13). In the kidney, tin-protoporphyrin (SnPP) induced
HO-1 protects the kidney from I/R injury (31). HO-1 also has a protective effect on renal
injury in several animal models of acute renal failure (12).
However, it is unknown whether HO-1 is involved in hypoxic preconditioning or remote
ischemic preconditioning of the kidney. In the present study, we examined the role of HO-1
in the protective effect of remote ischemic and/or hypoxic preconditioning on renal ischemia
insult in female Wistar rats.
6
Materials and Methods
Animal Care and Preparation
Female Wistar rats (10-12 weeks old, bodyweight: 200 to 250 g) were used for all
experiments. All animal experiments and care were performed in accordance with the Guide
for the Care and Use of Animals (National Academy Press, Washington, DC, 1996) and
allprotocols were approved by the Laboratory Animal Care Committee of the National
Taiwan University College of Medicine.
Rats were randomly divided into four groups: sea level (SL), hypoxia-adapted (HA), SL
with remote preconditioning (SLR), and HA with remote preconditioning (HAR). Then, rats
of each group were randomly divided into two subgroups. One subgroup, with 6 to 8 animals
per subgroup, was given renal ischemia/reperfusion (I/R) injury by left renal artery occlusion
for 45 min and the other subgroup were sham-operated (similar surgical procedure, but no
occlusion of the renal artery). Reperfusion was initiated by removal of the clamp for 4 h.
Remote preconditioning was performed by occlusion of the left femoral artery.
Experimental groups underwent 4 cycles of 10 min left femoral artery occlusion and 10 min
of reperfusion, followed by 2 h of reperfusion prior to data collection. HA rats were placed in
a hypobaric chamber (50.7 kPa, equivalent to an altitude of ~5500 m) for 15 h per day for 4
weeks prior to data collection.
Western Blot Analysis
7
After functional measurements and sacrifice of the rats via urethane (see below), left
kidneys were removed and the renal cortexes and medullas were isolated. For detection of
HO-1 immunoreactive proteins, the renal medulla and cortical tissue were homogenized in
ice-cold lysis buffer (20 mM Tris HCl, 137.5 mM NaCl, 1% Triton X-100, pH 8.0) with
protease inhibitors (10 µg/mL aprotinin, 1 mmol/L phenyl sulfonyl fluoride). The lysate was
mixed with an equal volume of double-strength sample buffer (250 mM Tris-HCl, 4%
sodium dodecyl sulfate, 10% glycerol, 2% β-mercaptoethanol, 0.006% bromophenol blue, pH
6.8), boiled for 10 min, sonicated for 30 s, then centrifuged at 10,000 g for 10 min. The
resulting supernatants were collected and subjected to 12% SDS polyacrylamide gel
electrophoresis. Proteins were transferred to nitrocellulose membranes using a Semiphor unit
(Hoefer Scientific Instruments, San Francisco, CA), and blocked in Tris-buffered saline (TBS)
with 0.05% polyoxyethylene-sorbitan monolaureate (Tween 20; TBS-T buffer) that contained
5% w/v nonfat dry milk powder. Then, the membrane was incubated with anti–HO-1 primary
antibodies (Stressgen Biotechnologies, Corp. BC Canada.; 1:500 dilution in TBS-T with 5%
dry milk powder) overnight at 4°C. After TBS-T washing for 3 times, the membrane was
incubated with goat biotinylated anti-rabbit IgG secondary antibodies (Vector, Burligame,
CA, USA.; 1:200 dilution) for 1 h at room temperature. An enhanced chemiluminescence
western blotting system (peroxidase substrate kit, DAB, Vector Laboratories, Inc.,
8
Burlingame, CA. USA) was used for detection. Quantification of protein signals was
performed by computer-assisted densitometry.
HO Activity Assay
HO activity in the renal medulla and cortical cells were measured by generation of
bilirubin. Briefly, microsomes from harvested cells were added to a reaction mixture that
contained NADPH, rat liver cytosol (a source of biliverdin reductase), and the substrate
hemin. This reaction was performed at 37°C in the dark for 1 h, and terminated by the
addition of 1 mL of chloroform. The extracted bilirubin was calculated as the difference in
absorbance between 464 nm and 530 nm (Δε = 40 mM-1 cm-1). HO activity is expressed as
picomoles/h/mg protein.
Surgical Procedures
Rats were anesthetized with i.p. urethane (1 g/kg bodyweight). Polyethylene cannulas
were placed in the trachea to aid ventilation. The right femoral vein was used for infusion of a
saline solution with inulin (Sigma Chemical Co., MO, USA) (0.25 mg/min/kg bodyweight);
the left femoral vein was used for administration of anesthesia; the left femoral artery was
used for blood sampling; the left carotid artery was used for measurement of mean arterial
pressure; and the left ureter was used for urine collection. The left kidney was exposed by a
frank incision and a flow probe (Probe# 0.5VBB517, Transonic Systems Inc., Ithaca, NY,
USA) was placed around the left renal artery for measurement renal blood flow, which was
9
recorded continuously with a T206 recording system (Transonic Systems), and displayed on
the PowerLab/16S Data Acquisition System (ADI Instruments, Pty Ltd, Castle Hill,
Australia).
Effect of HO inhibition on renal function
Our experiments were designed to determine whether inhibition of HO affects the renal
function of SL, SLR, HA, and HAR rats and to determine whether these rats experience renal
I/R. Animals were surgically prepared as described above, and renal function was measured
in the basal stage, vehicle stage, and in the presence of zinc protoporphoryn IX (ZnPP IX, an
HO inhibitor). The inulin-containing saline was infused continuously in the 60-min control
period and throughout all three stages. I/R was assessed after the control period and was
followed in the basal stage, vehicle stage, and ZnPP IX stage . Each stage was performed for
1 h (consisting of two 30-min periods), and renal function was assessed by measurement of
GFR and renal blood flow. ZnPP IX was infused at dose of 10 μmol/kg body weight via the
left femoral vein during two consecutive 30-min periods (ZnPP IX stage) in the sham and I/R
subgroups. The GFR was estimated from the measured renal clearance of inulin. For
assessment of GFR, a sustained inulin-containing saline solution was infused at a rate of 0.25
mg/min/kg bodyweight via the right femoral vein. Aterial blood samples were taken from the
left femoral arterial catheter at the mid-point of 30-min period in each 1 h stage. The left
ureter was cannulated with a PE-10 tube for urine collection from the left kidney.
10
Data Analysis
The functions of renal function were averaged over each stage. All data are expressed as
the means ± SEMs. Statistical analysis was performed using the Newman-Keuls test of
analysis of variance for multiple comparisons. Student’s t test was used for paired
comparisons between groups. A P value of 0.05 was considered statistically significant.
11
Results
Effect of remote ischemic and/or hypoxic preconditioning on renal HO-1
Our western blotting results indicate that remote ischemic preconditioning and hypoxic
preconditioning significantly increased the expression of HO-1 protein in the renal medulla
and cortex of rats (Figs. 1 and 2). In particular, rats in the SL group (lanes 1-3) had
significantly lower expression of renal medullar HO and cortical HO than rats in the SLR,
HA, and HAR groups.
In agreement with these results, remote ischemic and hypoxic preconditioning also
increased the HO enzymatic activity in these two renal regions (Fig. 3). Rats preconditioned
by remote ischemia (SLR, HO activity: 100.6 ± 25.0 pmol/mg protein/hr) and/or 4 weeks of a
hypoxic environment (HA, HO activity: 96.3 ± 21.4 pmol/mg protein/hr; HAR, HO activity:
100.5 ± 15.9 pmol/mg protein/hr) had significantly higher HO activity than rats in the SL
group.
Effect of remote ischemic and/or hypoxic preconditioning on renal function during
ischemia/reperfusion
In subgroups that did not undergo renal I/R, the basal levels of GFR in SL, SLR, HA and
HAR rats were not significantly different (Table 1). However, after intravenous infusion of
ZnPP IX, the GFRs of SLR, HA, and HAR rats were significantly lower than the GFR of SL
rats.
12
After 45 min of renal I/R, the levels of GFR decreased significantly in all four subgroups
(Fig. 4). However, the SLI subgroup had significantly lower GFR than the SLRI, HAI, and
HARI subgroups (Fig. 4). Furthermore, rats in the SLRI, HAI and HARI subgroups were
more sensitive to administration of ZnPP IX than rats in the SLI subgroup (Fig. 5). In
agreement with these results, renal blood flow decreased more in the SLI group than in the
SLRI, HAI, and HARI groups after renal I/R (Fig. 6 and Fig. 7).
Taken together, our results indicate that preconditioning by hypoxia or occlusion of the
left femoral artery reduce the injurious effects of subsequent I/R injury. Inhibition of HO
activity by ZnPP IX reduced the protective effects of preconditioning, suggesting that HO
plays a role in the protective effect of preconditioning.
13
Discussion
The main findings of our study of a rat I/R model are: (i) remote ischemic and/or hypoxic
preconditioning increased the level of HO-1 protein and enzyme activity, suggesting a role
for HO-1 in the cellular response to preconditioning; (ii) remote ischemic and/or hypoxic
preconditioning protects the kidney from subsequent I/R injury; and (iii) the protective effects
induced by remote ischemic and/or hypoxic preconditioning are reduced by inhibition of
HO-1 enzyme activity, further supporting a role for HO-1 in the response to preconditioning.
The locally protective effects of IP are well known (3). In particular, Riera et al. (14)
reported that 5, 10, 15 or 20 min of renal ischemia preconditioning, followed by 10 min of
reperfusion, results in protection against 40 min of renal ischemia, as reflected by reduced
levels of serum creatinine. Another study found that the renal protective effect of IP may
result from a decrease in renal cell apoptosis that is mediated by the production of NO or HO
(15). Recently, it has been suggested that IP also affects more distant organs (4, 5, 6). Ates et
al. (7) demonstrated functional protection of the kidney when rats were given remote
ischemic preconditioning by brief hepatic ischemia. They also found that preconditioning
reduced the renal cellular oxidative stress that followed renal ischemia injury, and that
preconditioning reduced the increases of BUN and creatinine that normally follow renal I/R.
Other studies of IP have indicated that receptor-mediated triggers may include adenosine
(16), opioid (17), bradykinin (18), and NO (19), the postreceptor protein kinase C (PKC)
14
signaling pathway (20), or an end-effector, such as ATP-sensitive potassium channels (20).
Our study demonstrated a direct protective role of HO-1 in renal ischemic preconditioning.
It was first shown in 1958 that ischemic tolerance of the heart increased following
long-term exposure of animals to intermittent hypoxia (21). The cardiac protective effects of
HA may persist longer than other hypoxia-induced adaptive responses, such as polycythemia
and pulmonary hypertension (22). Walker et al. (11) reported that long-term hypoxia induced
HO-1 and that CO (a metabolic product of HO) had vasodilation effects on the kidney.
Jernigan et al. (23) found that vascular HO-1 expression is elevated after 48 h of hypoxia and
that administration of ZnPP IX dramatically decreased renal blood flow. They also found that
the HO-1 renal protein level was elevated after 4 weeks of hypoxia, but that 48 h of hypoxia
had no effect. It has been suggested that different tissues undergo different changes in HO
expression following hypoxia, and that HO-1 has numerous roles in renal protection.
Thus, in contrast to remote ischemic renal preconditioning, the mechanism of cardiac
protection induced by HA does not involve the activation of adenosine receptors (24) or the
PKC pathway (25), suggesting a different signaling pathway. Recent studies have examined
the protective mechanisms of HA. Asemu et al. (26) found that the mitochondrial K-ATPase
plays a role in the protection afforded by HA. Ladilov et al. (27) reported that HP exerts
PKC-independent protection and that protein phosphatase 1 is a possible mediator. Sasaki et
al. (28) proposed that HA triggers angiogenesis and thereby enhances functional reserve of
15
the ischemic heart. In agreement with our results, Lai et al. (13) found that HO-1 is an
important mediator in the protection effects of HA on the liver.
Hypoxic stress is known to induce hypoxia-inducible factor-1 (HIF-1), a nuclear factor that
is required for hypoxic activation of transcription of erythropoietin, inducible nitric oxide
synthase, and vascular endothelial growth factor (29). In long-term hypoxia, HIF-1 regulates
HO-1 gene expression by binding to hypoxia response elements on the enhancer sequences
(29). Our study, which found that HO-1 is an important mediator of the protective effects
induced by remote ischemic preconditioning, suggests that HIF-1 may also be involved in
remote ischemic preconditioning, although there is no direct evidence for this at present.
Botros et al. (30) demonstrated that induction of renal HO-1 by hemin and SnCl2
attenuated afferent arteriolar autoregulation by increasing CO production from tubular
epithelial cells. Renal autoregulation maintains GFR and the afferent arterioles are the vessels
that provide the most resistance. Myogenic tone and tubuloglomerular feedback, which
contribute to renal autoregulation, were also modulated by HO-1. Another study (12) found
that induction of HO-1 by Sn-protoporphrin also had a protective effect on subsequent renal
I/R.
The renal medulla is most susceptible to hypoxia because the countercurrent mechanism of
the kidney preserves high medullary solute concentrations, resulting in low concentrations of
oxygen. Thus, anaerobic metabolism is predominant in medullary tissues, and the lowest PO2
16
is in the inner medulla. In rats, Zou (31) reported higher HO-1 expression in the renal
medulla and that CO (its metabolic product) controls renal medullary circulation. Our results
indicate that GFR and RBF decreased after renal I/R, and that remote ischemic and/or
hypoxic preconditioning protected the kidney from I/R injury. The differences in reduction
between GFR and RBF may be due to the differences in expression of vascular HO-1 and
renal HO-1.
HO-1 induction has been shown to protect against renal failure in numerous experimental
animal models, including the rat renal I/R model (12). Induction of renal HO-1 increases the
levels of CO and bilirubin, decreases NADPH oxidase-mediated oxidative stress, and inhibits
20-HETE synthase and thromboxane synthase (30). Shimizu et al. (9) demonstrated
significant induction of HO-1 in an animal model of unilateral renal I/R injury and that
inhibition of HO activity by Sn-mesoporphrin increased the level of microsomal heme and
aggravated renal injury.
The mechanism by which HO-1 induces cytoprotection against I/R injury of the kidney are
not yet well-established, but it has been shown that cells which over-express HO-1 have low
levels of free iron due to an increase of ferritin and excretion of iron into the extracellular
space (32). Cellular iron contributes to the formation of free radicals, resulting in damage of
DNA, proteins, and lipids (33). Thus, the elimination of iron from the cell may be the
mechanism by which HO-1 protects against oxidative stress. In addition, CO (a product of
17
HO-1) also confers protection against cellular injury (34, 35). It has been postulated that CO,
like NO, modulates intrarenal vessel tone and corrects microcirculatory dysfunction, leading
to increased blood flow to kidney (36). Bilirubin, another product of heme metabolism, is a
potent antioxidant and attenuates the inflammatory response by preventing oxidant-induced
microvascular leukocyte adhesion (12).
In summary, our study of a rat model indicates that remote ischemic or/and hypoxic
preconditioning leads to increased expression of HO, and protects against subsequent I/R
injury of the kidney. Our results suggest that HO-1 plays a role in the protective mechanism
of remote ischemic or/and hypoxic preconditioning. We suggest that future studies examine
the mechanism by which HO-1 confers protection against I/R injury.
18
Table 1
n
Baseline
Vehicle
ZnPP
(ml/min/g)
(ml/min/g)
(ml/min/g)
SL
7
0.58 ± 0.02
0.58 ± 0.02
0.54 ± 0.02
HA
6
0.65 ± 0.02†
0.64 ± 0.02
0.32 ± 0.02**††
SLR
6
0.59 ± 0.02
0.57 ± 0.02
0.31 ± 0.02**††
HAR
8
0.46 ±0.01††
0.45 ± 0.01††
0.27 ± 0.02**††
19
Figure 1.
(A)
12 11
10
9
8
7
6
5
4
3
2
1
HO-1
32kDa
Actin
(B)
Densitomeric unit
10
＊＊
8
＊＊
＊＊
6
4
2
0
SL
SLR
HA
HAR
20
Figure 2.
12
(A)
11
10
9
8
7
6
5
4
3
2
1
HO-1
32kDa
Actin
(B)
6
＊＊
Densitomeric unit
5
＊＊
＊＊
4
3
2
1
0
SL
SLR
HA
HAR
21
Figure 3.
Medulla
Cortex
(pmol/h/mg protein)
140
＊＊
＊＊
120
＊＊
＊＊
＊＊
100
＊＊
80
60
40
20
0
SL
HA
SLR
HAR
22
Figure 4.
(%)
0
SLI
HAI
SLRI
HARI
Glomerular filtration rate (% control)
-20
-40
＊＊
-60
＊＊
＊＊
-80
-100
23
Figure 5.
(%)
0
SLI
HAI
SLRI
HARI
Glomerular filtration rate (% control)
-20
-40
-60
-80
＊
＊
＊
-100
24
Figure 6.
(%)
0
SLI
Renal blood flow (% control)
HAI
SLRI
＊
＊
HARI
-20
-40
＊
-60
-80
-100
25
Figure 7.
(%)
0
SLI
HAI
Renal blood flow (% control)
SLRI
HARI
＊
＊
-20
-40
-60
-80
26
Table 1. Influence of zinc protoporphyrin IX (ZnPP IX, 10 μmol/kg bw) on GFR of rats
treated by hypoxic and/or remote ischemic preconditioning. SL: sea level (101.3 kPa); HA:
hypoxia (50.7 kPa) adapted, SLR: SL + remote preconditioning; HAR: HA + remote
preconditioning. Values are means ± SEs. *Each group after administration of ZnPP IX
compared to Baseline (**P < 0.01). †HA, SLR, and HAR compared to SL in the same period
(†P < 0.05, ††P < 0.01).
Fig. 1. Effects of hypoxic and/or ischemic preconditioning on HO-1 expression in rat renal
medulla. (A) Western blots of HO-1 protein expression (3 rats per group). SL: Lanes 1-3;
SLR: Lanes 4-6; HA: Lanes 7-9; HAR: Lanes 10-12. (B) Quantitation of HO-1 protein levels.
In this and all subsequent figures, SL: sea level (1.0 atm); HA: hypoxia (0.5 atm) adapted;
SLR: SL + remote preconditioning; HAR: HA + remote preconditioning. **P < 0.01 vs. SL.
Fig. 2. Effects of hypoxic and/or ischemic preconditioning on HO-1 expression in rat renal
cortex. (A) Western blots for HO-1 protein expression (3 rats per group). SL: Lanes 1-3; SLR:
Lanes 4-6; HA: Lanes 7-9; HAR: Lanes 10-12. (B) Quantitation of HO-1 protein levels. **P
< 0.01 vs. SL.
Fig. 3. HO enzymatic activity in the renal medulla and cortex of SL, HA, SLR, and HAR rats.
**P < 0.01 vs. SL.
Fig. 4. Effect of renal ischemia/reperfusion on glomerular filtration rate (GFR) of rats
preconditioned by hypoxia and/or remote preconditioning. **P < 0.01 vs. SLI.
Fig. 5. Effect of renal ischemia/reperfusion and zinc protoporphyrin IX (ZnPP IX, 10
27
μmol/kg bw) on the GFR of rats preconditioned by hypoxia and/or remote preconditioning.
*P < 0.05 vs. SLI.
Fig. 6. Effect of renal ischemia/reperfusion on the renal blood flow of rats preconditioned by
hypoxia and/or remote preconditioning. *P < 0.05 vs. SLI.
Fig. 7. Effect of renal ischemia/reperfusion and zinc protoporphyrin IX (ZnPP IX, 10
μmol/kg bw) on the renal blood flow of rats preconditioned by hypoxia and/or remote
preconditioning. *P < 0.05 vs. SLI.
28
References
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