Renal ischemia-reperfusion injury (IRI) is a common clinical cause of acute kidney injury (AKI).1 Although biochemical recovery is often observed with return to premorbid renal function, a number of observational studies show that these patients remain at increased risk of adverse short- and long-term outcomes. These include an increased risk of death, progression to chronic kidney disease (CKD), and the development of end stage kidney disease.2-5 In postoperative patients, even minor increases in serum creatinine were associated with a 2-fold increase in death.6 Furthermore, in the setting of renal transplantation, IRI contributes to acute dysfunction of the transplanted kidney and predisposes to deterioration in long-term allograft function. Deceased donor kidneys from uncontrolled circulatory death donors with prolonged renal ischemia of more than 10 minutes were associated with increased renal fibrosis and reduced estimated glomerular filtration rate at 1-year postrenal transplantation compared with donation after brain death.7 Therapies that reduce IRI have the potential to improve both short- and long-term outcomes, but treatment options are currently lacking.
Ischemia induces tissue hypoxia and microvascular injury in the affected organ. Although the reestablishment of blood flow is essential to halt ongoing organ damage, reperfusion itself further enhances inflammation, coagulation, and immune activation. At the same time, ischemia and reperfusion activate endogenous mechanisms of injury resolution that include the production of extracellular adenosine from adenosine triphosphate (ATP) released from the intracellular to the extracellular space after cellular injury, reviewed by Eltzschig et al.8 In the extracellular space, ATP signals via the P2X7 receptor leading to inflammasome activation and promotion of tissue injury.8,9 Hydrolysis of ATP to adenosine diphosphate (ADP) by the ectonucleotidase CD39 (ENTPDase1) is an important step in reducing the proinflammatory effects of ATP.10,11 CD39 also hydrolyses ADP to adenosine monophosphate (AMP), which is subsequently converted to adenosine by CD73 (5′ ectonucleotidase).12 Extracellular adenosine is taken up by cells through the equilibrative nucleoside transporters or converted to inosine by adenosine deaminase (ADA). Adenosine signaling via the adenosine A1, A2A, and A2B receptors reduces the susceptibility to renal IRI, but signaling via the adenosine A3 receptor exacerbates renal IRI, reviewed by Roberts et al.13
Augmenting CD39 activity is a potential therapy to improve both short- and long-term outcomes of IRI by reducing the extracellular concentration of proinflammatory ATP and promoting adenosine generation. In mouse models of IRI, treatment with soluble CD39 (apyrase)14,15 or expression of a human CD39 (hCD39) transgene in mice (CD39Tg mice)16,17 reduced the severity of AKI as evidenced by decreased serum creatinine and reduced tubular injury score at 24 hours, but long-term outcomes have not been examined.
Although adenosine is protective in acute ischemia-induced renal injury, chronically high adenosine levels can promote renal fibrosis.18,19 In a mouse model of angiotensin II-induced hypertension, upregulation of CD73 increased renal adenosine levels in association with increased A2BR expression and the development of renal fibrosis.18 Treatment of the animals with polyethyleneglycol-conjugated ADA reduced renal adenosine content and renal fibrosis. Similarly, administration of the A2BR antagonist PSB1115 was associated with lesser renal fibrosis compared with vehicle-treated mice.18 Recently, in vitro examination of renal fibroblasts demonstrated functional A2BR expression, which upon activation with the A2BR agonist, led to an increase in profibrotic markers such as TGFβ.20 The effects of augmented CD39 activity on renal adenosine content, long-term renal function and renal fibrosis have not been examined.
Apyrase administration has potential clinical application for the reduction of renal IRI during renal transplantation and in other situations of compromised renal circulation. We therefore examined the effect of apyrase administration on the acute (24 hours) and chronic (4 weeks) renal outcomes as well as renal purinergic nucleotide and nucleoside levels in wild-type (WT) mice after IRI. We also examined similar outcomes in hCD39 transgenic mice and their WT littermates after renal IRI.
MATERIALS AND METHODS
CD39Tg mice21 and C57BL/6 WT littermates were bred and maintained at the BioResources Centre, St Vincent’s Hospital Melbourne. WT C57BL/6 mice from Animal Resources Centre, Murdoch, Western Australia, were obtained for studies using apyrase and vehicle. The St. Vincent's Hospital Melbourne Animal Ethics Committee approved all procedures.
Unilateral Renal IRI Model
Male mice aged 10 to 14 weeks were anesthetized using ketamine and xylazine (16 and 8 mg/kg, respectively). A midline laparotomy was performed followed by right nephrectomy. A microvascular clamp (Roboz, Rockville, MD) was applied to the left renal pedicle for 23.5 minutes, whereas the mouse was placed in a temperature-controlled chamber at 37°C. The clamp was then removed, and the abdomen closed in 2 layers with silk 2.0 sutures. Sham-operated mice underwent right nephrectomy without ischemia. For hydration, each mouse received 200 μL of warmed normal saline by intraperitoneal injection after the procedure. An ischemic time of 23.5 minutes was optimal for the development of renal fibrosis at 4 weeks post-IRI. Ischemia for shorter periods did not result in renal fibrosis and ischemia for longer periods resulted in high mortality (data not shown). For the apyrase experiments, mice were given apyrase (Apyrase from potato, Sigma A6535, 0.4 U/g) or vehicle (normal saline) by intraperitoneal injection 20 minutes before ischemia. Cohorts of mice (n=3-8) were euthanized at baseline, at 23.5 minutes of ischemia, 24 hours, and 4 weeks postischemia for tissue and serum analysis.
Assessment of Renal Function
Whole blood was collected from the inferior vena cava of anesthetized mice and serum creatinine levels measured by the St. Vincent's Hospital Melbourne Department of Pathology using a modified Jaffe method on an Olympus AU2700 analyzer (Integrated Science, Chatswood, NSW, Australia).
The left ischemic kidney was harvested, cut in half lengthways, and one half fixed in 10% formalin and embedded in paraffin. Kidney tissue sections (3 μm) were stained with hematoxylin-eosin (H&E) to score the degree of tubular necrosis. Tubular injury was scored in a blinded fashion based on the percentage of tubular necrosis in the cortex according to the following metric16: score 1, less than 10%; score 2, 10% to less than 25%; score 3, 25% to 75%; score 4, greater than 75% of cortex with tubular necrosis. Renal fibrosis was quantified in a blinded fashion by scanning Masson trichrome-stained midsagittal kidney sections (3 μm) using an Aperio ScanScope (Leica Biosystems, North Ryde, NSW, Australia) to generate a digitized image of the whole section. Fibrosis was expressed as a positivity score (positive pixel count algorithm), which is the ratio of the area of positive staining compared with the total area of the section.
Immunohistochemical staining for hCD39 was performed on 4-μm frozen sections of ischemic kidneys that were embedded in OCT and stored at −80°C. Sections were fixed with 2% Paraformaldehyde for 10 minutes at room temperature before removal of endogenous peroxidase with 1% H2O2. After washing in phosphate-buffered saline, the sections were blocked with 10% rabbit serum for 1 hour at room temperature. Excess rabbit serum was removed followed by incubation with 50 μL of primary anti–CD39-fluorescein isothiocyanate antibody (Ancell) at the concentration of 1:100 for 1 hour. After washing with phosphate-buffered saline, sections were incubated with the secondary anti-fluorescein isothiocyanate antibody (Pierce) for 30 minutes at a concentration of 1:50 before development with 3,3′-diaminobenzidine.
Reverse Transcription and Quantitative Reverse-Transcriptase Polymerase Chain Reaction
RNA was extracted from a quarter of the ischemic left kidney using TRIzol (Invitrogen). RNA (5 μg) was converted to complementary DNA followed by real time polymerase chain reaction using Taqman (Life Technologies) primer-probe sets (Table S1, SDC,http://links.lww.com/TP/B405). Messenger ribonucleic acid levels, calculated as 2−[INCREMENT]Ct (where [INCREMENT] denotes the change in the threshold cycle [Ct]) are expressed relative to glyceraldehyde 3-phosphate dehydrogenase mRNA levels.
Quantification of Kidney Nucleotide and Nucleoside Levels
Kidney nucleotide and nucleoside levels were measured in a separate series of mice. The whole left kidney was rapidly collected into liquid nitrogen, then pulverized at the temperature of liquid nitrogen and the powder stored at −80°C. The powder was homogenized in 1 mL 0.5 mol/L perchloric acid (4°C) using a dismembrator (Sartorius). The homogenate was centrifuged (14 680 revolutions per minute, 20 238 g, Eppendorf 5424 centrifuge) and the supernatant was extracted with 2 mL chloroform:tri-n-octylamine (78/22, v/v) at 4°C, then analyzed by HPLC (Methods, SDC,http://links.lww.com/TP/B405). ATP, ADP, and AMP were analyzed as described by Bernocchi et al,22 adenosine as described by Saadjian et al23 and inosine as described by Severini and Aliberti.24
Data were expressed as means±SEM. Statistical comparisons were performed using Mann-Whitney U test or parametric tests with or without logarithmic transformation, as appropriate for the data distribution. All statistical analyses were performed using GraphPad Prism software (La Jolla, CA), and P less than 0.05 was considered significant.
Apyrase Treatment Reduces Acute and Chronic Renal IRIs
Apyrase administration before IRI significantly attenuated the increase in serum creatinine, tubular injury score and relative kidney injury molecule-1 (KIM-1) mRNA expression at 24 hours post-IRI, indicating reduced AKI (Figures 1A-E). At week 4 post-IRI, serum creatinine levels remained elevated in vehicle-treated mice, whereas serum creatinine levels in apyrase-treated mice were similar to the levels in the sham mice (Figure 2A). Apyrase-treated mice also had less renal fibrosis than vehicle-treated mice at 4 weeks post-IRI (Figures 2B, C, D).
Apyrase Does Not Modify Baseline ATP, ADP, AMP, Adenosine or Inosine Levels, But Reduces ATP, ADP, and AMP Levels During Ischemia
To ascertain the effect of apyrase on basal nucleotide and nucleoside levels, we measured renal ATP, ADP, AMP, adenosine, and inosine levels in kidneys that had not been subjected to ischemia. Kidneys were harvested at 45 minutes, 24 hours, and 2 weeks after vehicle or apyrase treatment (Table 1). ATP and ADP were present in abundance in whole kidney lysates from vehicle-treated mice, whereas the concentrations of AMP, adenosine, and inosine were significantly less. This is consistent with high intracellular concentrations of ATP and ADP and low pericellular concentrations of adenosine under resting conditions.25 Consistent with this, we did not observe any difference in concentration of ATP, ADP, AMP, adenosine, and inosine with apyrase treatment under basal conditions.
During ischemic injury renal ATP and ADP levels were markedly reduced and AMP, adenosine, and inosine levels increased in vehicle-treated mice, in keeping with the rapid release of ATP and ADP from ischemic cells and sequential hydrolysis in the extracellular space to AMP, adenosine, and inosine (Table 2).25 Apyrase administration further reduced renal ATP and ADP levels and also reduced AMP levels, but did not alter adenosine or inosine levels during ischemia (Table 2). ATP, ADP, AMP, adenosine, and inosine levels returned to baseline by 24 hours after IRI, in both vehicle- and apyrase-treated mice. Interestingly, at week 4 post-IRI, ATP, but not ADP or AMP, levels in apyrase-treated mice were lower than the levels in vehicle-treated mice. To exclude residual apyrase activity as a possible explanation, we measured renal nucleotide levels after a 23.5-minute ischemia 8 days after the administration of apyrase. Nucleotide levels approximated those of vehicle-treated mice, suggesting that the duration of apyrase activity in vivo was less than 8 days (Table S2, SDC,http://links.lww.com/TP/B405) and therefore, residual apyrase activity was unlikely to account for the lower ATP levels at 4 weeks post-IRI in apyrase-treated mice.
Apyrase Attenuates the Increase in A2BR mRNA Levels at Week 4 Post-IRI
We have previously shown that the renal expression of A2BR mRNA was increased after ischemic injury and sustained for at least 4 weeks.26 Consistent with these observations, both vehicle- and apyrase-treated mice had higher renal A2BR mRNA levels at week 4 post-IRI compared with sham mice (Figure 2E); however, apyrase treatment significantly attenuated the increase in A2BR mRNA compared with vehicle treatment.
Taken together, these data demonstrate that a single administration of apyrase before ischemic injury reduced both ischemic-induced AKI and chronic renal fibrosis in association with reduction in renal ATP levels during ischemia. Furthermore, the reduction in renal fibrosis was associated with lower renal A2BR mRNA levels than vehicle-treated mice at 4 weeks post-IRI.
hCD39 Transgene Expression Reduces Ischemia-Induced Acute Renal Injury, But Exacerbates Chronic Renal Injury
In accordance with our previously published data,16,17 we showed that renal injury was attenuated in CD39Tg mice compared with WT littermates at 24 hours post-IRI, as evidenced by significantly lower serum creatinine levels (Figure 3A), lower tubular injury score, and lower KIM-1 mRNA expression (Figures 3B, C, D, E). Despite this early protection, serum creatinine was increased in CD39Tg mice to a similar level as WT mice at week 4 post-IRI (Figure 4A). In keeping with this, biochemical evidence of CKD, both WT and CD39Tg mice had increased renal fibrosis compared with sham-operated mice. Furthermore, CD39Tg mice had a significantly higher fibrosis score than WT mice at week 4 post-IRI (Figures 4B, C, D).
hCD39 Transgene Expression Does Not Alter Renal ATP, ADP, AMP, or Adenosine Levels at Baseline, During Ischemia, or 24 Hours Post-IRI, But Increases Adenosine Levels at 4 Weeks Post-IRI
At baseline, in the absence of ischemia, ATP, ADP, AMP, and adenosine levels were similar in WT and CD39Tg mice, although inosine levels were higher in CD39Tg mice (Table 3). Ischemia was associated with reduction in ATP and ADP levels and an increase in AMP, adenosine, and inosine levels, with no differences between WT and CD39Tg mice (Table 3). These levels had largely recovered by 24 hours after IRI, with no differences between WT and CD39Tg mice. At 4 weeks post-IRI, ATP, ADP, and AMP levels were not different between WT and CD39Tg mice, whereas renal adenosine and inosine levels were higher in CD39Tg than WT mice (Table 3); the latter failing to reach statistical significance.
hCD39 Transgene Expression Is Increased After Renal IRI
Given our finding of increased renal fibrosis and increased renal adenosine levels in CD39Tg mice at week 4 post-IRI, the expression pattern of the hCD39 transgene after renal IRI was determined by immunohistochemistry. At 24 hours post-IRI, hCD39 expression was similar to that observed at baseline21 and largely restricted to the vasculature (Figure 5). However, hCD39 expression was more widespread at 4 weeks post-IRI and included the periglomerular region and within areas of fibrosis (Figure 5). These expression data were confirmed at the transcriptional level by measuring hCD39 mRNA levels, which were significantly increased at week 4 post-IRI (Figure 6A). hCD39 mRNA expression was undetectable in WT mice (not shown).
To examine the potential contribution of endogenous CD39 and CD73 to the increased renal adenosine levels in CD39Tg mice at week 4 post-IRI, the expression of murine CD39 and CD73 mRNA was evaluated at baseline, 24 hours, and 4 weeks post-IRI in both WT and CD39Tg mice. Murine CD39 and CD73 mRNA levels were similar at baseline, at 24 hours, and 4 weeks post-IRI, with no difference between WT and CD39Tg mice at any time point (Figures 6C, D).
Renal Expression of the hCD39 Transgene Does Not Attenuate the Increase in A2BR mRNA Levels at Week 4 Post-IRI
In keeping with the role of the A2BR in ischemia-induced renal fibrosis, A2BR mRNA expression was increased to a similar level in both WT and CD39Tg mice at 4 weeks post-IRI, compared with sham-operated mice (Figure 6B).
Taken together, these data show that although the overexpression of human CD39 mitigated acute ischemia-induced kidney injury, it promoted the progression of CKD in association with increased renal adenosine levels and A2BR mRNA expression.
Whereas the protective effects of apyrase administration and hCD39 transgene expression in acute ischemia-induced kidney injury are well established,14-16 this is the first report of the opposing effects of these 2 strategies on the chronic sequelae of renal IRI. These experiments confirmed that apyrase administration and CD39 transgene expression attenuate AKI after IRI, but found that the 2 strategies had opposite effects on chronic renal injury. Apyrase treatment reduced renal fibrosis whereas hCD39 transgene expression was associated with increased renal fibrosis. Protection from chronic renal injury at 4 weeks by apyrase was associated with reduced renal expression of the A2BR whereas the increased renal fibrosis in CD39Tg mice at 4 weeks was associated with increased hCD39 transgene expression, increased adenosine levels, and elevation of A2BR mRNA expression.
Apyrase reduced renal ATP, ADP, and AMP levels during ischemia. Administered apyrase remained in the extracellular compartment and the reduction in ATP, ADP, and AMP levels provided a measure of the release of these nucleotides from the intracellular to the extracellular compartment during ischemia where they were metabolized by apyrase. High levels of extracellular ATP promote apoptosis and programmed cell death.27-30 Although suggesting that protection by apyrase was due to reduction of the extracellular ATP levels during ischemia, we cannot discount the possibility that increased adenosine levels also contributed to the protective actions of apyrase.13 It was anticipated that accelerated metabolism of ATP and ADP by apyrase would have produced transient increase in AMP, adenosine, and inosine levels. Our failure to detect this increase at the end of 23.5 minutes of ischemia may have been due to rapid metabolism of AMP by ubiquitously expressed CD73, cellular uptake of adenosine through the equilibrative nucleoside transporters and metabolism of adenosine and inosine by ADA and purine nucleoside phosphorylase, respectively. Measurement of AMP, adenosine, and inosine levels at an earlier time point during ischemia may have shown an elevation in these levels. Our data confirmed that the enzymatic activity of apyrase was time-limited; using the ATP levels during ischemia as a guide, we found no evidence for residual apyrase activity at 8 days after administration. These data support the notion that a single apyrase treatment limited ischemic-induced AKI, which in turn limited chronic renal injury.
In the nonischemic kidney, apyrase administration did not influence the nucleotide or nucleoside levels, which is in contrast to previous reports by Grenz et al14 and Kohler et al.28 Our finding is consistent with the very low extracellular ATP and ADP levels and thus pericellular adenosine levels in the basal state.25,31
Adenosine is the only known endogenous ligand for the A2BR,32 but it is the least sensitive adenosine receptor.33 Despite its low sensitivity to adenosine, the A2BR plays a key role in pathological conditions when extracellular adenosine levels are increased, such as during hypoxia/ischemia34,35 and inflammation.36 In the kidney, the A2BR is expressed on vascular endothelium,37 renal tubules,18,38 mesangial cells,39 and renal fibroblasts.19,20 Mice with increased renal adenosine content, such as ADA deficiency or angiotensin II induced hypertension, have increased A2BR mRNA expression and develop renal fibrosis that is attenuated by A2BR inhibition or A2BR gene deletion.18,19 These studies imply that A2BR activation promotes renal fibrosis.
The hCD39 transgene is under the control of the mouse major histocompatibility complex H-2Kb promoter, which can be induced by exposure to interferon gamma40 or proliferative stimuli after IRI in a time dependent manner.41 At baseline and during ischemia, the hCD39 transgene has limited expression on the vascular endothelium,21 collocated with the A2BR.37 Our failure to demonstrate any differences between nucleotide and nucleoside levels in hCD39 transgenic mice and their WT littermates during ischemia is likely due to the restricted expression of hCD39 in the vascular compartment such that any local change in nucleotide or nucleoside levels produced by hCD39 could not be detected when measured in the total renal homogenate. The mechanism of protection by the hCD39 transgene is therefore unexplained by our data. The higher levels and broader distribution of transgene expression at 4 weeks post-IRI may have been responsible for the higher renal adenosine levels measured at this time point that, together with the increased A2BR mRNA expression, may have contributed to the increased renal fibrosis of the hCD39 transgenic mice at 4 weeks post-IRI. Clinically, it is noteworthy that African Americans with ENTPD1 (CD39) haplotypes associated with increased CD39 activity show increased incidence of diabetic nephropathy,42 although the expression of the A2BR was not examined in this cohort.
Although apyrase administration and hCD39 transgene expression both increased CD39 activity and attenuated acute renal injury after IRI, the major differences between these 2 experimental models preclude any extrapolation of possible mechanisms from one model to the other. These differences include the level and time course of CD39 activity in the different compartments of the kidney, the different effects of apyrase and transgene expression on renal nucleotide and nucleoside levels, and any extra-renal effects of systemic apyrase administration.
Acute ischemia-induced renal injury is a common clinical problem and there is need for therapies to reduce its incidence and arrest its progression to CKD.43 Our data show that a single administration of apyrase was able to attenuate both acute and chronic renal injuries after IRI. A recombinant human apyrase, APT102, with antiplatelet and anti-inflammatory properties but without increased risk of bleeding, has been engineered for use in humans and is under clinical investigation.44 Our studies provide support for investigation of the clinical application of this therapy before harvesting of kidneys for renal transplantation and in patients with renal IRI.
The authors thank the Bio Resources Centre for the care and management of the animals.
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