Vitamin D activates type A natriuretic peptide receptor gene transcription in inner medullary collecting duct cells
2007; Elsevier BV; Volume: 72; Issue: 3 Linguagem: Inglês
10.1038/sj.ki.5002274
ISSN1523-1755
AutoresS. Chen, Keith Olsen, Christopher L. Grigsby, David G. Gardner,
Tópico(s)Electrolyte and hormonal disorders
ResumoMany clinical and animal studies suggest that vitamin D and its metabolites have beneficial effects in the cardiovascular and renal systems. Using immunologic and enzymatic assays, vitamin D receptor and 25 hydroxyvitamin D3 1α-hydroxylase activity were found in inner medullary collecting duct (IMCD) cells suggesting an autocrine/paracrine role in this nephron segment. In this study, we examined the ability of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to regulate the expression of the vasculoprotective natriuretic peptide receptor-A gene in these cells in culture. Treatment of the cells with 1,25(OH)2D3 caused a doubling of natriuretic peptide-dependent cyclic guanosine monophosphate production and a significant increase in natriuretic peptide receptor-A protein expression. This was accompanied by significant increases in receptor mRNA levels and gene-promoter activity. Mutation of a vitamin D response element, positioned upstream from the gene start site, resulted in a complete loss of 1,25(OH)2D3-dependent induction but not the induction by hypertonic stimuli. Introduction of small interfering RNA directed against the vitamin D receptor into the IMCD cells resulted in decreased natriuretic peptide receptor-A gene promoter activity and protein. The increase in this receptor expression may account for some of the reported beneficial effect of 1,25(OH)2D3 on the cardiovascular system and kidney. Many clinical and animal studies suggest that vitamin D and its metabolites have beneficial effects in the cardiovascular and renal systems. Using immunologic and enzymatic assays, vitamin D receptor and 25 hydroxyvitamin D3 1α-hydroxylase activity were found in inner medullary collecting duct (IMCD) cells suggesting an autocrine/paracrine role in this nephron segment. In this study, we examined the ability of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to regulate the expression of the vasculoprotective natriuretic peptide receptor-A gene in these cells in culture. Treatment of the cells with 1,25(OH)2D3 caused a doubling of natriuretic peptide-dependent cyclic guanosine monophosphate production and a significant increase in natriuretic peptide receptor-A protein expression. This was accompanied by significant increases in receptor mRNA levels and gene-promoter activity. Mutation of a vitamin D response element, positioned upstream from the gene start site, resulted in a complete loss of 1,25(OH)2D3-dependent induction but not the induction by hypertonic stimuli. Introduction of small interfering RNA directed against the vitamin D receptor into the IMCD cells resulted in decreased natriuretic peptide receptor-A gene promoter activity and protein. The increase in this receptor expression may account for some of the reported beneficial effect of 1,25(OH)2D3 on the cardiovascular system and kidney. Vitamin D, or more specifically its polar metabolite 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), is thought to play a significant role in the regulation of growth and function in the cardiovascular system and kidney.1.Levin A. Li Y.C. Vitamin D and its analogues: do they protect against cardiovascular disease in patients with kidney disease?.Kidney Int. 2005; 68: 1973-1981Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar Vitamin D deficiency in rats is associated with hypertension and cardiac hypertrophy2.Weishaar R.E. Simpson R.U. Vitamin D3 and cardiovascular function in rats.J Clin Invest. 1987; 79: 1706-1712Crossref PubMed Scopus (151) Google Scholar and expansion of the interstitial compartment of the myocardium.3.Weishaar R.E. Kim S.N. Saunders D.E. et al.Involvement of vitamin D3 with cardiovascular function. III. Effects on physical and morphological properties.Am J Physiol. 1990; 258: E134-142PubMed Google Scholar Treatment of cultured neonatal cardiac myocytes with 1,25(OH)2D3 effectively reverses endothelin-induced hypertrophy in this in vitro model.4.Wu J. Garami M. Cheng T. et al.1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.J Clin Invest. 1996; 97: 1577-1588Crossref PubMed Scopus (215) Google Scholar Genetic deletion of the vitamin D receptor5.Li Y.C. Pirro A.E. Amling M. et al.Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.Proc Natl Acad Sci USA. 1997; 94: 9831-9835Crossref PubMed Scopus (753) Google Scholar results in a mouse with hyper-reninemic hypertension6.Li Y.C. Kong J. Wei M. et al.1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system.J Clin Invest. 2002; 110: 229-238Crossref PubMed Scopus (1615) Google Scholar and cardiac hypertrophy.7.Xiang W. Kong J. Chen S. et al.Cardiac hypertrophy in vitamin D receptor knockout mice: role of the systemic and cardiac renin-angiotensin systems.Am J Physiol Endocrinol Metab. 2005; 288: E125-E132Crossref PubMed Scopus (480) Google Scholar Subsets of human hypertensive patients have low 25-hydroxyvitamin D3 (25(OH)D3) levels,8.Lind L. Hanni A. Lithell H. et al.Vitamin D is related to blood pressure and other cardiovascular risk factors in middle-aged men.Am J Hypertens. 1995; 8: 894-901Crossref PubMed Scopus (258) Google Scholar as do patients with congestive heart failure9.Shane E. Mancini D. Aaronson K. et al.Bone mass, vitamin D deficiency, and hyperparathyroidism in congestive heart failure.Am J Med. 1997; 103: 197-207Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar,10.Zittermann A. Schleithoff S.S. Tenderich G. et al.Low vitamin D status: a contributing factor in the pathogenesis of congestive heart failure?.J Am Coll Cardiol. 2003; 41: 105-112Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar and peripheral vascular disease.11.Fahrleitner A. Dobnig H. Obernosterer A. et al.Vitamin D deficiency and secondary hyperparathyroidism are common complications in patients with peripheral arterial disease.J Gen Intern Med. 2002; 17: 663-669Crossref PubMed Scopus (74) Google Scholar Treatment of small groups of hypertensive patients with 1,25(OH)2D3 has been shown to lower blood pressure,12.Lind L. Wengle B. Ljunghall S. Blood pressure is lowered by vitamin D (alphacalcidol) during long-term treatment of patients with intermittent hypercalcaemia. A double-blind, placebo-controlled study.Acta Med Scand. 1987; 222: 423-427Crossref PubMed Scopus (42) Google Scholar, 13.Lind L. Wengle B. Wide L. et al.Reduction of blood pressure during long-term treatment with active vitamin D (alphacalcidol) is dependent on plasma renin activity and calcium status. A double-blind, placebo-controlled study.Am J Hypertens. 1989; 2: 20-25Crossref PubMed Scopus (94) Google Scholar, 14.Pfeifer M. Begerow B. Minne H.W. et al.Effects of a short-term vitamin D(3) and calcium supplementation on blood pressure and parathyroid hormone levels in elderly women.J Clin Endocrinol Metab. 2001; 86: 1633-1637Crossref PubMed Scopus (549) Google Scholar whereas treatment of patients with end-stage renal disease on dialysis with 1,25(OH)2D3 leads to regression of left ventricular hypertrophy in this patient population.15.Park C.W. Oh Y.S. Shin Y.S. et al.Intravenous calcitriol regresses myocardial hypertrophy in hemodialysis patients with secondary hyperparathyroidism.Am J Kidney Dis. 1999; 33: 73-81Abstract Full Text Full Text PDF PubMed Google Scholar Furthermore, retrospective analysis of dialysis patients treated with either 1,25(OH)2D316.Teng M. Wolf M. Ofsthun M.N. et al.Activated injectable vitamin D and hemodialysis survival: a historical cohort study.J Am Soc Nephrol. 2005; 16: 1115-1125Crossref PubMed Scopus (714) Google Scholar or paricalcitol,17.Teng M. Wolf M. Lowrie E. et al.Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy.N Engl J Med. 2003; 349: 446-456Crossref PubMed Scopus (851) Google Scholar a 1,25(OH)2D3 analogue with reduced calcemic potential, reduced mortality compared with controls. As cardiovascular disease is the leading cause of death in patients with end-stage renal disease on dialysis, it can be inferred that these vitamin D receptor (VDR) ligands are positively impacting cardiovascular disease in this patient group. 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.Zeidel M.L. Hormonal regulation of inner medullary collecting duct sodium transport.Am J Physiol. 1993; 265: F159-F173PubMed Google Scholar Sodium handling in this nephron segment is highly regulated by a number of hormones including atrial natriuretic peptide (ANP). ANP is produced in the heart and acts by binding to natriuretic peptide receptor type A (NPR-A) in target tissues and triggering increased synthesis of cyclic guanosine monophosphate (cGMP). Binding of circulating ANP or its locally produced homologue urodilatin19.Drummer C. Involvement of the renal natriuretic peptide urodilatin in body fluid regulation.Semin Nephrol. 2001; 21: 239-243Abstract Full Text PDF PubMed Scopus (14) Google Scholar to NPR-A on IMCD cells leads to an increase in urinary sodium excretion through a cGMP-dependent mechanism.20.Levin E.R. Gardner D.G. Samson W.K. Natriuretic peptides.N Engl J Med. 1998; 339: 321-328Crossref PubMed Scopus (1968) Google Scholar We have recently shown that treatment of rat aortic smooth muscle cells with 1,25(OH)2D3 leads to an increase in expression and activity of NPR-A in these cells.21.Chen S. Ni X.P. Humphreys M.H. et al.1,25 dihydroxyvitamin D amplifies type a natriuretic peptide receptor expression and activity in target cells.J Am Soc Nephrol. 2005; 16: 329-339Crossref PubMed Scopus (24) Google Scholar In this study, we demonstrate that VDR, present in IMCD cells, triggers a similar increase in NPR-A activity and gene expression through a transcriptional mechanism and that suppression of endogenous VDR levels reverses this response. We hypothesize that low levels of 1,25(OH)2D3 or VDR-dependent activity may contribute to sodium retention seen in disorders like congestive heart failure and salt-sensitive hypertension through a reduction in NPR-A activity. Vitamin D has been suggested to play a role in the regulation of renal calcium handling, possibly through induction of calcium transport proteins in different nephron segments.22.Glendenning P. Ratajczak T. Dick I.M. et al.Calcitriol upregulates expression and activity of the 1b isoform of the plasma membrane calcium pump in immortalized distal kidney tubular cells.Arch Biochem Biophys. 2000; 380: 126-132Crossref PubMed Scopus (29) Google Scholar To assess the presence of endogenous VDR and 25(OH)D3 1α-hydroxylase in cultured IMCD cells, we examined 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 1α-hydroxylase were readily demonstrable in these extracts. Interestingly, pretreatment with 1,25(OH)2D3 resulted in a dose-dependent increment in VDR levels (2–2.5-fold increase relative to control) over a physiologically relevant range of ligand concentrations. 1,25(OH)2D3 treatment had no effect on 25(OH)D3 1α-hydroxylase expression. To demonstrate 25(OH)D3 1α-hydroxylase activity in IMCD cells, we measured 1,25(OH)2D3 production and release in the medium of cultured IMCD cells following addition of 25(OH)D3 substrate. Compared with the substrate-free cultures, there was a dramatic increase in 1,25(OH)2D3 levels in the medium of the 25(OH)D3-treated group (3.63±0.47 vs 11.94±2.17 pg/ml, n=3 P<0.01), demonstrating the ability of IMCD cells to covert 25(OH)D3 to 1,25(OH)2D3. We next assessed the effect of 1,25(OH)2D3 treatment on the ability of these IMCD cells to respond to ANP, as a surrogate marker of NPR-A activity. As shown in Figure 2, pretreatment with 1,25(OH)2D3 or the 1,25(OH)2D3 analogue, RO-25-6760, elicited a doubling in ANP-dependent cGMP generation and 3–4-fold increase in NPR-A protein expression in these cells. The increase in NPR-A activity was accompanied by an increase in NPR-A mRNA levels in IMCD cells. Both 1,25(OH)2D3 and RO-25-6760 elucited a significant increase in NPR-A mRNA levels, which peaked ∼3-fold above the controls treated with vehicle alone (Figure 3). The increase in NPR-A transcript levels was linked to an increase in NPR-A gene transcriptional activity. As shown in Figure 4a, 1,25(OH)2D3 treatment led to ∼2.5-fold increase in NPR-A gene promoter activity over a physiological concentration range for the ligand. Transfection of additional exogenous receptor (Figure 4b) led to further amplification of the 1,25(OH)2D3-dependent stimulation of promoter activity. To explore the VDR dependence of this stimulatory activity, we used a small interfering RNA (siRNA) strategy directed against endogenous VDR-coding sequence. Treatment of IMCD cells with siRNAs directed against the VDR resulted in a decrease in VDR levels in these cells (Figure 5a) with two of the three siRNAs (V1 and V2, but not V3) and a reduction in 1,25(OH)2D3-dependent stimulation of NPR-A-promoter activity (Figure 5b). A negative control siRNA, which lacked targeting capability, was without effect. VD-dependent induction of NPR-A gene-promoter activity was virtually completely dependent on the presence of a VDR element (VDRE) located ∼495 bp upstream from the transcription start site in the promoter (Figure 6a). This was also true for RO-25-6760; however, the VDRE mutation had no effect on the osmotic induction of promoter activity by NaCl or sucrose.23.Chen S. Gardner D.G. Osmoregulation of natriuretic peptide receptor signaling in inner medullary collecting duct. A requirement for p38 MAPK.J Biol Chem. 2002; 277: 6037-6043Crossref PubMed Scopus (32) Google Scholar Furthermore, addition of 1,25(OH)2D3 to either NaCl or sucrose resulted in a modest, albeit not additive, increase in promoter activity which was only partially reduced in the VDRE-mutant promoter-driven reporter. Electrophoretic mobility shift assay of IMCD cell extracts demonstrated that 1,25(OH)2D3 promoted VDR/retinoid X receptor (RXR) binding to oligonucleotides harboring the NPR-A VDRE (Figure 6b). The binding was eliminated in the presence of unlabeled wild-type DNA sequence; however, unlabeled, mutated sequence was unable to compete. The findings presented above support our hypothesis that 1,25(OH)2D3 stimulates transcription of the gene encoding NPR-A in IMCD cells. It does so through a mechanism that requires the vitamin D receptor and the presence of a functional VDRE positioned ∼495 bp upstream from the NPR-A gene transcription start site. This supports our earlier demonstration of 1,25(OH)2D3-dependent stimulation of NPR-A gene regulation in rat aortic smooth muscle cells21.Chen S. Ni X.P. Humphreys M.H. et al.1,25 dihydroxyvitamin D amplifies type a natriuretic peptide receptor expression and activity in target cells.J Am Soc Nephrol. 2005; 16: 329-339Crossref PubMed Scopus (24) Google Scholar and the findings of Li et al.24.Li X. Zheng W. Li Y.C. Altered gene expression profile in the kidney of vitamin D receptor knockout mice.J Cell Biochem. 2003; 89: 709-719Crossref PubMed Scopus (42) Google Scholar describing reduced NPR-A gene expression in the kidneys of VDR -/- mice. Of note, we have shown here that IMCD cells express 25(OH)D3 1α-hydroxylase, the enzyme which is responsible for conversion of 25(OH)D3 to its more polar and bioactive metabolite, 1,25(OH)2D3. Endogenous 1,25(OH)2D3 in IMCD cells probably contributes to the maintenance of basal NPR-A levels. Local production of 1,25(OH)2D3 may accommodate the need for relatively high ligand concentrations (above circulating plasma concentrations) that we observed in this study (see Figures 2 and 3). Recent studies have suggested that 1,25(OH)2D3 may play an important role in the regulation of a variety of physiological processes, including skin and hair follicle development,25.Vegesna V. O'Kelly J. Uskokovic M. et al.Vitamin D3 analogs stimulate hair growth in nude mice.Endocrinology. 2002; 143: 4389-4396Crossref PubMed Scopus (35) Google Scholar,26.Kira M. Kobayashi T. Yoshikawa K. Vitamin D and the skin.J Dermatol. 2003; 30: 429-437Crossref PubMed Scopus (30) Google Scholar growth and function in the cardiovascular system,4.Wu J. Garami M. Cheng T. et al.1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.J Clin Invest. 1996; 97: 1577-1588Crossref PubMed Scopus (215) Google Scholar,27.Mitsuhashi T. Morris Jr, R.C. Ives HE: 1,25-dihydroxyvitamin D3 modulates growth of vascular smooth muscle cells.J Clin Invest. 1991; 87: 1889-1895Crossref PubMed Scopus (163) Google Scholar and the control of immune function and inflammatory response.28.Hayes C.E. Nashold F.E. Spach K.M. et al.The immunological functions of the vitamin D endocrine system.Cell Mol Biol (Noisy-le-grand). 2003; 49: 277-300PubMed Google Scholar,29.Mathieu C. Van Etten E. Gysemans C. et al.In vitro and in vivo analysis of the immune system of vitamin D receptor knockout mice.J Bone Miner Res. 2001; 16: 2057-2065Crossref PubMed Scopus (132) Google Scholar All of these are largely independent of the more classical, and better understood, effects of 1,25(OH)2D3 on mineral homeostasis and bone metabolism. Observational studies suggest that the presence of hypertension30.Rostand S.G. Ultraviolet light may contribute to geographic and racial blood pressure differences.Hypertension. 1997; 30: 150-156Crossref PubMed Scopus (359) Google Scholar and susceptibility to certain types of infections like tuberculosis,31.Liu P.T. Stenger S. Li H. et al.Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.Science. 2006; 311: 1770-1773Crossref PubMed Scopus (2735) Google Scholar, 32.Nursyam E.W. Amin Z. Rumende C.M. The effect of vitamin D as supplementary treatment in patients with moderately advanced pulmonary tuberculous lesion.Acta Med Indones. 2006; 38: 3-5PubMed Google Scholar, 33.Ustianowski A. Shaffer R. Collin S. et al.Prevalence and associations of vitamin D deficiency in foreign-born persons with tuberculosis in London.J Infect. 2005; 50: 432-437Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar correlate inversely with the ability to generate endogenous vitamin D. In the cardiovascular system, 1,25(OH)2D3 has been shown to regulate proliferative activity in vascular smooth muscle cells in culture,28.Hayes C.E. Nashold F.E. Spach K.M. et al.The immunological functions of the vitamin D endocrine system.Cell Mol Biol (Noisy-le-grand). 2003; 49: 277-300PubMed Google Scholar to antagonize cardiac myocyte hypertrophy in an in vitro model,4.Wu J. Garami M. Cheng T. et al.1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.J Clin Invest. 1996; 97: 1577-1588Crossref PubMed Scopus (215) Google Scholar to reduce blood pressure in selected groups of human hypertensives,13.Lind L. Wengle B. Wide L. et al.Reduction of blood pressure during long-term treatment with active vitamin D (alphacalcidol) is dependent on plasma renin activity and calcium status. A double-blind, placebo-controlled study.Am J Hypertens. 1989; 2: 20-25Crossref PubMed Scopus (94) Google Scholar,14.Pfeifer M. Begerow B. Minne H.W. et al.Effects of a short-term vitamin D(3) and calcium supplementation on blood pressure and parathyroid hormone levels in elderly women.J Clin Endocrinol Metab. 2001; 86: 1633-1637Crossref PubMed Scopus (549) Google Scholar and to reverse hypertrophy in patients with end-stage renal disease on dialysis.15.Park C.W. Oh Y.S. Shin Y.S. et al.Intravenous calcitriol regresses myocardial hypertrophy in hemodialysis patients with secondary hyperparathyroidism.Am J Kidney Dis. 1999; 33: 73-81Abstract Full Text Full Text PDF PubMed Google Scholar Furthermore, treatment of dialysis patients with 1,25(OH)2D3,16.Teng M. Wolf M. Ofsthun M.N. et al.Activated injectable vitamin D and hemodialysis survival: a historical cohort study.J Am Soc Nephrol. 2005; 16: 1115-1125Crossref PubMed Scopus (714) Google Scholar or a 1,25(OH)2D3 analogue with reduced calcemic potential,17.Teng M. Wolf M. Lowrie E. et al.Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy.N Engl J Med. 2003; 349: 446-456Crossref PubMed Scopus (851) Google Scholar leads to improvement in survival, a variable which is heavily dependent on the presence of cardiovascular disease in this population. Collectively, these studies support the notion that 1,25(OH)2D3, at least at nontoxic concentrations, exerts a beneficial effect in promoting 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 25(OH)D3 and 1,25(OH)2D39.Shane E. Mancini D. Aaronson K. et al.Bone mass, vitamin D deficiency, and hyperparathyroidism in congestive heart failure.Am J Med. 1997; 103: 197-207Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar,10.Zittermann A. Schleithoff S.S. Tenderich G. et al.Low vitamin D status: a contributing factor in the pathogenesis of congestive heart failure?.J Am Coll Cardiol. 2003; 41: 105-112Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar and treatment of heart failure patients with 1,25(OH)2D3 has been shown to diminish plasma levels of cytokines (e.g., tumor necrosis factor) that are associated with myocardial inflammation.34.Schleithoff S.S. Zittermann A. Tenderich G. et al.Vitamin D supplementation improves cytokine profiles in patients with congestive heart failure: a double-blind, randomized, placebo-controlled trial.Am J Clin Nutr. 2006; 83: 754-759PubMed Google Scholar Heart failure is characterized by resistance to natriuretic peptides, a phenomenon which may, in part, reflect altered NPR function.35.Charloux A. Piquard F. Doutreleau S. et al.Mechanisms of renal hyporesponsiveness to ANP in heart failure.Eur J Clin Invest. 2003; 33: 769-778Crossref PubMed Scopus (86) Google Scholar The excessive sodium retention associated with heart failure is exacerbated by inhibition of NPR-A activity36.Nishikimi T. Hagaman J.R. Takahashi N. et al.Increased susceptibility to heart failure in response to volume overload in mice lacking natriuretic peptide receptor-A gene.Cardiovasc Res. 2005; 66: 94-103Crossref PubMed Scopus (23) Google Scholar,37.Zhang P.L. Mackenzie H.S. Totsune K. et al.Renal effects of high-dose natriuretic peptide receptor blockade in rats with congestive heart failure.Circ Res. 1995; 77: 1240-1245Crossref PubMed Scopus (18) Google Scholar and myocardial NPR-A is thought to mediate a ligand-dependent antagonism of hypertrophy and fibrosis in the myocardial wall.38.Holtwick R. van Eickels M. Skryabin B.V. et al.Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A.J Clin Invest. 2003; 111: 1399-1407Crossref PubMed Scopus (280) Google Scholar By inference, 1,25(OH)2D3 insufficiency or bonafide 1,25(OH)2D3 deficiency could, through an indirect effect to reduce NPR-A activity, play an important role in accelerating development of heart failure, or salt-sensitive hypertension, and in contributing to the excessive sodium retention that occurs in those diseases. The cGMP radioimmunoassay kit was obtained from Perkin-Elmer Life Sciences (Boston, MA, USA). ANP was purchased from American Peptide (Sunnyvale, CA, USA). R0-25-6760 (1,25-dihydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-cholecalciferol) was a kind gift of M Uskokovic (Roche Pharmaceuticals, Nutley, NJ, USA). 1,25(OH)2D3 was obtained from Calbiochem Inc. (La Jolla, CA, USA). VDR antibody (cat no. sc-1008) and glyceraldehyde-3-phosphate dehydrogenase antibody (cat no. sc-32233) were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA). 25-hydroxyvitamin D 1α-hydoxylase antibody (cat no. PC290) was from The Binding Site (Birmingham, UK). NPR-A antibody (cat no. ab14356) was obtained from Abcam Inc. (Cambridge, MA, USA). Adult Sprague–Dawley rats were killed by CO2 narcosis, followed by bilateral thoracotomy in compliance with a protocol approved by the University of California at San Francisco Committee on Animal Research. The inner medullary tissue from each kidney was dissected free from the outer medulla, minced, and digested with 1 mg/ml collagenase at 37°C with gentle agitation during each 30-min cycle. IMCD cells were enriched in the preparation using hypotonic lysis as described previously.39.Grenier F.C. Rollins T.E. Smith W.L. Kinin-induced prostaglandin synthesis by renal papillary collecting tubule cells in culture.Am J Physiol. 1981; 241: F94-F104PubMed Google Scholar Cells were resuspended in medium-1 (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal bovine serum, 42 mM sodium bicarbonate, 100 IU/ml penicillin, and 100 μg/ml streptomycin)24.Li X. Zheng W. Li Y.C. Altered gene expression profile in the kidney of vitamin D receptor knockout mice.J Cell Biochem. 2003; 89: 709-719Crossref PubMed Scopus (42) Google Scholar and seeded onto culture plates. After 24 h, the cells were placed in K-1 medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic (HEPES) (pH 7.4), 42 mM sodium bicarbonate, 5 μg/ml insulin, 50 nM hydrocortisone, 5 μg/ml transferrin, 5 pM triiodothyronine, 100 IU/ml penicillin, and 100 μg/ml streptomycin)24.Li X. Zheng W. Li Y.C. Altered gene expression profile in the kidney of vitamin D receptor knockout mice.J Cell Biochem. 2003; 89: 709-719Crossref PubMed Scopus (42) Google Scholar and cultured for 3–4 days. Seventy percent confluent IMCD cells in six-well plates were treated with 10-7 M 25(OH)D3 for 24 h. Medium (2 ml) from cultured cells was collected and 25(OH)D3 1α-hydroxylase activity was assessed in 0.1 ml delipidated medium by measurement of 1,25(OH)2D3 using a 1,25(OH)2D3 enzyme-linked immunosorbent assay kit40.Feng J.Q. Ward L.M. Liu S. et al.Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.Nat Genet. 2006; 38: 1310-1315Crossref PubMed Scopus (859) Google Scholar,41.Fritsche J. Mondal K. Ehrnsperger A. et al.Regulation of 25-hydroxyvitamin D3-1α-hydroxylase and production of 1α, 25-dihydroxyvitamin D3 by human dendritic cells.Blood. 2003; 102: 3314-3316Crossref PubMed Scopus (196) Google Scholar (Immunodiagnostics Systems Inc.; Fountain Hills, AZ). This enzyme-linked immunosorbent assay kit is specific for 1,25(OH)2D3 and has minimal reactivity with other vitamin D metabolites (e.g., 0.009% cross-reactivity with 25(OH)D3). To control for cross-reactivity of 25(OH)D3 or nonenzymatic generation of 1,25(OH)2D3 in the assay, medium containing the same concentration of 25(OH)D3 was incubated under identical condition but without cells. The assay was performed by following the protocol provided by Immunodiagnostics Systems Inc. IMCD cells were grown to approximately 80% confluence and treated with 10-9–10–7 M 1,25(OH)2D3 for 48 h. For measurement of ANP-stimulated cGMP accumulation, cells were washed three times with prewarmed phosphate-buffered saline and incubated with 0.5 ml of Dulbecco's modified Eagle's medium that contained 0.5 mM isobutylmethylxanthine and 10 mM HEPES (pH 7.4) for 10 min at 37°C. The incubation was continued for another 10 min with the addition of 10–7 M ANP to the medium. The reaction was stopped by the removal of medium and addition of 0.3 ml of 10% trichloroacetic acid. The extraction was continued for 30 min at 4°C. The contents of the plate were collected and centrifuged to pellet particulate material. The supernatant fraction was extracted four times with 0.5 ml of water-saturated ether. cGMP levels were determined by radioimmunoassay after acetylation of the samples and standards using a commercial antibody and [125I]cGMP as tracer. IMCD cells were cultured and treated with 1,25(OH)2D3 or RO-25-6760 at different concentrations, as indicated in the individual figure legends. Total RNA was prepared using the RNeasy minikit (Qiagen, Valencia, CA, USA). Denatured RNA was separated on a gel, transferred to nitrocellulose filters, and hybridized to radiolabeled cDNA as described previously. A 1.2-kb EcoRI fragment of the rat NPR-A cDNA was radiolabeled using the primer-it RMT kit (Stratagene, LaDolla, CA, USA) and separated from free nucleotide using NucTrap push columns (Stratagene). The membranes were hybridized with 32P-labeled NPR-A cDNA for 1 h at 68°C in hybridization solution provided by Stratagene. All membranes were subsequently stripped and rehybridized with a radiolabeled 1150-bp BamHI/EcoRI fragment of 18S ribosomal DNA to permit normalization among samples for differences in RNA loading and/or transfer to the filter. Hybridization signal was detected by autoradiography and quantified using Kodak Scientific Imaging systems. A VDRE in the -1575 NPR-A promoter was identified (-472 5′-GAGCCAGAGTCCGGGTTACCCAGTCCATCAATG-3′ -504; element in italics and underlined) and mutations (-472 5′-GAGCCAGAGTCCGtGTTgCCCAtTCCATCAATG-3′ -504; mutated bases in lower case letters) were created in –1575 NPR-A-Luc by site-directed mutagenesis using a commercial kit (Stratagene), as reported previously.21.Chen S. Ni X.P. Humphreys M.H. et al.1,25 dihydroxyvitamin D amplifies type a natriuretic peptide receptor expression and activity in target cells.J Am Soc Nephrol. 2005; 16: 329-339Crossref PubMed Scopus (24) Google Scholar Cells were plated in six-well plates and grown to about 70% confluence. At that time, transfection was performed with lipofectin reagent (Invitrogen Corp, Carlsbad, CA, USA) using a protocol recommended by the manufacturer. One microgram of wild-type- or VDRE-mutated -1575 NPR-A-Luc together with 0.5 μg Renilla-Luc were introduced into each well. The DNA–liposome suspension was incubated with the cultures for 5–6 h at 37°C in Opti-MEMI reduced serum medium (Invitrogen Corp). The suspension was then removed and replaced with K-1 medium for the ensuing 24 h, at which point cells were treated with 1,25(OH)2D3 for 48 h. Luciferase activity was measured using the Dual-Luciferase® reporter assay system (Promega, Madison, WI, USA). Transfection efficiency was normalized for Renilla luciferase activity in the individual cultures. The cells were treated with 1,25(OH)2D3 at different concentrations for 48 h, then scraped into lysis buffer containing protease inhibitors (1 tablet/50 ml; Roche Applied Science, Indianapolis, IN, USA). Twenty micrograms of total protein was denatured at 100°C for 3 min, subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk in TBST (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and probed with a rabbit polyclonal antibody directed against VDR and a sheep anti-murine 25(OH)D3 1α-hydroxylase. Horseradish peroxidase-conjugated second antibodies were used to detect immunoreactive bands using the enhanced chemiluminescence Western blotting-detection system (Amersham Bioscience, Piscataway, NJ, USA). The same membranes were reblotted with anti-glyceraldehyde-3-phosphate dehydrogenase antibody. VDR and 25(OH)D3 1α-hydroxylase signals were quantified using Kodak Scientific Imaging systems and normalized for glyceraldehyde-3-phosphate dehydrogenase signal. To prepare membrane protein, cells were treated with 1,25(OH)2D3 or its analogue for 48 h and then scraped into ice-cold phosphate-buffered saline containing protease inhibitors. After brief centrifugation, cells were resuspended in buffer 1 (50 mM Tris, pH 7.5, 200 mM sucrose, 1 mM ethylenediaminetetraacetic acid, 3 mM MgCl2, and protease inhibitors) and sonicated. After centrifugation at 500 r.p.m. for 10 min, soluble and membrane fractions were separated by centrifugation at 80 000 r.p.m. for 1 h at 4°C. The particulate fractions were resuspended in lysis buffer. Fifty microgram of membrane protein were used for Western blot with 1:1000 NPR-A antibody. The same filter was washed and reprobed with β tubulin antibody (Santa Cruz, Biotechnology). NPR-A signal was quantified on a Kodak Image Station and normalized for beta tubulin signal. IMCD cells were transfected with 5 μg of human VDR and human RXR or sham transfected using lipofectin reagent (Invitrogen, Carlsbad, CA, USA) for 24 h. Cells were changed to K-1 media and treated with vehicle or 10-8 M 1,25(OH)2D3 for 48 h. Cells were lysed with buffer A (10 mM HEPES at pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5% nonidet P-40, 1 mM dithiothreitol, protease inhibitors) on ice for 10 min. Lysates were centrifuged for 5 min at 4°C. Particulates were resuspended in buffer C (20 mM HEPES at pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid, 25% glycerol, and the above protease inhibitors) and kept on ice for 30 min. Nuclear suspensions were centrifuged at 12 800 × g for 10 min and the supernatants (nuclear extracts) were used for electrophoretic mobility shift assay. Oligonucleotides used for electrophoretic mobility shift assay were as follows (sequence represents sense strand; italicized represents sequence the putative VDRE), mutated bases in lower case: NPR-A4 (5′-GAGCCAGAGTCCGGGTTACCCAGTCCATCAATG-3'), M2-NPR-A4 (5′-GAGCCAGAGTCCGtGTTgCCCAtTCCATCAATG-3′).21.Chen S. Ni X.P. Humphreys M.H. et al.1,25 dihydroxyvitamin D amplifies type a natriuretic peptide receptor expression and activity in target cells.J Am Soc Nephrol. 2005; 16: 329-339Crossref PubMed Scopus (24) Google Scholar Nuclear extracts (3–5 μg) were incubated in binding reaction buffer (10 mM HEPES at pH 7.9, 50 mM KCl, 0.2 mM ethylenediaminetetraacetic acid, 2.5 mM dithiothreitol, 10% glycerol, and 0.05% nonidet P-40) containing 1 μg of poly(dI-dC) and 32P-end-labeled double-stranded wild-type NPR-A4 oligonucleotide at room temperature for 30 min. For competition experiments, a 50-fold molar excess of unlabeled, double-stranded oligonucleotide was added to the binding reaction. All samples were resolved on 4% nondenaturing polyacrylamide gels. Gels were dried and exposed to X-ray film. Twenty one-nucleotide annealed duplex siRNAs directed against rat VDR gene sequence (V1 – GCGUAAGAGGAGAUGAUAtt, V2 – GGAUUCUGAUGACCCGUCUtt, and V3 – GGAGUUCAUCCUGACAGAUtt; all sense strand) were chemically synthesized and purified using a commercial source (Amibon, Austin, TX) as was a negative control siRNA (Ambion) containing a 19-bp scrambled sequence. The latter sequences have no significant homology to any known gene sequences from rat. IMCD cells were cultured to 60–70% confluence and then transfected with 100 pmol V1, V2, V3, or the negative control siRNA using lipofectamine (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. After transfection for 48 h, cells were washed and cellular lysates were prepared and analyzed for VDR expression by Western blot analysis. In a separate experiment, 1 μg -1575 rat NPR-A-Luc and 0.5 μg Renilla-Luc were cotransfected with 100 pmol V1, V2, V3, or control siRNA into IMCD cells using lipofectamine. After 48 h, cells were collected and lysates were generated for luciferase (firefly and Renilla) assay. Data were analyzed by analysis of variance, using the Newman–Keuls test to assess significance. This work was supported in part by HL45637 from the NIH.
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