Effect of angiotensin II on ammonia production and secretion by mouse proximal tubules perfused in vitro.

The effects of angiotensin II on total ammonia (tNH3) production and net secretion were investigated using in vitro microperfused mouse S2 proximal tubule segments incubated in Krebs-Ringer bicarbonate buffer containing 0.5 mM L-glutamine. Basolateral exposure of mouse S2 segments to 10(-11), 10(-10), and 10(-9) M angiotensin II stimulated tNH3 production rates by 23, 52, and 49%, respectively. Addition of 10(-6) M angiotensin II inhibited the tNH3 production rate by 34%. 10(-10) M angiotensin II inhibited net luminal secretion of tNH3 in the presence of enhanced luminal acidification and in the absence of altered luminal tNH3 efflux rates. Measurements of intracellular pH (pHi) and intracellular calcium concentration [( Ca2+]i) suggested that the effects of angiotensin II on tNH3 production were not mediated by changes in pHi but by the stimulatory effect of angiotensin II correlated with increased [Ca2+]i. Inhibition of the calcium-calmodulin-dependent pathway with W-7 blocked the stimulatory effect of 10(-10) M angiotensin II on tNH3 production and luminal acidification. These results indicate that angiotensin II has concentration-dependent effects on tNH3 production; that its action to stimulate tNH3 production may be mediated by rises in [Ca2+]i and the calcium-calmodulin pathway; and that angiotensin II, at concentrations that stimulate tNH3 production, inhibits net luminal ammonia secretion by a mechanism that is not mediated by diminished luminal acidification or by changes in luminal ammonia efflux rates.     

Effect of bath and luminal potassium concentration on ammonia production and secretion by mouse proximal tubules perfused in vitro.

To determine the effects of acute changes in K+ concentration in vitro on ammonia production and secretion by the proximal tubule, we studied mouse S2 segments perfused with and bathed in Krebs-Ringer bicarbonate buffers containing various K+ concentrations. All bath solutions contained L-glutamine as the ammoniagenic substrate. High bath and luminal K+ concentrations (8 mM), but not high luminal K+ concentration alone, inhibited total ammonia production rates by 26%, while low bath and luminal K+ concentrations (2 mM), but not low luminal K+ concentration alone, stimulated total ammonia production rates by 33%. The stimulation of ammonia production by low bath K+ concentration was not observed when L-glutamine was added to the luminal perfusion solution. On the other hand, high luminal K+ concentration stimulated, while low luminal K+ concentration inhibited, net luminal secretion of total ammonia in a way that was: (a) independent of total ammonia production rates, (b) independent of Na(+)-H+ exchange activity, and (c) not due to changes in transepithelial fluxes of total ammonia. These results suggest that luminal potassium concentration has a direct effect on cell-to-lumen transport of ammonia.     

Regulation of ammonia production by mouse proximal tubules perfused in vitro. Effect of luminal perfusion.

To investigate factors regulating ammonia (NH3) production by isolated defined proximal tubule segments, we examined the rates of total NH3 (NH3 + NH+4) production by individual proximal tubule segments perfused in vitro under a variety of perfusion conditions. Segments consisting of late convoluted and early straight portions of superficial proximal tubules were incubated at 37 degrees C in Krebs-Ringer bicarbonate (KRB) buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, pH 7.4. The rate of total ammonia production was calculated from the rate of accumulation of total NH3 in the bath. The total ammonia production rate by unperfused proximal segments was 6.0 +/- 0.2 (+/- SE) pmol/mm per minute, which was significantly lower than segments perfused at a flow rate of 22.7 +/- 3.4 nl/min with KRB buffer (21.5 +/- 1.4 pmol/mm per minute; P less than 0.001) or with KRB buffer containing 0.5 mM L-glutamine (31.9 +/- 2.5; P less than 0.001). The rate of NH3 production was higher in segments perfused with glutamine than in segments perfused without glutamine (P less than 0.01). The perfusion-associated stimulation of NH3 production was characterized further. Analysis of collected luminal fluid samples revealed that the luminal fluid total NH3 leaving the distal end of the perfused proximal segment accounted for 91% of the increment in NH3 production observed with perfusion. Increasing the perfusion flow rate from 3.7 +/- 0.1 to 22.7 +/- 3.4 nl/min by raising the perfusion pressure resulted in an increased rate of total NH3 production in the presence or absence of perfusate glutamine (mean rise in rate of total NH3 production was 14.9 +/- 3.7 pmol/mm per minute in segments perfused with glutamine and 7.8 +/- 0.9 in those perfused without glutamine). In addition, increasing the perfusion flow rate at a constant perfusion pressure increased the rate of luminal output of NH3. Total NH3 production was not affected by reducing perfusate sodium concentration to 25 mM and adding 1.0 mM amiloride to the perfusate, a condition that was shown to inhibit proximal tubule fluid reabsorption. These observations demonstrate that the rate of total NH3 production by the mouse proximal tubule is accelerated by perfusion of the lumen of the segment, by the presence of glutamine in the perfusate, and by increased perfusion flow rates. The increased rate of NH3 production with perfusion seems not to depend upon normal rates of sodium reabsorption. The mechanism underlying the stimulation of NH3 production by luminal flow is unknown and requires further study.     

Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule

The proximal convoluted tubule (PCT) reabsorbs most of the filtered bicarbonate. Proton secretion is believed to be mediated predominantly by an apical membrane Na(+)/H(+) exchanger (NHE). Several NHE isoforms have been cloned, but only NHE3 and NHE2 are known to be present on the apical membrane of the PCT. Here we examined apical membrane PCT sodium-dependent proton secretion of wild-type (NHE3(+/+)/NHE2(+/+)), NHE3(-/-), NHE2(-/-), and double-knockout NHE3(-/-)/NHE2(-/-) mice to determine their relative contribution to luminal proton secretion. NHE2(-/-) and wild-type mice had comparable rates of sodium-dependent proton secretion. Sodium-dependent proton secretion in NHE3(-/-) mice was approximately 50% that of wild-type mice. The residual sodium-dependent proton secretion was inhibited by 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Luminal sodium-dependent proton secretion was the same in NHE3(-/-)/NHE2(-/-) as in NHE3(-/-) mice. These data point to a previously unrecognized Na(+)-dependent EIPA-sensitive proton secretory mechanism in the proximal tubule that may play an important role in acid-base homeostasis.     

Transport of the organic cation N1-methylnicotinamide by the rabbit proximal tubule. II. Reabsorption and secretion in the isolated perfused tubule

The mechanisms involved in the transport of the organic cation N1-methylnicotinamide (NMN) were investigated in the isolated perfused rabbit S2 proximal tubule. NMN underwent a small reabsorptive transport which appeared to be passive. NMN secretory transport was saturable, inhibited by mepiperphenidol (10(-4) M) and presented a relatively low apparent affinity (apparent Km of 852 microM) for the organic cation transport system, with transport against a concentration gradient being observed only at low flow rates. Acidification of the perfusate, by either buffering it at pH 6.8 with MES, or by bubbling the bath with a mixture of 80% O2-20% CO2, resulted in a decrease, rather than an increase, of NMN secretion. Carbonic anhydrase inhibition with ethoxyzolamide (10(-4) M in the bath) did not modify NMN secretion. Addition of 20 to 40 microM NMN in the perfusate also did not change NMN secretion. Proton or organic cation counterexchange seemed therefore not to play a major role in NMN apical step of secretion, the basolateral step appearing to be the general organic cation system of transport for which NMN shows a low affinity.     

Indomethacin secretion in the isolated perfused proximal straight rabbit tubule. Evidence for two parallel transport mechanisms.

We studied indomethacin as a probe of anion transport across the isolated perfused proximal straight tubule of the rabbit and discovered that a substantial component of transport may occur by anion exchange at the basolateral membrane. Various perturbations involving direct or indirect dissipation of the cellular sodium gradient (ouabain, sodium- or potassium-free solutions, cooling to 18 degrees C) resulted in only a 50% inhibition of indomethacin transport, which raised the question of a co-existent alternative pathway for secretion. Similarly, the anion exchange inhibitor, 4,4'-diisothiocyanostilbene (DIDS), diminished indomethacin secretion by only 50%. Cooling followed by DIDS or the reverse sequence resulted in additive inhibition such that the combination abolished active secretion of indomethacin. We conclude that active secretion of indomethacin by the proximal straight tubule appears to be in part sodium gradient dependent; the remainder may be driven by an anion exchanger on the basolateral membrane.     

Urea secretion by the straight segment of the proximal tubule.

Studies utilizing in vitro microperfusion were designed to examine whether urea is actively or passively transported across superficial and juxtamedullary straight segments of rabbit proximal tubules. With perfusate and bath solutions containing 1 mM urea and electrolytes similar to normal plasma, the efflux (lumen-to-bath) isotopic permeability (X 10(-5) cm s-1) of superficial segments was 1.37 +/- 0.16 and of juxtamedullary segments was 2.14 +/- 0.20. In the same tubules, the influx (bath-to-lumen) isotopic permeability was 3.70 +/- 0.35 in superficial segments and 4.75 +/- 0.37 in juxtamedullary segments. Despite net water movement in the opposite direction (0.5 nl mm-1 min-1), the influx rate was significantly higher than the efflux rate of urea in both groups. With a low perfusion rate (2 nl/min) and equivalent specific activities of [14C]urea in bath and perfusate, the collected-to-perfused ratio of [14C]urea, corrected for volume marker change, was 1.07 +/- 0.01 in superficial and 1.09 +/- 0.01 in juxtamedullary nephrons, thus indicating net secretion in both segments. In separate studies urea influx was inhibited by hypothermia (decrease from 37 degrees to 28 degrees C), by phloretin (0.1 mM in bath), by cyanide (1 mM), but not by probenecid (0.2 mM). In each case the inhibition was highly significant and reversible. These data suggest that urea is actively secreted by the straight segments of both the superficial and juxtamedullary proximal tubules. These segments may, therefore, contribute significantly to the high urea concentration found at the bend of Henle's loop by micropuncture.     

Inhibitory effects of volume expansion performed in vivo on transport in the isolated rabbit proximal tubule perfused in vitro.

To examine the renal tubular sites and mechanisms involved in the effects of hypooncotic volume expansion (VE) on renal electrolyte excretion, we performed clearance and isolated tubular perfusion studies using intact and thyroparathyroidectomized (TPTX) rabbits. We also examined the effect of VE on luminal brush border transport. In the microperfusion studies, proximal convoluted (PCT) and straight (PST) tubules were taken from rabbits without prior VE or after 30 min of 6% (body wt) VE. Acute VE increased the percentage excretion of Na, Ca, and P in TPTX animals and the percentage and absolute excretions of these ions in intact rabbits. In PST from VE animals, fluid flux (Jv) was depressed compared with Jv in PST from nonVE rabbits: Jv = 0.18 +/- 0.03, (VE) vs. 0.31 +/- 0.03 nl/mm.min, (nonVE) P less than 0.02. Phosphate transport (Jp) in the PST from VE animals was also depressed: JP = 1.58 +/- 0.10 (VE) vs. 2.62 +/- 0.47 pmol/mm.min, (nonVE) P less than 0.05. Similar results were obtained with TPTX animals. In the PCT from VE animals, Jv was decreased (0.49 +/- 0.10 (VE) vs. 0.97 +/- 0.14 nl/mm.min, (nonVE) P less than 0.02), but JP was not affected significantly. Transport inhibition was stable over approximately 90 min of perfusion. In the brush border vesicle studies, sodium-dependent phosphate transport was inhibited compared with that in control animals, at the 9-, 30-, and 60-s time points. These findings indicate that the inhibition of renal ionic transport by VE occurs in both PCT and PST and is, in part, the result of a direct effect of VE on tubular transport mechanisms.     

Ammonia production by isolated mouse proximal tubules perfused in vitro. Effect of metabolic acidosis.

We examined the effects of metabolic acidosis in vivo and reduced bath and luminal pH in vitro on total NH3 (NH3 + NH+4) production rates by isolated mouse proximal tubule segments. Midproximal tubule segments were obtained from mice with NH4Cl-induced metabolic acidosis and from nonacidotic controls. The segments were perfused with modified Krebs-Ringer bicarbonate (KRB) buffer, incubated in KRB buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, and gassed with 95% O2 and 5% CO2. Isolated unperfused and perfused proximal tubules from acidotic mice produced total NH3 at higher rates than corresponding tubules from nonacidotic mice. Perfusion of the tubular lumen stimulated total NH3 production by tubules from both acidotic and nonacidotic mice. In contrast, lowering the bath pH to 7.0 by lowering the HCO3- concentration increased total NH3 production rates by tubules from nonacidotic mice but not by tubules from acidotic mice. Reducing the HCO3- concentration of the bath buffer to 10 mM while maintaining a pH of 7.4 had no significant effect on total NH3 production by tubules from nonacidotic mice. Lowering the luminal fluid pH by reducing the perfusate HCO-3 from 25 mM to 10, 5, or 1.2 mM while maintaining a bath pH of 7.4 lowered collected luminal fluid pH but had no effect on total NH3 production by proximal tubules from nonacidotic mice. These observations demonstrated that metabolic acidosis in vivo stimulated total NH3 production in isolated mouse proximal tubule segments and that low peritubular pH and HCO-3 stimulated total NH3 production by proximal tubule segments from nonacidotic mice in vitro.     

Inducible Cre/loxP recombination in the mouse proximal tubule

Transgenic technologies in mice became invaluable experimental tools to identify the in vivo function of proteins. However, conventional knockout technology often results in embryonic lethality and because genes are frequently expressed in multiple cell types, the resulting knockout phenotypes can be complex and difficult or impossible to dissect. These issues are particularly important for gene-targeting strategies used to examine renal function. The kidney contains quite a number of different cell types, the function of many of which impacts that of other renal cells. To avoid these limitations conditional knockout strategies have been designed. As one important part of this system we describe the development of a mouse line expressing the tamoxifen-activatable Cre recombinase Cre-ER(T2) specifically in renal proximal tubules. The expression of Cre-ER(T2) is driven by a promoter fragment of the mouse gamma-glutamyl transpeptidase type II gene resulting in the generation of the activatable recombinase in S3 segments of the proximal tubules from which over 80% were positive for Cre activity. In combination with loxP-based conditional mutant mice as a second tool this tamoxifen-inducible Cre-ER(T2) line allows functional analysis of a variety of genes important for renal development and function in a precisely controlled spatiotemporal manner.     

Transport of [3H]losartan across isolated perfused rabbit proximal tubule.

The transport of the angiotensin II receptor antagonist losartan and its interaction with organic anion transport were examined in the isolated perfused rabbit proximal tubule. Losartan reversibly inhibited the secretion of para-aminohippurate (PAH) in a concentration-dependent manner (IC50 = 15 +/- 0.5 microM). Other angiotensin II receptor antagonists also inhibited PAH secretion with similar potencies: eprosartan, 11 +/- 2.3 microM; irbesartan, 17 +/- 2.2 microM; and valsartan 3 +/- 0.6 microM. [3H]Losartan was secreted by the proximal tubule by a saturable and probenecid-sensitive mechanism. The affinity of losartan for the organic anion transporter (Km = 12.3 +/-1.8 microM) was significantly greater than that of PAH (Km = 88.5 +/- 10.7 microM). [3H]Losartan secretion was stimulated in the presence of alpha-ketoglutarate, suggesting that losartan, like PAH, enters the cell in exchange for a dicarboxylate. These results demonstrate that losartan and probably other nonpeptide angiotensin II receptor antagonists are secreted by an organic anion transporter that is similar to, if not identical with, the classic PAH transporter.     

Transport of methotrexate by the in vitro isolated rabbit proximal tubule

The tubular transport of [3H]methotrexate was studied in isolated nonperfused and perfused superficial proximal tubular segments of rabbit kidneys. Reabsorption represented only 5% of perfused methotrexate, and appeared to be mostly of passive nature inasmuch as it was not modified by reducing the temperature or by ouabain. Cellular accumulation in nonperfused segments and secretion in perfused tubules were highest in the S2 segment and lower in the S3 and S1 segments. Secretion against a bath-to-lumen concentration gradient was observed only in S2 segments (with a maximum methotrexate secretory rate of 478 +/- 48 fmol/mm.min and an apparent Km of transport of 363 +/- 32 microM), and was inhibited by probenecid and folate. The low capacity for methotrexate secretion may be explained by a low capacity of transport across the basolateral membrane of the proximal cell as methotrexate was accumulated only to a low extent in nonperfused tubules (tissue water to medium concentration ratio of 8.2 +/- 1 in S2 segments). During secretion a small amount of methotrexate was metabolized; the nature of the metabolite(s) remains to be defined.     

Acute effect of cadmium-metallothionein on glucose and amino acid transport across the apical membrane of the rabbit proximal tubule perfused in vitro

Acute as well as chronic exposure of cadmium (Cd) leads to proximal tubule injury. The exact cellular mechanism of this disorder and whether there is a contribution of cadmium-metallothionein (Cd-MT), a binding protein of Cd, remain unclear. We perfused isolated S2 segments of rabbit nephron, and the deflections of transmural voltage (DeltaV(t)) and apical membrane voltage (DeltaV(a)) on elimination of glucose or alanine from the perfusate were measured for the parameters of activity of Na(+)-glucose and Na(+)-amino acid cotransporters. The effects of Cd-MT or CdCl(2) to either bath or lumen for 10 min on these parameters were examined. We also measured the lumen-to-bath [(14)C]glucose flux. Addition of Cd-MT to lumen suppressed glucose- or alanine-dependent DeltaV(t) and DeltaV(a), as well as baseline V(t) and basolateral membrane voltage (V(b)), at approximately 10 min. [(14)C]glucose flux was inhibited by Cd-MT to lumen. The effects of Cd-MT to bath and CdCl(2) to either lumen or bath were 100-fold less potent than that of Cd-MT to lumen. Luminal Cd-MT immediately suppressed the glucose-dependent DeltaV(a), whereas the baseline V(a) and V(t) were unchanged. The early effect of luminal Cd-MT was simulated by addition of 10(-4) M phloretin. Addition of 10(-4) M ouabain to the bath simulated the later effect of Cd-MT. The protection of SH group by dithiothreitol prevented the early effect of Cd-MT, but not the later effect. We concluded that Cd-MT initially acts directly on Na(+)-glucose and Na(+)-amino acid cotransporters from the lumen by attacking SH group, followed by the later inhibition of Na(+)-K(+)-ATPase after entering the cell from the apical membrane.     

Effect of S-8666 on Cl- transport in the rabbit connecting tubule perfused in vitro.

S-8666, [6, 7-dichloro-5-(N, N-dimethylsulfamoyl)-2, 3-dihydrobenzofurancarboxylic acid] is a potent diuretic with uricosuric action. Although the major site of action of S-8666 has been proven by the in vitro microperfusion study to be the thick ascending limb of Henle's loop, clearance studies in the rat suggested that this drug has an additional thiazide-like action. To provide direct evidence that S-8666 acts also on distal nephron segments, we examined effect of S-8666 on Cl- flux across the rabbit connecting tubule perfused in vitro. The drug suppressed the lumen-to-bath Cl- flux by 96 +/- 41 (S.E.)pmol.mm-1.min-1 (n = 9) without affecting transmural voltage. To demonstrate that S-8666 acts on the connecting tubule cell, the target of thiazide diuretics, we compared effects of S-8666 and trichlormethiazide on the basolateral membrane voltage of the connecting tubule cell. Both drugs added to the lumen caused a small but significant hyperpolarization of the basolateral membrane without affecting transmural voltage. We conclude that S-8666 is a unique uricosuric diuretic having actions on both thick ascending limb of Henle's loop and connecting tubule.     

Effect of steviol on para-aminohippurate transport by isolated perfused rabbit renal proximal tubule

An inhibitory effect of steviol, metabolite of the natural sweetener stevioside, on transepithelial transport of p-aminohippurate (J(PAH)) was observed in isolated S(2) segments of rabbit renal proximal tubules using in vitro microperfusion. Addition of steviol (0.01--0.25 mM) to the bathing medium significantly depressed J(PAH) (approximately 50--90%). This inhibitory effect was dose-dependent and was maximum at a concentration of 0.05 mM. To further examine this effect, a steviol concentration (0.01 mM) that produced approximately 50% inhibition of J(PAH), was chosen. Addition of 0.01 mM steviol to the bathing medium significantly depressed J(PAH) by about 50 to 60%. Steviol at the same concentration (0.01 mM), when present in the tubule lumen, had no significant effect on J(PAH). Addition of 0.01 mM steviol to lumen and bath simultaneously, produced a slightly greater inhibitory effect compared with addition to bath alone (60 versus 70%). A higher concentration of steviol, 0.05 mM (which maximally inhibited J(PAH) when on the basolateral side), was required on the luminal side than on the basolateral side before an inhibitory effect was observed. To further examine the mechanism by which steviol inhibited J(PAH), its effect on Na(+)-K(+) ATPase activity and ATP content was determined. Steviol at concentrations of 0.01 and 0.05 mM had no effect on Na(+)-K(+) ATPase activity or cell ATP content. Kinetic analyses indicated that steviol can competitively inhibit PAH transport at the basolateral membrane. The present study clearly showed that steviol can have a direct inhibitory effect on renal tubular transport by competitive binding with organic anion transporter.     

Flow dependence of transtubular potential difference in isolated perfused segments of rabbit proximal convoluted tubule

Transmembrane potential difference (pd) was studied in isolated perfused segments of rabbit proximal convoluted tubules. At perfusion flow rates above 10 nl/min the pd was -5.80 +/-0.3 mv (lumen negative) when perfusing with isosmolal ultrafiltrate of same rabbit serum as the bath. That this pd is generated by transport activity of the tubule is supported by three separate observations: (a) pd reversibly decreased with cooling from 37 degrees C to 25 degrees C; (b) pd decreased when 10(-5) M ouabain was added to the bath and reversed to control levels when ouabain was removed; and (c) heating to 47 degrees C irreversibly decreased pd to zero. The magnitude of the pd was related to perfusion flow rate at slower rates than 10 nl/min. A decrease in flow rate was associated with a decrease in pd. The tubular geometry and transmembrane hydrostatic pressure were ruled out as the mediating factors governing the magnitude of observed pd.     

Transport of ammonia in the rabbit cortical collecting tubule.

Nonionic diffusion and diffusion equilibrium of ammonia have been generally accepted as the mechanism of urinary ammonium excretion. However, these characteristics have not been examined directly in vitro. In the present studies, nonionic diffusion and diffusion equilibrium of ammonia were examined in rabbit cortical collecting tubules perfused in vitro. Collected fluid ammonium and pH were measured in tubules exposed to chemical gradients of NH3/NH+4. In tubules perfused with an acid perfusate free of ammonia and bathed with solutions containing NH4Cl, collected fluid ammonia failed to equilibrate across the epithelium except at slow flow rates. The estimated apparent permeability coefficient to NH3 was approximately 5 X 10(-3) cm/s. Predominant nonionic diffusion of NH3, rather than transport of NH+4, was indicated by alkalinization of luminal fluid in tubules exposed to peritubular NH4Cl and by the relative influence of peritubular NH+4 and NH3 on ammonia entry. In tubules perfused with an acid solution containing NH4Cl, little loss of ammonium was detectable, indicating a low permeability to NH+4. In contrast to the restricted diffusion of NH3 in cortical collecting tubules, proximal convoluted tubules exhibited a much higher apparent permeability to NH3. In conclusion, nonionic diffusion of NH3 accounted for most ammonium transport in the proximal convoluted tubule and in the cortical collecting tubule. However, there was relatively restricted diffusion in the collecting tubules; this may account for the failure of whole kidney ammonium excretion to obey quantitatively the predictions of nonionic diffusion and diffusion equilibrium of ammonia.     

cAMP-adenosine pathway in the proximal tubule

The "extracellular cAMP-adenosine pathway" refers to the conversion of cAMP to AMP by ecto-phosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all the steps occurring in the extracellular compartment. This study investigated whether the extracellular cAMP-adenosine pathway exists in proximal tubules. Freshly isolated proximal tubules rapidly converted basolaterally administered cAMP to AMP and adenosine. Proximal tubular cells in culture (first passage) rapidly converted apically administered cAMP to AMP and adenosine. In both freshly isolated proximal tubules and cultured proximal tubular cells, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methyleneadenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In cultured proximal tubular cells, but not freshly isolated proximal tubules, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine plus inosine, and these changes were also modulated by the inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. Conversion of renal interstitial (basolateral) cAMP and AMP to adenosine in vivo was shown by microdialysis coupled with ion trap mass spectrometry. Western blot analysis showed A1, A2A, and A3 receptors on both apical and basolateral proximal tubular membranes, with A1 and A2A receptors more highly expressed on basolateral compared with apical membranes. We conclude that cAMP that reaches either the apical or basolateral membranes of proximal tubular cells is converted in part to adenosine that has ready access to adenosine receptors.