Netrin-1 controls sympathetic arterial innervation.

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Sphingosine-1-phosphate receptor signaling during acute kidney injury: the tissue is the issue.

Sphingosine-1-phosphate is a lipid mediator that has been implicated in protection from acute kidney injury (AKI) by activation of the sphingosine-1-phosphate 1 receptor (S1P1R). The research team of H. Thomas Lee demonstrates that mice with induced deletion of S1P1R on endothelial cells experience increased ischemia-induced AKI. These findings have important translational implications. Indeed, S1P1R agonists have been used for the treatment of patients suffering from autoimmune encephalitis. Endothelial S1P1R signaling could be targeted for AKI prevention in surgical patients.

Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression.

Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A(2B) adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA(-/-) and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.

CD73-dependent generation of adenosine and endothelial Adora2b signaling attenuate diabetic nephropathy.

Nucleotide phosphohydrolysis by the ecto-5'-nucleotidase (CD73) is the main source for extracellular generation of adenosine. Extracellular adenosine subsequently signals through four distinct adenosine A receptors (Adora1, Adora2a, Adora2b, or Adora3). Here, we hypothesized a functional role for CD73-dependent generation and concomitant signaling of extracellular adenosine during diabetic nephropathy. CD73 transcript and protein levels were elevated in the kidneys of diabetic mice. Genetic deletion of CD73 was associated with more severe diabetic nephropathy, whereas treatment with soluble nucleotidase was therapeutic. Transcript levels of renal adenosine receptors showed a selective induction of Adora2b during diabetic nephropathy. In a transgenic reporter mouse, Adora2b expression localized to the vasculature and increased after treatment with streptozotocin. Adora2b(-/-) mice experienced more severe diabetic nephropathy, and studies in mice with tissue-specific deletion of Adora2b in tubular epithelia or vascular endothelia implicated endothelial Adora2b signaling in protection from diabetic nephropathy. Finally, treatment with a selective Adora2b agonist (BAY 60-6583) conveyed potent protection from diabetes-associated kidney disease. Taken together, these findings implicate CD73-dependent production of extracellular adenosine and endothelial Adora2b signaling in kidney protection during diabetic nephropathy.

Perioperative organ injury.

Despite the fact that a surgical procedure may have been performed for the appropriate indication and in a technically perfect manner, patients are threatened by perioperative organ injury. For example, stroke, myocardial infarction, acute respiratory distress syndrome, acute kidney injury, or acute gut injury are among the most common causes for morbidity and mortality in surgical patients. In the current review, the authors discuss the pathogenesis of perioperative organ injury, and provide select examples for novel treatment concepts that have emerged over the past decade. Indeed, the authors are of the opinion that research to provide mechanistic insight into acute organ injury and identification of novel therapeutic approaches for the prevention or treatment of perioperative organ injury represent the most important opportunity to improve outcomes of anesthesia and surgery.

Signaling through hepatocellular A2B adenosine receptors dampens ischemia and reperfusion injury of the liver.

Ischemia and reperfusion significantly contributes to the morbidity and mortality of liver surgery and transplantation. Based on studies showing a critical role for adenosine signaling in mediating tissue adaptation during hypoxia, we hypothesized that signaling events through adenosine receptors (ADORA1, ADORA2A, ADORA2B, or ADORA3) attenuates hepatic ischemia and reperfusion injury. Initial screening studies of human liver biopsies obtained during hepatic transplantation demonstrated a selective and robust induction of ADORA2B transcript and protein following ischemia and reperfusion. Subsequent exposure of gene-targeted mice for each individual adenosine receptor to liver ischemia and reperfusion revealed a selective role for the Adora2b in liver protection. Moreover, treatment of wild-type mice with an Adora2b-selective antagonist resulted in enhanced liver injury, whereas Adora2b-agonist treatment was associated with attenuated hepatic injury in wild-type, but not in Adora2b(-/-) mice. Subsequent studies in mice with Adora2b deletion in different tissues--including vascular endothelia, myeloid cells, and hepatocytes--revealed a surprising role for hepatocellular-specific Adora2b signaling in attenuating nuclear factor NF-κB activation and thereby mediating liver protection from ischemia and reperfusion injury. These studies provide a unique role for hepatocellular-specific Adora2b signaling in liver protection during ischemia and reperfusion injury.

Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion.

UNLABELLED: Ischemia and reperfusion-elicited tissue injury contributes to morbidity and mortality of hepatic surgery and during liver transplantation. Previous studies implicated extracellular adenosine signaling in liver protection. Based on the notion that extracellular adenosine signaling is terminated by uptake from the extracellular towards the intracellular compartment by way of equilibrative nucleoside transporters (ENTs), we hypothesized a functional role of ENTs in liver protection from ischemia. During orthotopic liver transplantation in humans, we observed higher expressional levels of ENT1 than ENT2, in conjunction with repression of ENT1 and ENT2 transcript and protein levels following warm ischemia and reperfusion. Treatment with the pharmacologic ENT inhibitor dipyridamole revealed elevations of hepatic adenosine levels and robust liver protection in a murine model of liver ischemia and reperfusion. Studies in gene-targeted mice for Ent1 or Ent2 demonstrated selective protection from liver injury in Ent1(-/-) mice. Treatment with selective adenosine receptor antagonists indicated a contribution of Adora2b receptor signaling in ENT-dependent liver protection. CONCLUSION: These findings implicate ENT1 in liver protection from ischemia and reperfusion injury and suggest ENT inhibitors may be of benefit in the prevention or treatment of ischemic liver injury.

Protective role for netrin-1 during diabetic nephropathy.

Recent studies implicate neuronal guidance molecules in the orchestration of inflammatory events. For example, previous studies demonstrate a functional role for netrin-1 in attenuating acute kidney injury. Here, we hypothesized a kidney-protective role for netrin-1 during chronic kidney disease, such as occurs during diabetic nephropathy. To study the role of netrin-1 during diabetic nephropathy, we induced diabetes in mice at the age of 8 weeks by streptocotozin (STZ) treatment. Sixteen weeks after STZ treatment, we examined the kidneys. Initial studies in wild-type mice demonstrated robust induction of renal, urinary, and plasma netrin-1 protein levels during diabetic nephropathy. Subsequent genetic studies in mice with partial netrin-1 deficiency (Ntrn1(+/-) mice) revealed a more severe degree of diabetic nephropathy, including more severe loss of kidney function (albuminuria, glomerular filtration rate, histology). We subsequently performed pharmacologic studies with recombinant netrin-1 treatment given continuously via osmotic pump. Indeed, netrin-1 treatment was associated with attenuated albuminuria and improved histologic scores for diabetic nephropathy compared to controls. Consistent with previous studies implicating purinergic signaling in netrin-1-elicited tissue protection, mice deficient in the Adora2b adenosine receptor were not protected. Taken together, these studies demonstrate a functional role for endogenous netrin-1 in attenuating diabetic kidney disease.

Elevated ecto-5'-nucleotidase-mediated increased renal adenosine signaling via A2B adenosine receptor contributes to chronic hypertension.

RATIONALE: Hypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood. OBJECTIVE: We sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression. METHODS AND RESULTS: Using high-throughput quantitative reverse-transcription polymerase chain reaction profiling, we discovered that the expression level of 5'-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II-infused mice, an animal model of hypertensive nephropathy. Genetic and pharmacological studies in mice revealed that elevated CD73-mediated excess renal adenosine preferentially induced A2B adenosine receptor (ADORA2B) production and that enhanced kidney ADORA2B signaling contributes to angiotensin II-induced hypertension. Similarly, in humans, we found that CD73 and ADORA2B levels were significantly elevated in the kidneys of CKD patients compared with normal individuals and were further elevated in hypertensive CKD patients. These findings led us to further discover that elevated renal CD73 contributes to excess adenosine signaling via ADORA2B activation that directly stimulates endothelin-1 production in a hypoxia-inducible factor-α-dependent manner and underlies the pathogenesis of the disease. Finally, we revealed that hypoxia-inducible factor-α is an important factor responsible for angiotensin II-induced CD73 and ADORA2B expression at the transcriptional level. CONCLUSIONS: Overall, our studies reveal that angiotensin II-induced renal CD73 promotes the production of renal adenosine that is a prominent driver of hypertensive CKD by enhanced ADORA2B signaling-mediated endothelin-1 induction in a hypoxia-inducible factor-α-dependent manner. The inhibition of excess adenosine-mediated ADORA2B signaling represents a novel therapeutic target for the disease.

Transcriptional control of adenosine signaling by hypoxia-inducible transcription factors during ischemic or inflammatory disease.

Inflammatory lesions, ischemic tissues, or solid tumors are characterized by the occurrence of severe tissue hypoxia within the diseased tissue. Subsequent stabilization of hypoxia-inducible transcription factors-particularly of hypoxia-inducible factor 1α (HIF1A)--results in significant alterations of gene expression of resident cells or inflammatory cells that have been recruited into such lesions. Interestingly, studies of hypoxia-induced changes of gene expression identified a transcriptional program that promotes extracellular adenosine signaling. Adenosine is a signaling molecule that functions through the activation of four distinct adenosine receptors--the ADORA1, ADORA2A, ADORA2B, and ADORA3 receptors. Extracellular adenosine is predominantly derived from the phosphohydrolysis of precursor nucleotides, such as adenosine triphosphate or adenosine monophosphate. HIF1A-elicited alterations in gene expression enhance the enzymatic capacity within inflamed tissues to produce extracellular adenosine. Moreover, hypoxia-elicited induction of adenosine receptors--particularly of ADORA2B--results in increased signal transduction. Functional studies in genetic models for HIF1A or adenosine receptors implicate this pathway in an endogenous feedback loop that dampens excessive inflammation and promotes injury resolution, while at the same time enhancing ischemia tolerance. Therefore, pharmacological strategies to enhance HIF-elicited adenosine production or to promote adenosine signaling through adenosine receptors are being investigated for the treatment of acute inflammatory or ischemic diseases characterized by tissue hypoxia.

Adora2b adenosine receptor signaling protects during acute kidney injury via inhibition of neutrophil-dependent TNF-α release.

Renal ischemia is among the leading causes of acute kidney injury (AKI). Previous studies have shown that extracellular adenosine is a prominent tissue-protective cue elicited during ischemia, including signaling events through the adenosine receptor 2b (Adora2b). To investigate the functional role of Adora2b signaling in cytokine-mediated inflammatory pathways, we screened wild-type and Adora2b-deficient mice undergoing renal ischemia for expression of a range of inflammatory cytokines. These studies demonstrated a selective and robust increase of TNF-α levels in Adora2b-deficient mice following renal ischemia and reperfusion. Based on these findings, we next sought to understand the contribution of TNF-α on ischemic AKI through a combination of loss- and gain-of-function studies. Loss of TNF-α, through either Ab blockade or study of Tnf-α-deficient animals, resulted in significantly attenuated tissue injury and improved kidney function following renal ischemia. Conversely, transgenic mice with overexpression of TNF-α had significantly pronounced susceptibility to AKI. Furthermore, neutrophil depletion or reconstitution of Adora2b(-/-) mice with Tnf-α-deficient neutrophils rescued their phenotype. In total, these data demonstrate a critical role of adenosine signaling in constraining neutrophil-dependent production of TNF-α and implicate therapies targeting TNF-α in the treatment of ischemic AKI.

Use of a hanging-weight system for liver ischemia in mice.

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues. Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease.

Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A(2B)R) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A(2B)R or treatment with the A(2B)R antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A(2B)R attenuated vascular remodeling and hypertension in our model. Furthermore, direct A(2B)R activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A(2B)R antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.

Hypoxia signaling during intestinal ischemia and inflammation.

PURPOSE OF REVIEW: During critical illness, alterations of intestinal blood supply and inflammatory activation can result in severe intestinal hypoxia (limited oxygen availability). Conditions of hypoxia lead to the activation of a transcriptional program that is under the control of the transcription factor hypoxia-inducible factor (HIF). In many instances, HIF-dependent alterations of gene expression represent endogenous adaptive responses that dampen pathologic inflammation and could be targeted to treat intestinal injury. RECENT FINDINGS: Post-translational stabilization of the HIF transcription factor and corresponding changes in gene expression are central to the resolution of intestinal injury. Examples for such responses that we discuss in this review include hypoxia-elicited increases in extracellular adenosine production and signaling, particularly through the A2B adenosine receptor, and intestinal protection provided by hypoxia-inducible netrin-1. SUMMARY: The present review focuses on HIF-elicited anti-inflammatory pathways that result in intestinal protection during critical illness. Many of these pathways represent novel therapeutic targets for attenuating multiorgan failure and critical illness. Whereas these therapeutic approaches are currently being investigated in cell culture models or in genetic mouse models, we are optimistic that at least some of these novel targets can be translated from bench to bedside in the near future.

Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice.

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) - a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1-/- mice. Comprehensive examination of adenosine receptor-knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications.

Absence of equilibrative nucleoside transporter 1 in ENT1 knockout mice leads to altered nucleoside levels following hypoxic challenge.

AIMS: Equilibrative nucleoside transporters (ENT) modulate the flux of adenosine. The ENT1-null (KO) mouse heart is endogenously cardioprotected but the cellular basis of this phenotype is unknown. Therefore, we investigated the cellular mechanisms underlying ENT1-mediated cardioprotection. MAIN METHODS: Circulating adenosine levels were measured in WT and KO mice. Cellular levels of nucleosides and nucleotides were investigated in isolated adult cardiomyocytes from WT and KO mice using HPLC following hypoxic challenge (30 min, 2% O(2)). Changes in hypoxic gene expression were analyzed by PCR arrays and cAMP levels were measured to investigate contributions from adenosine receptors. KEY FINDINGS: Circulating adenosine levels were significantly higher in KO (416±42nmol/l, n=12) compared to WT animals (208±21, n=13, p<0.001). Absence of ENT1 led to an elevated expression of genes involved in cardioprotective pathways compared to WT cardiomyocytes. Following hypoxic challenge, extracellular adenosine levels were significantly elevated in KO (4360±1840 pmol/mg protein) versus WT cardiomyocytes (3035±730 pmol/mg protein, n≥12, p<0.05). This effect was enhanced in the presence of dipyridamole (30 µM), which inhibits ENT1 and ENT2. Enhanced extracellular adenosine levels in ENT1-null cardiomyocytes appeared to come from a pool of extracellular nucleotides including IMP, AMP and ADP. Adenosine receptor (AR) activation mimicked increases in cAMP levels due to hypoxic challenge suggesting that ENT1 modulates AR-dependent signaling. SIGNIFICANCE: ENT1 contributes to modulation of extracellular adenosine levels and subsequent purinergic signaling via ARs. ENT1-null mice possess elevated circulating adenosine levels and reduced cellular uptake resulting in a perpetually cardioprotected phenotype.

Interleukin 37 expression protects mice from colitis.

IL-37, a newly described member of the IL-1 family, functions as a fundamental inhibitor of innate inflammation and immunity. In the present study, we examined a role for IL-37 during experimental colitis. A transgenic mouse strain was generated to express human IL-37 (hIL-37tg), and these mice were subjected to dextran sulfate sodium (DSS)-induced colitis. Despite the presence of a CMV promoter to drive expression of IL-37, mRNA transcripts were not present in colons at the resting state. Expression was observed only upon disruption of the epithelial barrier, with a six- to sevenfold increase (P = 0.02) on days 3 and 5 after continuous exposure to DSS. During the development of colitis, clinical disease scores were reduced by 50% (P < 0.001), and histological indices of colitis were one-third less in hIL-37tg mice compared with WT counterparts (P < 0.001). Reduced inflammation was associated with decreased leukocyte recruitment into the colonic lamina propria. In addition, release of IL-1ß and TNFα from ex vivo colonic explant tissue was decreased 5- and 13-fold, respectively, compared with WT (P ≤ 0.005), whereas IL-10 was increased sixfold (P < 0.001). However, IL-10 was not required for the anti-inflammatory effects of IL-37 because IL-10-receptor antibody blockade did not reverse IL-37-mediated protection. Mechanistically, IL-37 originating from hematopoietic cells was sufficient to exert anti-inflammatory effects because WT mice reconstituted with hIL-37tg bone marrow were protected from colitis. Thus, IL-37 emerges as key modulator of intestinal inflammation.

Use of a hanging-weight system for isolated renal artery occlusion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30%. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis. The occurrence of AKI is associated with dramatic increases of morbidity and mortality. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed. To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

Partial netrin-1 deficiency aggravates acute kidney injury.

The netrin family of secreted proteins provides migrational cues in the developing central nervous system. Recently, netrins have also been shown to regulate diverse processes beyond their functions in the brain, incluing the ochrestration of inflammatory events. Particularly netrin-1 has been implicated in dampening hypoxia-induced inflammation. Here, we hypothesized an anti-inflammatory role of endogenous netrin-1 in acute kidney injury (AKI). As homozygous deletion of netrin-1 is lethal, we studied mice with partial netrin-1 deletion (Ntn-1(+/-) mice) as a genetic model. In fact, Ntn-1(+/-) mice showed attenuated Ntn-1 levels at baseline and following ischemic AKI. Functional studies of AKI induced by 30 min of renal ischemia and reperfusion revealed enhanced kidney dysfunction in Ntn-1(+/-) mice as assessed by measurements of glomerular filtration, urine flow rate, urine electrolytes, serum creatinine and creatinine clearance. Consistent with these findings, histological studies indicated a more severe degree kidney injury. Similarly, elevations of renal and systemic inflammatory markers were enhanced in mice with partial netrin-1 deficiency. Finally, treatment of Ntn-1(+/-) mice with exogenous netrin-1 restored a normal phenotype during AKI. Taking together, these studies implicate endogenous netrin-1 in attenuating renal inflammation during AKI.

A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation.

Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5'-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A(2B) receptor (A(2B)R) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A(2B)R signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A(2B)R in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A(2B)R activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.