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Krüppel–Like Factor 15 Mediates Glucocorticoid-Induced Restoration of Podocyte Differentiation Markers

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Krüppel–Like Factor 15 Mediates Glucocorticoid-Induced Restoration of Podocyte Differentiation Markers BASIC RESEARCH www.jasn.org Krüppel–Like Factor 15 Mediates Glucocorticoid- Induced Restoration of Podocyte Differentiation Markers † Sandeep K. Mallipattu,* Yiqing Guo,* Monica P. Revelo, Lucia Roa-Peña,* Timothy Miller,* ‡ Jason Ling,* Stuart J. Shankland, Agnieszka B. Bialkowska,§ Victoria Ly,* Chelsea Estrada,* | | Mukesh K. Jain, Yuan Lu, Avi Ma’ayan,¶ Anita Mehrotra,** Rabi Yacoub,** †† ‡‡ Edward P. Nord,* Robert P. Woroniecki, Vincent W. Yang,§ and John C. He¶** Division of Nephrology, Departments of *Medicine and ††Pediatrics and §Division of Gastroenterology, Department of Medicine, Stony Brook University, Stony Brook, New York; †Department of Pathology, University of Utah, Salt Lake City, Utah; ‡Division of Nephrology, Department of Medicine, University of Washington School of Medicine, Seattle, Washington; |Case Cardiovascular Institute Research Institute, Department of Medicine, Case Western Reserve University, Cleveland, Ohio; ¶Department of Pharmacology and Systems Therapeutics and **Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; and ‡‡Renal Section, James J. Peters Veterans Affairs Medical Center, New York, New York ABSTRACT Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulo- pathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel–like factor 15 (KLF15), a kidney–enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15. In three independent proteinuric murine models, podocyte- specificlossofKlf15 abrogated dexamethasone–induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Con- versely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, thelevelofKLF15expressioninthepodocytesandglomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte. J Am Soc Nephrol 28: 166–184, 2017. doi: 10.1681/ASN.2015060672 Podocytes, the visceral glomerular epithelial cells, in Received June 19, 2015. Accepted April 5, 2016. normal mature kidneys are regarded as highly differentiated and quiescent cells.1 In many pri- Published online ahead of print. Publication date available at www.jasn.org. mary glomerular diseases, such as minimal change disease (MCD) and FSGS, podocytes are injured Correspondence: Dr. Sandeep K. Mallipattu, Department of Medicine/Nephrology, Stony Brook University, 100 Nicolls Road, and undergo a major change in phenotype, result- HSCT17-090B, Stony Brook, NY 11780. Email: sandeep.mallipattu@ ing in a loss of their terminal differentiation mark- stonybrookmedicine.edu ers.2,3 Although the mechanism by which podocyte Copyright © 2016 by the American Society of Nephrology 166 ISSN : 1046-6673/2801-166 J Am Soc Nephrol 28: 166–184, 2017 www.jasn.org BASIC RESEARCH injury occurs in each of these diseases may differ, the RESULTS end result is a compromised filtration barrier with ensuing nephrosis. Dexamethasone Induces KLF15 Expression in Human inflammatory diseases are frequently treated with a Podocytes prolonged course of glucocorticoids (GCs). The immunomod- Because previous studies have shown that KLF15 may mediate ulatory effects of GCs are evident in these proinflammatory the GC-induced differentiation in murine embryonic fibro- states.4,5 However, GCs also exhibit a therapeutic benefit in pri- blasts,16 we initially treated cultured human podocytes with mary glomerulopathies, such as MCD, where a proinflammatory dexamethasone (DEX) and measured KLF15 expression. In milieu is not readily apparent. In fact, the initial treatment option comparison with vehicle–treated human podocytes, KLF15 for MCD is high–dose GC therapy, and in many instances, mRNA expression was increased within 3 hours of DEX treat- alternate immunosuppressive therapy is typically not consid- ment and peaked at 12 hours (Figure 1A). Immunostaining ered until patients have failed GC therapy.6 Previous studies and Western blot for KLF15 were performed in human have described the potential for GCs to have a direct effect on podocytes treated with DEX or vehicle for 12 hours and con- the podocyte by rearrangement of actin cytoskeleton, inhibit- firmed an increase in KLF15 expression in cells treated with ing apoptosis and regulating protein trafficking of critical slit DEXcomparedwiththosetreatedwithvehicle(Figure1,B diaphragm proteins in murine and human podocytes.7–11 andC).Inaddition,weobservedthatKlf15expressionwas In addition, glucocorticoid receptor (GR) as well as the major increased in podocytes as well as other glomerular cells in components of the GR complex are expressed in human mice treated with DEX compared with those treated with podocytes.8,11 Furthermore, GCs have also been implicated vehicle (Figure 1D). To further explore the potential of in ameliorating podocyte injury11,12 and improving podocyte KLF15 to mediate expression of GC target genes, we survival9 in murine and human cell culture models. Al- performed TRANSFAC promoter analysis18 to identify GC though there is some evidence to suggest that GCs may have target genes that possess transcriptional binding sites for an antiapoptotic effect on the podocyte,9–11 the mechanism KLF15. We subsequently performed gene set enrichment mediating this process remains largely unexplored. analysis on these genes with KLF15 binding sites using En- A comparative promoter analysis of podocyte slit dia- richr.19 The NCI-Nature Pathways Gene Set Library in En- phragm molecules revealed that Krüppel-Like Factor (KLF) is richr revealed a significant increase in the pathways involved one of four common transcriptional binding sites on many in GC signaling (Table 1). On the basis of these findings, we podocyte–specific genes.13 KLFs are a subclass of the zinc confirmed that GR binding to the promoter region of KLF15 finger family of DNA binding transcriptional regulators is enhanced in response to DEX treatment in differentiated that are involved in a broad range of cellular processes (i.e., human podocytes by ChIP followed by real-time PCR (Fig- cell differentiation, angiogenesis, and erythropoiesis).14 We ure 1E). To define the mechanism by which GR induces recently showed that KLF15, a kidney–enriched transcription KLF15 expression, we transfected human podocytes with re- factor, restores podocyte differentiation markers by tran- porter construct directed at the KLF15 promoter region scriptionally regulating podocyte-specificgenes,suchas (pGL4.20-hKLF15). KLF15 promoter activity was signifi- nephrin and podocin.15 Because GCs are a major treatment cantly increased in pGL4.20-hKLF15 cells with DEX (1 or for podocyte injury, we initially reviewed the literature for a 10 mM) treatment compared with vehicle-treated cells and possible interaction between GCs and KLF15. Interestingly, a DEX–treated pGL4.20-EV (control) cells (Figure 1F). These recent promoter analysis in combination with chromatin im- findings strongly suggest that KLF15 mediates DEX–induced munoprecipitation (ChIP) identified putative GR binding target gene expression and point to specific genes for addi- sites in the promoter region of KLF15.16 In addition, treat- tional analysis. ment with GCs increased KLF15 expression in multiple cell types, such as murine embryonic fibroblasts and airway Podocyte-Specific Loss of Klf15 Prevents DEX–Induced smooth muscle cells.17 Therefore, we hypothesized that Podocyte Recovery in LPS-Treated Mice GC-induced restoration of podocyte differentiation markers To assess whether the loss of Klf15 in podocytes abrogates is mediated by KLF15. Here, we show that treatment with DEX-induced attenuation of podocyte injury, Klf15 was spe- GCs induces the expression of KLF15 early in podocytes, cifically knocked down in podocytes using the Cre-loxP re- suggesting that KLF15 is an early inducible gene. We also combination system. Podocin-Cre mice (C57BL/6) were flox/flox observe that a podocyte-specificlossofKlf15 abrogates crossed with Klf15 (C57BL/6) to generate Podocin-Cre flox/flox the beneficial effect of GCs in cell culture as well as in three Klf15 mice (F2). Primary culture of glomerular epithelial +/+ proteinuric mouse models. In addition, the overexpression cells isolated from Podocin-Cre Klf15 mice and Podocin-Cre flox/flox of KLF15 in cultured human podocytes prevents the destabi- Klf15 mice revealed a significant reduction in Klf15 lization of the actin cytoskeleton
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