Aldosterone potentiates 1,25-dihydroxyvitamin D3 action in renal thick ascending limb via a nongenomic, ERK-dependent pathway

David W. Good, Thampi George, and Bruns A. Watts, III

Department of Medicine and Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555

Submitted 2 April 2003 ; accepted in final form 26 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, we demonstrated that aldosterone inhibits HCO3- absorption in the rat medullary thick ascending limb (MTAL) via a nongenomic pathway blocked by inhibitors of extracellular signal-regulated kinase (ERK) activation. Here we examined the effects on the MTAL of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which regulates cell functions through nongenomic mechanisms in nonrenal systems. Addition of 1 nM 1,25(OH)2D3 to the bath decreased HCO3- absorption by 24%, from 15.0 ± 0.3 to 11.4 ± 0.5 pmol· min-1· mm-1 (P < 0.001). This inhibition was maximal within 60 min and was eliminated by pretreatment with actinomycin D, cycloheximide, or inhibitors of protein kinase C. In MTAL bathed with 1 nM aldosterone [added 15-20 min before 1,25(OH)2D3], the absolute (5.6 ± 0.3 vs. 3.6 ± 0.3 pmol· min-1· mm-1) and fractional (49 ± 2 vs. 24 ± 2%) decreases in HCO3- absorption induced by 1,25(OH)2D3 were significantly greater than those in the absence of aldosterone (P < 0.05). The effect of aldosterone to potentiate inhibition by 1,25(OH)2D3 was not affected by spironolactone but was eliminated by the MAPK kinase/ERK inhibitor U-0126. U-0126 did not affect inhibition of HCO3- absorption by 1,25(OH)2D3 alone. Aldosterone induced rapid activation of ERK via a transcription-independent pathway. We conclude that 1) 1,25(OH)2D3 inhibits HCO3- absorption in the MTAL via a genomic pathway involving protein kinase C, which may contribute to 1,25(OH)2D3-induced regulation of urinary net acid and/or Ca2+ excretion and 2) aldosterone potentiates inhibition by 1,25(OH)2D3 through an ERK-dependent, nongenomic pathway. These results identify a novel regulatory interaction whereby aldosterone acts via nongenomic mechanisms to enhance the genomic response to 1,25(OH)2D3. Aldosterone may influence a broad range of biological processes, including epithelial transport, by modifying the response of target tissues to 1,25(OH)2D3 stimulation.

1,25-dihydroxycholecalciferol; kidney; calcium homeostasis; bicarbonate absorption; acid-base transport


IN ADDITION TO THEIR CLASSICAL actions mediated through regulation of nuclear transcription and protein synthesis, steroid hormones have been recognized recently to induce rapid cellular responses that occur independently of transcription and translation (12). Nongenomic effects have been identified for all groups of steroids, including aldosterone, glucocorticoids, progesterone and estrogen, and vitamin D3 (7-10, 12, 22, 33). The biological and clinical relevance of rapid steroid effects is not well understood. Nongenomic pathways have been implicated in a variety of important processes, including endothelial cell biology, cardiovascular function and blood pressure, fertility, neuronal excitability, and electrolyte homeostasis (7-10, 12, 22, 23, 33, 34). The rapid steroid effects likely are transmitted through cell-surface receptors (10, 12, 23, 36, 46), and there is increasing evidence that steroid hormones may control their own transcriptional regulation through the rapid activation of second messenger pathways (7-10, 12). However, the physiological significance of interactions between nongenomic and genomic mechanisms in steroid-induced regulation is poorly defined.

Aldosterone plays a major role in Na+, K+, and H+ homeostasis through transcriptional regulation of electrolyte transport across renal tubule epithelia (1, 37, 43). Recently, we demonstrated that aldosterone inhibits transepithelial HCO3- absorption in the medullary thick ascending limb (MTAL) of the rat via a pathway that is highly selective, operates independently of transcription and translation, and is not mediated through the classical mineralocorticoid receptor (20). This nongenomic regulation is observed with physiological aldosterone concentrations and is blocked by inhibitors of the extracellular signal-regulated kinase (ERK) signaling pathway (19, 20). These studies provided the first evidence that nongenomic mechanisms mediate steroid-induced regulation of the transport function of renal tubules. Whether steroid hormones in addition to aldosterone influence renal tubule function through nongenomic actions has not been determined. How nongenomic and genomic steroid pathways may interact to regulate renal tubule transport also is unknown.

1{alpha},25-Dihydroxyvitamin D3 [1,25(OH)2D3], the major active metabolite of vitamin D3, plays a crucial role in the regulation of Ca2+ and phosphate balance through its integrated actions on the intestine, kidney, bone, and parathyroid glands (7, 28, 42). Most of the biological actions of 1,25(OH)2D3 are mediated through transcriptional regulation of target genes after the hormone binds to the nuclear vitamin D receptor (VDR) (7, 28). However, 1,25(OH)2D3 also activates nongenomic signaling pathways in some systems (7, 12). 1,25(OH)2D3 influences a variety of renal functions, including reabsorption of Ca2+ and phosphate and excretion of acid (7, 14, 25, 41, 42). It is unclear to what extent these effects are mediated through direct actions of 1,25(OH)2D3 on the renal tubules or through indirect effects of the hormone on plasma Ca2+ concentration, parathyroid hormone (PTH) levels, or other confounding factors (7, 25, 42). The VDR is expressed in multiple nephron segments (26, 30), and 1,25(OH)2D3 can influence Ca2+ transport in renal cell lines and isolated membrane preparations (5, 6, 15, 42). However, whether 1,25(OH)2D3 regulates the transport function of renal tubules directly remains unclear. Whether 1,25(OH)2D3 may influence renal tubule function through nongenomic mechanisms is unknown.

The purpose of the present study was to examine directly the effects of 1,25(OH)2D3 on HCO3- absorption by the MTAL of the rat and to determine whether nongenomic mechanisms are involved in mediating 1,25(OH)2D3-induced transport regulation. Interactions between 1,25(OH)2D3- and aldosterone-induced regulatory pathways also were investigated.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All experimental procedures were approved by the University of Texas Medical Branch Animal Care and Use Committee.

Tubule Perfusion and Measurement of Net HCO3- Absorption

MTAL from male Sprague-Dawley rats (50-100 g body wt; Taconic, Germantown, NY) were isolated and perfused in vitro as described previously (18, 20, 49, 50). The rats had free access to standard chow (NIH 31 diet; Ziegler) and distilled H2O. Tubules were dissected from the inner stripe of the outer medulla at 10°C in control bath solution (see below), transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipettes for perfusion at 37°C. In all experiments, the lumen and bath solutions contained (in mM) 146 Na+, 4 K+, 122 Cl-, 25 HCO3-, 2.0 Ca2+, 1.5 Mg2+, 2.0 phosphate, 1.2 SO42-, 1.0 citrate, 2.0 lactate, and 5.5 glucose (290 mosmol/kgH2O). Bath solutions also contained 0.2% fatty acid-free bovine albumin. All solutions were equilibrated with 95% O2-5% CO2; pH was 7.45 at 37°C. Bath solutions were delivered to the perfusion chamber via a continuously flowing exchange system. To maintain constant pH and O2 tension surrounding the tubules, the bath solutions were delivered through lines jacketed with 95% O2-5% CO2, and the perfusion chamber was suffused continuously with the same gas mixture (21). Experimental agents were added to the bath solutions as described in RESULTS. 1,25(OH)2D3 (Sigma) was prepared as a stock solution in ethanol and diluted into bath solutions to final concentrations given in RESULTS. Solutions containing aldosterone and other experimental agents were prepared as described (18, 20, 50). Equal concentrations of vehicle were added to control solutions in all protocols.

The protocol for study of transepithelial HCO3- absorption has been described elsewhere (18, 20, 49). In most experiments, tubules were equilibrated for 20-30 min at 37°C in the initial perfusion and bath solutions, and the luminal flow rate (normalized per unit tubule length) was adjusted to 1.6-2.0 nl· min-1· mm-1. One to five 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 5-10 min after an experimental agent was added to or removed from the bath solution. In some experiments, longer treatment periods were used. The absolute rate of HCO3- absorption was calculated from the luminal flow rate, and the difference between total CO2 concentrations measured in perfused and collected fluids (18, 20). An average HCO3- absorption rate was calculated for each period studied in a given tubule. When repeat measurements were made at the beginning and end of an experiment (initial and recovery periods), the values were averaged. Single tubule values are presented in Figs. 1, 2, 3, 4, 5. Values are means ± SE; n is the number of tubules. The absolute decrease in HCO3- absorption was calculated for individual tubules as the difference between absorption rates measured in the absence and presence of 1,25(OH)2D3. The fractional decrease in HCO3- absorption is the absolute decrease expressed as a percentage of the control absorption rate measured in the same tubule. Differences between means were evaluated using Student's t-test for paired data or analysis of variance with Newman-Keuls multiple range test, as appropriate. P < 0.05 was considered statistically significant.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] inhibits HCO3- absorption in medullary thick ascending limb (MTAL). A: absolute rate of HCO3- absorption (JHCO3-) was measured under control conditions and after addition of 1 nM 1,25(OH)2D3 to the bath for 60 min. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P value is for paired t-test. Mean values are given in RESULTS. B: time dependence of inhibition of HCO3- absorption by 1 nM 1,25(OH)2D3. Results are expressed as percentage of control transport rate measured in the same tubule. Values are means ± SE for 3-7 tubules at each time point. *P < 0.05, 1,25(OH)2D3 vs. control.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Actinomycin D (Act D) and cycloheximide (Cyclohex) block inhibition by 1,25(OH)2D3. MTAL were bathed with actinomycin D (12.5 µg/ml) for 100 min (A) or cycloheximide (40 µg/ml) for 120 min (B) before addition of 1 nM 1,25(OH)2D3. Tubules were bathed with 1,25(OH)2D3 for >=60 min. JHCO3-, data points, lines, and P values are as described in Fig. 1A legend. NS, not significant.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Protein kinase C inhibitors block inhibition by 1,25(OH)2D3. Tubules were bathed with 10-7 M chelerythrine chloride or 10-7 M staurosporine, and then 1 nM 1,25(OH)2D3 was added to the bath solution. JHCO3-, data points, lines, and P value are as described in Fig. 1A legend. Mean values are given in RESULTS.

 


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4. A and B: aldosterone (Aldo) potentiates inhibition of HCO3- absorption by 1 nM 1,25(OH)2D3. Tubules were bathed for 15-20 min in the absence (A) or presence (B) of 1 nM aldosterone, and then 1,25(OH)2D3 was added to the bath solution. A: control data are repeated from Fig. 1A to facilitate comparison. Initial transport rates in B are less than those in A because of the previously described inhibitory effect of aldosterone (20). C and D: U-0126 blocks potentiation by aldosterone. Tubules were bathed with 15 µM U-0126 (C) or 15 µM U-0126 + 1 nM aldosterone (D), and then 1 nM 1,25(OH)2D3 was added to the bath solution. JHCO3-, lines, data points, and P values are as described in Fig. 1A legend. E and F: summary of results in A-D. Data show absolute (E) and fractional (F) decreases in HCO3- absorption induced by 1,25(OH)2D3. Values are means ± SE; n, number of tubules. Cont, control. *P < 0.05 vs. other 3 conditions.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Spironolactone (Spirono) does not prevent potentiation by aldosterone. MTAL were bathed with 1 nM aldosterone + 10 µM spironolactone, and then 1 nM 1,25(OH)2D3 was added to the bath solution. JHCO3-, data points, lines, and P value are as described in Fig. 1A legend. Mean HCO3 absorption rates and absolute and in HCO3- absorption are given in RESULTS.

 

Determination of ERK Activity

The inner stripe tissue preparation used to study ERK activity in the MTAL has been described (48, 50). Briefly, thin strips of tissue were dissected at 4°C from the inner stripe of the outer medulla, the region of the kidney highly enriched in MTAL (3, 48). The strips were divided into four samples of equal amount and incubated in vitro at 37°C in the same solutions used for HCO3- transport experiments. In one series of experiments, tissue samples were equilibrated in control solution for 90 min in the absence and presence of actinomycin D (12.5 µg/ml) and then maintained in control solution or treated with 1 nM aldosterone for 15 min. In a second series, tissue samples were equilibrated for 30 min in the absence and presence of 15 µM U-0126 before aldosterone treatment. After incubation, the tissue was lysed and ERK1/2 activity was measured in an immune complex kinase assay, with myelin basic protein used as substrate, as described previously (48, 50). Phosphorylated substrate was isolated by SDS-PAGE, visualized by autoradiography, and quantified by densitometry (48, 50). Equal amounts of ERK in different experimental conditions were verified in parallel samples by immunoblotting. We demonstrated previously that changes in protein kinase activities measured in the inner stripe accurately reproduce changes in the MTAL (3, 48, 50).


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of 1,25(OH)2D3 on HCO3- Absorption

1,25(OH)2D3 inhibits HCO3- absorption. Addition of 1 nM 1,25(OH)2D3 to the bath for 60 min decreased HCO3- absorption by 24%, from 15.0 ± 0.3 to 11.4 ± 0.5 pmol· min-1· mm-1 (P < 0.001; Fig. 1A). The time course (Fig. 1B) shows that 1,25(OH)2D3 decreased HCO3- absorption within 40 min, with maximum inhibition at 60 min. The HCO3- absorption rate returned to its control value within 60 min after removal of 1,25(OH)2D3 from the bath solution (Fig. 1A). For subsequent experiments (Figs. 2, 3, 4, 5), HCO3- absorption data show the maximum inhibition by 1 nM 1,25(OH)2D3. 1,25(OH)2D3 added to the bath at 0.1 nM decreased HCO3- absorption by 11%, from 15.3 ± 0.8 to 13.7 ± 0.9 pmol· min-1· mm-1 (n = 3, P < 0.005).

Effects of actinomycin D and cycloheximide. Inhibition of HCO3- absorption by aldosterone in the MTAL is not affected by inhibitors of transcription or translation (20). Similar protocols were used to assess the mechanism of inhibition by 1,25(OH)2D3. MTAL were bathed with the transcription inhibitor actinomycin D (12.5 µg/ml) for 100 min or the protein synthesis inhibitor cycloheximide (40 µg/ml) for 120 min before addition of 1 nM 1,25(OH)2D3. Actinomycin D or cycloheximide blocked completely the inhibition of HCO3- absorption by 1,25(OH)2D3 (Fig. 2). Thus, in contrast to aldosterone, inhibition by 1,25(OH)2D3 is dependent on gene transcription and protein synthesis. Actinomycin D and cycloheximide alone do not affect HCO3- absorption (data not shown).

Effects of protein kinase C inhibitors. Activation of protein kinase C (PKC) mediates certain cellular effects of 1,25(OH)2D3. The role of PKC in the inhibition of HCO3- absorption was examined using chelerythrine chloride and staurosporine, PKC inhibitors that selectively block PKC-dependent regulation of HCO3- absorption in the MTAL (3, 17-19). In tubules bathed with 10-7 M chelerythrine chloride or 10-7 M staurosporine, addition of 1 nM 1,25(OH)2D3 to the bath had no effect on HCO3- absorption: 11.6 ± 0.3, inhibitors vs. 11.7 ± 0.3 pmol· min-1· mm-1, inhibitors + 1,25(OH)2D3 (Fig. 3). These results suggest that 1,25(OH)2D3 inhibits HCO3- absorption via a PKC-dependent pathway.

Interaction of 1,25(OH)2D3 and Aldosterone

1,25(OH)2D3 inhibits HCO3- absorption in the MTAL through gene regulation, whereas inhibition by aldosterone is transcription and translation independent (20). Further experiments were carried out to investigate how these different pathways interact to regulate MTAL function.

Aldosterone potentiates inhibition by 1,25(OH)2D3. In MTAL bathed with 1 nM aldosterone for 15-20 min, 1 nM 1,25(OH)2D3 decreased HCO3- absorption by 48% (from 11.7 ± 0.5 to 6.1 ± 0.4 pmol· min-1· mm-1, P < 0.001) compared with a decrease of 24% observed under the same conditions in the absence of aldosterone (Fig. 4, A and B). The absolute and fractional decreases in HCO3- absorption induced by 1,25(OH)2D3 were significantly greater in the presence than in the absence of aldosterone (Fig. 4, E and F). Thus aldosterone potentiates the inhibitory effect of 1,25(OH)2D3. Actinomycin D blocked inhibition of HCO3- absorption by 1,25(OH)2D3 in the presence of aldosterone: 12.1 ± 0.8, aldosterone + actinomycin D vs. 11.3 ± 0.5 pmol· min-1· mm-1, aldosterone + actinomycin D + 1,25(OH)2D3 (n = 4, P = not significant).

Further studies were designed to determine whether aldosterone influences 1,25(OH)2D3-induced regulation through nongenomic mechanisms. We previously identified two key features of the nongenomic pathway activated by aldosterone in the MTAL: 1) it is blocked by inhibitors of ERK activation, and 2) it is not affected by spironolactone, a competitive antagonist of the classical mineralocorticoid receptor (19, 20). We took advantage of these properties to test whether this non-genomic pathway mediates aldosterone-induced potentiation of 1,25(OH)2D3 action. If it does, then the potentiation by aldosterone should be blocked by ERK inhibitors, but not by spironolactone.

U-0126 blocks potentiation by aldosterone. U-0126 is a selective inhibitor of the MAPK kinase MEK1/2, which directly activates ERK1/2 (13, 50). U-0126 blocks ERK activation in the MTAL and prevents the nongenomic inhibition of HCO3- absorption by aldosterone (19, 48, 50). We therefore tested whether U-0126 would influence the effect of aldosterone to potentiate inhibition by 1,25(OH)2D3. In MTAL bathed with 1 nM aldosterone + 15 µM U-0126, 1 nM 1,25(OH)2D3 decreased HCO3- absorption by 21% (from 14.5 ± 1.1 to 11.4 ± 1.1 pmol· min-1· mm-1, P < 0.001; Fig. 4D), an amount similar to that observed with 1,25(OH)2D3 alone (Fig. 4A). The effect of aldosterone to enhance the absolute and fractional inhibition of HCO3- absorption by 1,25(OH)2D3 was eliminated completely by U-0126 (Fig. 4, E and F). In contrast, U-0126 had no effect on the inhibition of HCO3- absorption by 1,25(OH)2D3 in the absence of aldosterone (Fig. 4, C, E, and F), indicating that ERK is not involved in mediating inhibition by 1,25(OH)2D3 and that the MEK/ERK inhibitor blocked specifically the regulatory action of aldosterone. These results indicate that aldosterone potentiates the inhibition by 1,25(OH)2D3 via an ERK-dependent mechanism and support the view that aldosterone acts via its nongenomic pathway.

Spironolactone does not block potentiation by aldosterone. In MTAL bathed with 1 nM aldosterone + 10 µM spironolactone, 1 nM 1,25(OH)2D3 decreased HCO3- absorption from 11.7 ± 0.5 to 5.5 ± 0.4 pmol· min-1· mm-1 (P < 0.001; Fig. 5). The absolute (6.2 ± 0.2 pmol· min-1· mm-1) and fractional (53 ± 2%) decreases in HCO3- absorption were similar to those induced by 1,25(OH)2D3 in the presence of aldosterone alone and were greater than the decreases observed with 1,25(OH)2D3 in the absence of aldosterone (Fig. 4, E and F). Thus the potentiation of 1,25(OH)2D3 action by aldosterone is not blocked by spironolactone and, therefore, not mediated through the classical mineralocorticoid receptor.

Aldosterone activates ERK via a transcription-independent mechanism. Results of the preceding experiments support the view that aldosterone acts through a nongenomic pathway involving ERK to potentiate inhibition by 1,25(OH)2D3. To confirm directly that aldosterone activates ERK via a nongenomic mechanism, inner stripe tissue was incubated in vitro in the absence and presence of aldosterone for 15 min, and then ERK activity was assessed by immune complex assay (48, 50). Incubation with 1 nM aldosterone increased ERK activity 1.6 ± 0.1-fold (n = 3, P < 0.05), and this increase was not prevented by pretreatment with actinomycin D (Fig. 6, A and B). These results demonstrate that aldosterone increases ERK activity via a transcription-independent pathway. The aldosterone-induced ERK activation was blocked completely by pretreatment with 15 µM U-0126 (Fig. 6C). Immunoblot analysis of inner stripe samples using antiphospho-ERK1/2 antibody showed that aldosterone increased ERK phosphorylation (results not shown).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. Aldosterone activates ERK via a transcription-independent pathway. A: inner stripe tissue was incubated for 90 min in the absence (Cont) and presence (Act D) of 12.5 µg/ml actinomycin D and then treated with 1 nM aldosterone for 15 min in the absence (Aldo) or continued presence (Act D + Aldo) of the inhibitor. ERK activity was measured by immune complex assay, with myelin basic protein (MBP) used as substrate. Phosphorylated substrate was detected after SDS-PAGE by autoradiography and quantified by densitometry. Results are representative of 3 separate experiments. Relative band intensities are given in RESULTS. B: parallel samples assessed by immunoblotting with anti-ERK1/2 antibody verify equal protein amounts in all conditions. C: U-0126 blocks activation of ERK by aldosterone. Inner stripe tissue was incubated for 30 min in the absence (Cont) and presence (U-0126) of 15 µM U-0126 and then treated with 1 nM aldosterone for 15 min in the absence (Aldo) or continued presence (U-0126 + Aldo) of the inhibitor. ERK activity was measured as described in A and is presented as a percentage of control activity measured in the same experiment. Values are means ± SE for 3 separate experiments. *P < 0.05 vs. other 3 conditions.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major findings of this study are that 1) 1,25(OH)2D3 inhibits HCO3- absorption in the MTAL via a genomic pathway likely involving PKC, and 2) aldosterone potentiates the inhibition by 1,25(OH)2D3 through a nongenomic pathway involving ERK activation. These results, to our knowledge, are the first in any system to identify a direct interaction between aldosterone and 1,25(OH)2D3 in the regulation of cell function and demonstrate that this interaction is the result of cross talk between nongenomic and genomic steroid hormone signaling pathways. Although nongenomic actions of aldosterone have been demonstrated in a variety of cell systems (12, 33), their physiological relevance to the regulation of epithelial transport has been unclear. Our results in the MTAL establish that nongenomic pathways activated by aldosterone not only regulate transepithelial transport directly (20) but also can modify the transport response to other, dissimilar steroid hormones.

1,25(OH)2D3 Inhibits HCO3- Absorption in the MTAL

1,25(OH)2D3 influences renal excretion of Ca2+, phosphate, SO42-, and net acid, but very little is known about the direct effects of this hormone on the transport function of individual nephron segments (7, 14, 25, 28, 41, 42). Our results are the first to identify the MTAL as a target for direct transport regulation by 1,25(OH)2D3. 1,25(OH)2D3 inhibited HCO3- absorption in the MTAL at 0.1 and 1.0 nM. These values encompass the range of circulating 1,25(OH)2D3 levels in serum (0.1-0.4 nM) (6, 16, 39, 47) and are similar to 1,25(OH)2D3 concentrations reported to stimulate Ca2+ transport in cultured distal tubule and collecting duct cells (5, 15). It is possible that intrarenal levels of 1,25(OH)2D3 may exceed concentrations in systemic plasma as the result of 1,25(OH)2D3 production by the proximal tubule. We found that 1,25(OH)2D3 inhibits HCO3- absorption in the MTAL at concentrations at or above systemic plasma levels, indicating that the transport effects are likely to represent physiologically relevant regulation.

1,25(OH)2D3 inhibits HCO3- absorption in the MTAL by an amount quantitatively similar to that observed with aldosterone; however, the mechanisms of action of the two steroids are different. Aldosterone inhibits HCO3- absorption via a nongenomic pathway that is blocked by MEK inhibitors, but not by PKC inhibitors (19, 20). In contrast, 1,25(OH)2D3 inhibits HCO3- absorption via a genomic pathway that is blocked by PKC inhibitors, but not by MEK inhibitors. Transport regulation by 1,25(OH)2D3 in the MTAL depends on gene transcription and protein synthesis, as evidenced by 1) its complete inhibition by actinomycin D or cycloheximide and 2) the time course, which demonstrates a lag period of up to 40 min for the 1,25(OH)2D3-induced inhibition. The MTAL expresses the VDR (26, 30), a member of the nuclear receptor superfamily that mediates 1,25(OH)2D3-induced transcriptional regulation (7, 28). Whether the VDR is involved in inhibition of HCO3- absorption in the MTAL remains to be determined.

The Na+/H+ exchanger isoform NHE3 is localized selectively in the apical membrane of certain renal and intestinal epithelial cells, where it mediates transepithelial reabsorption of NaCl and/or NaHCO3 (35, 44). It is unknown whether the activity of this transporter is regulated by 1,25(OH)2D3. NHE3 mediates virtually all the luminal H+ secretion necessary for HCO3- absorption in the MTAL (2, 4, 21, 49). Thus it is highly likely that 1,25(OH)2D3 inhibits NHE3 in the MTAL. Further direct studies of the effect of 1,25(OH)2D3 on the apical Na+/H+ exchanger are needed to determine whether the 1,25(OH)2D3-induced signaling pathway is coupled directly to inhibition of the exchanger or whether 1,25(OH)2D3 may act indirectly to decrease exchanger activity through effects on another transporter, such as basolateral HCO3- efflux pathways or the Na+-K+-ATPase. It is noteworthy that inhibition of HCO3- absorption by 1,25(OH)2D3 likely involves PKC, which has been reported to inhibit NHE3 in some systems (35, 44). However, in the MTAL, neither activation nor inhibition of PKC by itself leads to inhibition of HCO3- absorption (3, 18). Thus it is unlikely that PKC is a direct mediator of 1,25(OH)2D3-induced inhibition of apical Na+/H+ exchange activity.

Our results provide the first evidence for direct regulation of transepithelial H+ secretion by 1,25(OH)2D3 in a nephron segment. The inhibition of HCO3- absorption by 1,25(OH)2D3 in the MTAL may play a role in acid-base regulation. For example, metabolic acidosis can adversely affect the production of 1,25(OH)2D3 through several mechanisms: 1) decreasing the activity of the 1{alpha}-hydroxylase, which catalyzes the formation of 1,25(OH)2D3 from 25-hydroxyvitamin D3 in the proximal tubule, 2) blunting the action of PTH to stimulate renal 1{alpha}-hydroxylase activity, and 3) increasing the activity of the renal 24-hydroxylase that catalyzes 1,25(OH)2D3 degradation (7, 29, 31, 41). On the basis of results of the present study, decreased 1,25(OH)2D3 levels in acidosis could lead to increased H+ secretion and HCO3- absorption by the MTAL, which would aid in limiting or correcting the acidemia. In addition, 1,25(OH)2D3 may directly affect other MTAL transport functions. Recent studies in rats show that chronic hypercalcemia induced by 1,25(OH)2D3 administration reduces the abundance of the apical Na+-K+-2Cl- cotransporter (BSC-1 or NKCC2) and the apical K+ channel (ROMK), effects that would reduce MTAL NaCl absorption and contribute to the urinary concentrating defect associated with hypercalcemia (45). The adaptations in the two MTAL transporters were explained on the basis of renal and cellular responses to hypercalcemia (45). However, our results raise the additional possibility that the adaptations in BSC-1 and ROMK observed in these studies may involve direct receptor-mediated effects of 1,25(OH)2D3 on the MTAL.

Aldosterone Potentiates Inhibition by 1,25(OH)2D3

In the presence of aldosterone, the inhibition of HCO3- absorption by 1,25(OH)2D3 was markedly enhanced. The time course of inhibition by 1,25(OH)2D3 was not affected by aldosterone, and actinomycin D eliminated the 1,25(OH)2D3-induced inhibition in the absence or presence of aldosterone, supporting the view that aldosterone upregulates the genomic pathway responsible for 1,25(OH)2D3-induced regulation. Several lines of evidence support the conclusion that aldosterone potentiates 1,25(OH)2D3 action via a nongenomic, ERK-dependent pathway: 1) aldosterone activates ERK via a rapid, transcription-independent mechanism, 2) inhibitors of ERK activation prevent nongenomic inhibition of HCO3- absorption by aldosterone (19, 20), 3) inhibitors of ERK activation eliminate the potentiating effect of aldosterone on 1,25(OH)2D3 but have no effect on the inhibition of HCO3- absorption by 1,25(OH)2D3 alone, 4) 1,25(OH)2D3 action is potentiated by short-term (15 min) aldosterone treatment, and 5) potentiation by aldosterone is not prevented by spironolactone, indicating that it does not involve the classical mineralocorticoid receptor. Much recent attention has been focused on the possibility that steroid hormones may act through genomic and nongenomic mechanisms simultaneously, whereby rapid activation of nongenomic signals by a steroid may, in turn, modulate its genomic response (8-10, 12). Our results broaden this concept by demonstrating that the activation of nongenomic pathways by one steroid can modify the genomic response to another, dissimilar steroid. These findings identify novel mechanisms through which aldosterone may influence electrolyte transport in the kidney and through which tissue-specific regulation by aldosterone could be achieved. In addition, they present new therapeutic options whereby the biological response to a particular steroid such as 1,25(OH)2D3 could be modified through pharmacological manipulation of nongenomic mechanisms activated by a different steroid hormone in the same cell.

The mechanism(s) by which aldosterone potentiates regulation by 1,25(OH)2D3 remain to be defined, but some possibilities can be considered on the basis of findings in other systems. The interaction could involve cross talk between the aldosterone-induced ERK pathway and PKC, which appears to be an essential component of the 1,25(OH)2D3 regulatory pathway. Alternatively, the ERK-dependent pathway activated by aldosterone could alter the transcriptional activity of the VDR through receptor phosphorylation or by alteration of the activity of coactivator proteins (28). One such coactivator may be the retinoid X receptor, which heterodimerizes with the VDR and is essential for its DNA-binding and transcriptional activity (7, 28). Recent work suggests that the retinoid X receptor can be specifically phosphorylated and regulated through the Ras-MEK-ERK cascade (11, 27). These and other possible mechanisms need to be tested experimentally in the future. Our results suggest, however, that ERK is an important mediator of cross talk between nongenomic and genomic steroid regulatory pathways.

Physiological Significance and Clinical Implications

The direct action of 1,25(OH)2D3 to inhibit HCO3- absorption in the MTAL and the ability of aldosterone to enhance 1,25(OH)2D3-induced regulation have important implications on several levels. 1) As discussed above, increased MTAL HCO3- absorption in response to decreased 1,25(OH)2D3 levels may facilitate acid excretion in metabolic acidosis. 2) Infusion of HCO3- and induction of bicarbonaturia in acidotic animals decreased renal Ca2+ excretion and enhanced Ca2+ absorption by the distal convoluted tubule independent of PTH, consistent with an effect of luminal HCO3- to directly enhance Ca2+ absorption (42). It is therefore possible that the primary inhibition of HCO3- absorption by 1,25(OH)2D3 in the MTAL could secondarily enhance Ca2+ absorption in the distal convoluted tubule and other distal nephron segments by increasing luminal HCO3- delivery to those downstream segments. 3) Extracellular fluid volume has an important influence on renal Ca2+ handling, with volume depletion decreasing and volume expansion increasing Ca2+ excretion (42). Our findings suggest the novel possibility that direct aldosterone-1,25(OH)2D3 interactions could contribute to this relation. For example, 1,25(OH)2D3-dependent stimulation of Ca2+ absorption in distal nephron segments could be potentiated by increased aldosterone levels during volume depletion. Conceivably that interaction could promote hypercalcemia that may contribute to vasoconstriction in volume-contracted states. Direct tests of these hypotheses will require future studies of the interacting effects of aldosterone and 1,25(OH)2D3 on Ca2+ absorption in multiple nephron segments and direct analysis of the role of luminal HCO3- as a determinant of distal nephron Ca2+ absorption.

The discovery of a functional interaction between 1,25(OH)2D3 and aldosterone has additional, broader implications. In addition to its role in Ca2+ and phosphate homeostasis, 1,25(OH)2D3 influences a wide variety of other biological processes, including the immune response, insulin secretion, cardiovascular function, and blood pressure (7, 24, 28, 40, 47). Several 1,25(OH)2D3 target tissues, including intestinal epithelial cells and the cardiovascular system, also have been identified as sites of nongenomic aldosterone regulation (12, 22, 33). Our results raise the possibility that aldosterone could influence a broad range of physiological and pathophysiological processes, such as intestinal Ca2+ absorption, vascular inflammation and calcification, and blood pressure, by modifying the response of target tissues to 1,25(OH)2D3 stimulation. This possibility is supported by recent work indicating that aldosterone may influence blood pressure by acting through nongenomic mechanisms to modulate vascular responses to adrenergic stimulation (33, 38). Finally, 1,25(OH)2D3 has been identified recently as a regulator of the renin-angiotensin-aldosterone system (32). In normal mice, inhibition of 1,25(OH)2D3 synthesis increased, whereas 1,25(OH)2D3 administration decreased, renin production. Furthermore, in VDR-null mice, renin production was elevated, leading to increased angiotensin II levels and hypertension (32). Our results suggest that increased aldosterone levels induced by 1,25(OH)2D3 deficiency could enhance 1,25(OH)2D3 actions in the kidney and other target tissues, thereby maintaining 1,25(OH)2D3-dependent regulation despite reduced circulating hormone levels. In this way, aldosterone could serve as the effector in a feedback system whereby the renin-angiotensin-aldosterone system helps mitigate against the highly detrimental systemic consequences of 1,25(OH)2D3 deficiency.


    DISCLOSURES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38217 and American Heart Association, Texas Affiliate, Grant-in-Aid 0255406Y.


    ACKNOWLEDGMENTS
 
We thank W. Mitch for valuable discussion and critical reading of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. W. Good, 4.200 John Sealy Annex, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0562 (E-mail: dgood{at}utmb.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Alpern RJ. Renal acidification mechanisms. In: The Kidney, edited by Brenner BM. Philadelphia, PA: Saunders, 2000, vol. I, p. 455-519.

2. Amemiya M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, and Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney Int 48: 1206-1215, 1995.[ISI][Medline]

3. Aristimuño PC and Good DW. PKC isoforms in rat medullary thick ascending limb: selective activation of the {delta}-isoform by PGE2. Am J Physiol Renal Physiol 272: F624-F631, 1997.[Abstract/Free Full Text]

4. Biemesderfer D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson PS. Monoclonal antibodies for high-resolution localization of NHE-3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997.[Abstract/Free Full Text]

5. Bindels RJM, Hartog A, Timmerman J, and Van Os CH. Active Ca2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH. Am J Physiol Renal Fluid Electrolyte Physiol 261: F799-F807, 1991.[Abstract/Free Full Text]

6. Bouhtiauy I, Lajeunesse D, and Brunette MG. Effect of vitamin D depletion on calcium transport by the luminal and basolateral membranes of proximal and distal nephrons. Endocrinology 132: 115-120, 1993.[Abstract]

7. Brown AJ, Dusso A, and Slatopolsky E. Vitamin D. Am J Physiol Renal Physiol 277: F157-F175, 1999.[Abstract/Free Full Text]

8. Chen YZ and Qiu J. Possible genomic consequences of nongenomic action of glucocorticoids in neural cells. News Physiol Sci 16: 292-296, 2001.[Abstract/Free Full Text]

9. Coleman KM and Smith CL. Intracellular signaling pathways: nongenomic actions of estrogens and ligand-independent activation of estrogen receptors. Front Biosci 6: D1379-D1391, 2001.[ISI][Medline]

10. Davis PJ, Tillman HC, Davis FB, and Wehling M. Comparison of the mechanisms of nongenomic actions of thyroid hormone and steroid hormones. J Endocrinol Invest 25: 377-388, 2002.[ISI][Medline]

11. Dwivedi PP, Hii CS, Ferrante A, Tan J, Der CJ, Omdahl JL, Morris HA, and May BK. Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5. J Biol Chem 277: 29643-29653, 2002.[Abstract/Free Full Text]

12. Falkenstein E, Tillmann HC, Christ M, Feuring M, and Wehling M. Multiple actions of steroid hormones—a focus on rapid, nongenomic effects. Pharmacol Rev 52: 513-555, 2000.[Abstract/Free Full Text]

13. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Strodley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 18623-18632, 1998.[Abstract/Free Full Text]

14. Fernandes I, Hampson G, Cahours X, Morin P, Coureau C, Couette S, Prie D, Biber J, Murer H, Friedlander G, and Silve C. Abnormal sulfate metabolism in vitamin D-deficient rats. J Clin Invest 100: 2196-2203, 1997.[Abstract/Free Full Text]

15. Friedman PA and Gesek FA. Vitamin D3 accelerates PTH-dependent calcium transport in distal convoluted tubule cells. Am J Physiol Renal Fluid Electrolyte Physiol 265: F300-F308, 1993.[Abstract/Free Full Text]

16. Gepp H, Koch M, Schwille PO, Erben RG, Rumenapf G, Schmiedl A, and Fries W. Vagus-sparing gastric fundectomy in the rat: development of osteopenia, relationship to urinary phosphate and net acid excretion, serum gastrin, and vitamin D. Res Exp Med (Berl) 200: 1-16, 2000.[Medline]

17. Good DW. Hyperosmolality inhibits HCO3- absorption in rat medullary thick ascending limb via a protein tyrosine kinase-dependent pathway. J Biol Chem 270: 9883-9889, 1995.[Abstract/Free Full Text]

18. Good DW. PGE2 reverses AVP inhibition of HCO3- absorption in rat MTAL by activation of protein kinase C. Am J Physiol Renal Fluid Electrolyte Physiol 270: F978-F985, 1996.[Abstract/Free Full Text]

19. Good DW, George T, and Watts BA III. Aldosterone inhibits HCO3- absorption via a nongenomic pathway in rat medullary thick ascending limb (Abstract). J Am Soc Nephrol 12: 4A, 2001.

20. Good DW, George T, and Watts BA III. Aldosterone inhibits HCO3- absorption via a nongenomic pathway in medullary thick ascending limb. Am J Physiol Renal Physiol 283: F699-F706, 2002.[Abstract/Free Full Text]

21. Good DW and Watts BA III. Functional roles of apical membrane Na+/H+ exchange in rat medullary thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 270: F691-F699, 1996.[Abstract/Free Full Text]

22. Harvey BJ, Condliffe S, and Doolan CM. Sex and salt hormones: rapid effects in epithelia. News Physiol Sci 16: 174-177, 2001.[Abstract/Free Full Text]

23. Haseroth K, Christ M, Falkenstein E, and Wehling M. Aldosterone- and progesterone-membrane-binding proteins: new concepts of nongenomic steroid action. Curr Protein Pept Sci 1: 385-401, 2000.[Medline]

24. Holick MF. Sunlight and vitamin D: both good for cardiovascular health. J Gen Intern Med 17: 733-735, 2002.[ISI][Medline]

25. Hulter HN. Effects and interrelationships of PTH, Ca2+, vitamin D, and Pi in acid-base homeostasis. Am J Physiol Renal Fluid Electrolyte Physiol 248: F739-F752, 1985.[Abstract/Free Full Text]

26. Iida K, Taniguchi S, and Kurokawa K. Distribution of 1,25-dihydroxyvitamin D3 receptor and 25-hydroxyvitamin D3-24-hydroxylase mRNA expression along rat nephron segments. Biochem Biophys Res Commun 194: 659-664, 1993.[ISI][Medline]

27. Ishaq M, Fan M, and Natarajan V. Accumulation of RXR{alpha} during activation of cycling human T lymphocytes: modulation of RXRE transactivation function by mitogen-activated protein kinase pathways. J Immunol 165: 4217-4225, 2000.[Abstract/Free Full Text]

28. Jones G, Strugnell SA, and DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 78: 1193-1231, 1998.[Abstract/Free Full Text]

29. Kawashima H, Kraut JA, and Kurokawa K. Metabolic acidosis suppresses 25-hydroxyvitamin D3-1{alpha}-hydroxylase in the rat kidney. Distinct site and mechanism of action. J Clin Invest 70: 135-140, 1982.[ISI][Medline]

30. Kawashima H and Kurokawa K. Localization of receptors for 1,25-dihydroxyvitamin D3 along the rat nephron. J Biol Chem 257: 13428-13432, 1982.[Free Full Text]

31. Lee SW, Russell J, and Avioli LV. 25-Hydroxycholecalciferol to 1,25-dihydroxycholecalciferol: conversion impaired by systemic metabolic acidosis. Science 195: 994-996, 1977.[ISI][Medline]

32. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, and Cao LP. 1,25-Dihydroxyvitamin D3 is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest 110: 229-238, 2002.[Abstract/Free Full Text]

33. Losel RM, Feuring M, Falkenstein E, and Wehling M. Nongenomic effects of aldosterone: cellular aspects and clinical implications. Steroids 67: 493-498, 2002.[ISI][Medline]

34. Mendelsohn ME. Genomic and nongenomic effects of estrogen in the vasculature. Am J Cardiol 90: 3F-6F, 2002.[ISI][Medline]

35. Moe OW. Acute regulation of proximal tubule apical membrane Na+/H+ exchanger NHE-3: role of phosphorylation, protein trafficking, and regulatory factors. J Am Soc Nephrol 10: 2412-2425, 1999.[Free Full Text]

36. Norman AW, Ishizuka S, and Okamura WH. Ligands for the vitamin D endocrine system: different shapes function as agonists and antagonists for genomic and rapid response receptors or as a ligand for the plasma vitamin D binding protein. J Steroid Biochem Mol Biol 76: 49-59, 2001.[ISI][Medline]

37. Rossier BC and Palmer LG. Mechanisms of aldosterone actions on sodium and potassium transport. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. New York: Raven, 1992, p. 1373-1409.

38. Schmidt BM, Georgens HC, Martin N, Tillmann HC, Feuring M, Christ M, and Wehling M. Interaction of rapid nongenomic cardiovascular aldosterone effects with the adrenergic system. J Clin Endocrinol Metab 86: 761-767, 2001.[Abstract/Free Full Text]

39. Seely EW, Brown EM, DeMaggio DM, Weldon DK, and Graves SW. A prospective study of calciotropic hormones in pregnancy and post partum: reciprocal changes in serum intact parathyroid hormone and 1,25-dihydroxyvitamin D. Am J Obstet Gynecol 176: 214-217, 1997.[ISI][Medline]

40. Sigmund CD. Regulation of renin expression and blood pressure by vitamin D3. J Clin Invest 110: 155-156, 2002.[Free Full Text]

41. Stim JA, Bernardo AA, and Arruda JA. The role of parathyroid hormone and vitamin D in acid excretion and extrarenal buffer mobilization. Miner Electrolyte Metab 20: 60-71, 1994.[ISI][Medline]

42. Suki W, Lederer ED, and Rouse D. Renal transport of calcium, magnesium, and phosphate. In: The Kidney, edited by Brenner BM. Philadelphia, PA: Saunders, 2000, vol. I, p. 520-574.

43. Verrey F. Early aldosterone action: toward filling the gap between transcription and transport. Am J Physiol Renal Physiol 277: F319-F327, 1999.[Abstract/Free Full Text]

44. Wakabayashi S, Shigekawa M, and Pouyssegur J. Molecular physiology of vertebrate Na+/H+ exchangers. Physiol Rev 77: 51-74, 1997.[Abstract/Free Full Text]

45. Wang W, Kwon TH, Frokiaer LC, Knepper MA, and Nielsen S. Reduced expression of Na-K-2Cl cotransporter in medullary TAL in vitamin D-induced hypercalcemia in rats. Am J Physiol Renal Physiol 282: F34-F44, 2002.[Abstract/Free Full Text]

46. Watson CS and Gametchu B. Membrane estrogen and glucocorticoid receptors—implications for hormonal control of immune function and autoimmunity. Int Immunopharmacol 1: 1049-1063, 2001.[ISI][Medline]

47. Watson KE, Abrolat ML, Malone LL, Hoeg JM, Doherty T, Detrano R, and Demer LL. Active serum vitamin D levels are inversely correlated with coronary calcification. Circulation 96: 1755-1760, 1997.[Abstract/Free Full Text]

48. Watts BA III, Di Mari JF, Davis RJ, and Good DW. Hypertonicity activates MAP kinases and inhibits HCO3- absorption via distinct pathways in thick ascending limb. Am J Physiol Renal Physiol 275: F478-F486, 1998.[Abstract/Free Full Text]

49. Watts BA III and Good DW. Hyposmolality stimulates apical membrane Na+/H+ exchange and HCO3- absorption in renal thick ascending limb. J Clin Invest 104: 1593-1602, 1999.[Abstract/Free Full Text]

50. Watts BA III and Good DW. Extracellular signal-regulated kinase mediates inhibition of Na+/H+ exchange and HCO3- absorption by nerve growth factor in MTAL. Am J Physiol Renal Physiol 282: F1056-F1063, 2002.[Abstract/Free Full Text]