IL-11 Is Required for A1 Adenosine Receptor–Mediated Protection against Ischemic AKI

Joo Yun Kim; Mihwa Kim; Ahrom Ham; Kevin M Brown; Robert W Greene; Vivette D D’Agati; H Thomas Lee

doi:10.1681/ASN.2013010114

. 2013 Jun 27;24(10):1558–1570. doi: 10.1681/ASN.2013010114

IL-11 Is Required for A₁ Adenosine Receptor–Mediated Protection against Ischemic AKI

Joo Yun Kim ^*, Mihwa Kim ^*, Ahrom Ham ^*, Kevin M Brown ^*, Robert W Greene ^†, Vivette D D’Agati ^‡, H Thomas Lee ^*,^✉

PMCID: PMC3785280 PMID: 23813214

Abstract

A₁ adenosine receptor activation ameliorates ischemic AKI through the induction of renal proximal tubular sphingosine kinase-1. However, systemic adverse effects may limit A₁ adenosine receptor–based therapy for ischemic AKI, indicating a need to identify alternative therapeutic targets within this pathway. Here, we evaluated the function of renal proximal tubular IL-11, a clinically approved hematopoietic cytokine, in A₁ adenosine receptor–mediated induction of sphingosine kinase-1 and renal protection. Treatment of human proximal tubule epithelial (HK-2) cells with a selective A₁ adenosine receptor agonist, chloro-N(6)-cyclopentyladenosine (CCPA), induced the expression of IL-11 mRNA and protein in an extracellular signal–regulated kinase–dependent manner, and administration of CCPA in mice induced renal synthesis of IL-11. Pretreatment with CCPA protected against renal ischemia-reperfusion injury in wild-type mice, but not in IL-11 receptor–deficient mice. Administration of an IL-11–neutralizing antibody abolished the renal protection provided by CCPA. Similarly, CCPA did not induce renal IL-11 expression or protect against renal ischemia-reperfusion injury in mice lacking the renal proximal tubular A₁ adenosine receptor. Finally, treatment with CCPA induced sphingosine kinase-1 in HK-2 cells and wild-type mice, but not in IL-11 receptor–deficient or renal proximal tubule A₁ adenosine receptor–deficient mice. Taken together, these results suggest that induction of renal proximal tubule IL-11 is a critical intermediary in A₁ adenosine receptor–mediated renal protection that warrants investigation as a novel therapeutic target for the treatment of ischemic AKI.

AKI results in extremely high mortality and morbidity costing more than $10 billion per year in the United States.¹ Furthermore, patients with AKI frequently die of multiorgan failure and sepsis, and those who survive have an increased incidence of CKD. Renal ischemia and reperfusion (IR) injury or ischemic AKI is a frequent and serious complication for patients subjected to major cardiac, liver, vascular, or kidney surgery.² Attenuation of renal tubular cell necrosis, inflammation, and apoptosis ameliorates ischemic AKI in preclinical animal models.³ Unfortunately, there is no clinically effective therapy or drug to treat or prevent AKI.⁴

Adenosine is an endogenous paracrine molecule with potent physiologic and cytoprotective properties.⁵ Activation of cell surface adenosine receptors (ARs) powerfully attenuates all three components of cell death that contribute to ischemic AKI.⁶ In particular, we previously showed that activation of the A₁ AR protects against ischemic AKI in mice and rats by reducing necrosis, inflammation, and apoptosis.⁷^–⁹ However, A₁AR-based therapy for ischemic AKI may be limited by the extrarenal systemic adverse effects of cardiac and central nervous system A₁AR activation (bradycardia, hypotension, and sedation). In particular, A₁AR agonist therapy may not be tolerated by critically ill patients at risk for developing AKI. One way to mitigate this problem is to target the distal signaling molecules synthesized after renal tubular A₁AR activation.

IL-11 is a 20-kDa member of the IL-6–type cytokine family and is clinically approved to promote megakaryocyte maturation in patients receiving chemotherapy.¹⁰ In addition to its powerful hematopoietic properties, IL-11 protects against intestinal, cardiomyocyte, and endothelial cell death.¹¹ We recently showed that recombinant human IL-11 treatment before or after renal ischemia attenuated ischemic AKI in mice.¹² Specifically, IL-11 administration significantly attenuated all three components of renal cell death (necrosis, inflammation, and apoptosis) after ischemic AKI, closely mimicking the renal protective effects of A₁AR activation. This IL-11–mediated protection against ischemic AKI requires the downstream induction of sphingosine kinase-1 (SK-1) as recombinant IL-11 induced SK-1 synthesis and SK-1–deficient mice were not protected against renal IR with IL-11 treatment. We were intrigued by this finding as we recently discovered that A₁AR-mediated protection against ischemic AKI also requires SK-1 induction and activation.¹³

Collectively, our previous studies suggest that A₁AR activation and IL-11 therapy induce the identical downstream cytoprotective enzyme SK-1. Therefore, in this study we tested the hypothesis that A₁AR-mediated protection against ischemic AKI, as well as A₁AR-mediated induction of SK-1, requires the induction of renal tubular IL-11. We first tested whether A₁AR activation induces IL-11 synthesis in renal proximal tubule cells via extracellular signal–regulated kinase/mitogen-activated protein kinase (ERK MAPK) and/or protein kinase B (Akt) activation. We then tested whether A₁AR-mediated protection against renal IR is attenuated or abolished in mice lacking IL-11 signaling. Finally, to investigate the direct role of proximal tubule A₁AR in generating IL-11 and mediating renal protection in vivo, we generated mice with renal proximal tubule–specific deletion of A₁AR by crossing A₁AR floxed mice¹⁴ with mice expressing phosphoenolpyruvate carboxykinase promoter driven Cre recombinase.¹⁵

Results

A₁AR Activation Induces IL-11 in HK-2 Cells via ERK MAPK

Figure 1 shows a time-dependent (0–6 hours, part A) and dose-dependent (0.1–1 μM, part B) induction of IL-11 mRNA (top) and IL-11 protein (released into cell culture media, bottom) in human proximal tubule (HK-2) cells with chloro-N(6)-cyclopentyladenosine (CCPA, a selective A₁AR agonist) treatment. A₁AR phosphorylates ERK MAPK as well as Akt in renal proximal tubule cells.¹⁶ We therefore tested the hypothesis that A₁AR-mediated ERK and/or Akt phosphorylation directly induces IL-11 in HK-2 cells. We show that CCPA failed to induce IL-11 in HK-2 cells pretreated with a specific ERK MAPK inhibitor (PD98059; Figure 1B), whereas a selective Akt inhibitor (wortmannin) had no effect. Inhibitors alone had no significant effect on IL-11 levels in HK-2 cells.

A₁AR-Mediated Induction of Kidney IL-11 mRNA and Protein Synthesis In Vivo

We previously showed that the dose of CCPA used (0.1 mg/kg intraperitoneally) produced a small decrease (approximately 9%) in systolic BP without any effect on renal blood flow.¹⁶ Figure 2 shows that CCPA (0.1 mg/kg) significantly induced IL-11 mRNA (top) and protein expression (bottom) measured in mouse kidneys compared with vehicle-treated group. However, CCPA did not change liver or small intestine IL-11 mRNA expression (data not shown). We also determined that a single dose of CCPA did not change leukocyte (vehicle-treated mice: 5087±466 cells/μl, n=3; CCPA-treated mice: 7233±1868 cells/μl, n=3; P=0.37) or platelet count (vehicle-treated mice: 894,667±50,584 cells/μl, n=3; CCPA-treated mice: 935,333±132,942 cells/μl, n=3; P=0.80).

Figure 2. — A₁AR activation increases IL-11 mRNA and protein synthesis in mouse kidney. Representative bands for IL-11 mRNA (RT-PCR, A) and protein (ELISA, B) expression in mouse kidney (n=4 for each group) are shown. Mice were treated with vehicle (1% DMSO in saline) or with CCPA (0.1 mg/kg intraperitoneally); 6 hours later, kidneys (cortex and corticomedullary junction) were collected for analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as internal loading controls for RT-PCR. *P<0.05 versus vehicle-treated group.

Immunohistochemistry (Figure 3A) shows diffuse renal tubular IL-11 staining in mice anesthetized with pentobarbital and increased IL-11 staining in the kidneys of mice treated with 0.1 mg/kg CCPA 6 hours earlier. Interestingly, this increase in IL-11 staining was more intense in the cortex compared with medulla. IL-11 staining was not visible in the kidneys stained with negative isotype control antibody. Quantification of immunohistochemical staining (400×) confirmed a significant increase in renal tubular IL-11 immunoreactivity in kidneys from CCPA-treated mice (Figure 3B). Therefore, from these experiments we can conclude that A₁AR activation induces IL-11 mRNA and protein expression in vivo as well as in vitro.

Critical Role of IL-11 in A₁AR-Mediated Renal Protection In Vivo

IL-11R wild-type (WT) mice treated with vehicle or with 0.1 mg/kg CCPA and subjected to sham operation had similar plasma creatinine (Figure 4A). Similarly, IL-11R knockout (KO) mice or IL-11R WT mice pretreated with IL-11 neutralizing antibody had similar creatinine values after sham operation. Plasma creatinine significantly increased in vehicle-treated IL-11R WT mice subjected to 30 minutes of renal ischemia and 24-hour reperfusion compared with sham-operated mice (Figure 4A). However, IL-11R WT mice pretreated with 0.1 mg/kg CCPA before renal ischemia had significantly decreased plasma creatinine 24 hours after injury compared with vehicle-treated mice subjected to renal IR. Supporting a critical role for IL-11 in A₁AR-mediated renal protection against IR, IL-11R KO mice as well as IL-11R WT mice treated with IL-11–neutralizing antibody before renal ischemia were not protected against renal injury with CCPA treatment (Figure 4A). Next we tested whether endogenous IL-11 provides resistance against renal IR injury. Figure 4B shows that IL-11R KO mice had significantly worse renal injury after 20 minutes of renal ischemia and 24-hour reperfusion.

Kidney histologic assessment (Figure 5A [original magnification, ×200] and Figure 5B [original magnification, ×400]) demonstrated severe necrotic renal injury in vehicle-treated IL-11R WT or IL-11R KO mice subjected to renal IR. Compared with sham-operated vehicle-treated mice (not shown), the kidneys of IL-11R WT mice subjected to renal IR showed significant tubular necrosis, cast formation, and increased congestion. In contrast, consistent with the plasma creatinine data, CCPA-pretreated IL-11R WT mice had reduced renal tubular necrosis 24 hours after renal IR. Supporting a critical role of IL-11 in A₁AR-mediated renal protection against IR, CCPA pretreatment failed to reduce renal tubular necrosis in IL-11R WT mice pretreated with a neutralizing IL-11 antibody or in IL-11R KO mice. The Jablonski scale¹⁷ renal injury score was used to grade renal tubular necrosis in the corticomedullary junction 24 hours after renal IR (Figure 5C). Thirty minutes of renal ischemia and 24 hours of reperfusion resulted in severe acute tubular necrosis in vehicle-treated IL-11R WT and IL-1R KO mice. A₁AR activation significantly reduced the renal injury score in IL-11R WT mice but not in IL-11R KO mice or in IL11R WT mice pretreated with IL-11 neutralizing antibody.

Critical Role of IL-11 in A₁AR-Mediated Reduction in Kidney Neutrophil Infiltration and Apoptosis

Figure 5D shows representative images (from four experiments) of immunohistochemistry of neutrophils infiltrating the kidneys of mice subjected to 30 minutes of renal ischemia and 24-hour reperfusion (original magnification, ×200). In sham-operated mice, we were unable to detect any neutrophils in the kidney (data not shown). There was heavy neutrophil infiltration (dark brown) in the kidneys (corticomedullary junction) of vehicle-treated IL-11R WT and IL-11R KO mice subjected to renal IR. In contrast, IL-11R WT mice pretreated with a selective A₁AR agonist CCPA showed significantly reduced neutrophil infiltration in the kidney 24 hours after IR (Figure 5E). In contrast, CCPA treatment failed to reduce kidney neutrophil infiltration in IL-11R WT mice treated with IL-11–neutralizing antibody and in IL-11R KO mice.

TUNEL staining detected apoptotic renal tubular cells in kidneys of mice. In sham-operated mice, very few TUNEL-positive cells were detected in the kidney (data not shown). Vehicle-treated IL-11R WT and IL-11R KO mice subjected to renal IR demonstrated numerous renal tubule cell apoptosis detected with TUNEL staining (Figure 5F [original magnification, ×100] and Figure 5G). However, IL-11R WT mice pretreated with CCPA before renal ischemia had a significantly reduced number of apoptotic TUNEL-positive cells in the kidney 24 hours after IR. Again, CCPA treatment failed to reduce renal cell apoptosis in IL-11R WT mice treated with IL-11–neutralizing antibody and in IL-11R KO mice supporting a critical role for IL-11 in CCPA-mediated reduction of kidney apoptosis.

Critical Role of Renal Proximal Tubule A₁AR for Renal Protection and IL-11 Induction

Mating mice carrying the floxed A₁AR gene (A₁AR^f/f mice¹⁴) with phosphoenolpyruvate carboxykinase (PEPCK)^Cre recombinase mice¹⁵ resulted in mice carrying floxed A₁AR with (null) or without (WT) PEPCK Cre recombinase. Tail PCR confirmed the genotypes of proximal tubule A₁AR null mice (A₁AR^f/f PEPCK^Cre/−) and the WT control transgenic mice (A₁AR^f/fPEPCK^−/−) generated (Figure 6A).

Figure 6B shows plasma creatinine from WT (A₁AR^f/f PEPCK^−/−) or from proximal tubule A₁AR null mice (A₁AR^f/f PEPCK^Cre/-) subjected to 30 minutes of renal ischemia and 24 hours of reperfusion. Treatment with a selective A₁AR agonist (CCPA, 0.1 mg/kg intraperitoneally 15 minutes before renal ischemia) protected against ischemic AKI in WT mice. However, CCPA failed to protect proximal tubule A₁AR null mice against ischemic AKI. We also performed experiments to test whether proximal tubule A₁ARs provide endogenous resistance against a shorter period of renal IR injury. We show that proximal tubule A₁AR null mice had significantly worse renal injury after 20 minutes of renal ischemia and 24 hours of reperfusion.

Figure 6D shows that CCPA treatment (0.1 mg/kg) 6 hours earlier induced kidney IL-11 in WT mice. However, CCPA failed to induce kidney IL-11 in proximal tubule A₁AR null mice. We previously showed that A₁AR activation induced SK-1 in mouse renal proximal tubules.¹³ Consistent with this, we demonstrate here that SK-1 induction was also selectively abolished in proximal tubule A₁AR null mice (Figure 6D). Kidney immunohistochemistry from proximal tubule A₁AR null (A₁AR^f/f PEPCK^Cre/−) and WT (A₁AR^f/f PEPCK^−/−) mice (Figure 6E) shows that IL-11 staining was darker in kidneys of WT mice treated with CCPA. IL-11 staining did not increase in proximal tubule A₁AR null mice. Quantification of immunohistochemical staining confirmed that renal tubular IL-11 immunoreactivity significantly increased in CCPA-treated WT mice but not in proximal tubule A₁AR null mice (Figure 6F). These experiments suggest that renal proximal tubule A₁AR activation is required for induction of IL-11 mRNA and protein expression in vivo.

We also determined whether A₁AR-mediated IL-11 induction is attenuated in proximal tubules from proximal tubule A₁AR null mice. We first noted significantly reduced A₁AR mRNA in proximal tubule cells from proximal tubule A₁AR null mice compared with WT mice (Figure 6G). In addition, baseline proximal tubule IL-11 mRNA expression was lower in proximal tubule A₁AR null mice compared with WT mice. Furthermore, Figure 6G also shows that CCPA treatment (0.1 mg/kg) 6 hours earlier induced IL-11 mRNA in proximal tubules isolated from WT mice but not in proximal tubules from proximal tubule A₁AR null mice. These experiments further support the hypothesis that renal proximal tubule A₁AR activation is required for induction of IL-11 mRNA expression in vivo.

Critical Role of IL-11 in A₁AR-Mediated SK-1 Induction in HK-2 Cells and in Mouse Kidney

Figure 7 shows that CCPA induced SK-1 mRNA (Figure 7A) and protein (Figure 7B) in control isotype antibody–treated HK-2 cells. In contrast, IL-11–neutralizing antibody treatment significantly attenuated CCPA-mediated induction of SK-1 mRNA and protein in HK-2 cells. CCPA had no effects on SK2 expression. Consistent with these in vitro data, we also demonstrate that CCPA (0.1 mg/kg intraperitoneally) significantly induced SK-1 mRNA expression in the kidneys of IL-11R WT mice compared with kidneys from vehicle-treated mice. In contrast, CCPA failed to induce SK-1 mRNA expression in the kidneys from IL-11R KO mice (Figure 8). Again, CCPA had no effects on SK2 expression. Collectively, these in vivo and in vitro data suggest that IL-11 signaling is critical for A₁AR-mediated induction of cytoprotective SK-1.

Figure 8. — IL-11 is required for A₁AR-mediated induction of SK-1 mRNA in mouse kidney. Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in SK-1 or SK-2 expression over vehicle-treated mice. IL-11 WT or IL-11R–deficient (IL-11R KO) mice were treated with vehicle or with a selective A₁AR agonist (CCPA, 0.1 mg/kg) 6 hours earlier. *P<0.05 versus IL-11R WT mice treated with vehicle. #P<0.05 versus IL-11R WT mice treated with CCPA. n=4. Data presented as mean ± SEM.

Discussion

AKI is a disastrous clinical complication with high mortality, morbidity, and health care costs.⁴^,¹⁸ Surgical renal ischemia (e.g., kidney transplantation and partial nephrectomy) and renal hypoperfusion (e.g., due to cardiogenic shock or aortic surgery) are the leading causes of perioperative AKI.¹⁹ It is becoming clear that reduction in renal tubular necrosis, apoptosis, and inflammation during and after renal IR is required to attenuate the pathogenesis of ischemic AKI.³ Therapies aimed at attenuating these renal cell death pathways have great clinical significance because there is no clinically efficacious therapy for AKI.

Adenosine regulates the physiology and function of many organs and cell types. Adenosine regulates the function of the heart (heart rate, contractility), brain (sedation), and kidney (GFR and urine output), as well as vascular tone (vasomotor control), via four subtypes of G-protein–coupled purinergic receptors.²⁰^,²¹ In addition to its physiologic effects, we previously demonstrated that renal A₁AR activation reduced kidney IR injury in mice and rats.⁷^,⁹^,¹⁶^,²² This A₁AR-mediated protection against ischemic AKI was characterized by significantly lower plasma creatinine and reductions in three major components of renal tubular cell death: necrosis, apoptosis, and inflammation.²²

Although the A₁AR agonist–mediated protection against ischemic AKI has significant therapeutic potential, the extrarenal effects of A₁AR activation, especially at high doses (e.g., bradycardia, reduction in GFR, hypotension), may produce undesirable systemic adverse effects limiting its clinical applications.²³ For example, A₁AR agonist therapy may not be suitable for hypotensive patients in the intensive care unit at risk for developing AKI. Instead, exogenous application of the downstream signaling molecule generated with A₁AR activation (such as IL-11) devoid of systemic hemodynamic and central nervous system effects would increase the clinical applicability to treat ischemic AKI. We also show in this study that IL-11–deficient mice had increased injury after mild renal IR (Figure 4B). In addition, we determined that proximal tubule A₁AR null mice had significantly worse renal injury after 20 minutes of ischemic AKI (Figure 6C). These findings suggest that basal IL-11 production, as well as endogenous activation of proximal tubule A₁ARs, provides endogenous resistance against ischemic AKI.

Recombinant human IL-11 (oprelvekin) is clinically approved to treat chemotherapy-induced thrombocytopenia. We show in this study that the A₁AR activation induces IL-11 mRNA and protein synthesis in renal proximal tubule cells in vitro and mouse kidney in vivo. IL-11 is a member of the IL-6–type cytokine family isolated from bone marrow–derived stromal cells.²⁴ IL-11 regulates hematopoiesis by promoting megakaryocyte maturation through activation of cell surface IL-11 receptors.²⁵ In addition to its effect on platelets, recent studies suggest a cytoprotective role for IL-11, and exogenous IL-11 administration attenuates necrotic, inflammatory, and apoptotic cell death.¹²^,²⁶^–²⁸

We recently demonstrated that exogenous human recombinant IL-11 attenuated ischemic AKI in mice and reduced necrosis and apoptosis in HK-2 proximal tubule cells.¹² In mice, IL-11 treatment attenuated renal tubular necrosis and apoptosis as well as the influx of proinflammatory neutrophils after renal IR. Not only did A₁AR activation induced IL-11 in the kidney proximal tubule cells, but the antinecrotic, antiapoptotic, and anti-inflammatory effects of exogenous IL-11 treatment closely mimicked the renal protective effects of A₁AR activation in mice.⁹^,²² Furthermore, the A₁AR selective agonist CCPA failed to attenuate renal histologic injury, apoptosis, and influx of proinflammatory neutrophils in IL-11R–deficient mouse kidneys after ischemic AKI. Taken together, we propose that IL-11 signaling is critical for A₁AR-mediated protection against ischemic AKI. To our knowledge, the A₁AR-mediated induction of IL-11 synthesis has not been described previously, and it represents a new paradigm in understanding of the cytoprotective mechanisms of A₁AR activation.

The A₁AR-mediated induction of IL-11 appears to be specific for kidneys because CCPA failed to induce IL-11 mRNA expression in the liver or small intestine. Moreover, CCPA failed to induce kidney IL-11 in proximal tubule A₁AR null mice (Figure 6D). Consistent with this, we demonstrated significantly reduced proximal tubule IL-11 mRNA expression in proximal tubules in proximal tubule A₁AR null mice (Figure 6G). Finally, renal tubular IL-11 immunoreactivity did not increase in CCPA-treated proximal tubule A₁AR null mice (Figure 6E). Taken together, these findings suggest that proximal tubular induction of IL-11 represents a major component of A₁AR-mediated kidney IL-11 induction. In addition, we proposed that proximal tubular induction of IL-11 plays a major role in A₁AR-mediated renal protection against ischemic AKI. However, to conclusively define the role for tubular IL-11 in A₁AR-mediated protection against ischemic AKI, future studies with conditional deletion of proximal tubule IL-11 or IL-11 receptors will need to be performed.

We show in this study that A₁AR-mediated induction of IL-11 requires ERK MAPK signaling because a selective inhibitor of ERK PD98059 prevented CCPA-mediated induction of IL-11. Our findings are consistent with those of previous studies implicating a critical role of ERK MAPK in induction of IL-11 in human bronchial epithelial cells and intestinal myofibroblasts.²⁹^,³⁰ Furthermore, previous studies suggest that IL-11 protects against oxidant-induced necrosis and apoptosis via mechanisms involving ERK MAPK and induction of heat shock protein 27 in vascular endothelial and intestinal epithelial cells.²⁸^,³¹ Interestingly, we previously showed that the cytoprotective effects of renal A₁AR activation also involve ERK MAPK and heat shock protein 27 pathways.¹⁶^,³² These data suggest that the A₁AR and IL-11–mediated renal protection may share common signaling mechanisms in renal proximal tubule cells.

We recently showed that the A₁AR-mediated protection against renal tubular necrosis, apoptosis, and inflammation requires an induction of another cytoprotective enzyme sphingosine kinase-1 (SK-1) in renal proximal tubule cells.¹³ Interestingly, we show in this study that a selective A₁AR agonist CCPA fails to induce SK-1 in the kidneys of IL-11 receptor–deficient mice, suggesting that IL-11 signaling is required for A₁AR-mediated induction of SK-1. Furthermore, we recently showed that exogenous IL-11 produces renal protection by direct induction of SK-1 via nuclear translocation of HIF-1α.¹² These previous and current data strongly suggest that A₁AR-mediated induction of proximal tubule SK-1 requires an intermediate signaling molecule mediated by IL-11 and IL-11 receptor activation.

SK-1 phosphorylates sphingosine to make intracellular sphingosine 1-phosphate (S1P).³³ S1P is a potent lipid signaling molecule that activates five S1P receptors (S1PRs) to regulate diverse biological effects, including cell growth, survival, and modulation of inflammation.³⁴^–³⁶ S1P₁R activation in particular has been shown to reduce ischemic AKI via direct renal tubular protection against necrosis, as well as via reducing T lymphocyte–mediated renal inflammation.³⁷^,³⁸ Activation of the S1P₁Rs on endothelial cells also protects the integrity of the vascular endothelial cell barrier function.³⁹ Collectively, the previous and current findings suggest that renal tubular A₁AR activation produces two sequential renal protective signaling molecules, including IL-11, followed by induction of SK-1 and S1P.

Renal proximal tubules in the corticomedullary junction (mainly the S3 segments) are extremely susceptible to IR injury,⁴⁰ and our current and previous studies confirm severe proximal tubule necrosis and apoptosis in mice subjected to renal IR. The A₁AR- and IL-11–mediated renal protection are associated with significantly preserved corticomedullary junction architecture and reduction in proximal tubule necrosis and apoptosis. Therefore, we tested the hypothesis that proximal tubular A₁ARs are critical for A₁AR-mediated IL-11 induction as well as renal protection. We show in this study that mice deficient in proximal tubule A₁ARs cannot be protected against ischemic AKI with CCPA treatment. These findings suggest that proximal tubule A₁AR is critical for CCPA-mediated renal protection against ischemic AKI. In addition, our findings suggest that other cell types, including endothelial cells and leukocytes, are dispensable for A₁AR-mediated renal protection. Taken together, our findings indicate that renal proximal tubule A₁ARs are critical for A₁AR-mediated protection against ischemic AKI, as well as induction of additional cytoprotective signaling proteins, including IL-11 and SK-1.

In summary, we demonstrated here that a selective A₁AR agonist protects against renal tubular necrosis, apoptosis, and inflammation by inducing cytoprotective IL-11 generation. Figure 9 summarizes the potential mechanisms of A₁AR-mediated renal protection involving IL-11 and SK-1 synthesis. We propose that IL-11 synthesized by A₁AR activation binds to the IL-11 receptor, which then induces cytoprotective SK-1. S1P generated by SK-1 then produces additional renal tubular protection via activation of S1P₁R. Our studies may lead to new therapeutic approaches (IL-11, S1P) with a drug that can reduce all three pathways of renal cell death (necrosis, apoptosis, and inflammation) to lessen the clinical perils from AKI and have implications in organ protection strategies beyond the kidney. Furthermore, IL-11–based renal protection would avoid some of the systemic adverse effects (e.g., bradycardia, hypotension, hypothermia, sedation) of A₁AR agonist therapy in critically ill patients.

Figure 9. — The cytoprotective effects of A₁AR activation against ischemic AKI are dependent on IL-11 mediated SK1 induction and activation. Collectively, our data suggest that A₁AR activation results in increased IL-11 mRNA and protein synthesis *via* ERK MAPK–mediated transcription factor. We propose that IL-11 synthesized then subsequently activates IL-11R to induce additional cytoprotective signaling including SK-1 synthesis and increased S1P generation.

Concise Methods

Cell Culture and A₁AR Activation

Immortalized HK-2 cells (American Type Culture Collection, Manassas, VA) were grown and passaged as described.⁴¹ To determine whether A₁AR activation induces IL-11, HK-2 cells were treated with 0.1–1 μM CCPA, a selective A₁AR agonist (Tocris Bioscience, Minneapolis, MN),⁴²^,⁴³ or with vehicle (1% DMSO) for 0–6 hours. CCPA has approximately 10,000-fold selectivity and subnanomolar affinity for A₁ARs. To elucidate whether MAPK or Akt signaling pathways are involved in A₁AR-mediated IL-11 induction, some HK-2 cells were treated with 50 μM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059, a selective inhibitor of MAPK (Tocris Bioscience) or with 100 nM wortmannin (a specific, covalent inhibitor of phosphoinositide 3-kinases (Sigma, St. Louis, MO) 30 minutes before CCPA treatment. The doses of inhibitors were determined in preliminary experiments to selectively block MEK1 or PI3K to prevent ERK or Akt phosphorylation (data not shown). To determine whether A₁AR-mediated IL-11 induction subsequently leads to the synthesis of new SK-1, some HK-2 cells were preincubated with 10 μg/ml of anti–IL-11 (MAB218, R&D Systems, Minneapolis, MN) 30 minutes before CCPA treatment.

Modulation of Renal IR Injury in Mice with A₁AR Activation and IL-11 Deletion/Neutralization

After Columbia University Institutional Animal Care and Use Committee approval, we subjected adult male C57BL/6 (Harlan, Indianapolis, IN) and IL-11 receptor–deficient mice (IL-11R KO mice) to 20 minutes (mild) or 30 minutes (severe) of renal IR or sham operation (laparotomy, right nephrectomy without renal ischemia) as described.⁴⁴^,⁴⁵ Heterozygous breeding pairs of IL-11R KO mice on a C57BL/6 background (B6.129S1-Il11ra1^tm1Wehi/J) were obtained from Jackson Labs (Bar Harbor, ME). IL-11R WT or IL-11R KO mice were pretreated with 0.1 mg/kg CCPA (a selective A₁AR agonist) or vehicle (1% DMSO) intraperitoneally 15 minutes before renal ischemia. To neutralize IL-11 in vivo, some IL-11 WT mice were pretreated with 1 mg/kg intravenous monoclonal anti–IL-11 (MAB418, R&D Systems) 30 minutes before CCPA treatment. We collected kidney (cortex and corticomedullary junction) and plasma 6 or 24 hours after IR injury to examine the severity of renal dysfunction (plasma creatinine, renal histology, apoptosis, and neutrophil infiltration) and IL-11 mRNA and protein detection. Plasma creatinine was measured as described with an enzymatic creatinine reagent kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA).⁴⁶ We also collected kidney, liver, and intestine samples 6 hours after treatment with vehicle or with 0.1 mg/kg CCPA. Because IL-11 is a regulator of hematopoiesis and affects megakaryocyte maturation,²⁵ we performed complete blood counts in some mice to determine whether a single injection of A₁AR agonist modifies systemic platelet and leukocyte count.

Histologic Detection of Kidney Necrosis, Apoptosis, and Neutrophil Infiltration

We examined and quantified kidney morphology (hematoxylin and eosin staining), neutrophil infiltration, and apoptosis, as described previously in our studies,⁹^,²² based on the grading system developed by Jablonski et al.¹⁷

Generation and Genotyping of Mice with Proximal Tubule–Specific Deletion of A₁AR

We crossed mice carrying the floxed A₁AR gene (A₁AR^ff mice¹⁴) with PEPCK-Cre recombinase mice (provided by Dr. Volker Haase, Vanderbilt University¹⁵). A₁AR-floxed mice carrying the PEPCK-Cre gene (A₁AR^f/f PEPCK^Cre/-) and control mice (A₁AR^f/f PEPCK^−/−) were genotyped by tail biopsy PCR using Cre transgene and A₁AR^loxP specific primers (Table 1). PEPCK-Cre primers generated a 466-bp fragment. Flox primers amplified a 550-bp fragment for the A₁AR floxed allele and a 1350-bp fragment for the A₁AR WT allele. Some A₁AR^f/f PEPCK^Cre/- and A₁AR^f/f PEPCK^−/− (controls) mice were pretreated with 0.1 mg/kg CCPA or vehicle intraperitoneally 15 minutes before renal ischemia.

Table 1.

Primers used to amplify cDNAs based on published GenBank sequences for mice.

Primers	Accession Number	Sequence (Sense/Antisense)	Product Size (bp)	Cycle Number	Annealing Temp (°C)
Mouse IL-11	NM_008350.4	5′-AACTGTGTTTGTCGCCTGGT-3′	267	30	68
Mouse IL-11	NM_008350.4	5′-AAGCTGCAAAGATCCCAATG-3′	267	30	68
Mouse SK1	NM_011451	5′-GATGCATGAGGTGGTGAATG-3′	337	22	64
Mouse SK1	NM_011451	5′-GCCCACTGTGAAACGAATC-3′	337	22	64
Mouse SK2	NM_020011.5	5′-ACTGCTCGCTTCTTCTCTGC-3′	437	23	68
Mouse SK2	NM_020011.5	5′-ACCATTGAGGGACAGGTCAG-3′	437	23	68
Human IL-11	NM_000641.3	5′-CTGAAGACTCGGCTGTGACC-3′	300	22	66
Human IL-11	NM_000641.3	5′-CAGGGCAGAAGTCTGTGGAC-3′	300	22	66
Human SK1	NM_021972	5′-ATCTCCTTCACGCTGATGC-3′	330	26	66
Human SK1	NM_021972	5′-GTGCAGAGACAGCAGGTTCA-3′	330	26	66
Human SK2	NM_020126	5′-GGAGGAAGCTGTGAAGATGC-3′	482	22	66
Human SK2	NM_020126	5′-GCAGGTCAGACACAGAACGA-3′	482	22	66
GAPDH	M32599	5′-ACCACAGTCCATGCCATCAC-3′	450	15	65
GAPDH	M32599	5′-CACCACCCTGTTGCTGTAGCC-3′	450	15	65
A₁AR^loxP		5′-GCCTTCCGAATCCACAGTT-3′	Lox: 450	35	62
A₁AR^loxP		5′-AGCCTCTGTTCCACATACAC-3′	WT: 1350	35	62
Cre transgene		5′-TGGGCGGCATGGTGCAAGTT-3′	466	30	61
Cre transgene		5′-CGGTGCTAACCAGCGTTTTC-3′	466	30	61

Open in a new tab

GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

RT-PCR

With RT-PCR, we measured mRNAs encoding human (HK-2 cells) or mouse IL-11, SK-1, and SK-2 after vehicle or CCPA treatment, as described elsewhere,⁴⁷ using primers listed in Table 1. We also performed IL-11 RT-PCR in mouse liver and small intestine samples 6 hours after treatment with vehicle or with 0.1 mg/kg CCPA.

IL-11 ELISA and SK Immunoblotting

HK-2 cell supernatant or mouse kidney cortex lysate IL-11 protein expression was measured using human or mouse-specific sandwich IL-11 ELISA kit (R&D Systems), respectively. HK-2 cell lysates were collected for immunoblotting analyses of SK-1, SK2, and β-actin (internal protein loading control) as described previously.⁴⁸^,⁴⁹

IL-11 Immunohistochemistry

Immunohistochemistry detected mouse kidney IL-11 protein expression and localization 6 hours after vehicle or CCPA (0.1 mg/kg intraperitoneally) treatment with rat anti–IL-11 antibody (MAB418; 1:50 dilution; R&D Systems) and biotin-conjugated antirat IgG (1:100 dilution; Vector Laboratories, Burlingame, CA). Normal rat IgG2a (Vector Laboratories) was used at the same concentration as the primary antibody as a negative isotype control. Renal tubular IL-11 expression was quantified as described by Kristina et al., with some modifications.⁵⁰

Isolation of Mouse Kidney Proximal Tubules

Mouse kidney proximal tubules were isolated using the methods described by Vinay et al. (collagenase digestion followed by Percoll density gradient separation) as we showed previously.⁵¹^,⁵²

Statistical Analyses

The data were analyzed with t test when comparing means between two groups or one-way ANOVA plus Tukey post hoc multiple comparison test when comparing multiple groups. The ordinal values of the renal injury scores were analyzed by the Mann-Whitney nonparametric test. In all cases, P<0.05 was taken to indicate significance. All data are expressed throughout the text as means ± SEM. Power analysis was performed with G.power 3.1 software.⁵³

A complete description of the methods is available in the Supplemental Material.

Disclosures

None.

Supplementary Material

Supplemental Data

supp_24_10_1558__index.html^{(800B, html)}

Acknowledgments

We appreciate the surgical, analytical, and technical assistance provided by Sang Won Park. We thank Dr. Volker Haase of Vanderbilt University for providing us with breeding pairs of PEPCK-Cre recombinase mice.

This work was supported in part by Department of Anesthesiology, Columbia University, R01 DK-058547 and R01 GM-067081 (to H.T.L.) and by Department of Veterans Affairs and R01NS075545 (to R.W.G.).

An abstract form of this work was presented at the 2012 American Society of Nephrology meeting in San Diego, CA.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013010114/-/DCSupplemental.

References

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2.Elapavaluru S, Kellum JA: Why do patients die of acute kidney injury? Acta Clin Belg Suppl 326–331, 2007 [DOI] [PubMed] [Google Scholar]
3.Bonventre JV, Weinberg JM: Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol 14: 2199–2210, 2003 [DOI] [PubMed] [Google Scholar]
4.Jones DR, Lee HT: Perioperative renal protection. Best Pract Res Clin Anaesthesiol 22: 193–208, 2008 [DOI] [PubMed] [Google Scholar]
5.Haskó G, Linden J, Cronstein B, Pacher P: Adenosine receptors: Therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov 7: 759–770, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Bauerle JD, Grenz A, Kim JH, Lee HT, Eltzschig HK: Adenosine generation and signaling during acute kidney injury. J Am Soc Nephrol 22: 14–20, 2011 [DOI] [PubMed] [Google Scholar]
7.Lee HT, Emala CW: Protective effects of renal ischemic preconditioning and adenosine pretreatment: Role of A(1) and A(3) receptors. Am J Physiol Renal Physiol 278: F380–F387, 2000 [DOI] [PubMed] [Google Scholar]
8.Lee HT, Emala CW: Protein kinase C and G(i/o) proteins are involved in adenosine- and ischemic preconditioning-mediated renal protection. J Am Soc Nephrol 12: 233–240, 2001 [DOI] [PubMed] [Google Scholar]
9.Lee HT, Xu H, Nasr SH, Schnermann J, Emala CW: A1 adenosine receptor knockout mice exhibit increased renal injury following ischemia and reperfusion. Am J Physiol Renal Physiol 286: F298–F306, 2004 [DOI] [PubMed] [Google Scholar]
10.Goldman SC, Bracho F, Davenport V, Slack R, Areman E, Shen V, Lenarsky C, Weinthal J, Hughes R, Cairo MS: Feasibility study of IL-11 and granulocyte colony-stimulating factor after myelosuppressive chemotherapy to mobilize peripheral blood stem cells from heavily pretreated patients. J Pediatr Hematol Oncol 23: 300–305, 2001 [DOI] [PubMed] [Google Scholar]
11.Du X, Williams DA: Interleukin-11: Review of molecular, cell biology, and clinical use. Blood 89: 3897–3908, 1997 [PubMed] [Google Scholar]
12.Lee HT, Park SW, Kim M, Ham A, Anderson LJ, Brown KM, D’Agati VD, Cox GN: Interleukin-11 protects against renal ischemia and reperfusion injury. Am J Physiol Renal Physiol 303: F1216–F1224, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Park SW, Kim M, Kim JY, Brown KM, Haase VH, D’Agati VD, Lee HT: Proximal tubule sphingosine kinase-1 has a critical role in A1 adenosine receptor-mediated renal protection from ischemia. Kidney Int 82: 878–891, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Scammell TE, Arrigoni E, Thompson MA, Ronan PJ, Saper CB, Greene RW: Focal deletion of the adenosine A1 receptor in adult mice using an adeno-associated viral vector. J Neurosci 23: 5762–5770, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Rankin EB, Tomaszewski JE, Haase VH: Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor. Cancer Res 66: 2576–2583, 2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Joo JD, Kim M, Horst P, Kim J, D’Agati VD, Emala CW, Sr, Lee HT: Acute and delayed renal protection against renal ischemia and reperfusion injury with A1 adenosine receptors. Am J Physiol Renal Physiol 293: F1847–F1857, 2007 [DOI] [PubMed] [Google Scholar]
17.Jablonski P, Howden BO, Rae DA, Birrell CS, Marshall VC, Tange J: An experimental model for assessment of renal recovery from warm ischemia. Transplantation 35: 198–204, 1983 [DOI] [PubMed] [Google Scholar]
18.Faubel S: Acute kidney injury and multiple organ dysfunction syndrome. Minerva Urol Nefrol 61: 171–188, 2009 [PubMed] [Google Scholar]
19.Ikeda M, Prachasilchai W, Burne-Taney MJ, Rabb H, Yokota-Ikeda N: Ischemic acute tubular necrosis models and drug discovery: A focus on cellular inflammation. Drug Discov Today 11: 364–370, 2006 [DOI] [PubMed] [Google Scholar]
20.Linden J: Molecular approach to adenosine receptors: Receptor-mediated mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 41: 775–787, 2001 [DOI] [PubMed] [Google Scholar]
21.Vallon V,, Osswald H: Adenosine receptors and the kidney. Handb Exp Pharmacol 443–470, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Lee HT, Gallos G, Nasr SH, Emala CW: A1 adenosine receptor activation inhibits inflammation, necrosis, and apoptosis after renal ischemia-reperfusion injury in mice. J Am Soc Nephrol 15: 102–111, 2004 [DOI] [PubMed] [Google Scholar]
23.Baraldi PG, Iaconinoto MA, Moorman AR, Carrion MD, Cara CL, Preti D, López OC, Fruttarolo F, Tabrizi MA, Romagnoli R: Allosteric enhancers for A1 adenosine receptor. Mini Rev Med Chem 7: 559–569, 2007 [DOI] [PubMed] [Google Scholar]
24.Kaye JA: The clinical development of recombinant human interleukin 11 (NEUMEGA rhIL-11 growth factor). Stem Cells 14[Suppl 1]: 256–260, 1996 [DOI] [PubMed] [Google Scholar]
25.Reynolds CH: Clinical efficacy of rhIL-11. Oncology (Williston Park) 14[Suppl 8]: 32–40, 2000 [PubMed] [Google Scholar]
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27.Kuenzler KA, Pearson PY, Schwartz MZ: IL-11 pretreatment reduces cell death after intestinal ischemia-reperfusion. J Surg Res 108: 268–272, 2002 [DOI] [PubMed] [Google Scholar]
28.Waxman AB, Mahboubi K, Knickelbein RG, Mantell LL, Manzo N, Pober JS, Elias JA: Interleukin-11 and interleukin-6 protect cultured human endothelial cells from H2O2-induced cell death. Am J Respir Cell Mol Biol 29: 513–522, 2003 [DOI] [PubMed] [Google Scholar]
29.Bamba S, Andoh A, Yasui H, Makino J, Kim S, Fujiyama Y: Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways. Am J Physiol Gastrointest Liver Physiol 285: G529–G538, 2003 [DOI] [PubMed] [Google Scholar]
30.Lecureur V, Arzel M, Ameziane S, Houlbert N, Le Vee M, Jouneau S, Fardel O: MAPK- and PKC/CREB-dependent induction of interleukin-11 by the environmental contaminant formaldehyde in human bronchial epithelial cells. Toxicology 292: 13–22, 2012 [DOI] [PubMed] [Google Scholar]
31.Ropeleski MJ, Tang J, Walsh-Reitz MM, Musch MW, Chang EB: Interleukin-11-induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress. Gastroenterology 124: 1358–1368, 2003 [DOI] [PubMed] [Google Scholar]
32.Lee HT, Kim M, Jan M, Penn RB, Emala CW: Renal tubule necrosis and apoptosis modulation by A1 adenosine receptor expression. Kidney Int 71: 1249–1261, 2007 [DOI] [PubMed] [Google Scholar]
33.Leclercq TM, Pitson SM: Cellular signalling by sphingosine kinase and sphingosine 1-phosphate. IUBMB Life 58: 467–472, 2006 [DOI] [PubMed] [Google Scholar]
34.Allende ML, Dreier JL, Mandala S, Proia RL: Expression of the sphingosine 1-phosphate receptor, S1P1, on T-cells controls thymic emigration. J Biol Chem 279: 15396–15401, 2004 [DOI] [PubMed] [Google Scholar]
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41.Lee HT, Emala CW: Adenosine attenuates oxidant injury in human kidney proximal tubular cells via A(1) and A(2a) adenosine receptors. Am J Physiol Renal Physiol 282: F844–F852, 2002 [DOI] [PubMed] [Google Scholar]
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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data

supp_24_10_1558__index.html^{(800B, html)}

ASN.2013010114_ASN2013010114SupplementaryData.pdf^{(67.6KB, pdf)}

[B1] 1.Chertow GM, Burdick E, Honour M, Bonventre JV, Bates DW: Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J Am Soc Nephrol 16: 3365–3370, 2005 [DOI] [PubMed] [Google Scholar]

[B2] 2.Elapavaluru S, Kellum JA: Why do patients die of acute kidney injury? Acta Clin Belg Suppl 326–331, 2007 [DOI] [PubMed] [Google Scholar]

[B3] 3.Bonventre JV, Weinberg JM: Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol 14: 2199–2210, 2003 [DOI] [PubMed] [Google Scholar]

[B4] 4.Jones DR, Lee HT: Perioperative renal protection. Best Pract Res Clin Anaesthesiol 22: 193–208, 2008 [DOI] [PubMed] [Google Scholar]

[B5] 5.Haskó G, Linden J, Cronstein B, Pacher P: Adenosine receptors: Therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov 7: 759–770, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Bauerle JD, Grenz A, Kim JH, Lee HT, Eltzschig HK: Adenosine generation and signaling during acute kidney injury. J Am Soc Nephrol 22: 14–20, 2011 [DOI] [PubMed] [Google Scholar]

[B7] 7.Lee HT, Emala CW: Protective effects of renal ischemic preconditioning and adenosine pretreatment: Role of A(1) and A(3) receptors. Am J Physiol Renal Physiol 278: F380–F387, 2000 [DOI] [PubMed] [Google Scholar]

[B8] 8.Lee HT, Emala CW: Protein kinase C and G(i/o) proteins are involved in adenosine- and ischemic preconditioning-mediated renal protection. J Am Soc Nephrol 12: 233–240, 2001 [DOI] [PubMed] [Google Scholar]

[B9] 9.Lee HT, Xu H, Nasr SH, Schnermann J, Emala CW: A1 adenosine receptor knockout mice exhibit increased renal injury following ischemia and reperfusion. Am J Physiol Renal Physiol 286: F298–F306, 2004 [DOI] [PubMed] [Google Scholar]

[B10] 10.Goldman SC, Bracho F, Davenport V, Slack R, Areman E, Shen V, Lenarsky C, Weinthal J, Hughes R, Cairo MS: Feasibility study of IL-11 and granulocyte colony-stimulating factor after myelosuppressive chemotherapy to mobilize peripheral blood stem cells from heavily pretreated patients. J Pediatr Hematol Oncol 23: 300–305, 2001 [DOI] [PubMed] [Google Scholar]

[B11] 11.Du X, Williams DA: Interleukin-11: Review of molecular, cell biology, and clinical use. Blood 89: 3897–3908, 1997 [PubMed] [Google Scholar]

[B12] 12.Lee HT, Park SW, Kim M, Ham A, Anderson LJ, Brown KM, D’Agati VD, Cox GN: Interleukin-11 protects against renal ischemia and reperfusion injury. Am J Physiol Renal Physiol 303: F1216–F1224, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Park SW, Kim M, Kim JY, Brown KM, Haase VH, D’Agati VD, Lee HT: Proximal tubule sphingosine kinase-1 has a critical role in A1 adenosine receptor-mediated renal protection from ischemia. Kidney Int 82: 878–891, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Scammell TE, Arrigoni E, Thompson MA, Ronan PJ, Saper CB, Greene RW: Focal deletion of the adenosine A1 receptor in adult mice using an adeno-associated viral vector. J Neurosci 23: 5762–5770, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Rankin EB, Tomaszewski JE, Haase VH: Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor. Cancer Res 66: 2576–2583, 2006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Joo JD, Kim M, Horst P, Kim J, D’Agati VD, Emala CW, Sr, Lee HT: Acute and delayed renal protection against renal ischemia and reperfusion injury with A1 adenosine receptors. Am J Physiol Renal Physiol 293: F1847–F1857, 2007 [DOI] [PubMed] [Google Scholar]

[B17] 17.Jablonski P, Howden BO, Rae DA, Birrell CS, Marshall VC, Tange J: An experimental model for assessment of renal recovery from warm ischemia. Transplantation 35: 198–204, 1983 [DOI] [PubMed] [Google Scholar]

[B18] 18.Faubel S: Acute kidney injury and multiple organ dysfunction syndrome. Minerva Urol Nefrol 61: 171–188, 2009 [PubMed] [Google Scholar]

[B19] 19.Ikeda M, Prachasilchai W, Burne-Taney MJ, Rabb H, Yokota-Ikeda N: Ischemic acute tubular necrosis models and drug discovery: A focus on cellular inflammation. Drug Discov Today 11: 364–370, 2006 [DOI] [PubMed] [Google Scholar]

[B20] 20.Linden J: Molecular approach to adenosine receptors: Receptor-mediated mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 41: 775–787, 2001 [DOI] [PubMed] [Google Scholar]

[B21] 21.Vallon V,, Osswald H: Adenosine receptors and the kidney. Handb Exp Pharmacol 443–470, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Lee HT, Gallos G, Nasr SH, Emala CW: A1 adenosine receptor activation inhibits inflammation, necrosis, and apoptosis after renal ischemia-reperfusion injury in mice. J Am Soc Nephrol 15: 102–111, 2004 [DOI] [PubMed] [Google Scholar]

[B23] 23.Baraldi PG, Iaconinoto MA, Moorman AR, Carrion MD, Cara CL, Preti D, López OC, Fruttarolo F, Tabrizi MA, Romagnoli R: Allosteric enhancers for A1 adenosine receptor. Mini Rev Med Chem 7: 559–569, 2007 [DOI] [PubMed] [Google Scholar]

[B24] 24.Kaye JA: The clinical development of recombinant human interleukin 11 (NEUMEGA rhIL-11 growth factor). Stem Cells 14[Suppl 1]: 256–260, 1996 [DOI] [PubMed] [Google Scholar]

[B25] 25.Reynolds CH: Clinical efficacy of rhIL-11. Oncology (Williston Park) 14[Suppl 8]: 32–40, 2000 [PubMed] [Google Scholar]

[B26] 26.Kimura R, Maeda M, Arita A, Oshima Y, Obana M, Ito T, Yamamoto Y, Mohri T, Kishimoto T, Kawase I, Fujio Y, Azuma J: Identification of cardiac myocytes as the target of interleukin 11, a cardioprotective cytokine. Cytokine 38: 107–115, 2007 [DOI] [PubMed] [Google Scholar]

[B27] 27.Kuenzler KA, Pearson PY, Schwartz MZ: IL-11 pretreatment reduces cell death after intestinal ischemia-reperfusion. J Surg Res 108: 268–272, 2002 [DOI] [PubMed] [Google Scholar]

[B28] 28.Waxman AB, Mahboubi K, Knickelbein RG, Mantell LL, Manzo N, Pober JS, Elias JA: Interleukin-11 and interleukin-6 protect cultured human endothelial cells from H2O2-induced cell death. Am J Respir Cell Mol Biol 29: 513–522, 2003 [DOI] [PubMed] [Google Scholar]

[B29] 29.Bamba S, Andoh A, Yasui H, Makino J, Kim S, Fujiyama Y: Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways. Am J Physiol Gastrointest Liver Physiol 285: G529–G538, 2003 [DOI] [PubMed] [Google Scholar]

[B30] 30.Lecureur V, Arzel M, Ameziane S, Houlbert N, Le Vee M, Jouneau S, Fardel O: MAPK- and PKC/CREB-dependent induction of interleukin-11 by the environmental contaminant formaldehyde in human bronchial epithelial cells. Toxicology 292: 13–22, 2012 [DOI] [PubMed] [Google Scholar]

[B31] 31.Ropeleski MJ, Tang J, Walsh-Reitz MM, Musch MW, Chang EB: Interleukin-11-induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress. Gastroenterology 124: 1358–1368, 2003 [DOI] [PubMed] [Google Scholar]

[B32] 32.Lee HT, Kim M, Jan M, Penn RB, Emala CW: Renal tubule necrosis and apoptosis modulation by A1 adenosine receptor expression. Kidney Int 71: 1249–1261, 2007 [DOI] [PubMed] [Google Scholar]

[B33] 33.Leclercq TM, Pitson SM: Cellular signalling by sphingosine kinase and sphingosine 1-phosphate. IUBMB Life 58: 467–472, 2006 [DOI] [PubMed] [Google Scholar]

[B34] 34.Allende ML, Dreier JL, Mandala S, Proia RL: Expression of the sphingosine 1-phosphate receptor, S1P1, on T-cells controls thymic emigration. J Biol Chem 279: 15396–15401, 2004 [DOI] [PubMed] [Google Scholar]

[B35] 35.Chae SS, Proia RL, Hla T: Constitutive expression of the S1P1 receptor in adult tissues. Prostaglandins Other Lipid Mediat 73: 141–150, 2004 [DOI] [PubMed] [Google Scholar]

[B36] 36.Venkataraman K, Thangada S, Michaud J, Oo ML, Ai Y, Lee YM, Wu M, Parikh NS, Khan F, Proia RL, Hla T: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient. Biochem J 397: 461–471, 2006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37.Jo SK, Bajwa A, Awad AS, Lynch KR, Okusa MD: Sphingosine-1-phosphate receptors: Biology and therapeutic potential in kidney disease. Kidney Int 73: 1220–1230, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38.Lai LW, Yong KC, Igarashi S, Lien YH: A sphingosine-1-phosphate type 1 receptor agonist inhibits the early T-cell transient following renal ischemia-reperfusion injury. Kidney Int 71: 1223–1231, 2007 [DOI] [PubMed] [Google Scholar]

[B39] 39.Spiegel S: Sphingosine 1-phosphate: A ligand for the EDG-1 family of G-protein-coupled receptors. Ann N Y Acad Sci 905: 54–60, 2000 [DOI] [PubMed] [Google Scholar]

[B40] 40.Molitoris BA, Dahl R, Geerdes A: Cytoskeleton disruption and apical redistribution of proximal tubule Na(+)-K(+)-ATPase during ischemia. Am J Physiol 263: F488–F495, 1992 [DOI] [PubMed] [Google Scholar]

[B41] 41.Lee HT, Emala CW: Adenosine attenuates oxidant injury in human kidney proximal tubular cells via A(1) and A(2a) adenosine receptors. Am J Physiol Renal Physiol 282: F844–F852, 2002 [DOI] [PubMed] [Google Scholar]

[B42] 42.Klotz KN, Lohse MJ, Schwabe U, Cristalli G, Vittori S, Grifantini M: 2-Chloro-N6-[³H]cyclopentyladenosine ([³H]CCPA)—a high affinity agonist radioligand for A1 adenosine receptors. Naunyn Schmiedebergs Arch Pharmacol 340: 679–683, 1989 [DOI] [PubMed] [Google Scholar]

[B43] 43.Lohse MJ, Klotz KN, Schwabe U, Cristalli G, Vittori S, Grifantini M: 2-Chloro-N6-cyclopentyladenosine: Aa highly selective agonist at A1 adenosine receptors. Naunyn Schmiedebergs Arch Pharmacol 337: 687–689, 1988 [DOI] [PubMed] [Google Scholar]

[B44] 44.Kim M, Chen SW, Park SW, Kim M, D’Agati VD, Yang J, Lee HT: Kidney-specific reconstitution of the A1 adenosine receptor in A1 adenosine receptor knockout mice reduces renal ischemia-reperfusion injury. Kidney Int 75: 809–823, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45.Kim M, Park SW, Kim M, Chen SW, Gerthoffer WT, D'Agati VD, Lee HT: Selective renal overexpression of human heat shock protein 27 reduces renal ischemia-reperfusion injury in mice. Am J Physiol Renal Physiol 299: F347–F358, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46.Slot C: Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest 17: 381–387, 1965 [DOI] [PubMed] [Google Scholar]

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PERMALINK

IL-11 Is Required for A1 Adenosine Receptor–Mediated Protection against Ischemic AKI

Joo Yun Kim

Mihwa Kim

Ahrom Ham

Kevin M Brown

Robert W Greene

Vivette D D’Agati

H Thomas Lee

Abstract

Results

A1AR Activation Induces IL-11 in HK-2 Cells via ERK MAPK

Figure 1.

A1AR-Mediated Induction of Kidney IL-11 mRNA and Protein Synthesis In Vivo

Figure 2.

Figure 3.

Critical Role of IL-11 in A1AR-Mediated Renal Protection In Vivo

Figure 4.

Figure 5.

Critical Role of IL-11 in A1AR-Mediated Reduction in Kidney Neutrophil Infiltration and Apoptosis

Critical Role of Renal Proximal Tubule A1AR for Renal Protection and IL-11 Induction

Figure 6.

Critical Role of IL-11 in A1AR-Mediated SK-1 Induction in HK-2 Cells and in Mouse Kidney

Figure 7.

Figure 8.