Extracellular 2′,3′-cAMP and 3′,5′-cAMP stimulate proliferation of preglomerular vascular endothelial cells and renal epithelial cells

Abstract

Kidneys release into the extracellular compartment 3′,5′-cAMP and its positional isomer 2′,3′-cAMP. The purpose of the present study was to investigate the metabolism of extracellular 2′,3′-cAMP and 3′,5′-cAMP in preglomular vascular endothelial and proximal tubular epithelial cells and to determine whether these cAMPs and their downstream metabolites affect cellular proliferation. In preglomerular vascular endothelial and proximal tubular epithelial cells, 1) extracellular 2′,3′-cAMP increased extracellular levels of 3′-AMP and 2′-AMP, whereas extracellular 3′,5′-cAMP increased extracellular levels of 5′-AMP; 2) extracellular 5′-AMP, 3′-AMP, and 2′-AMP increased extracellular adenosine; 3) α,β-methylene-adenosine-5′-diphosphate (CD73 inhibitor) prevented the 5′-AMP-induced increase in extracellular adenosine in preglomerular vascular endothelial cells, but did not affect the 5′-AMP-induced increase in extracellular adenosine in proximal tubular cells or the 3′-AMP-induced or 2′-AMP-induced increase in extracellular adenosine in either cell type; 4) extracellular 2′,3′-cAMP, 3′-AMP, 2′-AMP, 3′,5′-cAMP, 5′-AMP, and adenosine stimulated proliferation of both preglomerular vascular endothelial and proximal tubular cells; and 5) MRS-1754 (selective A_2B receptor antagonist) abolished the progrowth effects of extracellular 2′,3′-cAMP, 3′-AMP, 2′-AMP, 3′,5′-cAMP, 5′-AMP, and adenosine in both cell types. Extracellular 2′,3′-cAMP and 3′,5′-cAMP stimulate proliferation of preglomerular vascular endothelial cells and proximal tubular cells. The mechanism by which the cAMPs increase cell proliferation entails 1) metabolism to their respective AMPs, 2) metabolism of their respective AMPs to adenosine (which for 5′-AMP in preglomerular vascular endothelial cells is mediated by CD73), and 3) activation of A_2B receptors. Both extracellular 2′,3′-cAMP and 3′,5′-cAMP may help restore architecture of the preglomerular microcirculation and tubular system following kidney injury.

the kidney expresses an extracellular 3′,5′-cAMP-adenosine pathway [i.e., the export of intracellular 3′,5′-cAMP to the extracellular compartment, followed by metabolism of extracellular 3′,5′-cAMP to 5′-AMP and then metabolism of extracellular 5′-AMP to adenosine (extracellular 3′,5′-cAMP → 5′-AMP → adenosine)] (10). This pathway is stimulated by intense hormonal activation of adenyly cyclase (10) as might occur during times of cardiorenal stress. More recent studies in isolated, perfused rat (14, 19) and mouse (11) kidneys show that kidneys can also produce a positional isomer of 3′,5′-cAMP, namely 2′,3′-cAMP, and provide support for the concept that 2′,3′-cAMP derives from mRNA degradation triggered by energy depletion and renal injury (14). Additional studies demonstrate that kidneys can release 2′,3′-cAMP into the extracellular compartment and metabolize extracellular 2′,3′-cAMP to 2′-AMP and 3′-AMP and can further metabolize extracellular 2′-AMP and 3′-AMP to adenosine (11, 14), thus supporting the concept of an extracellular 2′,3′-cAMP-adenosine pathway (extracellular 2′,3′-cAMP → 2′-AMP + 3′-AMP → adenosine) in addition to an extracellular 3′,5′-cAMP-adenosine pathway.

It is conceivable that adenosine generated by the two cAMP-adenosine pathways protects kidneys and helps maintain a healthy renal architecture by preventing the overproliferation of renal vascular smooth muscle cells and glomerular mesangial cells while encouraging the proliferation of renal vascular endothelial cells and renal epithelial cells. In this regard, recent studies demonstrate that both preglomerular vascular smooth muscle cells and glomerular mesangial cells metabolize extracellular 3′,5′-cAMP and 2′,3′-cAMP to their corresponding AMPs and their corresponding AMPs to adenosine (13). Moreover, extracellular 3′,5′-cAMP and 2′,3′-cAMP inhibit the proliferation of preglomerular vascular smooth muscle cells and glomerular mesangial cells via the production of adenosine that then activates adenosine A_2B receptors (13). Likewise, extracellular 2′-AMP, 3′-AMP, and 5′-AMP inhibit proliferation of preglomerular vascular smooth muscle cells and glomerular mesangial cells via A_2B receptors (8). Whether the two cAMP-adenosine pathways could enhance proliferation of renal vascular endothelial cells and renal epithelial cells is presently unknown. Therefore, the goal of the present study was to investigate the metabolism of 2′,3′-cAMP, 2′-AMP, 3′-AMP, 3′,5′-cAMP, and 5′-AMP in preglomerular vascular endothelial cells (PGVECs) and proximal tubular epithelial cells (PTCs), to determine the effects of these purines on proliferation of PGVECs and PTCs, and finally to define the role of A_2B receptors in the growth effects of 2′,3′-cAMP, 2′-AMP, 3′-AMP, 3′,5′-cAMP, and 5′-AMP in PGVECs and PTCs.

METHODS

Rat PGVECs and human PTCs.

Rat preglomerular microvessels were isolated, as described in detail by us previously (18), from male Wistar-Kyoto rats that were obtained from Taconic Farms (Germantown, NY), and PGVECs were cultured from these microvessels using the method described by Frye and Patrick (6). The Institutional Animal Care and Use Committee approved all procedures. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Human PTCs were obtained from Cell Applications (San Diego, CA).

Metabolism experiments.

Studies were conducted in confluent cells in early passage under standard cell culture conditions. Cells were washed twice with HEPES-buffered Hanks balanced salt solution and then incubated for 1 h in 0.5 ml of Dulbecco's phosphate-buffered saline with HEPES (25 mM) and NaHCO₃ (13 mM) in the presence and absence of 3′,5′-cAMP, 2′,3′-cAMP, 5′-AMP, 3′-AMP, or 2′-AMP with or without α,β-methylene-adenosine-5′-diphosphate [AMPCP; selective inhibitor of CD73 (21)], all from Sigma (St. Louis, MO). After a 1-h incubation, the medium was collected, heated for 90 s at 100°C to denature enzymes, and then frozen at −80°C until assayed by mass spectrometry.

Proliferation experiments.

For cell number experiments, cells were allowed to attach overnight, were growth-arrested for 48 h, and were treated with 3′,5′-cAMP, 2′,3′-cAMP, 5′-AMP, 3′-AMP, 2′-AMP, or adenosine with or without MRS-1754 [selective A_2B receptor antagonist (17); Sigma] in DMEM containing PDGF (25 ng/ml). Treatments were repeated every 24 h for 4 days. On day 5, cells were dislodged and counted with a Coulter counter.

Purine assay.

2′-AMP, 3′-AMP, 5′-AMP, and adenosine were obtained from Sigma. The internal standard (¹³C₁₀-adenosine) was from Medical Isotopes (Pelham, NH). Purines were resolved by reversed-phase liquid chromatography (Agilent Zorbax eclipse XDB-C-18 column, 3.5-μm beads; 2.1 × 100 mm) and quantified using a triple quadrupole mass spectrometer (TSQ Quantum-Ultra, ThermoFisher Scientific, San Jose, CA) operating in the selected reaction monitoring mode with a heated electrospray ionization source as previously described in detail (14).

Statistics.

Data were analyzed by one-factor ANOVA, with post hoc comparisons using a Fisher's least significant difference test. The criterion of significance was P < 0.05. All values in text and figures are means ± SE.

RESULTS

Experiments in rat PGVECs.

Incubation of rat PGVECs with 2′,3′-cAMP significantly and concentration-dependently increased medium levels of 2′-AMP and 3′-AMP, but not 5′-AMP, and the increase in 3′-AMP was greater than the increase in 2′-AMP (Fig. 1A). In contrast to 2′,3′-cAMP, 3′,5′-cAMP significantly and concentration-dependently increased levels of 5′-AMP, but had no effect on medium levels of 2′-AMP or 3′-AMP (Fig. 1B). Incubation of rat PGVECs with 5′-AMP (prototypical adenosine precursor; Fig. 2A), 3′-AMP (Fig. 2B), and 2′-AMP (Fig. 2C) significantly and concentration-dependently increased levels of adenosine in the medium. The effect of 5′-AMP on medium levels of adenosine was abolished by AMPCP (100 μM; CD73 inhibitor; Fig. 2D), whereas AMPCP did not alter the adenosine increase induced by either 3′-AMP (Fig. 2E) or 2′-AMP (Fig. 2F).

Fig. 1.

Line graphs illustrate the concentration-dependent effects of 2′,3′-cAMP (A) and 3′,5′-cAMP (B) on levels of 2′-AMP, 3′-AMP, and 5′-AMP in the medium of cultures of rat preglomerular vascular endothelial cells (PGVECs). ^aP < 0.05 compared with basal (0).

Fig. 2.

Line graphs illustrate the concentration-dependent effects of 5′-AMP (A), 3′-AMP (B), and 2′-AMP (C) on levels of adenosine in the medium of cultures from rat PGVECs. Bar graphs illustrate the effects of α,β-methylene-adenosine-5′-diphosphate (AMPCP; 100 μM; CD73 inhibitor) on adenosine levels in the medium of rat PGVECs incubated with 10 μM of either 5′-AMP (D), 3′-AMP (E), or 2′-AMP (F). ^aP < 0.05 compared with basal (0 or PBS) or AMPCP. ^bP < 0.05 compared with 5′-AMP in the absence of AMPCP.

In rat PGVECs, both 3′,5′-cAMP (Fig. 3A) and 2′,3′-cAMP (Fig. 3B) significantly and concentration-dependently stimulated cell proliferation (cell number). MRS-1754 at both concentrations tested (0.1 and 0.3 μM) abolished the proliferation-enhancing effects of both 3′,5′-cAMP (Fig. 3C) and 2′,3′-cAMP (Fig. 3D). Similar to the cAMPs, in rat PGVECs 5′-AMP (Fig. 4A), 3′-AMP (Fig. 4B), 2′-AMP (Fig. 4C), and adenosine (Fig. 4D) significantly and concentration-dependently increased cell proliferation. MRS-1754 at 0.1 μM attenuated the proliferation-enhancing effects of 5′-AMP (Fig. 5A), 3′-AMP (Fig. 5B), 2′-AMP (Fig. 5C), and adenosine (Fig. 5D). Because the effects of 5′-AMP, 2′-AMP, and adenosine on cell proliferation were not abolished by 0.1 μM MRS-1754, we also examined the proproliferative effects of the AMPs and adenosine in the presence of a higher concentration of MRS-1754. In this regard, MRS-1754 at a concentration of 0.3 μM abolished the proliferation-promoting effects of 5′-AMP (Fig. 6A), 3′-AMP (Fig. 6B), 2′-AMP (Fig. 6C), and adenosine (Fig. 6D).

Fig. 3.

Line graphs illustrate the concentration-dependent effects of 3′,5′-cAMP (A) and 2′,3′-cAMP (B) on cell number in rat PGVECs. Bar graphs illustrate effects of MRS-1754 (A_2B antagonist) on the proliferative effects (cell number) of 3′,5′-cAMP (C) and 3′,5′-cAMP (D) in rat PGVECs. ^aP < 0.05 compared with corresponding basal (0). ^bP < 0.05 compared with 3′,5′-AMP or 2′,3′-cAMP in the absence of MRS-1754.

Fig. 4.

Line graphs illustrate the concentration-dependent effects of 5′-AMP (A), 3′-AMP (B), 2′-AMP (C), and adenosine (D) on cell number in rat PGVECs. ^aP < 0.05 compared with basal (0).

Fig. 5.

Bar graphs illustrate the effects of MRS-1754 (0.1 μM; A_2B antagonist) on the proliferative effects (cell number) of 5′-AMP (A), 3′-AMP (B), 2′-AMP (C), and adenosine (D) in rat PGVECs. ^aP < 0.05 compared with basal (0). ^bP < 0.05 compared with corresponding AMP in the absence of MRS-1754.

Fig. 6.

Bar graphs illustrate the effects of MRS-1754 (0.3 μM; A_2B antagonist) on the proliferative effects (cell number) of 5′-AMP (A), 3′-AMP (B), 2′-AMP (C), and adenosine (D) in rat PGVECs. ^aP < 0.05 compared with basal (0). ^bP < 0.05 compared with corresponding AMP in the absence of MRS-1754.

Experiments in human PTCs.

Incubation of human PTCs with 2′,3′-cAMP significantly and concentration-dependently increased medium levels of 2′-AMP and 3′-AMP, but not 5′-AMP (Fig. 7A). In contrast to PGVECs, in PTCs 2′,3′-cAMP increased 2′-AMP more than 3′-AMP. In human PTCs, 3′,5′-cAMP significantly and concentration-dependently increased levels of 5′-AMP, but had no effect on medium levels of 2′-AMP or 3′-AMP (Fig. 7B). Incubation of human PTCs with 5′-AMP (Fig. 8A), 3′-AMP (Fig. 8B), and 2′-AMP (Fig. 8C) significantly and concentration-dependently increased levels of adenosine in the medium. AMPCP did not alter increases in adenosine levels induced by either 5′-AMP (Fig. 8D), 3′-AMP (Fig. 8E), or 2′-AMP (Fig. 8F).

Fig. 7.

Line graphs illustrate the concentration-dependent effects of 2′,3′-cAMP (A) and 3′,5′-cAMP (B) on levels of 2′-AMP, 3′-AMP, and 5′-AMP in the medium of cultures of human proximal tubular epithelial cells (PTCs). ^aP < 0.05 compared with basal (0).

Fig. 8.

Line graphs illustrate the concentration-dependent effects of 5′-AMP (A), 3′-AMP (B), and 2′-AMP (C) on levels of adenosine in the medium of cultures from human PTCs. Bar graphs illustrate the effects of AMPCP (100 μM; CD73 inhibitor) on adenosine levels in the medium of human PTCs incubated with 10 μM of either 5′-AMP (D), 3′-AMP (E), or 2′-AMP (F). ^aP < 0.05 compared with basal (0 or PBS) or AMPCP.

In human PTCs, both 2′,3′-cAMP (Fig. 9A) and 3′,5′-cAMP (Fig. 9B) significantly stimulated cell proliferation, and these effects were abolished by MRS-1754 at 0.1 μM. In human PTCs, 5′-AMP (Fig. 10A), 3′-AMP (Fig. 10B), 2′-AMP (Fig. 10C), and adenosine (Fig. 10D) significantly and concentration-dependently increased cell proliferation. MRS-1754 at 0.1 μM attenuated or abolished the proliferation-enhancing effects of 5′-AMP (Fig. 11A), 3′-AMP (Fig. 11B), 2′-AMP (Fig. 11C), and adenosine (Fig. 11D).

Fig. 9.

Bar graphs illustrate effects of MRS-1754 (A_2B antagonist) on the proliferative effects (cell number) of 2′,3′-cAMP (A) and 3′,5′-cAMP (B) in human PTCs. ^aP < 0.05 compared with corresponding basal (0). ^bP < 0.05 compared with 3′,5′-AMP or 2′,3′-cAMP in the absence of MRS-1754.

Fig. 10.

Line graphs illustrate the concentration-dependent effects of 5′-AMP (A), 3′-AMP (B), 2′-AMP (C), and adenosine (D) on cell number in human PTCs. ^aP < 0.05 compared with basal (0).

Fig. 11.

Bar graphs illustrate the effects of MRS-1754 (0.1 μM; A_2B antagonist) on the proliferative effects (cell number) of 5′-AMP (A), 3′-AMP (B), 2′-AMP (C), and adenosine (D) in human PTCs. ^aP < 0.05 compared with basal (0). ^bP < 0.05 compared with corresponding AMP in the absence of MRS-1754.

DISCUSSION

An important objective of the present study was to determine whether PGVECs express the extracellular 2′,3′-cAMP-adenosine and 3′,5′-cAMP-adenosine pathways, and the results of the present study confirm this hypothesis. In this regard, PGVECs convert exogenous 2′,3′-cAMP and 3′,5′-cAMP to their respective AMPs and metabolize 2′-AMP, 3′-AMP, and 5′-AMP to adenosine. The results of the present study also demonstrate that in PGVECs, the CD73 inhibitor AMPCP blocks the conversion of extracellular 5′-AMP to extracellular adenosine, but it has no effect on the metabolism of extracellular 3′-AMP or 2′-AMP to extracellular adenosine. We conclude, therefore, that in PGVECs the ecto-2′/3′-nucleotidase that mediates the conversion of 3′-AMP or 2′-AMP to adenosine is distinct from the ecto-5′-nucleotidase (CD73) that mediates the conversion of extracellular 5′-AMP to extracellular adenosine.

A second objective of this investigation was to determine whether the extracellular 2′,3′-cAMP-adenosine pathway and extracellular 3′,5′-cAMP-adenosine pathway affect the proliferation of PGVECs. In this regard, the present study establishes for the first time that both extracellular cAMP-adenosine pathways accelerate the proliferation of PGVECs. In support of this conclusion, application of extracellular 2′,3′-cAMP and its metabolites (2′-AMP, 3′-AMP, and adenosine) and application of extracellular 3′,5′-cAMP and its metabolites (5′-AMP and adenosine) significantly increase cell number in cultures of PGVECs. Importantly, our previous studies demonstrate that both cAMP-adenosine pathways inhibit proliferation of preglomerular vascular smooth muscle cells and glomerular mesangial cells (2, 8, 13). Thus, it appears that the cAMP-adenosine pathways are well-suited to maintain a healthy renal architecture by 1) preventing abnormal remodeling of renal microvessels (via inhibiting vascular smooth muscle cell proliferation), 2) preventing glomerulosclerosis (via inhibiting mesangial cell expansion), and 3) accelerating the rate of repair of renal microvessels (via stimulating vascular endothelial cell growth).

Importantly, the proproliferative effects of 2′,3′-cAMP and its metabolites (2′-AMP, 3′-AMP, and adenosine) and 3′,5′-cAMP and its metabolites (5′-AMP and adenosine) on PGVECs are effectively blocked by the highly selective A_2B receptor antagonist MRS-1754. This observation supports two conclusions: 1) the proproliferative effects of the cAMPs and AMPs in PGVECs are mediated by adenosine and 2) the A_2B receptor mediates the proproliferative effects of adenosine in PGVECs. This finding is entirely consistent with our previous report that adenosine via A_2B receptors stimulates the proliferation of porcine coronary and rat aortic endothelial cells (1).

A third objective of the present study was to determine whether PTCs express the 2′,3′-cAMP-adenosine and 3′,5′-cAMP-adenosine pathways, and the results of the present study confirm this concept. In support of this conclusion, PTCs convert exogenous 2′,3′-cAMP and 3′,5′-cAMP to their respective AMPs and metabolized 2′-AMP, 3′-AMP, and 5′-AMP to adenosine. These findings are consistent with our previous results showing that in rat proximal tubules and rat collecting ducts, 3′,5′-cAMP is metabolized to 5′-AMP and adenosine (9, 16). As in PGVECs, the CD73 inhibitor AMPCP does not block the conversion of extracellular 2′-AMP and 3′-AMP to adenosine. However, unlike PGVECs, in PTCs, AMPCP also does not block the metabolism of 5′-AMP to extracellular adenosine. Thus, we conclude that in PTCs ecto-5′-nucleotidase (CD73) plays little or no role in mediating the cAMP-adenosine pathways.

A fourth objective of this experimental series was to determine whether the extracellular 2′,3′-cAMP-adenosine pathway and extracellular 3′,5′-cAMP-adenosine pathway affect the proliferation of PTCs. The present study establishes for the first time that both extracellular cAMP-adenosine pathways accelerate the proliferation of PTCs. In this regard, application of extracellular 2′,3′-cAMP and its metabolites (2′-AMP, 3′-AMP, and adenosine) and application of extracellular 3′,5′-cAMP and its metabolites (5′-AMP and adenosine) significantly increase cell number in cultures of PTCs. This is important because it shows that the cAMP-adenosine pathways may not only modulate structure of the microcirculation and glomeruli, but also may influence renal tubular structure.

Interestingly, our previous studies show that A_2B receptors in preglomerular vascular smooth muscle cells and glomerular mesangial cells mediate the anti-proliferative effects of the cAMP-adenosine pathways (8, 13); yet here we show that A_2B receptors instead mediate progrowth effects of the cAMP-adenosine pathways in PGVECs and PTCs. Thus, the growth effects of A_2B receptors appear to be cell-type specific. The signal transduction mechanism by which A_2B receptors regulate cell proliferation is unknown. Important recent studies by Eckle et al. (4) identify the circadian rhythm protein period 2 (Per2) as a key mediator of A_2B receptor signaling in the heart. In this regard, A_2B receptor activation, via stimulation of the 3′,5′-cAMP-CREB pathway, increases Per2 gene transcription. Moreover, A_2B receptor activation decreases the neddylation status of cullin 1. When cullin 1 is deneddylated, the polyubiquitination of Per2 is reduced, which leads to Per2 stabilization. Thus, A_2B receptor signaling increases Per2 levels and Per2 in turn increases oxygen-efficient glycolysis, in part by stabilizing hypoxia-inducible factor-1α. Whether Per2 participates in A_2B receptor-mediated regulation of cell proliferation is an intriguing issue that merits investigation.

Evidence continues to emerge indicating an important role for A_2B receptors in kidney injury. However, whether A_2B receptors are beneficial or harmful may depend on the time frame of repair after injury. For example, all of the effects we observe with regard to modulation of proliferation of preglomerular vascular smooth muscle cells, glomerular mesangial cells, PGVECs, and PTCs would be predicted to aid rapid restoration of kidney structure and function following injury. Consistent with this concept, a comprehensive study by Grenz et al. (7) demonstrates that renal adenosine protects against acute kidney injury via activation of A_2B receptors on vascular endothelial cells thereby preventing the postischemic no-reflow phenomenon. In contrast, very recent studies by Zhang et al. (20) suggest that long-term activation of A_2B receptors contributes to renal fibrosis and chronic kidney disease. Thus, it appears that the overall function of A_2B receptors is directed toward repair; however, beneficial repair mechanisms that are engaged for too long a duration may become detrimental.

As reviewed by Eltzschig and Carmeliet (5), hypoxia, via activation of the prolyl hydroxylase (PHD)-hypoxia-inducible transcription factor (HIF) mechanism, activates the adenosine system by increasing extracellular adenosine synthesis, by inhibiting the disposition of extracellular adenosine, and by increasing the expression of adenosine receptors. Because adenosine restrains both the innate and adaptive immune systems, the hypoxia-PHD-HIF-adenosine axis protects organs, including the kidney, from inflammation and injury. Since 2′,3′-cAMP-adenosine and 3′,5′-cAMP-adenosine pathways may protect against kidney injury, it is conceivable that the PHD-HIF mechanism may also activate enzymes involved in the cAMP-adenosine pathways, and this hypothesis merits investigation.

In summary, the present study demonstrates that both PGVECs and PTCs can metabolize both 2′,3′-cAMP and 3′,5′-cAMP to their corresponding downstream AMPs and that these AMPs can be converted to adenosine. Our results also establish that adenosine generated from these cAMPs can stimulate the proliferation of both renovascular endothelial cells and tubular epithelial cells. Thus, it is conceivable that the cAMP-adenosine pathways contribute to the restoration of a healthy renal architecture following renal injury. The concept that the cAMP-adenosine pathways may hasten restoration of tissue health following injury may be extrapolated to other cardiovascular tissues and organ systems because both pathways inhibit proliferation of aortic and coronary vascular smooth muscle cells (3, 12) and A_2B receptor activation stimulates proliferation of aortic and coronary endothelial cell proliferation (1).

GRANTS

This work was supported by National Institutes of Health Grants DK091190, HL069846, DK068575, and DK079307.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: E.K.J. conception and design of research; E.K.J. analyzed data; E.K.J. interpreted results of experiments; E.K.J. prepared figures; E.K.J. drafted manuscript; E.K.J. and D.G.G. edited and revised manuscript; E.K.J. and D.G.G. approved final version of manuscript; D.G.G. performed experiments.