Absence of direct effects of prostaglandins on sodium chloride transport in the mammalian nephron

Transcription

1 Kidney International, Vol. 19 (1981), pp Absence of direct effects of prostaglandins on sodium chloride transport in the mammalian nephron LEON G. FINE and MICHAEL A. KIRSCHENBAUM Division of Nephroiogy, Department of Medicine, University of California, Los Angeles, Los Angeles, California A multitude of factors appear to regulate the transport of sodium chloride in the mammalian nephron. Considerable effort has been spent over the past 15 years in attempting to characterize the mechanisms of action of the various humoral, neurogenic, and "physical" factors that interact to regulate renal salt excretion. Included in this list are the renal prostaglandins. The observation that prostaglandins are natriuretic under certain experimental circumstances [11 has stimulated investigation directed at elucidating whether the renal prostaglandins have a direct effect on membrane transport of sodium chloride. The attractiveness of demonstrating such an effect lies in the fact that the existence of a locally acting hormone produced by the kidney itself could explain many of the enigmas related to the regulation of salt excretion by invoking feedback regulation linked to segmental delivery and blood flow distribution. With the wisdom of hindsight, it has become evident that much research in this area has been misdirected. Inappropriate prostaglandins, often used in pharmacologic doses, delivered to areas of renal parenchyma unaccustomed to their presence, together with the confusing effects of hemodynamic alterations in conscious versus anesthetized animals, have produced an array of conflicting data that are open to alternative interpretations [21. The solution to this confusion is obviously to design an "ideal experiment." The question we would like to answer, stated in its simplest terms, is as follows: Do prostaglandins directly stimulate or inhibit transport of sodium chloride in one or more segments of the nephron? Designing the experiment To approach this question, we must exclude from consideration the other important humoral and physical factors that alter sodium chloride transport. Studies on substances that directly alter membrane transport of sodium chloride such as diuretics [3], vasopressin [41, or the uremic natriuretic factor [5] would suggest that an in vitro preparation of perfused or nonperfused renal tubules is an ideal model for this purpose. Such systems allow for precise control of the peritubular and luminal environment and permit "control" and "experimental" measurements of fluxes to be made in the same tubule. Next, it is important to establish whether or not the tubule manufactures its own prostaglandins (P's). If it does not, it is conceivable that its transport could be regulated by PG's produced by extratubular cells, such as those that exist in the renomedullary interstitium [61. If the tubules do manufacture their own PG's, it would be necessary to determine whether alteration of endogenous synthesis by the tubule results in an altered rate of sodium chloride transport. If exogenous PG's are used as probes, they must show effects at concentrations that approximate those which exist in vivo (1O to io-' M) [7] in order to be able to relate their effects to combination with specific binding sites or receptors. At higher concentrations, their effects as lipid solute acids may relate more to their lipid solubility and/or their acidity and may be entirely nonspecific. Finally, any demonstrable effects should be completely reversible in order to exclude from consideration possible "toxic" effects of these substances. It is evident, therefore, that the following issues must be addressed: (1) identification of nephron Received for publication December 11, /81/ $ by the International Society of Nephrology 797

2 798 Fine and Kirschenbaum segment(s) that produce(s) PG's, (2) evaluation of the effect of inhibition or stimulation of PG synthesis in such a nephron segment on net sodium chloride transport, (3) evaluation of the effects of exogenous PG's at low concentrations on sodium chloride transport and the demonstration that such an effect is reversible, (4) demonstration that PG's selectively affect a specific transport process, that is, active sodium transport or active chloride transport and thus act on or in a specific segment of the nephron. Effects of exogenous prostaglandins on the collecting tubule There are two studies in the literature that demonstrate an inhibitory effect of PGE2 on sodium transport across the isolated perfused rabbit cortical collecting tubule (CCT). In the first, Stokes and Kokko [8] evaluated the effect of PGE2 on collecting tubules obtained from DOCA-treated rabbits in which baseline sodium chloride transport was greatly increased. These workers found that POE2 at a concentration of l M inhibited the negative potential of the CCT by >26% and reversibly inhibited net sodium transport out of the lumen by inhibiting efflux. A similar study was performed by lino and Imai [9] who examined the effects of PGE2, PGE1, and PGF2a at concentrations of i M or lower on CCT's and outer medullary collecting tubules from both DOCA-treated and non-doca-treated rabbits. None of the PG's had an effect when introduced into the lumen, but when introduced into the bath, they inhibited net sodium reabsorption in both segments and reversibly decreased luminal negativity Ȯur experience [11 contrasts with that described above. The effects of both low (1' and io M) and high (2.5 x l M) bath concentrations of PGE2, PGF2a, and PGA2 were examined in isolated perfused rabbit medullary collecting tubules studied in artificial solutions. The medullary segments were selected because they appear to be the most likely targets for the PG's that are found in highest concentrations in the renal medulla. The results are shown in Fig. 1, control values being computed as the mean of the pre- and post-experimental periods. No effects of PG's on potential difference (PD) or net sodium flux at either low or high concentrations were observed. The possibility was considered that differences between our results and those described above could be due to differences in basal transport rates 3 2 E 1 I)]'I'l C PGE2 C PGF2n C PGE2' C PGE2 PG 2n PG F2 PGA2 PGA2 Fig. 1. Effects of high- and low-dose prostaglandins on transepithelial potential difference (PD) and net sodium flux (.JN) of isolated perfused rabbit medullary collecting tubules. C denotes control. 1 Bath concentration PGE2 (N= 5) 2.5 X io M FH Bath concentration: 11M 17M I Q Coot. Exp. Recov. Coot. Exp. Recov. Fig. 2. Effect of io- M PGE2 and its vehicle alone in the bath on transepithelial potential difference (PD) of isolated perfused cortical collecting tubules obtained from DOCA -treated rabbits. induced by DOCA [8]. We consequently evaluated the effects of PGE (1- M) on the PD of CCT's obtained from rabbits maintained on DOCA (5 mg/day) for 5 to 7 days prior to the study [11]. The study was performed in a blind fashion. The results are shown in Fig. 2. No effect of POE2 Ofl PD could be demonstrated. We have recently repeated these studies in DOCA-treated and potassium-loaded rabbits using the identical bath and perfusate described by Stokes and Kokko [8] (except that 5% rabbit serum rather than 5% fetal calf serum was used), and, once again, were unable to demonstrate an inhibitory effect of POE2 (l M) on transepithelial PD. Of interest is the fact that if the 5% rabbit serum I Vehicle IN=5)

3 NaC1 transport in mammalian nephron 799 is omitted from the bath, this same concentration of PGE2 has an inhibitory effect on PD that is irreversible. No recovery of PD was observed in any of the tubules studied when the PGE2 was removed from the bath. If PG's do indeed inhibit transport of sodium out of the lumen of cortical and medullary collecting tubules and act from the peritubular surface, they would be expected to reduce the extrusion of sodium from the cell by inhibiting the active transport step at the peritubular surface in a manner analogous to ouabain. The intracellular sodium concentration should rise under these circumstances. Dunn and Howe examined the effects of PGE, PGA, and PGF2a (1O to 1_b M) on net fluxes of sodium between the intracellular and extracellular compartment of a suspension of rabbit renal cortical and medullary tubules 1112]. No effects of PG's could be demonstrated, and the authors concluded that the natriuretic effects of PG's could not be ascribed to direct inhibition of sodium transport at the contraluminal pump site. It is also possible, however, that PG's could inhibit net transport of sodium by inhibiting intracellular entry of sodium at the luminal membrane. The studies of Dunn and Howe [121 were not designed to evaluate this possibility. Despite the fact that all of the studies cited above meet many of the criteria that we feel are required for adequate interpretation of the data (that is, lowdose PG's and reversibility), the results from different laboratories are conflicting and cannot be ascribed to obvious differences in methodology. Modulation of endogenous prostaglandin synthesis by the collecting tubule If PG's are purported to inhibit collecting tubule sodium transport, it is important to know whether the collecting tubule itself synthesizes PG's and whether modulation of such synthesis can alter net sodium transport. There is evidence in the literature based on indirect methods such as cyclooxygenase antigenicity and cyclooxygenase-positive fluorescence [13] and histochemical localization of PG synthetase [14] that PG's are, in fact, synthesized by the collecting tubule. We have recently evaluated this more directly by incubating cortical and medullary collecting tubules with 3H-arachidonic acid and identifying the PG's synthesized using thin-layer chromatography (Kirschenbaum, Lowe, Trizna, Fine, unpublished observations). We have found that the rabbit collecting tubule synthesizes PGE2, PGF2a, and PG!2 and thromboxane B2 in vitro and that approximately 5% of this synthesis E 2 - C Indomethacin C Meciofenamate C RO (4mg/mi) (1mg/mi) (5mg/mi) Fig. 3. Effects of inhibitors of prostaglandin synthesis added to the bath for 1 hour on transepithelial potential difference (PD) and net sodium flux (JN) ofisolated perfused rabbit medullary collecting tubules. can be inhibited by indomethacin and meclofenamate. If PG's are indeed modulators of sodium chloride transport in the collecting tubule, inhibition of their synthesis should also alter the rate of transport. We have approached this question in two ways. First, we administered indomethacin to rabbits 2 hours prior to studying the collecting tubules in vitro [11. This was done in an attempt to lower endogenous levels and thus to sensitize the tubules to exogenous PG's in vitro. No effect of exogenous PG's was seen. Second, we added different inhibitors of PG synthesis directly to the bath and measured sodium chloride transport after 1 hour of exposure to these agents. Within this period of time, endogenous synthesis is inhibited by 5%. As depicted in Fig. 3, no effect was observed with three different inhibitors of PG synthesis [1]. From all the above, we believe that PG's do not directly modulate sodium transport by the collecting tubule. Effects of exogenous and endogenous prostaglandins on the thick ascending limb of Henle's loop We performed studies similar to those described for the collecting tubule on the medullary thick ascending limb of Henle's loop [1]. The segments were bathed in normal rabbit serum and perfused

4 8 Fine and Kirschenbaum 12 Bath concentration Bath concentration: 2.5 X io M 11 M 1 M 6 4 B 'F1rII C PGE2 C PGF2 C PGE C PGE2 PGF2 PGA2 PGF2 PGA Fig. 4. Effects of high- and low-dose prostaglandins on transepithelial potential difference (PD) and net sodium flux (JN,) of isolated perfused rabbit medullary thick ascending limbs, with an isosmolal ultrafiltrate of the serum. As depicted in Fig. 4, no effects of either high- or lowdose PG's on PD or net sodium transport were observed. Similarly, inhibitors of PG synthesis added to the bath were without effect (Fig. 5). Subsequent studies by Stokes [15] revealed an inhibitory effect of PGE2 on the medullary thick ascending limb, if the segments were perfused and bathed in hypertonic or isotonic artificial solutions. There was no such effect if serum was used as the bath. No effect was observed in the cortical thick ascending limb regardless of the solutions used. Of note is the fact that where PD and net chloride flux were inhibited by PGE2, there was no recovery of function when it was removed. These results must be interpreted with caution for a number of reasons. First, the lack of effect in serum in the face of an inhibitory effect that was reversible in artificial solutions only at low concentrations (lo M) strongly suggests that the effect was nonphysiologic. Second is the surprising fact that the same worker has demonstrated inhibition of net sodium transport in the collecting tubule (presumably inhibition of active sodium transport) and inhibition of chloride transport in the ascending limb, a segment in which active chloride transport is probably the predominant mechanism of net sodium chloride transport. This lack of specificity raises the possibility that the observed changes may not be, 2 8 IiIØi C Indomethacin C Meclofenamate C RO (4 mg/mi) (1 mg/mi) (5 mg/mi) Fig. S. Effects of inhibitors of prostaglandin synthesis added to the bath for 1 hour on transepithelial potential difference (PD) and net sodium flux (JN,) of isolated perfused rabbit medullary thick ascending limbs. mediated by a specific hormone-receptor interaction and that, even at the low concentrations used in these studies, prostaglandins may have nonspecific effects on cell membranes. Conclusions Based largely on our own experimental data, our conclusion is that sodium chloride transport in the mammalian nephron is neither inhibited nor stimulated by the renal PG's. Discrepancies that have arisen from work in different laboratories need to be resolved by additional studies, preferably performed in a double-blind fashion. Acknowledgments The work cited was supported by grants #AM 2698 and #HL from the National Institutes of Health. Dr. L. Fine is the recipient of a Research Career Development Award from the National Institutes of Health. Dr. M. Kirschenbaum is an Established Investigator of the American Heart Association. Reprint requests to Dr. L. G. Fine, Division of Nephrology, Department of Medicine, University of California at Los Angeles, Los Angeles, California 924, USA References 1. KIRSCHENBAUM MA, SERROS E: Are prostaglandins natriuretic? Mineral Electrolyte Metab 3: , DUNN MJ: Renal prostaglandins: Influences on excretion of sodium and water, the renin-angiotensin system, renal blood flow and hypertension, in Contemporary Issues in Nephrology: Hormonal Function and the Kidney, edited by BRENNER

5 NaC1 transport in mammalian nephron 81 BM, STEIN J, New York, Churchill Livingstone, 1979, pp BURG M, STONER L, CARDINAL J, GREEN N: Furosemide effect on isolated perfused tubules. Am J Physiol 225: , FRINDT G, BURG MB: Effect of vasopressin on sodium transport in renal cortical collecting tubules. Kidney mt 1: , FINE LG, BOURGOIGNIE JJ, HWANG KH, BRICKER NS: On the influence of the natriuretic factor from patients with chronic uremia on the bioelectric properties and sodium transport of the isolated mammalian collecting tubule. J Gun Invest 58:59 597, MUIRHEAD EE, GERMAIN G, LEACH BE, PITCOCK JA, STEPHENSON P, BROOKS B, BRoslUs WL, DANIELS EG, KINMAN JW: Production of renomedullary prostaglandins by renomedullary interstitial cells grown in tissue culture. Circ Res 3 31 (Suppl 2):16l 172, MCGIFF JC, CROWSHAW K, TERRAGNO NA, MALIK Ky, LONIGRO AJ: Differential effects of noradrenaline and renal nerve stimulation on vascular resistance in the dog kidney and the release of a prostaglandin E-like substance. Gun Sci 42: , STOKES JB, KOKKO JP: Inhibition of sodium transport by prostaglandin E2 across the isolated, perfused rabbit collecting tubule. J Clin Invest 59: , Ino Y, 1MM M: Effects of prostaglandins on Na transport in isolated collecting tubules. Pfluegers Arch 373: , FINE LG, TRIZNA W: Influence of prostaglandins on sodium transport of isolated medullary nephron segments. Am J Physiol 232:F383 F39, FINE LG, SERROS E, KIR5CHENBAUM M, TRIZNA W: Absence of an effect of PGE2 on transepithelial potential difference of cortical collecting tubules from DOCA-treated rabbits (abst). Am Soc Nephrol l2:43a, DUNN MJ, HOWE D: Prostaglandins lack a direct inhibitory action on electrolyte and water transport in the kidney and the erythrocyte. Prostaglandins 13: , SMITH WL, WILLEIM GP: Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenase: The detection of the cyclooxygenase in rat, rabbit and guinea pig kidneys by immunofluorescence. Prostaglandins 13: , JANSZEN FH, NUGTEREN DH: Histochemical localization of prostaglandin synthetase. Histochemistry 27: , STOKES JB: Effect of prostaglandin E2 on chloride transport across the rabbit thick ascending limb of Henle. Selective inhibition of the medullary portion. J Clin Invest 64:495 52, 1979