Clinical and Experimental Pharmacology and Physiology (2015) 42, 278–286
doi: 10.1111/1440-1681.12350
ORIGINAL ARTICLE
Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion-induced renal injury in rats Shuhei Kobuchi,*† Ryosuke Tanaka,† Takuya Shintani,† Rie Suzuki,† Hidenobu Tsutsui,†‡ Mamoru Ohkita,† Yasuo Matsumura† and Kazuhide Ayajiki* *Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Hyogo, †Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences, Takatsuki, and ‡ Laboratory of Clinical Pharmacology, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Osaka, Japan
ABSTRACT Excitation of the renal sympathetic nervous system is important for the development of ischaemic acute kidney injury (AKI) in rats. We reported that intravenous treatment with GABA has preventive effects against ischaemia/ reperfusion (I/R)-induced renal dysfunction with histological damage in rats; however, the mechanisms underlying these effects on renal injury remain unknown. Thus, the aim of the present study was to clarify how GABA mechanistically affects ischaemic AKI in rats. Ischaemic AKI was induced in rats by clamping the left renal artery and vein for 45 min and then reperfusing the kidney to produce I/Rinduced injury. Treatment with the GABAB receptor antagonist CGP52432 (100 nmol/kg, i.v., or 1 nmol/kg, i.c.v.) abolished the suppressive effects of 50 lmol/kg, i.v., GABA on enhanced renal sympathetic nerve activity (RSNA) during ischaemia, leading to elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with 0.5 lmol/ kg GABA or i.v. treatment with 1 lmol/kg baclofen, a selective GABAB receptor agonist, prevented the I/R-induced renal injury equivalent to i.v. treatment with GABA. Conversely, i.v. treatment with 10 lmol/kg bicuculline, a GABAA receptor antagonist, failed to affect the preventive effects of GABA against ischaemic AKI. We therefore concluded that GABAB receptor stimulation in the central nervous system, rather than peripheral GABAB receptor stimulation, mediates the preventive effect of GABA against ischaemic AKI by suppressing the enhanced RSNA induced by renal ischaemia. Key words: acute kidney injury, GABA, ischaemia/ reperfusion, renal sympathetic nerve activity.
Correspondence: S Kobuchi, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe, Hyogo 650-8530, Japan. Email: [email protected] Received 28 August 2014; revision 6 November 2014; accepted 2 December 2014. © 2014 Wiley Publishing Asia Pty Ltd
INTRODUCTION Acute kidney injury (AKI) is commonly encountered in the hospital setting and is associated with a high rate of mortality.1 Ischaemia followed by reperfusion is one of the major causes of AKI,2 with reperfusion of previously ischaemic renal tissue initiating a complex series of cellular events that result in injury and the eventual death of renal cells due to a combination of apoptosis and necrosis.3 Several causative factors, including ATP depletion, reactive oxygen species, phospholipase activation, neutrophil infiltration and vasoactive peptides, reportedly contribute to the pathogenesis of this renal damage;4,5 however, the mechanisms underlying ischaemia/reperfusion (I/R)-induced renal injury are not fully understood. We found that enhancement of renal sympathetic nerve activity (RSNA) and its consequent effect on noradrenaline (NA) overflow from nerve terminals are involved in the development of I/R-induced AKI and that RSNA is significantly augmented during renal ischaemia in rats.6 In addition, we reported that ischaemic AKI is ameliorated by ganglionic blockade and that this effect is accompanied by suppression of elevated NA levels in the renal vein after reperfusion.7 In addition to the central nervous system (CNS), GABA, an inhibitory neurotransmitter, is also found in peripheral tissues8 and is known to suppress electrical renal nerve stimulationinduced NA release from rat isolated kidney without affecting basal release.9 These findings indicate that GABA can modulate peripheral as well as CNS neurotransmission. It has been reported that GABA has renoprotective effects against glycerol-induced AKI,10 and we have further shown that pre-ischaemic treatment with GABA suppresses the enhancement of RSNA and consequent elevation in NA levels in the renal vein observed in ischaemic AKI rats, suggesting that GABA has renoprotective effects against I/R-induced renal injury.11 However, no studies to date have elucidated the precise mechanisms underlying the beneficial action of GABA on AKI and/or I/R-induced renal injury, especially in terms of receptor and the sites of action. Therefore, in the present study we investigated the effects of i.v. treatment with bicuculline, a GABAA receptor antagonist, and CGP52432, a GABAB receptor antagonist, on the renoprotective effects of GABA to clarify the receptor subtypes involved. Similarly, to clarify the site of GABA action, we
GABA and acute kidney injury examined the effects of i.c.v. treatment with CGP52432 on the renoprotective effects of GABA. Finally, we examined the effects of i.v. treatment with baclofen, a GABAB receptor agonist, and i.c.v. treatment with GABA on ischaemic AKI.
RESULTS Effects of i.v. bicuculline or CGP52432 on GABA-induced improvements in ischaemic AKI Pre-ischaemic treatment of GABA (50 lmol/kg, i.v.) markedly suppressed the enhanced RSNA during the ischaemic period (Fig. 1a,b,d). This suppressive effect was inhibited by the selective GABAB receptor antagonist CGP52432 (10 and 100 nmol/ kg, i.v.) in a dose-dependent manner (Fig. 1c,d). Conversely, treatment with the selective GABAA receptor antagonist bicuculline (1 and 10 lmol/kg, i.v.) failed to attenuate the suppressive effect of GABA on RSNA (Fig. 1d). As shown in Fig. 2, the renal function of rats subjected to 45 min ischaemia showed marked deterioration when measured 29 h after reperfusion. Compared with sham-operated rats,
279
vehicle-treated AKI rats showed significant increases in blood urea nitrogen (BUN), plasma creatinine (PCr) concentrations and urine flow (UF) and a significant decrease in creatinine clearance (CCr), indicating renal dysfunction. Intravenous injection of GABA (50 lmol/kg) to ischaemic AKI rats markedly attenuated the I/R-induced renal dysfunction and this improvement was reversed by 100 nmol/kg, i.v., CGP52432. However, the renoprotective effects of GABA were not affected by bicuculline (1 and 10 lmol/kg) or 10 nmol/kg CGP52432. In addition, we confirmed that 10 lmol/kg, i.v., bicuculline alone had no effect on I/ R-induced renal injury (data not shown). Histological examination revealed severe lesions in the kidney of vehicle-treated AKI rats 29 h after reperfusion. These changes were characterized by proteinaceous casts in the tubules of the inner medulla (Fig. 3b), medullary congestion and haemorrhage in the outer zone of the inner medullary stripe (Fig. 3g) and tubular necrosis in the outer zone of the outer medullary stripe (Fig. 3l) compared with kidneys from sham-operated rats (Fig. 3a,f,k). Intravenous injection of GABA to ischaemic AKI rats significantly attenuated the development of all lesions (Table 1; Fig. 3c,h,m). In addition, 100 nmol/kg CGP52432 (Fig. 3e,j,o) abolished these
(a)
Integrated RSNA (%)
RSNA (μV)
AKI + vehicle, i.v. AKI + GABA 50 μmol/kg, i.v.
100
AKI + GABA 50 μmol/kg, i.v., + bicuculline 1 μmol/kg, i.v. AKI + GABA 50 μmol/kg, i.v., + bicuculline 10 μmol/kg, i.v.
100 Ischaemia
AKI + GABA 50 μmol/kg, i.v., + CGP52432 10 nmol/kg, i.v.
5 min
Vehicle, i.V.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 100 nmol/kg, i.v.
RSNA (μV)
(b) (d)
100
150
100 Ischaemia GABA 50 μmol/kg, i.v.
5 min
Integrated RSNA (%)
RSNA (μV)
(c)
Δ Integrated RSNA ( % )
Integrated RSNA (%)
††
100
††
50
††
††
††
††
**
0
** **
100 -50 sal Ba
100 Ischaemia GABA 50 μmol/kg, i.v.
-10
-5
5 min
0
5
15
25
35
45
Ischaemia Time (min)
CGP52432 100 nmol/kg, i.v.
Fig. 1 Typical responses of renal sympathetic nerve activity (RSNA) and integrated RSNA to injection of (a) vehicle (0.9% saline, i.v.), (b) GABA (50 lmol/kg, i.v.) or (c) GABA (50 lmol/kg, i.v.) + CGP52432 (100 nmol/kg, i.v.) during the 45 min ischaemic period in anaesthetized rats. (d) Changes in % integrated RSNA in response to injection of vehicle or GABA, alone or in combination with bicuculline or CGP52432. Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). ††P < 0.01 compared with basal (vehicle-treated acute kidney injury (AKI) rats only). **P < 0.01.
© 2014 Wiley Publishing Asia Pty Ltd
S Kobuchi et al.
280
AKI + GABA 50 μmol/kg, i.v., + bicuculline 1 μmol/kg, i.v. Sham
AKI + GABA 50 μmol/kg, i.v., + bicuculline 10 μmol/kg, i.v.
AKI + vehicle, i.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 10 nmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 100 nmol/kg, i.v.
(a)
(b)
120
3
††
††
80
** **
60 40
PCr (mg/dL)
BUN (mg/dL)
100
**
**
**
2
** **
1
**
**
**
20 0
CCr (mL/min per kg)
6
(d)
**
5 4
** **
3 2
*
** ††
UF (μL/min per kg)
(c)
0
††
120 100 80
**
60 40
1
20
0
0
**
Fig. 2 Effects of intravenous treatment with bicuculline or CGP52432 on renal protection induced by intravenous treatment with GABA. Parameters of renal function were evaluated 29 h after reperfusion: (a) blood urea nitrogen (BUN), (b) plasma creatinine (PCr), (c) creatinine clearance (CCr) and (d) urinary flow (UF). Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). *P < 0.05, **P < 0.01 compared with vehicle (i.v.)-treated acute kidney injury (AKI) rats; ††P < 0.01 compared with GABA (50 lmol/kg, i.v.)-treated AKI rats.
GABA-induced improvements, whereas 10 lmol/kg, i.v., bicuculline had no effect on the effects of GABA (Fig. 3d,i,n). Effects of i.c.v. CGP52432 on GABA-induced improvements in ischaemic AKI As shown in Fig. 4, the suppressive effect of GABA (50 lmol/ kg) on RSNA was partially attenuated by 0.1 nmol/kg, i.c.v., CGP52432, whereas 1 nmol/kg, i.c.v., CGP52432 almost abolished the effects of GABA. Similarly, the GABA-induced improvements in renal dysfunction were partially attenuated by 0.1 nmol/kg, i.c.v., CGP52432, but were almost abolished by 1 nmol/kg, i.c.v., CGP52432 (Fig. 5). Bicuculline (10 nmol/kg, i.c.v.) failed to affect the GABA-induced renoprotective effects (data not shown). Effects of i.v. baclofen or i.c.v. GABA on ischaemic AKI The enhancement of RSNA during the ischaemic period was markedly suppressed by both baclofen (0.2 and 1 lmol/kg, i.v.),
a selective GABAB receptor agonist, and GABA (0.1 and 0.5 lmol/kg, i.c.v.) in a dose-dependent manner (Fig. 6). As indicated in Table 2, baclofen (0.2 and 1 lmol/kg, i.v.) significantly prevented the I/R-induced renal dysfunction, with the higher dose of baclofen (1 lmol/kg) having equivalent effectiveness to GABA (50 lmol/kg). In addition, GABA (0.1 and 0.5 lmol/kg, i.c.v.) dose-dependently attenuated the I/R-induced renal dysfunction, with the higher dose similarly effective to treatment 50 lmol/kg GABA (Table 2).
DISCUSSION The renal sympathetic nervous system and circulating catecholamines have been implicated in the pathogenesis of AKI based on pharmacological blockade of the sympathetic nervous system having a protective effect against AKI.6,12 Recently, we found that i.v. treatment of ischaemic AKI rats with GABA (10 and 50 lmol/kg) also suppressed the enhancement of RSNA during ischaemia and the increased NA overflows after reperfusion.11 In the present study, we investigated the mechanisms underlying
© 2014 Wiley Publishing Asia Pty Ltd
GABA and acute kidney injury
Proteinaceous casts in tubules (a)
281
Medullary congestion
Tubular necrosis
(f)
(k)
(b)
(g)
(l)
(c)
(h)
(m)
(d)
(i)
(n)
(e)
(j)
(o)
Sham 100μm
AKI + vehicle, i.v.
AKI + GABA 50 μmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v., + bicuculline 10 μmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 100 nmol/kg, i.v. Fig. 3 Light microscopy of the inner zone (a–e), the outer zone inner stripe (f–j) and the outer zone outer stripe (k–o) of the medulla in the kidney of acute kidney injury (AKI) rats treated with i.v. injection of vehicle (b,g,l), GABA alone (c,h,m), GABA + bicuculline (d,i,n) and GABA + CGP52432 (e,j,o) 29 h after reperfusion, as well as in sham-operated rats (a,f,k). Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Severe proteinaceous casts in tubules (b), congestion and haemorrhage (g) and tubular necrosis (l) were observed in vehicle (i.v.)-treated AKI rats. Arrows indicate severe proteinaceous casts in tubules (b–e), medullary congestion (g–j) and tubular necrosis (l,o). Original magnification 9200.
these renoprotective effects of GABA against AKI with respect to receptor subtypes and sites of action. There are two known receptor subtypes for GABA: GABAA and GABAB receptors. The GABAA receptor is coupled to
ligand-gated Cl channels, whereas the GABAB receptor is coupled to G-proteins.13,14 The GABAA receptors are widely distributed within the mammalian CNS and exhibit a differential topographical distribution,15 whereas GABAB receptors are
© 2014 Wiley Publishing Asia Pty Ltd
S Kobuchi et al.
282
Table 1 Effects of 10 lmol/kg, i.v., bicuculline or 0.1 lmol/kg, i.v., CGP52432 on renal tissue protection induced by 50 lmol/kg, i.v., GABA, as evaluated by histological grading‡ Experimental group AKI AKI AKI AKI
+ + + +
Proteinaceous casts in tubules
vehicle, i.v. GABA GABA + bicuculline GABA + CGP52432
3.50 1.17 1.67 2.83
Medullary congestion
0.34 0.31** 0.33** 0.31††
3.50 1.83 1.67 3.50
Tubular necrosis
0.22 0.17** 0.42** 0.22††
3.83 1.50 1.50 3.50
0.17 0.22** 0.34** 0.22††
Data are the meanSEM (n = 6). *P < 0.05, **P < 0.01 compared with vehicle (i.v.)-treated acute kidney injury (AKI) rats; ††P < 0.01 compared with GABA-treated AKI rats. ‡ Tissues were graded as follows: 0, no change; 1, mild; 2, moderate; 3, severe; 4, very severe (see text for details).
RSNA (μV)
(a)
AKI + vehicle, i.v. + vehicle, i.c.v. 100
Integrated RSNA
AKI + GABA 50 μmol/kg, i.v. AKI + GABA 50 μmol/kg, i.v., + CGP52432 0.1 nmol/kg, i.c.v. 100
Ischaemia
AKI + GABA 50 μmol/kg, i.v., + CGP52432 1 nmol/kg, i.c.v. 5 min
GABA 50 μmol/kg, i.v. CGP52432 1 nmol/kg, i.c.v.
(b)
150
Δ Integrated RSNA ( % )
†† 100
†† ††
50
††
††
††
**
** **
0
Ba sa l
-50 -10
-5
0
5
15
25
35
45
Ischaemia Time (min) Fig. 4 (a) Typical responses of renal sympathetic nerve activity (RSNA) and integrated RSNA following injection of GABA (50 lmol/kg, i.v.) + CGP52432 (1 nmol/kg, i.c.v.) during the 45 min ischaemic period in anaesthetized rats. (b) Changes in % integrated RSNA in response to injections of vehicle and GABA, alone or in combination with CGP52432. Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). ††P < 0.01 compared with basal (vehicle-treated acute kidney injury (AKI) rats only). **P < 0.01.
widely distributed within the CNS as well as in peripheral autonomic terminals.16 In the present study, we evaluated which of these receptor subtypes is involved in the suppressive effects of GABA on I/R-induced enhancement of RSNA and against the functional and histological renal injury in rats. Our findings
clearly indicate that the suppressive effects of GABA were abolished by blockade of GABAB receptors (CGP52432 treatment), but not by blockade of GABAA receptors (bicuculline treatment). In addition, i.v. baclofen, a GABAB receptor agonist, prevented the I/R-induced renal injury by suppressing the
© 2014 Wiley Publishing Asia Pty Ltd
GABA and acute kidney injury
283
Sham
AKI + GABA 50 μmol/kg, i.v.
AKI + vehicle, i.v., + vehicle, i.c.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 0.1 nmol/kg, i.c.v. AKI + GABA 50 μmol/kg, i.v., + CGP52432 1 nmol/kg, i.c.v.
(a) 120
(b)
3
††
††
100 80
PCr (mg/dL)
BUN (mg/dL)
* ** 60 40
**
**
2
** 1
**
20 0 6
**
(d) 120
5 4
**
3 2
††
UF (μL/min per kg)
CCr (mL/min per kg)
(c)
0
100 80 60
** 40
1
20
0
0
Fig. 5 Effects of i.c.v. CGP52432 on renal protection induced by GABA. Parameters of renal function were evaluated 29 h after reperfusion: (a) blood urea nitrogen (BUN), (b) plasma creatinine (PCr), (c) creatinine clearance (CCr) and (d) urinary flow (UF). Vehicle or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). *P < 0.05, **P < 0.01 compared with vehicle (i.v.)-treated acute kidney injury (AKI) rats; ††P < 0.01 compared with GABA (50 lmol/kg, i.v.)-treated AKI rats.
enhancement of RSNA. These findings suggest that activation of GABAB, but not GABAA, receptors is responsible for protecting against the development of I/R-induced renal dysfunction. A recent study found an increased concentration of GABA in the cerebrospinal fluid following i.v. infusion of GABA itself,17 indicating that i.v. injection of GABA may suppress the enhanced RSNA during ischaemia by acting on the CNS. In the present study, we found that i.c.v. treatment with CGP52432 almost abolished the suppressive effects of GABA on I/R-induced enhancement of RSNA and renal injury. Furthermore, we demonstrated that i.c.v. GABA (0.1 and 0.5 lmol/kg) prevented the I/ R-induced renal injury by suppressing the enhanced RSNA. However, i.v. treatment with the same dose of GABA (0.5 lmol/ kg) had no significant effect on I/R-induced renal injury (data not shown). Together, these results suggest that GABA exerts its preventive action against postischaemic AKI by inhibiting central sympathetic outflow. The paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) are responsible for controlling sympathetic outflow,18,19 and microinjection of GABA or a GABA receptor agonist into either of these structures decreases blood pressure
by inhibiting sympathetic nerve activity.20,21 In addition, Durgam et al.22 reported that microinjection of a GABA receptor agonist into the nucleus tractus solitarius (NTS), a central baroreflex regulator, increased blood pressure by enhancing sympathetic outflow. Therefore, suppression of RSNA by GABA seems to be mediated through the RVLM and/or PVN rather than the NTS. Several mechanisms have been proposed for the effects of GABA in suppressing the peripheral sympathetic nervous system, including ganglionic blockade and/or inhibition of transmitter release from the nerve terminals.9,23,24 In the present study, we did not examine whether GABA suppressed NA overflow from peripheral sympathetic nerves because we did not use isolated tissues. Indeed, in the peripheral sympathetic nerves of AKI rats, systemically applied GABA may prevent I/R-induced renal injury by inhibiting NA release from nerve terminals, even after abrogating the GABA effect using CGP52432. However, the present study revealed that systemically applied GABA failed to prevent I/R-induced renal injury in rats administered i.c.v. CGP52432. These findings suggest that the renoprotective effect of GABA appears to be much more dependent on CNS neurotransmission than that mediated through peripheral sympathetic nerves.
© 2014 Wiley Publishing Asia Pty Ltd
Ischaemia
Δ Integrated RSNA ( % )
150
(d)
100
††
100 Ischaemia GABA 0.5 μmol/kg, i.c.v.
††
††
100
5 min
Baclofen 1 μmol/kg, i.v. (c)
RSNA (μV)
100
††
††
††
50 ** **
0
-50
5 min
150
Δ Integrated RSNA ( % )
RSNA (μV)
(b)
100
Integrated RSNA (%)
(a)
Integrated RSNA (%)
S Kobuchi et al.
284
†† 100
††
††
††
††
††
50 ** **
0
-10 -5 0
5
15
25
35
45
Ba sa l
Ba sa l
-50 -10 -5 0
5
15
Ischaemia
25
35
45
Ischaemia
Time (min)
Time (min)
AKI + vehicle, i.v.
AKI + vehicle, i.c.v.
AKI + baclofen 0.2 μmol/kg, i.v.
AKI + GABA 0.1 μmol/kg, i.c.v.
AKI + baclofen 1 μmol/kg, i.v.
AKI + GABA 0.5 μmol/kg, i.c.v.
Fig. 6 (a,b) Typical responses of renal sympathetic nerve activity (RSNA) and integrated RSNA following injection of (a) baclofen (1 lmol/kg, i.v.) or (b) GABA (0.5 lmol/kg, i.c.v.) during the 45 min ischaemic period in anaesthetized rats. (c,d) Percentage changes in integrated RSNA in response to injections of vehicle or baclofen (c) and vehicle or GABA (d). Vehicle, baclofen or GABA were administered 5 min before ischaemia. Data are the mean SEM (n = 6). ††P < 0.01, compared with basal (vehicle-treated acute kidney injury (AKI) rats only). **P < 0.01.
Table 2 Effects of i.v. baclofen or i.c.v. GABA on parameters of kidney function after reperfusion Experimental group AKI AKI AKI AKI AKI AKI
+ + + + + +
vehicle, i.v. baclofen 0.2 lmol/kg, i.v. baclofen 1 lmol/kg, i.v. vehicle, i.c.v. GABA 0.1 lmol/kg, i.c.v. GABA 0.5 lmol/kg, i.c.v.
BUN (mg/dL) 104 76.3 53.0 103 67.6 51.3
5 5.4†† 5.6†† 6 12.6* 6.6**
PCr (mg/dL) 2.70 1.86 1.24 2.57 1.84 1.18
0.28 0.24† 0.08†† 0.14 0.29* 0.12**
CCr (mL/min per kg) 1.40 2.00 2.97 1.28 2.03 3.18
0.17 0.24 0.35†† 0.18 0.39 0.42**
UF (lL/min per kg) 117 76.8 64.2 105 79.3 70.6
8 13.8† 7.6†† 9 12.9 8.0*
Data are the meanSEM (n = 6). †P < 0.05, ††P < 0.01 compared vehicle (i.v.)-treated acute kidney injury (AKI) rats; *P < 0.05, **P < 0.01 compared vehicle (i.c.v.)-treated AKI rats. BUN, blood urea nitrogen; PCr, plasma creatinine; CCr, creatinine clearance; UF, urinary flow.
In conclusion, GABA suppressed the enhanced RSNA during ischaemia and increased NA overflow after I/R through activation of GABAB, but not GABAA, receptors and this effect was particularly focused on CNS activity rather than on peripheral nerve activity in the sympathetic nervous system. These inhibitory effects are presumably responsible for renoprotection against I/Rinduced renal injury.
METHODS Animals and experimental design Male Sprague-Dawley rats (10 weeks of age; Japan SLC, Shizuoka, Japan) were used in the present study. Rats were housed in a light-controlled room under a 12 h light–dark cycle and were allowed access to food and water ad libitum. The Experimental
© 2014 Wiley Publishing Asia Pty Ltd
GABA and acute kidney injury Animal Committee at Osaka University of Pharmaceutical Sciences (Osaka, Japan) approved all experimental protocols and animal care methods used in this study. Two weeks before the study commenced (8-week-old rats), the right kidney was removed from rats through a small flank incision under pentobarbital anaesthesia (40 mg/kg, i.p.). After a 2 week recovery period, uninephrectomized rats were divided into sham-operated control, vehicle-treated ischaemic AKI and drug-treated ischaemic AKI groups. To induce ischaemic AKI, the left kidney of pentobarbital (50 mg/kg, i.p.)-anaesthetized rats was exposed through a small flank incision and the left renal artery and vein were occluded with a non-traumatic clamp for 45 min. At the end of the ischaemic period, the clamp was released to allow reperfusion. Each drug used in this study or vehicle (0.9% saline) was administered into the left external jugular vein (i.v. treatment; 1 mL/kg) or into the right lateral cerebral ventricle (i.c.v. treatment; 3 lL/rat). Intracerebroventricular injections were performed using a 30-gauge stainless steel cannula implanted into the right lateral cerebral ventricle (stereotaxic coordinates: 0.9–1.0 mm posterior to Bregma; 1.4–1.6 mm lateral to midline; 3.2–3.3 mm ventral to dura), as described by Paxinos and Watson.25 The position of the cannula was confirmed by staining of all four ventricles by injection of 5 lL Pontamine sky blue dye at the end of the experiment. Drugs or vehicle, except bicuculline and CGP52432, were injected 5 min before the start of ischaemia, whereas bicuculline and CGP52432 were administered 10 min before ischaemia to examine the effects of these drugs on GABA-induced renal protection. In sham-operated control rats, the left kidney was treated as above, but without clamping. Rats exposed to 45 min ischaemia were housed in metabolic cages for 24 h after reperfusion and 5 h urine samples were collected. At the end of urine collection, blood samples were drawn from the thoracic aorta and then the left kidney was excised under pentobarbital anaesthesia (50 mg/kg, i.p.). Plasma was isolated from the blood by centrifugation (1630 g, 15 min, 4°C) and used to measure renal function, as described below, whereas the kidneys were examined by light microscopy for histological analysis.
285
activity are expressed as a percentage of the control resting spontaneous nerve activity. Electrical signals of renal neural activity were recorded directly for evaluation of changes in RSNA during the 45 min ischaemic period. Renal function parameters Blood urea nitrogen and creatinine levels in plasma or urine were determined using commercially available kits (BUN-test-Wako and Creatinine-test-Wako, respectively; Wako Pure Chemical Industries, Osaka, Japan). Creatinine clearance was calculated using the formula CCr = UCr 9 UF/PCr, where UCr and PCr are creatinine concentrations in the urine and plasma, respectively, and UF is urine flow. Histological studies Excised left kidneys were processed for light microscopic observation according to standard procedures. Briefly, kidneys were fixed in phosphate-buffered 10% formalin, chopped into small pieces, embedded in paraffin wax, sectioned at 4 lm and stained with haematoxylin and eosin. The histological changes assessed were tubular necrosis, proteinaceous casts and medullary congestion, as described by Caramelo et al.27 Tubular necrosis and proteinaceous casts were graded as follows: 0, no damage; 1, mild (unicellular, patchy isolated damage); 2, moderate (damage 50% damage). The degree of medullary congestion was defined as no congestion (0), mild (1; vascular congestion with identification of erythrocytes at 9400 magnification), moderate (2; vascular congestion with identification of erythrocytes at 9200 magnification), severe (3; vascular congestion with identification of erythrocytes at 9100 magnification) and very severe (4; vascular congestion with identification of erythrocytes at 940 magnification). The scoring of histological data was performed by independent observers in a double-blinded manner. Drugs
Renal nerve recording For measurement of RSNA, uninephrectomized rats were anaesthetized with pentobarbital (50 mg/kg, i.p.) and then prepared for surgical and basic experimental techniques as described previously.26 Renal sympathetic nerve activity was recorded from the left renal nerve branch before and during ischaemia. The nerve was isolated near the aortic–renal arterial junction through a left flank incision and placed on a Teflon-coated stainless steel bipolar electrode. Then, the nerve and electrode were covered with silicone rubber. Renal nerve discharges were amplified using a differential amplifier (AVB-11A; Nihon Kohden, Osaka, Japan) with a band-pass filter (low frequency, 50 Hz; high frequency, 1 kHz). The amplified and filtered signal was visualized on a dual-beam oscilloscope (VC-11; Nihon Kohden) and monitored by an audio speaker. The output from the amplifier was integrated (EI601G; Nihon Kohden) with 1 s resetting and the integrator output was recorded and analysed using PowerLab (ML750; ADInstruments, Castle Hill, NSW, Australia). For the quantification of RSNA, the integrated nerve discharge heights were measured for 20 s in each experiment. Changes in nerve
GABA and (–)-bicuculline methiodide were purchased from Sigma Chemical (St Louis, MO, USA). CGP52432 (3-[[(3,4-dichlorophenyl)methyl]amino]propyl] diethoxymethyl) phosphinic acid) and baclofen were purchased from Tocris Cookson (Ellisville, MO, USA). These drugs were dissolved in saline (0.9%). Other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries. Statistical analysis All values are expressed as the meanSEM. Relevant data were processed by Instat (Graph-PAD Software for Science, La Jolla, CA, USA). Nerve recording studies were analysed by one-way repeated-measures ANOVA followed by Dunnett’s multiple range test for within-group data. For among-group data from nerve recordings, two-way repeated measures ANOVA was used, followed by Fisher’s protected least significant difference comparison tests. For among-group data from renal function studies, oneway ANOVA followed by Dunnett’s multiple comparison test was used. Histological data were analysed using the Kruskal–Wallis
© 2014 Wiley Publishing Asia Pty Ltd
S Kobuchi et al.
286
non-parametric test combined with a Steel-type multiple comparison test. For all comparisons, differences were considered significant at two-tailed P < 0.05.
ACKNOWLEDGEMENTS This study was supported, in part, by a ‘High Technology Research Center’ Project for Private Universities, with a matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology of Japan (2002–06 and 2007–09). The authors thank Inter-Biotech Ltd (http://www.interbiotech.com) for English language editing of the manuscript.
DISCLOSURE The authors declare no conflicts of interest.
REFERENCES 1. Lattanzio MR, Kopyt NP. Acute kidney injury. New concepts in definition, diagnosis, pathophysiology, and treatment. J. Am. Osteopath. Assoc. 2009; 109: 13–9. 2. Thadhani R, Pascual M, Bonventre JV. Acute renal failure. N. Engl. J. Med. 1996; 334: 1448–60. 3. Lieberthal W, Levine JS. Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury. Am. J. Physiol. 1996; 271: F477–88. 4. Chatterjee PK. Novel pharmacological approaches to the treatment of renal ischemia–reperfusion injury: A comprehensive review. Naunyn Schmiedebergs Arch. Pharmacol. 2007; 376: 1–43. 5. Lien YH, Lai LW, Silva AL. Pathogenesis of ischemia/reperfusion injury: Lessons from knockout mice. Life Sci. 2003; 74: 543–52. 6. Kurata H, Fujii T, Tsutsui H et al. Renoprotective effects of L-carnosine on ischemia/reperfusion-induced renal injury in rats. J. Pharmacol. Exp. Ther. 2006; 319: 640–7. 7. Tanaka R, Tsutsui H, Ohkita M, Takaoka M, Yukimura T, Matsumura Y. Sex differences in ischemia/reperfusion-induced acute kidney injury are dependent on the renal sympathetic nervous system. Eur. J. Pharmacol. 2013; 714: 397–404. 8. Gladkevich A, Korf J, Hakobyan VP, Melkonyan KV. The peripheral GABAergic system as a target in endocrine disorders. Auton. Neurosci. 2006; 124: 1–8. 9. Fujimura S, Shimakage H, Tanioka H et al. Effect of GABA on noradrenaline release and vasoconstriction induced by renal nerve stimulation in isolated perfused rat kidney. Br. J. Pharmacol. 1999; 127: 109–14. 10. Kim HY, Yokozawa T, Nagasawa T, Sasaki S. Protective effect of c-aminobutyric acid against glycerol-induced acute renal failure in rats. Food Chem. Toxicol. 2004; 42: 2009–14.
11. Kobuchi S, Shintani T, Sugiura T et al. Renoprotective effects of gamma-aminobutyric acid on ischemia/reperfusion-induced renal injury in rats. Eur. J. Pharmacol. 2009; 623: 113–8. 12. Sugiura T, Tsutsui H, Takaoka M et al. Protective effect of agmatine on ischemia/reperfusion-induced renal injury in rats. J. Cardiovasc. Pharmacol. 2008; 51: 223–30. 13. Macdonald RL, Olsen RW. GABAA receptor channels. Annu. Rev. Neurosci. 1994; 17: 569–602. 14. Kerr DI, Ong J. GABAB receptors. Pharmacol. Ther. 1995; 67: 187–246. 15. Heldt SA, Ressler KJ. Forebrain and midbrain distribution of major benzodiazepine-sensitive GABAA receptor subunits in the adult C57 mouse as assessed with in situ hybridization. Neuroscience 2007; 150: 370–85. 16. Bowery NG, Bettler B, Froestl W et al. International Union of Pharmacology XXXIII. Mammalian gamma-aminobutyric acid (B) receptors: Structure and function. Pharmacol. Rev. 2002; 54: 247–64. 17. Awadi MA, Pavlik A, Sarraf A. Increased brain uptake and brain to blood efflux transport of 14C-GABA in spontaneously hypertensive rats. Life Sci. 2006; 79: 847–53. 18. Coote JH. A role for the paraventricular nucleus of the hypothalamus in the autonomic control of heart and kidney. Exp. Physiol. 2005; 90: 169–73. 19. Tjen-A-Looi SC, Li P, Longhurst JC. Processing cardiovascular information in the vlPAG during electroacupuncture in rats. Roles of endocannabinoids and GABA. J. Appl. Physiol. 2009; 106: 1793–9. 20. Avanzino GL, Ruggeri P, Blanchi D, Cogo CE, Ermirio R, Weaver LC. GABAB receptor-mediated mechanisms in the RVLM studied by microinjections of two GABAB receptor antagonists. Am. J. Physiol. 1994; 266: H1722–8. 21. Li DP, Pan HL. Role of c-aminobutyric acid (GABA)A and GABAB receptors in paraventricular nucleus in control of sympathetic vasomotor tone in hypertension. J. Pharmacol. Exp. Ther. 2007; 320: 615–26. 22. Durgam VR, Vitela M, Mifflin SW. Enhanced gamma-aminobutyric acid-B receptor agonist responses and mRNA within the nucleus of the solitary tract in hypertension. Hypertension 1999; 33: 530–6. 23. Hayakawa K, Kimura M, Kamata K. Mechanism underlying c-aminobutyric acid-induced antihypertensive effect in spontaneously hypertensive rats. Eur. J. Pharmacol. 2002; 438: 107–13. 24. Kimura M, Hayakawa K, Sansawa H. Involvement of c-aminobutyric acid (GABA) B receptors in the hypotensive effect of systemically administered GABA in spontaneously hypertensive rats. Jpn. J. Pharmacol. 2002; 89: 388–92. 25. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates, 4th edn. Academic Press, San Diego, 1998. 26. Shokoji T, Nishiyama A, Fujisawa Y et al. Renal sympathetic nerve response to tempol in spontaneously hypertensive rats. Hypertension 2003; 41: 266–73. 27. Caramero C, Espinosa G, Manzarbeitia F et al. Role of endothelium-related mechanism in the pathophysiology of renal ischemia/ reperfusion in normal rabbits. Circ. Res. 1996; 79: 1031–8.
© 2014 Wiley Publishing Asia Pty Ltd
Copyright of Clinical & Experimental Pharmacology & Physiology is the property of WileyBlackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
doi: 10.1111/1440-1681.12350
ORIGINAL ARTICLE
Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion-induced renal injury in rats Shuhei Kobuchi,*† Ryosuke Tanaka,† Takuya Shintani,† Rie Suzuki,† Hidenobu Tsutsui,†‡ Mamoru Ohkita,† Yasuo Matsumura† and Kazuhide Ayajiki* *Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Hyogo, †Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences, Takatsuki, and ‡ Laboratory of Clinical Pharmacology, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Osaka, Japan
ABSTRACT Excitation of the renal sympathetic nervous system is important for the development of ischaemic acute kidney injury (AKI) in rats. We reported that intravenous treatment with GABA has preventive effects against ischaemia/ reperfusion (I/R)-induced renal dysfunction with histological damage in rats; however, the mechanisms underlying these effects on renal injury remain unknown. Thus, the aim of the present study was to clarify how GABA mechanistically affects ischaemic AKI in rats. Ischaemic AKI was induced in rats by clamping the left renal artery and vein for 45 min and then reperfusing the kidney to produce I/Rinduced injury. Treatment with the GABAB receptor antagonist CGP52432 (100 nmol/kg, i.v., or 1 nmol/kg, i.c.v.) abolished the suppressive effects of 50 lmol/kg, i.v., GABA on enhanced renal sympathetic nerve activity (RSNA) during ischaemia, leading to elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with 0.5 lmol/ kg GABA or i.v. treatment with 1 lmol/kg baclofen, a selective GABAB receptor agonist, prevented the I/R-induced renal injury equivalent to i.v. treatment with GABA. Conversely, i.v. treatment with 10 lmol/kg bicuculline, a GABAA receptor antagonist, failed to affect the preventive effects of GABA against ischaemic AKI. We therefore concluded that GABAB receptor stimulation in the central nervous system, rather than peripheral GABAB receptor stimulation, mediates the preventive effect of GABA against ischaemic AKI by suppressing the enhanced RSNA induced by renal ischaemia. Key words: acute kidney injury, GABA, ischaemia/ reperfusion, renal sympathetic nerve activity.
Correspondence: S Kobuchi, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe, Hyogo 650-8530, Japan. Email: [email protected] Received 28 August 2014; revision 6 November 2014; accepted 2 December 2014. © 2014 Wiley Publishing Asia Pty Ltd
INTRODUCTION Acute kidney injury (AKI) is commonly encountered in the hospital setting and is associated with a high rate of mortality.1 Ischaemia followed by reperfusion is one of the major causes of AKI,2 with reperfusion of previously ischaemic renal tissue initiating a complex series of cellular events that result in injury and the eventual death of renal cells due to a combination of apoptosis and necrosis.3 Several causative factors, including ATP depletion, reactive oxygen species, phospholipase activation, neutrophil infiltration and vasoactive peptides, reportedly contribute to the pathogenesis of this renal damage;4,5 however, the mechanisms underlying ischaemia/reperfusion (I/R)-induced renal injury are not fully understood. We found that enhancement of renal sympathetic nerve activity (RSNA) and its consequent effect on noradrenaline (NA) overflow from nerve terminals are involved in the development of I/R-induced AKI and that RSNA is significantly augmented during renal ischaemia in rats.6 In addition, we reported that ischaemic AKI is ameliorated by ganglionic blockade and that this effect is accompanied by suppression of elevated NA levels in the renal vein after reperfusion.7 In addition to the central nervous system (CNS), GABA, an inhibitory neurotransmitter, is also found in peripheral tissues8 and is known to suppress electrical renal nerve stimulationinduced NA release from rat isolated kidney without affecting basal release.9 These findings indicate that GABA can modulate peripheral as well as CNS neurotransmission. It has been reported that GABA has renoprotective effects against glycerol-induced AKI,10 and we have further shown that pre-ischaemic treatment with GABA suppresses the enhancement of RSNA and consequent elevation in NA levels in the renal vein observed in ischaemic AKI rats, suggesting that GABA has renoprotective effects against I/R-induced renal injury.11 However, no studies to date have elucidated the precise mechanisms underlying the beneficial action of GABA on AKI and/or I/R-induced renal injury, especially in terms of receptor and the sites of action. Therefore, in the present study we investigated the effects of i.v. treatment with bicuculline, a GABAA receptor antagonist, and CGP52432, a GABAB receptor antagonist, on the renoprotective effects of GABA to clarify the receptor subtypes involved. Similarly, to clarify the site of GABA action, we
GABA and acute kidney injury examined the effects of i.c.v. treatment with CGP52432 on the renoprotective effects of GABA. Finally, we examined the effects of i.v. treatment with baclofen, a GABAB receptor agonist, and i.c.v. treatment with GABA on ischaemic AKI.
RESULTS Effects of i.v. bicuculline or CGP52432 on GABA-induced improvements in ischaemic AKI Pre-ischaemic treatment of GABA (50 lmol/kg, i.v.) markedly suppressed the enhanced RSNA during the ischaemic period (Fig. 1a,b,d). This suppressive effect was inhibited by the selective GABAB receptor antagonist CGP52432 (10 and 100 nmol/ kg, i.v.) in a dose-dependent manner (Fig. 1c,d). Conversely, treatment with the selective GABAA receptor antagonist bicuculline (1 and 10 lmol/kg, i.v.) failed to attenuate the suppressive effect of GABA on RSNA (Fig. 1d). As shown in Fig. 2, the renal function of rats subjected to 45 min ischaemia showed marked deterioration when measured 29 h after reperfusion. Compared with sham-operated rats,
279
vehicle-treated AKI rats showed significant increases in blood urea nitrogen (BUN), plasma creatinine (PCr) concentrations and urine flow (UF) and a significant decrease in creatinine clearance (CCr), indicating renal dysfunction. Intravenous injection of GABA (50 lmol/kg) to ischaemic AKI rats markedly attenuated the I/R-induced renal dysfunction and this improvement was reversed by 100 nmol/kg, i.v., CGP52432. However, the renoprotective effects of GABA were not affected by bicuculline (1 and 10 lmol/kg) or 10 nmol/kg CGP52432. In addition, we confirmed that 10 lmol/kg, i.v., bicuculline alone had no effect on I/ R-induced renal injury (data not shown). Histological examination revealed severe lesions in the kidney of vehicle-treated AKI rats 29 h after reperfusion. These changes were characterized by proteinaceous casts in the tubules of the inner medulla (Fig. 3b), medullary congestion and haemorrhage in the outer zone of the inner medullary stripe (Fig. 3g) and tubular necrosis in the outer zone of the outer medullary stripe (Fig. 3l) compared with kidneys from sham-operated rats (Fig. 3a,f,k). Intravenous injection of GABA to ischaemic AKI rats significantly attenuated the development of all lesions (Table 1; Fig. 3c,h,m). In addition, 100 nmol/kg CGP52432 (Fig. 3e,j,o) abolished these
(a)
Integrated RSNA (%)
RSNA (μV)
AKI + vehicle, i.v. AKI + GABA 50 μmol/kg, i.v.
100
AKI + GABA 50 μmol/kg, i.v., + bicuculline 1 μmol/kg, i.v. AKI + GABA 50 μmol/kg, i.v., + bicuculline 10 μmol/kg, i.v.
100 Ischaemia
AKI + GABA 50 μmol/kg, i.v., + CGP52432 10 nmol/kg, i.v.
5 min
Vehicle, i.V.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 100 nmol/kg, i.v.
RSNA (μV)
(b) (d)
100
150
100 Ischaemia GABA 50 μmol/kg, i.v.
5 min
Integrated RSNA (%)
RSNA (μV)
(c)
Δ Integrated RSNA ( % )
Integrated RSNA (%)
††
100
††
50
††
††
††
††
**
0
** **
100 -50 sal Ba
100 Ischaemia GABA 50 μmol/kg, i.v.
-10
-5
5 min
0
5
15
25
35
45
Ischaemia Time (min)
CGP52432 100 nmol/kg, i.v.
Fig. 1 Typical responses of renal sympathetic nerve activity (RSNA) and integrated RSNA to injection of (a) vehicle (0.9% saline, i.v.), (b) GABA (50 lmol/kg, i.v.) or (c) GABA (50 lmol/kg, i.v.) + CGP52432 (100 nmol/kg, i.v.) during the 45 min ischaemic period in anaesthetized rats. (d) Changes in % integrated RSNA in response to injection of vehicle or GABA, alone or in combination with bicuculline or CGP52432. Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). ††P < 0.01 compared with basal (vehicle-treated acute kidney injury (AKI) rats only). **P < 0.01.
© 2014 Wiley Publishing Asia Pty Ltd
S Kobuchi et al.
280
AKI + GABA 50 μmol/kg, i.v., + bicuculline 1 μmol/kg, i.v. Sham
AKI + GABA 50 μmol/kg, i.v., + bicuculline 10 μmol/kg, i.v.
AKI + vehicle, i.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 10 nmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 100 nmol/kg, i.v.
(a)
(b)
120
3
††
††
80
** **
60 40
PCr (mg/dL)
BUN (mg/dL)
100
**
**
**
2
** **
1
**
**
**
20 0
CCr (mL/min per kg)
6
(d)
**
5 4
** **
3 2
*
** ††
UF (μL/min per kg)
(c)
0
††
120 100 80
**
60 40
1
20
0
0
**
Fig. 2 Effects of intravenous treatment with bicuculline or CGP52432 on renal protection induced by intravenous treatment with GABA. Parameters of renal function were evaluated 29 h after reperfusion: (a) blood urea nitrogen (BUN), (b) plasma creatinine (PCr), (c) creatinine clearance (CCr) and (d) urinary flow (UF). Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). *P < 0.05, **P < 0.01 compared with vehicle (i.v.)-treated acute kidney injury (AKI) rats; ††P < 0.01 compared with GABA (50 lmol/kg, i.v.)-treated AKI rats.
GABA-induced improvements, whereas 10 lmol/kg, i.v., bicuculline had no effect on the effects of GABA (Fig. 3d,i,n). Effects of i.c.v. CGP52432 on GABA-induced improvements in ischaemic AKI As shown in Fig. 4, the suppressive effect of GABA (50 lmol/ kg) on RSNA was partially attenuated by 0.1 nmol/kg, i.c.v., CGP52432, whereas 1 nmol/kg, i.c.v., CGP52432 almost abolished the effects of GABA. Similarly, the GABA-induced improvements in renal dysfunction were partially attenuated by 0.1 nmol/kg, i.c.v., CGP52432, but were almost abolished by 1 nmol/kg, i.c.v., CGP52432 (Fig. 5). Bicuculline (10 nmol/kg, i.c.v.) failed to affect the GABA-induced renoprotective effects (data not shown). Effects of i.v. baclofen or i.c.v. GABA on ischaemic AKI The enhancement of RSNA during the ischaemic period was markedly suppressed by both baclofen (0.2 and 1 lmol/kg, i.v.),
a selective GABAB receptor agonist, and GABA (0.1 and 0.5 lmol/kg, i.c.v.) in a dose-dependent manner (Fig. 6). As indicated in Table 2, baclofen (0.2 and 1 lmol/kg, i.v.) significantly prevented the I/R-induced renal dysfunction, with the higher dose of baclofen (1 lmol/kg) having equivalent effectiveness to GABA (50 lmol/kg). In addition, GABA (0.1 and 0.5 lmol/kg, i.c.v.) dose-dependently attenuated the I/R-induced renal dysfunction, with the higher dose similarly effective to treatment 50 lmol/kg GABA (Table 2).
DISCUSSION The renal sympathetic nervous system and circulating catecholamines have been implicated in the pathogenesis of AKI based on pharmacological blockade of the sympathetic nervous system having a protective effect against AKI.6,12 Recently, we found that i.v. treatment of ischaemic AKI rats with GABA (10 and 50 lmol/kg) also suppressed the enhancement of RSNA during ischaemia and the increased NA overflows after reperfusion.11 In the present study, we investigated the mechanisms underlying
© 2014 Wiley Publishing Asia Pty Ltd
GABA and acute kidney injury
Proteinaceous casts in tubules (a)
281
Medullary congestion
Tubular necrosis
(f)
(k)
(b)
(g)
(l)
(c)
(h)
(m)
(d)
(i)
(n)
(e)
(j)
(o)
Sham 100μm
AKI + vehicle, i.v.
AKI + GABA 50 μmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v., + bicuculline 10 μmol/kg, i.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 100 nmol/kg, i.v. Fig. 3 Light microscopy of the inner zone (a–e), the outer zone inner stripe (f–j) and the outer zone outer stripe (k–o) of the medulla in the kidney of acute kidney injury (AKI) rats treated with i.v. injection of vehicle (b,g,l), GABA alone (c,h,m), GABA + bicuculline (d,i,n) and GABA + CGP52432 (e,j,o) 29 h after reperfusion, as well as in sham-operated rats (a,f,k). Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Severe proteinaceous casts in tubules (b), congestion and haemorrhage (g) and tubular necrosis (l) were observed in vehicle (i.v.)-treated AKI rats. Arrows indicate severe proteinaceous casts in tubules (b–e), medullary congestion (g–j) and tubular necrosis (l,o). Original magnification 9200.
these renoprotective effects of GABA against AKI with respect to receptor subtypes and sites of action. There are two known receptor subtypes for GABA: GABAA and GABAB receptors. The GABAA receptor is coupled to
ligand-gated Cl channels, whereas the GABAB receptor is coupled to G-proteins.13,14 The GABAA receptors are widely distributed within the mammalian CNS and exhibit a differential topographical distribution,15 whereas GABAB receptors are
© 2014 Wiley Publishing Asia Pty Ltd
S Kobuchi et al.
282
Table 1 Effects of 10 lmol/kg, i.v., bicuculline or 0.1 lmol/kg, i.v., CGP52432 on renal tissue protection induced by 50 lmol/kg, i.v., GABA, as evaluated by histological grading‡ Experimental group AKI AKI AKI AKI
+ + + +
Proteinaceous casts in tubules
vehicle, i.v. GABA GABA + bicuculline GABA + CGP52432
3.50 1.17 1.67 2.83
Medullary congestion
0.34 0.31** 0.33** 0.31††
3.50 1.83 1.67 3.50
Tubular necrosis
0.22 0.17** 0.42** 0.22††
3.83 1.50 1.50 3.50
0.17 0.22** 0.34** 0.22††
Data are the meanSEM (n = 6). *P < 0.05, **P < 0.01 compared with vehicle (i.v.)-treated acute kidney injury (AKI) rats; ††P < 0.01 compared with GABA-treated AKI rats. ‡ Tissues were graded as follows: 0, no change; 1, mild; 2, moderate; 3, severe; 4, very severe (see text for details).
RSNA (μV)
(a)
AKI + vehicle, i.v. + vehicle, i.c.v. 100
Integrated RSNA
AKI + GABA 50 μmol/kg, i.v. AKI + GABA 50 μmol/kg, i.v., + CGP52432 0.1 nmol/kg, i.c.v. 100
Ischaemia
AKI + GABA 50 μmol/kg, i.v., + CGP52432 1 nmol/kg, i.c.v. 5 min
GABA 50 μmol/kg, i.v. CGP52432 1 nmol/kg, i.c.v.
(b)
150
Δ Integrated RSNA ( % )
†† 100
†† ††
50
††
††
††
**
** **
0
Ba sa l
-50 -10
-5
0
5
15
25
35
45
Ischaemia Time (min) Fig. 4 (a) Typical responses of renal sympathetic nerve activity (RSNA) and integrated RSNA following injection of GABA (50 lmol/kg, i.v.) + CGP52432 (1 nmol/kg, i.c.v.) during the 45 min ischaemic period in anaesthetized rats. (b) Changes in % integrated RSNA in response to injections of vehicle and GABA, alone or in combination with CGP52432. Bicuculline or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). ††P < 0.01 compared with basal (vehicle-treated acute kidney injury (AKI) rats only). **P < 0.01.
widely distributed within the CNS as well as in peripheral autonomic terminals.16 In the present study, we evaluated which of these receptor subtypes is involved in the suppressive effects of GABA on I/R-induced enhancement of RSNA and against the functional and histological renal injury in rats. Our findings
clearly indicate that the suppressive effects of GABA were abolished by blockade of GABAB receptors (CGP52432 treatment), but not by blockade of GABAA receptors (bicuculline treatment). In addition, i.v. baclofen, a GABAB receptor agonist, prevented the I/R-induced renal injury by suppressing the
© 2014 Wiley Publishing Asia Pty Ltd
GABA and acute kidney injury
283
Sham
AKI + GABA 50 μmol/kg, i.v.
AKI + vehicle, i.v., + vehicle, i.c.v.
AKI + GABA 50 μmol/kg, i.v., + CGP52432 0.1 nmol/kg, i.c.v. AKI + GABA 50 μmol/kg, i.v., + CGP52432 1 nmol/kg, i.c.v.
(a) 120
(b)
3
††
††
100 80
PCr (mg/dL)
BUN (mg/dL)
* ** 60 40
**
**
2
** 1
**
20 0 6
**
(d) 120
5 4
**
3 2
††
UF (μL/min per kg)
CCr (mL/min per kg)
(c)
0
100 80 60
** 40
1
20
0
0
Fig. 5 Effects of i.c.v. CGP52432 on renal protection induced by GABA. Parameters of renal function were evaluated 29 h after reperfusion: (a) blood urea nitrogen (BUN), (b) plasma creatinine (PCr), (c) creatinine clearance (CCr) and (d) urinary flow (UF). Vehicle or CGP52432 were administered to rats 10 min before ischaemia; vehicle or GABA were administered 5 min before ischaemia. Data are the meanSEM (n = 6). *P < 0.05, **P < 0.01 compared with vehicle (i.v.)-treated acute kidney injury (AKI) rats; ††P < 0.01 compared with GABA (50 lmol/kg, i.v.)-treated AKI rats.
enhancement of RSNA. These findings suggest that activation of GABAB, but not GABAA, receptors is responsible for protecting against the development of I/R-induced renal dysfunction. A recent study found an increased concentration of GABA in the cerebrospinal fluid following i.v. infusion of GABA itself,17 indicating that i.v. injection of GABA may suppress the enhanced RSNA during ischaemia by acting on the CNS. In the present study, we found that i.c.v. treatment with CGP52432 almost abolished the suppressive effects of GABA on I/R-induced enhancement of RSNA and renal injury. Furthermore, we demonstrated that i.c.v. GABA (0.1 and 0.5 lmol/kg) prevented the I/ R-induced renal injury by suppressing the enhanced RSNA. However, i.v. treatment with the same dose of GABA (0.5 lmol/ kg) had no significant effect on I/R-induced renal injury (data not shown). Together, these results suggest that GABA exerts its preventive action against postischaemic AKI by inhibiting central sympathetic outflow. The paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) are responsible for controlling sympathetic outflow,18,19 and microinjection of GABA or a GABA receptor agonist into either of these structures decreases blood pressure
by inhibiting sympathetic nerve activity.20,21 In addition, Durgam et al.22 reported that microinjection of a GABA receptor agonist into the nucleus tractus solitarius (NTS), a central baroreflex regulator, increased blood pressure by enhancing sympathetic outflow. Therefore, suppression of RSNA by GABA seems to be mediated through the RVLM and/or PVN rather than the NTS. Several mechanisms have been proposed for the effects of GABA in suppressing the peripheral sympathetic nervous system, including ganglionic blockade and/or inhibition of transmitter release from the nerve terminals.9,23,24 In the present study, we did not examine whether GABA suppressed NA overflow from peripheral sympathetic nerves because we did not use isolated tissues. Indeed, in the peripheral sympathetic nerves of AKI rats, systemically applied GABA may prevent I/R-induced renal injury by inhibiting NA release from nerve terminals, even after abrogating the GABA effect using CGP52432. However, the present study revealed that systemically applied GABA failed to prevent I/R-induced renal injury in rats administered i.c.v. CGP52432. These findings suggest that the renoprotective effect of GABA appears to be much more dependent on CNS neurotransmission than that mediated through peripheral sympathetic nerves.
© 2014 Wiley Publishing Asia Pty Ltd
Ischaemia
Δ Integrated RSNA ( % )
150
(d)
100
††
100 Ischaemia GABA 0.5 μmol/kg, i.c.v.
††
††
100
5 min
Baclofen 1 μmol/kg, i.v. (c)
RSNA (μV)
100
††
††
††
50 ** **
0
-50
5 min
150
Δ Integrated RSNA ( % )
RSNA (μV)
(b)
100
Integrated RSNA (%)
(a)
Integrated RSNA (%)
S Kobuchi et al.
284
†† 100
††
††
††
††
††
50 ** **
0
-10 -5 0
5
15
25
35
45
Ba sa l
Ba sa l
-50 -10 -5 0
5
15
Ischaemia
25
35
45
Ischaemia
Time (min)
Time (min)
AKI + vehicle, i.v.
AKI + vehicle, i.c.v.
AKI + baclofen 0.2 μmol/kg, i.v.
AKI + GABA 0.1 μmol/kg, i.c.v.
AKI + baclofen 1 μmol/kg, i.v.
AKI + GABA 0.5 μmol/kg, i.c.v.
Fig. 6 (a,b) Typical responses of renal sympathetic nerve activity (RSNA) and integrated RSNA following injection of (a) baclofen (1 lmol/kg, i.v.) or (b) GABA (0.5 lmol/kg, i.c.v.) during the 45 min ischaemic period in anaesthetized rats. (c,d) Percentage changes in integrated RSNA in response to injections of vehicle or baclofen (c) and vehicle or GABA (d). Vehicle, baclofen or GABA were administered 5 min before ischaemia. Data are the mean SEM (n = 6). ††P < 0.01, compared with basal (vehicle-treated acute kidney injury (AKI) rats only). **P < 0.01.
Table 2 Effects of i.v. baclofen or i.c.v. GABA on parameters of kidney function after reperfusion Experimental group AKI AKI AKI AKI AKI AKI
+ + + + + +
vehicle, i.v. baclofen 0.2 lmol/kg, i.v. baclofen 1 lmol/kg, i.v. vehicle, i.c.v. GABA 0.1 lmol/kg, i.c.v. GABA 0.5 lmol/kg, i.c.v.
BUN (mg/dL) 104 76.3 53.0 103 67.6 51.3
5 5.4†† 5.6†† 6 12.6* 6.6**
PCr (mg/dL) 2.70 1.86 1.24 2.57 1.84 1.18
0.28 0.24† 0.08†† 0.14 0.29* 0.12**
CCr (mL/min per kg) 1.40 2.00 2.97 1.28 2.03 3.18
0.17 0.24 0.35†† 0.18 0.39 0.42**
UF (lL/min per kg) 117 76.8 64.2 105 79.3 70.6
8 13.8† 7.6†† 9 12.9 8.0*
Data are the meanSEM (n = 6). †P < 0.05, ††P < 0.01 compared vehicle (i.v.)-treated acute kidney injury (AKI) rats; *P < 0.05, **P < 0.01 compared vehicle (i.c.v.)-treated AKI rats. BUN, blood urea nitrogen; PCr, plasma creatinine; CCr, creatinine clearance; UF, urinary flow.
In conclusion, GABA suppressed the enhanced RSNA during ischaemia and increased NA overflow after I/R through activation of GABAB, but not GABAA, receptors and this effect was particularly focused on CNS activity rather than on peripheral nerve activity in the sympathetic nervous system. These inhibitory effects are presumably responsible for renoprotection against I/Rinduced renal injury.
METHODS Animals and experimental design Male Sprague-Dawley rats (10 weeks of age; Japan SLC, Shizuoka, Japan) were used in the present study. Rats were housed in a light-controlled room under a 12 h light–dark cycle and were allowed access to food and water ad libitum. The Experimental
© 2014 Wiley Publishing Asia Pty Ltd
GABA and acute kidney injury Animal Committee at Osaka University of Pharmaceutical Sciences (Osaka, Japan) approved all experimental protocols and animal care methods used in this study. Two weeks before the study commenced (8-week-old rats), the right kidney was removed from rats through a small flank incision under pentobarbital anaesthesia (40 mg/kg, i.p.). After a 2 week recovery period, uninephrectomized rats were divided into sham-operated control, vehicle-treated ischaemic AKI and drug-treated ischaemic AKI groups. To induce ischaemic AKI, the left kidney of pentobarbital (50 mg/kg, i.p.)-anaesthetized rats was exposed through a small flank incision and the left renal artery and vein were occluded with a non-traumatic clamp for 45 min. At the end of the ischaemic period, the clamp was released to allow reperfusion. Each drug used in this study or vehicle (0.9% saline) was administered into the left external jugular vein (i.v. treatment; 1 mL/kg) or into the right lateral cerebral ventricle (i.c.v. treatment; 3 lL/rat). Intracerebroventricular injections were performed using a 30-gauge stainless steel cannula implanted into the right lateral cerebral ventricle (stereotaxic coordinates: 0.9–1.0 mm posterior to Bregma; 1.4–1.6 mm lateral to midline; 3.2–3.3 mm ventral to dura), as described by Paxinos and Watson.25 The position of the cannula was confirmed by staining of all four ventricles by injection of 5 lL Pontamine sky blue dye at the end of the experiment. Drugs or vehicle, except bicuculline and CGP52432, were injected 5 min before the start of ischaemia, whereas bicuculline and CGP52432 were administered 10 min before ischaemia to examine the effects of these drugs on GABA-induced renal protection. In sham-operated control rats, the left kidney was treated as above, but without clamping. Rats exposed to 45 min ischaemia were housed in metabolic cages for 24 h after reperfusion and 5 h urine samples were collected. At the end of urine collection, blood samples were drawn from the thoracic aorta and then the left kidney was excised under pentobarbital anaesthesia (50 mg/kg, i.p.). Plasma was isolated from the blood by centrifugation (1630 g, 15 min, 4°C) and used to measure renal function, as described below, whereas the kidneys were examined by light microscopy for histological analysis.
285
activity are expressed as a percentage of the control resting spontaneous nerve activity. Electrical signals of renal neural activity were recorded directly for evaluation of changes in RSNA during the 45 min ischaemic period. Renal function parameters Blood urea nitrogen and creatinine levels in plasma or urine were determined using commercially available kits (BUN-test-Wako and Creatinine-test-Wako, respectively; Wako Pure Chemical Industries, Osaka, Japan). Creatinine clearance was calculated using the formula CCr = UCr 9 UF/PCr, where UCr and PCr are creatinine concentrations in the urine and plasma, respectively, and UF is urine flow. Histological studies Excised left kidneys were processed for light microscopic observation according to standard procedures. Briefly, kidneys were fixed in phosphate-buffered 10% formalin, chopped into small pieces, embedded in paraffin wax, sectioned at 4 lm and stained with haematoxylin and eosin. The histological changes assessed were tubular necrosis, proteinaceous casts and medullary congestion, as described by Caramelo et al.27 Tubular necrosis and proteinaceous casts were graded as follows: 0, no damage; 1, mild (unicellular, patchy isolated damage); 2, moderate (damage 50% damage). The degree of medullary congestion was defined as no congestion (0), mild (1; vascular congestion with identification of erythrocytes at 9400 magnification), moderate (2; vascular congestion with identification of erythrocytes at 9200 magnification), severe (3; vascular congestion with identification of erythrocytes at 9100 magnification) and very severe (4; vascular congestion with identification of erythrocytes at 940 magnification). The scoring of histological data was performed by independent observers in a double-blinded manner. Drugs
Renal nerve recording For measurement of RSNA, uninephrectomized rats were anaesthetized with pentobarbital (50 mg/kg, i.p.) and then prepared for surgical and basic experimental techniques as described previously.26 Renal sympathetic nerve activity was recorded from the left renal nerve branch before and during ischaemia. The nerve was isolated near the aortic–renal arterial junction through a left flank incision and placed on a Teflon-coated stainless steel bipolar electrode. Then, the nerve and electrode were covered with silicone rubber. Renal nerve discharges were amplified using a differential amplifier (AVB-11A; Nihon Kohden, Osaka, Japan) with a band-pass filter (low frequency, 50 Hz; high frequency, 1 kHz). The amplified and filtered signal was visualized on a dual-beam oscilloscope (VC-11; Nihon Kohden) and monitored by an audio speaker. The output from the amplifier was integrated (EI601G; Nihon Kohden) with 1 s resetting and the integrator output was recorded and analysed using PowerLab (ML750; ADInstruments, Castle Hill, NSW, Australia). For the quantification of RSNA, the integrated nerve discharge heights were measured for 20 s in each experiment. Changes in nerve
GABA and (–)-bicuculline methiodide were purchased from Sigma Chemical (St Louis, MO, USA). CGP52432 (3-[[(3,4-dichlorophenyl)methyl]amino]propyl] diethoxymethyl) phosphinic acid) and baclofen were purchased from Tocris Cookson (Ellisville, MO, USA). These drugs were dissolved in saline (0.9%). Other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries. Statistical analysis All values are expressed as the meanSEM. Relevant data were processed by Instat (Graph-PAD Software for Science, La Jolla, CA, USA). Nerve recording studies were analysed by one-way repeated-measures ANOVA followed by Dunnett’s multiple range test for within-group data. For among-group data from nerve recordings, two-way repeated measures ANOVA was used, followed by Fisher’s protected least significant difference comparison tests. For among-group data from renal function studies, oneway ANOVA followed by Dunnett’s multiple comparison test was used. Histological data were analysed using the Kruskal–Wallis
© 2014 Wiley Publishing Asia Pty Ltd
S Kobuchi et al.
286
non-parametric test combined with a Steel-type multiple comparison test. For all comparisons, differences were considered significant at two-tailed P < 0.05.
ACKNOWLEDGEMENTS This study was supported, in part, by a ‘High Technology Research Center’ Project for Private Universities, with a matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology of Japan (2002–06 and 2007–09). The authors thank Inter-Biotech Ltd (http://www.interbiotech.com) for English language editing of the manuscript.
DISCLOSURE The authors declare no conflicts of interest.
REFERENCES 1. Lattanzio MR, Kopyt NP. Acute kidney injury. New concepts in definition, diagnosis, pathophysiology, and treatment. J. Am. Osteopath. Assoc. 2009; 109: 13–9. 2. Thadhani R, Pascual M, Bonventre JV. Acute renal failure. N. Engl. J. Med. 1996; 334: 1448–60. 3. Lieberthal W, Levine JS. Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury. Am. J. Physiol. 1996; 271: F477–88. 4. Chatterjee PK. Novel pharmacological approaches to the treatment of renal ischemia–reperfusion injury: A comprehensive review. Naunyn Schmiedebergs Arch. Pharmacol. 2007; 376: 1–43. 5. Lien YH, Lai LW, Silva AL. Pathogenesis of ischemia/reperfusion injury: Lessons from knockout mice. Life Sci. 2003; 74: 543–52. 6. Kurata H, Fujii T, Tsutsui H et al. Renoprotective effects of L-carnosine on ischemia/reperfusion-induced renal injury in rats. J. Pharmacol. Exp. Ther. 2006; 319: 640–7. 7. Tanaka R, Tsutsui H, Ohkita M, Takaoka M, Yukimura T, Matsumura Y. Sex differences in ischemia/reperfusion-induced acute kidney injury are dependent on the renal sympathetic nervous system. Eur. J. Pharmacol. 2013; 714: 397–404. 8. Gladkevich A, Korf J, Hakobyan VP, Melkonyan KV. The peripheral GABAergic system as a target in endocrine disorders. Auton. Neurosci. 2006; 124: 1–8. 9. Fujimura S, Shimakage H, Tanioka H et al. Effect of GABA on noradrenaline release and vasoconstriction induced by renal nerve stimulation in isolated perfused rat kidney. Br. J. Pharmacol. 1999; 127: 109–14. 10. Kim HY, Yokozawa T, Nagasawa T, Sasaki S. Protective effect of c-aminobutyric acid against glycerol-induced acute renal failure in rats. Food Chem. Toxicol. 2004; 42: 2009–14.
11. Kobuchi S, Shintani T, Sugiura T et al. Renoprotective effects of gamma-aminobutyric acid on ischemia/reperfusion-induced renal injury in rats. Eur. J. Pharmacol. 2009; 623: 113–8. 12. Sugiura T, Tsutsui H, Takaoka M et al. Protective effect of agmatine on ischemia/reperfusion-induced renal injury in rats. J. Cardiovasc. Pharmacol. 2008; 51: 223–30. 13. Macdonald RL, Olsen RW. GABAA receptor channels. Annu. Rev. Neurosci. 1994; 17: 569–602. 14. Kerr DI, Ong J. GABAB receptors. Pharmacol. Ther. 1995; 67: 187–246. 15. Heldt SA, Ressler KJ. Forebrain and midbrain distribution of major benzodiazepine-sensitive GABAA receptor subunits in the adult C57 mouse as assessed with in situ hybridization. Neuroscience 2007; 150: 370–85. 16. Bowery NG, Bettler B, Froestl W et al. International Union of Pharmacology XXXIII. Mammalian gamma-aminobutyric acid (B) receptors: Structure and function. Pharmacol. Rev. 2002; 54: 247–64. 17. Awadi MA, Pavlik A, Sarraf A. Increased brain uptake and brain to blood efflux transport of 14C-GABA in spontaneously hypertensive rats. Life Sci. 2006; 79: 847–53. 18. Coote JH. A role for the paraventricular nucleus of the hypothalamus in the autonomic control of heart and kidney. Exp. Physiol. 2005; 90: 169–73. 19. Tjen-A-Looi SC, Li P, Longhurst JC. Processing cardiovascular information in the vlPAG during electroacupuncture in rats. Roles of endocannabinoids and GABA. J. Appl. Physiol. 2009; 106: 1793–9. 20. Avanzino GL, Ruggeri P, Blanchi D, Cogo CE, Ermirio R, Weaver LC. GABAB receptor-mediated mechanisms in the RVLM studied by microinjections of two GABAB receptor antagonists. Am. J. Physiol. 1994; 266: H1722–8. 21. Li DP, Pan HL. Role of c-aminobutyric acid (GABA)A and GABAB receptors in paraventricular nucleus in control of sympathetic vasomotor tone in hypertension. J. Pharmacol. Exp. Ther. 2007; 320: 615–26. 22. Durgam VR, Vitela M, Mifflin SW. Enhanced gamma-aminobutyric acid-B receptor agonist responses and mRNA within the nucleus of the solitary tract in hypertension. Hypertension 1999; 33: 530–6. 23. Hayakawa K, Kimura M, Kamata K. Mechanism underlying c-aminobutyric acid-induced antihypertensive effect in spontaneously hypertensive rats. Eur. J. Pharmacol. 2002; 438: 107–13. 24. Kimura M, Hayakawa K, Sansawa H. Involvement of c-aminobutyric acid (GABA) B receptors in the hypotensive effect of systemically administered GABA in spontaneously hypertensive rats. Jpn. J. Pharmacol. 2002; 89: 388–92. 25. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates, 4th edn. Academic Press, San Diego, 1998. 26. Shokoji T, Nishiyama A, Fujisawa Y et al. Renal sympathetic nerve response to tempol in spontaneously hypertensive rats. Hypertension 2003; 41: 266–73. 27. Caramero C, Espinosa G, Manzarbeitia F et al. Role of endothelium-related mechanism in the pathophysiology of renal ischemia/ reperfusion in normal rabbits. Circ. Res. 1996; 79: 1031–8.
© 2014 Wiley Publishing Asia Pty Ltd
Copyright of Clinical & Experimental Pharmacology & Physiology is the property of WileyBlackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
