View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 66 (2004), pp. 242–250 Aldosterone enhances renal calcium reabsorption by two types of channels MARIE LECLERC,MICHELE` G. BRUNETTE, and DENIS COUCHOUREL Maisonneuve-Rosemont Hospital and University of Montreal, Canada Aldosterone enhances renal calcium reabsorption by two types transepithelial potential difference in the late segments of channels. of the nephron, reflecting a stimulation of the amiloride- Background. Aldosterone has been known for many years + + sensitive Na transport by the apical membranes. to increase sodium (Na ) reabsorption by the distal nephron. The present in vitro experiments investigated the effect of the In contrast to the abundant literature dealing with the + hormone on calcium (Ca2 ) transport by the luminal membrane antinatriuretic effect of aldosterone, data concerning its + of the rabbit nephron, independent of any systemic influence. action on Ca2 transport are relatively scarce. Clearance Methods. Proximal and distal tubules were incubated with studies showed either an increase in calciuria [2] or an + either aldosterone or the carrier. The luminal membranes of absence of significant change of Ca2 excretion [3, 4] af- these tubules were purified, vesiculated, and 45Ca uptake by these vesicles was subsequently measured. ter aldosterone administration. In a randomized study in Results. Treatment of the distal tubules with 10−8 mol/L al- male volunteers, Van Hamersvelt et al [5] compared the + + dosterone enhanced both 0.1 and 0.5 mmol/L Ca2 transport. effect of the Ca2 channel blocker felodipine, alone or The hormone action was abolished by tyrosine kinase inhibitors. in association with aldosterone. Felodipine alone had a + 2+ The presence of Na in the medium decreased both Ca up- natriuretic effect, which was attenuated by aldosterone. take and the effect of aldosterone. This hormone action was already significant after a 5-minute incubation, with a half- The action of felodipine was attributed to the proximal maximal efficient concentration of approximately 10−10 mol/L. tubule. However, in this study, calciuria was not mea- + Ca2 transport by the distal membranes presents a dual kinetics. sured. Resnick et al [6] reported the cases of seven hyper- Aldosterone enhanced the Vmax values of both components of tensive patients with primary aldosteronism whose serum these kinetics. Mibefradil abolished the action of aldosterone on 2+ + Ca rose significantly after removal of the aldosterone- 0.5 mmol/L but not on 0.1 mmol/L Ca2 uptake, suggesting that the targeted low affinity channel belongs to the T-type, whereas producing tumor. This rise was thought to be the result diltiazem prevented the hormone action exclusively at the low of a secondary hyperparathyroidism, rather than a direct + + Ca2 concentration (0.1 mmol/L), indicating an effect on a high effect of aldosterone suppression on Ca2 reaborption. affinity L-type channel. + Finally,Kabadi [7] observed a patient with primary hyper- Conclusion. Aldosterone increases Ca2 transport by the dis- 2+ aldosteronism and renal calculi who presented a decline tal luminal membranes through L- and T-type Ca channels, 2+ and this action requires tyrosine kinase activity. in the excretion of Ca and uric acid after spironolactone administration. The author concluded that aldosterone + decreases Ca2 reabsorption. The purpose of the present study was to investigate the It has been known for several decades that 2+ mineralocorticoid-depleted patients have a tendency to effect of aldosterone on Ca transport by the nephron, present hyponatremia, acidosis, and volume contrac- independent of any systemic changes and independent of tion, while mineralocorticoid excess provokes opposite the antinatriuretic action. The hormone did not influence metabolic abnormalities [1]. electrolyte transport by the proximal membranes. In con- trast and unexpectedly, treatment of rabbit distal tubules A number of experiments using several techniques + 2+ showed that aldosterone has a highly significant effect on with aldosterone enhanced both Na and Ca uptake by the corresponding luminal membranes. Key words: aldosterone, renal calcium reabsorption, mineral transport METHODS regulation. Tubule preparation Received for publication October 27, 2003 and in revised form January 19, 2004 Fresh rabbit kidneys were directly obtained from Accepted for publication February 5, 2004 the slaughterhouse. The rabbit strains were heteroge- neous (New Zealand, Satin, Chinchilla, and California) C 2004 by the International Society of Nephrology equally males and females, 9 to 13 weeks old. The renal 242 Leclerc et al: Aldosterone enhances calcium reabsorption by two channels 243 cortical tissue was minced and first digested for 5 min- Table 1. Enzyme activities in the membrane preparations ◦ utes at 35 C in HAMF-12 culture medium equilibrated Alkaline phosphatase Na-K ATPase with 95% O2, and containing 1 mg/mL collagenase type Preparation nmol/lg/15 min nmol/lg/20 min V clostridium histolyticum with 5 mg/mL bovine serum Cortex 2.65 ± 0.24 3.40 ± 0.34 albumin (BSA). The tissue was rinsed with the culture PT homogenates 2.66 ± 0.32 2.98 ± 0.34 medium in a tea strainer and again digested for 20 min- Luminal membranes 14.59 ± 2.01 (×5.48) 2.05 ± 0.27 (×0.68) ◦ Cortex 1.82 ± 0.17 1.70 ± 0.11 utes at 35 C in the same medium containing collagenase, DT homogenates 0.40 ± 0.04 (×0.19) 1.09 ± 0.17 (×0.64) BSA, and 10,000 U/mL DNAse. This new suspension was DT LUM membranes 1.39 ± 0.10 (×0.76) 0.92 ± 0.09 (×0.54) filtered, and the filtrate was centrifuged for 20 seconds Data are mean values ± SEM, N = 12. in a table centrifuge. The collected tubules were rinsed with Krebs-Henseleit buffer (KHB) and BSA, and fi- nally suspended in the culture medium containing 45% and the suspension rapidly filtered through Millipore filters. In 22Na transport experiments, the incubation Percoll equilibrated with 95% O2. After centrifugation for 30 minutes at 28,000g, the distal and proximal tubule medium contained 278 mmol/L mannitol, 20 mmol/L Tris- 22 enriched bands were collected separately and washed HEPES (pH 7.4) and 1 mmol/L NaCl, and the uptake in KHB with BSA to remove the Percoll. The distal was stopped by 1 mL of a solution containing 140 mmol/L tubule suspensions, visualized under the microscope, con- LiCl2 and 20 mmol/L Tris-HEPES (pH 7.4). In each ex- tained a mixture of distal convoluted tubules, connecting periment, the nonspecific binding was measured at time tubules, and cortical collecting ducts. 0 of incubation and substracted from the total uptake. Incubation of the tubules with aldosterone Enzyme marker measurements The tubules were incubated with aldosterone or the The purity of the tubule suspensions and membranes carrier in HAMF-12 at 35◦C for 5 minutes unless other- was monitored by measurement of the specific enzyme wise specified. Incubation was stopped by rapid dilution markers (Table 1). Alkaline phosphatase (the proximal in ice-cold KHB/BSA. The tubules were washed twice tubule luminal membrane specific enzyme) activity was in this solution, and an additional time with KHB with- determined according to the technique of Kelly and out BSA, and then were frozen in 10 mmol/L mannitol, Hamilton [8], and Na-K-ATPase (the basolateral mem- 20 mmol/L Tris-HEPES (pH 7.4) at −80◦C. brane marker) by the technique of Post and Sen [9]. Pre- vious experiments performed with membranes prepared Luminal membrane purification in same manner showed very low activities of succinate- dehydrogenase and glucose-phosphatase, indicating neg- On the day of the experiment, the frozen proximal and ligible contamination with mitochondrial or endoplasmic distal tubules were thawed and homogenized, and the reticulum membranes [10]. luminal membranes purified using the MgCl2 precipita- tion technique: 12 mmol/L MgCl2 (final concentration) was added to the suspension, which was stirred on ice Statistics and calculation for 20 minutes (proximal tubules) or 10 minutes (distal All of the uptake measurements were carried out in tubules) and centrifuged at 3000g for 20 minutes. The su- duplicate, and the figures reflect the mean values ± SEM pernatants were collected and centrifuged at 28,000g for of three to six experiments as indicated. The results were 20 minutes at 4◦C. The membrane pellets were suspended analyzed by the Student unpaired t test. The kinetic values + in 280 mmol/L mannitol with 20 mmol/L Tris-HEPES of Ca2 uptake were calculated according to the nonlin- (pH 7.4), and allowed to vesiculate at 4◦C for one hour. ear regression analysis of Huntson [11]. Passive diffusion + was evaluated by the slope of Ca2 transport at saturating Calcium and sodium transport measurement concentrations. 45Ca and 22Na uptakes by the vesicles were measured by the rapid Millipore filtration technique. Uptakes were Materials ◦ 45 initiated by adding 25 lL of incubation medium at 35 C CaCl2 (carrier free) was obtained from Perkin-Elmer to 5 lL of membrane suspension (approximately 25 lg Life Science (Boston, MA, USA). Aldosterone, can- + protein). In Ca2 transport experiments, the incubation renone, spironolactone, cycloheximide, collagenase V, 45 medium contained, in addition to the CaCl2 at the indi- and other chemicals were obtained from Sigma Aldrich cated concentration, 20 mmol/L Tris-HEPES (pH 7.4) Company (St. Louis, MO, USA). x-Conotoxin MVIIC with either 120 mmol/L NaCl and 20 mmol/L choline was purchased from Calbiochem (San Diego, CA, USA), chloride or 140 mmol/L choline chloride. Each uptake the filters from Millipore Co. (Bedford, MA, USA), and was stopped by addition of 1 mL of 150 mmol/L KCl, Mibefradil was kindly provided by Hoffman Laroche Co. 20 mmol/L Tris-HEPES (pH 7.4), and 2 mmol/L EGTA, (Basel, Switzerland).