Retinoic acid improves nephrotoxic serum–induced glomerulonephritis through activation of podocyte retinoic acid receptor α
2017; Elsevier BV; Volume: 92; Issue: 6 Linguagem: Inglês
10.1016/j.kint.2017.04.026
ISSN1523-1755
AutoresYan Dai, Anqun Chen, Ruijie Liu, Leyi Gu, Shuchita Sharma, Weijing Cai, Fadi Salem, David J. Salant, Jeffrey W. Pippin, Stuart J. Shankland, Marcus J. Moeller, Norbert B. Ghyselinck, Xiaoqiang Ding, Peter Y. Chuang, Kyung Lee, John Cijiang He,
Tópico(s)Reproductive System and Pregnancy
ResumoProliferation of glomerular epithelial cells, including podocytes, is a key histologic feature of crescentic glomerulonephritis. We previously found that retinoic acid (RA) inhibits proliferation and induces differentiation of podocytes by activating RA receptor-α (RARα) in a murine model of HIV-associated nephropathy. Here, we examined whether RA would similarly protect podocytes against nephrotoxic serum–induced crescentic glomerulonephritis and whether this effect was mediated by podocyte RARα. RA treatment markedly improved renal function and reduced the number of crescentic lesions in nephritic wild-type mice, while this protection was largely lost in mice with podocyte-specific ablation of Rara (Pod-Rara knockout). At a cellular level, RA significantly restored the expression of podocyte differentiation markers in nephritic wild-type mice, but not in nephritic Pod-Rara knockout mice. Furthermore, RA suppressed the expression of cell injury, proliferation, and parietal epithelial cell markers in nephritic wild-type mice, all of which were significantly dampened in nephritic Pod-Rara knockout mice. Interestingly, RA treatment led to the coexpression of podocyte and parietal epithelial cell markers in a small subset of glomerular cells in nephritic mice, suggesting that RA may induce transdifferentiation of parietal epithelial cells toward a podocyte phenotype. In vitro, RA directly inhibited the proliferation of parietal epithelial cells and enhanced the expression of podocyte markers. In vivo lineage tracing of labeled parietal epithelial cells confirmed that RA increased the number of parietal epithelial cells expressing podocyte markers in nephritic glomeruli. Thus, RA attenuates crescentic glomerulonephritis primarily through RARα-mediated protection of podocytes and in part through the inhibition of parietal epithelial cell proliferation and induction of their transdifferentiation into podocytes. Proliferation of glomerular epithelial cells, including podocytes, is a key histologic feature of crescentic glomerulonephritis. We previously found that retinoic acid (RA) inhibits proliferation and induces differentiation of podocytes by activating RA receptor-α (RARα) in a murine model of HIV-associated nephropathy. Here, we examined whether RA would similarly protect podocytes against nephrotoxic serum–induced crescentic glomerulonephritis and whether this effect was mediated by podocyte RARα. RA treatment markedly improved renal function and reduced the number of crescentic lesions in nephritic wild-type mice, while this protection was largely lost in mice with podocyte-specific ablation of Rara (Pod-Rara knockout). At a cellular level, RA significantly restored the expression of podocyte differentiation markers in nephritic wild-type mice, but not in nephritic Pod-Rara knockout mice. Furthermore, RA suppressed the expression of cell injury, proliferation, and parietal epithelial cell markers in nephritic wild-type mice, all of which were significantly dampened in nephritic Pod-Rara knockout mice. Interestingly, RA treatment led to the coexpression of podocyte and parietal epithelial cell markers in a small subset of glomerular cells in nephritic mice, suggesting that RA may induce transdifferentiation of parietal epithelial cells toward a podocyte phenotype. In vitro, RA directly inhibited the proliferation of parietal epithelial cells and enhanced the expression of podocyte markers. In vivo lineage tracing of labeled parietal epithelial cells confirmed that RA increased the number of parietal epithelial cells expressing podocyte markers in nephritic glomeruli. Thus, RA attenuates crescentic glomerulonephritis primarily through RARα-mediated protection of podocytes and in part through the inhibition of parietal epithelial cell proliferation and induction of their transdifferentiation into podocytes. Crescentic glomerulonephritis (GN), characterized by multilayered accumulation of proliferating cells in the Bowman space, is a rapidly progressive disease that leads to end-stage renal failure. Immunosuppressive therapy, which has a significant adverse effect profile, is the mainstay treatment of GN. Thus, there is a need to identify more specific therapies with few significant adverse effects for GN. Although podocytes are terminally differentiated quiescent cells in normal kidneys, proliferative crescent cells were shown to express podocyte-specific markers in a murine model of antiglomerular basement membrane (anti-GBM) GN1Le Hir M. Keller C. Eschmann V. et al.Podocyte bridges between the tuft and Bowman's capsule: an early event in experimental crescentic glomerulonephritis.J Am Soc Nephrol. 2001; 12: 2060-2071Crossref PubMed Google Scholar, 2Moeller M.J. Soofi A. Hartmann I. et al.Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.J Am Soc Nephrol. 2004; 15: 61-67Crossref PubMed Scopus (157) Google Scholar and in human crescentic GN, wherein cells coexpressed cell proliferation markers.3Bariety J. Bruneval P. Meyrier A. et al.Podocyte involvement in human immune crescentic glomerulonephritis.Kidney Int. 2005; 68: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar These data suggest that proliferative podocytes are a component of cellular crescents in GN. In addition, mice with podocyte-specific deletion of the von Hippel-Lindau gene specifically develop crescentic GN with proliferative podocytes,4Ding M. Cui S. Li C. et al.Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice.Nat Med. 2006; 12: 1081-1087Crossref PubMed Scopus (172) Google Scholar suggesting that genetically induced podocyte proliferation results in kidney disease that is similar to crescentic GN. In agreement, inhibition of podocyte proliferation improved renal function in a murine model of crescentic GN.5Griffin S.V. Krofft R.D. Pippin J.W. Shankland S.J. Limitation of podocyte proliferation improves renal function in experimental crescentic glomerulonephritis.Kidney Int. 2005; 67: 977-986Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar However, recent lineage-tracing experiments demonstrated that glomerular parietal epithelial cells (PECs) are a major component of crescents in GN and of pseudocrescents in collapsing glomerulosclerosis.6Smeets B. Uhlig S. Fuss A. et al.Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 2604-2615Crossref PubMed Scopus (201) Google Scholar Studies also suggest that PEC injury leads to cellular activation and the proliferation of the remaining PECs, which ultimately form cellular crescents.7Sicking E.M. Fuss A. Uhlig S. et al.Subtotal ablation of parietal epithelial cells induces crescent formation.J Am Soc Nephrol. 2012; 23: 629-640Crossref PubMed Scopus (54) Google Scholar In addition, PECs expressed stem cell markers and were transdifferentiated into podocytes.8Ronconi E. Sagrinati C. Angelotti M.L. et al.Regeneration of glomerular podocytes by human renal progenitors.J Am Soc Nephrol. 2009; 20: 322-332Crossref PubMed Scopus (429) Google Scholar Recent data further revealed that although podocyte generation from PECs is mainly confined to the glomerular development period and does not occur in aging kidneys or in response to nephron loss, it may occur after acute glomerular injury.9Berger K. Schulte K. Boor P. et al.The regenerative potential of parietal epithelial cells in adult mice.J Am Soc Nephrol. 2014; 25: 693-705Crossref PubMed Scopus (86) Google Scholar, 10Wanner N. Hartleben B. Herbach N. et al.Unraveling the role of podocyte turnover in glomerular aging and injury.J Am Soc Nephrol. 2014; 25: 707-716Crossref PubMed Scopus (140) Google Scholar Although the mechanisms underlying cell injury in crescentic GN require further clarification, podocyte injury likely triggers glomerular cell proliferation and subsequent crescent formation. Retinoic acids (RAs) are vitamin A derivatives and have multiple cellular functions, including inhibiting proliferation and inflammation and inducing cell differentiation.11Evans T.R. Kaye S.B. Retinoids: present role and future potential.Br J Cancer. 1999; 80: 1-8Crossref PubMed Scopus (186) Google Scholar In addition to established benefits in the treatment of various types of cancers, RA also provided protection against kidney disease and podocyte injury in multiple experimental models.12Xu Q. Lucio-Cazana J. Kitamura M. et al.Retinoids in nephrology: promises and pitfalls.Kidney Int. 2004; 66: 2119-2131Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13Lehrke 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, 14Suzuki 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 (79) Google Scholar, 15De Lema G.P. Lucio-Cazana F.J. Molina A. et al.Retinoic acid treatment protects MRL/lpr lupus mice from the development of glomerular disease.Kidney Int. 2004; 66: 1018-1028Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 16Wagner 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 We previously found that RA reduces proteinuria and glomerulosclerosis in a murine model of HIV-associated nephropathy, wherein pseudocrescent formations were present.17He J.C. Lu T.C. Fleet M. et al.Retinoic acid inhibits HIV-1-induced podocyte proliferation through the cAMP pathway.J Am Soc Nephrol. 2007; 18: 93-102Crossref PubMed Scopus (81) Google Scholar RA also inhibited HIV-induced glomerular cell proliferation and preserved the expression of podocyte differentiation markers.17He J.C. Lu T.C. Fleet M. et al.Retinoic acid inhibits HIV-1-induced podocyte proliferation through the cAMP pathway.J Am Soc Nephrol. 2007; 18: 93-102Crossref PubMed Scopus (81) Google Scholar, 18Vaughan 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 (82) Google Scholar These studies suggest that RA is an effective therapeutic option for proliferative glomerular diseases such as crescentic GN and HIV-associated nephropathy. Retinoids exert their biological effects by binding two families of nuclear receptors, namely the retinoic acid receptors (RARs) and retinoid X receptors, which have distinct cellular functions and are expressed in various tissues, including the kidney.12Xu Q. Lucio-Cazana J. Kitamura M. et al.Retinoids in nephrology: promises and pitfalls.Kidney Int. 2004; 66: 2119-2131Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Although all-trans-retinoic acid (atRA) binds and activates all RAR subtypes (RAR-α, -β, and -γ),19Allenby G. Bocquel M.T. Saunders M. et al.Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.Proc Natl Acad Sci U S A. 1993; 90: 30-34Crossref PubMed Scopus (664) Google Scholar our recent data suggest that the protective effects of RA against podocyte injury and glomerular disease require the retinoic acid receptor-α (RARα), which was the most predominant RAR subtype expressed in podocytes.20Ratnam K.K. Feng X. Chuang P.Y. et al.Role of the retinoic acid receptor-alpha in HIV-associated nephropathy.Kidney Int. 2011; 79: 624-634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21Zhong Y. Wu Y. Liu R. et al.Novel retinoic acid receptor alpha agonists for treatment of kidney disease.PLoS One. 2011; 6: e27945Crossref PubMed Scopus (33) Google Scholar However, because previous studies were performed using a global deletion of Rara, it remains unclear how much of the renoprotection conferred by atRA was directly mediated by podocytes. In addition, many studies have shown the important roles of regulatory T cells in the pathogenesis of crescentic GN,22Tipping P.G. Holdsworth S.R. T cells in crescentic glomerulonephritis.J Am Soc Nephrol. 2006; 17: 1253-1263Crossref PubMed Scopus (150) Google Scholar and because atRA can induce regulatory T cells and inhibit the differentiation of Th17 cells,23Raverdeau M. Mills K.H. Modulation of T cell and innate immune responses by retinoic Acid.J Immunol. 2014; 192: 2953-2958Crossref PubMed Scopus (149) Google Scholar the beneficial effects of atRA or RAR agonists in vivo may also reflect modulated immune responses. Therefore, in this study, we investigated the direct contribution of RA signaling on podocytes through podocyte RARα. Using mice with podocyte-specific knockout of Rara, we revealed that the marked amelioration of kidney injury by RA in nephrotoxic serum–induced GN (NTS-GN) is largely dependent on podocyte-specific expression of RARα, confirming the direct protective effects of RA on podocytes. These renoprotective effects were also associated with the inhibition of glomerular cell proliferation and the restoration of differentiated podocyte marker expression. However, RA also induced the simultaneous expression of podocyte and PEC markers in a small subset of glomerular cells in NTS-GN kidneys, suggesting the presence of transitional cells undergoing transdifferentiation from PECs into podocytes. Using in vitro and in vivo lineage-tracing experiments, we confirmed that RA enhances the transdifferentiation of PECs into podocytes. Altogether, the present data indicate that RA attenuates crescentic GN primarily through RARα-mediated protection of podocytes, and in part, through the inhibition of PEC proliferation and enhancement of transdifferentiation into podocytes. To generate podocyte-specific RARα knockout mice, mice carrying homozygous floxed Rara allele (RARαL2/L2)24Chapellier B. Mark M. Garnier J.M. et al.A conditional floxed (loxP-flanked) allele for the retinoic acid receptor alpha (RARalpha) gene.Genesis. 2002; 32: 87-90Crossref PubMed Scopus (71) Google Scholar were crossed with transgenic mice expressing Cre recombinase driven by the podocin promoter (podocin-Cre).25Moeller M.J. Sanden S.K. Soofi A. et al.Podocyte-specific expression of cre recombinase in transgenic mice.Genesis. 2003; 35: 39-42Crossref PubMed Scopus (249) Google Scholar Polymerase chain reaction (PCR) analysis of genomic DNA obtained from isolated glomeruli showed the presence of a floxed L2 allele and an excised L-allele (Supplementary Figure S1A and B) in podocyte-specific RARα-knockout (Pod-Rara−/−) mice, and these alleles were not present in the Rara wild-type (Pod-Rara+/+) mice. To confirm the specific deletion of RARα in podocytes, we isolated primary podocytes from Pod-Rara−/− and Pod-Rara+/+ mice and cultured them in vitro, as previously described.26Chuang P.Y. Xu J. Dai Y. et al.In vivo RNA interference models of inducible and reversible Sirt1 knockdown in kidney cells.Am J Pathol. 2014; 184: 1940-1956Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Western blot analyses showed the loss of RARα expression in primary podocytes from Pod-Rara−/− mice compared with those from Pod-Rara+/+ mice (Supplementary Figure S1C). Weak residual expression of RARα was observed in some primary podocyte samples of Pod-Rara−/− mice, possibly because of contamination with other glomerular cells during podocyte isolation and/or incomplete excision of floxed Rara in podocytes. No gross defects were observed in Pod-Rara−/− mice, with no changes observed in kidney function up to 8 months of age (data not shown). To investigate the roles of RA and RARα in podocytes during glomerular injury, we established a murine model of NTS-GN at 7 weeks of age by injecting anti-GBM antibodies after preimmunization with sheep IgG as previously described.27Salant D.J. Cybulsky A.V. Experimental glomerulonephritis.Methods Enzymol. 1988; 162: 421-461Crossref PubMed Scopus (121) Google Scholar, 28Dai Y. Gu L. Yuan W. et al.Podocyte-specific deletion of signal transducer and activator of transcription 3 attenuates nephrotoxic serum-induced glomerulonephritis.Kidney Int. 2013; 84: 950-961Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Control mice were administered phosphate-buffered saline (PBS; vehicle) after preimmunization. To assess protective effects of RA against NTS-GN, mice were treated daily with RA or vehicle from 1 day before NTS injections for a total of 7 days. Representative images of kidney histology are shown in Figure 1a. As anticipated, NTS treatments led to glomerular crescent formation in both Pod-Rara+/+ and Pod-Rara−/− mice (Figure 1a, arrowheads; Figure 1b). Although RA treatments markedly reduced the numbers of crescents in Pod-Rara+/+ kidneys, this effect was limited in Pod-Rara−/− kidneys. NTS also induced significant albuminuria (Figure 1c, Supplementary Figure S2) and resulted in declining kidney function, as indicated by blood urea nitrogen levels in both Pod-Rara−/− and Pod-Rara+/+ mice (Figure 1d). RA treatments markedly diminished albuminuria and improved renal function in nephritic Pod-Rara+/+ mice but had limited effects in Pod-Rara−/− mice. We further confirmed that the beneficial effects of RA are not mediated by the suppression of immune complex deposition because NTS-induced immune complex deposition (mouse IgG, sheep IgG, and C3) in glomeruli was not different between NTS-injected mice with or without RA treatment (Supplementary Figure S3). Altogether, these results suggest that RA protects against NTS-induced glomerular injury, which is directly conferred by activating podocyte RARα. Although these protective effects were limited in Pod-Rara−/− GN mice, partial protection was observed after RA treatments, suggesting that residual podocyte RARα is present (Supplementary Figure S1C) due to incomplete ablation of Rara by podocin-Cre. RA may also affect other glomerular cell types such as PECs, potentially contributing to the observed partial protection in Pod-Rara−/− mice. RA administration also led to marked decreases in the presence of infiltrating inflammatory cells in nephritic mice of both genotypes (Supplementary Figures S4–S6), suggesting that the immunomodulatory effects of RA contribute to the partial protection observed in nephritic Pod-Rara−/− mice. We and others previously demonstrated that RA reduces proliferation markers and preserves podocyte-specific differentiation markers in vitro and in vivo.17He J.C. Lu T.C. Fleet M. et al.Retinoic acid inhibits HIV-1-induced podocyte proliferation through the cAMP pathway.J Am Soc Nephrol. 2007; 18: 93-102Crossref PubMed Scopus (81) Google Scholar, 18Vaughan 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 (82) Google Scholar Therefore, we determined whether RA affected the expression of podocyte differentiation markers in NTS-GN, and if so, whether the effect was directly mediated through podocyte RARα. NTS markedly suppressed synaptopodin protein and mRNA expressions in mice of both genotypes (Figure 2a, b, and d ). However, whereas RA treatment significantly restored expression in nephritic Pod-Rara+/+ mice, this restoration was limited in nephritic Pod-Rara−/− mice. CD44 expression, which is typically enhanced in renal injuries including crescentic GN,29Nakamura H. Kitazawa K. Honda H. Sugisaki T. Roles of and correlation between alpha-smooth muscle actin, CD44, hyaluronic acid and osteopontin in crescent formation in human glomerulonephritis.Clin Nephrol. 2005; 64: 401-411Crossref PubMed Google Scholar, 30Sano N. Kitazawa K. Sugisaki T. Localization and roles of CD44, hyaluronic acid and osteopontin in IgA nephropathy.Nephron. 2001; 89: 416-421Crossref PubMed Scopus (38) Google Scholar, 31Roy-Chaudhury P. Khong T.F. Williams J.H. et al.CD44 in glomerulonephritis: expression in human renal biopsies, the Thy 1.1 model, and by cultured mesangial cells.Kidney Int. 1996; 50: 272-281Abstract Full Text PDF PubMed Scopus (39) Google Scholar was markedly increased in both protein and mRNA expression levels in nephritic mice of both genotypes (Figure 2a, c, and e). However, CD44 mRNA expression was significantly greater in nephritic Pod-Rara−/− glomeruli than in nephritic Pod-Rara+/+. RA treatment significantly suppressed CD44 mRNA expression in nephritic Pod-Rara+/+ glomeruli but to a much lesser extent in nephritic Pod-Rara−/− glomeruli, further indicating that podocyte RARα is necessary for the complete protective effects of RA against NTS-induced injuries. We next determined the effects of RA on cell proliferation in NTS-GN mice and confirmed that these were dependent on podocyte RARα. Immunohistological staining revealed numerous Ki-67–positive nuclei in the glomeruli of NTS-injected mice compared with those of vehicle-injected mice (Figure 3a and b ). Moreover, Ki-67 mRNA expression was considerably increased in nephritic glomeruli (Figure 3c), and RA treatments led to a near complete repression of glomerular Ki-67 expression in nephritic Pod-Rara+/+ mice but had only modest effects in nephritic Pod-Rara−/− mice. Hence, RA may inhibit glomerular cell proliferation via mechanisms that are dependent on and independent of the activation of podocyte RARα. Although we did not determine which glomerular cell types were Ki-67 positive, these data suggest that decreased podocyte injury in the presence of RA affects glomerular cell proliferation in NTS-GN. Consistent with this notion, similar to what was observed with CD44 expression above, Ki-67 mRNA expression in nephritic Pod-Rara−/− glomeruli was significantly greater than that in nephritic Pod-Rara+/+ glomeruli, suggesting an association between the degree of injury and that of cell proliferation in GN. Ki-67 expression may also be increased by endogenous RA ligands that limit proliferation after binding RARα in nephritic Pod-Rara+/+ mice but not in Pod-Rara−/− mice, as indicated by previously demonstrated roles of endogenous RA in glomerular disease.32Li X. Dai Y. Chuang P.Y. He J.C. Induction of retinol dehydrogenase 9 expression in podocytes attenuates kidney injury.J Am Soc Nephrol. 2014; 25: 1933-1941Crossref PubMed Scopus (11) Google Scholar, 33Peired A. Angelotti M.L. Ronconi E. et al.Proteinuria impairs podocyte regeneration by sequestering retinoic acid.J Am Soc Nephrol. 2013; 24: 1756-1768Crossref PubMed Scopus (105) Google Scholar Because PECs are shown to be a major component of proliferating cells in crescentic GN, we investigated the contributions of PECs to RA-mediated inhibition of cell proliferation by comparing the expressions of claudin-1 and nephrin, which are PEC and podocyte markers, respectively, in control and nephritic mice. Consistent with observations of synaptopodin expression (Figure 2), nephrin expression was markedly decreased and claudin-1 expression was conversely increased in nephritic mice of both genotypes (Figure 4a–c ). RA treatments largely restored nephrin expression and effectively prevented the NTS-induced claudin-1 upregulation in nephritic Pod-Rara+/+ mice. However, RA had no effect on nephrin expression and led to the partial inhibition of claudin-1 in nephritic Pod-Rara−/− mice. Real-time PCR analyses of nephrin and claudin-1 in isolated glomeruli showed similar results (Figure 4d and e), indicating that RA/RARα signaling in podocytes prevents cellular injury, likely leading to decreased PEC proliferation in NTS-GN. However, because RA treatments partially reduced claudin-1 expression in nephritic Pod-Rara−/− mice, RA likely affects PEC proliferation directly, even in the presence of ongoing podocyte injury, as indicated by poor recovery of nephrin expression. A small number of claudin-1–positive cells were also nephrin-positive in RA-treated GN glomeruli (Figure 4f) from both Pod-Rara+/+ and Pod-Rara−/− mice, suggesting that RA induces transdifferentiation of PECs toward a podocyte phenotype, independently of RARα function in preexisting podocytes. Accordingly, Zhang et al. previously showed that RA increases the podocyte marker expressions in PECs under conditions of experimental glomerular disease.34Zhang J. Pippin J.W. Vaughan M.R. et al.Retinoids augment the expression of podocyte proteins by glomerular parietal epithelial cells in experimental glomerular disease.Nephron Exp Nephrol. 2012; 121: e23-e37Crossref PubMed Scopus (73) Google Scholar RA treatments reduced the expression of the PEC marker claudin-1 and led to the coexpression of nephrin in a small subset of claudin-1-positive glomerular cells in vivo. Thus, to confirm these data in vitro, we cultured conditionally immortalized mouse PECs (mPECs; from the laboratory of Dr. Stuart Shankland) as described in Ohse et al.35Ohse T. Pippin J.W. Vaughan M.R. et al.Establishment of conditionally immortalized mouse glomerular parietal epithelial cells in culture.J Am Soc Nephrol. 2008; 19: 1879-1890Crossref PubMed Scopus (68) Google Scholar and allowed the differentiation at 37°C for 14 days in the absence of interferon gamma to inactivate the T antigen. To determine whether RA directly inhibited PEC proliferation through RARα, differentiated mPECs were treated with vehicle (dimethylsulfoxide), atRA (5 μM), or the RARα-specific agonist AM580 (200 nM) and the proliferation was assessed using a crystal violet staining method.36Feoktistova M. Geserick P. Leverkus M. Crystal violet assay for determining viability of cultured cells.Cold Spring Harb Protoc. 2016; (pdb prot087379): 2016Google Scholar Addition of either atRA or AM580 significantly decreased proliferation, which was detectable at 48 hours and was more pronounced by 72 to 96 hours in the culture (Figure 5a). Furthermore, by 72 hours of incubation with atRA or AM580, there was a marked enhancement in the mRNA expression levels of podocin, nephrin, and synaptopodin compared with those in vehicle-treated cells; the PEC marker Pax2 remained unchanged (Figure 5b). Using immunofluorescence, we further confirmed that atRA enhanced the podocyte marker expression in mPECs. Although a small percentage of differentiated mPECs expressed both Pax2 and synaptopodin at basal levels (21.25% ± 1.38%), atRA treatment led to substantial increases in the number of cells that coexpressed these markers (73.25% ± 3.68%; Figure 5c, arrowheads). Altogether, these results support the current in vivo finding that RA directly inhibits PEC proliferation and enhances their transdifferentiation into podocytes. To further validate the in vitro findings, we performed lineage-tracing experiments of PECs in vivo under conditions of NTS-GN. In these experiments, Cre reporter mice that express red fluorescent protein (RFP), tdTomato, upon recombination37Madisen L. Zwingman T.A. Sunkin S.M. et al.A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (3846) Google Scholar were crossed with mice carrying PEC-rtTA and TetO-Cre transgenes.6Smeets B. Uhlig S. Fuss A. et al.Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 2604-2615Crossref PubMed Scopus (201) Google Scholar In the resulting triple transgenic mice (PEC-rtTA; TetO-Cre; tdTomato), PECs were first fluorescently labeled after feeding the mice doxycycline-supplemented chow (625 mg/kg chow) for 4 weeks before NTS injections. Doxycycline feeding induced robust tdTomato RFP expression in PECs (Figure 6a), and RA treatments in NTS-injected mice induced the migration of RFP-labeled PECs into glomerular tufts, where they exhibited podocyte morphology and concomitantly expressed synaptopodin. An average of 3 to 4 RFP–synaptopodin double–positive cells were observed per glomerulus in RA-treated GN mice, and these cells were not present in the glomeruli of untreated nephritic mice (Figure 6b), further indicating that RA induces PEC transdifferentiation. Altogether, our data indicate that RA protects against NTS-GN by directly activating RARα in podocytes and in part, by inhibiting PEC proliferation and inducing transdifferentiation into podocytes. Because our data suggested that RA is an effective therapeutic agent for treating renal injury in crescentic GN, we aimed to confirm that there were no alterations in the expression patterns of RARα in human crescentic GN. We obtained patient biopsy samples from 4 patients with minimal change disease and 5 patients with crescentic GN (clinical information of the patients is summarized in Supplementary Table S1). Immunostaining for RARα showed broadly distributed expression in all glomerular and tubular cells in the kidneys of patients with minimal change disease and GN, suggesting that RARα expression levels are not significantly altered in crescentic GN and normal kidneys (Figure 7). These findings are consistent with our previous studies that show no differences in RARα expression between human HIV-associated nephropathy kidneys and control kidneys.20Ratnam K.K. Feng X. Chuang P.Y. et al.Role of the retinoic acid receptor-alpha in HIV-associated nephropathy.Kidney Int. 2011; 79: 624-634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Here, we demonstrated that RARα is a key molecule that mediates beneficial effects of RA and that its expression remains unaltered in diseased kidneys. Hence, RARα agonists may provide a novel therapeutic approach for treating patients with crescentic GN. In addition to the critical roles in kidney development, RA has been shown to preserve differentiation markers duri
Referência(s)