Matrix Metalloproteinase-7 Is a Urinary Biomarker and Pathogenic Mediator of Kidney Fibrosis

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1 BASIC RESEARCH Matrix Metalloproteinase-7 Is a Urinary Biomarker and Pathogenic Mediator of Kidney Fibrosis Dong Zhou,* Yuan Tian, Ling Sun, Lili Zhou, Liangxiang Xiao, Roderick J. Tan, Jianwei Tian, Haiyan Fu,* Fan Fan Hou, and Youhua Liu* *Department of Pathology and Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, China ABSTRACT Matrix metalloproteinase-7 (MMP-7), a secreted zinc and calcium dependent endopeptidase, is a transcriptional target of canonical Wnt/b-catenin signaling. Because Wnt/b-catenin is activated in diseased kidney, we hypothesized that urinary MMP-7 level may be used as a noninvasive surrogate biomarker for fibrotic lesions. To test this hypothesis, we conducted a cross-sectional study, measuring urinary MMP-7 levels in a cohort of 102 patients with CKD. Compared with normal subjects, patients with various kidney disorders had markedly elevated urinary levels of MMP-7. Furthermore, urinary MMP-7 levels closely correlated with renal fibrosis scores in patients. In mice, knockout of MMP-7 ameliorated the fibrotic lesions and expression of matrix genes induced by obstructive injury. Genetic ablation of MMP-7 also preserved E-cadherin protein expression and substantially reduced the expression of total and dephosphorylated b-catenin and the de novo expression of vimentin and fibroblast-specific protein 1 in renal tubules of obstructed kidneys. In vitro, MMP-7 proteolytically degraded E-cadherin in proximal tubular cells, leading to b-catenin liberation and nuclear translocation and induction of b-catenin target genes by a mechanism independent of Wnt ligands. Finally, pharmacologic inhibition of MMP-7 immediately after obstructive injury reduced renal fibrosis in vivo. These results suggest that MMP-7 not only can serve as a noninvasive biomarker but also is an important pathogenic mediator of kidney fibrosis. J Am Soc Nephrol 28: ccc ccc, doi: /ASN CKD currently afflicts.10% of the adult population worldwide, and its incidence is continuing to rise. 1,2 There is no effective cure for CKD, and progression to ESRD necessitates dialysis or kidney transplantation. 3,4 CKD is manifested by persistent functional decline and progressive tissue fibrosis characterized by the relentless accumulation of extracellular matrix, leading to scar formation in the renal parenchyma. Although kidney function can be evaluated by serum creatinine level and an egfr, assessment of renal fibrosis is only achievable with kidney biopsies, which are taken from patients by an invasive procedure. 5,6 In this context, developing a noninvasive surrogate biomarker that can detect and monitor the development and progression of renal fibrotic lesions is urgently needed. 7 9 We have recently reported that urinary matrix metalloproteinase-7 (MMP-7) is a surrogate marker for predicting intrarenal activity of Wnt/b-catenin, a key fibrogenic signaling pathway, in animal models of CKD. 10 However, whether Received March 25, Accepted August 7, Published online ahead of print. Publication date available at. Correspondence: Dr. Fan Fan Hou, Division of Nephrology, Nanfang Hospital, 1838 North Guangzhou Avenue, Guangzhou , China, or Dr. Youhua Liu, Department of Pathology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA ffhouguangzhou@163.com or liuy@ upmc.edu Copyright 2016 by the American Society of Nephrology J Am Soc Nephrol 28: ccc ccc, 2016 ISSN : /2803-ccc 1

2 BASIC RESEARCH MMP-7 in the urine can be used as a noninvasive marker for kidney fibrosis in patients with CKD is unknown. MMP-7, also known as matrilysin, is a secreted zinc and calcium dependent endopeptidase. 11 Structurally, MMP-7 is one of the smallest MMPs, and it is synthesized as an inactive zymogen consisting of a prodomain and a catalytic domain. 12 Activation of the proenzyme involves proteolytic removal of a 9-kD N terminal proregion containing the cysteine switch motif, which results in the final 19-kD active enzyme. Functionally, MMP-7 degrades a broad range of extracellular matrix components, cleaves other substrates, such as cell associated Fas ligand and E-cadherin, and activates other proteinases The ability of MMP-7 to act on such a broad spectrum of substrates renders MMP-7 a critical player in regulating a diverse array of cellular processes, such as matrix remodeling, cell apoptosis, and epithelial-mesenchymal transition (EMT). 13,17 Whether MMP-7 plays any role in the pathogenesis of kidney fibrosis in vivo, however,remainstobe investigated. Little or no MMP-7 expression is detected in normal adult kidney; however, its expression is markedly induced in a multitude of CKDs, including diabetic nephropathy, adriamycin nephropathy, obstructive nephropathy, and chronic allograft nephropathy. 10,18 The regulation of MMP-7 in the kidney is primarily controlled transcriptionally by b-catenin, 10 the principal downstream mediator of canonical Wnt signaling. 19 Because activation of Wnt/b-catenin is a common pathologic finding in a variety of CKD, we hypothesized that MMP-7, a direct downstream target of this signaling pathway, is upregulated and plays a role in the pathogenesis of kidney fibrosis in patients with CKD. In this study, we examined MMP-7 expression in the urine and biopsy specimens of a cohort of patients with CKD. Using MMP-7 null mice, we showed that endogenous MMP-7 promoted kidney fibrosis by activating b-catenin via a Wntindependent pathway. Furthermore, we showed that pharmacologic inhibition of MMP-7 reduced renal fibrotic lesions in vivo after obstructive injury. Our studies illustrate that MMP-7 not only can serve as a noninvasive biomarker but also, is a pathologic mediator and therapeutic target of kidney fibrosis. RESULTS Urinary MMP-7 Levels Are Elevated in Human CKD MMP-7 is detectable and increased in the urine of animal models of CKD. 10 To assess the clinical relevance of this finding to human CKD, we measured urinary MMP-7 levels in a cohort of 102 patients with various CKDs and 20 healthy subjects by a specific ELISA. The diagnosis and demographic data of the patients are presented in Supplemental Tables 1 and 2. As shown in Figure 1A, urinary MMP-7 was markedly elevated in patients with various CKDs. Notably, the levels of urinary MMP-7 were closely correlated with the severity of kidney dysfunction (Figure 1B). In the patients who had impaired renal function (egfr,90 ml/min per 1.73 m 2 ), MMP-7 levels were increased dramatically compared with in healthy subjects or patients with relatively normal renal function (egfr.90 ml/min per 1.73 m 2 ) (Figure 1B). Serum MMP-7 levels were also slightly elevated in patients with CKD compared with healthy subjects. However, the magnitude of its elevation was trivial ( ng/ml in 68 patients with CKD versus ng/ml in 20 healthy subjects; P=0.03). We further examined renal expression of MMP-7 protein by immunohistochemical staining of kidney biopsy specimens. As shown in Figure 1C, MMP-7 protein was barely detectable in normal human kidneys (two patients tested). However, MMP- 7 was markedly induced in all 10 biopsy specimens from patients with CKD who were tested. Figure 1C shows the representative micrographs of MMP-7 staining in the kidney sections from patients with IgA nephropathy, membranous nephritis, FSGS, SLE, and thrombotic microangiopathy. MMP-7 protein was predominantly localized in renal tubular epithelium of the diseased kidneys (Figure 1C, yellow arrows in the enlarged areas). Other than tubular cells, glomerular podocytes were also positive for MMP-7 staining. These results suggest that renal MMP-7 expression is induced in patients with CKD, which ultimately leads to elevation of urinary MMP-7 levels. Urinary MMP-7 Levels Correlate with Kidney Fibrosis in Humans Because MMP-7 is the direct target of Wnt/b-catenin, a signaling pathway that plays a central role in renal fibrogenesis, 24,25 we reasoned that urinary MMP-7 levels may be used as a noninvasive surrogate biomarker for predicting and monitoring kidney fibrosis. To test this hypothesis, we assessed renal collagen deposition in kidney biopsy specimens of the patients with CKD by Masson trichrome staining (MTS). Figure 2A shows representative micrographs of the stained kidney biopsy sections, in which deposited collagen in the tubulointerstitium stains blue (Figure 2A, arrows). Urinary MMP-7 levels were given at the top of each respective micrograph in Figure 2A. Of the 30 patients with CKD who had biopsy specimens, there was a close correlation between urinary MMP-7 levels and kidney fibrotic score (Figure 2B). We also analyzed the correlation of MMP-7 expression with egfr in patients with CKD. As shown in Figure 2C, urinary MMP-7 inversely correlated with renal function. Similarly, urinary MMP-7 levels were also associated with serum creatinine (Figure 2D), serum levels of cystatin C (Figure 2E), and proteinuria (Figure 2, F and G). The correlation between urinary MMP-7 levels and renal function decline was particularly clear in the patients with renal impairment (egfr,90 ml/min per 1.73 m 2 ) (Supplemental Figure 1) but was not clear in the patients with relatively normal renal function (egfr.90 ml/min per 1.73 m 2 ) (Supplemental Figure 2). These data suggest that urinary MMP-7 may serve as a noninvasive biomarker for estimating the severity of kidney fibrosis and renal dysfunction in humans. 2 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc ccc, 2016

3 BASIC RESEARCH Figure 1. Urinary MMP-7 levels are elevated in human CKD. (A) Urinary MMP-7 levels in patients with CKD (n=102) and healthy subjects (n=20). Urinary MMP-7 levels were presented as nanograms per 1 mg urinary creatinine (Ucre.). Graph shows urinary MMP-7 value in individual patients with CKD and healthy subjects. *P,0.05. (B) Urinary MMP-7 levels inversely correlate with kidney function. Significant difference in urinary MMP-7 levels was observed in the patients with CKD with egfr.90 or,90 ml/min per 1.73 m 2.Ctrl, control. *P,0.05. **P,0.01 (C) Representative immunohistochemical staining shows an increased expression of MMP-7 protein in various human CKD. Box areas were enlarged to show the predominant tubular localization of MMP-7. Arrows indicate positively stained renal tubules. Control, nontumor kidney tissue from the patients who had renal cell carcinoma used as normal controls; IgAN, IgA nephropathy; MN, membranous nephritis; TMA, thrombotic microangiopathy. Scale bar, 50 mm. Endogenous MMP-7 Promotes Kidney Fibrosis To investigate the potential role of MMP-7 in the pathogenesis of renal fibrosis, we used MMP-7 knockout (KO) mice in which MMP-7 gene is globally deleted. Mice with MMP-7 ablation were phenotypically normal without overt physical or morphologic abnormalities. Age and sex matched wild type (WT; MMP- 7+/+) and MMP-7 null mice (MMP-72/2) in the same genetic background were subjected to unilateral ureteral obstruction (UUO) for 7 days. As shown in Figure 3A, MMP-7 protein was markedly induced in MMP-7+/+ mice at 7 days after UUO, predominantly in renal tubular epithelium. As expected, no MMP-7 protein was detected in MMP-72/2 mice after obstructive injury. Similar results were obtained when wholekidney lysates were examined by Western blot analysis (Figure 3, B and C). Of note, KO of MMP-7 did not affect renal expression of other MMP genes after UUO (Figure 3D). Other than MMP-7, several other MMPs, including MMP-3 (Figure 3E) and MMP-10 (Figure 3F) as well as MMP-2, MMP-4, MMP-8, MMP-9, and MMP-14 (data not shown), were induced in MMP-7+/+ mice at 7 days after UUO compared with sham controls, whereas MMP-13 expression was inhibited (Figure 3G). However, ablation of MMP-7 in the KO mice did not affect the expression of all other MMPs in the obstructed kidneys after UUO compared with WT controls (Figure 3, D G). We next examined renal fibrosis after obstructive injury in MMP-7 null mice and their WT counterparts. As shown in Figure 4, A and B, MTS revealed significant collagen deposition and renal interstitial fibrosis in the obstructed kidney of WT mice at 7 days after UUO (Figure 4A, arrows). However, ablation of MMP-7 in null mice substantially reduced renal fibrotic lesions (Figure 4, A and B). Analyses of gene expression by quantitative real time RT-PCR showed that type 1 collagen mrna expression was markedly induced in WT mice after UUO and that deletion of MMP-7 attenuated its induction (Figure 4C). Western blot analyses of whole-kidney lysates also revealed that the expression of fibronectin and a-smooth muscle actin (a-sma) proteins was decreased in the obstructed kidneys in MMP-72/2 mice compared with WT controls (Figure 4, D F). Ablation of MMP-7 also reduced the deposition of fibronectin in the obstructed kidneys at 7 days after UUO (Figure 4G). These data indicate that MMP-7 not only is a biomarker for kidney fibrosis but also, acts as a pathogenic mediator that promotes renal fibrosis. Endogenous MMP-7 Impairs Tubular Cell Integrity In Vivo Earlier in vitro studies show that MMP-7 proteolytically sheds the extracellular domain of E-cadherin, 10 leading to the impairment of epithelial cell integrity. Along this line, we explored the potential relationship between MMP-7 and E-cadherin in the obstructed kidneys in vivo. Asshownin Figure 5, A and B, Western blot analysis of whole-kidney J Am Soc Nephrol 28: ccc ccc, 2016 MMP-7 Is a Kidney Fibrosis Biomarker 3

4 BASIC RESEARCH Figure 2. Urinary MMP-7 levels correlate with kidney fibrosis in human CKD. (A) Representative micrographs show renal collagen deposition by MTS in kidney biopsies of the patients with CKD. Arrows indicate positive staining of collagen deposition (blue). Urinary MMP-7 levels were given at the top of each micrograph. (B) Scatter plot with linear regression shows a correlation between urinary MMP7 levels and kidney fibrosis. Fibrosis was quantitatively assessed on the MTS kidney sections by three individuals who were blinded to urinary MMP-7 data. (C) Linear regression shows an inverse correlation between urinary MMP-7 levels and kidney egfr. (D and E) Linear regression shows a correlation between urinary MMP-7 levels and (D) serum creatinine (Scr) and (E) serum cystatin C. (F and G) Linear regression shows a correlation between urinary MMP-7 levels and (F) total urine protein per 24 hours and (G) urine albumin per 24 hours. The Spearman correlation coefficient (rs) and P value are shown. Ucr, urinary creatinine. lysates revealed that loss of MMP-7 in KO mice resulted in an increased renal expression of E-cadherin protein at 7 days after UUO compared with WT controls. Immunofluorescence staining showed that, although renal E-cadherin protein 4 Journal of the American Society of Nephrology vanished after UUO in MMP-7+/+ mice, its expression and localization were largely preserved in MMP-72/2 mice (Figure 5C). Of interest, despite a marked increase of E-cadherin protein in MMP-72/2 mice, renal E-cadherin mrna levels J Am Soc Nephrol 28: ccc ccc, 2016

5 BASIC RESEARCH Figure 3. MMP-7 is induced in fibrotic kidneys after UUO, and its deficiency does not affect renal expression of other MMPs. (A) Representative immunohistochemical staining show an increased expression of MMP-7, predominantly in renal tubules of the obstructed kidneys, at 7 days after UUO in MMP-7+/+ mice but not in MMP-72/2 mice. Boxed areas were enlarged. Arrows indicate renal tubules. Scale bar, 50 mm. (B and C) Western blot analyses of renal MMP-7 protein in obstructive nephropathy. Kidney lysates were prepared from MMP-7+/+ (WT) and MMP-72/2 (KO) mice at 7 days after UUO and immunoblotted with specific antibody against MMP-7 and actin, respectively. Representative (B) Western blot and (C) quantitative data are presented. Both pro MMP-7 and active MMP-7 was shown. Numbers (1 and 2) indicate each individual animal in a given group (n=5). **P,0.01 versus WT. (D) RT-PCR analyses show that deletion of MMP-7 did not affect renal expression of other MMPs at 7 days after UUO. (E G) Quantitative real time RT-PCR analyses confirmed little difference in renal (E) MMP-3, (F) MMP-10, and (G) MMP-13 mrna expression between MMP-7+/+ and MMP-72/2 mice at 7 days after UUO. in these mice did not significantly differ from MMP-7+/+ controls (Figure 5D). These results suggest that the preservation of E-cadherin in MMP-72/2 mice after UUO primarily results from diminished proteolytic shedding of E-cadherin protein rather than an increased expression of its mrna. J Am Soc Nephrol 28: ccc ccc, 2016 Loss of E-cadherin would disrupt tubular epithelial cell integrity, which is often accompanied by acquisition of mesenchymal markers.10,26 Therefore, we also examined the expression of fibroblast-specific protein 1 (Fsp1), also known as S100A4, a cytoplasmic calcium binding protein that is MMP-7 Is a Kidney Fibrosis Biomarker 5

6 BASIC RESEARCH Figure 4. Endogenous MMP-7 promotes renal fibrosis after obstructive injury. (A and B) Ablation of MMP-7 in mice (MMP-72/2) ameliorated renal fibrosis after UUO. (A) Representative micrographs show the MTS of the obstructed kidneys at 7 days after UUO in various groups as indicated. Arrows indicate collagen deposition. Scale bar, 50 mm. Quantitative determination of renal fibrotic lesions in various groups is presented in B. Renal fibrotic lesions (defined as percentage of MTS positive fibrotic area) were quantified on MTS sections by computer aided morphometric analyses. *P,0.05 versus WT controls (n=5). (C) Quantitative real time RT-PCR analyses show a reduced mrna expression of type 1 collagen in MMP-72/2 mice at 7 days after UUO compared with MMP-7+/+ controls. *P,0.05 versus MMP-7+/+ controls (n=5). (D F) Western blot analyses show that deletion of MMP-7 reduced renal expression of fibronectin (Fn) and a-sma at 7 days after UUO. Representative (D) Western blot and (E and F) quantitative data are presented. Numbers (1 4) indicate each individual animals in a given group. *P,0.05 versus MMP-7+/+ (WT) controls (n=5). (G) Representative micrographs show Fn protein expression in various groups as indicated. Kidney sections were immunostained with specific antibody against Fn. Arrows indicate positive staining. Scale bar, 50 mm. activation. To test this hypothesis, we investigated the activation of b-catenin in the obstructed kidneys from both WT and MMP-7 KO mice. As shown in Figure 6, A, B, and D, b-catenin was dramatically upregulated in WT mice, predominantly in renal tubular epithelium, at 7 days after UUO, whereas genetic ablation of MMP-7 in KO mice abolished b-catenin induction in the obstructed kidneys. Similar results were obtained when the dephosphorylated active b-catenin was assessed (data not shown), suggesting that preservation of E-cadherin in MMP-7 null mice prevents renal b-catenin activation after obstructive injury. Double-immunofluorescence staining further confirmed colocalization of MMP-7 and b-catenin in renal tubular epithelium at 7 days after UUO in WTmice (Supplemental Figure 3). Recent studies have shown that activation of b-catenin plays a crucial role in promoting fibroblast activation and kidney fibrosis. 23,24 To examine this possibility, we examined the expression of vimentin, a marker for fibroblast activation. 3 As shown in Figure 6, A and C, genetic ablation of MMP-7 in KO mice largely abolished vimentin expression induced by obstructive injury. Of particular interest, immunohistochemical staining showed that vimentin largely localized to the tubular epithelium of diseased kidneys after UUO in WT mice (Figure 6D, arrows) but not in MMP-7 KO mice. Consistently, the expression of other fibrosis related proteins, such as Snail1 and plasminogen activator inhibitor-1 (PAI-1), was also inhibited in MMP-72/2 mice at 7 days after UUO compared with WT controls (Figure 6, E and F). These results suggest that ablation of MMP-7 substantially inhibits b-catenin activation and fibrosis related gene expression. highly expressed in fibroblasts and epithelial cells undergoing EMT. 27 As shown in Figure 5C, UUO caused substantial induction of Fsp1 in renal tubular epithelium in WTmice. However, ablation of MMP-7 in KO mice largely abolished tubular expression of Fsp1 after obstructive injury. Similar data were obtained when Fsp1 mrna was assessed (data not shown). Endogenous MMP-7 Promotes b-catenin Activation Because E-cadherin is physically associated with b-catenin in normal epithelial cells, MMP-7 mediated proteolytic degradation of E-cadherin could result in the dissociation of this complex, thereby leading to b-catenin liberation and MMP-7 Activates b-catenin Signaling In Vitro To elucidate the mechanism by which MMP-7 promotes renal fibrosis, we studied the b-catenin activation by MMP-7 in vitro by using cultured human kidney proximal tubular epithelial (HKC-8) cells, because mounting evidence suggests that Wnt/ b-catenin plays a key role in renal fibrogenesis. 24 As shown in Figure 7A, incubation of HKC-8 cells with recombinant MMP-7 protein dramatically diminished E-cadherin staining, presumably as a result of proteolytic shedding of its extracellular domains as reported previously. 10 Meanwhile, MMP-7 induced b-catenin redistribution, leading to its nuclear accumulation in HKC-8 cells (Figure 7A, arrows). Colocalization 6 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc ccc, 2016

7 BASIC RESEARCH Figure 5. Endogenous MMP-7 impairs tubular cell integrity in obstructive nephropathy. (A and B) Ablation of MMP-7 in mice preserved tubular E-cadherin protein. Renal E-cadherin protein expression was assessed by Western blot analyses at 7 days after UUO. Representative (A) Western blot and (B) quantitative data are presented. Numbers (1 4) indicate each individual animal in a given group. *P,0.05 versus WT (n=5). (C) Representative micrographs show renal expression of E-cadherin and Fsp1 in various groups as indicated. E-cadherin was assessed by immunofluorescence staining, whereas Fsp1 was detected by immunohistochemical staining. Arrows indicate positive staining. Scale bar, 50 mm. (D) Quantitative real time RT-PCR analyses show that ablation of MMP-7 in mice did not affect renal E-cadherin mrna expression at 7 days after UUO. *P,0.05 versus sham controls (n=5). of MMP-7 and b-catenin was also observed in the HKC-8 cells after stimulation with TGF-b1 (Supplemental Figure 4). We further examined b-catenin localization in different cellular compartments by Western blot analyses after subcellular fractionation. As shown in Figure 7, B and C, MMP-7 drastically induced the nuclear accumulation of b-catenin, whereas it concurrently decreased cytoplasmic b-catenin abundance. Accordingly, incubation with MMP-7 decreased E-cadherin/ b-catenin complex formation in HKC-8 cells as shown by coimmunoprecipitation (Figure 7D). Consistently, MMP-7 increased the binding of b-catenin to nuclear transcription factor lymphoid enhancer-binding factor 1 (LEF1) (Figure 7E). We further assessed the effect of MMP-7 on the expression ofb-catenin downstream target genes. As shown in Figure 7F, MMP-7 significantly promoted the mrna expression of b-catenin target genes, such as PAI-1 and MMP-7 itself. This action of MMP-7 was clearly dependent on its proteolytic activity, because an MMP-7 specific inhibitor (MMP inhibitor 2) but not an MMP-2/MMP-9 inhibitor (GM6001) completely abolished the mrna induction of PAI-1 and MMP-7 (Figure 7F). Similar results were obtained when MMP-7 and PAI-1 proteins were assessed by Western blot analyses (Figure 7G). Taken together, as shown in Figure 7H, MMP-7, via its proteolytic activity, degrades E-cadherin, which liberates b-catenin and induces its translocation into the nuclei where it binds to LEF1 and drives the expression of its target genes. Activation of b-catenin by MMP-7 Is Independent of Wnt Because b-catenin is the chief intracellular mediator of canonical Wnt signaling, we next tested whether Wnt is involved in MMP-7 mediated b-catenin activation by using a TOPFlash luciferase reporter assay. As shown in Figure 8A, incubation of HKC-8 cells with MMP-7 activated b-catenin and induced luciferase reporter activity as expected. Treatment of HKC-8 cells with ICG-001, a small molecule inhibitor that blocks b-catenin mediated gene transcription, 28,29 effectively abrogated MMP-7 induced gene transcription. However, we found that soluble Klotho (sklotho), an endogenous Wnt antagonist that binds to and sequesters Wnt ligands, 30 did not significantly affect MMP-7 mediated luciferase expression, suggesting that MMP-7 mediated expression of b-catenin target genes does not require Wnt participation. Similarly, J Am Soc Nephrol 28: ccc ccc, 2016 MMP-7 Is a Kidney Fibrosis Biomarker 7

8 BASIC RESEARCH Figure 6. Endogenous MMP-7 activates b-catenin and its downstream target genes in obstructive nephropathy. (A C) Western blot analyses show a reduced b-catenin protein and vimentin expression in MMP-72/2 mice at 7 days after UUO compared with MMP-7+/+ controls. Representative (A) Western blot and (B and C) quantitative data are presented (n=5). Numbers (1 4) indicate each individual animal in a given group. *P,0.05 versus WT controls. (D) Representative micrographs show renal expression of b-catenin and vimentin in various groups as indicated. The b-catenin and vimentin proteins were detected by immunohistochemical staining. Arrows indicate positive staining in renal tubules. Scale bar, 50 mm. (E and F) KO of MMP-7 also inhibited PAI-1 and Snail1 expression. Quantitative data showing renal (E) PAI-1 and (F) Snail1 protein levels at 7 days after UUO are presented. *P,0.05 versus WT controls (n=5). the expression of b-catenin target genes induced by MMP-7 could be inhibited by ICG-001 but could not be inhibited by sklotho (Figure 8, B and C). In addition, MMP-7 could induce a-sma expression in HKC-8 cells, which was abolished by ICG-001 and MMP inhibitor 2 (Figure 8). Taken together, these results suggest that MMP-7 activates b-catenin by a Wnt-independent mechanism. Pharmacologic Inhibition of MMP-7 Attenuates Renal Fibrosis In Vivo In view of the important role of endogenous MMP-7 in the evolution of renal fibrosis, we finally investigated whether inhibition of MMP-7 can reduce fibrotic lesions after obstructive injury. To this end, mice were injected intraperitoneally with MMP inhibitor II, a small molecule inhibitor of MMP-7, immediately after UUO. As shown in Figure 9, A and B, inhibition of MMP-7 substantially repressed renal expression of collagen 1 mrna at 7 days after UUO compared with vehicle controls. Similarly, inhibition of MMP-7 in vivo also reduced protein expression of fibronectin and a-sma in the obstructed kidneys as revealed by Western blot analyses of whole-kidney lysates (Figure 9, C and D). This inhibition of renal fibrosis by an MMP-7 inhibitor was associated with reduction of b-catenin in vivo (Figure 9, C and D). Immunofluorescence staining also showed an increased deposition of fibronectin and collagen 1 proteins in the obstructed kidneys at 7 days after UUO, which was substantially reduced by inhibition of MMP-7 (Figure 9E). These results suggest that MMP-7 is a novel therapeutic target as well, and its inhibition confers renal protection against development of kidney fibrosis. DISCUSSION At present, assessment of kidney fibrosis in patients with CKD requires renal biopsy specimens, which are obtained only by an 8 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc ccc, 2016

9 BASIC RESEARCH Figure 7. MMP-7 activates b-catenin by triggering E-cadherin degradation in vitro. (A)Immunofluorescence staining shows that exogenous MMP-7 degraded E-cadherin and promoted b-catenin nuclear translocation in vitro. HKC-8 cells were incubated with recombinant MMP-7 protein as indicated. Cells were then immunostained for b-catenin and E-cadherin. Cell nuclei were visualized by 49,6-Diamidino-2-Phenylindole (DAPI) staining. Arrows indicate positive b-catenin staining. (B and C) MMP-7 induced b-catenin nuclear translocation. (B) Nuclear and (C) cytoplasmic proteins were prepared by subcellular fractionation from HKC-8 cells incubated without or with MMP-7 as indicated. The (B) nuclear and (C) cytoplasmic lysates were immunoblotted with antibodies against b-catenin, TATA binding protein (TBP), or pan-specific actin. c-b-catenin, Cytoplasmic b-catenin; Ctrl, control; n-b-catenin, nuclear b-catenin. (D and E) Coimmunoprecipitation shows that MMP-7 reduced b-catenin/e-cadherin association and promoted b-catenin/lef1 complex formation. The lysates from HKC-8 cells incubated without or with MMP-7 were immunoprecipitated (IP) with antibodies against E-cadherin or LEF1 followed by immunoblotting (IB) with antibodies against b-catenin, E-cadherin, or LEF1 as indicated. (F and G) The enzymatic activity is required for MMP-7 to induce b-catenin target genes in vitro. (F) RT-PCR analyses and (G) Western blot showed that MMP-7 induced the mrna and protein expression of the b-catenin downstream target genes MMP-7 and PAI-1, which were blocked by MMP inhibitor II but not GM6001. (H) Schematic diagram depicts the potential pathway by which MMP-7 activates b-catenin. TCF, T cell factor. invasive procedure. 5,6 As such, it is extremely difficult, if not impossible, to monitor the dynamics of the fibrotic process, which may predict patient outcomes and allow for the evaluation of treatment efficacy in clinical settings. In this study, we show that urinary MMP-7 levels are closely correlated with the severity of renal fibrotic lesions, thereby providing a noninvasive, surrogate biomarker for kidney fibrosis in patients with CKD. Furthermore, using MMP-72/2 KO mice, in which the endogenous MMP-7 gene is completely deleted, we have provided unambiguous evidence that MMP-7 is an important pathogenic mediator that triggers the activation of b-catenin signaling and promotes renal fibrosis. Finally, we show that pharmacologic inhibition of MMP-7 activity by a small molecule inhibitor protects kidneys against development of fibrotic lesions in vivo. These studies establish that MMP-7, a secreted protease and a transcriptional target of Wnt/b-catenin signaling, not only can serve as a noninvasive biomarker but also, is a pathologic mediator and therapeutic target of kidney fibrosis. MMP-7 is one the smallest MMPs, predominantly localizes in renal tubular epithelium, and can be easily excreted into urine. Under pathologic conditions, MMP-7 expression is dramatically induced (Figures 1 and 3), which is primarily controlled at the transcriptional level by Wnt/b-catenin, 10 a signaling pathway that is activated in virtually all kinds of CKDs. 19,20 We have J Am Soc Nephrol 28: ccc ccc, 2016 MMP-7 Is a Kidney Fibrosis Biomarker 9

10 BASIC RESEARCH Figure 8. b-catenin activation induced by MMP-7 is independent of Wnt signaling. (A) MMP-7 activates b-catenin mediated gene transcription by a Wnt-independent mechanism. HKC-8 cells were cotransfected with TOP-Flash reporter plasmid in the absence or presence of secreted Klotho expression plasmid (pv5-sklotho) or ICG-001. Luciferase activity was assessed and reported as fold induction over the controls. *P,0.05 versus controls; P,0.05 versus MMP-7 alone or MMP-7 plus sklotho (n=3). (B) RT-PCR analyses show that MMP-7 mrna expression induced by MMP-7 was not inhibited by sklotho. (C) Western blot analyses show that blockade of Wnt signaling by sklotho did not abolish MMP-7 mediated autoinduction. Cell lysates were immunoblotted with antibodies against MMP-7 and a-tubulin. (D) Representative micrographs show that MMP-7 induced a-sma in HKC-8 cells. Cells were immunostained for a-sma after various treatments as indicated. Arrows indicate positive staining. previously shown that urinary MMP-7 is a marker for predicting intrarenal Wnt/b-catenin activity in animal models of CKD. 10 Consistent with this notion, urinary MMP-7 levels are also elevated in patients with CKD, regardless of the initial etiology (Figure 1). Furthermore, urinary MMP-7 dependably mirrors its expression in renal parenchyma, particularly in tubular epithelium (Figure 1). It is of interest to point out that urinary MMP-7 is already elevated substantially in patients when renal impairment is mild (egfr.90 ml/min per 1.73 m 2 ) and free of robust tissue fibrosis. Therefore, it is conceivable to speculate that MMP-7 in the urine could be an early biomarker that precedes fibrotic lesions. The limitations of this study include the relatively small sample size and the nature of a cross-sectional observation, which precludes the longitudinal assessment of the correlation between MMP-7 and kidney fibrosis after various treatments. Clearly, more vigorous, large scale, and longitudinal studies on the validation of MMP-7 as a noninvasive biomarker for kidney fibrosis are warranted in the future. One of the novel findings in this study is that MMP-7 is not only a biomarker for renal fibrosis but also, a major pathogenic mediator in the evolution of fibrotic lesions. Mice with genetic ablation of MMP-7 are largely protected against development of renal fibrotic lesions after UUO, with much less accumulation and deposition of collagen 1 and fibronectin in renal parenchyma and reduced myofibroblast activation (Figure 4). This result is in harmony with earlier observations that MMP-9 is deleterious in renal fibrogenesis. 31,32 Although MMPs are often presumed to be beneficial by promoting matrix degradation in fibrotic disorders, increasing evidence suggests that these proteases can affect the fibrotic process either positively or negatively by targeting different sets of substrate proteins for degradation. 11,17 It is worthwhile to point out that, because MMP-7 is a downstream target of Wnt/b-catenin, our findings on the detrimental role of endogenous MMP-7 underscore a novel mechanism by which Wnt/b-catenin signaling promotes renal fibrosis. The molecular mechanism behind the pathogenic action of MMP-7 is most likely related to its ability to promote EMT by degrading E-cadherin, an adherence receptor that is essential for the maintenance of epithelial cell integrity. KO of MMP-7 does not affect E-cadherin mrna expression but substantially preserves E-cadherin protein and its tubular localization after UUO (Figure 5), consistent with earlier in vitro observations that MMP-7 induces E-cadherin ectodomain shedding. 10,13 Loss of epithelial E-cadherin has been proposed as the initial step in EMT, 26 and therefore, MMP-7 mediated E-cadherin degradation is expected to promote tubular epithelial cell phenotypic transition. Indeed, de novo expression of Fsp1 and vimentin, two mesenchymal markers that are often used in EMT field, 26,27 is evident in tubular epithelium in MMP-7+/+ mice but not in MMP-72/2 mice (Figures 5 and 6). These data indicate that tubular epithelial cells in MMP-7+/+ mice 10 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc ccc, 2016

11 BASIC RESEARCH Figure 9. Pharmacologic inhibition of MMP-7 attenuates renal fibrosis after obstructive injury. (A, and B) Injections of MMP-7 inhibitor repressed renal collagen 1 mrna expression at 7 days after UUO. (A) RT-PCR and (B) quantitative data are presented (n=6 7). Numbers (1 7) indicate each individual animal in a given group. *P,0.05 versus sham controls; P,0.05 versus UUO controls. (C and D) Western blot analyses show that inhibition of MMP-7 repressed renal fibronectin, a-sma, and b-catenin protein expression at 7 days after UUO. Representative (C) Western blot and (D) quantitative data are presented. Numbers (1 3) indicate each individual animal in a given group. *P,0.05 versus sham controls; P,0.05 versus UUO controls (n=6 7). (E) Representative micrographs show that inhibition of MMP-7 reduced renal expression of fibronectin and a-sma proteins at 7 days after UUO. Cells were immunostained for fibronectin and a-sma after various treatments as indicated. Arrows indicate positive staining. Scale bar, 50 mm. after UUO at least undergo a partial EMT, in which they express markers of both epithelial and mesenchymal cells but remain in the tubular compartment. 33,34 It becomes clear that endogenous MMP-7 plays a role in this partial EMT process, because its deficiency preserves tubular cell integrity (Figures 5 and 6). Of note, although it remains controversial whether there is a complete EMT in fibrotic kidneys, recent studies have provided evidence for partial EMT phenotypic changes. 33,34 Such a partial EMT is crucial and required for driving fibrosis development after various injuries. 38 One consequence of MMP-7 mediated E-cadherin degradation is the liberation of b-catenin, because it normally associates with E-cadherin in the cellular adhesion complex. The liberated b-catenin then translocates into the nucleus as a transcription regulator and drives the expression of its target genes, such as PAI-1 and MMP-7 itself. 10,39 This series of events can be recapitulated in vitro, because MMP-7 causes an increased b-catenin/lef1 interaction and activates MMP-7 and PAI-1 mrna expression (Figure 7). As depicted in Figure 7G, by virtue of the ability to induce its own expression, MMP-7 contributes to a self-propagation cycle that leads to autoamplification of b-catenin signaling. As expected, this mode of b-catenin activation requires the protease activity of MMP-7 but is independent of Wnt ligands (Figure 8). Therefore, our studies highlight that, apart from Wnt ligands, endogenous MMP-7 is an important regulator that mediates the activation and amplification of b-catenin signaling in diseased kidneys. Our studies also indicate that MMP-7 could be a therapeutic target for the treatment of fibrotic CKD. Using a selective small molecule MMP-7 inhibitor, we show that blockade of MMP-7 activity inhibits renal expression of collagen 1 and fibronectin and reduces myofibroblast activation (Figure 9). Although more studies are needed, taken together, our results provide proof of principle that MMP-7 not only can serve as a noninvasive biomarker but also, is a pathogenic mediator and therapeutic target of kidney fibrosis. CONCISE METHODS Human Urine and Kidney Biopsies Samples Human urine and serum samples as well as kidney specimens were obtained from diagnostic renal biopsies performed at the Nanfang Hospital, Southern Medical University. Some urine and serum samples were also obtained from healthy volunteers. Urine samples, after centrifugation to remove urinary debris, were aliquoted and stored at 280 C. Paraffin embedded human kidney biopsy sections (2.5-mm thickness) were prepared using a routine procedure. Nontumor kidney tissue from the patients who had renal cell carcinoma and underwent nephrectomy was used as normal controls. All studies involving human kidney samples were approved by the Ethics Committee at the Southern Medical University and the Institutional Review Board at the University of Pittsburgh. Animal Models MMP-7 KO mice with C57BL/6J genetic background were obtained from The Jackson Laboratory (Stock ; Bar Harbor, ME). Age and sex matched C57BL/6J mice from the same vendor were used as WT controls. UUO was performed by using an established protocol as described elsewhere. 24 Sham-operated mice had their ureters exposed and manipulated but not ligated. Mice were killed at 7 days after UUO (n=5), and the kidney tissues were collected for various analyses. For assessing the therapeutic efficacy of MMP-7 inhibition on kidney fibrosis, three groups of mice were used: (1) sham control (n=6), (2) UUO injected with vehicle (n=7), and (3) UUO injected J Am Soc Nephrol 28: ccc ccc, 2016 MMP-7 Is a Kidney Fibrosis Biomarker 11

12 BASIC RESEARCH with MMP inhibitor II (n=6). Mice were daily injected intraperitoneally with vehicle (PBS) or MMP inhibitor II (444247; EMD Chemicals, Gibbstown, NJ) at 1 mg/kg body wt starting from the second day after surgery. Mice were euthanized at 7 days after UUO, and kidney tissues were analyzed. All animal studies were performed by use of the procedures approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and the Animal Ethics Committee at the Nanfang Hospital of the Southern Medical University. Cell Culture and Treatment The HKC-8 cell line was provided by L. Racusen of the Johns Hopkins University (Baltimore, MD). Cells were cultured in DMEM/F12 medium supplemented with 10% calf serum as described previously. 40 Serum starved HKC-8 cells were stimulated by human recombinant MMP-7 protein (444270; EMD Chemicals) at varying dosages in the absence or presence of different inhibitors, such as ICG-001-phosphate (provided by M. Kahn, University of Southern California, Los Angeles, CA), GM6001 (CC1000; EMD Millipore, Billerica, MA), and MMP inhibitor II (444247; EMD Chemicals). The cells were then subjected to immunofluorescence staining, RT- PCR, immunoprecipitation, and Western blot analyses. MMP-7 ELISA The Human Total MMP-7 Quantikine ELISA Kit was purchased from R&D Systems (DMP700; Minneapolis, MN). This assay uses the quantitative sandwich enzyme immunoassay technique. An mab specific for human MMP-7 was precoated onto a microplate. Standards and samples were pipetted into the wells, and MMP-7 present in a sample was bound to the wells by the immobilized antibody. After washing away unbound substances, an enzyme linked polyclonal antibody specific for human MMP-7 was added to the wells. After a wash to remove unbound antibody-enzyme reagent, a substrate solution was added to the wells, and color developed in proportion to the amount of total MMP-7 (pro and/or active) bound in the initial step. The color development was stopped, and the intensity of the color was measured. Urine MMP-7 levels were calculated after normalized to urinary creatinine and expressed as nanograms per 1 mg creatinine. Serum MMP-7 was also assayed and expressed as nanograms per milliliter. Semiquantitation Scoring of MTS Kidney biopsies from patients with CKD with different etiologies were subjected to MTS by routine procedures. Stained slides were observed under a microscope, and images from randomly selected fields were taken. Each image (3400; cm [length] cm [width]) was split into 100 squares, with the area of each square being 3.77 cm 2. Tissue fibrosis as defined by blue staining was scored by three experienced observers in a blinded fashion. The percentage of blue staining in each image was calculated, and the mean values of the fibrosis scores from three observers were reported. Histology and Immunohistochemical Staining Paraffin embedded mouse kidney sections (3-mm thickness) were prepared by a routine procedure. The sections were stained with Masson trichrome reagent. Immunohistochemical staining was performed using routine protocols. Antibodies used were as follow: rabbit polyclonal anti MMP-7 (GTX11603; GeneTex, Irvine, CA), mouse monoclonal anti MMP-7 (GTX17854; GeneTex), rabbit polyclonal antivimentin (5741s; Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti FSP1 (S100A4; A5114; DAKO, Carpinteria, CA), rabbit polyclonal anti b-catenin (ab15180; Abcam, Inc., Cambridge, MA), and mouse anti a-sma (A2547; Sigma-Aldrich, St. Louis, MO). After incubation with primary antibodies at 4 C overnight, the slides were then stained with Horseradish Peroxidase conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Nonimmune normal IgG was used to replace primary antibodies as a negative control, and no staining occurred. Slides were viewed under a Nikon Eclipse E600 Microscope equipped with a digital camera (Nikon, Tokyo, Japan). Immunofluorescence Staining and Confocal Microscopy Kidney cryosections were fixed with 3.7% paraformalin for 15 minutes at room temperature. HKC-8 cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 10 minutes at 220 C. After blocking with 10% donkey serum for 1 hour, the slides were immunostained with primary antibodies against fibronectin (F3648), a-sma (A2547; Sigma- Aldrich), collagen type 1 (234168; EMD Millipore), E-cadherin (3195; Cell Signaling Technology), and b-catenin (ab15180; Abcam, Inc.). These slides were then stained with Cy2 or Cy3 conjugated secondary antibody (Jackson ImmunoResearch Laboratories). For showing MMP- 7 and b-catenin colocalization, double-immunofluorescence staining on kidney cryosections and cultured HKC-8 cells was performed. The stained slides were viewed under a Nikon Eclipse E600 Microscope (Nikon) or a Leica TCS-SL Confocal Microscope (Leica Microsystems, Buffalo Grove, IL). Western Blot Analyses Protein expression was analyzed by Western blot analysis as described previously. 41 The primary antibodies used were as follows: anti MMP-7 (GTX11603; GeneTex), antiactive b-catenin (05 665; EMD Millipore), anti b-catenin (610154; BD Transduction Laboratories, San Jose, CA), anti PAI-1(sc-5297;SantaCruzBiotechnology,SantaCruz,CA),anti- Snail1 (ab17732; Abcam, Inc.), antifibronectin (F3648), anti a-sma (A2547), antivimentin (V2258), anti a-tubulin (T9026; Sigma-Aldrich), anti E-cadherin (3195; Cell Signaling Technology), anti-lef1 (2230; Cell Signaling Technology), and mouse monoclonal antipan specific actin (MAB1501; EMD Millipore). Coimmunoprecipitation The interaction of b-catenin with E-cadherin or LEF1 in HKC-8 cells was determined by coimmunoprecipitation as previously described. 30 Cell lysates were immunoprecipitated overnight at 4 C with anti Ecadherin antibody (610182; BD Transduction Laboratories), anti- LEF1 (2230; Cell Signaling Technology), and protein A/G plus agarose (sc-2003; Santa Cruz Biotechnology). The precipitated complexes were washed with lysis buffer and boiled for 5 minutes in SDS sample buffer followed by immunoblotting with anti b-catenin (610154; BD Transduction Laboratories), anti E-cadherin (3195; Cell Signaling Technology), or anti-lef1 antibody (2230; Cell Signaling Technology,), respectively. An aliquot of cell lysates was also immunoblotted with 12 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc ccc, 2016

13 BASIC RESEARCH anti a-tubulin antibody (T9026; Sigma-Aldrich) to confirm equal amount of cellular proteins (input) used. Nuclear and Cytoplasmic Fractionation HKC-8 cells were harvested and washed twice with cold PBS. Nuclear and cytoplasmic protein was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the protocols specified by the manufacturer (Thermo Fisher Scientific, Vernon Hills, IL). The nuclear protein was normalized with mouse monoclonal anti TATA binding protein antibody (ab818; Abcam, Inc.). Real Time RT-PCR Total RNA isolation and quantitative real time RT-PCR were carried out by the procedures described previously. 30 Briefly, the first strand cdna synthesis was carried out by using a Reverse Transcription System Kit according to the instructions of the manufacturer (Promega, Madison, WI). Real time RT-PCR was performed on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) as described previously. 30 The PCR reaction mixture in a 25-ml volume contained 12.5 ml 23 SYBR Green PCR Master Mix (Applied Biosystems), 5 ml diluted reverse transcription product (1:10), and 0.5 mm sense and antisense primer sets. PCR reaction was run by using standard conditions. After sequential incubations at 50 C for 2 minutes and 95 C for 10 minutes, the amplification protocol consisted of 40 cycles of denaturing at 95 C for 15 seconds, annealing, and extension at 60 C for 60 seconds. The standard curve was made from series dilutions of template cdna. The mrna levels of various genes were calculated after normalizing with b-actin. Primer sequences used for amplifications are presented as Supplemental Table 3. Transfection and Luciferase Assay The effect of MMP-7 on b-catenin mediated gene transcription was assessed by using the TOPFlash reporter plasmid containing two sets of three copies of the T cell factor binding site in the upstream of the thymidine kinase (TK) minimal promoter and luciferase cdna (EMD Millipore). HKC-8 cells were transfected using Lipofectamine 2000 Reagent with TOPFlash plasmid and incubated with human recombinant MMP-7 protein in the absence or presence of ICG- 001 as indicated. Some cells were cotransfected with sklotho expression plasmid (pv5-sklotho). An internal control plasmid (0.1 mg) Renilla reniformis luciferase driven under TK promoter (prl-tk; Promega) was also cotransfected for normalizing the transfection efficiency. Luciferase assay was performed using a dual luciferase assay system kit according to the manufacturer s protocols (Promega). Relative luciferase activity (arbitrary units) was reported as fold induction over the controls after normalizing for transfection efficiency. Statistical Analyses All data examined were expressed as means6sem. Statistical analyses of the data were carried out using SigmaStat software (Jandel Scientific, San Rafael, CA). Comparison between groups was made using one-way ANOVA followed by the Newman Keuls test. Spearman (nonparametric) correlation analysis was used to assess the relationship between urinary MMP-7 and other variables. P,0.05 was considered significant. ACKNOWLEDGMENTS We thank the Center for Biologic Imaging at the University of Pittsburgh for the use of their core facilities. This work was supported by the National Institutes of Health grants DK064005, DK091239, and DK106049; National Natural Science Foundation of China grants and ; Guangdong Science Foundation Innovative Group grant 2014A ; and Guangzhou Research Fund R.J.T. was supported by American Heart Association Fellow-to-Faculty transition grant 13FTF DISCLOSURES None. REFERENCES 1. Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, Van Lente F, Levey AS: Prevalence of chronic kidney disease in the United States. JAMA 298: , Zhang L, Wang F, Wang L, Wang W, Liu B, Liu J, Chen M, He Q, Liao Y, Yu X, Chen N, Zhang JE, Hu Z, Liu F, Hong D, Ma L, Liu H, Zhou X, Chen J, Pan L, Chen W, Wang W, Li X, Wang H: Prevalence of chronic kidney disease in China: A cross-sectional survey. Lancet 379: , Liu Y: Cellular and molecular mechanisms of renal fibrosis. Nat Rev Nephrol 7: , Boor P, Ostendorf T, Floege J: Renal fibrosis: Novel insights into mechanisms and therapeutic targets. 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