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Vitamin D Activates Type a Natriuretic Peptide Receptor Gene Transcription in Inner Medullary Collecting Duct Cells S Chen1, K Olsen1, C Grigsby1 and DG Gardner1,2

Vitamin D Activates Type a Natriuretic Peptide Receptor Gene Transcription in Inner Medullary Collecting Duct Cells S Chen1, K Olsen1, C Grigsby1 and DG Gardner1,2

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provided by Elsevier - Publisher Connector original article http://www.kidney-international.org & 2007 International Society of Nephrology

see commentary on page 237 D activates type A receptor gene transcription in inner medullary collecting duct cells S Chen1, K Olsen1, C Grigsby1 and DG Gardner1,2

1Diabetes Center, University of California at San Francisco, San Francisco, California, USA and 2Department of Medicine, University of California at San Francisco, San Francisco, California, USA

Many clinical and animal studies suggest that and Vitamin D, or more specifically its polar metabolite 1,25 its metabolites have beneficial effects in the cardiovascular dihydroxyvitamin D3 (1,25(OH)2D3), is thought to and renal systems. Using immunologic and enzymatic assays, play a significant role in the regulation of growth and and 25 hydroxyvitamin D3 1a-hydroxylase function in the cardiovascular system and .1 Vitamin D activity were found in inner medullary collecting duct (IMCD) deficiency in rats is associated with and cells suggesting an autocrine/paracrine role in this nephron cardiac hypertrophy2 and expansion of the interstitial segment. In this study, we examined the ability of 1,25 compartment of the myocardium.3 Treatment of cultured dihydroxyvitamin D3 (1,25(OH)2D3) to regulate the neonatal cardiac myocytes with 1,25(OH)2D3 effectively expression of the vasculoprotective natriuretic peptide reverses endothelin-induced hypertrophy in this in vitro receptor-A gene in these cells in culture. Treatment of the model.4 Genetic deletion of the vitamin D receptor5 results in 6 cells with 1,25(OH)2D3 caused a doubling of natriuretic a mouse with hyper-reninemic hypertension and cardiac peptide-dependent cyclic guanosine monophosphate hypertrophy.7 production and a significant increase in natriuretic peptide Subsets of hypertensive patients have low 25- receptor-A protein expression. This was accompanied by hydroxyvitamin D3 (25(OH)D3) levels,8 as do patients with significant increases in receptor mRNA levels and gene- congestive failure9,10 and peripheral vascular disease.11 promoter activity. Mutation of a vitamin D response element, Treatment of small groups of hypertensive patients with 12–14 positioned upstream from the gene start site, resulted in a 1,25(OH)2D3 has been shown to lower , complete loss of 1,25(OH)2D3-dependent induction but not whereas treatment of patients with end-stage renal disease on the induction by hypertonic stimuli. Introduction of small dialysis with 1,25(OH)2D3 leads to regression of left interfering RNA directed against the vitamin D receptor into ventricular hypertrophy in this patient population.15 Further- the IMCD cells resulted in decreased natriuretic peptide more, retrospective analysis of dialysis patients treated with 16 17 receptor-A gene promoter activity and protein. The increase either 1,25(OH)2D3 or , a 1,25(OH)2D3 in this receptor expression may account for some of the analogue with reduced calcemic potential, reduced mortality reported beneficial effect of 1,25(OH)2D3 on the compared with controls. As is the cardiovascular system and kidney. leading cause of death in patients with end-stage renal disease Kidney International (2007) 72, 300–306; doi:10.1038/sj.ki.5002274; on dialysis, it can be inferred that these vitamin D receptor published online 18 April 2007 (VDR) ligands are positively impacting cardiovascular disease KEYWORDS: vitamin D; cardiovascular disease; cyclic GMP; renal cell biology; in this patient group. gene transcription; The inner medullary collecting duct (IMCD) is positioned at the distal terminus of the nephron and, despite the fact that it samples less than 5% of filtered sodium load, it plays a pivotal role in determining the final urinary sodium concentration.18 Sodium handling in this nephron segment is highly regulated by a number of including atrial natriuretic peptide (ANP). ANP is produced in the heart and acts by binding to natriuretic peptide receptor type A *Correspondence: DG Gardner, Center and Department of (NPR-A) in target tissues and triggering increased synthesis Medicine, 1119 HSW, University of California at San Francisco, San Francisco, of cyclic guanosine monophosphate (cGMP). Binding of CA 94143-0540, USA. E-mail: [email protected] circulating ANP or its locally produced homologue urodila- 19 Received 14 June 2006; revised 14 February 2007; accepted 27 February tin to NPR-A on IMCD cells leads to an increase in urinary 20 2007; published online 18 April 2007 sodium excretion through a cGMP-dependent mechanism.

300 Kidney International (2007) 72, 300–306 S Chen et al.: Vitamin D activates NPR-A gene transcription in IMCD cells original article

We have recently shown that treatment of rat aortic 1a-hydroxylase expression. To demonstrate 25(OH)D3 smooth muscle cells with 1,25(OH)2D3 leads to an increase 1a-hydroxylase activity in IMCD cells, we measured 21 in expression and activity of NPR-A in these cells. In this 1,25(OH)2D3 production and release in the medium of study, we demonstrate that VDR, present in IMCD cells, cultured IMCD cells following addition of 25(OH)D3 triggers a similar increase in NPR-A activity and gene substrate. Compared with the substrate-free cultures, there expression through a transcriptional mechanism and that was a dramatic increase in 1,25(OH)2D3 levels in the suppression of endogenous VDR levels reverses this response. medium of the 25(OH)D3-treated group (3.6370.47 vs We hypothesize that low levels of 1,25(OH)2D3 or VDR- 11.9472.17 pg/ml, n ¼ 3 Po0.01), demonstrating the ability dependent activity may contribute to sodium retention seen of IMCD cells to covert 25(OH)D3 to 1,25(OH)2D3. in disorders like congestive heart failure and salt-sensitive We next assessed the effect of 1,25(OH)2D3 treatment on hypertension through a reduction in NPR-A activity. the ability of these IMCD cells to respond to ANP, as a surrogate marker of NPR-A activity. As shown in Figure 2, RESULTS pretreatment with 1,25(OH)2D3 or the 1,25(OH)2D3 Vitamin D has been suggested to play a role in the regulation analogue, RO-25-6760, elicited a doubling in ANP-depen- of renal handling, possibly through induction of dent cGMP generation and 3–4-fold increase in NPR-A calcium transport proteins in different nephron segments.22 protein expression in these cells. To assess the presence of endogenous VDR and 25(OH)D3 The increase in NPR-A activity was accompanied by 1a-hydroxylase in cultured IMCD cells, we examined an increase in NPR-A mRNA levels in IMCD cells. Both extracts of cells cultured for 48 h in the presence or absence of 1,25(OH)2D3 by Western blot analysis. As shown in Figure 1, VDR and 25(OH)D3 1a-hydroxylase were readily demonstrable in these extracts. Interestingly, pretreatment a – ANP + ANP with 1,25(OH) D3 resulted in a dose-dependent increment 2 20 * * in VDR levels (2–2.5-fold increase relative to control) over * * a physiologically relevant range of ligand concentrations. 15 * 1,25(OH)2D3 treatment had no effect on 25(OH)D3 10

Fold induction 5

a 1,25(OH)2D3 (log M) 0 –8 –9 –10 –11 –12 VDR 0 0 –7 –8 –9 –8 –9 1,25(OH) D3 RO 25-6760 GAPDH 2 (log M)

b 1,25(OH)2D3 (log M) 0 –8 –9 –10 –11 –12 b 1,25(OH) D3 RO 25-6760 25(OH)D3 1- hydroxylase 2 (log M) 0 –7 –8 –8 –9 GAPDH NPR-A

c 3 VDR 25(OH)D3 1- hydroxylase Tubulin * * * 6 * 2 5

4 *

3 1 * * 2

Fold induction (VDR/GAPDH) 1 0

0 –8 –9 –10 –11 –12 Fold induction (NPR-A/tubulin) 1,25(OH) D3 (log M) 0 2 0 –7 –8 –8 –9 Figure 1 | VDR and 25(OH)D3 1a-hydroxylase are expressed in 1,25(OH)2D3 RO 25-6760 IMCD cells and VDR is upregulated by 1,25(OH)2D3 (D3) in dose- (log M) dependent fashion. VDR and 25(OH)D3 1a-hydroxylase expression levels were normalized for glyceraldehyde-3- Figure 2 | 1,25(OH)2D3 stimulates NPRA expression and activity. dehydrogenase protein level. Representative autoradiographs 1,25(OH)2D3 and its analogue, RO-27-6760, increase (a) ANP-induced (a for VDR, b for 25(OH)D3 1a-hydroxylase) and pooled data (c) from cGMP level and (b) NPR-A protein expression in IMCD cells. Data from 3–5 independent experiments are shown. *Po0.01 vs control. 3–5 independent experiment are shown. *Po0.01 vs control.

Kidney International (2007) 72, 300–306 301 original article S Chen et al.: Vitamin D activates NPR-A gene transcription in IMCD cells

1,25(OH)2D3 and RO-25-6760 elucited a significant increase promoter activity (Figure 5b). A negative control siRNA, in NPR-A mRNA levels, which peaked B3-fold above the which lacked targeting capability, was without effect. controls treated with vehicle alone (Figure 3). VD-dependent induction of NPR-A gene-promoter activ- The increase in NPR-A transcript levels was linked to an ity was virtually completely dependent on the presence of a increase in NPR-A gene transcriptional activity. As shown in VDR element (VDRE) located B495 bp upstream from the Figure 4a, 1,25(OH)2D3 treatment led to B2.5-fold increase transcription start site in the promoter (Figure 6a). This was in NPR-A gene promoter activity over a physiological also true for RO-25-6760; however, the VDRE mutation had concentration range for the ligand. Transfection of additional no effect on the osmotic induction of promoter activity by 23 exogenous receptor (Figure 4b) led to further amplification NaCl or sucrose. Furthermore, addition of 1,25(OH)2D3 of the 1,25(OH)2D3-dependent stimulation of promoter to either NaCl or sucrose resulted in a modest, albeit not activity. additive, increase in promoter activity which was only To explore the VDR dependence of this stimulatory partially reduced in the VDRE-mutant promoter-driven activity, we used a small interfering RNA (siRNA) strategy reporter. Electrophoretic mobility shift assay of IMCD cell directed against endogenous VDR-coding sequence. Treat- extracts demonstrated that 1,25(OH)2D3 promoted VDR/ ment of IMCD cells with siRNAs directed against the VDR X receptor (RXR) binding to oligonucleotides resulted in a decrease in VDR levels in these cells (Figure 5a) harboring the NPR-A VDRE (Figure 6b). The binding was with two of the three siRNAs (V1 and V2, but not V3) and a eliminated in the presence of unlabeled wild-type DNA reduction in 1,25(OH)2D3-dependent stimulation of NPR-A- sequence; however, unlabeled, mutated sequence was unable to compete.

DISCUSSION a b The findings presented above support our hypothesis that 1,25(OH)2D3 (log M) 0 –7 –8 –9 RO 25-6760 (log M) 0 –7 –8 –9 NPR-A NPR-A 1,25(OH)2D3 stimulates transcription of the gene encoding NPR-A in IMCD cells. It does so through a mechanism that 18S 18S requires the vitamin D receptor and the presence of a 4 4 * * functional VDRE positioned B495 bp upstream from the 3 * * 3 NPR-A gene transcription start site. This supports our earlier 2 * 2 * 1 1 demonstration of 1,25(OH)2D3-dependent stimulation of 21 Fold induction Fold induction 0 NPR-A gene regulation in rat aortic smooth muscle cells 0 –7 –8 –9 0 0 –8 –9 –10 and the findings of Li et al.24 describing reduced NPR-A gene 1,25(OH)2D3 (log M) RO 25-6760 (log M) expression in the kidneys of VDR À/À mice. Of note, Figure 3 | 1,25(OH)2D3 stimulates NPR-A gene expression. Northern blot analysis of RNA from cultured IMCD cells treated with we have shown here that IMCD cells express 25(OH)D3 (a) 1,25(OH)2D3 or (b) RO-27-6760 using NPR-A cDNA probe. NPR-A 1a-hydroxylase, the enzyme which is responsible for conver- signal was normalized to 18S ribosomal RNA signal following rehybridization of the same membrane with 18S probe. Representative pictures and pooled data (n ¼ 3) are demonstrated. abCV1V2V3 *Po0.01 vs control. VDR 1.5 GAPDH 1.5 1 a 3 * b 8 * Vehicle 1 * 6 1,25(OH)2D3 * * 2 * 0.5

* Fold induction 0.5 * 4 * Fold induction (NPR-A-Luc/Renilla) (VDR/GAPDH) 1 *

Fold induction 2 0

Fold induction 0 CV1V2V3 C V1V2V3 0 0 0 –7 –8 –9 C VDR VDR Figure 5 | VDR siRNA reduces basal VDR expression and   NPR-A-Luc activity in IMCD cells. Three different VDR siRNAs 1,25(OH)2D3 (log M) 0.1 g 0.5 g (V1, V2, and V3) and control siRNA (C) were individually transfected Figure 4 | 1,25(OH)2D3 amplifies NPR-A transcription by into IMCD cells. Forty-eight hours later, cells were harvested and total increasing its promoter activity.(a) One microgram of À1575 protein was assayed for VDR or glyceraldehyde-3-phosphate NPR-A-Luc with 0.5 mg Renilla-Luc were cotransfected into IMCD dehydrogenase expression by Western blot analysis. VDR expression cells. After transfection for 24 h, cells were treated with different was normalized for glyceraldehyde-3-phosphate dehydrogenase concentrations of 1,25(OH)2D3 for 48 h. (b) IMCD cells were protein levels. (a) Representative results and pooled data (n ¼ 3). transfected with NPR-A-Luc and Renilla-Luc with or without 0.1 or In a separate experiment, the VDR siRNAs and control siRNA were 0.5 mg of VDR expression vector. Twenty-four hours after transfection, individually cotransfected with À1575 NPR-A-Luc and Renilla-Luc À8 cells were treated with 10 M 1,25(OH)2D3 for 48 h. NPR-A luciferase into IMCD cells and cultured for 48–72h. Cells were collected activity was normalized for Renilla-Luc activity in the individual and luciferase activities were measured. NPR-A-Luc activity was cultures. The experiments were repeated three times. *Po0.01 vs normalized for Renilla luciferase activity in individual samples. control. (b) Pooled data (n ¼ 3). *Po0.01 vs control.

302 Kidney International (2007) 72, 300–306 S Chen et al.: Vitamin D activates NPR-A gene transcription in IMCD cells original article

a 4 Recent studies have suggested that 1,25(OH)2D3 may Wild-type NPR-A-Luc play an important role in the regulation of a variety of * * NPR-A-Luc VDRE mutant physiological processes, including and hair follicle 3 development,25,26 growth and function in the cardiovascular * 4,27 * * * system, and the control of immune function and * * * * inflammatory response.28,29 All of these are largely indepen- 2 dent of the more classical, and better understood, effects of 1,25(OH)2D3 on homeostasis and . Fold induction Observational studies suggest that the presence of hyper- 1 tension30 and susceptibility to certain types of infections like ,31–33 correlate inversely with the ability to generate endogenous vitamin D. 0 C In the cardiovascular system, 1,25(OH)2D3 has been D3 D3 D3 2 2 2 NaCl shown to regulate proliferative activity in vascular smooth Sucrose muscle cells in culture,28 to antagonize cardiac myocyte

RO 25-6760 4 1,25(OH) hypertrophy in an in vitro model, to reduce blood pressure in selected groups of human hypertensives,13,14 and to reverse NaCl+1,25(OH) hypertrophy in patients with end-stage renal disease on Sucrose+1,25(OH) dialysis.15 Furthermore, treatment of dialysis patients with × 16 b 50 cold NPR-A4 ––– ––+ – 1,25(OH)2D3, or a 1,25(OH)2D3 analogue with reduced 50 ×cold M2-NPR-A4 –––––– + calcemic potential,17 leads to improvement in survival, Transfected with VDR ––– + +++ Transfected with RXR ––– + +++ a variable which is heavily dependent on the presence of Sham transfection – ++ ––– – cardiovascular disease in this population. Collectively, these 1,25(OH) D3 ––+ –+ + + 2 studies support the notion that 1,25(OH)2D3, at least at nontoxic concentrations, exerts a beneficial effect in promot- ing cardiovascular health. Heart failure is a complex disease of pump function and neurohormonal dysregulation that is characterized by inadequate delivery of blood flow to vascularized tissues. Heart failure is associated with reduced plasma levels of 9,10 25(OH)D3 and 1,25(OH)2D3 and treatment of heart failure patients with 1,25(OH)2D3 has been shown to diminish plasma levels of (e.g., tumor necrosis 29 factor) that are associated with myocardial inflammation.34

Figure 6 | Effects of sucrose, NaCl, and 1,25(OH)2D3 or Heart failure is characterized by resistance to natriuretic RO-25-6760 on NPR-A promoter activity and the effect of peptides, a phenomenon which may, in part, reflect altered mutation of the VDRE on this promoter. (a) IMCD cells were NPR function.35 The excessive sodium retention asso- transfected with wild-type or VDRE-mutated À1575 NPR-A Luc, À8 ciated with heart failure is exacerbated by inhibition of together with Renilla-Luc, and incubated with 10 M 1,25(OH)2D3 À9 36,37 or 10 M RO-27-6760 for 48 h, or 75 mM NaCl or 150 mM sucrose NPR-A activity and myocardial NPR-A is thought to for 24 h or a combination of 1,25(OH)2D3 and hypertonic agents. mediate a ligand-dependent antagonism of hypertrophy Firefly luciferase activity was normalized for Renilla luciferase activity. and fibrosis in the myocardial wall.38 By inference, Pooled data (n ¼ 3–5) are shown. *Po0.01 vs control. (b) 1,25(OH)2D3 promotes binding of VDR to VDRE on NPR-A promoter in IMCD 1,25(OH)2D3 insufficiency or bonafide 1,25(OH)2D3 defi- cells. Three micrograms of nuclear extract from sham- vs VDR/ ciency could, through an indirect effect to reduce NPR-A À8 RXR-transfected IMCD cells in the presence or absence of 10 M 32 activity, play an important role in accelerating development 1,25(OH)2D3 were incubated with P-labeled NPR-A4 of heart failure, or salt-sensitive hypertension, and in oligonucleotide alone or in combination with unlabeled NPR-A4 or M2-NPR-A4 oligonucleotides and separated by electrophoretic contributing to the excessive sodium retention that occurs mobility shift assay. Representative autoradiograph from three in those diseases. different experiments is shown.

MATERIALS AND METHODS sion of 25(OH)D3 to its more polar and bioactive metabolite, Materials 1,25(OH) D3. Endogenous 1,25(OH) D3 in IMCD cells 2 2 The cGMP radioimmunoassay kit was obtained from Perkin-Elmer probably contributes to the maintenance of basal NPR-A Life Sciences (Boston, MA, USA). ANP was purchased from levels. Local production of 1,25(OH)2D3 may accommodate American Peptide (Sunnyvale, CA, USA). R0-25-6760 (1,25- the need for relatively high ligand concentrations (above dihydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-) circulating plasma concentrations) that we observed in this was a kind gift of M Uskokovic (Roche Pharmaceuticals, Nutley, NJ, study (see Figures 2 and 3). USA). 1,25(OH)2D3 was obtained from Calbiochem Inc. (La Jolla,

Kidney International (2007) 72, 300–306 303 original article S Chen et al.: Vitamin D activates NPR-A gene transcription in IMCD cells

CA, USA). VDR antibody (cat no. sc-1008) and glyceraldehyde-3- RNA isolation and Northern blot analysis. phosphate dehydrogenase antibody (cat no. sc-32233) were IMCD cells were cultured and treated with 1,25(OH)2D3 or RO-25- purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA). 6760 at different concentrations, as indicated in the individual figure 25-hydroxyvitamin D 1a-hydoxylase antibody (cat no. PC290) was legends. Total RNA was prepared using the RNeasy minikit (Qiagen, from The Binding Site (Birmingham, UK). NPR-A antibody (cat no. Valencia, CA, USA). Denatured RNA was separated on a gel, ab14356) was obtained from Abcam Inc. (Cambridge, MA, USA). transferred to nitrocellulose filters, and hybridized to radiolabeled cDNA as described previously. A 1.2-kb EcoRI fragment of the rat Isolation and culture of rat IMCD cells NPR-A cDNA was radiolabeled using the primer-it RMT kit Adult Sprague–Dawley rats were killed by CO2 narcosis, followed (Stratagene, LaDolla, CA, USA) and separated from free nucleotide by bilateral thoracotomy in compliance with a protocol approved using NucTrap push columns (Stratagene). The membranes were by the University of California at San Francisco Committee on hybridized with 32P-labeled NPR-A cDNA for 1 h at 681Cin Animal Research. The inner medullary tissue from each kidney was hybridization solution provided by Stratagene. All membranes were dissected free from the outer medulla, minced, and digested with subsequently stripped and rehybridized with a radiolabeled 1150-bp 1 mg/ml collagenase at 371C with gentle agitation during each BamHI/EcoRI fragment of 18S ribosomal DNA to permit normal- 30-min cycle. IMCD cells were enriched in the preparation using ization among samples for differences in RNA loading and/or hypotonic lysis as described previously.39 Cells were resuspended in transfer to the filter. Hybridization signal was detected by medium-1 (1:1 mixture of Dulbecco’s modified Eagle’s medium and autoradiography and quantified using Kodak Scientific Imaging Ham’s F-12 medium supplemented with 10% fetal bovine serum, systems. 42 mM sodium bicarbonate, 100 IU/ml penicillin, and 100 mg/ml streptomycin)24 and seeded onto culture plates. After 24 h, the cells Site-directed mutagenesis were placed in K-1 medium (1:1 mixture of Dulbecco’s modified A VDRE in the À1575 NPR-A promoter was identified (À472 50-GA Eagle’s medium and Ham’s F-12 medium supplemented with 10 mM GCCAGAGTCCGGGTTACCCAGTCCATCAATG-30 À504; element N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic (HEPES) (pH in italics and underlined) and mutations (À472 50-GAGCCAGAGT 7.4), 42 mM sodium bicarbonate, 5 mg/ml , 50 nM hydro- CCGtGTTgCCCAtTCCATCAATG-30 À504; mutated bases in lower , 5 mg/ml transferrin, 5 pM triiodothyronine, 100 IU/ml case letters) were created in –1575 NPR-A-Luc by site-directed penicillin, and 100 mg/ml streptomycin)24 and cultured for 3–4 days. mutagenesis using a commercial kit (Stratagene), as reported previously.21 Measurement of 25(OH)D3 1a-hydroxylase activity in IMCD cells Transfection and luciferase assay Seventy percent confluent IMCD cells in six-well plates were treated Cells were plated in six-well plates and grown to about 70% À7 with 10 M 25(OH)D3 for 24 h. Medium (2 ml) from cultured cells confluence. At that time, transfection was performed with lipofectin was collected and 25(OH)D3 1a-hydroxylase activity was assessed in reagent (Invitrogen Corp, Carlsbad, CA, USA) using a protocol 0.1 ml delipidated medium by measurement of 1,25(OH)2D3 using recommended by the manufacturer. One microgram of wild-type- 40,41 a 1,25(OH)2D3 enzyme-linked immunosorbent assay kit (Im- or VDRE-mutated À1575 NPR-A-Luc together with 0.5 mg Renilla- munodiagnostics Systems Inc.; Fountain Hills, AZ). This enzyme- Luc were introduced into each well. The DNA–liposome suspension linked immunosorbent assay kit is specific for 1,25(OH)2D3 and has was incubated with the cultures for 5–6h at 371C in Opti-MEMI minimal reactivity with other vitamin D metabolites (e.g., 0.009% reduced serum medium (Invitrogen Corp). The suspension was cross-reactivity with 25(OH)D3). To control for cross-reactivity of then removed and replaced with K-1 medium for the ensuing 24 h, 25(OH)D3 or nonenzymatic generation of 1,25(OH)2D3 in the at which point cells were treated with 1,25(OH)2D3 for 48 h. assay, medium containing the same concentration of 25(OH)D3 was Luciferase activity was measured using the Dual-Luciferases incubated under identical condition but without cells. The assay was reporter assay system (Promega, Madison, WI, USA). Transfection performed by following the protocol provided by Immunodiagnos- efficiency was normalized for Renilla luciferase activity in the tics Systems Inc. individual cultures.

Measurement of ANP-stimulated cGMP levels Immunoblotting

IMCD cells were grown to approximately 80% confluence and The cells were treated with 1,25(OH)2D3 at different concentrations À9 –7 treated with 10 –10 M 1,25(OH)2D3 for 48 h. For measurement of for 48 h, then scraped into lysis buffer containing protease inhibitors ANP-stimulated cGMP accumulation, cells were washed three times (1 tablet/50 ml; Roche Applied Science, Indianapolis, IN, USA). with prewarmed phosphate-buffered saline and incubated with Twenty micrograms of total protein was denatured at 1001C for 0.5 ml of Dulbecco’s modified Eagle’s medium that contained 3 min, subjected to 10% sodium dodecyl sulfate–polyacrylamide gel 0.5 mM isobutylmethylxanthine and 10 mM HEPES (pH 7.4) for electrophoresis, and transferred onto polyvinylidene fluoride 10 min at 371C. The incubation was continued for another 10 min membranes. The membranes were blocked with 5% nonfat –7 with the addition of 10 M ANP to the medium. The reaction was in TBST (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) stopped by the removal of medium and addition of 0.3 ml of 10% and probed with a rabbit polyclonal antibody directed against VDR trichloroacetic acid. The extraction was continued for 30 min at 41C. and a sheep anti-murine 25(OH)D3 1a-hydroxylase. Horseradish The contents of the plate were collected and centrifuged to pellet peroxidase-conjugated second antibodies were used to detect particulate material. The supernatant fraction was extracted four immunoreactive bands using the enhanced chemiluminescence times with 0.5 ml of water-saturated ether. cGMP levels were Western blotting-detection system (Amersham Bioscience, Piscat- determined by radioimmunoassay after acetylation of the samples away, NJ, USA). The same membranes were reblotted with anti- and standards using a commercial antibody and [125I]cGMP as glyceraldehyde-3-phosphate dehydrogenase antibody. VDR and tracer. 25(OH)D3 1a-hydroxylase signals were quantified using Kodak

304 Kidney International (2007) 72, 300–306 S Chen et al.: Vitamin D activates NPR-A gene transcription in IMCD cells original article

Scientific Imaging systems and normalized for glyceraldehyde-3- Western blot analysis. In a separate experiment, 1 mg À1575 rat phosphate dehydrogenase signal. NPR-A-Luc and 0.5 mg Renilla-Luc were cotransfected with To prepare membrane protein, cells were treated with 100 pmol V1, V2, V3, or control siRNA into IMCD cells using 1,25(OH)2D3 or its analogue for 48 h and then scraped into ice- lipofectamine. After 48 h, cells were collected and lysates were cold phosphate-buffered saline containing protease inhibitors. After generated for luciferase (firefly and Renilla) assay. brief centrifugation, cells were resuspended in buffer 1 (50 mM Tris, pH 7.5, 200 mM sucrose, 1 mM ethylenediaminetetraacetic acid, 3 mM Statistical analyses MgCl2, and protease inhibitors) and sonicated. After centrifu- Data were analyzed by analysis of variance, using the Newman–Keuls gation at 500 r.p.m. for 10 min, soluble and membrane fractions test to assess significance. were separated by centrifugation at 80 000 r.p.m. for 1 h at 41C. The particulate fractions were resuspended in lysis buffer. Fifty ACKNOWLEDGMENTS microgram of membrane protein were used for Western blot with This work was supported in part by HL45637 from the NIH. 1:1000 NPR-A antibody. The same filter was washed and reprobed with b tubulin antibody (Santa Cruz, Biotechnology). NPR-A signal REFERENCES was quantified on a Kodak Image Station and normalized for beta 1. Levin A, Li YC. Vitamin D and its analogues: do they protect against tubulin signal. cardiovascular disease in patients with kidney disease? Kidney Int 2005; 68: 1973–1981. 2. Weishaar RE, Simpson RU. Vitamin D3 and cardiovascular function in rats. 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(3–5 mg) were incubated in binding reaction buffer (10 mM HEPES 11. Fahrleitner A, Dobnig H, Obernosterer A et al. Vitamin D deficiency and at pH 7.9, 50 mM KCl, 0.2 mM ethylenediaminetetraacetic acid, secondary hyperparathyroidism are common complications in patients with peripheral arterial disease. J Gen Intern Med 2002; 17: 663–669. 2.5 mM dithiothreitol, 10% glycerol, and 0.05% nonidet P-40) 12. Lind L, Wengle B, Ljunghall S. Blood pressure is lowered by vitamin D 32 containing 1 mg of poly(dI-dC) and P-end-labeled double-stranded (alphacalcidol) during long-term treatment of patients with intermittent wild-type NPR-A4 oligonucleotide at room temperature for 30 min. . A double-blind, placebo-controlled study. Acta Med For competition experiments, a 50-fold molar excess of unlabeled, Scand 1987; 222: 423–427. 13. Lind L, Wengle B, Wide L et al. Reduction of blood pressure during double-stranded oligonucleotide was added to the binding reaction. long-term treatment with active vitamin D (alphacalcidol) is All samples were resolved on 4% nondenaturing polyacrylamide dependent on plasma renin activity and calcium status. A double-blind, gels. Gels were dried and exposed to X-ray film. placebo-controlled study. Am J Hypertens 1989; 2: 20–25. 14. Pfeifer M, Begerow B, Minne HW et al. Effects of a short-term vitamin D(3) and calcium supplementation on blood pressure and parathyroid siRNA silencing of VDR gene levels in elderly women. J Clin Endocrinol Metab 2001; 86: Twenty one-nucleotide annealed duplex siRNAs directed against 1633–1637. rat VDR gene sequence (V1 – GCGUAAGAGGAGAUGAUAtt, 15. Park CW, Oh YS, Shin YS et al. Intravenous regresses myocardial hypertrophy in hemodialysis patients with secondary V2 – GGAUUCUGAUGACCCGUCUtt, and V3 – GGAGUUCAUCC hyperparathyroidism. Am J Kidney Dis 1999; 33: 73–81. UGACAGAUtt; all sense strand) were chemically synthesized and 16. Teng M, Wolf M, Ofsthun MN et al. Activated injectable vitamin D and purified using a commercial source (Amibon, Austin, TX) as was a hemodialysis survival: a historical cohort study. J Am Soc Nephrol 2005; negative control siRNA (Ambion) containing a 19-bp scrambled 16: 1115–1125. 17. Teng M, Wolf M, Lowrie E et al. Survival of patients undergoing sequence. The latter sequences have no significant homology to any hemodialysis with paricalcitol or calcitriol therapy. N Engl J Med 2003; known gene sequences from rat. IMCD cells were cultured to 349: 446–456. 60–70% confluence and then transfected with 100 pmol V1, V2, V3, 18. Zeidel ML. Hormonal regulation of inner medullary collecting duct or the negative control siRNA using lipofectamine (Invitrogen Life sodium transport. Am J Physiol 1993; 265: F159–F173. 19. Drummer C. Involvement of the renal natriuretic peptide urodilatin in Technologies, Carlsbad, CA, USA) according to the manufacturer’s body fluid regulation. Semin Nephrol 2001; 21: 239–243. instructions. After transfection for 48 h, cells were washed and 20. Levin ER, Gardner DG, Samson WK. Natriuretic peptides. N Engl J Med cellular lysates were prepared and analyzed for VDR expression by 1998; 339: 321–328.

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