Screening and identification of substances that regulate nephrin gene expression using engineered reporter podocytes
2006; Elsevier BV; Volume: 70; Issue: 5 Linguagem: Inglês
10.1038/sj.ki.5001625
ISSN1523-1755
AutoresKazushi Yamauchi, Y Takano, Ayumi Kasai, Kunihiro Hayakawa, Naoki Hiramatsu, Naoya Enomoto, Jian Yao, Masanori Kitamura,
Tópico(s)Renal and related cancers
ResumoDownregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was stably transfected with a gene encoding secreted alkaline phosphatase (SEAP) under the control of the 5.4 or 8.3 kb nephrin gene promoter. The established reporter cells REPON5.4 and REPON8.3 were exposed to various pathophysiologic substances, and culture media were subjected to SEAP assay to identify regulators of nephrin gene expression. Among the bioactive substances tested, three physiological ligands of nuclear receptors including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. These effects were observed in both REPON5.4 and REPON8.3 and were associated with upregulation of nephrin mRNA. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. In contrast, inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α as well as phorbol ester significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression. These results elucidated the bidirectional regulation of nephrin by distinct pathophysiologic substances and may provide molecular bases for explaining how proteinuria is induced under pathologic situations and why some ligands for nuclear receptors have the anti-proteinuric potential. Downregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was stably transfected with a gene encoding secreted alkaline phosphatase (SEAP) under the control of the 5.4 or 8.3 kb nephrin gene promoter. The established reporter cells REPON5.4 and REPON8.3 were exposed to various pathophysiologic substances, and culture media were subjected to SEAP assay to identify regulators of nephrin gene expression. Among the bioactive substances tested, three physiological ligands of nuclear receptors including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. These effects were observed in both REPON5.4 and REPON8.3 and were associated with upregulation of nephrin mRNA. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. In contrast, inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α as well as phorbol ester significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression. These results elucidated the bidirectional regulation of nephrin by distinct pathophysiologic substances and may provide molecular bases for explaining how proteinuria is induced under pathologic situations and why some ligands for nuclear receptors have the anti-proteinuric potential. Proteinuria is a typical clinical feature of renal diseases, especially glomerular diseases. Previous reports demonstrated that (1) increase in the level of urinary protein is toxic to the renal tubules and causes activation of tubulointerstitial cells, and (2) the degree of proteinuria is closely correlated with the severity of renal diseases.1.Zandi-Nejad K. Eddy A.A. Glassock R.J. Brenner B.M. Why is proteinuria an ominous biomarker of progressive kidney disease?.Kidney Int Suppl. 2004: S76-S89Abstract Full Text Full Text PDF Scopus (131) Google Scholar Because of this reason, control of proteinuria is a critical, therapeutic target in various kidney diseases. Under physiologic situations, structural integrity of the glomerular filtration barrier (basement membrane and slit diaphragm) is maintained by podocytes that regulate passage of macromolecules in plasma into the urinary space.2.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1117) Google Scholar In contrast, the integrity of the filtration barrier is often disrupted under pathological situations, leading to development of proteinuria. Currently, however, information is limited regarding how the function of the filtration barrier is regulated by endogenous bioactive substances under pathophysiologic circumstances. In particular, endogenous regulators for podocyte function are largely unknown. Various mechanisms may be involved in the regulation of glomerular filtration, but recent investigation identified nephrin, a podocyte-specific protein, as the key regulator involved in the structure and function of the slit diaphragm.3.Tryggvason K. Wartiovaara J. Molecular basis of glomerular permselectivity.Curr Opin Nephrol Hypertens. 2001; 10: 543-549Crossref PubMed Scopus (191) Google Scholar Nephrin is a 185 kDa transmembrane protein with eight immunogloblin-like domains and one fibronectin III-like domain in the extracellular region. Normally, it exists as complexes with other podocyte-specific proteins including NEPH1, podocin, and CD2-associated protein.4.Liu G. Kaw B. Kurfis J. et al.Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability.J Clin Invest. 2003; 112: 209-221Crossref PubMed Scopus (174) Google Scholar,5.Schwarz K. Simons M. Reiser J. et al.Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin.J Clin Invest. 2001; 108: 1621-1629Crossref PubMed Scopus (477) Google Scholar Loss of nephrin fails to form functional complexes in the slit diaphragm, resulting in dysfunction of the filtration barrier. In humans, massive proteinuria and progressive renal failure are caused by mutation of the nephrin gene in Finnish-type congenital nephrotic syndrome.6.Kestila M. Lenkkeri U. Mannikko M. et al.Positionally cloned gene for a novel glomerular protein – nephrin – is mutated in congenital nephrotic syndrome.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar Similarly, disruption of the nephrin gene in mice results in abnormal foot process formation in podocytes and causes consequent nephrosis.7.Putaala H. Soininen R. Kilpelainen P. et al.The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death.Hum Mol Genet. 2001; 10: 1-8Crossref PubMed Scopus (400) Google Scholar,8.Rantanen M. Palmen T. Patari A. et al.Nephrin TRAP mice lack slit diaphragms and show fibrotic glomeruli and cystic tubular lesions.J Am Soc Nephrol. 2002; 13: 1586-1594Crossref PubMed Scopus (96) Google Scholar Previous reports also showed that the level of nephrin is attenuated in human nephrotic syndrome.9.Furness P.N. Hall L.L. Shaw J.A. Pringle J.H. Glomerular expression of nephrin is decreased in acquired human nephrotic syndrome.Nephrol Dial Transplant. 1999; 14: 1234-1237Crossref PubMed Scopus (116) Google Scholar, 10.Huh W. Kim D.J. Kim M.K. et al.Expression of nephrin in acquired human glomerular disease.Nephrol Dial Transplant. 2002; 17: 478-484Crossref PubMed Scopus (58) Google Scholar, 11.Wang S.X. Rastaldi M.P. Patari A. et al.Patterns of nephrin and a new proteinuria-associated protein expression in human renal diseases.Kidney Int. 2002; 61: 141-147Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Furthermore, decreased expression of nephrin mRNA is often observed in glomeruli from patients with a wide range of proteinuric glomerular diseases including minimal change nephropathy, membranous nephropathy, focal segmental glomerulosclerosis, immunoglobulin A nephropathy, and diabetic nephropathy.9.Furness P.N. Hall L.L. Shaw J.A. Pringle J.H. Glomerular expression of nephrin is decreased in acquired human nephrotic syndrome.Nephrol Dial Transplant. 1999; 14: 1234-1237Crossref PubMed Scopus (116) Google Scholar, 12.Kim B.K. Hong H.K. Kim J.H. Lee H.S. Differential expression of nephrin in acquired human proteinuric diseases.Am J Kidney Dis. 2002; 40: 964-973Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 13.Gagliardini E. Benigni A. Tomasoni S. et al.Targeted downregulation of extracellular nephrin in human IgA nephropathy.Am J Nephrol. 2003; 23: 277-286Crossref PubMed Scopus (41) Google Scholar, 14.Toyoda M. Suzuki D. Umezono T. et al.Expression of human nephrin mRNA in diabetic nephropathy.Nephrol Dial Transplant. 2004; 19: 380-385Crossref PubMed Scopus (64) Google Scholar Based on the current knowledge described above, altered expression of nephrin is one of the major mechanisms involved in the development of proteinuria. Currently, however, little is known about endogenous substances that regulate expression of nephrin in podocytes. It is mainly because podocytes are terminally differentiated cells with little mitogenic potential,15.Kriz W. Gretz N. Lemley K.V. Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar,16.Pabst R. Sterzel R.B. Cell renewal of glomerular cell types in normal rats. An autoradiographic analysis.Kidney Int. 1983; 24: 626-631Abstract Full Text PDF PubMed Scopus (199) Google Scholar which does not allow for establishment of stable cell lines required for in vitro assays. To overcome this problem, some groups have reported establishment of conditionally immortalized podocytes from transgenic mice harboring a gene coding for the temperature-sensitive simian virus 40 (SV40) large T antigen under the control of the interferon-γ (IFN-γ)-inducible promoter.17.Mundel P. Reiser J. Zuniga Mejia Borja A. et al.Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (723) Google Scholar,18.Schiwek D. Endlich N. Holzman L. et al.Stable expression of nephrin and localization to cell–cell contacts in novel murine podocyte cell lines.Kidney Int. 2004; 66: 91-101Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar Using the conditionally immortalized podocytes, we aimed in the present study to establish an in vitro assay system that allows for sensitive screening and identification of substances that regulate the glomerular filtration barrier. For this purpose, a secreted alkaline phosphatase (SEAP) gene was fused to the promoter of the nephrin gene and introduced into conditionally immortalized podocytes. Using the established sensor cells, screening and identification of endogenous bioactive substances involved in the regulation of proteinuria were attempted. We especially focused on putative anti-inflammatory substances including retinoic acid, vitamin D3, and glucocorticoid19.Choi W.H. Ji K.A. Jeon S.B. et al.Anti-inflammatory roles of retinoic acid in rat brain astrocytes: suppression of interferon-gamma-induced JAK/STAT phosphorylation.Biochem Biophys Res Commun. 2005; 329: 125-131Crossref PubMed Scopus (70) Google Scholar, 20.Bouillon R. Okamura W.H. Norman A.W. Structure–function relationships in the vitamin D endocrine system.Endocr Rev. 1995; 16: 200-257Crossref PubMed Google Scholar, 21.Chrousos G.P. The hypothalamic–pituitary–adrenal axis and immune-mediated inflammation.N Engl J Med. 1995; 332: 1351-1362Crossref PubMed Scopus (2116) Google Scholar as well as inflammation-associated cytokines and growth factors involved in glomerular diseases. Nephrin reporting podocytes were established by stable transfection of conditionally immortalized murine podocytes with pN5.4-SEAP or pN8.5-SEAP (Figure 1a). Under a non-permissive condition (37–38°C in the absence of IFN-γ), eight clones exhibited substantial levels of SEAP activity. Typical morphologic feature of the established cells, resembling that of the parental podocytes,18.Schiwek D. Endlich N. Holzman L. et al.Stable expression of nephrin and localization to cell–cell contacts in novel murine podocyte cell lines.Kidney Int. 2004; 66: 91-101Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar is shown in Figure 1b. In HeLa cells, all-trans-retinoic acid (ATRA) is known to induce activation of the nephrin gene promoter.22.Suzuki A. Ito T. Imai E. et al.Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats.J Am Soc Nephrol. 2003; 14: 981-991Crossref PubMed Scopus (77) Google Scholar We examined responses of the established clones to ATRA under the non-permissive condition. Cells were treated with or without 10 μM ATRA for 24 h, and the activity of SEAP in culture medium was evaluated. After stimulation with ATRA, all eight clones exhibited modest increases in the level of SEAP (1.9- to 2.9-fold). A typical result is shown in Figure 1c. The elevation of SEAP activity was correlated with increased expression of nephrin mRNA when examined by reverse transcriptase-polymerase chain reaction (RT-PCR) (Figure 1d). We selected three representative clones and designated as reporter podocytes for nephrin (REPON): REPON5.4 (clone 5 (C5) and clone 17 (C17) transfected with pN5.4-SEAP) and REPON8.3 (clone 2 (C2) transfected with pN8.5-SEAP). These cells were used for the following experiments. For the screening and identification of exogenous and endogenous regulators of proteinuria, REPON5.4(C5) were seeded onto 96-well plates and cultured for 48 h under the non-permissive condition. The cells were then treated for 24 h with various substances including ATRA (10 μM), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10−7 M), dexamethasone (1 μM), interleukin-1β (IL-1β) (20 ng/ml), tumor necrosis factor-α (TNF-α; 250 U/ml), 12-o-tetradecanoylphorbol-13-acetate (TPA; 50 nM), and platelet-derived growth factor (PDGF; 20 ng/ml). After the treatment, 5 μl of culture medium was collected from each well and subjected to SEAP assay to evaluate the activity of the nephrin gene promoter. As shown in Figure 2, three physiological ligands for nuclear receptors including ATRA, 1,25(OH)2D3, and dexamethasone significantly activated the nephrin promoter. In contrast, inflammatory cytokines IL-1β and TNF-α as well as TPA significantly downregulated the activity of the promoter of the nephrin gene. In this experimental setting, PDGF had little impact. Figure 3a shows a dose-dependent effect of ATRA in REPON5.4(C5). Significant induction of SEAP was observed at concentrations higher than 0.1 μM, a relatively low concentration, and the effect was dose-dependent within the range from 0.1 to 10 μM. ATRA >10 μM was found to be toxic to the reporter podocytes (data not shown). We performed time-lapse experiments to examine how quickly REPON respond to ATRA and report activation of the nephrin promoter. REPON5.4(C5) were treated with 10 μM ATRA, and culture media were collected periodically. As demonstrated in Figure 3b, compared with untreated control, significant elevation of SEAP was observed within 4 h, and the level of induction was increased for at least 48 h in a time-dependent manner. The effect of ATRA was observed similarly in another N5.4-based reporter clone REPON5.4(C17) and also observed in N8.3-based clone REPON8.3(C2) (Figure 3c). Figures 4a and Figures 5a show dose-dependent effects of 1,25(OH)2D3, and dexamethasone in REPON5.4(C5). Significant induction of SEAP was observed at concentrations higher than 10−9 M of 1,25(OH)2D3 and 0.1 μM of dexamethasone. These effects were dose-dependent, peaked at 10−7 M of 1,25(OH)2D3 and 1 μM of dexamethasone and diminished at higher concentrations. RT-PCR confirmed that the induction of SEAP by these substances was correlated with upregulation of nephrin mRNA (Figures 4b and 5b). The effects of 1,25(OH)2D3 and dexamethasone on nephrin were also confirmed using other reporter cell lines, REPON5.4(C17) and REPON8.5(C2) (Figures 4c and 5c).Figure 5Activation of the nephrin gene promoter by dexamethasone. (a) REPON5.4(C5) cells precultured under a non-permissive condition were treated for 24 h with 1,25(OH)2D3 at different concentrations (0–100 μM), and culture media were subjected to SEAP assay. (b) REPON5.4(C5) cells were treated with (+) or without (−) 1 μM of dexamethasone for 24 h, and expression of nephrin mRNA was evaluated by RT-PCR. (c) REPON5.4(C5), REPON5.4(C17), and REPON8.3(C2) cells were treated with 1 μM of dexamethasone for 24 h and subjected to SEAP assay. Data were expressed as means±s.d., and asterisks indicate statistically significant differences (P<0.05).View Large Image Figure ViewerDownload (PPT) ATRA, 1,25(OH)2D3, and dexamethasone are known to exert their biological effects via the nuclear receptor family of molecules that may interact with each other.23.Nakajima H. Kizaki M. Ueno H. et al.All-trans and 9-cis retinoic acid enhance 1,25-dihydroxyvitamin D3-induced monocytic differentiation of U937 cells.Leuk Res. 1996; 20: 665-676Abstract Full Text PDF PubMed Scopus (45) Google Scholar We therefore examined combinational effects of these agents on the promoter activity of nephrin. REPON5.4(C5) were treated with ATRA (10 μM), 1,25(OH)2D3 (10−7 M), and dexamethasone (1 μM), individually or in combination, and subjected to SEAP assay. Among three compounds tested, 1,25(OH)2D3 was the most potent for activation of the nephrin gene. However, induction of SEAP by any combination of these agents was significantly higher than that by any single compound, and the maximum effect was achieved by the combination of three agents (Figure 6). Of note, the effects of some combination were not additive but synergistic; for example, induction by 1,25(OH)2D3 plus dexamethasone was 2.7±0.1- and 1.6±0.1-fold respectively, whereas the induction by their combination was 7.3±0.5-fold. The downregulation of SEAP by IL-1β, TNF-α, and TPA, demonstrated in Figure 2, might be caused by nonspecific damage of the reporter podocytes. To exclude this possibility, the identical cultures used for SEAP assay were subsequently subjected to a formazan assay to evaluate the number of viable cells. The result showed that none of IL-1β, TNF-α, or TPA as well as PDGF significantly decreased cell viability (Figure 7a). To confirm that the suppression of the nephrin promoter was not due to nonspecific effects, conditionally immortalized podocytes were stably transfected with the SEAP gene under the control of the SV40 promoter. The established cells constitutively secreting SEAP were treated with IL-1β, TNF-α, TPA, or PDGF for 24 h, and the activity of SEAP was evaluated. As shown in Figure 7b, none of these agents downregulated the level of SEAP. The suppressive effects of IL-1β, TNF-α, and TPA on nephrin gene expression were further confirmed by RT-PCR. Consistent with the findings on the activity of the nephrin promoter, the level of nephrin mRNA was depressed by IL-1β, TNF-α, and TPA (Figure 7c), confirming our conclusion. In the present study, we used reporter podocytes for experiments after depletion of IFN-γ for 48 h. However, the original work in which the conditionally immortalized podocytes were established used longer deprivation of IFN-γ to induce full differentiation of podocytes.18.Schiwek D. Endlich N. Holzman L. et al.Stable expression of nephrin and localization to cell–cell contacts in novel murine podocyte cell lines.Kidney Int. 2004; 66: 91-101Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar We suspected that the duration of IFN-γ deprivation could affect expression of nephrin and responses to stimuli of the established reporter podocytes. To examine this possibility, we compared levels of nephrin mRNA and nephrin protein as well as activity of the nephrin gene promoter using REPON5.4(C5) cells preincubated without IFN-γ for 48 h or 9 days. As shown in Figure 8a, the activity of the nephrin gene promoter was not affected by the deprivation term for IFN-γ. Consistently, depletion of IFN-γ for an additional 7 days only slightly increased the level of nephrin mRNA when examined by RT-PCR (Figure 8b). Western blot analysis also revealed that the level of nephrin protein was unaltered by the longer depletion of IFN-γ (Figure 8c). These results clearly showed that, in our reporter podocytes REPON, depletion of IFN-γ for 48 h was sufficient, and longer incubation was not necessary, for substantial induction of nephrin gene and protein. To confirm that longer deprivation of IFN-γ does not affect responses of REPON to stimuli, we compared responses of the reporter podocytes to ATRA, 1,25(OH)2D3, dexamethasone, IL-1β, and TNF-α using the cells preincubated without IFN-γ for 48 h or 9 days. As shown in Figure 9a, depletion of IFN-γ for an additional 7 days did not influence activation of the nephrin gene promoter by ATRA, 1,25(OH)2D3, or dexamethasone. Furthermore, suppression of the nephrin promoter by IL-1β and TNF-α was of the same extent regardless of the duration of IFN-γ deprivation (Figure 9b). These results evidenced that depletion of IFN-γ for longer than 48 h does not affect responses of REPON to the stimuli that regulate nephrin expression. Urinary protein is toxic to the tubulointerstitium in the kidney, and persistent proteinuria facilitates progression of renal injury and fibrosis.24.Nangaku M. Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure.Intern Med. 2004; 43: 9-17Crossref PubMed Scopus (262) Google Scholar Recent investigation revealed that nephrin is a key regulator involved in the regulation of proteinuria.3.Tryggvason K. Wartiovaara J. Molecular basis of glomerular permselectivity.Curr Opin Nephrol Hypertens. 2001; 10: 543-549Crossref PubMed Scopus (191) Google Scholar To date, however, little has been understood about endogenous molecules involved in the regulation of nephrin gene expression. In the present report, we established reporter podocytes REPON that allow for sensitive and continuous monitoring of nephrin gene expression. Using this method, we screened several bioactive substances that may be involved in glomerular pathophysiology and identified that three ligands of nuclear receptors, retinoic acid, vitamin D3, and glucocorticoid, trigger expression of nephrin. We also revealed that proinflammatory cytokines IL-1β and TNF-α downregulate the nephrin gene expression. ATRA is an active metabolite of vitamin A and a ligand of the nuclear receptor, retinoic acid receptor (RAR). After binding of ATRA to RAR, RAR forms homodimers or heterodimers with retinoid X receptor, and the resultant complexes exert biological effects via binding to the particular cis element, the retinoic acid response elements.25.Kato S. Sasaki H. Suzawa M. et al.Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors.Mol Cell Biol. 1995; 15: 5858-5867Crossref PubMed Scopus (142) Google Scholar,26.Wang J. Yen A. A novel retinoic acid-responsive element regulates retinoic acid-induced BLR1 expression.Mol Cell Biol. 2004; 24: 2423-2443Crossref PubMed Scopus (37) Google Scholar In the present study, we found that ATRA activated the murine nephrin promoters N8.3 and N5.4 and upregulated expression of nephrin mRNA. It is consistent with a recent report showing that ATRA induced differentiation of cultured podocyte with increased expression of nephrin and podocin.27.Vaughan M.R. Pippin J.W. Griffin S.V. et al.ATRA induces podocyte differentiation and alters nephrin and podocin expression in vitro and in vivo.Kidney Int. 2005; 68: 133-144Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar A previous report showed that three putative retinoic acid response elements are present in the regulatory region of the human nephrin gene.22.Suzuki A. Ito T. Imai E. et al.Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats.J Am Soc Nephrol. 2003; 14: 981-991Crossref PubMed Scopus (77) Google Scholar These elements are functional in HeLa cells treated with ATRA when examined by a reporter assay.22.Suzuki A. Ito T. Imai E. et al.Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats.J Am Soc Nephrol. 2003; 14: 981-991Crossref PubMed Scopus (77) Google Scholar Our current findings indicated that the murine nephrin promoter may also contain retinoic acid response elements or retinoic acid response element-like sequences responsive to ATRA. Using database analyses by Genomatix PromoterInspector (http://www.genomatix.de)28.Scherf M. Klingenhoff A. Werner T. Highly specific localization of promoter regions in large genomic sequences by PromoterInspector: a novel context analysis approach.J Mol Biol. 2000; 297: 599-606Crossref PubMed Scopus (233) Google Scholar and TFSEARCH based on TransFac (http://mbs.cbrc.jp/research/db/TFSEARCH.html),29.Heinemeyer T. Chen X. Karas H. et al.Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms.Nucleic Acids Res. 1999; 27: 318-322Crossref PubMed Scopus (269) Google Scholar we found single binding site for RAR at -5998 to -6016 of the murine nephrin promoter. However, this site is present within N8.3 but outside of N5.4, which responded similarly to ATRA in murine podocytes (Figure 3c). This result indicated that unidentified elements responsive to ATRA may exist in the promoter region of the mouse nephrin gene. Of note, 25 putative RAR-RXR half-sites (5′-[A/G]G[G/T][T/A]CA-3′) are present in N5.4,25.Kato S. Sasaki H. Suzawa M. et al.Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors.Mol Cell Biol. 1995; 15: 5858-5867Crossref PubMed Scopus (142) Google Scholar and these sites might be responsible for induction of the nephrin gene by ATRA. Alternatively, the effect of ATRA observed in this report may be indirect and mediated by induction of other ATRA-responsive proteins. Further investigation will be required to clarify the mechanism involved. Previous reports showed that retinoic acid was a potential therapeutic agent for the treatment of glomerular diseases.30.Liebler S. Uberschar B. Kubert H. et al.The renal retinoid system: time-dependent activation in experimental glomerulonephritis.Am J Physiol Renal Physiol. 2004; 286: F458-F465Crossref PubMed Scopus (22) Google Scholar, 31.Moreno-Manzano V. Mampaso F. Sepulveda-Munoz J.C. et al.Retinoids as a potential treatment for experimental puromycin-induced nephrosis.Br J Pharmacol. 2003; 139: 823-831Crossref PubMed Scopus (55) Google Scholar, 32.Kinoshita K. Yoo B.S. Nozaki Y. et al.Retinoic acid reduces autoimmune renal injury and increases survival in NZB/W F1 mice.J Immunol. 2003; 170: 5793-5798Crossref PubMed Scopus (74) Google Scholar, 33.Oseto S. Moriyama T. Kawada N. et al.Therapeutic effect of all-trans retinoic acid on rats with anti-GBM antibody glomerulonephritis.Kidney Int. 2003; 64: 1241-1252Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Several reports have demonstrated the anti-proteinuric effect of retinoic acid,30.Liebler S. Uberschar B. Kubert H. et al.The renal retinoid system: time-dependent activation in experimental glomerulonephritis.Am J Physiol Renal Physiol. 2004; 286: F458-F465Crossref PubMed Scopus (22) Google Scholar, 34.Wagner J. Dechow C. Morath C. et al.Retinoic acid reduces glomerular injury in a rat model of glomerular damage.J Am Soc Nephrol. 2000; 11: 1479-1487PubMed Google Scholar, 35.Lehrke I. Schaier M. Schade K. et al.Retinoid receptor-specific agonists alleviate experimental glomerulonephritis.Am J Physiol Renal Physiol. 2002; 282: F741-F751Crossref PubMed Scopus (57) Google Scholar, 36.Schaier M. Lehrke I. Schade K. et al.Isotretinoin alleviates renal damage in rat chronic glomerulonephritis.Kidney Int. 2001; 60: 2222-2234Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar but its precise mechanisms were largely unknown. Our current data, together with a recent report,22.Suzuki A. Ito T. Imai E. et al.Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats.J Am Soc Nephrol. 2003; 14: 981-991Crossref PubMed Scopus (77) Google Scholar raise a possibility that retinoic acid exerts the anti-proteinuric effect via induction or preservation of nephrin expression in podocytes. In the present study, we elucidated that 1,25(OH)2D3 activated the nephrin promoters N8.3 and N5.4 and induced expression of nephrin mRNA. 1,25(OH)2D3 is a biologically active metabolite of vitamin D that acts via its nuclear receptor, vitamin D receptor (VDR).37.Dusso A.S. Brown A.J. Slatopolsky E. Vitamin D.Am J Physiol Renal Physiol. 2005; 289: F8-F28Crossref PubMed Scopus (995) Google Scholar After binding of 1,25(OH)2D3 to VDR, the resultant complexes exert biological effects via the vitamin D response element.37.Dusso A.S. Brown A.J. Slatopolsky E. Vitamin D.Am J Physiol Renal Physiol. 2005; 289: F8-F28Crossref PubMed Scopus (995) Google Scholar Using Genomatrix, we found three putative VDR binding sites in N5.4 and five sites in N8.3. Together with the fact that podocytes express VDR constitutively,38.Stumpf W.E. Sar
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