Adenosine attenuates oxidant injury in human proximal tubular cells via A1 and A2a adenosine receptors

H. T. Lee and Charles W. Emala

Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have recently demonstrated protection against renal ischemic-reperfusion injury in vivo by A1- and A2a-adenosine receptor (AR) modulations. To further elucidate the signaling cascades of AR-induced cytoprotection against reperfusion/oxidant-mediated injury, immortalized human proximal tubule (HK-2) cells were treated with H2O2. H2O2 caused dose- and time-dependent HK-2 cell death that was measured by lactate dehydrogenase release and trypan blue dye uptake. Adenosine protected against H2O2-induced HK-2 cell death by means of A1- and A2a-AR activation. A1-AR-mediated protection involves pertussis toxin-sensitive G proteins and protein kinase C, whereas A2a-AR-mediated protection involves protein kinase A activation by means of cAMP and activation of the cAMP response element binding protein. Moreover, protein kinase A activators (forskolin and Sp-isomer cAMP) also protected HK-2 cells against H2O2 injury. De novo gene transcription and protein synthesis are required for both A1- and A2a-AR-mediated cytoprotection as actinomycin D and cycloheximide, respectively, blocked cytoprotection. Chronic treatments with a nonselective AR agonist abolished the protection by adenosine. Moreover, chronic treatments with a nonselective AR antagonist increased the endogenous tolerance of HK-2 cells against H2O2. We concluded that A1- and A2a-AR activation protects HK-2 cells against H2O2-induced injury by means of distinct signaling pathways that require new gene transcription and new protein synthesis.

adenosine 3',5'-cyclic monophosphate; immortalized human proximal tubule cells; hydrogen peroxide; pertussis toxin-sensitive G proteins; protein kinase A; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE RENAL FAILURE SECONDARY to ischemic-reperfusion (I/R) injury continues to be a significant clinical problem (22, 32, 45). The onset of acute renal failure implies a poor prognosis and is frequently complicated by many other life-threatening complications, including sepsis and multiorgan failure (3, 31, 32). In high-risk patients undergoing high-risk surgery, the mortality and morbidity rate from perioperative acute renal failure has changed little over the past 30 years (3, 13, 20, 45).

The A1 and A2a adenosine receptors (ARs) serve to protect against I/R injury in many organ systems, including the heart, brain, and kidney (16, 23-26, 36). We have recently demonstrated that pre- and postischemic activation of renal A1- and A2a-ARs, respectively, protected renal function against I/R injury in vivo (24-26). The in vivo mechanism of preischemic A1-AR-mediated renal protection involves pertussis toxin-sensitive G proteins and protein kinase C (PKC), whereas postischemic A2a-AR-mediated protection is through protein kinase A (PKA) activation by means of cAMP.

There are many limitations in using in vivo models of renal I/R injury to elucidate the detailed signaling cascades of AR-mediated renal cytoprotection. In vivo approaches must deal with multiple cell types within an organ and also with complex physiological control within an animal. In vitro studies with a pure population of a single cell type eliminate these complex external physiological influences and allow the direct study of signaling cascades.

The proximal tubules (especially, the straight, distal portion or S3 segment) located in the outer medulla of the kidney are the primary site of injury in renal ischemia and reperfusion (28, 48). Immortalized adult human proximal tubular cells (HK-2) have been transfected with E6/E7 genes of human papilloma virus type 16 (41). Transfection with human papilloma virus type 16 has been shown to immortalize epithelial cells of diverse origin without significantly altering their phenotypes or functions. HK-2 cells have been shown to retain the phenotypic expression and functional characteristics of human proximal tubules (39, 41). Extensive studies have utilized HK-2 cells to study in vitro renal physiology and pathology (18, 19, 54). Presently, there are four subtypes of identified ARs (A1, A2a, A2b, and A3) (12). We have recently verified the presence of all four subtypes of ARs and demonstrated several key signaling intermediates in HK-2 cells (26a).

There are few studies of in vitro renal cell protection with AR modulations. Adenosine protected against hypoxia-reoxygenation injury in a porcine kidney cell line (LLC-PK1) by means of an A2a-ARright-arrow cAMP-mediated mechanism (52). However, AR-mediated protection in a human renal cell line has not been reported to date. In this study, we injured HK-2 cells with H2O2. Oxygen free radicals with resultant oxidant tissue stress are a key mediator of renal reperfusion injury (15, 37). During reperfusion after ischemia, reactive oxygen species, such as superoxide anion, hydroxyl radical, and H2O2, are generated. These reactive oxygen species cause lipid peroxidation of the renal cell membrane, with a resultant intracellular calcium overload and subsequent necrotic cell death (37, 42, 43). We hypothesized that as we observed in vivo, A2a-AR activation would protect against oxidant-mediated injury in human renal cells. We also aimed to determine the signaling pathways of AR-mediated protection against oxidant injury in HK-2 cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HK-2 Cell Culture

HK-2 cells (American Type Culture Collection, Manassas, VA) were grown and passaged in 75-cm2 cell culture flasks containing culture medium (keratinocyte serum-free medium + 5 ng/ml epidermal growth factor and 40 mg/ml bovine pituitary extract) and antibiotics (100 U/ml of penicillin G, 100 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B) at 37°C in a 100% humidified atmosphere of 5% CO2-95% air. They were plated in 6- or 24-well plates when 80% confluent and used in the experiments described in Specific Protocols when confluent.

Methods for Inducing Oxidant Injury: H2O2-Induced Injury for HK-2 Cells

Oxidant injury in HK-2 cells was induced with H2O2. After indicated pretreatments (e.g., with AR agonists or with vehicle), confluent monolayers of HK-2 cells grown in 6- or 24-well plates were treated with H2O2 diluted in serum-free media for 1-3 h.

Measurement of Cell Viability and Cell Death

Cell viability assays were performed by using the trypan blue dye exclusion method. After the treatment protocols (e.g., H2O2 ± AR agonists), cells were trypsinized and stained with 0.4% trypan blue dye for 5 min. The proportion of cells remaining nonviable (unable to exclude trypan blue) was counted by using a hemocytometer. The number of cells remaining nonviable was expressed as a percentage of the total number of cells.

Lactate dehydrogenase (LDH) released into the media was also measured as a marker of cellular injury by using a commercially available colorimetric method (Sigma, St. Louis, MO). In some experiments, LDH released into the media was expressed as the percentage of total cellular LDH per well measured after the cells were lysed with 1% Triton X. Otherwise, LDH release after the various treatment protocols (e.g., H2O2 ± AR agonists) was expressed as the percentage of the LDH release by the H2O2 group within the same experiment.

Immunoblot Analyses

We measured activation of cAMP response element binding (CREB) proteins in HK-2 cells by immunoblotting with antibodies to the phosphorylated forms of CREB. The HK-2 cells in 6-well plates were washed twice with Hanks' balanced salt solution and scraped with 100 µl of calcium-free Hanks' balanced salt solution plus protease inhibitors (2 µg/ml leupeptin and 2 µg/ml aprotinin). Aliquots were used for protein assay, and the remainder were mixed with an equal volume of sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 5% beta -mercaptoethanol, final concentration). Twenty to forty micrograms of each sample were electrophoresed at room temperature through discontinuous 10% SDS-polyacrylamide gels at 80 V for 4 h and subjected to immunoblot analysis as described previously (17). The primary polyclonal antibody for phospho-CREB (New England Biolabs) was diluted 1:1,000 in Tris-buffered saline-0.1% Tween 20 containing 1% nonfat dry milk and 0.02% sodium azide. The secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase at 1:5,000 dilution) was detected with enhanced chemiluminescence immunoblotting detection reagents (Amersham), with subsequent exposure to autoradiography film. The intensities of the immunoblots were quantified with a scanner coupled to a personal computer with MacBas 2.2 software.

Up- and Downregulation of ARs in HK-2 Cells

We have previously demonstrated that treatment for 48 h with a nonselective AR antagonist, 8-phenyltheophylline (8-PT), upregulated A1- and A2a-ARs, whereas 48-h treatment with a nonselective AR agonist 5'-N-ethylcarboxamidoadenosine (NECA), downregulated all four subtypes of ARs (26a). When confluent, HK-2 cells grown in 6- or 24-well plates were incubated with 100 µM of nonselective AR antagonist 8-PT or 10 µM of nonselective agonist NECA for 48 h in complete cell culture media.

Protein Determination

Protein content was determined with the Pierce Chemical (Rockford, IL) bicinchoninic acid protein assay reagent with BSA as a standard.

Specific Protocols

We first determined the protective roles of adenosine and AR subtype-specific agonists against H2O2-mediated oxidant injury in HK-2 cells.

H2O2-induced injury. HK-2 cells in serum-free cell culture media were subjected to 1-3 h of 2, 5, and 10 mM H2O2. [The 1-h time point was chosen for the subsequent studies with 5 mM of H2O2 as this time and dose of H2O2-induced moderate (~50-60%) cellular injury.]

Adenosine before H2O2-induced injury. HK-2 cells were incubated with 1-100 µM of adenosine for 30 min in serum-free cell culture media and then subjected to 1 h of 5 mM H2O2 injury.

Selective AR agonists before H2O2-induced injury. HK-2 cells were incubated with 1 nM-10 µM of R-N6-phenyl-isopropyladenosine (R-PIA), 4-[(N-ethyl-5'-carbamoyadenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS-21680), or N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA), highly selective A1-, A2a-, and A3-AR agonists, respectively, for 30 min in serum-free cell culture media and then subjected to 1 h of 5 mM H2O2 injury.

Selective AR antagonists before adenosine. HK-2 cells were incubated with 1-10 µM of 1,3-dipropyl-8-cyclopentylxanthine, 8-(3-chlorostyryl)caffeine, or 9-chloro-2-(2-fury)[1,2,4] triazolo[1,5-c]quinazolin-5-phenylacetamide, highly selective A1-, A2a-, and A3-AR antagonists, respectively, for 30 min in serum-free cell culture media before 30 min of 100 µM adenosine followed by 1 h of 5 mM H2O2 injury.

We have previously described that PKC and pertussis toxin-sensitive G proteins (Gi/o) play important roles in signal transduction of A1-AR-mediated renal protection in vivo (24). To determine the potential roles of Gi/o and PKC in adenosine-mediated protection against oxidant injury, HK-2 cells were subjected to the following protocols.

Adenosine or selective AR agonists + pertussis toxin before H2O2-induced injury. HK-2 cells were pretreated with 100 ng/ml pertussis toxin for 14 h before adenosine or selective A1-, A2a-, or A3-AR agonist treatment followed by 1 h of 5 mM H2O2 injury.

PKC antagonist before adenosine or AR agonists. HK-2 cells were incubated with 100 nM of GF-109203X, a highly selective PKC antagonist, for 30 min in serum-free cell culture media before adenosine or selective A1-, A2a-, or A3-AR agonist treatment followed by 1 h of 5 mM H2O2 injury.

The A2a-ARs in HK-2 cells couple to adenylyl cyclase to activate the cAMP and PKA pathway (26a). In addition, we demonstrated that A2a-ARs protect against reperfusion injury in vivo by means of activation of PKA by cAMP (26). To determine the potential roles of PKA and cAMP in adenosine-induced protection against oxidant injury, HK-2 cells were subjected to the following protocols.

PKA antagonist before adenosine or AR agonist. HK-2 cells were incubated with 100 µM of Rp-isomer (Rp)-cAMP for 30 min in serum-free cell culture media before adenosine or selective A1-, A2a-, or A3-AR agonist treatment followed by 1 h of 5 mM H2O2 injury.

PKA agonist instead of adenosine. HK-2 cells were incubated with 100 µM of Sp-isomer (Sp)-cAMP for 30 min in serum-free cell culture media before 1 h of 5 mM H2O2 injury.

To determine the roles for new gene transcription and new protein synthesis in adenosine-induced renal protection against oxidant injury, HK-2 cells were subjected to the following protocols.

Actinomycin D before adenosine. HK-2 cells were incubated with 10 µg/ml actinomycin D, an inhibitor of new gene transcription, for 60 min in serum-free media before 30 min of 100 µM adenosine followed by 1 h of 5 mM H2O2 injury.

Cycloheximide before adenosine. HK-2 cells were incubated with 10 µg/ml cycloheximide, an inhibitor of new protein synthesis, for 60 min in serum-free media before 30 min of 100 µM adenosine followed by 1 h of 5 mM H2O2 injury.

After these treatment protocols, cell injury was quantified by measuring cellular release of LDH and trypan blue dye exclusion.

Materials

Adenosine was dissolved in saline. All other drugs were dissolved first in DMSO and then further diluted in water such that the final concentration of DMSO in each experimental condition was <0.01%. Solutions were made daily. All chemicals used were of the purest analytical grade and obtained from Sigma.

Statistics and Data Analysis

The data were analyzed with Student's t-test when means between two groups were compared or with one-way analysis of variance plus Dunnett's post hoc multiple comparison test to compare mean values across multiple treatment groups.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

H2O2 Injures HK-2 Cells

Exogenous H2O2 was used to induce free radical-mediated oxidant injury in HK-2 cells. Figure 1 shows that H2O2 injures HK-2 cells in a time (5 mM H2O2, n = 3; Fig. 1A)- and dose-dependent (1 h, n = 3; Fig. 1B) manner. HK-2 cell injury was quantified by the amount of LDH released into the cell culture media and by the percentage of trypan blue dye exclusion. Figure 1B also shows the protection against H2O2-mediated (5 mM) injury by catalase (9,000 U/ml). The 1-h time point was chosen for the subsequent studies with 5 mM of H2O2, because this time and dose of H2O2 induced moderate (~50-60%) cellular injury.


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Fig. 1.   H2O2 (5 mM) injures HK-2 cells in a time (n = 3; A)- and dose-dependent (1-h incubation, n = 3; B) manner. Cell injuries were quantified by measuring the percentage of total cellular lactate dehydrogenase (LDH) released into the culture media after a specified incubation period and dose. Catalase (9,000 U/ml) prevented HK-2 cell death after H2O2. Error bars, SE.

Adenosine Protects Against H2O2 Injury

Figures 2 and 3 show that adenosine pretreatment for 30 min significantly protects against H2O2-mediated cell death. With 100 µM of adenosine pretreatment, significantly less LDH (66.7 ± 5.3% of 5 mM H2O2-alone treated group, n = 6, P < 0.05; Fig. 2) was released into the cell culture media and more cells excluded trypan blue dye (6.1+0.9% trypan blue-positive cells vs. 14.8 ± 1.1% for 5 mM H2O2-alone-treated group, n = 6, P < 0.05; Fig. 3).


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Fig. 2.   Adenosine (ADO) potently attenuates H2O2-mediated HK-2 cell injury (n = 6). Cell injuries were quantified by measuring LDH released into the culture media from cells treated with 1-100 µM adenosine or vehicle for 30 min before the addition of 5 mM H2O2 for 1 h. Error bars, SE. *P < 0.05 vs. vehicle + H2O2-treated group (Vehicle).



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Fig. 3.   Adenosine (100 µM, n = 6), A1-adenosine receptor (AR) agonist [R-N6-phenyl-isopropyladenosine (R-PIA), 10 µM, n = 6] and A2a-AR agonist {4-[(N-ethyl-5'-carbamoyadenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS-21680), 10 µM, n = 6} added 30 min before a 1-h treatment with 5 mM H2O2 attenuated H2O2-mediated HK-2 cell injury. Cell injuries were quantified by measuring the percentage of cells taking up trypan blue dye. Error bars, SE. *P < 0.05 vs. vehicle + H2O2-treated group.

A1- and A2a-ARs Are Involved in Adenosine-Mediated Protection Against H2O2

In HK-2 cells, adenosine-mediated protection against H2O2-mediated injury involves the A1- and A2a-ARs as A1-AR agonist R-PIA (Figs. 3 and 4A) and A2a-AR agonist CGS-21680 (Figs. 3 and 4B) pretreatment provided significant protection against H2O2-induced cell injury. The LDH release in 10 µM R-PIA- and 10 µM CGS-21680-pretreated HK-2 cells was 78.6 ± 3.1% (n = 15, P < 0.05) and 67.7 ± 3.6% (n = 18, P < 0.05) of the H2O2-alone-treated group. Similar results were observed for trypan blue uptake when investigators were blinded to experimental conditions. The trypan blue uptake of R-PIA (9.3 ± 0.2%, n = 6)- and CGS-21680-treated (5.1 ± 0.5%, n = 6) cells was also significantly reduced compared with the H2O2-alone-treated group (14.8 ± 1.1%, n = 6, P < 0.05; Fig. 3). The A3-AR agonist IB-MECA (10 µM) failed to protect against oxidant injury (LDH release of 115.3 ± 5.4% of the H2O2-alone-treated group, n = 12).


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Fig. 4.   A1-AR agonist [R-PIA (PIA), n = 6: A] and A2a-AR agonist [CGS-21680 (CGS), n = 6; B] attenuate H2O2-mediated HK-2 cell injury. Cell injuries were quantified by measuring LDH released into the culture media from cells treated with 0.01-10 µM R-PIA or 0.01-10 µM CGS-21680 or vehicle for 30 min before the addition of 5 mM H2O2 for 1 h. Error bars, SE. *P < 0.05 vs. vehicle + H2O2-treated group.

A1-ARs Protect Via Gi/o and PKC Pathway

We utilized specific inhibitors of Gi/o proteins and PKC to determine the involvement of these signaling intermediates in A1- and A2a-AR-mediated protection against oxidant injury. Pertussis toxin treatment (100 ng/ml, 14 h) or pretreatment for 30 min with a PKC inhibitor (GF-109203X, 100 nM) blocked A1-AR agonist (10 µM R-PIA)-mediated protection against H2O2 (LDH = 105.6 ± 8.0%, n = 6, and LDH = 97.4 ± 3.6%, n = 6, of H2O2-alone-treated group, respectively; Fig. 5A). However, pertussis toxin and GF-109203X failed to block A2a-AR agonist (10 µM CGS-21680)-mediated protection against oxidant injury (LDH = 75.0 ± 6.4%, n = 6, and LDH = 79.9 ± 2.7%, n = 6, of H2O2-alone- treated group, respectively; Fig. 5A).


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Fig. 5.   A1-AR-mediated protection against H2O2 injury is mediated by pertussis toxin (PTX)-sensitive G proteins and is protein kinase C dependent (n = 6; A), whereas the A2a-AR-mediated protection involves protein kinase A (PKA) activation by cAMP (n = 6; B). Cell injuries were quantified by measuring LDH released into the culture media from cells pretreated with an inhibitor [of Gi/o, protein kinase C (PKC) or PKA] for 30 min before R-PIA or CGS-21680, which were applied 30 min before treatment with 5 mM H2O2 for 1 h. PTX 100 ng/ml, 14-h pretreatment; GF, GF-109203X PKC antagonist (100 nM, 30-min pretreatment); Sp-cAMP, PKA agonist (100 µM, 30-min pretreatment); Rp-cAMP, PKA antagonist (100 µM, 30-min pretreatment); Forsk, forskolin (10 µM, 30-min pretreatment); IB-MECA, N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine. Error bars, SE. *P < 0.05 vs. vehicle + H2O2-treated group.

A2a-ARs Protect Via cAMPright-arrow PKA Pathway

The A2a-AR-mediated cytoprotection against oxidant injury involves adenylyl cyclase activation and generation of cAMP to stimulate PKA, because the PKA agonist Sp-cAMP (100 µM, 30-min pretreatment) and the PKA antagonist Rp-cAMP (100 µM, 30-min pretreatment) mimicked and blocked, respectively, the A2a-AR agonist-induced protection against oxidant injury (LDH = 57.9 ± 6.3%, n = 6, and LDH = 91.0 ± 3.2%, n = 6, of H2O2-alone-treated group, respectively; Fig. 5B). A1-AR-mediated cellular protection was not blocked by Rp-cAMP. Moreover, forskolin (10 µM, 30-min pretreatment), an agonist that increases cAMP levels, mimicked the cytoprotection provided by the A2a-AR agonist (LDH = 69.8 ± 6.7%, n = 6; Fig. 5B).

Upregulation of A1- and A2a-ARs Modulates Protection Against H2O2

We have previously demonstrated that treatment for 48 h with a nonselective AR antagonist (8-PT) upregulated A1- (~1.9-fold) and A2a- (~1.5-fold) ARs, whereas 48-h treatment with a nonselective AR agonist (NECA) downregulated all four subtypes of ARs (26a). In this study, we demonstrate that chronic treatments with a nonselective AR antagonist conferred significant endogenous protection against oxidant injury (Fig. 6). After 48 h of treatment with 8-PT, 5 mM H2O2 killed significantly fewer HK-2 cells (LDH = 50.5 ± 3.1% of H2O2 group, n = 6). Conversely, chronic NECA treatment significantly attenuated the protection by 100 µM adenosine pretreatment (LDH = 90.8 ± 1.2%, n = 6).


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Fig. 6.   Chronic pretreatment with a nonselective AR antagonist 8-phenyltheophylline (8-PT), 100 µM for 48 h, known to increase expression of A1- (~1.9-fold) and A2a- (~1.5-fold) ARs (26a) resulted in endogenous protection against acute H2O2 injury (n = 6). Conversely, chronic pretreatment with a nonselective AR agonist [5'-N-ethylcarboxamidoadenosine (NECA), 10 µM for 48 h, known to decrease expression of all 4 subtypes of ARs] significantly attenuated the cytoprotective effect of acute adenosine (100 µM for 30 min, n = 6). Cell injuries were quantified by measuring LDH released into the culture media after addition of 5 mM H2O2 for 1 h compared with LDH released by the H2O2 group. Error bars, SE. *P < 0.05 vs. H2O2-alone group; #P < 0.05 vs. vehicle-treated acute adenosine treatment group.

Role of CREB in Oxidant Injury

We have previously demonstrated the expression of both phosphorylated (activated) and nonphosphorylated forms of CREB in HK-2 cells (26a). Oxidant injury with H2O2 significantly decreased the phosphorylated (activated) form of CREB. The A2a-AR agonist (CGS-21680) and Sp-CAMP rescued the decreased phosphorylation of CREB induced by H2O2. Activation of the CREB transcription factor correlates with cytoprotection by the A2a-AR agonist and Sp-CAMP (Fig. 7).


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Fig. 7.   Cytoprotective effects of PKA activation measured by phospho-specific antibody. H2O2 (5 mM) profoundly attenuated cAMP response element binding (CREB) phosphorylation (total n = 6). Catalase (Catal; 9,000 U/ml), A2a-AR agonist (CGS-21680, 10 µM, 30-min pretreatment), and Sp-cAMP (100 µM, 30-min pretreatment) rescued CREB phosphorylation. A1-AR agonist (R-PIA, 10 µM, 30-min pretreatment) had no effect on CREB activity. Representative immunoblot of HK-2 cell lysates with phosphospecific CREB (top) is also shown [(n = 2 for control (Cont), H2O2, catalase groups and n = 3 for R-PIA and CGS-21680]. Bands for Sp-cAMP are not shown. Error bars, SE. *P < 0.05 vs. vehicle-treated control group.

Role of New Gene Transcription and Protein Synthesis in Adenosine-Induced Cytoprotection

Actinomycin D and cycloheximide blocked the protection by the A1-AR agonist (LDH = 98.9 ± 7.5, n = 6, and 101.4 ± 4.7, n = 4, respectively, of the H2O2-alone-treated group), the A2a-AR (LDH = 97.3 ± 2.3, n = 6, and 104.6 ± 2.9, n = 3, respectively) agonist, and Sp-CAMP (LDH = 100.2 ± 2.6, n = 6, and 103.2 ± 5.8, n = 3, respectively), indicating that new transcription and protein synthesis, respectively, are required for A1- and A2a-AR-mediated protection against H2O2 oxidant injury (Fig. 8).


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Fig. 8.   Cytoprotection mediated by both A1- and A2a is dependent on new gene and protein synthesis. Cell injuries were quantified by measuring LDH released into the culture media after addition of 5 mM H2O2 for 1 h and compared with the LDH released by the H2O2-alone group. R-PIA and CGS-21680 = 10 µM of A1-and A2a-AR-selective agonist, respectively (30-min pretreatment); Sp-cAMP = 100 µM, 30-min pretreatment; n = 6 for actinomycin D experiments, and n = 4 for cycloheximide experiments. Error bars, SE. *P < 0.05 vs. H2O2-alone group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adenosine protects against H2O2-mediated oxidant injury in HK-2 cells by means of both A1- and A2a-AR activation. The A1-AR-mediated protection involves PKC by means of pertussis toxin-sensitive G proteins (Gi/o), whereas the A2a-AR-mediated protection is by means of PKA activation by cAMP. Our findings complement our previous studies in which differential AR modulations protected against global renal I/R injury in vivo (24-26).

The detrimental effect of renal I/R injury that results in acute renal failure is a serious and unresolved clinical challenge (47). Surgical procedures involving the aorta and renal arteries (e.g., supra- and juxtarenal abdominal aortic aneurysms and renal transplantation, in particular) display significant postoperative renal complications in the form of acute tubular necrosis and acute renal failure (3, 31, 32). Although ischemia can cause renal cell death and injury, significant renal tubular and vascular damage develop during the reperfusion period secondarily to oxygen free radical-mediated cellular injury (2, 34). Oxygen free radicals are considered to be important mediators of reperfusion injury, because reperfusion of a previously ischemic kidney results in robust generation of oxygen free radicals (34). H2O2 is formed in mitochondria as a dismutation product of the superoxide radical (O<UP><SUB>2</SUB><SUP>−</SUP></UP>) under physiological conditions. However, under ischemic stress, there is proteolytic modification of xanthine dehydrogenase to xanthine oxidase, which effectively produces a profoundly increased burst of O<UP><SUB>2</SUB><SUP>−</SUP></UP> and H2O2 when oxygen is reintroduced during reperfusion. H2O2 and its far more toxic metabolite, the hydroxyl radical, contribute significantly to renal injury during reperfusion. Cytotoxic oxidant free radicals produce cellular lipid peroxidation, cytotoxic enzyme activation, impaired energy metabolism, protein oxidation, and a massive rise in intracellular Ca2+ concentration (34, 43).

Renal cells with proximal tubular characteristics, including LLC-PK1 (pig), NHK-C (human), and opossum kidney cells, are much more susceptible to oxidant-reperfusion injury than those with distal tubular characteristics (Madin-Darby canine kidney cells) (50, 51). In this study, we utilized H2O2-mediated oxidant injury as an in vitro model of reperfusion injury. In initial pilot studies, our attempt to produce in vitro hypoxia-reoxygenation injury by using a hypoxic chamber was unsuccessful. Incubation (24 h) in a 95% N2-5% CO2 chamber followed by reoxygenation failed to produce significant HK-2 cell death measured by LDH release (26a). Epithelial cells such as HK-2 can utilize amino acid by means of gluconeogenesis to produce glucose and, therefore, are not susceptible to hypoxic cell death. Moreover, achieving true anoxia by removing O2 from the cell culture environment is often impractical. Many in vivo and in vitro models of renal oxidant injury utilized exogenous application of oxygen free radicals such as H2O2 or t-butylhydroperoxide to mimic the free radical-mediated injury of reperfusion (29, 53).

The proximal tubules (especially, the straight, distal portion or S3 segment) located in the outer medulla of the kidney are the primary site of injury in renal ischemia and reperfusion (28, 48). This region is marginally oxygenated under normal physiological conditions, with a high basal metabolic demand (11, 28). Therefore, with hypoxic or ischemic insult, proximal tubules in the outer medullary zone suffer the most damage.

To show that HK-2 cells are a valid cellular model to study adenosine-mediated protection of renal proximal tubule cells, we have demonstrated that HK-2 cells express all four subtypes (A1, A2a, A2b, and A3) of ARs and display key signaling intermediates, including several PKC isoforms (alpha , delta , and epsilon ), G proteins (Gi, Gs, and Gq), and mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase, and p38] (26a). We also verified that A1- and A3-ARs inhibit forskolin-stimulated adenylyl cyclase activity and that A2a-ARs stimulate adenylyl cyclase activity. In addition, chronic (48-h) antagonist (8-PT) and agonist (NECA) treatment led to up- and downregulation of various ARs and G protein subtypes.

Adenosine has cytoprotective effects in several cell types, including renal cells (5, 10). AR activation, specifically the A1 and A2a subtypes, attenuates several factors responsible for generating I/R injury (27, 38, 49). A1-AR activation attenuates I/R injury when given before the ischemic insult in cerebral (38), cardiac (9, 49), and renal (25) cells and has been implicated to mediate ischemic preconditioning. Conversely, postischemic A2a-AR also protects against tissue injury by attenuating the reperfusion phase of the injury process in pulmonary (1), cardiac (7), and renal cells (26).

In this study, we demonstrated that both A1- and A2a-AR agonists protected against the direct cytotoxic effects of H2O2 (nonreceptor-mediated cytotoxicity) by means of distinct receptor-mediated cellular mechanisms in HK-2 cells. We demonstrated in this study that A2a-ARs, by means of cAMP-dependent mechanisms, protected HK-2 cells against H2O2-mediated cell death. The A2a-AR-mediated protection from H2O2 injury was blocked by Rp-cAMP, an inhibitor of PKA. Additionally, agents that increase cAMP, isoproterenol and forskolin, or direct activation of PKA by Sp-cAMP also protected against H2O2-induced cell injury. These in vitro results agree with in vivo results previously described in which A2a-AR activation protected against renal I/R injury in vivo (26, 35). Stimulation of A2a-ARs, including those present in renal tubule cells and vasculature, results in increased cellular cAMP to activate PKA (4, 40). Our study agrees with previous studies that suggest that increased intracellular cAMP protects against renal reoxygenation-oxidant injury in vivo (6) and in vitro (21, 52). Our study also agrees with previous studies showing that agents that increase intracellular cAMP also attenuate reperfusion injury in the heart, lung, and kidney in vivo and in vitro (8, 44).

After oxidant injury, CREB activity decreased significantly. We have previously demonstrated in HK-2 cells that A2a-ARs activate CREB via cAMP- and PKA-dependent pathways (26a). Moreover, improved preservation of HK-2 CREB activity after A2a-AR activation or with Sp-cAMP correlates with improved cellular survival after H2O2-mediated injury. We propose that increased HK-2 cell survival and activation of CREB is one of the potential mechanistic links after A2a-AR activation by means of cAMP and PKA.

We also demonstrated that activation of A1-ARs in HK-2 cells also protects against H2O2-induced cellular injury via signaling pathways involving PKC and Gi/o proteins. This was shown by attenuating the protection induced by the A1-AR agonist R-PIA with pertussis toxin and GF-109203X. These findings agree with our previous in vivo studies in which both Gi/o and PKC were intermediates in A1-AR-mediated protection of renal I/R injury (24). This study agrees with heart studies in which A1-ARs are involved in protection against H2O2-induced oxidative injury in vivo and in vitro (33, 46) by modulation of the detrimental increases in intracellular calcium concentration and by means of activation of cardiomyocyte KATP channels after H2O2 exposure (14, 46). After exposure to H2O2, intracellular ATP decreases significantly (~30% of baseline, data not shown), and the A1-AR activation may attenuate this component of cellular injury. Therefore, whereas the A2a-ARs attenuate the free radical-mediated cellular injury (e.g., lipid peroxidation and membrane damage), the A1-ARs may attenuate the cellular injury caused by a lethal drop in intracellular ATP.

In summary, this is the first report of an in vitro protective effect of adenosine in HK-2 cells by means of A1- and A2a-AR activation. We have systematically deciphered the signal pathways of A1- and A2a-AR-mediated renal cytoprotection. These findings have potentially profound clinical significance in the protection of the kidney in the perioperative care of patients subjected to renal ischemia.


    ACKNOWLEDGEMENTS

This work was funded in part by intramural grant support from the Department of Anesthesiology, Columbia University College of Physicians and Surgeons, and by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO-1-DK-58547.


    FOOTNOTES

Address for reprint requests and other correspondence: H. T. Lee, Dept. of Anesthesiology, Columbia Presbyterian Medical Center, P&S Box 46 (PH-5), 630 West 168th St., New York, NY 10032-3784 (E-mail: tl128{at}columbia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 20, 2001;10.1152/ajprenal.00195.2001

Received 26 June 2001; accepted in final form 16 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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