BASIC RESEARCH www.jasn.org TWEAK-Fn14 Signaling Activates Myofibroblasts to Drive Progression of Fibrotic Kidney Disease † † † Ivan G. Gomez,* Allie M. Roach,* Naoki Nakagawa, Aldo Amatucci,* † ‡ ‡ Bryce G. Johnson,* Kadeshia Dunn, Mark C. Kelly, Gamze Karaca,* Timothy S. Zheng,* ‡ † Suzanne Szak,* Claire M. Peppiatt-Wildman, Linda C. Burkly,* and Jeremy S. Duffield* *Research & Development, Biogen, Cambridge, Massachusetts; †Division of Nephrology, Departments of Medicine & Pathology, and Institute of Stem Cell & Regenerative Medicine, University of Washington, Seattle, Washington; and ‡Medway School of Pharmacy, University of Kent, Chatham, Kent, United Kingdom ABSTRACT The identification of the cellular origins of myofibroblasts has led to the discovery of novel pathways that potentially drive myofibroblast perpetuation in disease. Here, we further investigated the role of innate immune signaling pathways in this process. In mice, renal injury-induced activation of pericytes, which are myofibroblast precursors attached to endothelial cells, led to upregulated expression of TNF receptor su- perfamily member 12a, also known as fibroblast growth factor-inducible 14 (Fn14), by these cells. In live rat kidney slices, administration of the Fn14 ligand, TNF-related weak inducer of apoptosis (TWEAK), promoted pericyte-dependent vasoconstriction followed by pericyte detachment from capillaries. In vitro, administra- tion of TWEAK activated and differentiated pericytes into cytokine-producing myofibroblasts, and further activated established myofibroblasts in a manner requiring canonical and noncanonical NF-kB signaling path- ways. Deficiency of Fn14 protected mouse kidneys from fibrogenesis, inflammation, and associated vascular instability after in vivo injury, and was associated with loss of NF-kB signaling. In a genetic model of sponta- neous CKD, therapeutic delivery of anti-TWEAK blocking antibodies attenuated disease progression, pre- served organ function, and increased survival. These results identify the TWEAK-Fn14 signaling pathway as an important factor in myofibroblast perpetuation, fibrogenesis, and chronic disease progression. J Am Soc Nephrol 27: 3639–3652, 2016. doi: 10.1681/ASN.2015111227 Fibrosis is a central problem in many forms of including types VI, VIII, and XII, and other matrix chronic disease affecting internal organs including proteins including fibrillin, hyaluronan, and fibro- the liver, lung, heart, central nervous system, and nectin.2–4 Recently, genetic fate-mapping studies kidney. The deposition of pathologic fibrotic ma- have demonstrated that in virtually all organs, dis- terial between and within organ units, as well as the crete populations of resident mesenchymal cells cellular activation required for such deposition, form the cellular link between initial tissue injury have been strongly implicated in the perpetuation and excessive, pathologic matrix production by mi- and progression of chronic diseases. In the kidney, grating from their niche and depositing pathologic fibrosis manifests histologically as interstitial fibro- matrix as (myo)fibroblasts.2,5–12 In the kidney, sis, glomerulosclerosis, and arteriosclerosis.1 The numerical expansion and activation of myofibro- blasts is central to fibrosis. Although a number of Received November 13, 2015. Accepted February 16, 2016. cell types have the potential to produce and secrete Published online ahead of print. Publication date available at extracellular matrix (ECM) proteins, including ep- www.jasn.org. ithelial cells, endothelial cells, and leukocytes, it is Correspondence: Dr. Jeremy S. Duffield or Dr. Linda C. Burkly, myofibroblasts that produce the pathogenic, fibril- Biogen, Building 6, 115 Broadway, Cambridge, MA, 02142. lar collagenous matrix in vivo, especially collagen Email: jeremy.duffi[email protected] or [email protected] types I, III, V, VII, and XI, microfibril collagens Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 27: 3639–3652, 2016 ISSN : 1046-6673/2712-3639 3639 BASIC RESEARCH www.jasn.org many of these mesenchymal cells are closely associated with Furthermore, release of TWEAK by increased blood flow capillaries, perform vascular functions, have mesenchymal in the kidney may be an early feature of the development stem cell characteristics, and may be referred to as pericytes of kidney disease.34 Since such microvascular problems are or perivascular cells.1,3,9,13,14 In the mouse kidney, many of recognized to drive kidney disease,14 and since pericytes are these perivascular cells lack expression of NG2 but express directly implicated in the fibrogenic process, we hypothe- PDGFRb, whereas in rat and human kidneys the expression sized that Fn14 may contribute to fibrogenesis by regulating of NG2 is more variable.15–17 pericyte functions directly. Here, we evaluate the role of The activation of pericytes results in migration from the Fn14 in the activation of kidney myofibroblast precursors, capillary to the interstitial space. This detachment from the as well as in the persistence of established myofibroblasts, capillary is associated with vascular instability, which may and determined its contribution to renal fibrogenesis and result in pathologic angiogenesis or capillary loss.3,13,18 Studies end organ failure. suggest that vascular instability and pericyte activation are di- rectly linked due the loss of survival signals, quiescence sig- nals, and basement membrane maintenance. Failure to resolve RESULTS these responses, either as a result of repetitive injury or loss of normal restorative mechanisms, appears to be central to the Fn14 is Expressed by Pericytes and Myofibroblasts in progression to chronic disease. the Kidney Although several secreted factors that contribute to cellular Tnfrsf12 (Fn14) and Tnfsf12 (TWEAK) were highly upregu- crosstalk and perpetuation of the fibrotic response have been lated in the setting of kidney injury with fibrosis caused by identified, including TGFb,VEGF,CTGF,WNTligands, surgical unilateral ureteral obstruction (UUO) (Figure 1, A Hedgehog ligands, and PDGFs, additional factors likely play and B). Flow cytometry-sorted pericytes from normal kid- critical roles in fibrogenesis. In particular, myofibroblasts neys, and activated pericytes (which progressively differentiate show activation of innate immune signaling pathways, sug- into myofibroblasts) sorted from diseased kidneys were eval- gesting that additional cytokine/receptor interactions contrib- uated for expression of Tnfrsf12 by quantitative PCR (qPCR). ute to their appearance and maintenance in a pathologic Tnfrsf12 was present in healthy pericytes and expression pro- state.3,4,11,19,20 gressively increased with disease (Figure 1B). In keeping with TNFreceptor and TNF superfamily receptor–signaling have high expression of Fn14 by myofibroblasts, kidney tissue been identified as potentially contributing to the pathogenesis sections from the UUO model were stained with anti-Fn14 of kidney diseases, including diabetic nephropathy, IgA ne- antibodies and costained with markers for myofibroblasts. phropathy, and lupus nephritis.21–25 TNF-related weak in- Fn14 was detected at high levels in interstitial myofibro- ducer of apoptosis (TWEAK) and its receptor fibroblast blasts, but was also detected in endothelial cells (Figure growth factor-inducible 14 (Fn14) are TNF superfamily 1C). A minority of injured epithelial cells also expressed a (TNFSF) members and have been implicated in models of low level of Fn14. Notably, only a subpopulation of myofi- kidney injury and disease, including models featuring renal broblasts appeared to express Fn14 and these were in the fibrosis, particularly where autoimmunity is thought to drive areas of greatest myofibroblast accumulation. Fn14 staining the pathogenesis. Such links with autoimmunity may derive in kidneys lacking Fn14 did not detect any specificsignal, from reports that TWEAK overexpression in vivo leads to the highlighting the specificity of this method for detecting pro- development of autoimmunity. However, the receptor and tein expression (Figure 1C, Supplemental Figure 1). A sim- ligand are upregulated in human chronic fibrosing diseases ilar pattern of staining was detected in biopsies from human of the kidney, such as FSGS and diabetic nephropathy, where CKD, where the majority of staining was detected in the there is no autoimmunity26,27; yet a role for this receptor and interstitium in myofibroblasts and endothelium (Supple- ligand directly in fibrogenesis remains undetermined.28–30 mental Figure 2). Fn14 expression was not detected in hu- Fn14 signaling via TNF receptor-associated factors has been man glomeruli. Further validation of these observations was reported to stabilize NF-kB–inducing kinase (NIK), thereby made in primary cultures of mouse kidney cells. Pericytes activating the P52/RelB NF-kB complex and causing down- expressed Fn14, seen as a single band by Western blotting. stream activation of NF-kB target genes, which have been Myofibroblasts, purified from chronically diseased and fi- suggested to be important factors in human kidney dis- brotic kidneys, expressed the receptor at significantly higher ease.31 Recently, we identified Fn14 (TNFRSF12)asanupre- levels. Epithelial and endothelial cells weakly expressed the gulated receptor on activated fibroblasts precursors, known receptor in vitro. Cultured macrophages did not express the as pericytes, and their pathologic counterparts in diseased receptor (Figure 1D). A similar pattern of expression was kidney, known as myofibroblasts. Transcripts