Role of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the renal 2',3'-cAMP-adenosine pathway

Abstract

Energy depletion increases the renal production of 2',3'-cAMP (a positional isomer of 3',5'-cAMP that opens mitochondrial permeability transition pores) and 2',3'-cAMP is converted to 2'-AMP and 3'-AMP, which in turn are metabolized to adenosine. Because the enzymes involved in this "2',3'-cAMP-adenosine pathway" are unknown, we examined whether 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) participates in the renal metabolism of 2',3'-cAMP. Western blotting and real-time PCR demonstrated expression of CNPase in rat glomerular mesangial, preglomerular vascular smooth muscle and endothelial, proximal tubular, thick ascending limb and collecting duct cells. Real-time PCR established the expression of CNPase in human glomerular mesangial, proximal tubular and vascular smooth muscle cells; and the level of expression of CNPase was greater than that for phosphodiesterase 4 (major enzyme for the metabolism of 3',5'-cAMP). Overexpression of CNPase in rat preglomerular vascular smooth muscle cells increased the metabolism of exogenous 2',3'-cAMP to 2'-AMP. Infusions of 2',3'-cAMP into isolated CNPase wild-type (+/+) kidneys increased renal venous 2'-AMP, and this response was diminished by 63% in CNPase knockout (-/-) kidneys, whereas the conversion of 3',5'-cAMP to 5'-AMP was similar in CNPase +/+ vs. -/- kidneys. In CNPase +/+ kidneys, energy depletion (metabolic poisons) increased kidney tissue levels of adenosine and its metabolites (inosine, hypoxanthine, xanthine, and uric acid) without accumulation of 2',3'-cAMP. In contrast, in CNPase -/- kidneys, energy depletion increased kidney tissue levels of 2',3'-cAMP and abolished the increase in adenosine and its metabolites. In conclusion, kidneys express CNPase, and renal CNPase mediates in part the renal 2',3'-cAMP-adenosine pathway.

Keywords: 2′,3′-cyclic adenosine monophosphate; 2′,3′-cyclic nucleotide 3′-phosphodiesterase; 2′-adenosine monophosphate; 3′-adenosine monophosphate; CNPase; adenosine.

Fig. 1.

Western blots and threshold cycle (Ct) from real-time PCR (n = 8; means ± SE) showing expression of CNPase in whole kidney, glomerular mesangial cells (GMCs), and preglomerular vascular smooth muscle cells (PGVSMCs). CNPase exists as 2 isoforms, CNPase 1 (46 kDa) and CNPase 2 (48 kDa). MW markers, molecular weight markers.

Fig. 2.

Western blots and Ct from real-time PCR (n = 2; means ± SE) showing expression of CNPase in thick ascending limb cells (TALCs), proximal tubular cells (PTCs), collecting duct cells (CDCs), and preglomerular vascular endothelial cells (PGVECs). CNPase exists as 2 isoforms, CNPase 1 (46 kDa) and CNPase 2 (48 kDa).

Fig. 3.

Bar graphs compare the expression of phosphodiesterease type 4 (PDE4) mRNA and CNPase mRNA (real-time PCR) using the 2^−ΔΔCt method as described by the method of Livak and Schmittgen (35). Results are for human GMCs (A), renal PTCs (B), aortic VSMCs (C), and coronary artery VSMCs (D). Values represent means ± SE.

Fig. 4.

A: Western blot shows the increased expression of CNPase in PGVSMCs induced by transfection with either 2.0 or 1.5 μg of a CNPase 1-expressing plasmid. B: PGVSMCs transfected with 2.0 μg of a CNPase 1-expressing plasmid and incubated for 1 h with 2′,3′-cAMP (10 μmol/l) produced more 2′-AMP than PGVSMCs transfected with a vector control plasmid, yet neither vector control nor CNPase 1-transfected cells converted 3′,5′-cAMP to 2′-AMP. C: PGVSMCs transfected with 2.0 μg of a CNPase 1-expressing plasmid and incubated for 1 h with 2′,3′-cAMP (10 μmol/l) produced less 3′-AMP than PGVSMCs transfected with a vector control plasmid, yet neither vector control nor CNPase 1-transfected cells converted 3′,5′-cAMP to 3′-AMP. Values represent means ± SE.

Fig. 5.

Kidney and brain tissue were harvested from CNPase +/+ and −/− mice and subjected to an immunoprecipitation (IP) procedure in which an Abcam anti-CNPase antibody was employed to IP CNPase and a Millipore anti-CNPase antibody was used to detect CNPase by Western blotting. The film was exposed for either 2 min or 10 s. A single band at ∼46 kDa was strongly detected in both kidneys and brains from CNPase +/+ mice, and this band was nearly absent in kidneys and brains from CNPase −/− mice.

Fig. 6.

Bar graphs illustrate the effects of 3′,5′-cAMP (30 μmol/l) in the arterial perfusate on renal venous perfusate levels of 3′,5′-cAMP (A), 5′-AMP (B), adenosine (C), and inosine (D) in isolated, perfused kidneys from CNPase +/+ and −/− mice. Values represent means ± SE.

Fig. 7.

Bar graphs illustrate the effects of 2′,3′-cAMP (30 μmol/l) in the arterial perfusate on renal venous perfusate levels of 2′,3′-cAMP (A), 2′-AMP (B), 3′-AMP (C), adenosine (D), inosine (E), hypoxanthine (F), xanthine (G), and uric acid (H) in isolated, perfused kidneys from CNPase +/+ and −/− mice. Values represent means ± SE.

Fig. 8.

Bar graphs illustrate the effects of metabolic toxins (iodoacetate plus 2,4-dinitrophenol, each at 50 μmol/l) in the arterial perfusate on renal venous levels of 2′,3′-cAMP (A), 3′,5′-cAMP (B), adenosine (C), inosine (D), hypoxanthine (E), xanthine (F), uric acid (G), and 5′-AMP (H) in isolated, perfused kidneys from CNPase +/+ and −/− mice. Values represent means ± SE. ^aSignificantly different (P < 0.05) compared with same genotype without metabolic toxins. ^b−/− Is significantly different from +/+ in the presence of metabolic toxins.

Fig. 9.

Bar graphs illustrate the effects of metabolic toxins (iodoacetate plus 2,4-dinitrophenol, each at 50 μmol/l) in the arterial perfusate on kidney tissue levels of 2′,3′-cAMP (A), 3′,5′-cAMP (B), adenosine (C), inosine (D), hypoxanthine (E), xanthine (F), uric acid (G), and 5′-AMP (H) in isolated, perfused kidneys from CNPase +/+ and −/− mice. Values represent means ± SE. ^aSignificantly different (P < 0.05) compared with the other 3 groups.

Fig. 10.

Chromatograms from high-performance liquid chromatography-tandem mass spectrometry monitoring the mass transition from 330 to 136 m/z in extracts of CNPase +/+ (A) and CNPase −/− (B) kidneys treated with metabolic toxins (iodoacetate plus 2,4-dinitrophenol, each at 50 μmol/l).