A 1 adenosine receptor knockout mice exhibit increased renal injury following is - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:17 |只看该作者 |倒序浏览 |打印

作者：H. Thomas Lee, Hua Xu, Samih H. Nasr, Jurgen Schnermann, and Charles W. Emala作者单位：Departments of 1 Anesthesiology and 2 Pathology, College of Physicians and Surgeons of Columbia University, New York, New York 10032; and 3 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 / W2 P7 a" h/ _# C$ T& c
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      【摘要】
   Controversy exists regarding the effect of A 1 adenosine receptor (AR) activation in the kidney during ischemia and reperfusion (I/R) injury. We sought to further characterize the role of A 1 ARs in modulating renal function after I/R renal injury using both pharmacological and gene deletion approaches in mice. A 1 AR knockout mice (A 1 KO) or their wild-type littermate controls (A 1 WT) were subjected to 30 min of renal ischemia. Some A 1 WT mice were subjected to 30 min of renal ischemia with or without pretreatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or 2-chrolo-cyclopentyladenosine (CCPA), selective A 1 AR antagonist and agonist, respectively. Plasma creatinine and renal histology were compared 24 h after renal injury. A 1 KO mice exhibited significantly higher creatinines and worsened renal histology compared with A 1 WT controls following renal I/R injury. A 1 WT mice pretreated with the A 1 AR antagonist or agonist demonstrated significantly worsened or improved renal function, respectively, after I/R injury. In addition, A 1 WT mice pretreated with DPCPX or CCPA showed significantly increased or reduced markers of renal inflammation, respectively (renal myeloperoxidase activity, renal tubular neutrophil infiltration, ICAM-1, TNF-, and IL-1 mRNA expression), while demonstrating no differences in indicators of apoptosis. In conclusion, we demonstrate that endogenous or exogenous preischemic activation of A 1 ARs protects against renal I/R injury in vivo via mechanisms leading to decreased necrosis and inflammation. 2 C- l* o- G' T- A# i3 F( E
      【关键词】 acute renal failure inflammation ischemicrepefusion injury
   ACUTE RENAL FAILURE (ARF) secondary to ischemic reperfusion (I/R) injury continues to be a significant perioperative problem. ARF is frequently complicated by many other life-threatening complications including sepsis and multiorgan failure ( 1 ). The prognosis for ARF is poor (with mortality 50%) and has changed little over the past 40 years ( 11, 21 )., Q2 r# H+ I0 G6 l- ?4 _

All four known adenosine receptor (AR) subtypes (A 1, A 2a, A 2b,A 3 ) are expressed in the kidney ( 16, 25 ). We demonstrated previously that pharmacological AR modulations significantly affect renal function after I/R injury in rats ( 12, 14, 15 ). In particular, we showed that preischemic activation of A 1 ARs attenuates renal failure after I/R injury in vivo ( 14 ). We also demonstrated the cytoprotective effects of A 1 AR activation in cultured proximal tubule cells injured with H 2 O 2 or with severe ATP depletion ( 13, 17 ).8 F% ^6 |5 k6 z- u. [; \
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In contrast, other investigators reported that a nonselective AR antagonist (theophylline) or selective A 1 AR antagonists [1,3-dipropyl-8-cyclopentylxanthine (DPCPX), KW-3902] improve renal function, urinary output, and renal hemodynamics against renal injury induced by other insults such as cisplatin, gentamicin, or glycerol ( 2, 5, 10, 23 ). Therefore, we sought to further substantiate the role of the A 1 AR in renal I/R injury using a mouse model genetically deficient in this receptor subtype, an approach that complements and extends the conclusions reached from pharmacological studies. Furthermore, we used a selective A 1 agonist [2-chrolo-cyclopentyladenosine (CCPA)] or antagonist (DPCPX) in separate groups of wild-type littermate controls (A 1 WT). As our previous studies indicated that preischemic A 1 AR activation was renal protective, we hypothesized that A 1 AR knockout mice (A 1 KO) mice would have worsened renal function after I/R injury compared with wild-type controls. We further hypothesized that preischemic activation or inhibition of A 1 ARs would protect against or worsen, respectively, ARF following I/R injury in wild-type mice. Furthermore, we questioned whether A 1 AR-mediated renal protection from I/R injury was associated with a change in markers of inflammation and whether protection occurred by a reduction in necrotic vs. apoptotic injury.  z- Z$ h6 ]- j% U/ L* U) I: B

METHODS. b5 H2 {. w# B) C0 s$ B
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A 1 KO Mice

The generation and initial characterization of the A 1 KO and A 1 WT mice with a C57BL/6 background have been described previously ( 22 ).
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Renal Injury Protocol
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To determine the role of endogenous A 1 ARs in renal I/R injury, we used A 1 KO mice and their appropriate wild-type controls (A 1 WT). To determine the role of exogenous manipulations of A 1 ARs in renal I/R injury, we pretreated wild-type mice with an A 1 AR agonist or antagonist. Male A 1 KO or A 1 WT mice weighing 25-30 g were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg or to effect) and placed supine on a heating pad under a warming light to maintain body temperature between 36 and 38°C. Additional pentobarbital sodium was given as needed based on response to tail pinch. Bilateral flank incisions were made and the left kidney was subjected to 30 min of ischemia with a microaneurysm clip after right nephrectomy. The duration of ischemia was chosen to maximize reproducibility of renal injury and to minimize mortality in these mice. Some A 1 WT mice were pretreated either with DPCPX (1 mg/kg ip), a selective A 1 AR antagonist, or with CCPA (0.1 mg/kg ip), a selective A 1 AR agonist, 15 min before renal ischemia. Some mice in each group underwent anesthesia and laparotomy without renal ischemia to serve as sham-operated controls.8 A/ X, J$ v* D, |

We showed previously that A 3 AR activation before renal ischemia exacerbates whereas A 3 AR antagonism protects against ARF due to I/R injury ( 14, 18 ). To determine whether exacerbation of renal dysfunction in A 1 KO mice can be rescued with A 3 AR antagonism, some A 1 KO mice were pretreated with 1 mg/kg MRS-1191 (a selective A 3 AR antagonist) before renal I/R injury.8 X+ r$ m$ n" I9 ?/ \+ d. l
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Assessment of Renal Function After I/R Injury9 g. b" _7 r2 W6 J3 _

Renal function was assessed by measuring plasma creatinine 24 h after renal ischemia using a commercially available colorimetric method (Sigma).' y+ V' J8 R- v
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Histological Examinations to Detect Necrosis
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For histological preparations, explanted kidneys were bisected along the long axis and were fixed in 10% formalin solution overnight. After automated dehydration through a graded alcohol series, transverse kidney slices were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin (H&E). Morphological assessment was performed by an experienced renal pathologist (S. H. Nasr) who was blinded to the treatment for each animal. A grading scale of 0-4, as outlined by Jablonski et al. ( 8 ), was used for the histopathological assessment of I/R-induced damage of the proximal tubules.; E; g8 H* W8 O" e4 `/ W

Assessment of Renal Inflammation# X% ?7 c& a* J. j( @

Renal inflammation after I/R injury was determined using multiple techniques.
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Renal cortical myeloperoxidase assay. Myeloperoxidase (MPO) is an enzyme present in neutrophils and macrophages and is an index of tissue neutrophil/macrophage infiltration ( 24 ). Twenty-four hours after renal ischemic injury, renal cortex ( 100 mg) was dissected and homogenized for 30 s in 1 ml of 50 mM potassium phosphate buffer, pH 7.4, at 4°C. The samples were centrifuged for 15 min at 16,000 g at 4°C, and the resultant pellet was resuspended in 1 ml of 50 mM potassium phosphate buffer, pH 7.4, with 0.5% hexadecyltrimethyl ammonium bromide at 4°C. The samples were sonicated for 30 s and centrifuged at 16,000 g for 15 min at 4°C. Fifty microliters of supernatant were mixed with 375 µl of 45 mM potassium phosphate buffer, pH 6.0, containing 0.167 mg/ml o -dianisidine and 0.3% H 2 O 2. The remaining supernatant was used to determine protein concentrations. Absorbance (460 nm) was measured over a period of 5 min, and the MPO enzyme activity was expressed as optical density (OD) per minute per milligram of protein.

Histological quantification of neutrophil infiltration. Postischemic infiltration of neutrophils contributes to the inflammatory process and injury in the kidney ( 3, 6 ). Neutrophils were identified by the morphology of the nucleus and localization of the cell under light microscopy of H&E stains by an experienced pathologist (S. H. Nasr) who was blinded to the treatment groups. Neutrophils were quantified in 75 randomly chosen microscope fields (magnification x 400) in the corticomedullary junction, and results were expressed as neutrophils counted per millimeter squared.

Immunohistochemistry for neutrophils. We also detected renal neutrophil infiltration using immunohistochemistry. Fixed mouse kidney sections were deparaffinized in xylene and rehydrated through graded ethanols to water. After blocking with 10% normal horse serum/PBS solution, the slides were stained for neutrophils by sequential incubation with rat anti-mouse neutrophil (mAb 7/4; Serotec, Raleigh, NC) at a 1:60 dilution for 30 min followed by HRP-conjugated rabbit anti-rat IgG at a 1:60 dilution for 30 min and diaminobenzidine reagent (Vector Laboratories, Burlingame, CA) for 10 min.' t* j, J' Z2 a0 y& }" L

Semiquantitative RT-PCR for TNF-, IL-1, and ICAM-1. Twenty-four hours after renal ischemic injury, renal cortices were dissected and total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) reagent. Total RNA concentrations were determined by spectrophotometric absorbance at 260 nm, and aliquots were analyzed by agarose gel electrophoresis to verify equal RNA inputs and RNA quality. RT-PCR was performed to analyze the expression of proinflammatory (TNF-, IL-1, and ICAM-1) genes. Primers were designed based on published GenBank sequences for the mouse ( Table 1 ). Primer pairs were chosen to yield expected PCR products of 200-600 bp and to amplify a genomic region that spans one or two introns to eliminate the confounding effect of amplifying contaminating genomic DNA. Primers were purchased from Sigma Genosys (The Woodlands, TX). RT-PCR was performed using the Access RT-PCR System (Promega, Madison, WI), which is designed for a single tube reaction for first-strand cDNA synthesis (48°C for 45 min) using AMV reverse transcriptase, and subsequent PCR using Tfl DNA polymerase. PCR cycles included a denaturation step of 94°C for 30 s followed by an optimized annealing temperature ( Table 1 ) for 1 min followed by a 1-min extension period at 68°C. All PCR reactions were completed with a 7-min incubation at 68°C to allow enzymatic completion of incomplete cDNAs. The PCR cycle number for each primer pair was optimized to yield linear increases in the densitometric measurements of resulting bands with increasing cycles of PCR (15 to 30 cycles). The starting amount of RNA was also optimized to yield linear increases in the densitometric measurements of resulting bands at an established number of PCR cycles. Based on these preliminary experiments, 0.5-1.0 µg of total RNA were used as the template for all RT-PCR reactions. The number of PCR cycles yielding linear results was 21, 24, and 22 for ICAM-1, TNF-, and IL-1, respectively. For each experiment, we also performed semiquantitative RT-PCR under conditions yielding linear results for GAPDH (15 cycles) to confirm equal RNA input. The products were resolved in a 6% polyacrylamide gel and stained with Syber Green (Roche, Indianapolis, IN), and the band intensities were quantified using a Fluor-S Multi Imager (Bio-Rad, Hercules, CA).9 |$ U! Y, Z' {, X2 {3 Y

Table 1. Primer sequence
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Assessmsnt of Renal Apoptosis; W) c" l. x5 e6 K0 z
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Histological examinations to detect apoptosis. Renal tubular apoptosis was assessed by counting the number of apoptotic bodies in proximal tubules in the outer stripe of the corticomedullary junction (expressed as mean number of apoptotic bodies per tubule) on H&E-stained kidney sections. This area is the most severely injured area after renal I/R injury. Apoptosis was identified visually in H&E-stained kidney sections (magnification x 400) as nuclear condensation and fragmentation with an intact plasma membrane. An average of 25-30 tubules per high-power field was counted and six fields were examined per slide. The morphological features used to identify apoptosis include cellular shrinkage and rounding, cytoplasmic eosinophilia, and chromatin condensation and fragmentation.2 I/ b% b! c4 p$ c
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DNA laddering. Renal apoptosis was also assessed by detecting DNA laddering (fragmentation). Twenty-four hours after renal ischemic injury, renal cortices were dissected and extracted DNA (Wizard, Promega) was electrophoresed at 70 V in a 2.0% agarose gel in Tris-acetate-EDTA buffer. The gel was stained with ethidium bromide and photographed under UV illumination. DNA ladder markers (100 bp) were added to each gel as a reference for the analysis of internucleosomal DNA fragmentation.
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In situ terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling assay. We also used terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) staining to detect DNA fragmentation in apoptosis. Fixed mouse kidney sections obtained at 24 h after renal injury were deparaffinized in xylene and rehydrated through graded ethanols to water. In situ labeling of fragmented DNA was performed with TUNEL (green fluorescence) using a commercially available in situ cell death detection kit (Roche) according to the manufacturer's instruction. To visualize the total number of cells in the field, kidney sections were also stained with propidium iodide (red fluorescence).2 {5 X: V# Z' w

Genotyping of Mice for Wild-Type or Knockout of A 1 ARs
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We analyzed the presence of the wild-type or mutant A 1 AR genomic DNA in A 1 WT and A 1 KO mice by PCR. To differentially genotype the A 1 KO mice from A 1 WT mice, primers were designed that recognize the 5' and 3' end of the wild-type A 1 AR (sense: 5'-ATGGAGTACATGGTCTACTTC-3' and antisense: 5'-GGAAGAGGATGAGGGCCAGCG-3') and the PGKneo insert (sense: 5'-CTATGACTGGGCACAACAGACAAT-3' and antisense: 5'-ATCAGCCATGATGGATACTTTCTC-3') in A 1 KO mice ( 22 ). All primers were purchased from Sigma Genosys. Genomic DNA was isolated from mouse tails using the Wizard Genomic DNA extraction kit (Promega). DNA concentrations were determined by spectrophotometric absorbance at 260/280. Genomic DNA (0.2 ug) was used as a template and amplified for 30 cycles ( Taq polymerase; Promega) using a PTC-200 thermal cycler (MJ Research, Waltham, MA). The products were resolved in a 6% polyacrylamide gel and stained with Syber Green (Roche) for analysis with a FluroS Multi Imager (Bio-Rad).6 @. B) a% U. d* ]& F

Statistics and Data Analysis

The data were analyzed with one-way analysis of variance plus Dunnett's post hoc multiple comparison test to compare mean values across multiple treatment groups.

Reagents) _: q# N5 Z& c

Unless otherwise specified, all reagents were purchased from Sigma (St. Louis, MO).5 Z4 R( d& G1 J
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Protein Determination" A* w7 G  {/ `# V  G
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Protein content was determined with a Pierce Chemical (Rockford, IL) bicinchoninic acid protein assay reagent with BSA as a standard.

RESULTS1 U, l+ a1 i8 y7 f( e

Genotyping of A 1 WT and A 1 KO Mice

To confirm the genotypes of mice used in these studies, genomic DNA extracted from tails was analyzed by PCR for the expression of the intact A 1 AR (A 1 WT mice) or the plasmid insert (PGKneo) originally used to disrupt the A 1 AR gene (A 1 KO mice) ( 22 ). Amplified PCR products corresponding to the full-length A 1 AR were only detected in A 1 WT mice, whereas PCR products corresponding to the PGKneo insert were only detected in the A 1 KO mice ( Fig. 1 ).  E, \7 I6 f$ U

Fig. 1. Representative PCR products using specific primers of mouse A 1 adenosine receptor (A 1 WT mice, expected size of 190 bp; A ) and PKGneo (A 1 KO mice, expected size of 290 bp; B ). DNA encoding the full-length A 1 adenosine receptor was detected in all A 1 WT mice, whereas the PKGneo insert was detected in all homozygous A 1 KO mice.# y5 N8 j2 S9 L+ h& B3 O2 S: P8 K

Endogenous Deletion or Exogenous Blockade of A 1 ARs Increased Mortality After I/R Injury
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Vehicle-, CCPA-, or DPCPX-treated sham-operated A 1 WT mice had 0% 24-h mortality. All of the A 1 WT mice subjected to renal I/R with or without pretreatment with CCPA survived 24 h after renal ischemic injury. However, 24-h mortality after I/R injury increased for A 1 KO mice (mortality = 20%) or A 1 WT pretreated with the A 1 AR antagonist DPCPX (mortality = 28.5%) before I/R injury.

Endogenous Deletion or Exogenous Blockade of A 1 ARs Worsens Renal Function After I/R Injury
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A 1 WT and A 1 KO mice that underwent sham operations had similar baseline renal function (Cr = 0.3 ± 0.1 mg/dl for A 1 WT, n = 6, and 0.3 ± 0.1 mg/dl for A 1 KO, n = 5; Fig. 2 ). However, 24 h after renal ischemic injury A 1 KO mice had significantly higher plasma creatinine (Cr = 2.2 ± 0.3 mg/dl, n = 10) compared with A 1 WT mice (Cr = 1.0 ± 0.1 mg/dl, n = 6, P 9 ~: ~  f# P! [! F2 ?3 g9 {

Fig. 2. Comparison of mean creatinine measured from sham-operated A 1 WT and A 1 KO mice (A 1 WT sham, n = 6; A 1 KO sham, n = 5), A 1 WT or A 1 KO subjected to ischemia and reperfusion (A 1 WT I/R, n = 6; A 1 KO I/R, n = 10), A 1 WT mice pretreated with DPCPX (A 1 WT I/R DPCPX, n = 6) or CCPA (A 1 WT I/R CCPA, n = 8) and subjected to ischemia and reperfusion, A 1 KO mice pretreated with MRS-1191 and subjected to ischemia and reperfusion (A 1 KO I/R MRS, n = 5). * P
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Endogenous Deletion or Exogenous Blockade of A 1 ARs Worsens Renal Histology After I/R Injury  l' t7 I" C2 Y4 A! a

Significantly worsened renal function in A 1 KO mice or DPCPX-pretreated wild-type mice subjected to I/R injury correlated with histological evaluations for necrosis. Furthermore, A 1 WT mice pretreated with the A 1 AR agonist CCPA showed less evidence of necrosis by histological evaluation. Representative histological slides are shown in Fig. 3. The Jablonski scale histology grading scores ( 8 ) are shown in Fig. 4. Twenty-four hours after 30 min of renal ischemia in A 1 WT mice resulted in mild to moderate acute tubular necrosis compared with wild-type sham-operated mice (A 1 WT I/R; grade: 1.5 ± 0.3, n = 4, and sham controls grade: 0.3 ± 0.1, n = 4). More severe renal injury developed in A 1 KO mice (grade: 2.7 ± 0.4, n = 4) or in DPCPX-pretreated A 1 WT mice (grade: 3.5 ± 0.3, n = 4) as evidenced by increased tubular necrosis, medullary congestion and hemorrhage, and the development of protein-aceous casts. A 1 WT mice pretreated with the A 1 AR agonist CCPA before I/R injury showed significantly improved renal morphology (grade: 0.5 ± 0.3, n = 4) compared with A 1 WT mice subjected to I/R only (grade: 1.5 ± 0.3, n = 4).
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Fig. 3. Representative photomicrographs of the outer medulla of the kidney. A : sham-operated A 1 WT mice. B : sham-operated A 1 KO mice. C : A 1 WT mice subjected to ischemia and reperfusion (I/R). D : A 1 KO mice subjected to I/R. E : A 1 WT mice pretreated with DPCPX. F : A 1 WT mice pretreated with CCPA and subjected to I/R (hematoxylin and eosin staining, magnification x 400).6 h; }8 w5 _# l8 ~4 p

Fig. 4. Jablonski grading scale scores of histological appearance of acute tubular necrosis from sham-operated A 1 WT ( n = 4) and A 1 KO mice ( n = 4), A 1 WT mice subjected to I/R injury (A 1 WT I/R, n = 4), A 1 KO mice subjected to I/R injury (A 1 KO I/R, n = 4), A 1 WT mice pretreated with DPCPX and then subjected to I/R injury (A 1 WT I/R DPCPX, n = 4), A 1 WT mice pretreated with CCPA and then subjected to I/R injury (A 1 WT I/R CCPA, n = 4). * P

Endogenous Deletion or Exogenous Blockade of A 1 ARs Increases Renal Inflammation After I/R Injury, K  g$ O9 `! G5 C# `7 d5 ^

Renal cortical MPO assay. A 1 WT mice subjected to I/R injury did not show increased MPO activity (1.1 ± 0.1 OD·min -1 ·mg protein -1, n = 6; Fig. 5 ) compared with A 1 WT sham-operated mice (0.9 ± 0.1 OD·min -1 ·mg protein -1, n = 6, P = 0.28). In contrast, A 1 KO mice (1.8 ± 0.1 OD·min -1 ·mg protein -1, n = 6) or A 1 WT mice treated with the A 1 AR antagonist DPCPX (1.9 ± 0.3 OD·min -1 ·mg protein -1, n = 6) and subjected to renal I/R had significantly higher MPO activity compared with A 1 WT subjected to I/R injury alone (1.1 ± 0.1 OD·min -1 ·mg protein -1, n = 6).3 Q2 h7 i3 a5 W) z

Fig. 5. Myeloperoxidase (MPO) activity in renal cortex of mice in sham-operated A 1 WT mice (A 1 WT sham, n = 6), A 1 KO mice (A 1 KO sham, n = 6), A 1 WT mice subjected to I/R injury (A 1 WT I/R, n = 6), A 1 KO mice subjected to I/R injury (A 1 KO I/R, n = 6), A 1 WT mice pretreated with DPCPX and then subjected to I/R injury (A 1 KO I/R DPCPX, n = 4). * P
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RT-PCR of proinflammatory genes. We performed semiquantitative RT-PCR to measure the expression of mRNA-encoding proinflammatory proteins (ICAM-1, TNF-, and IL-1; Fig. 6 ). Total RNA was isolated from renal cortices and the quantitative accuracy of our RT-PCR technique was first established (see METHODS ). A 1 KO or DPCPX-pretreated wild-type mice subjected to I/R injury showed increased expression of the mRNA-encoding ICAM-1, IL-1, and TNF- compared with A 1 WT subjected to injury alone or to sham-operated animals ( Fig. 6 ).
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Fig. 6. A : representative ICAM-1 ( bottom right ), IL-1 ( top ), and TNF- ( bottom left ) mRNA expression measured by semiquantitative RT-PCR in mouse kidney cortex from sham-operated A 1 WT mice (A 1 WT sham), A 1 WT mice subjected to I/R injury (A 1 WT I/R), sham-operated A 1 KO mice (A 1 KO sham), A 1 KO mice subjected to I/R injury (A 1 KO I/R), A 1 WT mice pretreated with DPCPX and then subjected to I/R injury (A 1 KO I/R DPCPX). One-hundred-bp RNA size marker (M) is shown for each gel. B : densitometric quantifications of band intensities from RT-PCR reactions for ICAM-1, IL-1, and TNF- ( n = 4). Data in bar graphs are means ± SE. * P
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Immunohistochemical and histological detection of neutrophils. Figure 7 shows representative immunohistochemical detection of neutrophil infiltration into the corticomedullary junction 24 h after sham operation or I/R injury in mice. Renal I/R injury results in recruitment of neutrophils that cause further renal injury. In sham A 1 WT mice, we detected 0.7 ± 0.8 neutrophils/mm 2 ( n = 4) in the corticomedullary junction by histological examination of H&E stains that increased to 13.5 ± 6.3 neutrophils/mm 2 ( n = 4) after I/R injury. A 1 KO mice subjected to I/R injury had a higher neutrohil count in the corticomedullary junction (32.5 ± 5.0 neutrophils/mm 2, n = 4, P
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Fig. 7. Representative photomicrograph of neutrophil accumulation in the renal cortex for sham-operated A 1 WT mice (A 1 WT sham; A ), sham-operated A 1 KO mice (A 1 KO sham; B ), A 1 WT mice subjected to I/R injury (A 1 WT I/R; C ), A 1 KO mice subjected to I/R injury (A 1 KO I/R; D ), A 1 WT mice pretreated with DPCPX and then subjected to I/R injury (A 1 KO I/R DPCPX; E ), or A 1 WT mice pretreated with CCPA and then subjected to I/R injury (A 1 KO I/R CCPA; F ). Few neutrophils are present in sham mice. After I/R injury, marked neutrophil accumulation was found in the renal cortex of A 1 KO ( D ) or DPCPX-pretreated A 1 WT ( E ) mice (magnification x 200).
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Endogenous Deletion or Exogenous Blockade of A 1 ARs Does Not Increase Renal Apoptosis After I/R Injury

Assessment of renal tubular apoptotic bodies. The degree of renal tubular apoptosis was quantified by counting the number of apoptotic bodies in proximal tubules in the corticomedullary area of the kidney (expressed as apoptotic bodies/tubule). Twenty-five to 30 tubules were counted per field from each treatment group and kidneys from four experiments were examined. Sham-operated A 1 WT (0.02 ± 0.01) or A 1 KO (0.02 ± 0.01) mice had no morphological evidence of apoptosis. Renal I/R injury increased the number of apoptotic bodies within proximal tubules in both A 1 WT (0.34 ± 0.04, P % L) t# t: |6 d. l0 {
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DNA laddering. Although all mice subjected to I/R injury demonstrated DNA laddering in DNA isolated from renal cortices, there were no differences between A 1 WT or A 1 KO mice ( Fig. 8 ).
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Fig. 8. Representative experiment (of 4) illustrating DNA fragmentation (laddering) in an ethidium bromide-stained agarose gel. Equal amounts of DNA were loaded in each lane. No laddering was visible in DNA extracted from sham-operated kidneys of A 1 WT (A 1 WT sham) and A 1 KO (A 1 KO sham) mice. DNA from A 1 WT (A 1 WT I/R) and A 1 KO (A 1 KO I/R) mice subjected to I/R injury showed similar degree of DNA fragmentation. Each lane represents DNA from individual animals. One-hundred-bp DNA size marker (M) was loaded in the first lane.

TUNEL assay. We failed to detect TUNEL-positive cells in kidney sections (corticomedullary junction) from sham-operated mice ( Fig. 9, A and B ). A 1 WT ( Fig. 9 C ) and A 1 KO ( Fig. 9 D ) mice subjected to 30 min of renal ischemia and 24 h of reperfusion showed equivalent degree of TUNEL-positive cells in the corticomedullary junction consistent with the visual inspection of apoptotic bodies and DNA laddering (representative figure of 4 experiments for each treatment group).) ]$ W7 q) Z0 S1 B9 T2 B: g5 i
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Fig. 9. Representative fluorescent photomicrographs of kidney sections from identical fields illustrate apoptotic nuclei [terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) fluorescence stain; left ] or total nuclei (propidium iodide fluorescence stain; right ). Sham-operated A 1 WT mice ( A ) and sham-operated A 1 KO mice ( B ) showed no apoptotic nuclei. C : A 1 WT mice subjected to I/R injury showed TUNEL-positive cells at 24 h. D : A 1 KO mice subjected to I/R injury showed a similar degree of TUNEL-positive cells at 24 h. Corticomedullary areas are shown (magnification x 200) and arrows indicate TUNEL-positive cells. Photomicrographs are representative of 4 independent experiments.

DISCUSSION3 n# m& u+ s9 [

The major findings of our study are that 1 ) mice deletionally lacking A 1 ARs (A 1 KO) demonstrate worse renal function after I/R renal injury compared with wild-type mice (A 1 WT), 2 ) preischemic pharmacological blockade of A 1 ARs worsened renal I/R injury in A 1 WT mice, 3 ) exogenous activation of A 1 ARs before renal ischemia protected against renal I/R injury in A 1 WT mice, 4 ) A 1 KO mice pretreated with an A 3 AR antagonist showed an improvement in renal function after I/R injury, and 5 ) exacerbation of renal function in A 1 KO or DPCPX-pretreated wild-type mice was associated with increased inflammation but not increased apoptosis. Therefore, both endogenous and exogenous A 1 AR activation in association with renal I/R injury contribute a cytoprotective role.% h' L, O8 @8 `' |- i- n

In this study, we used both pharmacological and gene deletion approaches in mice. We used mice that lack A 1 ARs to further probe the role of A 1 ARs in renal I/R injury. These A 1 KO mice have been shown to have equivalent renal blood flow and glomerular filtration rate (GFR) compared with A 1 WT mice ( 4 ). In addition, A 1 KO mice have equivalent mRNA expressions of A 2a and A 2b ARs. In our previous studies, we showed that preischemic A 1 or postischemic A 2a AR activation protects against renal I/R injury in rats ( 12, 14, 15 ). In addition, we demonstrated in rats that A 3 AR activation with IB-MECA or inhibition with MRS-1191 worsened and protected, respectively, against I/R-induced renal failure ( 14 ). We also showed that mice lacking A 3 ARs are endogenously protected against ischemic and myoglobinuric forms of ARF. The current study in mice agrees with our previous studies in rats ( 14 ) in that pretreatment with a highly selective A 1 AR agonist (CCPA) protected renal function after I/R injury. We also demonstrated in this study that A 3 AR antagonism prevented the exacerbation of renal dysfunction in A 1 KO mice. Furthermore, we now demonstrated that A 1 AR-mediated protection from I/R injury is associated with decreased inflammation and necrosis without a change in apoptosis.
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Pharmacological blockade of endogenous A 1 ARs with DPCPX in A 1 WT mice exacerbated renal failure after I/R injury. Moreover, we show that mice deletionally lacking A 1 ARs demonstrate worse renal function after I/R injury. Therefore, not only does exogenous A 1 AR activation confer cyto-protection but endogenous A 1 ARs are crucial in attenuating renal injury after I/R. These findings demonstrate that endogenous tonic activation of A 1 ARs serves to protect renal function.. J& s8 p8 d( g2 z

Several investigators have reported that a nonselective AR antagonist (theophylline) or selective A 1 AR antagonists (DPCPX, KW-3902) improved renal function, urinary output, and renal hemodynamics in models of nephrotoxic ARF induced by cisplatin, gentamicin, or glycerol ( 2, 5, 10, 23 ). In addition, Lin et al. ( 19, 20 ) demonstrated that theophylline increased renal plasma flow and GFR after ischemic renal injury. These studies were performed with the hypothesis that A 1 AR antagonism would improve renal function (as measured by increased urinary output, solute transport, and improved renal blood flow). Indeed, A 1 AR antagonists reversed these indexes of renal injury in toxin and ischemic models of ARF.; D" d) s: Z# `0 U6 `: c

At first glance, renal effects of A 1 AR activation appear to be detrimental to its function because A 1 AR activation produces effects that appear to "worsen" renal function: reduced GFR and afferent cortical blood flow as well as impaired solute transport. However, when the renal effects of A 1 ARs are looked at more closely, several renal protective attributes against renal I/R injury of A 1 ARs are evident, such as a reduction in GFR, reduced renin release and sympathetic outflow, and decreased active solute transport, all of which would reduce renal oxygen consumption during ischemic and nephrotoxic renal injury. Our finding of renal protection against ischemic ARF by pretreatment with the A 1 AR agonist (CCPA) and A 1 KO or A 1 AR antagonist-pretreated (DPCPX) wild-type mice showing exacerbation of renal dysfunction contradicts previous studies demonstrating renal protection from ischemic injury with A 1 AR antagonism. However, there are several fundamental differences between our study and the previous studies. We studied ischemic renal injury, whereas most of the studies demonstrating the renal protective effects of A 1 AR antagonism used models of toxin-induced renal failure (cisplatin, glycerol, or gentamicin). The indexes of renal function measured were also different. In our study, the primary determinants of renal function were plasma creatinine, renal necrosis scores, and indexes of renal inflammation, whereas most studies suggesting a beneficial effect of A 1 AR antagonism focused on renal blood flow, urinary output, and urinary electrolyte excretion. The studies by Lin et al. ( 19, 20 ) showed that theophylline treatment increased renal blood flow, urinary output, and urinary electrolyte excretion after renal ischemic injury. However, their studies did not indicate whether there was improvement in creatinine values with theophylline treatment. Moreover, theophylline is a nonselective AR antagonist, whereas we used a selective A 1 AR antagonist (DPCPX). Our current and previous studies show that A 3 AR antagonism, instead of previously proposed A 1 AR antagonism, produces renal protective effects.- N+ E. `; @. p, M

We show in our previous and current studies that DPCPX (an A 1 AR antagonist) worsens and MRS-1191 (an A 3 AR antagonist) protects against ARF in rats and in mice ( 14, 18 ). Although high doses of DPCPX or MRS-1191 can be nonselective for A 1 or A 3 ARs, respectively, our findings of opposing renal effects of these two antagonists are consistent with the antagonism of A 1 and A 3 ARs by DPCPX and MRS-1191, respectively. However, selectivity of pharmacological agents for a given receptor subtype (A 1 vs. A 3 ARs) is a continuous concern in pharmacological studies. Therefore, the study of mice with targeted gene deletion has become a tool that complements and extends the conclusions reached from pharmacological studies. For this reason, the data derived from the use of A 1 and A 3 KO mice are important to substantiate conclusions based on the use of AR antagonists. Taken together, our studies show that there are endogenous opposing effects of activation of subtypes of AR; preischemic activation of the A 1 AR protects renal function measured by creatinine concentrations, whereas preischemic activation of the A 3 AR exacerbates renal injury.

Preischemic A 1 AR activation has been shown to protect against I/R injury in many organs including the heart, kidney, and brain ( 7, 9, 13 ). Modulation of inflammatory responses after I/R injury is an important component of renal protection as inflammation is a major component of cell death associated with renal injury. Significant necrosis occurs with I/R injury, and necrotic tissue itself initiates an inflammatory signaling cascade, which causes further necrosis. In this study, we show that A 1 activation or blockade (by either pharmacological manipulation or endogenous genetic deletion) is associated with reduced or enhanced inflammatory responses, respectively, after renal I/R injury in mice. Increased markers of inflammation (MPO activity, neutrophil infiltration, and proinflammatory mRNA expression) were demonstrated after I/R injury with endogenous or exogenous A 1 AR blockade.
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In conclusion, we demonstrate that mice lacking A 1 AR show worsened renal function after I/R injury. Moreover, an A 1 AR agonist and an antagonist protected and worsened, respectively, against I/R-mediated renal injury in wild-type mice. Our study demonstrates that endogenous A 1 ARs serve as a cytoprotective receptor. These findings further support the potential role of a selective A 1 AR agonist to protect against perioperative renal failure.
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This work was supported by National Institutes of Health Grant RO1 DK-58547.  V4 m! s& |. k7 b" O5 s
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