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NFkB-Induced Periostin Activates -b3 Signaling to Promote Renal Injury in GN

† †‡ Niki Prakoura,* Panagiotis Kavvadas,* Raphaёl Kormann,* Jean-Claude Dussaule,* † † Christos E. Chadjichristos,* and Christos Chatziantoniou*

*Institut National de la Santé Et de la Recherche Médicale, Unité Mixte de Recherche Scientifique 1155, Tenon Hospital, Paris, France; †Sorbonne Université, Université Pierre-et-Marie-Curie Paris 6, Paris, France; and ‡Department of Physiology, Saint-Antoine Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France

ABSTRACT De novo expression in the kidney of periostin, a involved in odontogenesis and osteogenesis, has been suggested as a biomarker of renal disease. In this study, we investigated the mechanism(s) of induction and the role of periostin in renal disease. Using a combination of bioinformatics, reporter assay, and chromatin immunoprecipitation analyses, we found that NFkB and other proinflammatory transcription factors induce periostin expression in vitro and that binding of these factors on the periostin promoter is enriched in glo- meruli during experimental GN. Mice lacking expression of periostin displayed preserved renal function and structure during GN. Furthermore, delayed administration of periostin antisense oligonucleotides in wild-type animals with GN reversed already established proteinuria, diminished tissue inflammation, and improved renal structure. Lack of periostin expression also blunted the de novo renal expression of integrin-b3and phosphorylation of focal adhesion kinase and AKT, known mediators of integrin-b3 signaling that affect cell motility and survival, observed during GN in wild-type animals. In vitro, recombinant periostin increased the expression of integrin-b3 and the concomitant phosphorylation of focal adhesion kinase and AKT in podo- cytes. Notably, periostin and integrin-b3 were highly colocalized in biopsy specimens from patients with inflammatory GN. These results demonstrate that interplay between periostin and renal inflammation orches- trates inflammatory and fibrotic responses, driving podocyte damage through downstream activation of integrin-b3 signaling. Targeting periostin may be a novel therapeutic strategy for treating CKD.

J Am Soc Nephrol 28: ccc–ccc, 2016. doi: 10.1681/ASN.2016070709

CKD is a major burdenworldwide with currently no highly induced in vitro by TGF-b1,1,5,8 and it was also available early prognostic marker or efficient ther- found to be upregulated by angiotensin II in cardiac apeutic treatment. Although various stimuli can fibroblasts.8 IL-4 and IL-13 increased periostin in lung lead to CKD, there are common mechanisms char- fibroblasts and bronchial epithelial cells, associating acterized by chronic inflammation and accumulation its induction with asthma.9 of within the kidney, resulting in Periostin has been found to be upregulated in decline of renal function and finally, ESRD. renal disease models and kidney biopsy specimens. Periostin is a matricellular protein abundant in periosteum, periodontal ligament, bone, skin, and de- veloping heart.1,2 Periostin interacts with extracellular Received July 1, 2016. Accepted October 31, 2016. matrix components, including collagen I, tenascin-C, Published online ahead of print. Publication date available at and fibronectin, promoting collagen fibrillogenesis www.jasn.org. and increased strength performance of the extracellu- Correspondence: Dr. Christos Chatziantoniou, Institut National 3–5 lar matrix. Interaction of periostin with cell-surface de la Santé et de la Recherche Médicale, Unité Mixte de Re- avb3andavb5 has been associated with in- cherche Scientifique 1155, Tenon Hospital, 4 rue de la Chine, creased adhesion and migration of cancer cells and 75020 Paris, France. Email: [email protected] vascular smooth muscle cells (SMCs).6,7 Periostin is Copyright © 2016 by the American Society of Nephrology

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It was found highly overexpressed in experimental polycystic transfection with the transcription factor plasmids, confirm- kidney disease promoting cyst growth and fibrosis.10 Periostin ing the results of the luciferase assay (Figure 1E). tissue expression and urinary levels were inversely correlated We performed chromatin immunoprecipitation (ChIP) ex- with renal function in patients with lupus nephritis and type 2 periments to verify the in vivo binding of these transcription diabetes, respectively.11,12 Periostin mRNA expression was factors on periostin promoter in a chronic renal disease highly upregulated in both glomeruli and tubulointerstitium model, the NTS-induced GN. Indeed, p65 was highly enriched in patients with different nephropathies.13 We previously re- on its respective binding sites on periostin promoter after NTS ported that periostin expression is highly correlated with dis- administration (Figure 1F). p-c-Jun and p-Stat1 were also en- ease progression in a model of hypertensive nephropathy, and riched on several of their predicted binding sites on periostin we subsequently showed that mice lacking periostin are pro- promoter in the NTS group (Figure 1, G and H). On the con- tected against the structural alterations induced by unilateral trary, p-Smad3 was not enriched on periostin promoter after ureteral obstruction.14,15 NTS, in agreement with its low efficiency to induce periostin Despite these findings, the mechanisms of induction and promoter activity or mRNA expression (Figure 1I). function of periostin in renal disease are largely unknown. In this study we investigated these mechanisms in an established Genetic Deletion of Periostin Preserves Renal chronic renal disease model induced by nephrotoxic serum Structure and Function in the NTS Model (NTS) administration. We found that periostin is induced We used periostin null mice in the C57BL/6 background and by key proinflammatory transcription factors, with the major confirmed whether periostin expressionwas induced after NTS factor being NFkB, and showed that periostin colocalizes with administration. Transcriptional and protein analysis showed integrin-b3 at sites of renal damage driving activation of increased renal expression of periostin in wild-type NTS mice p-FAK– and p-AKT–dependent pathways. Finally, using a compared with control littermates treated with PBS, whereas pharmacogenetic approach of in vivo antisense administration knockout (KO) mice did not display any periostin expression after severe proteinuria and the disease onset, we showed that (Figure 2, A and B). Next, we analyzed functional and struc- inhibition of periostin can be used as a therapeutic strategy to tural alterations indicative of disease progression. The increase alleviate renal disease progression. To our knowledge, we de- in weight caused by edema and the severe increase of protein- scribe for the first time how periostin is induced to mediate the uria were markedly attenuated in KO mice, showing that lack progression of renal disease and underline periostin targeting of periostin resulted in preservation of kidney function (Fig- as therapeutic treatment against CKD. ure 2, C and D). Histologic examination of kidney sections stained with Masson trichrome showed that mice lacking per- iostin developed less fibrotic lesions and had highly preserved RESULTS kidney structure together with decreased number of injured glomeruli (Figure 2, E and F). Periostin Is Induced by Proinflammatory Transcription We next examined the expression of known proinflamma- Factors in the Model of NTS-Induced GN tory mediators and fibrotic molecules, as well as the inflam- To find transcription factors that may induce periostin expres- matory cell infiltration profile of the diseased kidneys. Both sion, we performed bioinformatics analysis of a 2-kb region of collagens I and III and profibrotic TGF-b1 were upregulated in periostin promoter using the Genomatix software. We focused wild-type NTS mice, but their expression was highly attenu- on known proinflammatory and profibrotic transcription fac- ated in KO mice (Figure 2, G–I). In parallel, the induction of tors and mapped their predicted binding sites on the periostin proinflammatory mediators, such as MCP-1, Rantes, and promoter sequence (Figure 1A). VCAM-1, in wild-type mice after NTS was completely blunted To examine the efficiency of these transcription factors to in mice lacking periostin (Figure 2, J–L). Infiltration of mac- affect periostin expression, we performed luciferase assays rophages and lymphocytes was markedly reduced in KO NTS using 2 and 1 kb constructs of human or mouse periostin mice (Figure 2, M and N), establishing a close connection promoter, together with expression plasmids of these tran- between periostin and inflammation probably leading to de- scription factors. The large subunit of NFkB, p65, highly velopment of fibrosis and renal damage. induced the activity of both human periostin promoter con- structs and, even more impressively, that of mouse promoter Delayed Inhibition of Periostin Prevents the constructs (Figure 1B). Other proinflammatory transcription Development of Renal Disease in the NTS Model factors (c-Jun, Stat1a, and Stat6) induced a two-fold increase To assess whether inhibition of periostin can be used as ther- in the activity of both human and mouse constructs (Figure apeutic approach in renal disease, we inhibited periostin 1C). In contrast, from the group of profibrotic transcription expression via constant administration of antisense oligonu- factors, only Smad4 induced a sustained two-fold promoter cleotides (ODN) after induction of GN, and scrambled non- activity with Smad3 and Smad5, resulting in a much smaller specific ODN was used as control. induction of promoter activity (Figure 1D). The increase of Antisense administration in NTS mice efficiently inhibited periostin mRNA paralleled the promoter activation after the mRNA and protein upregulation of periostin (Figure 3, A

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Figure 1. Periostin is upregulated by proinflammatory transcription factors in the model of NTS-induced GN. (A) Mouse periostin promoter scheme with putative transcriptions factor binding sites, as resulted by bioinformatics analysis. (B–D) Luciferase-assay results in HEK293 cells showing that both human and mouse periostin promoter activity was highly induced by p65, a NFkΒ subunit (B), and to a lesser extent by other proinflammatory transcription factors like c-Jun, STAT1, and STAT6 (C), or profibrotic transcription factors like Smad3, Smad4, and Smad5 (D). (E) Endogenous periostin mRNA expression in HEK293 cells was induced after transfection with the indicated transcription factors to a level comparable to the luciferase assay results (F–I). ChIP analysis performed in the model of NTS- induced GN, showing high enrichment of the proinflammatory transcription factors p65 (F), p-c-Jun (G), and p-STAT1 (H) on several positions of periostin promoter in vivo after NTS. On the contrary, p-Smad3, a profibrotic transcription factor, was not enriched on periostin promoter after NTS (I). For mice, n=4 per group. For cell cultures, quantifications of three independent experiments per- formed in triplicates are shown. *P,0.05; **P,0.01 versus control or wild-type PBS.

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Figure 2. Periostin KO mice are protected from the development of NTS-induced GN. (A and B) Periostin mRNA (A) and protein (B) are increased in NTS-induced GN. (C and D) Periostin KO mice are highly protected from the increase in weight (C) and proteinuria (D) caused by NTS. (E) Masson trichrome staining showing reduced fibrosis and tissue damage in periostin KO mice (upper panels, 3200; lower panels, 3400). (F) Quantification of glomerular lesions. (G–I) mRNA expression of the fibrotic molecules COL1 (G), COL3 (H), and TGF-b1 (I) is highly decreased in periostin KO mice after NTS. (J–L) mRNA expression of the inflammatory mediators MCP-1 (J), RANTES (K), and VCAM-1 (L) is diminished in periostin KO mice after NTS. (M and N) F4/80 (M) and CD3 (N) staining showing markedly decreased accumulation of macrophages and lymphocytes, respectively, in periostin KO mice after NTS. Quantification of the staining is shown in the graphs below. mRNA and protein results from the whole kidney are shown (A, B, G–L). Animals were euthanized after 14 days of NTS administration. Scale bars, 100 mm (E, M, and N). n=6–7 per group. *P,0.05; **P,0.01 versus WT PBS or WT NTS; #P,0.05 versus WT NTS. WT, wild type. and B). The renal function of NTS scrambled mice was largely resulted in better preservation of kidney structure character- compromised, characterized by high increase in weight, pro- ized by restricted formation of fibrotic, glomerular, and tubu- teinuria, and BUN levels. These changes were reversed in mice lar lesions compared with severe structural alterations in NTS receiving antisense: body weight and BUN increases were scrambled mice (Figure 3, F–H). The preservation of kidney blunted and proteinuria was decreased to almost control levels structure and function in antisense-treated mice was accom- (Figure 3, C–E). Treatment with antisense against periostin panied by decreased expressions of profibrotic (COL1, COL3,

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Figure 2. Continued.

TGF-b1) and proinflammatory mediators (MCP-1, VCAM-1, de novo expressed in NTS mice, with its expression being CSF-1) (Figure 3, I–N), and by lesser infiltration of macro- nearly blunted in KO or antisense-treated mice (Figure 4, B phages and lymphocytes compared with NTS scrambled mice and E) and was also localized in vessels and glomeruli of dam- (Figure 3, O and P). aged kidneys (Figure 4, C and F). Staining of serial sections verified that integrin-b3 and periostin were coexpressed at the Periostin Is Colocalized with Integrin-b3 at Sites of same sites in diseased kidneys (Figure 4G). Further double Renal Damage and Drives Integrin-b3 Downstream costaining with nestin or CD44 showed that both Signaling are overexpressed in podocytes and activated parietal epithe- We performed immunohistochemical analysis to localize per- lial cells in the NTS model (Figure 4, H and I). On the contrary, iostin expression in the diseased kidney. In both NTSprotocols, costaining of periostin with a-SMA or CD146 did not reveal periostin showed a basic expression in control vessels and a any colocalization with glomerular mesangial or endothelial high upregulation in vessels and injured glomeruli of damaged cells, respectively (Figure 4I). Notably, coimmunoprecipita- kidneys. Periostin expressionwas null in KO kidneys and highly tion analysis demonstrated that periostin interacts with its diminished in antisense-treated kidneys (Figure 4, A and D). receptor, integrin-b3, in vivo during development of renal Interestingly, integrin-b3, a proposed periostin receptor, was disease (Figure 4J).

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Figure 3. Delayed treatment with antisense against periostin reverses the development of NTS-induced GN. (A and B) Periostin mRNA (A) and protein (B) is increased in NTS scrambled mice but is decreased in mice treated with antisense ODN. (C–E) Initiation of antisense treatment at D3 after NTS reversed the increase in weight (C), proteinuria (D), and BUN (E) observed in NTS scrambled mice. (F) Masson trichrome staining showing reduced fibrosis and tissue damage in NTS antisense-treated mice (upper panels, 3200; lower panels, 3400). (G and H) Quantification of structural glomerular (G) and tubular (H) alterations showing highly reduced crescent formation, fibrin deposition, glomerulosclerosis, and tubular dilation in NTS antisense-treated mice. (I–K) mRNA expression of the fibrotic mol- ecules COL1 (I), COL3 (J), and TGF-b1 (K) is attenuated in antisense-treated mice after NTS. (L–N) mRNA expression of the

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Figure 3. Continued.

Tofurther investigate the connection between periostin and survival and growth, was de novo expressed in renal vessels integrin-b3, we analyzed the expression of known mediators and glomeruli after NTS, whereas its expression was negligible of integrin-b3 downstream signaling in the NTS model. We in KO kidneys (Figure 5B). Costaining with CD44 and nestin found that p-FAK, known for mediating and revealed that both integrin mediators p-FAK and p-AKT were motility, was induced several-fold in wild-type mice after expressed by activated parietal cells and podocytes after NTS NTS, whereas its expression remained low in the KO NTS (Figure 5C), as was shown with periostin and integrin-b3 group (Figure 5A). Similarly, p-AKT, a mediator of cell (Figure 4, H and I). inflammatory mediators MCP-1 (L), VCAM-1 (M), and CSF-1 (N) is diminished in antisense-treated mice after NTS. (O and P) F4/80 (O) and CD3 (P) staining showing markedly decreased accumulation of macrophages and lymphocytes, respectively, in NTS mice treated with antisense against periostin. Quantification of the staining is shown in the graphs on the right. Animals were euthanized after 9 days of NTS administration. Scale bars, 100 mm (F, O, and P). n=5 per group. *P,0.05; **P,0.01 versus PBS; #P,0.05; ##P,0.01 versus NTS scrambled.

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Figure 4. Periostin and integrin-b3 are colocalized at the sites of injury in the model of NTS-induced GN. (A and D) Immunohisto- chemical staining for periostin in WT/KO (A) and scrambled/antisense (D) mice showing increased expression of periostin in glomeruli and vessels of WT NTS mice that is absent in KO and ameliorated in antisense mice. (B and E) Integrin-b3 mRNA expression is induced in WT NTS (B) and NTS scrambled (E) mice but is highly ameliorated in KO NTS and NTS antisense mice, respectively. (C and F) Immunohistochemical localization of integrin-b3 in WT/KO (C) and scrambled/antisense (F) mice showing increased expression in glomeruli and vessels of WT NTS mice that is highly attenuated in KO and antisense mice, respectively. (G) Immunohistochemical staining of integrin-b3 and periostin in serial sections of WT NTS mice, showing localization of both proteins at the same sites (arrows indicating expression of both proteins in the same structures). (H) Double immunofluorescence for periostin (green)/nestin (red) and integrin-b3 (green)/nestin (red) in WT NTS mice, showing that both periostin and integrin-b3 are expressed by podocytes in glomeruli of NTS mice. (I) Double immunofluorescence for periostin or integrin-b3 (green) with CD44 (red) in WT NTS mice showed that both proteins are strongly expressed by activated parietal epithelial cells. Double immunofluorescence for periostin (green) with a-SMA or CD146 (red) showed no colocalization with mesangial and endothelial cells, respectively. (J) Coimmunoprecipitation analysis using anti–integrin-b3 antibody successfully precipitated both integrin-b3 and periostin, showing that the two proteins interact in vivo in the NTS model. The samples were run on the same gel but were discontinuous. Animals were euthanized after 14 or 9 days of NTS in the series of experiments with the KO (A–C) or the antisense (D–J), respectively. Scale bars, 100 mm (G) or 50 mm (A, C, D, F, H, and I). Representative images are shown. n=5–7 per group. *P,0.05 versus WT PBS; #P,0.05 versus WT NTS or NTS scrambled.

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Figure 4. Continued.

To examine whether periostin can activate integrin-b3and sections of renal biopsies from patients with ANCA vasculitis, its downstream signaling, we incubated an immortalized po- an inflammatory glomerulopathy. A strong staining for both docyte cell line, one of the cell types being injured in the NTS periostin and integrin-b3 was observed in the examined model, with recombinant periostin. In a time-course experi- biopsies, and the proteins were found to colocalize within ment, periostin strongly induced the expression of integrin- damaged glomeruli and tubules (Figure 6). In addition, a com- b3, along with its effectors p-FAK and p-AKT, with a peak at 6 parison of expression profiles from renal disease datasets hours (Figure 5, D and E). deposited in the publicly available platform Nephroseq revealed a strong correlation and a comparable upregulation of Periostin and Integrin-b3 Colocalize in Renal Biopsies periostin and integrin-b3 in several of these datasets (Supple- To investigate whether periostin and integrin-b3 are coex- mental Figure 1, Supplemental Material). These results support pressed in cases of human renal disease, we performed immu- thenotionthatperiostinandintegrin-b3 cooperate to mediate the nohistochemical localization of both proteins in consecutive development of renal disease.

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Figure 5. Periostin promotes integrin-b3 signaling in vivo and in vitro. (A) pFAK expression in WT/KO NTS mice is shown by Western blot. The increase in FAK phosphorylation observed in WT mice after NTS is blunted in KO mice. (B) Immunohistochemical staining of pAKT shows upregulation in glomeruli and vessels in WT mice after NTS, which is blunted in KO mice. (C) Double immunofluorescence for p-FAK and p-AKT (green) with CD44 or nestin (red) shows that both proteins are expressed by activated parietal epithelial cells and podocytes in the glomeruli of NTS mice. (D) E11 immortalized podocytes were incubated with recombinant mouse periostin

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Figure 6. Periostin and integrin-b3 are coexpressed in human renal biopsies. (A) Immunohistochemical staining of integrin-b3and periostin in consecutive sections from a biopsy specimen of a patient with ANCA vasculitis, depicting a strong expression and a high degree of colocalization between periostin and integrin-b3 both in damaged glomeruli and tubules. (B) Magnified images of the panels drawn in (A), showing in more detail the coexpression of periostin and integrin-b3 in damaged glomeruli and surrounding tubules. Arrows indicate colocalization sites. Scale bars, 100 mm(A)or30mm (B). Representative images are shown.

DISCUSSION induce periostin promoter activity in preosteoblasts, whereas YY1 was found to bind on a conserved enhancer region on The increasing prevalence of CKD and the lack of efficient treat- periostin promoter, regulating tissue-specific periostin expres- ment necessitate the discovery of novel prognostic markers and sion in the cardiac cushion .18,19 Our bioinfor- therapeutic targets. Periostin, a matricellular protein associated matics analysis revealed the presence of several putative with mesenchymal phenotype and fibrous structures, was found proinflammatory transcription factor binding sites on perios- by us andother investigators tobe highly inducedin thekidney in tin promoter, whereas transcription factor binding sites of the several types of renal disease, both in animal models and patient classic TGF-b1 signaling pathway, which has been shown to biopsies.16,17 Nevertheless, the mechanism(s) of induction and upregulate periostin in vitro,1,5,8 were rare. In luciferase assays the actual role of periostin during disease progression have not and at the endogenous gene level, we showed that proinflamma- been examined. This study aimed to unravel these mechanisms tory transcription factors such as NFkΒ (p65), c-Jun, and STAT1 in an established model of NTS-induced renal disease. Our directly induced periostin promoter activity or gene expression. results showed for the first time that proinflammatory transcrip- The active forms of these transcription factors were found to be tion factors induce periostin in renal disease. Moreover, perios- highly enriched in vivo on periostin promoter in NTS-induced tin was found to drive both the expression and downstream GN, indicating that periostin was upregulated in response to signaling of integrin-b3, a proposed periostin receptor, causing these transcription factors in the NTS model. The fact that phenotypical changes associated with disease progression. Fi- p-Smad3 was not enriched on periostin promoter implies that nally, we showed that delayed in vivo inhibition of periostin after either periostin is not induced by TGF-b1 in the NTS model or the disease onset can efficiently alleviate the progression of severe that TGF-b1 may control periostin expression by an Smad- renal disease. The proposed mechanisms of induction and func- independent pathway, involving the activation of proinflamma- tion of periostin in renal disease are summarized in Figure 7. tory transcription factors like NFkB, Jun, and STAT.20 Little is presently known about the transcription factors that The induction of periostin by proinflammatory pathways control periostin gene expression. Twist was shown to bind and correlates with its observed role to promote inflammatory

(400 ng/ml) for the indicated time points and Western blot analysis was performed for integrin-b3, pFAK, pAKT, and GAPDH, with quantification graphs shown on the right. At 6 hours of periostin incubation integrin-b3 and its downstream effectors pFAK and pAKT were upregulated. (E) E11 cells were incubated for 6 hours with increasing doses of recombinant mouse periostin (400 and 1000 ng/ml), fol- lowed by Western blot analysis of integrin-b3, pFAK, FAK, pAKT, AKT, and GAPDH. Quantifications are shown on the right. pFAK and pAKT remain elevated after incubation with increasing doses of periostin. Scale bars, 100 mm (B and C). n=6–7 mice per group. Animals were euthanized after 14 days of NTS administration. For cell cultures, quantifications of three independent experiments performed in triplicates are shown. *P,0.05 versus control; **P,0.01 versus WT PBS or control.

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Figure 7. Proposed mechanism of how periostin mediates renal damage. Proinflammatory transcription factors, with the major factor being NFkΒ, induce expression of periostin, which activates integrin-b3, inducing integrin downstream signaling and leading to cell motility, survival, and podocyte foot process effacement. In parallel, periostin promotes the tissue expression of several chemokines (MCP-1, RANTES, VCAM-1, and CSF-1) and profibrotic molecules (COL1, COL3, and TGF-b1), inducing inflammatory cell infiltration and matrix stabilization, and podocyte damage further amplifies these effects, causing a vicious circle. reactions in several disease contexts. In allergen-induced skin of collagens and TGF-b1, and improved renal structure and inflammation, IL-4 and IL-13 were shown to induce periostin, function. Periostin has been associated with fibrosis in several which further amplified Th-2 type immune responses.21 Fur- tissues. Analysis of periostin expression in human skin pathol- ther, periostin-deficient lung fibroblasts showed lower pro- ogies demonstrated that it is overexpressed during scar duction of chemokines in response to TNF-a, accounting formation and associated with wound healing and pathologic for the lack of inflammatory response in pulmonary fibro- remodeling.26 Accordingly, periostin null dermal fibroblasts sis.22 In a model of glioblastoma xenografts, periostin secreted showed reduced expression of Acta2 and Col1a1 transcripts by stem cells was found to recruit tumor-associated macro- upon TGF-b1 treatment,27 whereas mesenchymal cells treated phages promoting malignant growth.23 These studies are in with exogenous periostin showed increased collagen production accordance with our observations that lack or inhibition of and wound closure.28 In a model of acute myocardial infarction, periostin reduced the production of proinflammatory cyto- periostin null mice exhibited impaired cardiac healing as a result kines and the subsequent macrophage and lymphocyte infil- of reduced myocardial stiffness and collagen fibril formation, tration to the injured kidneys. To examine whether the whereas inducible overexpression of periostin protected mice difference in the inflammatory response was because of strain from cardiac rupture.29,30 The ability of periostin to interact difference in the activation of inflammatory cells, we com- and stabilize fibrillar collagens and other extracellular matrix pared the activated monocytes/macrophages of wild-type molecules is probably a key event by which periostin controls and KO mice after thioglycolate-induced peritonitis. Numer- extracellular matrix deposition and development of fibrosis.3–5 ation of the infiltrated peritoneal monocytes after thioglyco- We found de novo expressed periostin to colocalize and in- late treatment did not show any differences between wild-type teract with integrin-b3 in glomeruli and vascular SMCs of NTS and KO mice (Supplemental Figure 2A). Leukocyte cell anal- mice. Overexpression of periostin in aortic SMCs induced mi- ysis before and after thioglycolate treatment did not reveal any gration together with increased integrin-b3 expression and FAK variations in the leukocyte subtypes (Supplemental Figure phosphorylation.7 In addition, periostin expression was in- 2B). In migration assays toward chemoattractant MCP-1, creased in vascular SMCs subjected to mechanical strain, and the isolated peritoneal monocytes of wild-type and KO mice this increase was followed by FAK activation and MCP-1 secre- showed the same migratory capacities, also explained by the tion, which were inhibited by a periostin-neutralizing anti- similar expression levels of various receptors and cytokines body.31 In agreement with these studies, induction of periostin (Supplemental Figure 2, C and D). Notably, periostin expres- in the NTS model was determinant for the de novo expression of sion was null in the isolated wild-type monocytes. Thus, peri- integrin-b3 followed by downstream FAK and AKT activation. ostin expression by the damaged tissue is crucial in inducing Thus, periostin may induce and activate integrin-b3, promoting production of chemokines to attract inflammatory cells at the motility, permeability, and secretion of chemokines essential for sites of injury. Accordingly, recombinant periostin was found remodeling and inflammatory cell infiltration.32 to directly enhance adhesion of eosinophils and monocytes to Activation of integrin avb3 in podocytes has been shown to fibronectin and function as a chemoattractant to primary mediate podocyte dysfunction, motility, and proteinuria in monocytes and macrophages in migration assays.24,25 FSGS and LPS-induced renal disease.33,34 In the NTS model, Chronic inflammation plays a central role in development of induction of integrin-b3 and its downstream signaling in pa- fibrosis and kidney damage. Concomitantly to the attenuation rietal cells and podocytes was dependent on the expression of of inflammation, periostin deficiency diminished the expression periostin. Treatment of cultured podocytes with recombinant

12 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2016 www.jasn.org BASIC RESEARCH periostin was sufficient to increase integrin-b3 expression fol- antibodies (N-Histofine; Nichirei Biosciences) and AEC (Dako) as lowed by FAK and AKTactivation. Activation of FAK and AKT substrate were used. Slides were counterstained with hematoxylin QS in the injured podocytes and activated parietal cells induces (Vector Laboratories, Burlingame, CA) and mounted with perma- cell motility, invasion,35 and crosstalk between the two glo- nent aqueous mounting medium (ScyTek). Biopsies from four pa- merular cell types, resulting ultimately in foot process efface- tients were used for this set of experiments. ment, proteinuria, and kidney damage. We found strong expression and a high degree of colocal- Animal Models ization between periostin and integrin-b3 in renal biopsies All procedures regarding animal experimentation were in accordance from patients with ANCA vasculitis. In addition to their glo- with the European Union Guidelines for the Care and Use of Labo- merular expression, both proteins were also localized in tubu- ratory Animals and approved by the local ethics committee of the lar cells in the human biopsies. This difference with the NTS National Institute for Health and Medical Research (Institut National model may reflect the distinct stimuli causing the disease, de la Santé et de la Recherche Médicale). Animals were housed at which in the case of the NTS model, target primarily the glo- constant temperature with free access to water and food. merulus, whereas they affect the whole kidney in the case of The strain of periostin KO mice was created at the laboratory of Dr. ANCA vasculitis. To further investigate the coexpression pat- Simon Conway and has been previously described.15 For the model of tern of periostin and integrin-b3 in patients with renal disease, NTS-induced GN, decomplemented NTS was prepared as previously we performed a meta-analysis of gene expression profiles from described.37,38 Female wild-type and KO littermate mice of the C57BL/6 renal disease datasets deposited in the Nephroseq platform background (8–10 weeks old) received intravenous injections of 15 ml (www.nephroseq.org). We compared the expression levels of NTS per g body wt over two consecutive days (days 0 and 1) to induce periostin and integrin-b3 in a similar disease dataset as the crescentic GN. Control mice were injected with PBS. Mice were eutha- biopsies used in our study (patients with vasculitis versus nized 14 days after the first injection (n=6–7 per group). Blood, urine, healthy donors, n=52), and found a strong correlation and renal tissues were collected for subsequent analyses. between periostin and integrin-b3(R=0.54, P,0.001). More- For the antisense experiments, SV129 male wild-type mice aged 8– over, both proteins were significantly upregulated to a com- 10 weeks were used. The mice received intravenous injections of 12 ml parable level in several other datasets (Supplemental Figure 1). NTS per g body wt over two consecutive days (days 0 and 1) to induce Presently, only two studies in animal models of pulmonary crescentic GN, whereas control mice were injected with PBS. Perios- fibrosis and ovarian cancer have shown that inhibition of peri- tin expression was inhibited with a cocktail of two different ODNs ostin via blocking antibodies could be used as a therapy against specifically targeting periostin mRNA, designed using the Integrated the disease progression.28,36 We used an alternative strategy of DNA Technologies platform, also previously described.15 Scrambled in vivo antisense administration after establishment of severe nonspecific ODNs were used as control. The ODN sequences were proteinuria and found that inhibition of periostin could re- modified with phosphorothioate to prevent their in vivo hydrolysis by markably diminish the inflammation and reverse the increase nucleases (Sigma-Aldrich, St. Louis, MO; Supplemental Table 1). For in proteinuria. The ability of periostin to play distinctive roles administration, the ODNs were diluted in normal saline and placed in crucial events during development of renal disease by promot- in osmotic mini-pumps (model 1002; Alzet, Cupertino, CA), which ing tissue inflammatory response, accumulation of extracellular were subsequently implanted subcutaneously, constantly releasing a matrix and podocyte damage underline periostin targeting as a dose of 0.25 ml/h corresponding to a release of 150 pmol of ODN per potential efficient future treatment against CKD. day. The implantation of mini-pumps was performed at day 3 after administration of NTS, taking 1 day for the pump to start function- ing. Mice were euthanized 9 days after the first injection (n=5 per CONCISE METHODS group). Blood, urine, and renal tissues were collected.

Immunohistochemical Staining of Patient Biopsies Proteinuria and BUN Measurements All procedures and use of human tissue were performed according to Urine was collected at several time points during the mouse protocols the national ethical guidelines and were in accordance with the Dec- and blood was collected at the end of each protocol. Proteinuria was laration of Helsinki. Written informed consent was given by the pa- measured using a Konelab analyzer (Thermo Fisher Scientific, Vernon tients for use of part of their biopsy specimen for scientific purposes Hills, IL), and was normalized to urine creatinine. BUN levels were before inclusion in the study. measured with an enzymatic method (Konelab analyzer) and ex- Renal biopsies from patients were retrospectively analyzed. Exper- pressed in millimoles per liter. iments were performed on biopsies from patients with ANCA vascu- litis. The biopsies were characterized by diffuse glomerular lesions, Histologic Evaluation fibrin deposits, weak lesions of acute tubular necrosis, and extraca- Formalin-fixed, paraffin-embedded, 4-mm thick kidney sections were pillary crescentic proliferation. Formalin-fixed, paraffin-embedded stained with Masson trichrome. Quantifications were performed by consecutive kidney sections (4-mm thick) were stained with primary examining at least ten microphotographs of random, nonoverlapping antibodies for periostin (ab14041; Abcam, Inc., Cambridge, MA) or fields at 2003 magnification per mouse. Crescents, fibrin deposits, integrin-b3 (ab75872; Abcam, Inc.). Appropriate secondary and glomerulosclerosis were expressed as percentage of the total

J Am Soc Nephrol 28: ccc–ccc, 2016 Periostin Targeting Reverses CKD 13 BASIC RESEARCH www.jasn.org glomeruli evaluated. Tubular dilation was examined using a zero (no For periostin immunostaining, 5-mm frozen tissue sections were dilation) to three (high dilation) injury scale per photograph and the fixed in 4% PFA for 15 minutes, blocked in 10% FBS, and incubated mean value was calculated for each mouse. with primary anti-periostin antibody (PA5–34641; Thermo Fisher The evaluation of glomerular injury on the resistant C57BL/6 Scientific). Integrin-b3 (ab75872; Abcam, Inc.) and p-AKT immu- background was on the basis of the combined examination of me- nostainings (ab81283; Abcam, Inc.) were performed on 4-mm sangial matrix expansion, endothelial wall thickening, capillary rup- formalin-fixed, paraffin-embedded kidney sections. Incubation ture, and glomerular infiltration and was expressed as percentage of with secondary antibodies and signal detection was performed as injured glomeruli per total glomeruli examined. described above. Dual immunolabeling was performed on 5-mm frozen or 4-mm Western Blot Analysis paraffin tissue sections incubated with primary antibodies to perios- Proteins from half kidneys or cell culture were extracted in a com- tin, integrin-b3, nestin (556309; BD Pharmingen), CD44 (11–624- mercial RIPA lysis buffer supplemented with PMSF, a protease C100; Exbio), a-SMA (A5228; Sigma-Aldrich), CD146 (kind gift inhibitor cocktail and sodium orthovanadate (Santa Cruz Biotech- from Professor Françoise Dignat-George), pAKT and pFAK nologies, Santa Cruz, CA), and total protein concentration was mea- (ab55335; Abcam, Inc.). The signal was detected with Alexa Fluor sured using the Bradford assay. Equal amounts of proteins were loaded –488 and –546 secondary antibodies followed by DAPI counterstaining on a NuPAGE 4%–12% gradient gel (Invitrogen, Carlsbad, CA) and and mounting in PermaFluor medium (Thermo Fisher Scientific). transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). Immunoblotting was performed for periostin (MOSF20; R&D Sys- E11 Podocyte Cell Culture tems, Minneapolis, MN), integrin-b3 (ab75872; Abcam, Inc.), pFAK TheE11immortalizedmousepodocytecelllinewasculturedaspre- (#3283; Cell Signaling Technology, Danvers, MA), FAK (sc-557; Santa viously described.17 Briefly, cells were maintained in RPMI 1640 Cruz Biotechnologies), pAKT (#9271; Cell Signaling Technology), medium supplemented with 10% FBS, 100 U/ml penicillin streptomy- and AKT 1/2/3 (sc-8312; Santa Cruz Biotechnologies). Gapdh cin, and 10 U/ml recombinant mouse INFg (Peprotech) to induce syn-

(Sigma-Aldrich) was used as loading control. thesis of the immortalizing T antigen at 33°C and 5% CO2.Toinitiate differentiation, cells were trypsinized and thermoshifted to 37°C in me- Quantitative Real-Time PCR dium without INFg for 10–15 days. For treatment with recombinant Total RNA was extracted from half kidneys using TRI Reagent (MRC) mouse periostin, differentiated cells in six-well plates were serum starved and from cell cultures using the EZ-10 Spin Column Total RNA Mini- for 24 hours and incubated with recombinant periostin (R&D Systems) preps Super kit (Bio Basic Inc). RNAquality was verified by measuring at a concentration of 400 or 1000 ng/ml for the indicated time points. the OD 260:280 ratio and residual genomic DNA was removed by DNase I treatment (Thermo Fisher Scientific) for 30 minutes at 37°C. Cell Culture and Transfection A total of 1 mg RNA was transcribed to cDNA using the Maxima First The HEK293 cell line was maintained in DMEM 4.5 g/L glucose supple- Strand cDNA Synthesis Kit from Thermo Fisher Scientificperman- mented with 10% FBS, 100 U/ml penicillin streptomycin, and 2 mM ufacturer’s instructions. Real-time PCR was performed with the Ro- L-glutamine at 37°C/5% CO2. Transfection was performed in 12-well che Light Cycler 480 detection system using SYBR Green PCR Master plates via the CaCl2 method. Briefly, 2 hours before transfection, cells Mix (Roche Diagnostics, Indianapolis, IN). Specific primers for target were replenished with fresh culture medium. Each well was transfected mRNAs (Supplemental Table 2) were used for amplification under the with a mixture of 0.35 mg pGL3 plasmid, 0.35 mgCMV–b-galactosidase following program: 95°C for 5 minutes, 45 cycles at 95°C for 15 seconds plasmid (used for normalization), and 1.25 mg transcription factor ex- and 60°C for 15 seconds, and 72°C for 15 seconds. For quantitative anal- pression plasmid added to 14 ml 2 M CaCl2 and 111 ml23 HBS solution ysis, experimental were normalized to Gusb mRNA or Hprt mRNA (Sigma-Aldrich) in a final volume of 222 ml. The day after transfection, expression using the DDCT method. Dissociation curves were analyzed cell medium was replaced with fresh medium and the cells were incu- in order to determine that a single product was amplified. bated for another day before being tested in the luciferase assay or sub- jected to RNA extraction followed by quantitative real-time PCR. Immunohistochemistry and Double Immunofluorescence Plasmid Construction For immunostaining of macrophages and lymphocytes, 4-mmthick The pGL3-basic luciferase reporter vector (Promega, Madison, WI), formalin-fixed, paraffin-embedded kidney sections were stained with the CMV-b-galactosidase expression plasmid, and the expression primary antibodies to F4/80 (MCA497R; Abd Serotec) or CD3 plasmids for Smad3, Smad4, and Smad5 were kindly provided by (A0452; Dako), respectively. Appropriate secondary antibodies Dr. A. Charonis (Biomedical Research Foundation of the Academy (N-Histofine; Nichirei Biosciences) and AEC (Dako) as substrate of Athens). Expression plasmids for RelA/p65 (#21984), c-Jun were used. Slides were counterstained with hematoxylin QS (Vector (#47443), Stat1a (#8690), and Stat6 (#35482) were obtained from Laboratories) and mounted with permanent aqueous mounting me- Addgene. PUC19 encoding for an inactive fragment of b-galactosidase dium (ScyTek). The F4/80 and CD3 positive area was quantified in at was used as control plasmid for transfection. The human and mouse least ten photographs at 2003 magnification per animal, using pub- periostin promoter regions hProm 2 kb (22003+113), hProm 1 kb licly available image processing software (ImageJ; Fiji), and expressed (2888+113), mProm 2 kb (22037+20), and mProm 1 kb (2876+20), as percentage of the total tissue area. taking +1 as the transcription start site, were amplified by PCR from

14 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2016 www.jasn.org BASIC RESEARCH human or mouse DNA and cloned into the multiple cloning site se- orthovanadate, and total protein concentration was measured using the quence of pGL3-basic vector. The translation start site (ATG) for the Bradford assay. Equal amounts of proteins were precipitated with a rabbit human and mouse periostin gene is at +119 and +35 after the tran- anti–integrin-b3 antibody (ab119992; Abcam, Inc.) and rabbit IgG was scription start site, respectively. used as a control. The precipitated complexes were purified with the use of Protein G Magnetic Beads (#8740; Cell Signaling Technology) and loaded Bioinformatics Analysis on a denaturating SDS-PAGE followed by Western blotting. Equal Bioinformatics analysis of human and mouse periostin promoters was amounts of input were also loaded onthesamegel.Immunoblotting performed by the Genomatix software (www.genomatix.de). The de- was performed for periostin (MOSF20; R&D Systems) and the membrane sired promoter sequence was retrieved by the Genomatix database was stripped using Restore Western Blot Stripping Buffer (#21059; Thermo using the tool Gene2Promoter followed by multiple alignment to Fisher Scientific) and reblotted for integrin-b3 (ab75872; Abcam, Inc.) check for conserved sequences and detection of putative transcrip- and Gapdh (Sigma-Aldrich). Veriblot was used for immunoprecipitation tion factor binding sites per sequence similarity. analysis of secondary antibody (ab131366; Abcam, Inc.), preferentially detecting the nonreduced form of IgG to avoid nonspecific signals from Luciferase Assays coeluted reduced forms of the antibodies used for immunoprecipitation. For luciferase activity measurement, the Luciferase Assay System from Promega (E4030) was used according to manufacturer’s instructions. Statistical Analyses Briefly, the cells were lysed in Reporter Lysis Buffer, the extract was Data are expressed as mean6SEM. Data were analyzed using one-way collected after centrifugation and removal of cell debris, and luciferase ANOVA followed by a Fisher test. Values of P,0.05 were considered activity was measured immediately in a luminometer after mixing 15 ml significant. extract with 70 ml Luciferase Assay Reagent. To normalize luciferase measurements, the activity of b-galactosidase was measured in a b-galactosidase assay. For each sample, 30 mlcellextractweremixed ACKNOWLEDGMENTS with 3 ml magnesium solution (0.1 M MgCl2,4.5Mb-mercaptoethanol), 66 ml ONPG (substrate), and 201 ml sodium phosphate (pH 7.5) and We thank Dr. Aristidis Charonis and Dr. Panagiotis Politis (from incubated at 37°C for several minutes until the development of a faint Biomedical Research Foundation of the Academy of Athens) for yellow color. The reaction was stopped by addition of 1 M Na2CO3 and providing plasmid expression vectors. We also thank Dr. Nicole absorbance was measured at 420 nm. The results were expressed as the Endlich for providing the podocyte cell line and Caroline Martin for ratio of the relative luciferase units to b-galactosidase measurements. valuable help with animal breading. The CD146 antibody was a generous gift from Professor Françoise Dignat-George. ChIP This work was supported by grants from the Institut National de la ChIP was performed in kidneys from wild-type SV129 mice injected Santé et de la Recherche Médicale. N.P. was the recipient of a long- with NTS or PBS (control), using the SimpleChIP Plus Enzymatic term fellowship from the Society of European Renal Association- ’ Chromatin IP Kit (#9005; Cell Signaling Technology) per manufacturer s European Dialysis and Transplantation. instructions. Briefly, whole kidneys were finely minced and crosslinked with 1.5% formaldehyde for 20 minutes under constant rotation, fol- lowed by addition of 0.1 M glycine to stop the reaction. The tissue was DISCLOSURES disaggregated using a Dounce homogenizer, nuclei were prepared, and None. chromatin was digested, sonicated, and analyzed by gel electrophoresis and OD260 measurement to determine DNA concentration. Precipita- tion was performed with the following ChIP-graded antibodies from Cell REFERENCES Signaling Technology: NF-kB p65 (#8242), p-c-Jun (#3270), p-Stat1 1. Horiuchi K, Amizuka N, Takeshita S, Takamatsu H, Katsuura M, Ozawa (#7649), and p-Smad3 (#9520). Rabbit IgG was used as control. The fi fi H, Toyama Y, Bonewald LF, Kudo A: Identi cation and characterization precipitated complexes were puri ed with the use of Protein G Magnetic of a novel protein, periostin, with restricted expression to periosteum Beads, crosslinks were reversed, and DNA was eluted and purified. and periodontal ligament and increased expression by transforming Sequence-specific primers for selected regions of mouse periostin pro- growth factor beta. JBoneMinerRes14: 1239–1249, 1999 moter (Supplemental Table 3) were used in subsequent quantitative 2. Kruzynska-Frejtag A, Machnicki M, Rogers R, Markwald RR, Conway SJ: fi real-time PCR analysis to check for enrichment of precipitated DNAs Periostin (an osteoblast-speci c factor) is expressed within the embryonic mouse heart during valve formation. Mech Dev 103: 183–188, 2001 in the NTS over the PBS group. The results were expressed as percentage 3. 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