PSB-1115

Abnormal growth of glomerular mesangial cells (GMCs) contributes to the pathophysiology of many types of nephropathy. Because adenosine is an autocrine/paracrine factor that potentially could regulate GMC proliferation and because the extracellular 3',5'-cAMP-adenosine pathway (i.e., the conversion of extracellular 3',5'-cAMP to 5'-AMP and adenosine on the cell surface) could generate adenosine in the biophase of GMC receptors, we investigated the role of the 3',5'-cAMP-adenosine pathway in modulating growth [cell proliferation, DNA synthesis ([(3)H]thymidine incorporation), collagen synthesis ([(3)H]proline incorporation), and mitogen-activated protein kinase activity] of GMCs. The addition of exogenous 3',5'-cAMP to human GMCs increased extracellular levels of 5'-AMP, adenosine, and inosine, and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), 1,3-dipropyl-8-p-sulfophenylxanthine (ecto-phosphodiesterase inhibitor), and alpha,beta-methylene-adenosine-5'-diphosphate (ecto-5'-nucleotidase inhibitor) attenuated the increases in adenosine and inosine. Forskolin augmented extracellular 3',5'-cAMP and adenosine concentrations, and 2',5'-dideoxyadenosine (adenylyl cyclase inhibitor) blocked these increases. Exogenous 3',5'-cAMP and forskolin inhibited all indices of cell growth, and antagonism of A(2) [(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine, KF17837] or A(1)/A(2) (1,3-dipropyl-8-p-sulfophenylxanthine, DPSPX), but not A(1) (8-cyclopentyl-1,3-dipropylxanthine), or A(3){N-(2-methoxyphenyl)-N'-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF5574}, adenosine receptors blocked the growth-inhibitory actions of exogenous 3',5'-cAMP, but not the effects of 8-bromo-3',5'-cAMP (stable 3',5'-cAMP analog). Erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor) plus 5-iodotubercidin (adenosine kinase inhibitor) enhanced the growth inhibition by exogenous 3',5'-cAMP and forskolin, and A(2) receptor antagonism blocked this effect. In rat GMCs, down-regulation of A(2B) receptors with antisense, but not sense or scrambled, oligonucleotides abrogated the inhibitory effects of 3',5'-cAMP and forskolin on cell growth. The extracellular 3',5'-cAMP-adenosine pathway exists in GMCs and attenuates cell growth via A(2B) receptors. Pharmacological augmentation of this pathway could abate pathological glomerular remodeling.

Although adenosine contributes importantly to the regulation of renin release, renal vascular resistance and renal tubular reabsorption, the metabolic pathways that control the intrarenal production rate of adenosine remain ill defined. The objective of this study was to determine whether extracellular metabolism of cyclic AMP to AMP by extracellular phosphodiesterase and hence to adenosine by ecto-5'-nucleotidase can occur in the intact kidney. To test this hypothesis, five experimental series were conducted in kidneys from male Sprague-Dawley rats perfused in a nonrecirculating system (5 ml/min) in vitro with oxygenated Tyrode's solution at 37 degrees C. In each experimental series, cyclic AMP was added to the Tyrode's solution and the renal secretion rates (i.e., the renal venous concentration of purine x the perfusion flow rate) of AMP, adenosine and inosine were determined using high-performance liquid chromatography. In the first experimental series, only cyclic AMP was added to the perfusate. In the second, third, fourth and fifth experimental series, kidneys were perfused with Tyrode's solution containing both cyclic AMP and either 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), alpha,beta-methyleneadenosine-5'-diphosphate (an ecto-5'-nucleotidase inhibitor), dilazep (an adenosine transport inhibitor) or 1,3-dipropyl-8-p-sulfophenylxanthine (a xanthine that is restricted to the extracellular compartment). In the first experimental series (n = 8), addition of cyclic AMP to the perfusate resulted in significant concentration-related increases in the renal secretion rates of AMP, adenosine and inosine, with the increase in AMP secretion being significantly greater than the increases in adenosine or inosine secretions (delta adenosine secretion/delta AMP secretion = 0.38 +/- 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)