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Protein Kinase C and G_i/o Proteins Are Involved in Adenosine- and Ischemic Preconditioning—Mediated Renal Protection

Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York.

Correspondence to Dr. H. Thomas Lee, Department of Anesthesiology, Columbia Presbyterian Medical Center, P&S Box 46, 630 West 168th Street, New York, NY 10032. Phone: 212-305-0586; Fax: 212-305-8980; E-mail: tl128{at}columbia.edu

	Abstract

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Abstract. Renal ischemic reperfusion (IR) injury is a significant clinical problem in anesthesia and surgery. Recently, it was demonstrated that both renal ischemic preconditioning (IPC) and systemic adenosine pretreatment protect against renal IR injury. In cardiac IPC, pertussis toxin-sensitive G-proteins (i.e., G_i/o), protein kinase C (PKC), and ATP-sensitive potassium (K⁺ _ATP) channels are implicated in this protective signaling pathway. The aim of this study was to elucidate the signaling pathways that are responsible for renal protection mediated by both IPC and adenosine pretreatment. In addition, because A₁ adenosine receptor antagonist failed to block renal IPC, whether activation of bradykinin, muscarinic, or opioid receptors can mimic renal IPC was tested because these receptors have been implicated in cardiac IPC. Rats were acutely pretreated with chelerythrine or glibenclamide, selective blockers of PKC and K⁺ _ATP channels, respectively, before IPC or adenosine pretreatment. Some rats were pretreated with pinacidil (K⁺ _ATP channel opener), bradykinin, methacholine, or morphine before renal ischemia. Twenty-four h later, plasma creatinine was measured. Separate groups of rats received pertussis toxin intraperitoneally 48 h before being subjected to the above protective protocols. IPC and adenosine pretreatment protected against renal IR injury. Pretreatment with pertussis toxin and chelerythrine abolished the protective effects of both renal IPC and adenosine. However, glibenclamide pretreatment had no effect on either renal IPC or adenosine-induced renal protection, indicating no apparent role for K⁺ _ATP channels. Moreover, pinacidil, bradykinin, methacholine, and morphine failed to protect renal function. Therefore, the conclusion is that cellular signal transduction pathways of renal IPC and adenosine pretreatment in vivo involve G_i/o proteins and PKC but not K⁺ _ATP channels. Unlike cardiac IPC, bradykinin, muscarinic, and opioid receptors do not mediate renal IPC.

	Introduction

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Renal dysfunction secondary to ischemic reperfusion (IR) injury contributes to frequent and grave clinical morbidity in aortovascular surgery and anesthesia (1,2). Postoperative acute renal failure implies a poor clinical prognosis and is frequently associated with many other life-threatening complications, including sepsis and multiorgan failure (1,2,3). In high-risk patients undergoing major vascular surgery and anesthesia, the mortality and morbidity rate from perioperative acute renal failure has changed little during the past 30 yr; the incidence of renal dysfunction after aortovascular surgery is reported to be as high as 50% (3).

Murry (4), in 1986, first reported the protective effects of "ischemic preconditioning (IPC)" against IR injury in cardiac muscle by showing that multiple brief ischemic periods before a prolonged ischemic period lessened myocardial dysfunction and infarction size after the reperfusion period. We recently demonstrated that IPC protects renal function and morphology in rats after 45 min of ischemia and 24 h of reperfusion (5).

Extensive studies of cardiac IPC have implicated pre-ischemic activation of adenosine receptors (AR), specifically, A₁, AR, as a predominate mechanism that mediates protection (6,7). In addition, it is hypothesized in cardiac models of IPC that stimulation of A₁ AR results in activation and translocation of protein kinase C (PKC) from the cytosolic to the membrane compartment, resulting in phosphorylations of unknown cytoprotective proteins (8,9). In addition to PKC, many studies of cardiac protection with IPC and A₁ AR activation suggest that activation and opening of ATP-sensitive potassium (K⁺ _ATP) channels are involved (6,10). In models of cardiac protection, PKC and K⁺ _ATP channel activation are thought to be coupled to cell surface A₁ AR via pertussis toxin-sensitive G-proteins (i.e., G_i/o, (11,12). Therefore, we hypothesized that G_i/o, PKC, and K⁺ _ATP channels are intermediate signaling proteins involved in adenosine- and IPC-mediated renal protection from IR injury as has been demonstrated in the heart.

We recently demonstrated that systemic adenosine pretreatment protects renal function via A₁ AR activation and mimics renal IPC (5). However, unlike most models of cardiac preconditioning, renal IPC was not blocked by an A₁ AR antagonist. This suggests either that IPC- and adenosine-mediated protection follow completely different cellular signaling pathways with a common physiologic end point or that multiple endogenous agonists with common intermediate signaling pathways are involved in renal IPC. Evidence from cardiac preconditioning suggests that endogenously released agonists other than adenosine, e.g., bradykinin, acetylcholine, and opioid agonists, can also mimic IPC (13,14,15,16,17).

The distal portion (S₃ segment) of the proximal tubule located in the outer medulla of the kidney is the primary site of injury in renal ischemia and reperfusion (18,19) because of its marginal oxygenation under normal physiologic conditions coupled with high basal metabolic demand (19,20,21). These renal tubular cells express the A₁ AR (22) as well as the bradykinin (23,24), muscarinic (25,26), and opioid (27,28) receptors. Therefore, the second hypothesis of the current study was that bradykinin, muscarinic, or opioid receptors may mimic the protection induced by A₁ AR activation.

	Materials and Methods

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General Surgical Preparation
All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Surgical procedures have been previously described (5). Briefly, adult male Wistar rats were anesthetized with pentobarbital intraperitoneally (45 mg/kg body wt or to effect) and allowed to breathe room air spontaneously. After 500 U of heparin (intraperitoneally) and maintaining the body temperature at 37°C, the right femoral artery and vein were cannulated with heparinized (10 U/ml) polyethylene tubing (PE-50). After a 10-min stabilization period, a midline laparotomy was performed. A right nephrectomy was performed, and the left renal artery and vein were isolated.

IPC and Adenosine Pretreatment
For IPC and adenosine pretreatment protocols, rats were subjected to the following protocols after right nephrectomy as described previously (5): (1) control group (SHAM), isolation of left renal artery and vein only; (2) ischemia-reperfusion group (IR), 45 min of left renal ischemia followed by reperfusion; (3) ischemic preconditioning group (IPC), four cycles of 8 min of left renal ischemia separated by 5 min of reperfusion periods before 45 min of left renal ischemia followed by reperfusion; and (4) adenosine pretreatment group (ADO), adenosine (1.75 mg/kg per min x 10 min, intravenously) 2 min before 45 min of left renal ischemia followed by reperfusion. Twenty-four h later, animals were killed and serum creatinine (Cr) levels were measured.

Role of PKC in IPC- and Adenosine-Mediated Renal Protection
To determine the potential role of PKC in renal IPC- or adenosine-induced renal protection, we subjected the rats to the following protocols after right nephrectomy: (1) PKC antagonist (chelerythrine) controls (Che+Sham), chelerythrine (5 mg/kg, intraperitoneally) 15 min before sham operations; (2) chelerythrine and ischemia-reperfusion (Che+IR), chelerythrine 15 min before being subjected to 45 min of left renal ischemia followed by reperfusion; (3) chelerythrine before adenosine (Che+ADO), chelerythrine 15 min before 10 min of adenosine pretreatment followed by 45 min of left renal ischemia followed by reperfusion; and (4) chelerythrine before IPC (Che+IPC), chelerythrine 15 min before IPC treatment followed by 45 min of left renal ischemia followed by reperfusion. Twenty-four h later, animals were killed and serum Cr levels were measured.

Role of K⁺ _ATP Channels in IPC- and Adenosine- Mediated Renal Protection
To determine the potential role of K⁺ _ATP channels in renal IPC- or adenosine-induced renal protection, we subjected the rats to the following protocols after right nephrectomy: (1) high dose K⁺ _ATP channel antagonist (glibenclamide) controls (H-Glib+Sham), glibenclamide (6 mg/kg intravenously) 30 min before sham operations; (2) high dose glibenclamide and ischemia-reperfusion (H-Glib+IR), glibenclamide (6 mg/kg intravenously) 30 min before 45 min of left renal ischemia followed by reperfusion; (3) low dose glibenclamide before adenosine (L-Glib+ADO), glibenclamide (1 mg/kg intravenously) 30 min before 10 min of adenosine pretreatment followed by 45 min of left renal ischemia followed by reperfusion; (4) high dose glibenclamide before adenosine (H-Glib+ADO), glibenclamide (6 mg/kg intravenously) 30 min before 10 min of adenosine pretreatment followed by 45 min of left renal ischemia followed by reperfusion; (5) high dose glibenclamide before IPC (H-Glib+IPC), glibenclamide (6 mg/kg intravenously) 30 min before IPC treatment followed by 45 min of left renal ischemia followed by reperfusion; and (6) pinacidil pretreatment group, pinacidil, a K⁺ _ATP channel opener, (100 µg/kg per min x 10 min intravenously) 2 min before 45 min of left renal ischemia followed by reperfusion. Twenty-four h later, animals were killed and serum Cr levels were measured.

Potential Roles of Muscarinic, Bradykinin, and Opioid Receptors in Renal Protection
To determine whether other agonists with similar signaling pathways in renal cells mimic renal protection afforded by adenosine or IPC, methacholine (a muscarinic agonist), bradykinin, or morphine was given to rats before ischemia and reperfusion. Rats were divided to the following groups after right nephrectomy: (1) methacholine pretreatment group (MCh), methacholine (2 mg/kg, intraperitoneally) 15 min before 45 min of left renal ischemia followed by reperfusion; (2) bradykinin pretreatment group (BK), bradykinin (500 µg/kg per min x 10 min intravenously) terminating 2 min before 45 min of left renal ischemia followed by reperfusion; and (3) morphine pretreatment group (Morph), morphine (5 mg/kg intraperitoneally) 15 min before 45 min of left renal ischemia followed by reperfusion. Twentyfour h later, animals were killed and serum Cr levels were measured. The doses of chelerythrine, glibenclamide, methacholine, bradykinin, and morphine were selected based on previous in vivo studies (15,28,29,30,31).

Role of Pertussis Toxin—Sensitive G-Proteins in Renal Protection
To determine the potential role of pertussis toxin—sensitive G-proteins in renal IPC- and adenosine-induced renal protection, we initially pretreated rats for 48 h with pertussis toxin 25 µg/kg intraperitoneally. Pilot studies demonstrated that all of the rats that were pretreated intraperitoneally with 25 µg/kg pertussis toxin and subjected to 45 min of renal ischemia died within 8 h during the reperfusion period with evidence of elevated body temperature and distended fluid-filled small bowel. Therefore, three separate and independent changes in the protocol were made in an attempt to enhance survival after pertussis toxin treatment and ischemia-reperfusion: (1) The dose of pertussis toxin was reduced to 10 µg/kg intraperitoneally, (2) the reperfusion period was shortened to 6 h in some rats, and (3) the ischemic period was shortened from 45 to 30 min. Despite lowering the pertussis toxin dose to 10 µg/kg, none of the rats survived more than 10 h after 45 min of renal ischemia. The Cr measured at 6 h of reperfusion indicated that pertussis toxin pretreatment blocked IPC- and adenosine-mediated renal protection (see the Results section). In an attempt to prolong the survival after pertussis toxin (10 µg/kg intraperitoneally) and renal ischemia, the ischemic time interval was reduced to 30 min, resulting in a greater that 60% survival during 24 h of reperfusion. Subsequently, the following protocols were performed: (1) ischemia-reperfusion group with modified ischemia protocol (IR30), 30 min of left renal ischemia and reperfusion; (2) IPC group with modified ischemia protocol (IPC30), four cycles of 8 min of left renal ischemia separated by 5 min of reperfusion periods before 30 min of left renal ischemia followed by reperfusion; (3) adenosine pretreatment group with modified ischemia protocol (ADO30), adenosine (1.75 mg/kg per min x 10 min intravenously) until 2 min before 30 min of left renal ischemia followed by reperfusion; (4) pertussis controls (PTX+SHAM), pertussis toxin (10 µg/kg intraperitoneally) 48 h before the sham operation; (5) pertussis toxin and ischemia-reperfusion group (PTX+IR30), pertussis toxin (10 µg/kg intraperitoneally) 48 h before 30 min of left renal ischemia followed by reperfusion; (6) pertussis toxin before IPC group (PTX+IPC30), pertussis toxin (10 µg/kg intraperitoneally) 48 h before being subjected to four cycles of 8 min of left renal ischemia separated by 5 min of reperfusion periods before 30 min of left renal ischemia followed by reperfusion; (7) pertussis toxin before adenosine group (PTX+ADO30), pertussis toxin (10 µg/kg intraperitoneally) 48 h before receiving an systemic intravenous infusion of adenosine (1.75 mg/kg per min x 10 min) until 2 min before 30 min of left renal ischemia followed by reperfusion.

Effectiveness of K⁺ _ATP Channel Blockade by Intravenous Glibenclamide In Vivo
To test the effectiveness of glibenclamide in blocking K⁺ _ATP channels in vivo, we measured hemodynamic and metabolic parameters. Rats received 0.3 mg/kg pinacidil (K⁺ _ATP channel opener) intravenously while a second group was pretreated with 6 mg/kg of glibenclamide intravenously 30 min before receiving pinacidil (0.3 mg/kg intravenously). Maximal changes in mean arterial BP were recorded for each animal. In addition, blood glucose was measured colorimetrically by Antec Diagnostics (Farmingdale, NY) 60 min after receiving glibenclamide (6 mg/kg intravenously).

Measurement of Cr
Plasma Cr levels were measured spectrophotometrically using a commercially available quantitative colorimetric assay (Sigma, St. Louis, MO).

Materials
Adenosine, pertussis toxin, and methacholine were dissolved in sterile, isotonic saline. All other drugs were dissolved in 50% DMSO. Solutions were made daily. Pentobarbital was purchased from Henry Schein Veterinary Co. (Indianapolis, IN). All other drugs were obtained from Sigma Chemical Company.

Statistical Analyses
A one-way ANOVA was used to compare mean values across multiple treatment groups with a Dunnett post hoc multiple comparison test, e.g., SHAM versus IPC. In all cases, a probability statistic less than 0.05 was taken to indicate significance. All data are expressed throughout the text as mean ± SEM.

	Results

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Protective Effects of Renal IPC and Systemic Adenosine Pretreatment
Forty-five min of renal ischemia and 24 h of reperfusion (IR) resulted in significant rises in Cr (4.6 ± 0.3 mg/dl [n = 9]) compared with the sham-operated group (Cr = 0.8 ± 0.1 mg/dl [n = 12]; P < 0.01; Figure 1). IPC significantly improved renal function (Cr = 2.5 ± 0.4 mg/dl [n = 9]; P < 0.05) after 45 min of renal ischemia and 24 h of reperfusion compared with animals that were subjected to IR injury alone (Figure 1). Systemic adenosine (ADO) pretreatment also resulted in significant improvements in renal function (Cr = 1.7 ± 0.4 mg/dl [n = 12]; P < 0.01; Figure 1) compared with animals that were subjected to IR injury alone.

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Pretreatment with a protein kinase C (PKC) antagonist blocks adenosine- and ischemic precondition (IPC)-mediated renal protection from ischemic reperfusion (IR) injury. Comparison of mean creatinine (Cr) measured from sham-operated (Sham, n = 12), chelerythrine (5 mg/kg intraperitoneally) treatment before sham operation (Che+Sham, n = 4), ischemic-reperfusion (IR, n = 9), ischemic-preconditioned (IPC, n = 9), adenosine-pretreated (ADO, n = 9), chelerythrine treatment before ischemia and reperfusion (Che+IR, n = 3), chelerythrine treatment before IPC (Che+IPC, n = 9), and chelerythrine treatment before adenosine (Che+ADO, n = 10) animals. *, P < 0.05 versus sham; #, P < 0.05 versus IR. Error bars represent 1 SEM.

Roles of PKC and K⁺ _ATP Channels in Renal IPC- and Adenosine-Induced Renal Protection
Chelerythrine (Che, 5 mg/kg, intraperitoneally), a PKC antagonist, given 15 min before IPC or adenosine infusion abolished the renal protection induced by IPC (Cr = 4.1 ± 0.4 mg/dl [n = 9]) or adenosine (Cr = 4.4 ± 0.5 mg/dl [n = 10]; Figure 1). Chelerythrine itself had no effect on renal function of sham-operated rats (Cr = 0.7 ± 0.1 mg/dl [n = 4]) or of rats that underwent 45 min of renal ischemia and 24 h of reperfusion (Cr = 4.4 ± 0.8 mg/dl [n = 3]).

In contrast to the effects of chelerythrine, both the low (L-Glib, 1 mg/kg) and high (H-Glib, 6 mg/kg) doses of glibenclamide, a selective antagonist for K⁺ _ATP channels, given 30 min before adenosine failed to block the renal protection by systemic adenosine pretreatment (low dose: Cr = 1.8 ± 0.5 mg/dl [n = 4]; high dose: Cr = 1.7 ± 0.4 mg/dl [n = 6]; Figure 2). High-dose glibenclamide also failed to block the renal protection by IPC (Cr = 1.9 ± 0.3 mg/dl [n = 6]; Figure 2). Glibenclamide (6 mg/kg intravenously) given alone had no effect on renal function of sham-operated rats (Cr = 1.1 ± 0.1 mg/dl [n = 3]) or of rats that were subjected to ischemia and reperfusion (Cr = 4.0 ± 0.2 mg/dl [n = 4]). Moreover, pretreatment with pinacidil, a K⁺ channel opener, failed to protect renal function (Cr = 4.0 ± 0.3 mg/dl [n = 3]).

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. K+ ATP channels are not involved in adenosine- or IPC- mediated renal protection from IR injury. Comparison of mean Cr measured from sham-operated (Sham, n = 12), high-dose glibenclamide (6 mg/kg intravenously) treatment before sham operation (H-Glib+Sham, n = 4), ischemic-reperfusion (IR, n = 9), ischemic-preconditioned (IPC, n = 9), adenosine-pretreated (ADO, n = 9), high-dose glibenclamide treatment before ischemia and reperfusion (H-Glib+IR, n = 4), high-dose glibenclamide treatment before IPC (H-Glib+IPC, n = 6), low-dose glibenclamide (1 mg/kg intravenously) treatment before adenosine (L-Glib+ADO, n = 6), high-dose glibenclamide treatment before adenosine (H-Glib+ADO, n = 6), and pinacidil treatment before ischemia and reperfusion (PIN+IR, n = 3) animals. *, P < 0.05 versus sham; #, P < 0.05 versus IR.

Effectiveness of K⁺ _ATP Channel Antagonism by Glibenclamide In Vivo
Activation of vascular K⁺ _ATP channels leads to hypotension in vivo. Figure 3 shows the hypotensive response (maximum drop in mean arterial BP = 78 ± 16 mmHg [n = 3]) to pinacidil (0.3 mg/kg intravenously), a K⁺ _ATP channel opener. Pretreatment with glibenclamide (6 mg/kg intravenously) 30 min before pinacidil bolus significantly attenuated the hypotension (maximum drop in mean arterial BP = 13 ± 6 mmHg [n = 3]; P < 0.05), indicating an effective in vivo blockade of vascular K⁺ _ATP channels.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effectiveness of glibenclamide in antagonizing vascular K+ ATP channels as demonstrated by attenuated pinacidil-induced reductions in mean arterial BP (mmHg). Glibenclamide pretreatment (H-Glib, 6 mg/kg intravenously [n = 3]) significantly attenuated the hypotension induced by pinacidil (0.3 mg/kg intravenously [n = 3]). *, P < 0.05 versus pinacidil-only group.

We also measured blood glucose levels before and 60 min after glibenclamide (6 mg/kg intravenously). Blockade of pancreatic K⁺ _ATP channels results in an increased release of insulin and subsequent fall in blood glucose. Glibenclamide significantly decreased the blood glucose level from 224 ± 40 mg/dl (n = 3) to 31 ± 11 mg/dl 60 min after injection (P < 0.01). Control rats that were not treated with glibenclamide had significantly higher blood glucose levels (152 ± 15 mg/dl [n = 3]) 60 min after initiation of the surgical procedure (preoperative blood glucose = 208 ± 12 mg/dl [n = 3]). Taken together, these physiologic effects of glibenclamide suggest an effective blockade of K⁺ _ATP channels in the present study.

Pertussis Toxin Abolished Protective Effects of Renal IPC and Adenosine Pretreatment
The bradycardic responses to adenosine and R-phenyliso-propyladenosine (R-PIA) are known to be mediated via activation of A₁ AR that couple to G_i/o (32,33). Forty-eight h of either 10 or 25 µg/kg pertussis toxin treatment abolished the bradycardic effects of R-PIA and adenosine (data not shown), indicating effective in vivo blockade of G_i/o by our pertussis toxin pretreatment regimen.

Pertussis toxin pretreatment alone had no effect on animals' hemodynamic profile, apparent well-being, or appearance. Moreover, pertussis toxin—treated rats that underwent sham operations had similar renal function (Cr = 1.1 ± 0.1 mg/dl [n = 2]) compared with the sham-operated controls (Cr = 0.8 ± 0.1 mg/dl [n = 12]). However, none of the pertussis toxin—treated animals (either 10 or 25 µg/kg) survived more than 8 h after being subjected to 45 min of renal ischemia. Therefore, in the initial group of experiments, the reperfusion period was shortened to 6 h in both the pertussis toxin—treated and control groups. Forty-five min of renal ischemia and 6 h of reperfusion resulted in significant rises in Cr (2.7 ± 0.2 mg/dl [n = 6]), although these rises as expected were much less than those of rats that were subjected to 45 min of renal ischemia and 24 h of reperfusion (Cr = 4.6 ± 0.3 mg/dl [n = 9]). IPC (Cr = 1.4 ± 0.1 mg/dl [n = 6]) and R-PIA (A₁ AR agonist) pretreatment (Cr = 1.9 ± 0.1 mg/dl [n = 6]) also protected renal function after 45 min of renal ischemia and the shorter reperfusion period of 6 h. However, pretreatment with pertussis toxin (25 µg/kg intraperitoneally) 48 h before renal IPC (Cr = 3.0 ± 0.4 mg/dl [n = 6]) and A₁ AR agonist (Cr = 2.7 ± 0.4 mg/dl, [n = 6]) abolished renal protection by either A₁ AR activation or IPC.

In an attempt to enhance survival beyond 24 h, both the dose of pertussis toxin and the duration of renal ischemia were reduced in the next series of experiments, which resulted in more than 60% of the animals surviving 24 h of reperfusion. Thirty min of renal ischemia and 24 h of reperfusion resulted in significant rises in Cr (4.1 ± 0.2 mg/dl [n = 3]; Figure 4). IPC (Cr = 1.5 ± 0.2 mg/dl [n = 3]) and adenosine pretreatment (Cr = 1.3 ± 0.1 mg/dl [n = 3]) protected renal function in these rats (Figure 4). The rats that were pretreated for 48 h with 10 µg/kg pertussis toxin intraperitoneally and then subjected to 30 min of renal ischemia and 24 h of reperfusion also exhibited significant impairments of renal function (Cr = 4.4 ± 0.5 mg/dl [n = 4]). However, pretreatment with 10 µg/kg pertussis toxin 48 h before renal IPC (Cr = 5.0 ± 0.3 mg/dl [n = 3]) or systemic adenosine (Cr = 5.3 ± 0.5 [n = 3]) abolished their renal protective effects (Figure 4). These data suggest that pertussis toxin—sensitive G-proteins are signaling intermediates in both adenosine- and IPC-mediated protection of renal IR injury.

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Pretreatment with pertussis toxin blocks adenosine- and IPC-mediated renal protection from IR injury. Comparison of mean Cr measured from sham-operated (Sham, n = 4), 48-h pertussis toxin treatment before sham operation (PTX+Sham, n = 2), ischemic-reperfusion (IR30, n = 3), ischemic-preconditioned (IPC30, n = 3), adenosine-pretreated (ADO30, n = 3), 48-h pertussis toxin treatment before ischemia and reperfusion (PTX+IR30, n = 4), 48-h pertussis toxin treatment before IPC (PTX+IPC30, n = 3), and 48 h pertussis toxin treatment before adenosine (PTX+ADO30, n = 3) animals. For this series of study, all groups underwent 30 min of global renal ischemia and 24 h of reperfusion. *, P < 0.05 versus sham; #, P < 0.05 versus IR.

Methacholine, Bradykinin, and Morphine Failed to Protect Renal Function
Intravenous bradykinin at 500 µg/kg per min caused a similar degree of hypotension (systolic BP, approximately 60 to 70 mmHg) as observed with intravenous adenosine (5). Systemic methacholine caused transient reduction in BP but was associated with markedly increased salivary and lacrimal secretions. Pretreatments with systemic methacholine (Cr = 3.8 ± 0.1 mg/dl [n = 6]), bradykinin (Cr = 4.6 ± 0.1 mg/dl [n = 5]), or morphine (Cr = 4.3 ± 0.2 mg/dl [n = 4]) failed to protect renal function (Figure 5). This is in contrast to protections obtained with either 10 min of systemic adenosine pretreatment or with renal IPC (5).

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Bradykinin, methacholine, or morphine did not mimic adenosine-mediated renal protection from IR injury. Comparison of mean Cr measured from sham-operated (Sham, n = 12), IR (IR, n = 9), adenosine-pretreated (ADO, n = 9), bradykinin-pretreated (BK, n = 6), methacholine-pretreated (MCh, n = 6), and morphine-pretreated (Morph, n = 4) animals before ischemia and reperfusion. *, P < 0.05 versus sham; #, P < 0.05 versus IR.

	Discussion

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The current in vivo study in the rat kidney demonstrates that renal IPC- and adenosine-mediated renal protection from IR injury involve both pertussis toxin—sensitive G-proteins and PKC. In contrast to the findings in most models of cardiac and skeletal muscle preconditioning (6,10,34), K⁺_ATP channels are not involved in renal protection. In addition, unlike adenosine (6,7) and unlike some models of cardiac preconditioning (13,14,15,35), pretreatments with bradykinin, methacholine, or morphine, agonists that are known to mimic IPC in cardiac muscle, failed to protect renal function after ischemia and reperfusion. The current study is the first to identify signaling intermediates that are responsible for renal IPC- and adenosine-induced renal protection.

The results of our studies have similarities and differences to IPC studies in the heart. In our previous study (5), we were able to protect renal function with pre-ischemic A₁ AR activation and mimic renal IPC. However, we were unable to block the protective effects of renal IPC with an A₁ AR antagonist. We hypothesized either that IPC- and adenosine-induced protection in the kidney follows completely different cellular signaling pathways or that multiple endogenous agonists are involved in renal IPC. This second hypothesis led us to test whether activation of other endogenous receptors such as muscarinic, bradykinin, or opioid receptors protected renal function against IR injury. In some studies of cardiac IR injury, other receptors that demonstrate common intracellular signaling intermediates with adenosine, such as bradykinin (14,15), acetylcholine (13), phenylephrine (36), and opioids (35), mimic IPC. That multiple agonists (bradykinin, morphine, acetylcholine, and phenylephrine) are able to induce cardiac preconditioning suggested to us that A₁ AR activation per se may not be the only agonist inducing protection but that any agonist that stimulates common second messenger signaling intermediates (e.g., G_i/o and PKC), may also protect the heart against IR injury. However, unlike studies in the heart, we did not find that activation of muscarinic, bradykinin, or opioid receptors mimicked renal IPC, suggesting that activation of G_i/o alone was not sufficient to mediate renal protection. Alternatively, it is possible that the rat renal cells that are protected by IPC and adenosine pretreatment may not express muscarinic, bradykinin, or opioid receptors that couple to G_i/o.

The current study demonstrated that PKC plays a role in renal IPC- and adenosine-induced renal protection in vivo. We used chelerythrine, a highly selective and specific PKC antagonist that inhibits the PKC catalytic domain (K_i = 0.7 µM (37)), to block the physiologic effects of PKC to determine whether PKC activation is required for renal IPC- and adenosine-induced renal protection. It is accepted that PKC plays a critical role in mediating IPC- and A₁ AR-mediated cardiac protection (8,30). PKC modulates both short- and long-term cellular responses after IR injury, such as ion channel regulation, new protein synthesis, and cellular proliferation. Coupling of A₁ AR to PKC via G_i/o in renal tubules has been confirmed previously (38,39,40). Currently, it is not conclusively known which subtypes of PKC are involved in mediating cardiac preconditioning, although evidence exists that the and/or isoforms may be involved in the heart (8).

A₁ AR, including those present in the kidney, couple to intracellular effectors via G_i/o (pertussis toxin—sensitive G-proteins (22,32). In cardiac IPC from a number of species including the rat, G_i/o are intermediates in modulating adenosine's protective effect (11). Moreover, cardiac preconditioning in vivo is abolished after the pertussis toxin treatment (12,41). Therefore, we hypothesized that by blocking G_i/o with pertussis toxin, we may prevent the renal protective effects of IPC and adenosine. The doses of pertussis toxin used in this study (25 and 10 µg/kg) were based on previous studies of cardiac IPC in rats (11,12,41). Pertussis toxin treatment for 48 h abolished the protective effects of renal IPC and adenosine pretreatment, supporting a role for G_i/o proteins as intermediates in renal IPC- and adenosine-induced renal protection from IR injury. Although we did not measure the degree of adenosine diphosphate ribosylation of G_i/o in this study, that A₁ AR-mediated bradycardic responses to R-PIA and adenosine were abolished in pertussis toxin—treated rats suggests that A₁ AR-G_i/o coupling was blocked with pertussis toxin treatment. Endon et al. (42) also demonstrated that pertussis toxin at doses ranging from 1.25 to 10 µg/kg dose-dependently attenuated the negative inotropic and chronotropic effects of atrial muscarinic receptor activity; the pertussis toxin dose of 10 µg/kg maximally inhibited the receptor-G_i/o interactions.

That pertussis toxin treatment associated with in vivo renal ischemia led to significant mortality and morbidity is a limitation in our study. None of the rats survived for 24 h after 45 min of global renal ischemia with either 10 or 25 µg/kg of pertussis toxin. We were able to improve the survival rate by reducing the dose of pertussis toxin to 10 µg/kg and the ischemic time to 30 min. With this reduced dose of pertussis toxin and reduced ischemic interval, we enhanced the survival at 24 h of reperfusion and again showed that pertussis toxin pretreatment blocked both adenosine- and IPC-mediated protection from renal IR injury. It is unclear why the combination of pertussis toxin and renal ischemia in vivo is detrimental to rat survival. Sham-operated rats that received 10 to 25 µg/kg of pertussis toxin seemed normal before and after the surgical procedures.

K⁺_ATP channels are present in various cell types in the kidney, including the proximal tubule, the thick ascending limb of Henle's loop, and the cortical collecting duct (43). Under physiologic conditions, these channels have a high open probability and function to regulate renal blood flow, reabsorption of electrolytes and solutes, renin release, K⁺ secretion, and diuresis (43,44,45). In the heart and skeletal muscle, glibenclamide, a selective K⁺_ATP channel blocker, at doses ranging from 0.3 mg/kg to 3 mg/kg blocked the protective effects of IPC and adenosine (31,34,46). Moreover, the K⁺_ATP channels play a role to protect the brain against IR injury (47). These data suggest that IPC in these tissues may be mediated by the activation of K⁺_ATP channels coupled to A₁ AR and that these channels may serve an endogenous protective role. However, the current study does not support the hypothesis that renal protection by IPC or adenosine pretreatment involves the activation of K⁺_ATP channels. Neither the high (6 mg/kg) nor the low (1 mg/kg) doses of glibenclamide abolished the renal protection afforded by adenosine or IPC. Moreover, pinacidil, a K⁺_ATP channel activator, failed to protect renal function. Our hemodynamic and blood glucose data show that 6 mg/kg glibenclamide intravenously effectively blocked the K⁺_ATP channels in vivo. This finding makes the kidney unique compared with the brain, heart, and skeletal muscle, where K⁺_ATP channels are known to play an important role in tissue protection. The role of K⁺_ATP channels in excitable tissues such as brain, heart, and skeletal muscle seems to be different from the role of K⁺_ATP channels in cells of epithelial origin, e.g., renal tubules, in terms of cytoprotection.

In summary, we extended our in vivo studies in the rat to show that both G_i/o and PKC are signaling intermediates involved in renal protection induced by either IPC or adenosine pretreatment. Unlike the findings in cardiac models of protection from IR injury, K⁺_ATP channels are not signaling intermediates in renal protection. Moreover, bradykinin, methacholine, or opioid receptor activation does not induce renal protection from IR injury. These studies offer a mechanistic insight into the signaling intermediates that are responsible for adenosine-induced and IPC-induced renal protection from IR injury.

	Acknowledgments

 
This work was funded in part by intramural grant support from the Department of Anesthesiology, College of Physicians and Surgeons of the Columbia University.

	References

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