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Renal action of progesterone: effect on calcium reabsorption
Molecular and Cellular Endocrinology 194 (2002) 183 /190 www.elsevier.com/locate/mce
Renal action of progesterone: effect on calcium reabsorption Miche`le G. Brunette *, Marie Leclerc Maisonneuve-Rosemont Hospital, Guy-Bernier Research Center and University of Montreal, 5415 l’Assomption Boulevard, Montreal, Quebec, Canada H1T 2M4 Received 28 January 2002; accepted 16 April 2002
Abstract Recently, the kidney has been reported to be the site of receptors for progesterone. Although the exact segment of the nephron has not been precisely determined, the cortical collecting tubule was suspected, since the hormone displaces bound 3H aldosterone. The aim of the present study was to investigate the effect of progesterone on calcium (Ca2 ) transport by the renal luminal membranes and to determine the site and mechanisms of action. Incubation of proximal tubules from rabbit kidney with progesterone did not influence Ca2 or Na transport by the luminal membranes. In the distal tubules (DT), a 5 min treatment with 10 11 M of the hormone enhanced 0.5 mM 45Ca uptake from 0.609/0.02 to 0.849/0.08 pmol/mg per 10 s (P B/0.05) in the absence of Na and from 0.269/0.02 to 0.419/0.02 pmol/mg per 10 s (P B/0.01) in the presence of 100 mM Na . The dose /response curve showed a biphasic action with a peak at 10 11 M. Ca2 uptake by DT membranes presents dual kinetics. The hormone enhanced the Vmax value of the high affinity component from 0.419/0.05 to 0.579/0.06 pmol/mg per 10 s (P B/0.05). In contrast, incubation of DT with 10 8 M progesterone decreased 1 mM Na uptake from 0.689/0.03 to 0.539/0.07 pmol/mg per 10 s (P B/0.05). Finally, 10 11 M progesterone also enhanced Ca2 uptake by the DT membranes through a direct nongenomic mechanism. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Progesterone; Renal calcium transport; Calciuria
1. Introduction Progesterone and estrogen control the functions of female reproductive organs. Both hormones play major roles in uterus endometrium modifications, allowing embryo implantation and protection (Katzenellenbogen, 1980). However, the uterus is not the only target organ of these hormones. In mammals, progesterone and estrogen receptor expression has been described in the thymus (Fujii-Hanamoto et al., 1985; Nilsson et al., 1990), while in the chicken, similar receptors were reported in the brain (Sterling et al., 1987) and the gastro-intestinal tract (Salomaa et al., 1989). The kidney seems also to be the site of receptors for progesterone, as reported in various studies. Shughrue et al. (1988) injected 125I-progestin in pregnant mice and studied the target cells in the foetus. Nuclear uptake was
* Corresponding author. Tel.: /1-514-252-3400x3334; fax: /1-514252-3569 E-mail address: [email protected] (M.G. Brunette).
observed in numerous tissues, including the kidney. However, the segment involved was not clearly established. Since progesterone antagonized the action of aldosterone on sodium retention by the kidney (Landau and Lugibihl, 1958), a distal site was suspected. Confirming this hypothesis, Rafestin-Oblin et al. (1991) investigated the renal action of two synthesized progesterone derivates, the 18 vinyl-progesterone and the 18 ethynyl-progesterone, in adrenalectomized rats. The two compounds displaced 3H aldosterone binding, suggesting that the target segment was the cortical collecting tubule. More recently, in vivo experiments confirmed a competition binding mechanism between progesterone and aldosterone in guinea pigs (Myles and Funder, 1996). However, Pasanen et al. (1997) studied the distribution of progesterone receptors in chickens. They found binding in many organs including the kidney, although receptor expression in this organ was localized exclusively in capsular cells and smooth muscles of blood vessels. Tubules were negative. Another unanswered question: if the kidney is a site of progesterone receptors, does the hormone influence
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electrolyte physiology, in particular Ca2 reabsorption? And finally, because Na and Ca2 reabsorption by the kidney luminal membrane of the distal nephron are tightly related, any hypothetical action on Ca2 transfer may be correlated with an effect on Na reabsorption. In the present study, we investigated the action of progesterone on Ca2 and Na transport by the luminal membranes of proximal and distal tubules of the rabbit kidney.
2. Material and method 2.1. Tubule suspension The experiments were performed on rabbit kidneys which were removed at the slaughter house immediately after decapitation of the animals and rapidly transported on ice to the laboratory. Slices of 1 /3-mm thickness were cut through the cortex and immersed in modified Krebs /Henseleit solution (MKH). Digestion was then performed in Dulbecco’s Modified Eagle’s Medium nutrient-mixture F-12 HAM (DMEN F-12 HAM, Sigma, St. Louis, MI) containing 1 mg/ml of collagenase fraction V, (clostridium histolyticum; Sigma) and 5 mg/ml bovine serum albumin (BSA fraction V). Following a 20-min digestion at 37 8C, the suspension was rapidly filtered through a metal tea strainer. The filtrate was washed three times in MKH with 5 mg/ml BSA and suspended in DMEN F-12 HAM containing 45% Percoll previously equilibrated with 95% O2 for 20 min. After centrifugation for 30 min at 28,000 /g , three bands were clearly formed corresponding to the distal tubules, glomeruli and proximal tubules. The tubules were collected separately and washed in MKH until free of Percoll. Whereas the proximal tubule bands consisted essentially of convoluted segments, the distal tubule suspensions contained a mixture of convoluted distal tubules and cortical connecting tubules. 2.2. Incubation of tubules with progesterone Proximal and distal tubules were incubated at 37 8C in the cell culture medium (DMEN F-12 HAM) with 2% (vol/vol) fetal bovine serum, 0.1 mM phenyl methylsulfonate fluoride (PMSF) and progesterone at the indicated concentration. Following incubation, the tubules were washed three times with MKH solution, suspended in 10 mM mannitol and 20 mM Tris-Hepes pH 7.4 and frozen at /80 8C until the day of the experiment. 2.3. Luminal membrane preparation On the day of experiment, the tubules were thawed and homogenized with ten strokes of a Potter homo-
genizer. The luminal membranes from the proximal and distal tubules were purified using the Mg2 precipitation technique: 12 mM MgCl2 (final concentration) was added to the suspension which was stirred on ice for 10 min (20 min for proximal tubules) and centrifuged at 3000 /g for 20 min. The supernatant was collected and centrifuged at 28,000 /g for 20 min at 4 8C. The pellet was washed twice in 280 mM mannitol, 20 mM TrisHepes, pH 7.4, passed three times through a 22 gauge needle and left for 1 h on ice to complete the vesiculization. Enrichment of alkaline phosphatase was measured using the technique of Kelly and Hamilton (1970), the Na /K ATPase, the technique of Post and Sen (1967) (Tables 1 and 2) and protein contents by the technique of Lowry et al. (1951).
2.4. Ca2 and Na transport measurement Ca2 and Na transport were measured by the rapid filtration technique. The incubation medium for Ca2 uptake contained 141 mM choline chloride, 20 mM TrisHepes, pH 7.4 and 45CaCl2 at the indicated concentrations and for Na uptake, 278 mM mannitol, 20 mM Tris-Hepes pH 7.4 and 1 mM 22NaCl. In some Ca2 transport experiments, the 141 mM choline chloride was replaced by 100 mM NaCl and 41 mM choline chloride. Uptake was initiated by adding 25 ml of incubation medium at 35 8C to 5 ml of membrane suspension (:/20 mg protein). At the indicated time, transport was stopped by the addition of 1 ml of ice-cold stop solution. The suspension was filtered through Millipore filters (HAWP 0.45 mM pore size). The filters were rinsed with an additional 5 ml of stop-solution and radioactivity was counted. The stop-solution contained 150 mM KCl, 20 mM Tris-Hepes, pH 7.4 and 2 mM EGTA for Ca2 uptake, or 150 mM LiCl, 20 mM Tris-Hepes, pH 7.4 for Na uptake experiments. The kinetic parameters of Ca2 uptake by the membrane vesicles were calculated by a non-linear regression analysis using the equations described by Huntson (1975). Table 1 Enzyme marker activities Preparations
Alkaline phosphatase (nmol/mg per 15 min)
Na-K ATPase (nmol/mg per 20 min)
Cortex PT homogenates PT LUM membranes DT homogenates LUM membranes
2.1290.11 1.3590.09 11.6292.02 (8.61x) 0.2890.05 1.7590.26 (6.25x)
3.2190.35 2.5590.22 2.0390.13 (0.80x) 0.5890.11 0.5090.08 (0.86x)
x
Enrichment compared to the corresponding homogenate values.
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Table 2 Effect of distal tubules incubation with progesterone on kinetic parameters of Ca2 by distal luminal membranes
3, NS) in control and experimental conditions, respectively.
Component
Affinity
Control
Progesterone
Km (mM)
Low High
0.8490.26 0.03190.006
0.6090.11 0.03190.007
3.2. Progesterone influences Ca2 uptake by the distal membranes
Vmax (pmol/mg per 10 s)
Low High
1.2190.26 0.4190.046
1.2990.15 0.57490.06*
* P B 0.05 compared to the control values, n 5.
2.5. Statistics Each experiment was carried out at least in triplicate. The radioactive background was measured at time zero and subtracted from each value of Ca2 uptake. The results were analyzed using Student’s unpaired or paired t-test depending upon the experiments.
In contrast, progesterone significantly enhanced Ca2 transport by the distal tubule membranes. Fig. 2 represents the time-course of 0.5 mM Ca2 uptake by membranes obtained from distal tubules treated or not with 10 11 M progesterone. This uptake was performed in a buffered medium containing either 41 mM choline Cl, 100 mM NaCl (/Na) and 20 mM Tris-Hepes, pH 7.4 or 141 mM choline Cl and 20 mM Tris-Hepes, pH 7.4 (-Na). Treatment of tubules with 10 11 M progesterone for 5 min increased the initial 10 s uptake by the luminal membranes from 0.609/0.02 to 0.849/0.08 pmol/mg in the absence of Na (P B/0.05, n /4) and from 0.269/0.02 to 0.419/0.02 pmol/mg per 10 s in the presence of 100 mM Na (P B/0.01, n /3). After a 30min incubation of the vesicles, the effect was no longer visible.
3. Results 3.1. Effect of progesterone on Ca2 and Na transport by the proximal tubule Fig. 1 represents the time-course of Ca2 and Na uptake by the luminal membranes of proximal tubules incubated during 5 min with either 10 11 M (for Ca2 uptake) or 10 9 M (for Na uptake) progesterone. Neither of the two electrolyte transports were influenced by the hormone: 0.5 mM Ca2 uptakes were 0.519/0.04 vs. 0.459/0.02 pmol/mg per 10 s (n /3) and 1.0 mM Na uptakes 0.829/0.16 vs. 0.909/0.12 pmol/mg per 10 s (n /
3.3. How fast is the action of progesterone on the luminal membrane? This action was very rapid, indeed. In the following experiments, distal tubules were incubated with 10 11 M progesterone during 1, 5, 15, 30 or 60 min. Then the luminal membranes were purified from these various preparations and 0.5 mM Ca2 uptake measured. Results are presented in Fig. 3. Ca2 uptake was already significantly enhanced after a 1-min incubation. However, within 15 min of incubation, the effect was no longer apparent.
Fig. 1. Effect of 10 11 M progesterone on 0.5 mM Ca2 (left side) and 10 9 M progesterone on 1 mM Na (right side) uptakes by the luminal membranes of control proximal tubules (m) and vesicles treated with progesterone (k). The vesicles were loaded with 280 mM mannitol and 20 mM Tris-Hepes, pH 7.4. The incubation medium contained 0.5 mM 45Ca2 , 141 mM choline chloride and 20 mM Tris-Hepes, pH 7.4. for Ca2 uptake and 278 mM mannitol, 20 mM Tris-Hepes pH 7.4 and 1 mM 22Na for Na uptake, n /3.
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Fig. 2. Effect of 10 11 M progesterone on 0.5 mM Ca2 uptake by the luminal membranes of distal tubules. (m) Vesicles from control tubules. (k) Vesicles from treated tubules. The incubation medium contained 20 mM Tris-Hepes, pH 7.4, 0.5 mM CaCl2 and either 100 mM NaCl and 41 mM choline chloride (left side, n /3) or 141 mM choline chloride (right side, n /4). *P B/0.05 and **P B/0.01 compared to the control values.
3.5. Does progesterone directly influence the distal luminal membrane?
Fig. 3. Effect of the time of incubation of distal tubules with 10 11 M progesterone on 0.5 M Ca2 uptake by the luminal membranes in the absence of Na in the incubation medium. *P B/0.05 and **P B/0.01, n /3.
In various types of cells other than kidney cells, progesterone (Turner and Meizel, 1995; Blackmore, 1998), androstenedione (Machelon et al., 1998) and aldosterone (Moura and Worgel, 1984) have been shown to have nongenomic effects upon these cells. We investigated whether progesterone was able to directly influence Ca2 transport by the distal luminal membrane vesicles. We previously reported that estrogen does not (Brunette and Leclerc, 2001). But unexpectedly, a 1 min incubation with 10 11 M progesterone in cis-position, in the absence of Na did influence Ca2 uptake by the vesicles, enhancing 0.5 mM Ca2 from 0.549/0.07 to 0.799/0.08 pmol/mg per 10 s (P B/0.02). Here again, the effect was biphasic (Fig. 5) and as when the entire tubule was incubated with the hormone, the peak action was obtained with 10 11 M progesterone.
3.6. Effect of progesterone on the kinetic parameters of Ca2 uptake by the distal luminal membranes 3.4. The dose /response curve effect of progesterone on Ca2 uptake Confirming the effect of progesterone on Ca2 transport by the distal membrane, the response to this hormone was dose-dependent. Interestingly, the curve was biphasic, with a maximal effect being observed with 10 11 M progesterone (Fig. 4). For comparison, a similar dose/response curve was performed with luminal membrane vesicles from proximal tubules. As in the previous experiments, progesterone failed to influence the Ca2 uptake by the brush border membrane vesicles at any of the concentrations studied.
We repeatedly reported dual kinetics of Ca2 transport by the luminal membrane of the distal tubules. The low affinity component is influenced by calcitonin (Zuo et al., 1997) while the high affinity component is stimulated by parathyroid hormone (Bouhtiauy et al., 1994), vitamin D dependent calbindin D 28 K (Bouhtiauy et al., 1991) and estrogen (Brunette and Leclerc, 2001). Therefore, we questioned on which of these components progesterone was acting. Incubation of distal tubules for 5 min with 10 11 M progesterone very clearly enhanced Ca2 uptake by the high affinity component, increasing (in the absence of Na ) the Vmax values from 0.419/0.05 to 0.579/0.06 pmol/mg per
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Fig. 4. Dose /response curve of the effect of progesterone on 0.5 mM Ca2 uptake by luminal membranes from proximal (PT) and distal (DT) tubules in the absence of Na in the incubation medium.
10 s, (P B/0.05, n /5) and leaving the Km unchanged: 0.319/0.006 vs. 0.319/0.007 mM (Fig. 6). 3.7. Progesterone also influences Na transport by the distal tubule
Fig. 5. Direct effect of progesterone in cis-position on 0.5 mM Ca2 uptake by the luminal membranes of distal tubules, in the absence of Na . *P B/0.05 and **P B/0.02, n /4.
Fig. 6. Kinetics of the effect of 10 11 M progesterone on Ca2 uptake by the distal luminal membranes: from control tubules (m) and tubules treated with 10 11 M progesterone (k), n /5.
Ca2 and Na reabsorption is tightly related in the distal segments of the nephron. Na strongly curtails Ca2 transport (Brunette et al., 1992) and conversely, Ca2 decreases Na reabsorption (Brunette et al., 1999). Most, if not all of the Ca2 regulating hormones, such as parathyroid hormone, calcitonin and calbindin 28K, negatively influence Na transport. Therefore, we investigated whether progesterone also influences the two electrolyte uptakes in opposite directions. Again, this was the case: incubation of distal tubules with 10 8 M progesterone for 5 min significantly decreased 1 mM Na transport from 0.689/0.03 to 0.539/0.07 pmol/mg per 10 s (P B/0.05, n /4). As shown in Fig. 7, this effect was dose-dependent with a maximal response at
Fig. 7. Effect of progesterone on 1 mM Na uptake by the distal luminal membrane: dose /response curve. **P B/0.02 and ***P B/ 0.01.
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10 9 M. However, at higher concentrations, the effect declined to return to the control levels at 10 7 M. 3.8. Does Na prevent the effect of progesterone on Ca2 transport by the distal membrane? The presence of Na in the incubation medium decreases Ca2 transport by the distal luminal membranes and enhances the effect of parathyroid hormone (Lajeunesse et al., 1994), calcitonin (Zuo et al., 1997) and calbindin D 28 K (Bouhtiauy et al., 1991) on this transport. In other words, these hormones enhance Ca2 uptake only in the presence of Na . Interestingly, this is not the case with progesterone. Fig. 8 shows the influence of increasing concentrations of NaCl on Ca2 uptake by membranes from tubules treated with either 10 11 M progesterone or the carrier. At all of the NaCl concentrations, the positive effect of progesterone remained identical.
4. Discussion 4.1. Progesterone influences electrolyte reabsorption by the distal nephron As previously mentioned, the presence of receptors for progesterone in the renal tubule was not clearly demonstrated. Such receptors have been localized in the aldosterone-sensitive segment (Rafestin-Oblin et al., 1991; Myles and Funder, 1996), whereas immunochemistry rather detected them in mesothelial and stroma cells (Pasanen et al., 1997). Our results demonstrate the presence of receptors in the distal part of the nephron, since the hormone stimulated Ca2 reabsorption at this site; and if the target cells are also sensitive to aldosterone, then the progesterone responsive segment is probably the cortical
Fig. 8. Effect of 10 11 M progesterone on 0.5 mM Ca2 uptake by the distal luminal membrane in the presence of increasing concentrations NaCl. *P B/0.05 and **P B/0.02, n /4.
collecting duct which is included in our distal tubule suspensions. In contrast, the proximal tubule seems to be devoid of receptors. Alternatively, it is possible that an absence of some intracellular pathway prevents the hormone in influencing the channels in the luminal membrane of this segment.
4.2. Progesterone and estrogen have opposite effects on Ca2 and Na transport by the distal nephron We recently reported that, as progesterone, estrogen does not influence electrolyte transport by the proximal tubule. However, incubation of distal segments with estrogen significantly decreased Ca2 and increased Na uptakes by the luminal membranes (Brunette and Leclerc, 2001). The effect on Ca2 transport was shown to involve exclusively the high affinity component of the kinetics. We now report that progesterone has the opposite effect! These results were unexpected because this hormone had been previously shown to have a Na retaining property in adrenalectomized rats (Burton et al., 1995; Galigniana et al., 2000). Indeed, in vitro and in vivo actions are not necessarily identical due to the complex regulation of electrolyte excretion in vivo. However, our in vitro experiments detected a factor which certainly contributes to the final clinical status and it is possible that the combined action of the two hormones during an important proportion of the sex hormone cycle in women, finally, does not produce any significant loss or gain in either of these electrolytes; but a lack of balance between the two hormone secretions, or receptors, in the kidney may be responsible for uncomfortable situations, such as premenstrual tension.
4.3. The dose /response curve of the effect of progesterone on Ca2 uptake Our dose/response curve indicates a peak of action on Ca2 uptake between 10 12 and 10 9 M. In the human, plasma concentrations vary from 0.89 to 3.88 10 9M in men and from 1.37 to 47.13 10 9 M in women (Centaur, 1999). In urine, progesterone excretion reaches a maximum level of 10 6 M during the luteal phase and even 100 times more during pregnancy. We are not aware of any data concerning the plasma concentrations in the rabbit. Furthermore, our rabbit population was a mixture of males and females. At first glance, the blood and urine concentrations found in the human are far above those that produced a maximal effect in our in vitro experiments. This apparent discrepancy might result from several factors, such as species differences. It is also possible that part of the hormone in the blood is bound to some circulating proteins and therefore, not readily available.
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4.4. The site of action of progesterone along the distal nephron Progesterone enhances Ca2 and decreases Na uptakes. Rafestin-Oblin et al. (1991) observed that the progesterone derivates displaced 3H-aldosterone binding, to a lower extent, however, than aldosterone. These findings suggest that progesterone acts, at least partially, in the cortical collecting duct. However, the two hormones influence Na transport in opposite directions: aldosterone increases whereas progesterone decreases Na uptake. Therefore, if progesterone and aldosterone really share the same receptors, then their influences on subsequent events (specific G proteins or messengers involved) should differ. 4.5. The presence of Na does not prevent the effect of progesterone on Ca2 uptake by the distal membranes This was an unexpected finding. Generally the hormones which influence Ca2 transport by the distal luminal membrane act in the inverse direction on Ca2 and Na transports: PTH, calcitonin, calbindin 28 K and angiotensin II increase Ca2 and decrease Na uptakes, whereas estrogen decreases Ca2 and increases Na uptakes. Progesterone is another example of this phenomenon. However, except for angiotensin II (Charbonneau et al., 2001), the effects of hormones which stimulate Ca2 transport are usually more efficient in the presence of Na . Conversely, the effects of those which, like estrogen, curtail Ca2 transport, are more pronounced in the absence of Na . Hypothetically, these channels are not totally dedicated to Ca2 reabsorption. They are also permeable to Na and the task of these hormones is to facilitate uptake of one of the cations at the expense of the other. Progesterone escapes this rule. As angiotensin II, this hormone increases Ca2 uptake independently of the Na content of the incubation medium. This unusual character proves that the type of channels targeted by progesterone and probably angiotensin II are different from those influenced by the PTH, calcitonin and calbindin 28 K. 4.6. The nongenomic effect of progesterone There is increasing evidence that in several types of cells, steroids might influence electrolyte transport through a nongenomic mechanism. For example, aldosterone has been shown to directly stimulate the transmembrane Na efflux from arterial smooth muscles (Moura and Worgel, 1984) and to increase intracellular Ca2 in human kidney cells (Koppel et al., 1999). A similar nongenomic effect was detected in lymphocytes (Wehling et al., 1991, 1992, 1993). Androstenedione also directly influences the human granulosa luteinizing cells
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(Machelon et al., 1998) and finally, progesterone has already been reported to have a nongenomic effect on spermatozoa (Turner and Meizel, 1995; Blackmore, 1998). It is the first time, however, that such a mechanism is observed with progesterone in the kidney.
5. Conclusion In conclusion, the incubation of rabbit proximal tubules with progesterone had no influence on Ca2 or Na transport by brush border membrane vesicles. In contrast, the hormone significantly increased Ca2 and decreased Na uptake by the distal tubule luminal membranes. These effects were significant following 1 min of incubation. Progesterone influenced the high affinity component of Ca2 transport. The hormone increased 0.5 mM Ca2 uptake according to a bell shaped dose /response curve, whereas the effect on Na uptake showed a biphasic decline of this transport with increasing hormone concentrations. Finally, a nongenomic effect was observed when the membranes were directly incubated with the hormone. Since it has been previously shown that estrogen has the opposite actions on Ca2 and Na reabsorption, it is probable that the combined activity of the two hormones on these action reabsorption by the nephron are relatively modest.
Acknowledgements This study was supported by grants from the Medical Research Council of Canada (MT-11307) and the Kidney Foundation of Canada. The authors are grateful to Jean-Marie Be´langer for providing the rabbit kidneys and to Claudette Plante and Jocelyne Laflamme for secretarial assistance.
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Myles, K., Funder, J.W., 1996. Progesterone binding to mineralocorticoid receptors: in vitro and in vivo studies. Am. J. Physiol. 270 (Endocrinology 33), 601 /607. Nilsson, B., Ferno¨, M., VonSchoultz, B., 1990. Estrogen and progesterone receptors in the human thymus. Gynecol. Obst. Invest. 29, 289 /291. Pasanen, S., Ylikomi, T., Syvala, H., Tuohimaa, P., 1997. Distribution of progesterone receptors in chicken: model target organs for progesterone and estrogen action. Mol. Cell. Endocrinol. 135, 79 / 91. Post, R.S., Sen, A.K., 1967. Sodium and potassium ATPase. Methods Enzymol. 10, 762 /768. Rafestin-Oblin, M.E., Couette, B., Barlet-Bas, C., Cheval, L., Viger, A., Doucet, A., 1991. Renal action of progesterone and 18substituted derivatives. Am. J. Physiol. 260 (Renal Fluid Elect. 29), F828 /F832. Salomaa, S., Pekki, A., Sannisto, T., Ylikomi, T., Tuohimaa, P., 1989. Progesterone receptor is constitutively expressed in chicken intestinal mesothelium and smooth muscle. J. Steroid Biochem. 29, 297 /306. Shughrue, P.J., Stumpf, W.E., Sar, M., 1988. The distribution of progesterone receptor in the 20 day old foetal mouse: an autoradiographic study with 125I-progestin. Endocrinology 123, 2382 /2389. Sterling, R.J., Gase, J.M., Sharp, P.J., Renoir, J.M., Tuohimaa, P., Beaulieu, E.E., 1987. The distribution of nuclear progesterone receptors in the hypothalamus and forebrain of the domestic hen. Cell Tissue Res. 248, 201 /205. Turner, K.O., Meizel, S., 1995. Progesterone-mediated efflux of cytosolic chloride during the human sperm acrosome reaction. Biochem. Biophys. Res. Commun. 213, 774 /780. Wehling, M., Kasmay, R.J., Theisen, K., 1991. Evidence for rapid non genomic effects of mineralocorticoids in lymphocytes involving the sodium-protein exchanger of the cell membrane. Am. J. Physiol. 260 (Endocrinol. Metab. 23), E719 /E726. Wehling, M., Christ, M., Theisen, K., 1992. Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action. Am. J. Physiol. 263 (Endocrinol. Metab. 26), E974 /E979. Wehling, M., Eisen, C., Christ, M., 1993. Membrane receptors for aldosterone: a new concept of non genomic mineralocorticoid action. News Physiol. Sci. (NIPS) 8, 241 /244. Zuo, Q., Claveau, D., Hilal, G., Leclerc, M., Brunette, M.G., 1997. Effect of calcitonin on calcium transport by the luminal and basolateral membranes of the rabbit nephron. Kidney Int. 51, 11991 /11999.
Renal action of progesterone: effect on calcium reabsorption Miche`le G. Brunette *, Marie Leclerc Maisonneuve-Rosemont Hospital, Guy-Bernier Research Center and University of Montreal, 5415 l’Assomption Boulevard, Montreal, Quebec, Canada H1T 2M4 Received 28 January 2002; accepted 16 April 2002
Abstract Recently, the kidney has been reported to be the site of receptors for progesterone. Although the exact segment of the nephron has not been precisely determined, the cortical collecting tubule was suspected, since the hormone displaces bound 3H aldosterone. The aim of the present study was to investigate the effect of progesterone on calcium (Ca2 ) transport by the renal luminal membranes and to determine the site and mechanisms of action. Incubation of proximal tubules from rabbit kidney with progesterone did not influence Ca2 or Na transport by the luminal membranes. In the distal tubules (DT), a 5 min treatment with 10 11 M of the hormone enhanced 0.5 mM 45Ca uptake from 0.609/0.02 to 0.849/0.08 pmol/mg per 10 s (P B/0.05) in the absence of Na and from 0.269/0.02 to 0.419/0.02 pmol/mg per 10 s (P B/0.01) in the presence of 100 mM Na . The dose /response curve showed a biphasic action with a peak at 10 11 M. Ca2 uptake by DT membranes presents dual kinetics. The hormone enhanced the Vmax value of the high affinity component from 0.419/0.05 to 0.579/0.06 pmol/mg per 10 s (P B/0.05). In contrast, incubation of DT with 10 8 M progesterone decreased 1 mM Na uptake from 0.689/0.03 to 0.539/0.07 pmol/mg per 10 s (P B/0.05). Finally, 10 11 M progesterone also enhanced Ca2 uptake by the DT membranes through a direct nongenomic mechanism. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Progesterone; Renal calcium transport; Calciuria
1. Introduction Progesterone and estrogen control the functions of female reproductive organs. Both hormones play major roles in uterus endometrium modifications, allowing embryo implantation and protection (Katzenellenbogen, 1980). However, the uterus is not the only target organ of these hormones. In mammals, progesterone and estrogen receptor expression has been described in the thymus (Fujii-Hanamoto et al., 1985; Nilsson et al., 1990), while in the chicken, similar receptors were reported in the brain (Sterling et al., 1987) and the gastro-intestinal tract (Salomaa et al., 1989). The kidney seems also to be the site of receptors for progesterone, as reported in various studies. Shughrue et al. (1988) injected 125I-progestin in pregnant mice and studied the target cells in the foetus. Nuclear uptake was
* Corresponding author. Tel.: /1-514-252-3400x3334; fax: /1-514252-3569 E-mail address: [email protected] (M.G. Brunette).
observed in numerous tissues, including the kidney. However, the segment involved was not clearly established. Since progesterone antagonized the action of aldosterone on sodium retention by the kidney (Landau and Lugibihl, 1958), a distal site was suspected. Confirming this hypothesis, Rafestin-Oblin et al. (1991) investigated the renal action of two synthesized progesterone derivates, the 18 vinyl-progesterone and the 18 ethynyl-progesterone, in adrenalectomized rats. The two compounds displaced 3H aldosterone binding, suggesting that the target segment was the cortical collecting tubule. More recently, in vivo experiments confirmed a competition binding mechanism between progesterone and aldosterone in guinea pigs (Myles and Funder, 1996). However, Pasanen et al. (1997) studied the distribution of progesterone receptors in chickens. They found binding in many organs including the kidney, although receptor expression in this organ was localized exclusively in capsular cells and smooth muscles of blood vessels. Tubules were negative. Another unanswered question: if the kidney is a site of progesterone receptors, does the hormone influence
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 1 1 3 - 2
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electrolyte physiology, in particular Ca2 reabsorption? And finally, because Na and Ca2 reabsorption by the kidney luminal membrane of the distal nephron are tightly related, any hypothetical action on Ca2 transfer may be correlated with an effect on Na reabsorption. In the present study, we investigated the action of progesterone on Ca2 and Na transport by the luminal membranes of proximal and distal tubules of the rabbit kidney.
2. Material and method 2.1. Tubule suspension The experiments were performed on rabbit kidneys which were removed at the slaughter house immediately after decapitation of the animals and rapidly transported on ice to the laboratory. Slices of 1 /3-mm thickness were cut through the cortex and immersed in modified Krebs /Henseleit solution (MKH). Digestion was then performed in Dulbecco’s Modified Eagle’s Medium nutrient-mixture F-12 HAM (DMEN F-12 HAM, Sigma, St. Louis, MI) containing 1 mg/ml of collagenase fraction V, (clostridium histolyticum; Sigma) and 5 mg/ml bovine serum albumin (BSA fraction V). Following a 20-min digestion at 37 8C, the suspension was rapidly filtered through a metal tea strainer. The filtrate was washed three times in MKH with 5 mg/ml BSA and suspended in DMEN F-12 HAM containing 45% Percoll previously equilibrated with 95% O2 for 20 min. After centrifugation for 30 min at 28,000 /g , three bands were clearly formed corresponding to the distal tubules, glomeruli and proximal tubules. The tubules were collected separately and washed in MKH until free of Percoll. Whereas the proximal tubule bands consisted essentially of convoluted segments, the distal tubule suspensions contained a mixture of convoluted distal tubules and cortical connecting tubules. 2.2. Incubation of tubules with progesterone Proximal and distal tubules were incubated at 37 8C in the cell culture medium (DMEN F-12 HAM) with 2% (vol/vol) fetal bovine serum, 0.1 mM phenyl methylsulfonate fluoride (PMSF) and progesterone at the indicated concentration. Following incubation, the tubules were washed three times with MKH solution, suspended in 10 mM mannitol and 20 mM Tris-Hepes pH 7.4 and frozen at /80 8C until the day of the experiment. 2.3. Luminal membrane preparation On the day of experiment, the tubules were thawed and homogenized with ten strokes of a Potter homo-
genizer. The luminal membranes from the proximal and distal tubules were purified using the Mg2 precipitation technique: 12 mM MgCl2 (final concentration) was added to the suspension which was stirred on ice for 10 min (20 min for proximal tubules) and centrifuged at 3000 /g for 20 min. The supernatant was collected and centrifuged at 28,000 /g for 20 min at 4 8C. The pellet was washed twice in 280 mM mannitol, 20 mM TrisHepes, pH 7.4, passed three times through a 22 gauge needle and left for 1 h on ice to complete the vesiculization. Enrichment of alkaline phosphatase was measured using the technique of Kelly and Hamilton (1970), the Na /K ATPase, the technique of Post and Sen (1967) (Tables 1 and 2) and protein contents by the technique of Lowry et al. (1951).
2.4. Ca2 and Na transport measurement Ca2 and Na transport were measured by the rapid filtration technique. The incubation medium for Ca2 uptake contained 141 mM choline chloride, 20 mM TrisHepes, pH 7.4 and 45CaCl2 at the indicated concentrations and for Na uptake, 278 mM mannitol, 20 mM Tris-Hepes pH 7.4 and 1 mM 22NaCl. In some Ca2 transport experiments, the 141 mM choline chloride was replaced by 100 mM NaCl and 41 mM choline chloride. Uptake was initiated by adding 25 ml of incubation medium at 35 8C to 5 ml of membrane suspension (:/20 mg protein). At the indicated time, transport was stopped by the addition of 1 ml of ice-cold stop solution. The suspension was filtered through Millipore filters (HAWP 0.45 mM pore size). The filters were rinsed with an additional 5 ml of stop-solution and radioactivity was counted. The stop-solution contained 150 mM KCl, 20 mM Tris-Hepes, pH 7.4 and 2 mM EGTA for Ca2 uptake, or 150 mM LiCl, 20 mM Tris-Hepes, pH 7.4 for Na uptake experiments. The kinetic parameters of Ca2 uptake by the membrane vesicles were calculated by a non-linear regression analysis using the equations described by Huntson (1975). Table 1 Enzyme marker activities Preparations
Alkaline phosphatase (nmol/mg per 15 min)
Na-K ATPase (nmol/mg per 20 min)
Cortex PT homogenates PT LUM membranes DT homogenates LUM membranes
2.1290.11 1.3590.09 11.6292.02 (8.61x) 0.2890.05 1.7590.26 (6.25x)
3.2190.35 2.5590.22 2.0390.13 (0.80x) 0.5890.11 0.5090.08 (0.86x)
x
Enrichment compared to the corresponding homogenate values.
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Table 2 Effect of distal tubules incubation with progesterone on kinetic parameters of Ca2 by distal luminal membranes
3, NS) in control and experimental conditions, respectively.
Component
Affinity
Control
Progesterone
Km (mM)
Low High
0.8490.26 0.03190.006
0.6090.11 0.03190.007
3.2. Progesterone influences Ca2 uptake by the distal membranes
Vmax (pmol/mg per 10 s)
Low High
1.2190.26 0.4190.046
1.2990.15 0.57490.06*
* P B 0.05 compared to the control values, n 5.
2.5. Statistics Each experiment was carried out at least in triplicate. The radioactive background was measured at time zero and subtracted from each value of Ca2 uptake. The results were analyzed using Student’s unpaired or paired t-test depending upon the experiments.
In contrast, progesterone significantly enhanced Ca2 transport by the distal tubule membranes. Fig. 2 represents the time-course of 0.5 mM Ca2 uptake by membranes obtained from distal tubules treated or not with 10 11 M progesterone. This uptake was performed in a buffered medium containing either 41 mM choline Cl, 100 mM NaCl (/Na) and 20 mM Tris-Hepes, pH 7.4 or 141 mM choline Cl and 20 mM Tris-Hepes, pH 7.4 (-Na). Treatment of tubules with 10 11 M progesterone for 5 min increased the initial 10 s uptake by the luminal membranes from 0.609/0.02 to 0.849/0.08 pmol/mg in the absence of Na (P B/0.05, n /4) and from 0.269/0.02 to 0.419/0.02 pmol/mg per 10 s in the presence of 100 mM Na (P B/0.01, n /3). After a 30min incubation of the vesicles, the effect was no longer visible.
3. Results 3.1. Effect of progesterone on Ca2 and Na transport by the proximal tubule Fig. 1 represents the time-course of Ca2 and Na uptake by the luminal membranes of proximal tubules incubated during 5 min with either 10 11 M (for Ca2 uptake) or 10 9 M (for Na uptake) progesterone. Neither of the two electrolyte transports were influenced by the hormone: 0.5 mM Ca2 uptakes were 0.519/0.04 vs. 0.459/0.02 pmol/mg per 10 s (n /3) and 1.0 mM Na uptakes 0.829/0.16 vs. 0.909/0.12 pmol/mg per 10 s (n /
3.3. How fast is the action of progesterone on the luminal membrane? This action was very rapid, indeed. In the following experiments, distal tubules were incubated with 10 11 M progesterone during 1, 5, 15, 30 or 60 min. Then the luminal membranes were purified from these various preparations and 0.5 mM Ca2 uptake measured. Results are presented in Fig. 3. Ca2 uptake was already significantly enhanced after a 1-min incubation. However, within 15 min of incubation, the effect was no longer apparent.
Fig. 1. Effect of 10 11 M progesterone on 0.5 mM Ca2 (left side) and 10 9 M progesterone on 1 mM Na (right side) uptakes by the luminal membranes of control proximal tubules (m) and vesicles treated with progesterone (k). The vesicles were loaded with 280 mM mannitol and 20 mM Tris-Hepes, pH 7.4. The incubation medium contained 0.5 mM 45Ca2 , 141 mM choline chloride and 20 mM Tris-Hepes, pH 7.4. for Ca2 uptake and 278 mM mannitol, 20 mM Tris-Hepes pH 7.4 and 1 mM 22Na for Na uptake, n /3.
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Fig. 2. Effect of 10 11 M progesterone on 0.5 mM Ca2 uptake by the luminal membranes of distal tubules. (m) Vesicles from control tubules. (k) Vesicles from treated tubules. The incubation medium contained 20 mM Tris-Hepes, pH 7.4, 0.5 mM CaCl2 and either 100 mM NaCl and 41 mM choline chloride (left side, n /3) or 141 mM choline chloride (right side, n /4). *P B/0.05 and **P B/0.01 compared to the control values.
3.5. Does progesterone directly influence the distal luminal membrane?
Fig. 3. Effect of the time of incubation of distal tubules with 10 11 M progesterone on 0.5 M Ca2 uptake by the luminal membranes in the absence of Na in the incubation medium. *P B/0.05 and **P B/0.01, n /3.
In various types of cells other than kidney cells, progesterone (Turner and Meizel, 1995; Blackmore, 1998), androstenedione (Machelon et al., 1998) and aldosterone (Moura and Worgel, 1984) have been shown to have nongenomic effects upon these cells. We investigated whether progesterone was able to directly influence Ca2 transport by the distal luminal membrane vesicles. We previously reported that estrogen does not (Brunette and Leclerc, 2001). But unexpectedly, a 1 min incubation with 10 11 M progesterone in cis-position, in the absence of Na did influence Ca2 uptake by the vesicles, enhancing 0.5 mM Ca2 from 0.549/0.07 to 0.799/0.08 pmol/mg per 10 s (P B/0.02). Here again, the effect was biphasic (Fig. 5) and as when the entire tubule was incubated with the hormone, the peak action was obtained with 10 11 M progesterone.
3.6. Effect of progesterone on the kinetic parameters of Ca2 uptake by the distal luminal membranes 3.4. The dose /response curve effect of progesterone on Ca2 uptake Confirming the effect of progesterone on Ca2 transport by the distal membrane, the response to this hormone was dose-dependent. Interestingly, the curve was biphasic, with a maximal effect being observed with 10 11 M progesterone (Fig. 4). For comparison, a similar dose/response curve was performed with luminal membrane vesicles from proximal tubules. As in the previous experiments, progesterone failed to influence the Ca2 uptake by the brush border membrane vesicles at any of the concentrations studied.
We repeatedly reported dual kinetics of Ca2 transport by the luminal membrane of the distal tubules. The low affinity component is influenced by calcitonin (Zuo et al., 1997) while the high affinity component is stimulated by parathyroid hormone (Bouhtiauy et al., 1994), vitamin D dependent calbindin D 28 K (Bouhtiauy et al., 1991) and estrogen (Brunette and Leclerc, 2001). Therefore, we questioned on which of these components progesterone was acting. Incubation of distal tubules for 5 min with 10 11 M progesterone very clearly enhanced Ca2 uptake by the high affinity component, increasing (in the absence of Na ) the Vmax values from 0.419/0.05 to 0.579/0.06 pmol/mg per
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187
Fig. 4. Dose /response curve of the effect of progesterone on 0.5 mM Ca2 uptake by luminal membranes from proximal (PT) and distal (DT) tubules in the absence of Na in the incubation medium.
10 s, (P B/0.05, n /5) and leaving the Km unchanged: 0.319/0.006 vs. 0.319/0.007 mM (Fig. 6). 3.7. Progesterone also influences Na transport by the distal tubule
Fig. 5. Direct effect of progesterone in cis-position on 0.5 mM Ca2 uptake by the luminal membranes of distal tubules, in the absence of Na . *P B/0.05 and **P B/0.02, n /4.
Fig. 6. Kinetics of the effect of 10 11 M progesterone on Ca2 uptake by the distal luminal membranes: from control tubules (m) and tubules treated with 10 11 M progesterone (k), n /5.
Ca2 and Na reabsorption is tightly related in the distal segments of the nephron. Na strongly curtails Ca2 transport (Brunette et al., 1992) and conversely, Ca2 decreases Na reabsorption (Brunette et al., 1999). Most, if not all of the Ca2 regulating hormones, such as parathyroid hormone, calcitonin and calbindin 28K, negatively influence Na transport. Therefore, we investigated whether progesterone also influences the two electrolyte uptakes in opposite directions. Again, this was the case: incubation of distal tubules with 10 8 M progesterone for 5 min significantly decreased 1 mM Na transport from 0.689/0.03 to 0.539/0.07 pmol/mg per 10 s (P B/0.05, n /4). As shown in Fig. 7, this effect was dose-dependent with a maximal response at
Fig. 7. Effect of progesterone on 1 mM Na uptake by the distal luminal membrane: dose /response curve. **P B/0.02 and ***P B/ 0.01.
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10 9 M. However, at higher concentrations, the effect declined to return to the control levels at 10 7 M. 3.8. Does Na prevent the effect of progesterone on Ca2 transport by the distal membrane? The presence of Na in the incubation medium decreases Ca2 transport by the distal luminal membranes and enhances the effect of parathyroid hormone (Lajeunesse et al., 1994), calcitonin (Zuo et al., 1997) and calbindin D 28 K (Bouhtiauy et al., 1991) on this transport. In other words, these hormones enhance Ca2 uptake only in the presence of Na . Interestingly, this is not the case with progesterone. Fig. 8 shows the influence of increasing concentrations of NaCl on Ca2 uptake by membranes from tubules treated with either 10 11 M progesterone or the carrier. At all of the NaCl concentrations, the positive effect of progesterone remained identical.
4. Discussion 4.1. Progesterone influences electrolyte reabsorption by the distal nephron As previously mentioned, the presence of receptors for progesterone in the renal tubule was not clearly demonstrated. Such receptors have been localized in the aldosterone-sensitive segment (Rafestin-Oblin et al., 1991; Myles and Funder, 1996), whereas immunochemistry rather detected them in mesothelial and stroma cells (Pasanen et al., 1997). Our results demonstrate the presence of receptors in the distal part of the nephron, since the hormone stimulated Ca2 reabsorption at this site; and if the target cells are also sensitive to aldosterone, then the progesterone responsive segment is probably the cortical
Fig. 8. Effect of 10 11 M progesterone on 0.5 mM Ca2 uptake by the distal luminal membrane in the presence of increasing concentrations NaCl. *P B/0.05 and **P B/0.02, n /4.
collecting duct which is included in our distal tubule suspensions. In contrast, the proximal tubule seems to be devoid of receptors. Alternatively, it is possible that an absence of some intracellular pathway prevents the hormone in influencing the channels in the luminal membrane of this segment.
4.2. Progesterone and estrogen have opposite effects on Ca2 and Na transport by the distal nephron We recently reported that, as progesterone, estrogen does not influence electrolyte transport by the proximal tubule. However, incubation of distal segments with estrogen significantly decreased Ca2 and increased Na uptakes by the luminal membranes (Brunette and Leclerc, 2001). The effect on Ca2 transport was shown to involve exclusively the high affinity component of the kinetics. We now report that progesterone has the opposite effect! These results were unexpected because this hormone had been previously shown to have a Na retaining property in adrenalectomized rats (Burton et al., 1995; Galigniana et al., 2000). Indeed, in vitro and in vivo actions are not necessarily identical due to the complex regulation of electrolyte excretion in vivo. However, our in vitro experiments detected a factor which certainly contributes to the final clinical status and it is possible that the combined action of the two hormones during an important proportion of the sex hormone cycle in women, finally, does not produce any significant loss or gain in either of these electrolytes; but a lack of balance between the two hormone secretions, or receptors, in the kidney may be responsible for uncomfortable situations, such as premenstrual tension.
4.3. The dose /response curve of the effect of progesterone on Ca2 uptake Our dose/response curve indicates a peak of action on Ca2 uptake between 10 12 and 10 9 M. In the human, plasma concentrations vary from 0.89 to 3.88 10 9M in men and from 1.37 to 47.13 10 9 M in women (Centaur, 1999). In urine, progesterone excretion reaches a maximum level of 10 6 M during the luteal phase and even 100 times more during pregnancy. We are not aware of any data concerning the plasma concentrations in the rabbit. Furthermore, our rabbit population was a mixture of males and females. At first glance, the blood and urine concentrations found in the human are far above those that produced a maximal effect in our in vitro experiments. This apparent discrepancy might result from several factors, such as species differences. It is also possible that part of the hormone in the blood is bound to some circulating proteins and therefore, not readily available.
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4.4. The site of action of progesterone along the distal nephron Progesterone enhances Ca2 and decreases Na uptakes. Rafestin-Oblin et al. (1991) observed that the progesterone derivates displaced 3H-aldosterone binding, to a lower extent, however, than aldosterone. These findings suggest that progesterone acts, at least partially, in the cortical collecting duct. However, the two hormones influence Na transport in opposite directions: aldosterone increases whereas progesterone decreases Na uptake. Therefore, if progesterone and aldosterone really share the same receptors, then their influences on subsequent events (specific G proteins or messengers involved) should differ. 4.5. The presence of Na does not prevent the effect of progesterone on Ca2 uptake by the distal membranes This was an unexpected finding. Generally the hormones which influence Ca2 transport by the distal luminal membrane act in the inverse direction on Ca2 and Na transports: PTH, calcitonin, calbindin 28 K and angiotensin II increase Ca2 and decrease Na uptakes, whereas estrogen decreases Ca2 and increases Na uptakes. Progesterone is another example of this phenomenon. However, except for angiotensin II (Charbonneau et al., 2001), the effects of hormones which stimulate Ca2 transport are usually more efficient in the presence of Na . Conversely, the effects of those which, like estrogen, curtail Ca2 transport, are more pronounced in the absence of Na . Hypothetically, these channels are not totally dedicated to Ca2 reabsorption. They are also permeable to Na and the task of these hormones is to facilitate uptake of one of the cations at the expense of the other. Progesterone escapes this rule. As angiotensin II, this hormone increases Ca2 uptake independently of the Na content of the incubation medium. This unusual character proves that the type of channels targeted by progesterone and probably angiotensin II are different from those influenced by the PTH, calcitonin and calbindin 28 K. 4.6. The nongenomic effect of progesterone There is increasing evidence that in several types of cells, steroids might influence electrolyte transport through a nongenomic mechanism. For example, aldosterone has been shown to directly stimulate the transmembrane Na efflux from arterial smooth muscles (Moura and Worgel, 1984) and to increase intracellular Ca2 in human kidney cells (Koppel et al., 1999). A similar nongenomic effect was detected in lymphocytes (Wehling et al., 1991, 1992, 1993). Androstenedione also directly influences the human granulosa luteinizing cells
189
(Machelon et al., 1998) and finally, progesterone has already been reported to have a nongenomic effect on spermatozoa (Turner and Meizel, 1995; Blackmore, 1998). It is the first time, however, that such a mechanism is observed with progesterone in the kidney.
5. Conclusion In conclusion, the incubation of rabbit proximal tubules with progesterone had no influence on Ca2 or Na transport by brush border membrane vesicles. In contrast, the hormone significantly increased Ca2 and decreased Na uptake by the distal tubule luminal membranes. These effects were significant following 1 min of incubation. Progesterone influenced the high affinity component of Ca2 transport. The hormone increased 0.5 mM Ca2 uptake according to a bell shaped dose /response curve, whereas the effect on Na uptake showed a biphasic decline of this transport with increasing hormone concentrations. Finally, a nongenomic effect was observed when the membranes were directly incubated with the hormone. Since it has been previously shown that estrogen has the opposite actions on Ca2 and Na reabsorption, it is probable that the combined activity of the two hormones on these action reabsorption by the nephron are relatively modest.
Acknowledgements This study was supported by grants from the Medical Research Council of Canada (MT-11307) and the Kidney Foundation of Canada. The authors are grateful to Jean-Marie Be´langer for providing the rabbit kidneys and to Claudette Plante and Jocelyne Laflamme for secretarial assistance.
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