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Am J Physiol Renal Physiol 293: F601-F606, 2007. First published June 6, 2007; doi:10.1152/ajprenal.00085.2007
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Calcium regulation of endothelin-1 synthesis in rat inner medullary collecting duct

Kevin A. Strait, Peter K. Stricklett, Jessica L. Kohan, Margaux B. Miller, and Donald E. Kohan

Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City, Utah

Submitted 16 February 2007 ; accepted in final form 1 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Collecting duct-derived endothelin-1 (ET-1) reduces blood pressure and inhibits Na and water reabsorption. Collecting duct ET-1 production is increased by volume expansion; however, the mechanism by which this occurs is unknown. We hypothesized that intracellular calcium, which is likely to be increased by volume expansion, regulates collecting duct ET-1 synthesis. Rat inner medullary collecting ducts (IMCD) were studied in primary culture. ET-1 release was decreased by 50–70% after chelation of intracellular calcium (BAPTA) or inhibition of CaM (W7) or CaMK (KN-93). These agents reduced ET-1 mRNA to a similar degree. CaM inhibition did not affect ET-1 mRNA stability. Transfection of IMCD with rat ET-1 promoter-luciferase constructs revealed maximal activity within 1.7 kb 5' to the transcription start site; 5, 20, 35, and 90% of this activity were in the 0.08-, 0.37-, 1.0-, and 3.0-kb promoter regions, respectively. W7 markedly inhibited activity of the 3.0-kb but not 0.37- or 1.0-kb promoter regions. In contrast, W7 did not affect ET-1 release by rat aortic endothelial cells. Furthermore, transfected endothelial cells had maximal activity in the 0.37-kb region (as compared with the 1.7- and 3.0-kb regions), whereas W-7 had no effect on the activity of any of these promoter regions. In summary, IMCD ET-1 synthesis is regulated by calcium/CaM/CaMK-dependent pathways. The calcium/CaM-sensitive pathway is active in IMCD, but not endothelial cells. This suggests that IMCD-specific enhancer elements exist within the ET-1 promoter that confer unique calcium responsiveness.

calmodulin; mRNA; promoter

ENDOTHELIN-1 (ET-1) is an autocrine inhibitor of collecting duct Na and water reabsorption. This nephron segment produces and binds relatively large amounts of the peptide (reviewed in Ref. 9). In vitro studies indicate that ET-1 can inhibit collecting duct water reabsorption (30) and Na-K-ATPase activity (34), as well as reduce epithelial Na channel activity in distal nephron cells (17). Mice with collecting duct-specific disruption of the ET-1 gene are hypertensive and have impaired renal Na and water excretion (1). Similarly, mice with collecting duct-specific knockout of the endothelin B receptor gene develop hypertension as well as reduced ability to excrete an acute Na load (4). Decreased collecting duct ET-1 production is associated with hypertension in animal models, while urinary ET-1 excretion has been reported to be reduced in patients with essential hypertension (reviewed in Ref. 10). Taken together, the above data indicate that collecting duct-derived ET-1 is an important regulator of systemic blood pressure and renal salt and water excretion.

Numerous studies showed that renal medullary and urinary ET-1 production is increased by salt or water loading, suggesting that collecting duct-derived ET-1 is stimulated in response to expanded extracellular fluid volume or some related process (reviewed in Ref. 10). However, the factors regulating collecting duct ET-1 production are not well understood. Circulating hormones that modify renal Na and/or water reabsorption are potential candidates for regulating collecting duct ET-1 synthesis. However, none of these hormones has been shown to mediate an increased ET-1 under volume-expanded states. Atrial natriuretic peptide, which is elevated during volume expansion, either modestly decreased (12) or did not change (22) ET-1 release by inner medullary collecting duct (IMCD). Hormones which are decreased during volume expansion could potentially exert a tonic inhibitory effect on IMCD ET-1 production, but current data do not support this. ANG II infusion does not alter urinary ET-1 excretion (8). Vasopressin does not change ET-1 release from porcine or rat IMCD (11, 22). Aldosterone, which would be expected to exert an inhibitory effect on IMCD ET-1 production, rapidly induced ET-1 mRNA accumulation in a mouse IMCD cell line (mIMCD-3 cells) (5); this suggests that ET-1 may serve as a negative feedback regulator of aldosterone-stimulated Na reabsorption. Another possibility is that renal nerve activity could inhibit collecting duct ET-1 production; decreased renal nerve activity, as occurs during volume expansion, could mediate increased collecting duct ET-1 release. However, neither epinephrine (11) nor norepinephrine (unpublished observations by our group) had any effect on ET-1 release by rat IMCD. Hence, there is no evidence to date that circulating hormones or renal nerves mediate the volume expansion-associated increase in collecting duct ET-1 production.

Physical factors that are altered during volume expansion might also mediate changes in collecting duct ET-1 production. High Na intake has been demonstrated to increase medullary tonicity which, in turn, augments thick ascending limb ET-1 production, leading to increased endothelial nitric oxide synthase activity and inhibition of chloride transport (6, 7). The effect of extracellular tonicity on collecting duct ET-1 production is, however, controversial. Media made hypertonic with NaCl or other relatively impermeant solutes have been reported to increase Madin-Darby canine kidney (MDCK; a distal nephron cell line) ET-1 production (14). In contrast, media hypertonicity due to NaCl or mannitol reduced collecting duct ET-1 synthesis in our hands and in others (13, 29). Increased tubule flow rate, as occurs during Na or water loading, is an interesting possibility for stimulating collecting duct ET-1 release. Recent studies indicate that elevated luminal flow rate can be detected by collecting duct cells through bending of primary cilia leading to increased intracellular calcium concentration (18, 19, 27). Direct examination of the effect of luminal flow on collecting duct ET-1 production has not been successfully performed, most likely due to technical difficulties in detecting changes in ET-1 production. However, examination of the effects of intracellular calcium, as well as related signaling pathways [calmodulin (CaM)], could be more readily accomplished and could well provide clues as to how extracellular fluid volume might regulate collecting duct ET-1 synthesis. Consequently, the current study was undertaken to examine whether, and how, changes in intracellular calcium concentration, and associated signaling pathways, might modify collecting duct ET-1 production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Types 1 and 2 collagenase were obtained from Worthington (Lakewood, NJ); penicillin, streptomycin, and glutamine from Invitrogen (Carlsbad, CA); culture dishes from BD Falcon (Franklin Lakes, NJ); pGEM-T cloning vector from Promega (Madison, WI); G-50 columns from Roche (Indianapolis, IN); and A23187 [GenBank] , ionomycin, KN-62, KN-93, actinomycin D, and W-7 from Calbiochem (San Diego, CA). All other reagents and materials were obtained from Sigma (St. Louis, MO) unless stated otherwise.

Cell culture. Rat IMCD fragments were isolated by removing the inner medulla, mincing, and incubating in HBSS containing 1 mg/ml type I collagenase + 0.1 mg/ml DNase at 37°C for 40 min. Our protocols were submitted to, and approved by, the University of Utah Institutional Animal Care and Use Committee. The digest was shaken vigorously several times during the incubation, osmotically shocked by addition of 1.5 volumes water, and centrifuged at 400 g for 5 min. The pellet was washed in HBSS + 10% BSA, centrifuged, resuspended in renal epithelial growth media (REGM; Cambex, Watersville, MD), and plated on plastic culture plates. Cells were grown at 37°C in 5% CO2 until confluence. After being confluent for no more than a day, cells were growth arrested in DMEM/F12 without phenol red + 1,000 U/ml penicillin, 1,000 µg/ml streptomycin, and 0.292 mg/ml glutamine for 24 h and then studied immediately. IMCD cell culture was modified for transfection experiments as described in that section.

Primary cultures of rat aorta endothelial cells were prepared by a modification of the method of Kwan et al. (15). Briefly, the vasculature was perfused via cardiac puncture with HBSS, the thoracic aorta was removed, sliced open lengthwise, cut into 2-mm sections, and incubated in 0.2% type II collagenase/HBSS at 37°C with gentle agitation for 15 min. The aortic pieces were then removed and the supernatant was centrifuged at 1,500 rpm for 5 min. The resulting pellet was resuspended in 10% BSA/HBSS and recentrifuged at 800 rpm for 5 min. The final pellet was resuspended in RPMI 1640/10% FBS, aliquoted onto 24-well culture plates previously coated with 0.2% gelatin, and grown in at 37°C in 5% CO2 until confluent (~5–6 days).

ET-1 release. In general, IMCD or aortic endothelial cells were incubated with vehicle or test compound for 1 h in growth arrest media. The media was then discarded and fresh media plus vehicle, agonist, or antagonist was added for 4 h at 37°C in 5% CO2. After incubation, media was transferred to a siliconized microfuge tube and stored frozen. On the day of analysis, media was vacuum concentrated to dryness and ET-1 content was determined using a QuantiGlo ET-1 enzyme immunoassay (R&D Systems, Minneapolis, MN). Luminescence was measured using a Molecular Devices LMaxII Luminometer and Softmax Pro 4.7 data analysis program. Cells were dissolved in 0.1 N NaOH and protein content was determined using the Bradford protein assay.

RNA analysis. Riboprobes were made from primary cultures of rat IMCD. RNA was reverse transcribed and the resultant cDNA was used as template for PCR amplification of the ET-1 coding region using primers designed to amplify 552 bp of rat ET-1 cDNA–ET-1 5'-primer 5'-GGC TTT CCA AGG AGC TCC AGA-3' and ET-1 3'-primer 5'-ATC AAC TTC TGG TCT CTG TAG AG-3'. The amplified product was purified and cloned into pGEM-T cloning vector. The insert was sequenced to ensure cloning fidelity and orientation. The probe was digested to give the antisense strand and riboprobes made using 32P-UTP and T7 RNA polymerase. Radioactive probe was purified over a G-50 column and specific activity was calculated. The probe was used at ~10 ng/ml with a specific activity ≥109 dpm/µg.

Total RNA was isolated from IMCD after exposure to vehicle or agent for 4 h. RNA was electrophoresed on a 0.9% formaldehyde gel and transferred to a nylon membrane (Amersham, Piscataway, NJ). The blot was prehybridized at 60°C in 50% formamide, 5x SSC, 5x Denhardts, 1% SDS, and 100 µg/ml sheared denatured salmon sperm DNA. Fresh solution was added for hybridization along with radio actively labeled probe and incubated overnight at 60°C. The blot was washed and subjected to autoradiography and densitometry (NucleoTech gel documentation system). A human U1 small nuclear riboprotein (snRNP) riboprobe was used for normalizing loading (28). RNA product sizes were 2.1 kb (ET-1) and 0.75 kb (snNRP).

For mRNA half-life, IMCD were preincubated with compounds for 4 h before the addition of 5 µg/ml actinomycin D. At varying time points up to 2 h later, total RNA was isolated and Northern analysis for ET-1 and snRNP mRNA was conducted as above.

ET-1 promoter-luciferase reporter constructs. 3.2 kb of the ET-1 upstream promoter region encompassing 189 bp of the 5'-untranslated region of the mRNA were amplified from rat genomic DNA. PCR reactions employed high-fidelity Taq DNA polymerase (Platinum Taq, Invitrogen) and ET-1-specific primers: 3'-primer 5'-TATA- T(GTCGAC)TCTGCAAAGGGATCAGAAGAAG-3' and 5'-primer 5'-TATAT(ACTAGT)CTACTTGTCCTAGGCTGGTGA-3'. The underlined sequence in each primer is complimentary to sequences in the ET-1 promoter region. For cloning purposes, a SalI restriction enzyme site was added to the 3'-primer and a SpeI site to the 5'-primer (shown in parentheses). The 5-bp sequence TATAT was added to the 5'-end of both primers to facilitate restriction enzyme digestion. The final PCR product was digested with SalI/SpeI, gel-isolated, and subcloned into the XhoI/NheI sites of the pGL3-Basic Vector (Promega) immediately upstream of the luciferase gene to generate the (–3,048 to +189) ET-1-pGL3 construct (sequenced to ensure authenticity).

Serial deletions of the 5'-end of (–3,048 to +189) ET-1pGL3 construct were generated in a series of separate restriction enzyme reactions using the following enzymes: SacI (–1725), SacII (–1026), NheI (–366), and MluI (–75). The fragment to be deleted was liberated from the vector using the pGL3 multiple cloning site cutter KpnI. The shortened ET-1pGL3 constructs were subsequently gel-isolated, blunt ended, recircularized by ligation, and ultimately transformed into bacteria. All constructs were sequenced to ensure authenticity.

Transient transfection assays. DNA constructs were transiently transfected into primary cultures of IMCD or aortic endothelial cells using Lipofectamine 2000 Reagent (Invitrogen). Cells were grown on 24-well tissue culture plates to greater than 90% confluence. Transfections were carried out for 18 h according to the manufacturer's protocol, using pRL-TK Renilla luciferase as a control for transfection efficiency. The various compounds to be tested were added 1 day posttransfection and allowed to incubate overnight. The following day, the culture medium was changed and the cells were placed back into a 37°C, 5% CO2 incubator for an additional 4 h in the presence or absence of the test compounds. Initially, the test compounds were not preincubated overnight, being added for only a total of 4 h before analysis of promoter activity. However, due to the relatively long half-life of luciferase, it was necessary to expose the cells to test compounds overnight.

Cells were lysed in passive lysis buffer (Promega) and subjected to freezing and thawing to ensure complete lysis. Luciferase activity in the cell lysates was determined using the Dual-Luciferase reporter assay system (Promega) to allow sequential determination, in the same sample, of both the firefly luciferase activity from the ET-1 promoter constructs and the transfection efficiency from the pRL-TK Renilla luciferase. All assays were carried out in a DRC-1 single sample luminometer (DIGENE Diagnostics). All reported firefly luciferase values were normalized for transfection efficiency using the pRL-TK, Renilla luciferase value.

The transfection efficiency was 27% in >90% confluent primary cultures of IMCD cells. Optimal conditions for transfection were determined using the pEGFP-C1 vector (Clontech), containing a CMV promoter-driven coding region for green fluorescent protein. For optimization, the DNA concentrations in the transfection system were varied from 0.5 to 5 µg and Lipofectamine 2000 from 2 to 12 µl. After 24 h, the percentage of cells transfected for each condition was analyzed by fluorescent microscopy.

Statistics. All data were analyzed by ANOVA using the Bonferroni correction. Data are expressed as means ± SE. P < 0.05 was taken as significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ET-1 release. Basal ET-1 release from primary cultures of rat IMCD cells was 2.1 pg ET-1/mg total cell protein over 1 h, 7.7 ± 0.5 pg ET-1/mg total cell protein over 4 h, and 15.7 ± 0.8 pg ET-1/mg total cell protein over 24 h. These determinations were made from cells 24 h following growth arrest; cells were growth-arrested to facilitate evaluation of the effects of potential stimulators or inhibitors of ET-1 release in the absence of confounding effects of added growth factors or serum. For purposes of comparison, ET-1 release from proliferating cells was determined and found to be 23.5 ± 1.6 pg ET-1/mg total cell protein over 4 h, approximately three times that seen when growth-arrested.

The effects of blocking various components of calcium signaling on IMCD ET-1 release were evaluated. Chelation of intracellular calcium with BAPTA markedly reduced IMCD ET-1 release (Fig. 1). In addition, inhibition of CaM with W7 reduced ET-1 release to 45% of control levels (Fig. 1). Inhibition of CaM kinases (CaMKs) with KN-93 or KN-62 also decreased IMCD ET-1 release, although KN-93 was a more effective inhibitor than KN-62 (Fig. 1). The spectrum of KN-93 and KN-62 inhibition of CaMKs has not been fully ascertained, hence it is not possible to say which CaMKs are primarily involved. Treatment with calcium ionophores resulted in significant cell toxicity at concentrations that are well tolerated by other cell types (0.1–1 µM A23187 [GenBank] or 0.1–1 µM ionomycin); ET-1 production was markedly reduced, but these results were deemed uninterpretable. Similarly, 1–5 µM EGTA caused variable cell detachment, obviating data interpretation.


Figure 1
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  		Fig. 1. Endothelin-1 (ET-1) release by rat inner medullary collecting duct (IMCD) cells. Regulation by BAPTA (50 µM), W7 (20 µM), KN93 (10 µM), and KN62 (10 µM). Cells were incubated with inhibitor for 1 h, the media was discarded, and then fresh inhibitor was added for 4 h. Data are expressed as percent of control without inhibitor. Control cell ET-1 release was 6.9 ± 0.5 pg ET-1·mg total cell protein–1·4 h–1. *P < 0.001 vs. control. **P < 0.01 vs. control; n = 6–12 each data point.

 
ET-1 mRNA metabolism. The above data indicated that calcium/CaM significantly regulated IMCD ET-1 release. To see whether such regulation occurred at the mRNA level, steady-state ET-1 mRNA content was assessed. BAPTA markedly reduced ET-1 mRNA, while W7 and KN-93 decreased mRNA content by ~40–50% (Fig. 2). This inhibitory effect of W7 was not due to alterations in mRNA stability since ET-1 mRNA half-life was not altered (Fig. 3). Thus calcium/CaM increases ET-1 release, at least in part, through increases in mRNA content, the latter being due to augmented ET-1 gene transcription and not reduced mRNA degradation.


Figure 2
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  		Fig. 2. Steady-state ET-1 mRNA content in rat IMCD cells. Regulation by BAPTA (50 µM), W7 (20 µM), and KN93 (10 µM). Cells were incubated with inhibitor for 4 h. Data are expressed as percent of control without inhibitor. ET-1 mRNA content was normalized to small nuclear riboprotein (snRNP) content; snRNP content was unaffected by any treatment. *P < 0.01 vs. control; n = 6 each data point.

 

Figure 3
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  		Fig. 3. ET-1 mRNA stability in rat IMCD cells. Regulation by W7 (20 µM). Cells were incubated with inhibitor for 4 h, followed by addition of 5 µg/ml actinomycin D and determination of mRNA levels over the following 2 h. Data were normalized to snRNP content; snRNP content was unaffected by inhibitor treatment and did not decrease over the 2-h time period following addition of actinomycin D. Arrows indicate time at which 50% of ET-1 mRNA remained; n = 3 each data point.

 
ET-1 promoter activity in IMCD cells. To more directly determine how ET-1 production is regulated at the transcriptional level, rat ET-1 promoter-firefly luciferase DNA constructs were generated and transfected in rat IMCD cells. Five different sized ET-1 promoter regions were studied, extending from +189 (numbered 3' to the transcriptional start site) to –75 (minimal promoter), –366, –1026, –1725, and –3048 (all numbered 5' to the transcriptional start site). The minimal promoter drove very little luciferase activity (Fig. 4). The –366 region exhibited increased activity while the –1026 region further increased luciferase activity (Fig. 4). There was a marked increase in reporter activity using the –1725 region; extending this to –3048 caused a comparable increase in reporter activity compared with –1725 (Fig. 4).


Figure 4
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  		Fig. 4. Rat ET-1 gene promoter activity in rat IMCD cells. Effect of promoter size on reporter activity. Cells were transfected with various sized ET-1 promoter sequences coupled to firefly luciferase reporters. All data were normalized to Renilla luciferase activity (cells were cotransfected with pRL-TK Renilla luciferase). Numbers indicate length of construct beginning at the transcription start site and extending 5' into the promoter. Data are expressed as percent of –366 construct values. *P < 0.01 vs. –366 construct values. **P < 0.001 vs. –366 construct values and P < 0.01 vs. –1026 construct values; n = 8 each data point.

 
To directly examine whether ET-1 promoter activity is regulated by CaM/CaMK, the effect of W7 on activity of the –366, –1026, and –3048 constructs was examined. This range of promoter constructs covers all degrees of promoter activity seen, from modest (–366), to moderate (–1026), to maximal (–3048). Since no difference was seen between the –1725 and –3048 constructs, only one size was utilized. W7 markedly decreased promoter activity of the –3048, but not activity of the smaller sized promoters (Fig. 5A).


Figure 5
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  		Fig. 5. Rat ET-1 gene promoter activity in rat IMCD (A) and rat aortic endothelial cells (B). Basal activity and effect of W7 (20 µM). Cells were transfected with various sized ET-1 promoter sequences coupled to firefly luciferase reporters. All data were normalized to Renilla luciferase activity (cells were cotransfected with pRL-TK Renilla luciferase). Numbers indicate length of construct beginning at the transcription start site and extending 5' into the promoter. Data are expressed as percent of –366 construct values. Inhibitor was added the day after transfection and left on the cells for 16–18 h, n = 8 each data point. *P < 0.05 vs. –366 basal. **P < 0.001 vs. –366 basal. #P < 0.001 vs. –3048 basal.

 
ET-1 production and promoter activity in rat aortic endothelial cells. The finding that ET-1 promoter regions 5' to –1026 conferred markedly enhanced activity in IMCD is in contrast to that reported in other cell types, particularly endothelial cells, where the first 150 bp 5' to the transcriptional start site contain the main regulatory regions (see DISCUSSION). To determine whether IMCD can regulate ET-1 promoter activity in a manner different to other cell types, and particularly in our hands under similar experimental conditions, ET-1 production and ET-1 promoter activity, and their regulation by CaM, were assessed in primary cultures of rat aortic endothelial cells. Baseline ET-1 release from aortic endothelial cells was 1.5 ± 0.2 pg ET-1·mg total cell protein–1·4 h–1 (n = 6), ~20% of that released by IMCD cells. In contrast to IMCD, treatment with W7 did not affect ET-1 release by aortic endothelial cells (1.8 ± 0.2 pg ET-1·mg total cell protein–1·4 h–1, n = 6). Also, in contrast to IMCD, transfection of the –366, –1026, and –3048 ET-1 promoter-reporter constructs revealed no difference in firefly luciferase activity (relative to Renilla luciferase) in endothelial cells (Fig. 5B). In addition, W7 had no effect on promoter activity for any of the three constructs (Fig. 5B). Thus, at least compared with endothelial cells, ET-1 promoter activity is uniquely regulated by CaM in IMCD. Furthermore, regions of the ET-1 promoter 5' to –366 confer unique regulation of ET-1 gene transcription in IMCD.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
One of the more extensively studied factors in regulation of ET-1 production is calcium. Despite this, the effects of calcium (as well as CaM and/or CaMK) on ET-1 synthesis appear to be quite variable, differing between and even within cell types. In human mesangial cells, BAPTA has no effect on basal or thrombin-stimulated ET-1 mRNA; similarly, a calcium inonophore (A23187 [GenBank] ) had no effect on ET-1 production by rat pulmonary artery smooth muscle cells (32). In human umbilical vein endothelial cells (HUVEC), thrombin-induced increases in ET-1 mRNA were inhibited by BAPTA, EDTA, W7, or KN-62, while A23187 [GenBank] increased basal and thrombin-stimulated ET-1 mRNA levels (21). In contrast, another group found that ionomycin and A23187 [GenBank] decreased ET-1 production by HUVEC (23). In rat aortic endothelial cells, heparin inhibition of ET-1 mRNA expression was blunted by W7 (33). Hence, it was not possible to predict what effect, if any, calcium would have on collecting duct ET-1 synthesis [albeit one study found that A23187 [GenBank] modestly increased ET-1 production by porcine IMCD (22)]. The current study found that ET-1 synthesis by IMCD is markedly calcium/CaM/CaMK dependent, providing strong evidence that this system can play an important role in modulating collecting duct ET-1 release, and ultimately ET-1 actions.

The mechanism by which calcium and CaM regulate IMCD ET-1 production is clearly at the transcriptional level. Reduction or inhibition of these factors reduced steady-state mRNA levels but did not affect mRNA stability. Most importantly, CaM inhibition greatly reduced activity of the –3048 ET-1 promoter construct. Notably, CaM inhibition did not reduce activity of the –1026 or –366 ET-1 promoter fragments. Furthermore, maximal ET-1 promoter activity was conferred by regions distal to the first proximal 1-kb region of the promoter. These findings have no precedent. Although no data are available in collecting duct cells, ET-1 promoter elements primarily involved in regulation of gene transcription by other cell types are located within the first 150 bp 5' to the transcriptional start site. For example, bovine aortic endothelial cells transfected with ET-1 promoter constructs exhibited maximal promoter activity in the proximal 143-bp promoter fragment; regions containing the proximal 0.2, 0.7, 1.7, and 4.4 promoter fragments did not confer increased (or decreased) activity (16). Similarly, the proximal 120–143 bp of the ET-1 promoter had maximal activity in transfected mesangial cells, compared with 1.7- and 4.4-kb proximal promoter regions (3). To our knowledge, the effect of CaM inhibition or calcium on ET-1 promoter activity has not been well studied. A putative calcium response element has been described at –590 to –599 in the ET-1 promoter (16); however, the ability of calcium to regulate activity of this region has not been studied. Furthermore, our data indicate that this region is not responsible for CaM regulation of ET-1 promoter activity. Thus collecting duct ET-1 promoter activity appears, at least based on comparisons to previously published studies, to be under relatively unique control.

To more conclusively demonstrate the potential differential characteristics of ET-1 synthesis between collecting duct and other cell types, ET-1 synthesis and promoter activity were examined in aortic endothelial cells. Unlike IMCD, ET-1 promoter fragment activity was maximal in the region –366 to the transcription start site. Furthermore, W-7 did not affect ET-1 release or promoter activity. These data conclusively show that collecting duct ET-1 synthesis is under differential regulation, with regard to both the region of the promoter and the nature of calcium/CaM interaction, at least compared with endothelial cells. Analysis of the ET-1 promoter between 1 and 3 kb 5' to the transcription start site does not reveal any regions with known responsiveness to calcium or CaM, nor are other likely cis-acting regions evident. Elucidation of such sites will require careful promoter analysis and identification of relevant trans-acting factors.

The finding that calcium/CaM regulates IMCD ET-1 production may be an important clue as to how extracellular fluid volume is coupled to collecting duct ET-1 synthesis. Collecting duct principal cells express primary cilia that extend into the lumen and have been implicated as flow sensors (18, 19). Studies using MDCK cells showed that bending the primary cilium increases intracellular calcium concentration, while removal of the primary cilia abolishes flow sensing (25, 26). In addition, mutation of genes leading to loss of primary cilia abolishes regulation of calcium entry into collecting duct cells (27). Since Na or water loading increase collecting duct luminal fluid flow rate, and likely cause increased ET-1 production by this nephron segment, it seems quite possible that primary cilia are involved in coupling flow rate to ET-1 release, and likely through alteration of intracellular calcium concentration. Notably, increased flow rate or shear stress can increase ET-1 production by nonrenal cell types (2, 20, 24, 31), although the role of calcium in this response has not been clarified. Studies are needed that will examine the direct effect of flow on collecting duct ET-1 production and whether such an effect is calcium/CaM dependent, although completing such studies will be technically quite challenging.

In summary, the current study demonstrates that IMCD ET-1 production is markedly regulated by calcium/CaM, that this occurs at the transcriptional level, and involves pathways that are at least relatively unique to collecting duct cells. Further elucidation of this system may shed substantial light on how renal Na and water excretion, and systemic blood pressure, are regulated.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grant R01-DK-96392 (to D. E. Kohan).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. E. Kohan, Division of Nephrology, Univ. of Utah Health Sciences Center, 1900 East, 30 North, Salt Lake City, UT 84132 (e-mail: donald.kohan{at}hsc.utah.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ahn D, Ge Y, Stricklett P, Gill P, Taylor D, Hughes A, Yanagisawa M, Miller L, Nelson R, Kohan D. Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention. J Clin Invest 114: 504–511, 2004.[CrossRef][Web of Science][Medline]
  2. Dancu M, Berardi D, Vanden Heuvel J, Tarbell J. Asynchronous shear stress and circumferential strain reduces endothelial NO synthase and cyclooxygenase-2 but induces endothelin-1 gene expression in endothelial cells. Arterioscler Thromb Vasc Biol 24: 2088–2094, 2004.[Abstract/Free Full Text]
  3. Foschi M, Sorokin A, Pratt P, McGinty A, La Villa G, Franchi F, Dunn M. PreproEndothelin-1 expression in human mesangial cells: evidence for a p38 mitogen-activated protein kinase/protein kinases-C-dependent mechanism. J Am Soc Nephrol 12: 1137–1150, 2001.[Abstract/Free Full Text]
  4. Ge Y, Bagnall A, Stricklett P, Strait K, Webb D, Kotelevtsev Y, Kohan D. Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention. Am J Physiol Renal Physiol 291: F1274–F1280, 2006.[Abstract/Free Full Text]
  5. Gumz ML, Popp MP, Wingo CS, Cain BD. Early transcriptional effects of aldosterone in a mouse inner medullary collecting duct cell line. Am J Physiol Renal Physiol 285: F664–F673, 2003.[Abstract/Free Full Text]
  6. Herrera M, Garvin J. Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb. Am J Physiol Renal Physiol 287: F231–F235, 2004.[Abstract/Free Full Text]
  7. Herrera M, Garvin J. A high-salt diet stimulates thick ascending limb eNOS expression by raising medullary osmolality and increasing release of endothelin-1. Am J Physiol Renal Physiol 288: F58–F64, 2005.[Abstract/Free Full Text]
  8. Klein H, Abassi Z, Keiser HR. Effects of angiotensin II and phenylephrine on urinary endothelin in normal female volunteers. Metabolism 44: 115–118, 1995.[CrossRef][Web of Science][Medline]
  9. Kohan D. Endothelins: renal tubule synthesis and actions. Clin Exp Pharmacol Physiol 23: 337–344, 1996.[Web of Science][Medline]
  10. Kohan DE. The renal medullary endothelin system in control of sodium and water excretion and systemic blood pressure. Curr Opin Nephrol Hypertens 15: 34–40, 2006.[Web of Science][Medline]
  11. Kohan DE, Fiedorek FTJ. Endothelin synthesis by rat inner medullary collecting duct cells. J Am Soc Nephrol 2: 150–155, 1991.[Abstract]
  12. Kohan DE, Padilla E. Endothelin-1 production by rat inner medullary collecting ducts: effect of nitric oxide, cGMP, and immune cytokines. Am J Physiol Renal Fluid Electrolyte Physiol 266: F291–F297, 1994.[Abstract/Free Full Text]
  13. Kohan DE, Padilla E. Osmolar regulation of endothelin-1 production by rat inner medullary collecting duct. J Clin Invest 91: 1235–1240, 1993.[Web of Science][Medline]
  14. Kramer H, Hashemi T, Backer A, Bokemeyer D. Hyperosmolality induced by betaine or urea stimulates endothelin synthesis by differential activation of ERK and p38 MAP kinase in MDCK cells. Kidney Blood Press Res 25: 65–70, 2002.[CrossRef][Web of Science][Medline]
  15. Kwan HY, Huang Y, Yao X. Store-operated calcium entry in vascular endothelial cells is inhibited by cGMP via a protein kinase G-dependent mechanism. J Biol Chem 275: 6758–6763, 2000.[Abstract/Free Full Text]
  16. Lee ME, Bloch KD, Clifford JA, Quertermous T. Functional analysis of the endothelin-1 gene promoter. Evidence for an endothelial cell-specific cis-acting sequence. J Biol Chem 265: 10446–10450, 1990.[Abstract/Free Full Text]
  17. Ling BN. Luminal endothelin-1 inhibits apical Na+ and Cl– channels in cultured rabbit CCT principal cells. J Am Soc Nephrol 5: 292, 1994.
  18. Liu W, Murcia NS, Duan Y, Weinbaum S, Yoder BK, Schwiebert E, Satlin LM. Mechanoregulation of intracellular Ca2+ concentration is attenuated in collecting duct of monocilium-impaired orpk mice. Am J Physiol Renal Physiol 289: F978–F988, 2005.[Abstract/Free Full Text]
  19. Liu W, Xu S, Woda C, Kim P, Weinbaum S, Satlin LM. Effect of flow and stretch on the [Ca2+]i response of principal and intercalated cells in cortical collecting duct. Am J Physiol Renal Physiol 285: F998–F1012, 2003.[Abstract/Free Full Text]
  20. Malek AM, Greene AL, Izumo S. Regulation of endothelin 1 gene by fluid shear stress is transcriptionally mediated and independent of protein kinase C and cAMP. Proc Natl Acad Sci USA 90: 5999–6003, 1993.[Abstract/Free Full Text]
  21. Marsen TA, Simonson MS, Dunn MJ. Roles of calcium and kinases in regulation of thrombin-stimulated preproendothelin-1 transcription. Am J Physiol Heart Circ Physiol 271: H1918–H1925, 1996.[Abstract/Free Full Text]
  22. Migas I, Bäcker A, Meyer-Lehnert H, Kramer HJ. Endothelin synthesis by porcine inner medullary collecting duct cells. Am J Hypertens 8: 748–752, 1995.[CrossRef][Web of Science][Medline]
  23. Mitchell MD, Branch DW, Lamarche S, Dudley DJ. The regulation of endothelin production in human umbilical vein endothelial cells: unique inhibitory action of calcium ionophores. J Clin Endocrinol Metab 75: 665–668, 1992.[Abstract]
  24. Morgera S, Schlenstedt J, Hambach P, Giessing M, Deger S, Hocher B, Neumayer H. Combined ETA/ETB receptor blockade of human peritoneal mesothelial cells inhibits collagen I RNA synthesis. Kidney Int 64: 2033–2040, 2003.[CrossRef][Web of Science][Medline]
  25. Praetorius HA, Frokiaer J, Nielsen S, Spring KR. Bending the primary cilium opens Ca2+-sensitive intermediate-conductance K+ channels in MDCK cells. J Membr Biol 191: 193–200, 2003.[CrossRef][Web of Science][Medline]
  26. Praetorius HA, Spring KR. Removal of the MDCK cell primary cilium abolishes flow sensing. J Membr Biol 191: 69–76, 2003.[CrossRef][Web of Science][Medline]
  27. Siroky BJ, Ferguson WB, Fuson AL, Xie Y, Fintha A, Komlosi P, Yoder BK, Schwiebert EM, Guay-Woodford LM, Bell PD. Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells. Am J Physiol Renal Physiol 290: F1320–F1328, 2006.[Abstract/Free Full Text]
  28. Thorburn J, Frost J, Thorburn A. Mitogen-activated protein kinases mediate changes in gene expression, but not cytoskeletal organization associated with cardiac muscle cell hypertrophy. J Cell Biol 126: 1565–1572, 1994.[Abstract/Free Full Text]
  29. Todd-Turla KM, Zhu XL, Shu X, Chen M, Yu T, Smart A, Killen PD, Fejes-Toth G, Briggs JP, Schnermann JB. Synthesis and secretion of endothelin in a cortical collecting duct cell line. Am J Physiol Renal Fluid Electrolyte Physiol 271: F330–F339, 1996.[Abstract/Free Full Text]
  30. Tomita K, Nonoguchi H, Marumo F. Inhibition of Fluid Transport by Endothelin Through Protein Kinase C in Collecting Duct of Rats. Basel: Karger, 1991.
  31. Walshe T, Ferguson G, Connell P, O'Brien C, Cahill P. Pulsatile flow increases the expression of eNOS, ET-1, and prostacyclin in a novel in vitro coculture model of the retinal vasculature. Invest Opthalmol Vis Sci 46: 375–382, 2005.[Abstract/Free Full Text]
  32. Yi SL, Kantores C, Belcastro R, Cabacungan J, Tanswell AK, Jankov RP. 8-Isoprostane-induced endothelin-1 production by infant rat pulmonary artery smooth muscle cells is mediated by Rho-kinase. Free Radic Biol Med 41: 942–949, 2006.[CrossRef][Web of Science][Medline]
  33. Yokokawa K, Kohno M, Minami M, Yasunari K, Mandal AK, Yoshikawa J. Heparin suppresses cyclosporine-induced endothelin-1 synthesis in rat endothelial cells. J Cardiovasc Pharmacol 31, Suppl 1: S460–S463, 1998.[CrossRef][Web of Science][Medline]
  34. Zeidel ML, Brady HR, Kone BC, Gullans SR, Brenner BM. Endothelin, a peptide inhibitor of Na+-K+-ATPase in intact tubular epithelial cells. Am J Physiol Cell Physiol 257: C1101–C1107, 1989.[Abstract/Free Full Text]




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