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Pericyte TIMP3 and ADAMTS1 Modulate Vascular Stability after Kidney Injury

†‡ †‡ †‡ ‡ Claudia Schrimpf,* Cuiyan Xin, Gabriella Campanholle, Sean E. Gill, William Stallcup,§ | ‡ Shuei-Liong Lin, George E. Davis,¶ Sina A. Gharib, Benjamin D. Humphreys,* †‡ and Jeremy S. Duffield*

*Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; †Division of Nephrology and ‡Center for Lung Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington; §Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California; |National Taiwan University Hospital, Taipei, Taiwan; and ¶Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri

ABSTRACT Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased ap- proach to identify in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a and metalloprotease with motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rar- efaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.

J Am Soc Nephrol 23: 868–883, 2012. doi: 10.1681/ASN.2011080851

Recent studies have identified pericytes of the peri- (ECs) to stimulate vascular basement membrane tubular capillaries of the kidney as a major precursor matrix assembly.5,6 Each tissue bed has pericytes de- population of scar-forming myofibroblasts,1,2 and rived from organ-specified mesenchyme,7 and thus studies from embryonic development and cancer there are likely to be organ-specific differences in growth demonstrate vital angiogenic and vascular pericyte functions that are poorly understood. The stabilizing functions for pericytes.3,4 Prompted by these studies, we sought to understand the role of kidney pericytes in homeostasis and the molecular Received August 24, 2011. Accepted January 9, 2012. mechanisms regulating pericyte attachment to Published online ahead of print. Publication date available at capillaries in the kidney and detachment and migra- www.jasn.org. tion from capillaries in response to injury. Correspondence: Dr. Jeremy S. Duffield, Department of Ne- Pericytes are mesenchyme-derived mural cells of phrology and Lung Biology, Institute for Stem Cell and Re- capillaries that have processes embedded within generative Medicine, University of Washington, 815 Mercer capillary basement membrane and they may play Street, Seattle, WA 98105. Email: [email protected] a direct role in conjunction with endothelial cells Copyright © 2012 by the American Society of Nephrology

868 ISSN : 1046-6673/2305-868 J Am Soc Nephrol 23: 868–883, 2012 www.jasn.org BASIC RESEARCH direct interaction of pericyte processes with endothelium is be- RESULTS lieved to be the location of cell: cell signaling.5 Pericytes of the kidney derive during embryogenesis from a layer of embryonic Microarray Analyses of Pericytes in Early Injury cap metanephric mesenchyme lying outside of the mesenchyme Responses in Kidney Identify Enriched Pathways and that specifies epithelium. This mesenchyme activates the fork- Regulated Genes head transcription factor Foxd1, specifically during embryonic To explore mechanisms that orchestrate attachment and de- specification.8 In normal adult kidney, pericytes are Foxd1- tachment of pericytes, we used an unbiased approach to identify derived, PDGF receptor b (PDGFRb+), CD73+, Coll1a1+, regulated genes and activated pathways. Most biologic processes CD45-, a smooth muscle actin (aSMA-) cells.2,9 entail coordinated interaction among functionally coherent During angiogenesis in other organs, heparan sulfate– groups of genes known as modules.21 We postulated that peri- tethered PDGF-BB released by tip ECs is thought to signal cyte differentiation is characterized by the temporal activation of via PDGFRb to promote pericyte migration along the form- distinct biologic modules and that key regulators of this process ing capillary and stable attachment to endothelial cells.10 reside within these coherent pathways. We purified pericytes However, signaling via pericyte PDGFRb in formed kidney from normal Coll-GFP kidneys (day 0) by flow cytometry and vessels, in response to microvascular injury, stimulates pericyte at early time points after onset of injury (day 2 and day 7) in the detachment, migration, and differentiation into myofibro- murine unilateral ureteral obstruction (UUO) model in which blasts.9,11 Similarly, vascular endothelial growth factor A pericyte detachment is well defined, using well established meth- (VEGFA) signaling from pericytes to angioblasts in organ de- ods of cell purification1,22–24 (Supplemental Figure 1) and as- velopment is important in successful angiogenesis; however, sessed their transcriptome by microarray analysis. The Coll-GFP in response to injury, expression of alternate VEGFA splice reporter mouse used in this disease model specifically labels variant by kidney pericytes stimulates pericyte detachment pericytes and myofibroblasts, and there are no significant and differentiation into myofibroblasts.9,12 Therefore, it is fibrocytes contaminating the collection (Supplemental Fig- likely that multiple signaling pathways and extracellular inter- ure 1).1,22–24 In addition to pericytes, small numbers of peri- actions that are poorly explored are important in determining vascular fibroblasts are collected because they also express pericyte attachment, migration, and activation. Coll-GFP, but these represent ,5% of all cells expressing GFP The kidney is particularly susceptible to ischemic and toxic under regulation of the collagen1a1 promoter (Coll-GFP+ injuries, due in part to the concentrating functions of the cells) in normal kidney.1 Principal component analysis of the nephron and to its unusual vasculature. The kidney vascula- transcriptomes revealed grouping of biologic replicates that ture comprises a primary capillary network in the glomer- corresponded to the time of injury, implying global perturba- uli specialized for filtration followed by a secondary capillary tions in temporal expression as pericytes detach and dif- plexus that supplies the remainder of the kidney, resorbs sol- ferentiate into myofibroblasts (Supplemental Figure 2A). More utes, and uptakes water being returned to the circulation. The than 800 genes were differentially expressed when control peri- specialized functions of this secondary capillary plexus, peri- cytes (day 0) were compared with postinjury myofibroblasts tubular capillaries (PTCs), are facilitated by its low oxygen (days 2 and 7) (Supplemental Figure 2B and Supplemental tension, with low flow. Moreover, the secondary plexus is at the Table 1). To elucidate the temporal patterns of gene expres- vagaries oftheglomerular plexus.Becausethe kidneycortexand sion and pathway activation in response to injury, we im- medulla perform complex transport functions, they have plemented a clustering algorithm optimized for analysis of significant oxygen and nutrient demands. Hence, the kidney short time series25 and identified two highly significant gene is susceptible to ischemia if PTC flow/supply is insufficient. clusters—each mapping to very distinct functional categories Subtle kidney ischemia due to loss of PTCs is widely believed (Figure 1). One cluster corresponded to upregulated genes and to be a major cause of essential hypertension and a major driv- was highly enriched in processes involved in matrix production, ing force in the progression of CKD and in the failure of AKI cell proliferation, migration, innate immune responses, and che- to resolve.13–15 motaxis. In addition, genes mapping to angiogenesis and vascu- Previous studies, particularly in cancer growth and wound lar development were members of this upregulated group. The repair, have implicated proteinase-mediated degradation of second cluster was composed of genes downregulated during the capillary basement membrane and surrounding extracel- injury, and was functionally enriched in processes involved in lular matrix as critical for vascular instability and vascular re- mitochondrial function, redox status, and metabolism. A small gression (rarefaction).16,17 Moreover, proteinases and factors number of genes regulating extracellular matrix production and that regulate their activity are now recognized to serve as key angiogenesis were also downregulated. A disintegrin and metal- regulators of cell migration and spreading in cancer biology loproteinase with thrombospondin motif-1 (ADAMTS1) was and development, model systems that may be instructive in highly significantly upregulated early after injury onset and studying kidney injury.18–20 among the 15 most upregulated genes early after injury in peri- In this study, we investigated the role of kidney pericytes in cytes (Figure 1 and Supplemental Figure 2C). Moreover, it was a vascular stabilization and the central role that pericytes play in strong candidate effector molecule in regulating detachment and regulating activity of the vasculature. migration. ADAMTS1 has been reported to cleave capillary

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Figure 1. Global analysis of kidney pericyte transcriptome in response to UUO injury. Temporal changes in map to two distinct clusters characterized by progressively increasing or decreasing patterns. In each graph, the dark gray line corresponds to the average change in gene expression in response to injury relative to baseline expression (day 0) and the light gray lines identify the 90th and 10th percentiles of each group. The temporal expression patterns of two genes, Adamts1 and Timp3, are depicted with red lines. Gene members of each cluster underwent functional analysis based on database annotations. Enriched functional categories among upregulated and downregulated genes are shown using color-coded bars corresponding to enrichment P values with the most highly significant shown in red and the least significant shown in dark blue. basement membrane proteins and inhibit angiogenesis and there- (MMPs) such as MMP14, and has been reported to regulate vas- fore may reasonably facilitate pericyte detachment.26,27 Tissue culogenesis and angiogenesis and to promote tissue regeneration inhibitor of 3 (TIMP3), an endogenous after injury in the lung.29–31 Thus, the downregulation of TIMP3 inhibitor of ADAMTS1, is highly expressed at baseline in peri- as pericytes transition to myofibroblasts in vivo may have impor- cytes28 but is significantly downregulated as disease progresses tant consequences for the kidney microvasculature. Given the (Figure 1 and Supplemental Figure 2C). TIMP3 has been dem- marked regulation of these two functionally related genes and onstrated or implicated in silencing the activity of metalloprotein- candidacy as regulators of pericyte function in response to injury, ases including ADAM17 and some matrix we aimed to study their function in kidney pericytes.

870 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 868–883, 2012 www.jasn.org BASIC RESEARCH

Kidney Pericytes Prevent Capillary Tubular Network number of metalloproteinases that were regulated in pericytes Collapse in response to injury (Table 1), Adamts1 transcripts were To study pericyte function, we generated primary pure cultures highly regulated early and most markedly. We postulated by magnetic bead immunoaffinity from mouse kidneys. Primary that the dynamic regulation of these kidney pericyte genes mouse pericytes expressed typical pericyte markers (Figure 2, A served as important determinants of the detachment of peri- and B) and morphology, which share characteristics with bone cytes and vascular instability that occurs in response to kidney marrow mesenchymal stromal cells and lacked epithelial, endo- injury.1,9,40 To validate the microarray findings, we performed thelial, and leukocyte markers and lacked the podocyte markers in situ hybridization and immunofluorescence studies to lo- WT1 and synaptopodin.32,33 Compared with primary cultures calize gene expression (Figure 4, A and B). Timp3 transcripts of myofibroblasts, pericytes expressed more angiogenic factors were readily seen in kidney pericytes of normal adult mouse including Angiopoietin-1 (Angpt1)andAngiopoietin-2 (Angpt2), kidney. Timp3 transcripts were detectable at day 2 after kidney fewer Collagen1a1, and intermediate filament transcripts. injury onset in the interstitium. At day 7 after injury onset, the One of the defining features of pericytes is their ability to bind interstitium in which myofibroblasts are located was almost to and stabilize capillaries.12,34–36 We tested their capacity to devoid of signal for Timp3 (Figure 4A). Timp3 expression was stabilize a capillary tubular network formed by three-dimensional seen in some injured epithelium and also localized to podo- (3D) EC culture. Human ECs spontaneously form a capillary cytes, but not mesangium in the glomerulus (Figure 4A). Coll- tube network in collagen gel (Figure 3, A–C). The capillary tube GFP+ cells were purified from normal kidneys (pericytes) or network can be destabilized and undergoes regression in the kidneys day 7 after disease (myofibroblasts). Timp3 transcripts presence of coagulation , which function by a number were downregulated in Coll-GFP+–purified myofibroblasts of mechanisms including activation of endothelial matrix metal- compared with Coll-GFP+ pericytes purified from normal loproteinases.37,38 Because the lumina of the forming capillary kidneys (Figure 4C). Consistent with these observations, pri- tubes do not contain any collagen gel, tube regression can be vi- mary cultures of kidney pericytes generated higher levels of sualized by gel retraction (collapse) (Figure 3D). In 3D co-culture, TIMP3 protein compared with primary cultures of myofibro- mouse kidney pericytes bind to EC capillary tubes (Figure 3E blasts (Figure 4D). By contrast, in situ hybridization of normal and Supplemental Movie 1). Moreover, the cells have long pro- adult kidney did not detect any transcripts for Adamts1 (Figure cesses that form invaginations on the tube abluminal surface 4A). However, by day 2 after kidney injury, Adamts1 was readily similar to the peg and socket processes seen by electron micros- detected both interstitial cells and epithelial cells, and it was seen copy.9,39 When EC capillary tubes were exposed to the serine in epithelium and to a lesser extent in interstitial cells on day 7 proteinase kallikrein (KLK), both capillary tube regression and (Figure 4A). Immunostaining of kidney tissues with antibodies gel collapse were stimulated in a time- and concentration- against ADAMTS1 revealed a similar pattern (Figure 4B) with dependent manner (Figure 3F). In the absence of KLK, EC cap- expression in the vast majority (83%65.6%) of Coll-GFP+ cells illary tubes did not regress. When kidney pericytes were added to at day 2 after injury onset and 96%66.1% after day 7. ADAMTS1 the gel in a 10%/20% ratio (1 PC for every 10 ECs/2 PCs for every protein was also clearly detected in epithelial cells, predominantly 10 ECs), the collapse of the gel in the presence of KLK was re- at the apical surface in the region of the brush border. As ex- tarded (Figure 3G). When 30% (3 PCs for every 10 ECs) kidney pected, when kidney pericytes or myofibroblasts were purified pericytes were co-cultured, vascular regression was completely from normal or diseased (day 2) kidneys, respectively, Adamts1 prevented and the gel did not collapse (Figure 3G). Similar ob- transcripts were highly upregulated (Figure 4E) in the myofi- servations were made when human vascular brain pericytes were broblasts. In keeping with this observation, primary cultures substituted for kidney pericytes (Figure 3H); however, kidney of pericytes expressed much lower levels of Adamts1 transcript myofibroblasts, which are derived in vivo from kidney pericytes, compared with primary myofibroblasts (Figure 4F). To deter- did not stabilize the capillary network (Figure 3H). Kidney peri- mine the relationship between Timp3 transcripts and protein, cytes suppressed the amount of active EC-derived MMP9 in the immunoblots of whole kidney lysates confirmed the downregu- collagen gels as determined by gelatin zymography (Figure 3I), lation of TIMP3 protein levels in mouse kidney in response to and suppressed activation (phosphorylation) at the EC- disease (day 2) and cultured myofibroblasts expressed lower lev- restricted vascular endothelial growth factor receptor 2 els of TIMP3 protein compared with pericytes (Figure 4G). (VEGFR2) (Figure 3J) triggered by KLK. These observations in- dicate that kidney pericytes and brain pericytes have similar ca- ADAMTS1 Destabilizes and TIMP3 Stabilizes Capillary pacity to stabilize capillary tubes, and that pericytes lose vascular Tubular Networks stabilizing functions when they differentiate into myofibroblasts. TIMP3 has multiple biologic activities that may contribute to vascular stability. Foremost, it inhibits activity of a number of TIMP3 and ADAMTS1 Are Regulated during Kidney metalloproteinasesincludingADAMTS1andendothelialMMPs. Pericyte Detachment In Vivo In addition, in high concentrations, it may regulate signaling The microarray studies (Figure 1) indicated that Timp3 tran- directly at endothelial VEGFR2.28,29,41To explore the role of scripts were highly expressed in kidney pericytes and down- TIMP3 in vascular integrity further, we applied recombinant regulated in response to kidney injury. By contrast, among a TIMP3 to the capillary tube regression assay. In lower

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Figure 2. Characterization of primary kidney pericyte cultures. (A) Fluorescent confocal images of kidney pericyte cultures for typical pericyte markers and markers of endothelium or podocytes (bar=25 mm). (B) Histogram plots and dot plot from FACS analysis of primary kidney pericyte cultures for typical pericyte markers and markers of potential contaminating cells (gray areas are the isotype controls and white areas are the specific antibody). (C) Quantitative PCR for typical pericyte markers and myofibroblast markers in primary kidney pericyte compared with myofibroblast cultures (n=3–6/group; ***P,0.001).

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Figure 3. Kidney pericytes stabilize capillary tubes in a 3D gel assay. (A) Schema showing the addition of ECs (red) to gel in wells that spontaneously form capillary networks. Addition of pericytes to this assay permits migration and binding of pericytes to capillary tubes. (B) Toluidine blue–stained gel showing capillary tube network (ECs only) within the gel (bar=100 mm). (C) Kidney pericyte (GFP+) in culture (bar=25 mm). Note cell processes extending the length of several cell bodies. (D) Low-power light images of gels containing capillary tubes (ECs only). Under the influence of the coagulation cascade serine protease, KLK, endothelial tubes are destabilized in the gel and vessels become disorganized leading to progressive collapse of the gel (bar=100 mm). (E) Confocal image of 3D gel with YZ and XZ stacks showing capillary tube (red, CD34) and kidney pericyte (green, Coll-GFP). Note the attachment of kidney pericytes to the capillary tube and nu- merous processes attached to the capillary tube. Z stacks (arrowheads) show yellow color at points of direct interaction of pericyte pro- cesses with capillary tubes, suggestive of peg and socket junctions (bar=25 mm). (F) Dose-response curves measuring collapse of collagen gel (ECs only) induced by KLK. (G) Gel collapse curves induced by KLK (625 ng/ml) in the presence of increasing numbers of kidney pericytes. Note 30% kidney pericytes completely prevent gel collapse (n=12–16/timepoint). (H) Gel collapse curves in response to KLK (625 ng/ml) in the presence of 30% human vascular brain pericytes (HVBPs) or 30% mouse kidney myofibroblasts (kMF). (I) Gelatin zymography of supernatants from collagen gels in the presence of KLK showing multiple bands of activity. Note that an approximately 72-kD band of activity, representing MMP9, is completely lost by the addition of 20% or 30% pericytes and the major approximately 60-kD band of MMP2 is attenuated by the presence of 30% pericytes. (J) Phosphoblot for proteins from the collagen gels of KLK-activated gels de- tecting phospho-VEGFR2. Note that kidney pericytes complete suppress signaling at VEGFR2.

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Table 1. Quantitative PCR of metalloproteinases and reperfusion injury (IRI), and the function of pericytes in these 2 2 inhibitors regulated in pericytes during detachment at vascular responses. Adult Timp3 / mice were smaller than day 0, day 2, and day 7 after UUO injury littermate controls but had normal renal function and no al- Metalloproteinases Day 2 after Day7 after buminuria (Table 2). The microvascular density of kidney Day 0 and Inhibitors Injury Onset Injury Onset PTCs of adult mice, assessed by two different methods, was Adamts-1 0.8958 9.8249 10.3441a increased with areas of abnormal capillaries (Figure 6, A and Adamts-2 0.4564 0.6883 6.42167b E and Supplemental Figure 3) and a greater number of PTC Adamts-4 0.2562 1.3983 1.94952 ECs were in the cell cycle (Figure 6F). There were discrete ex- Adamts-5 0.2412 0.4097 0.94389 pansions of Pdgfrb+, CD73+, aSMA2 microvascular peri- Mmp2 2.85 2.58 2.87 cytesinPTCareasandalsoaSMA+ myofibroblasts in the Mmp7 0.33 0.87 1.48a 2/2 +/+ b Timp3 kidneys (Figure 6B) not seen in Timp3 kidneys. Mmp9 2.14 3.03 4.33 Small increases of interstitial fibrillar collagen deposition were Mmp11 1.0536 2.1585 7.43393 2 2 detected in normal Timp3 / adult kidneys (Figure 6, C, H, and Mmp14 4.92 12.96 22.00 fi 42 Mmp23 0.7695 0.8386 8.73210 I), con rming previous reports. Furthermore, unlike pericytes 2/2 Timp-3 137.4332 66.172 45.0715a of wild-type normal kidneys, pericytes of Timp3 normal Timp-1 0.1280 8.1431 26.6397a kidneys expressed ADAMTS1 (Figure 6, D, J, and L). In addi- aP,0.01. tion to the interstitial changes, there were mild changes to 2 2 bP,0.05. glomeruli of Timp3 / normal kidneys comprising mild mesangial hypercellularity and matrix expansion (not shown). Given that the major site of expression of Timp3 in the kidney is 2 2 concentrations, TIMP3 retarded capillary tube regression; how- the pericyte, these findings collectively suggest that Timp3 / ever, at higher concentrations, it completely blocked capillary pericytes in vivo are more activated. 2 2 tube regression (Figure 5, A and B). Thus, TIMP3 can com- To determine whether Timp3 / pericytes were intrinsically pletely replace pericyte function (Figure 3) in this assay. By overactivated as opposed to activated in response to vascular contrast, application of recombinant ADAMTS1 to the vascu- changes, we studied primary cultures of pericytes in prolifera- lar assay in the absence of pericytes weakly accelerated capillary tion and migration assays (Figure 6M) and expression of aSMA. 2 2 tube regression in the presence of KLK (Figure 5C), but did not Timp3 / pericytes showed enhanced aSMA expression, trigger regression (not shown). Much more strikingly, how- increased proliferation, and enhanced motility, in keeping ever, is the finding that kidney pericytes were no longer able with an overactivated phenotype. Enhanced migration by 2 2 2 2 to stabilize capillary tubes in the capillary tube regression assay Timp3 / pericytes was cell specific because cultured Timp3 / in the presence of recombinant ADAMTS1 (Figure 5D). These macrophages migrated normally (not shown). findings indicate that ADAMTS1 was sufficient to overcome Totest the effect of injury responses in the microvasculature, the vascular stabilizing functions of pericytes (Figure 5D). To we used a mild kidney IRI model that has a well recognized test specifically whether TIMP3 in pericytes stabilizes capillary microvascular injury, rarefaction, followed by incomplete tubes, TIMP3 protein expression was silenced using specific angiogenesis.9,40,44 We observed that ischemic injury resulted 2 2 small interfering RNA (siRNA) (Figure 5E). Using sequential in a greater rarefaction of the vasculature in Timp3 / kidneys silencing methods, a .70% reduction in protein level was compared with wild-type kidneys in this model (Figure 6, A achieved in pericytes. Whereas pericytes with control gene and E), and IRI stimulated more ECs into the cell cycle in 2 2 silencing completely stabilized capillary tubes, pericytes with Timp3 / kidneys (Figure 6F). In concert with this observation, TIMP3 silencing showed a nonsignificant, weakly impaired ca- there was excessive expansion of pericyte/myofibroblast popu- pacity to stabilize capillary tubes during KLK-mediated regres- lation (Figure 6, B, G, H, and K) after IRI. Using the aSMA sion (Figure 5F), in keeping with pericyte-derived TIMP3 marker to define pericyte transition to myofibroblasts, there playing a direct role in vascular stability. was increased expansion of these myofibroblasts, excessive ex- pression of aSMA in response to injury, and increased inter- 2 2 TIMP3 Mutant Mice Have a Kidney Microvascular stitial fibrosis in Timp3 / kidneys compared with Timp3+/+ Phenotype kidneys byday 5 after IRI (Figure 6, C, I, and K). After kidney 2 2 Our findings suggested that TIMP3 may play a role in vivo in IRI, there was a persistent microvascular leak in Timp3 / regulating vascular stability in the kidney and perhaps more kidneys, as detected by Evans blue dye assay (Figure 6, N and broadly in pericyte function. Timp3-deficient mice survive O) and this leak extended to vascular beds outside of the kidney, into adult life but have been noted to have microvascular defects suggesting that generalized microvascular injury responses in in the retina and in cancer growth, and also fail to resolve lung these mice results in persistent increase in microvascular perme- inflammation after injury. Furthermore, recent reports suggest ability. The enhanced microvascular leak suggests that pericyte 2 2 that Timp3-deficient mice have kidney fibrosis.31,42,43 We therefore functions were more compromised in Timp3 / mice compared 2 2 investigated the microvasculature of normal Timp3 / kidneys, with Timp3+/+ mice after vascular injury. Consistent with this the response of the kidney microvasculature to acute ischemia compromised pericyte function, phosphorylated VEGFR2 was

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Figure 4. Characterization of TIMP3 and ADAMTS1 expression in pericytes, in vivo and in vitro.(A)In situ hybridization of kidney sections from normal kidney (day 0) and on day 2 and day 7 after injury onset (disease) for Timp3, Adamts1, and control epithelial marker Ngfa. Positive-stained pericytes and interstitial cells are shown (arrows) and positive-stained epithelial cells are shown (arrowheads) (bar=50 mm). (B) Split panels showing immunostaining for Adamts1 in normal kidneys (day 0) and on day 2 and day 7 after injury kidney sections from Coll-GFP mice. Coll-GFP+ pericytes do not express Adamts1 (arrows) but early after injury onset (day 2) pericytes and cellular processes (arrowheads) now express Adamts1. Kidney tubule expression is also increased. (C and D) Quantitative PCR for Timp3 comparing (C) Coll-GFP+ purified pericytes (normal kidney) with Coll-GFP+ purified myofibroblasts (disease kidney day 2) or (D) primary pericyte cultures with primary myofibroblast cultures. (E and F) Quantitative PCR for Adamts1 comparing (E) Coll-GFP+ purified pericytes (normal kidney) with Coll-GFP+ purified myofibroblasts (disease kidney day 2) or (F) primary pericyte cultures with primary myofibroblast cultures. (G) Western blot showing expression of TIMP3 (upper blot) in normal kidney compared with diseased kidney (day 7) or (lower blot) pericyte cultures compared with myofibroblasts. Note that the 24-kD Timp3 band is expressed at higher levels in pericytes (n=3–5/group; *P, 0.05, ***P,0.001).

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Figure 5. Characterization of TIMP3 and ADAMTS1 functions on capillary tube stability in a 3D tube assay. (A and B) Graph of KLK-induced capillary tube regression (ECs only) showing (A) retardation of collapse in the presence of low concentrations of recombinant TIMP3 and (B) complete prevention of collapse (ECs only) in the presence of high concentrations of recombinant TIMP3 (doses stated in ng/ml). (C) Graph of KLK-induced capillary tube regression (ECs only) showing modest acceleration by recombinant ADAMTS1 (dose stated in ng/ml). (D) Graph of KLK-induced capillary tube regression in the presence of kidney pericytes and recombinant ADAMTS1. Thirty percent pericyte addition is no longer able to stabilize capillaries when ADAMTS1 is present (doses stated in ng/ml). (E) Western blots showing Timp3 levels in pericytes at time points after introduction of siRNA by transfection. (F) Graph of KLK-induced capillary tube regression in the presence of 30% kidney pericytes after Timp3 silencing or mock silencing (n=12–16/timepoint; *P,0.05, **P,0.01, ***P,0.001).

2 2 increased in the injured Timp3 / kidneys compared with in- as highly regulated kidney pericyte genes that play roles in jured Timp3+/+ kidneys indicative of destabilized microvascu- microvascular stability of the peritubular capillaries. Moreover, lature surrounded by overactivated pericytes (Figure 6P).9,12 ADAMTS1 expression is a marker of activated pericytes. Because these studies indicated that the injury responses in Tissueischemiaisincreasinglybelievedtobeacentralproblem 2 2 Timp3 / kidneys resulted in deleterious microvascular re- in failure to regenerate the kidney normally after injury, a driving sponses, we tested whether acute IRI resulted in greater functional force for progressive organ dysfunction after injury, and a cause deficiency. Twenty four hours after 21 minutes of kidney IRI, for hypertension and possiblycardiovascular disease.45 Central to 2 2 Timp3 / mice (n=6) had plasma creatinine levels of 0.4961.2 kidney tissue ischemia is loss of the microvasculature, which is 1 1 mg/dl, whereas Timp3 / mice (n=6) had plasma creatinine levels also known as capillary rarefaction. The recent identification of a of 0.3160.03 mg/dl (P=0.06). Mice exposed to sham surgery had relatively large population of pericytes in the cortex and medulla creatinine levels of 0.1760.03 and 0.1560.02 mg/dl, respectively. of normal kidney and the observation that they detach from capillaries in response to injury, migrate into the interstitial space, and transition to scar-forming myofibroblasts have raised DISCUSSION the possibility that chronic diseases in the kidney are a state of functional pericyte deficiency. Studies have suggested that in- These studies describe novel vascular stabilizing functions for jury of the kidney is a state of vascular instability that can easily kidney pericytes, and identify genes involved in regulation of lead to rarefaction despite endothelial proliferation and that fac- pericyte detachment and activation. Specifically, they identify tors that prevent pericyte detachment can prevent this instabil- the protease regulator TIMP3 and one of its targets, ADAMTS1, ity.9,11 Thefactthatthefindings presented here reveal kidney

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Table 2. Characterization of Timp3-deficient mice particularly in response to dysangiogenic VEGFA isoforms, +/+ 2/2 fi Timp3 Male 12 wks Timp3 Male 12 wks released by activated pericytes/myo broblasts in the kidney, 9,11 Weight (g) 29.960.9a 24.760.8 as a major factor in microvascular instability. Therefore, “ ” Albuminuria ND ND dialing down the degree of signaling at this receptor may be ND, not detected. desirable. Our studies in vitro and in vivo indicate that kidney aP,0.01. pericytes and TIMP3 function to limit endothelial VEGFR2 sig- naling potentially by both direct and indirect mechanisms. pericytes to have a similar capacity to pericytes from other Timp3 mutation in humans is associated with retinal micro- organs to prevent microvascular instability ex vivo strongly vascular disease and Timp3-deficient mice exhibit increased suggests that interventions that will promote pericyte attach- dysangiogenesis (increased angiogenesis with leaky, poorly ment in the kidney will lead to preservation of capillaries. In functioning vessels) in growth of some tumors and increased keeping with the hypothesis that chronic diseases have func- angiogenesis in growth of others.29,46–49 Consistent with these tional pericyte deficiency due to pericyte transition to my- findings, we identified dysangiogenesis in the kidney after injury ofibroblasts, kidney myofibroblasts were unable to stabilize when Timp3 was mutated, particularly in response to microvas- vessels in the capillary tube assay. cular injury, and showed that pericytes are more activated and The microarray analysis indicated that there is a marked exhibit excessive migration and proliferation. Although further phenotypic change in pericytes when they become myofibro- studies are required, measurement of plasma creatinine in acute blasts. The analysis we performed was based on collecting cells injury of Timp3-deficient kidneys suggests that TIMP3 may that express Coll-GFP. We previously characterized these cells serve to protect kidneys from acute injury, as we previously in the kidney and showed that they are pericytes. We identified showed for the lung.31 Further studies are required to under- very few circulating fibrocytes (leukocytes that produce collagen stand whether TIMP3 regulates pericyte properties through reg- matrix) in this kidney injury model and our purification method ulating MMPs or through regulating cleavage of other factors excludes all leukocytes.1,2,9,39 We previously identified perivas- such as semaphorins.29 The finding that TIMP3 can mimic peri- cular fibroblasts around arterioles of the kidney that also express cytes in vascular stabilization in vitro but that TIMP3 silencing Coll-GFP and expand to become myofibroblasts. These cells were had a weak effect on pericyte function in vascular stabilization in also purified along with pericytes because there is currently no vitro was surprising but may be instructive. Timp3-deficient marker described to distinguish them. Although the microarray mice are born healthy and have a subtle microvascular pheno- analysis requires validation due to potential contamination from type only. Therefore, it may not be surprising that only a modest other kidney cells that may have been inadvertently collected defect was seen in the short vascular stabilization assay. These during the flow cytometric sorting procedure, it is striking that findings also indicate that pericytes rely on factors other than activated pericytes are involved in innate immune responses, che- TIMP3 to stabilize capillary tubes. These other factors are cur- motaxis, and represent a major source of cytokines. This is highly rently unknown. In the capillary tube stabilization assay, peri- relevant because recent studies that target the pericyte directly by cytes inhibit MMP production primarily from ECs, but the blocking PDGFRb or PDGFRa signalinginthekidneynotonly signal that mediates this inhibition is unknown. Although attenuate fibrosis and capillary rarefaction but also markedly at- TIMP3 may be a factor in this regulation, other factors must tenuate the inflammatory response.11 Therefore, it is likely that be contributing. TIMP3-deficient pericytes have a number of activated pericytes are a major contributing cell to the inflamma- functional abnormalities, including enhanced migration prolif- tory response. Further studies are required to understand eration and activated phenotype as judged by aSMA expression whether this response can be specifically targeted in the kidney. and ADAMTS1 expression. It is unclear how TIMP3 regulates all Our microarray studies identified Timp3 as a pericyte factor of these processes, but these studies suggest that TIMP3 must be that is lost during pericyte transition to myofibroblasts, and our able to affect transcription by either direct or indirect means. in vitro and in vivo studies showed TIMP3 to function as a mo- Although a number of metalloproteinases including lecular factor promoting microvascular stability. Although ADAMTS2 and MMP11 are activated in the microvasculature TIMP3 is an inhibitor of metalloproteinases, it is also a pleio- in response to injury, Adamts1 was most remarkably regulated tropic, extracellular matrix–anchored molecule that is able in pericytes of the kidney. ADAMTS1, like TIMP3, is highly teth- to inhibit a number of that breakdown capillary base- ered to the extracellular matrix and acts locally. We identified ment membrane, including ADAMTS1, and other matrix ADAMTS1 regulation in epithelial cells, as well as pericytes, in components. In addition, TIMP3 is able to specifically inhibit the kidney. ADAMTS1 expression in kidney development was signaling at the endothelial receptor VEGFR2, initiated by all previously reported.44,50 Because ADAMTS1 acts locally, the sig- VEGFA isoforms. Interestingly, TIMP2 is also capable of in- nificance of epithelial expression of ADAMTS1, particularly, at hibiting VEGFR2 signaling in a manner independent of its the brush border proximal tubule, is unclear. However, it is likely proteinase inhibitor activity, and previous work demonstrated that pericyte expression of ADAMTS1 early after injury exerts a role for both pericyte-derived TIMP3 and EC-derived TIMP2 activity at the pericyte-endothelial interface. ADAMTS1 has been in capillary tube stabilization.16 In recent studies, we identi- reported to cleave capillary basement membrane components, fied excessive signaling in response to injury at VEGFR2, including aggrecan and versican; to play an anti-angiogenic role

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Figure 6. TIMP3 deficiency in vivo predisposes to microvascular instability of kidney peritubular capillaries due to overactivated pericytes. (A) Confocal images showing microvascular density and pericyte coverage of normal and day 5 post-IRI kidneys. (B) Confocal 2 2 images showing areas of expanded pericytes (Pdfgfr§+) in normal kidneys of Timp3 / mice, some of which express aSMA (arrows) and some of which do not (arrowheads). Five days after IRI, the pericyte/myofibroblasts (Pdgfrb+) show greater expansion and higher levels 2 2 of aSMA, particularly in Timp3 / kidneys. (C) Low-power images of Sirius red–stained kidneys. (D) Fluorescence images of Adamts1. 2 2 Note that normal Timp3 / kidneys have pericytes that express Adamts1 (arrowheads). (E through K) Graphs showing quantification of (E) vascular density, (F) endothelial cell proliferation, (G) pericytes, (H) myofibroblasts, and (I) fibrosis in interstitial (J) Adamts1+ cells. 2 2 (K) Western blot showing aSMA and Pdgfrb expression in kidneys from Timp3 / and Timp3+/+ mice. (L) Western blot quantifying Adamts1 in kidney lysates after IRI. (M) Graphs showing primary pericyte cultures undergoing migration in a scratch closure assay, proliferation, and quantitative PCR expression of Acta2. (N and O) Images of (N) Evans blue–labeled mouse skin and sectioned kidneys and (O) Evans blue quantification indicating a microvascular leak 5 days after IRI. (P) pVEGFR2 expression in normal plus day 5 post-IRI kidneys (bar=50 mm; n=6–8/group; *P,0.05, **P,0.01, ***P,0.001). g, glomerulus; a, arteriole.

878 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 868–883, 2012 www.jasn.org BASIC RESEARCH in both in vivo and in vitro angiogenesis assays; and to regulate Pericyte or Myofibroblast Isolation from Kidney migratory capacity of a number of cells through cleavage of a For pericyte isolation from normal kidney of C57BL6 WT or Coll-GFP number of substrates, including syndecans and semaphorins. It mice, the kidney was decapsulated, diced, and then incubated at 37°C is therefore a protease that may mediate detachment of pericytes. for 30 minutes with Liberase (0.5 mg/ml; Roche) and DNase (100 U/ml; Moreover, it selectively binds to and sequesters VEGFA164 but Roche) in DMEM/F12. Digestion was curtailed by the addition of 10% not dysangiogenic VEGFA isomers.51 We recently showed that FCS to the single cell suspension. The suspension was filtered (40 mm) when pericytes detach from capillaries in the kidney, they switch several times, centrifuged (3003g, 5 minutes), and resuspended in from VEGF164 to VEGF120 and VEGF188 production. The MACS (Miltenyi Biotech, Bergisch Gladbach, Germany) buffer or 1% latter two VEGFA forms contribute to unstable angiogenesis. BSA buffer containing EDTA. In some experiments, cells were incu- Therefore, it is likely that ADAMTS1 also promotes unstable bated with anti-rabbit Pdgfrb polyclonal antibodies generated against a angiogenesis by regulating VEGFR2-mediated signaling. In segment of the full-length receptor54 (800 ng per kidney on ice) for 15 keeping with this, Timp3-deficient mice show overexpression minutes. After washing, cells were incubated with goat anti-rabbit of ADAMTS1 in pericytes, have increased but dysfunctional vas- IgG microbeads (Miltenyi Biotech) (15 minutes at 4°C) and resus- cularity under physiologic conditions, and have increased vessel pended and positive selection was performed by MACS magnetic rarefaction after injury compared with littermate controls. Our bead separation. Selected cells were resuspended in RLT buffer (Qia- studies predict therefore that ADAMTS1 blockade would bene- gen) or cultured in DMEM/F12 with 10% FBS and 1% penicillin/strep- ficially affect the kidney by limiting pericyte detachment after tomycin and 1% ITS, in six-well plates precoated with gelatin. Primary injury, promoting vessel stability, and reducing interstitial fibrosis. cultured cells used in all experiments were between passages 2 and 5. Adamts1 mutant mice have microvascular defects as well as a severe For other purposes, RNA was isolated using the RNeasy Mini Kit kidney developmental phenotype resembling hydroureter.52,53 (Qiagen). To note, this purification does not distinguish pericytes The severe hydroureter phenotype renders them unsuitable for from perivascular fibroblasts. In other experiments, single cells from study of pericyte function in response to injury. One possible Coll-GFP mice were separated by green fluorescent protein fluores- explanation for the developmental kidney phenotype is im- cence by FACS Aria cell sorting, directly into RLT buffer for RNA anal- paired migration of kidney stroma during nephrogenesis leading ysis or into medium (cell culture). In these experiments, kidney single to impaired nephron lengthening and papilla formation.53 Fur- cell suspensions were also incubated with anti-CD45-APC, anti-CD31- ther studies will be required to determine whether Adamts1 is a PE, anti-F4/80-APC,or anti-Tim1 antibodies (eBioscience) as de- – targetable gene in kidney injury to prevent pericyte detachment. scribed1,22 24 to exclude these cell populations from contaminating In summary, kidney pericytes stabilize capillaries ex vivo the Coll-GFP cells. To purify kidney myofibroblasts, single cell prepa- and detach from capillaries in interstitial disease, and pericyte ration and purification were achieved as described above but kidneys factors TIMP3 and ADAMTS1 are key regulators of these peri- from day 2 or day 7 after UUO were used. cyte functions in vivo. Cell Culture Human umbilical cord ECs (HUVECs) (American Type Culture Collection) were used from P3 to P6 and grown with endothelial cell CONCISE METHODS basal medium (American Type Culture Collection) including one Endothelial Cell Growth Kit BBE (American Type Culture Collection) Materials per 500 ml medium. Human brain pericytes (ScienCell) were cultured All reagents were from Sigma Aldrich, all plastic ware was from BD in DMEM (Cellgro) containing 10% FCS (Invitrogen) and used from Falcon, and all media were from Cellgro unless otherwise stated. P3 to P6. Primary mouse kidney pericytes (see below) were cultured on 0.1% gelatin-coated T75 flasks (BD Falcon) or six-well plates (BD Animals Falcon) and used from P2 to P5. Mouse kidney–derived myofibroblasts a Coll1 1-GFP (Coll-GFP) transgenic mice were generated and validated were established as previously described1,9 and were maintained under 1,9,23 2/2 as previously described on the C57BL6 background. Timp3 the same conditions as pericyte cultures and used from P3 to P7. gene–targeted homozygous mice and littermate controls were gen- erated as previously described31,43 and maintained on the C57BL6 Three-Dimensional Vascular Tube Regression Assay background. ECs were placed in 3D vascular tube regression assays following a modified protocol of Saunders et al.30,37 HUVECs (106 cells/ml) were Animal Models of Kidney Injury suspended in 3.75 mg/ml of rat tail collagen type I (4°C) (Invitrogen), UUO and unilateral and bilateral kidney IRI models were performed transferred to a 37°C incubator in which polymerization occurred, in adult mice (aged 10–12 weeks) as previously described.1,22,23 The and were allowed to form 3D networks in the presence of VEGF165 ischemic time was 21 minutes. In some experiments, BrdU (50 mg/g) (40 ng/ml) and FGF2 (40 ng/ml) (Peprotech),38 in 384-well plates or or Evans blue dye (1%) was injected intraperitoneally or intravenously, 96-well 1/2 plates. For quantitative analysis of gel contraction, 15 ml respectively, 1 hour before euthanasia. All animal experiments were of ECs alone or ECs and varying amounts of primary kidney PCs performed under protocols approved by the Animal Resources and (as indicated) in collagen matrix were placed in 384-well plates (BD Comparative Medicine Committee of Harvard University and the De- Falcon) (n.10/condition). Addition of medium with or without hu- partment of Comparative Medicine at the University of Washington. man plasma KLK ( Research Laboratories) denoted the

J Am Soc Nephrol 23: 868–883, 2012 Pericytes Stabilize Capillaries 879 BASIC RESEARCH www.jasn.org starting point of the assay. Cultures were monitored every 4 hours for FACS sorting into RLT buffer as described in the section above. RNA gel contraction. Upon initiation of gel contraction, the regressed area was purified by the RNeasy method (Qiagen). The quality of RNA was was quantified using an ocular grid. In every experiment, at least one tested by a Agilent Bioanalyzer and high-quality samples underwent condition (n=5–10) with HUVECs in collagen matrix without the RNA amplification by in vitro transcription methods at Harvard Uni- addition of KLK was used as an internal control. In some experi- versity Core Microarray Biotechnology Center. cRNA samples were ments, recombinant mouse Adamts1 or recombinant human hybridized to CodeLink mouse whole-genome microarrays using the TIMP3 (R&D Systems) was applied to the media. For experiments manufacturer’s protocols (Applied Microarrays) and scanned using in which rmAdamts1 was applied, HUVECs were allowed to form tubes Axon GenePix scanner (Molecular Devices). Image processing, back- for 24 hours before addition of KLK and recombinant protein. Toluidine ground subtraction, and median-based normalization of probe set blue staining of gels was performed as described previously.30 Condi- intensities were performed using CodeLink Expression Analysis Soft- tioned media or whole gels were collected to examine differential pro- ware (Applied Microarrays). tein expression at various time points and were analyzed by SDS-PAGE gels and Western blotting or zymography. Microarray Data Analyses Principal component analysis using the entire transcriptome of fi Pericyte Migration and Proliferation Assays pericytes and myo broblasts was performed based on the covariance Forty-eight-well plates (BD Falcon) were coated with 0.1% gelatin for matrix of normalized gene expression data55 (Supplemental Figure 2 hours at 37°C. Cells were seeded with the same density of cells per 2A). Temporal profiles of gene expression (day 0, day 2, and day 7) were well and incubated with DMEM/F12 10% FCS until confluent. A produced using Short Time-series Expression Miner clustering— scratch “wound” was performed using a 200-ml pipet tip, wells were an algorithm developed for analyzing short time series microarray washed twice with PBS, and DMEM/F12 with 2% FCS was applied. At data.25 Replicate gene expression values were averaged at day 0 and 28 pre-fixed points along the scratches, photomicrographs were performed day 2. Highly significant clusters (P values ranging from 5.4310 to 2199 at 0 hours and 12 hours after initialization of the wound. Migration of 5.9310 ) were merged if they displayed similar patterns, resulting cells and quantification of the wound area and its closure after 12 hours in two distinct temporal profiles (Figure 1). Functional analysis of the were analyzed using ImageJ software. To quantify proliferation, 105 gene members of these two clusters was performed using the Database 2 2 Timp3 / and Timp3+/+ pericytes were seeded onto gelatin-coated glass for Annotation, Visualization, and Integrated Discovery, a web-based 1.5-cm diameter cover slips (n=9 cover slips/group). BrdU (Sigma) (10 program based on the Gene Ontology database.56 To demonstrate dif- mM/ml) was applied to the medium and incubated for 3 hours. Cells ferential gene expression between pericytes (day 0, no surgery) versus were fixed in acid ethanol, washed, and incubated with 2M HCL, then myofibroblasts (day 2 and day 7 after UUO surgery), we applied a Bayes- 0.1M sodium borate, and then 0.2% Triton in 3% BSA. After addi- ian implementation of the parametric t test57 and corrected for multiple tional washing, cells were incubated with an anti-BrdU FITC anti- comparisons using the Q value method.58 A Q value ,0.01 was chosen body (1:200; eBioscience) in 3% BSA and mounted with Vectashield for identifying significant differential expression, and genes meeting and 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories), and this cutoff underwent two-dimensional hierarchical clustering using BrdU+ nuclei were quantified in 15 random fields per coverslip. Pearson’s correlation (Supplemental Figure 2B and Supplemental Table 1). 55 Gene Silencing in Primary Kidney Pericytes with siRNA siRNA for Timp3 and control siRNA (Santa Cruz Biotechnology) Quantitative Analyses were resuspended in universal buffer and stored (280°C, 10 mM). We used Western blot analysis to quantify protein concentrations in For efficient transfection of primary kidney pericytes, cells were washed, tissues. Total cellular protein extracted using radioimmunoprecipitation trypsinized, and harvested. A single cell suspension was generated assay buffer containing Complete Mini Enzyme Inhibitor Cocktail and cells were distributed into a six-well plate with 200,000 cells/well. (Roche Diagnostics) fromwhole tissue, cells, or supernatants or collagen siRNA (final concentration of 100 nM/well) was resuspended in serum- gel from wells of the vascular stabilization assay. Equal protein amounts free medium, vortexed, and incubated for 10 minutes at room tem- were subjected to SDS-PAGE and Western blotting using previously 59 perature with HiPerfect Transfection Reagent (Qiagen). Medium described methods. The following primary antibodies were used to fi containing siRNA was added dropwise to the wells and plates were detect the speci c protein: anti-TIMP3-C-terminus (rabbit 1:100; Mil- swirled gently. Cells were incubated under normal conditions for 3 lipore), anti-ADAMTS-1 (rabbit 1:100; Santa Cruz Biotechnology), b hours, followed by substitution of additional culture medium. After anti- -actin (goat 1:1000; Santa Cruz Biotechnology), and anti- b 48 hours, cells were analyzed for gene silencing. In experiments in which phospho-VEGFR2 and anti-PDGFR (rabbit 1:1000). To measure TIMP3 was silenced, cells were transfected again with siRNA as de- MMP activity after regression in 3D cultures treated with KLK, we scribed above 48 hours after initial transfection to achieve higher levels used a 10% gelatin zymogram ready gel (BioRad) and loaded super- of gene silencing from 72 to 96 hours. natant of regressed gels for electrophoresis under nonreducing con- ditions. Protein amounts were normalized by the Bradford method. Microarray Experiments After electrophoresis, gels were washed twice in a 2.5% Triton X-100 Kidney pericytes or kidney myofibroblasts were purified from 12-week- including 0.02% NaN3 buffer for 20 minutes, switched to an incuba- old adult kidneys of Coll-GFP mice that had no surgery (pericytes, day 0, tion buffer (50 mM Tris HCl pH 8.0, % mM CaCl2, 0.02% NaN3) for n=2) or had undergone UUO surgery 2 days (n=2) or 7 days (n=1) 10 minutes and incubated in fresh incubation buffer at 37°C for 24 to 48 previously (myofibroblasts) by generating single cell preparation and hours. After incubation, gels were fixed and stained in fresh Coomassie

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40 blue solution and destained in MeOH:AcOH:H2O (5:1:4) followed by of vascular density was performed as previously described, and mor- 10% AcOH. Gels were scanned and MMP activity was measured by phometry of Sirius red stain or other fluorescent labels was performed densitometry of destained areas. as previously reported.1,22–24 In some studies, extravasated Evans blue Total RNA was extracted using RNeasy Mini Kit. Purity was dye (1%) was detected in whole kidney measuring absorbance.62 Digital determined by the A260/A280 ratio. cDNAwas synthesized using Iscript images were captured by confocal microscopy. Sirius red staining or (BioRad). Conventional and quantitative PCR was performed using periodic acid–Schiff staining were performed on 3-mm paraffin sec- previously described methods.1 The specific primer pairs used for PCR tions as previously described.63 Positive cells or area of positive stain/ are listed in Supplemental Table 2. fluorescence in images were quantified as previously described.1,38

In Situ Hybridization Statistical Analyses Riboprobes were designed by linearization pCytoMegaloVirus sport6 Comparisons of two groups were performed using the t test, whereas vectors containing the specificcDNAforTimp3, Adamts1,orNgf several groups were analyzed by one-way ANOVA followed by multiple alpha (control) (Invitrogen) via restriction enzyme digestion. RNA comparison testing, using GraphPad Prism software. Groups were con- probes were then generated using a DIG labeling kit (Roche) con- sidered significant if P values were ,0.05. Error bars indicate SEM. taining Sp6 or T7 polymerases to generate probes from the 39 end. Probe sizes were confirmed by RNA agarose gel electrophoresis and ACKNOWLEDGMENTS digoxigenin incorporation was determined by membrane spotting. Hy- bridizations were performed following methods adapted from in situ Wethank Dr. Li Li Hsiao (Harvard Medical School, MA), Dr. Roderick hybridization in kidney embryos.60 In brief, paraformaldehyde (PFA) Jensen (Virginia Tech, VA), and Michael J. Lombardi (Microarray fixed kidney cryosections (8 mm) were used within 48 hours, postfixed, Biotechnology Center, Harvard Medical School) for assistance with proteinase-K digested, postfixed again, acetylated, and then incubated microarrays; Dr. William Parks and Dr. Yujiro Kida (University with 750 ng/ml riboprobes at 68°C for 16 hours in hybridization buffer of Washington, WA) and Dr. Anastasia Sacharidou (University of (ENZ-33808; Enzo Life Sciences). Sections were digested by RNase, and Missouri, MI) for advice and assistance with the vascular assay, re- then washed again with solutions of increasing stringency. After incu- spectively; Dr. Rama Khokha (University of Toronto, Toronto, ON, bation with anti-digoxigenin antibody (Roche) for 2 hours at room 2 2 Canada) for providing the Timp3 / mice; and the Lynne and Mike temperature, sections were washed and developed with nitro blue tet- Garvey Microscopy Suite at South Lake Union for microscopy sup- razolium chloride (Roche) for up to 24 hours. Color reactions were port. We also thank Dr. Herman Haller for ongoing support and stopped with fixation and sections were mounted with ProLong Gold advice. The Duffield laboratory is funded by the National Institutes of (Invitrogen). Health (Grants DK73299, DK84077, and DK87389), University of Washington, Genzyme (Research in Progress Grant), and the Nephcure Tissue Preparation, Histology, and Analyses Foundation, as well as by a research agreement with Regulus Thera- Mouse kidneys were flushed with ice-cold PBS to remove blood, and peutics. C.S. was partly funded by Medizinische Hochschule, were harvested and fixed with paraformaldehyde, L-lysine, periodate Hannover, Germany. solution for 2 hours before cryopreservation for immunofluorescence studies or with 10% formalin solution for 12 to 16 hours for paraffin sections.61 Mouse kidneys for in situ hybridization were fixed overnight DISCLOSURES with 4% PFA before cryopreservation.60 Human kidney biopsies were J.S.D. is on the scientific advisory board for Promedior Inc and Regulus either prepared for electron microscopy (EM) by standard methods9 or Therapeutics, has stock options with Promedior Inc, and has consulted for fixed with paraformaldehyde, L-lysine, periodate solution for 1 hour, Amira Pharamceuticals, Gilead, Abbott Pharmaceuticals, Takeda Pharma- followed by cryopreservation.61 Human or mouse cryosections (7 mm) ceuticals, and Boehringer Ingelheim. for immune fluorescence studies were blocked in 10% normal serum and then incubated with primary antibody (2 hours at room tempera- REFERENCES ture or 16 hours at 4°C) in blocking serum using the following primary antibodies for mouse kidneys: anti-ADAMTS1 (rabbit 1:100; Santa 1. Lin SL, Kisseleva T, Brenner DA, Duffield JS: Pericytes and perivascular Cruz Biotechnology), anti-PDGFRb (rabbit 1:400 collaborator), fibroblasts are the primary source of collagen-producing cells in ob- anti-CD31 (rat 1:250; R&D Systems), and anti-aSMA-cy3 (mouse structive fibrosis of the kidney. Am J Pathol 173: 1617–1627, 2008 1:250; Sigma Aldrich). Anti-mouse PDGFRb antibodies were gener- 2. Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre fi 54 JV, Valerius MT, McMahon AP, Duf eld JS: Fate tracing reveals the ated and characterized as previously described. The following anti- pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. bodies were used for human kidneys: anti-CD34 (1:250 Ab30375; Am J Pathol 176: 85–97, 2010 Abcam) (1:300; BD Biosciences), anti-PDGFRb (1:250; Cell Signal- 3. Benjamin LE, Hemo I, Keshet E: A plasticity window for blood vessel ing), and anti-aSMA-Cy3 (mouse 1:250; Sigma Aldrich). As necessary, remodelling is defined by pericyte coverage of the preformed endo- sections were incubated with appropriate affinity purified secondary thelial network and is regulated by PDGF-B and VEGF. Development 125: 1591–1598, 1998 Cy3-conjugated antibodies (1:500; Jackson Immunoresearch) for 1 hour 4. Benjamin LE, Golijanin D, Itin A, Pode D, Keshet E: Selective ablation of fi at room temperature, post xedin1%PFAfor2minutes,andmounted immature blood vessels in established human tumors follows vascular with Vectashield plus DAPI or ProLong Gold plus DAPI. Quantification endothelial growth factor withdrawal. JClinInvest103: 159–165, 1999

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5. Armulik A, Abramsson A, Betsholtz C: Endothelial/pericyte inter- 23. Castaño AP, Lin SL, Surowy T, Nowlin BT, Turlapati SA, Patel T, Singh A, actions. Circ Res 97: 512–523, 2005 Li S, Lupher ML Jr, Duffield JS: Serum amyloid P inhibits fibrosis through 6. Armulik A, Genové G, Mäe M, Nisancioglu MH, Wallgard E, Niaudet C, Fc gamma R-dependent monocyte-macrophage regulation in vivo. Sci He L, Norlin J, Lindblom P, Strittmatter K, Johansson BR, Betsholtz C: Transl Med 1: 5ra13, 2009 Pericytes regulate the blood-brain barrier. Nature 468: 557–561, 2010 24. Lin SL, Castaño AP, Nowlin BT, Lupher ML Jr, Duffield JS: Bone marrow 7. Majesky MW, Dong XR, Hoglund V, Mahoney WM Jr, Daum G: The Ly6Chigh monocytes are selectively recruited to injured kidney and dif- adventitia: A dynamic interface containing resident progenitor cells. ferentiate into functionally distinct populations. JImmunol183: 6733– Arterioscler Thromb Vasc Biol 31: 1530–1539, 2011 6743, 2009 8. Levinson RS, Batourina E, Choi C, Vorontchikhina M, Kitajewski J, 25. Ernst J, Bar-Joseph Z: STEM: A tool for the analysis of short time series Mendelsohn CL: Foxd1-dependent signals control cellularity in the gene expression data. BMC Bioinformatics 7: 191, 2006 renal capsule, a structure required for normal renal development. De- 26. Jönsson-Rylander AC, Nilsson T, Fritsche-Danielson R, Hammarström A, velopment 132: 529–539, 2005 Behrendt M, Andersson JO, Lindgren K, Andersson AK, Wallbrandt P, 9. Lin SL, Chang FC, Schrimpf C, Chen YT, Wu CF, Wu VC, Chiang WC, Rosengren B, Brodin P, Thelin A, Westin A, Hurt-Camejo E, Lee-Søgaard Kuhnert F, Kuo CJ, Chen YM, Wu KD, Tsai TJ, Duffield JS: Targeting CH: Role of ADAMTS-1 in atherosclerosis: Remodeling of carotid artery, endothelium-pericyte cross talk by inhibiting VEGF receptor signaling immunohistochemistry, and proteolysis of versican. Arterioscler Thromb attenuates kidney microvascular rarefaction and fibrosis. Am J Pathol Vasc Biol 25: 180–185, 2005 178: 911–923, 2011 27. Vázquez F, Hastings G, Ortega MA, Lane TF, Oikemus S, Lombardo M, 10. Stratman AN, Schwindt AE, Malotte KM, Davis GE: Endothelial-derived Iruela-Arispe ML: METH-1, a human ortholog of ADAMTS-1, and PDGF-BB and HB-EGF coordinately regulate pericyte recruitment METH-2 are members of a new family of proteins with angio-inhibitory during vasculogenic tube assembly and stabilization. Blood 116: 4720– activity. JBiolChem274: 23349–23357, 1999 4730, 2010 28. Rodríguez-Manzaneque JC, Westling J, Thai SN, Luque A, Knauper V, 11. Chen YT, Chang FC, Wu CF, Chou YH, Hsu HL, Chiang WC, Shen J, Murphy G, Sandy JD, Iruela-Arispe ML: ADAMTS1 cleaves aggrecan at Chen YM, Wu KD, Tsai TJ, Duffield JS, Lin SL: Platelet-derived growth multiple sites and is differentially inhibited by metalloproteinase in- factor receptor signaling activates pericyte-myofibroblast transition in hibitors. Biochem Biophys Res Commun 293: 501–508, 2002 obstructive and post-ischemic kidney fibrosis. Kidney Int 80: 1170– 29. Qi JH, Ebrahem Q, Moore N, Murphy G, Claesson-Welsh L, Bond M, 1181, 2011 Baker A, Anand-Apte B: A novel function for tissue inhibitor of 12. Greenberg JI, Shields DJ, Barillas SG, Acevedo LM, Murphy E, Huang J, metalloproteinases-3 (TIMP3): Inhibition of angiogenesis by blockage Scheppke L, Stockmann C, Johnson RS, Angle N, Cheresh DA: A role of VEGF binding to VEGF receptor-2. Nat Med 9: 407–415, 2003 for VEGF as a negative regulator of pericyte function and vessel mat- 30. Saunders WB, Bohnsack BL, Faske JB, Anthis NJ, Bayless KJ, Hirschi KK, uration. Nature 456: 809–813, 2008 Davis GE: Coregulation of vascular tube stabilization by endothelial cell 13. Wühl E, Schaefer F: Managing kidney disease with blood-pressure TIMP-2 and pericyte TIMP-3. JCellBiol175: 179–191, 2006 control. Nat Rev Nephrol 7: 434–444, 2011 31. Gill SE, Huizar I, Bench EM, Sussman SW, Wang Y, Khokha R, Parks 14. Jung YJ, Kim DH, Lee AS, Lee S, Kang KP, Lee SY, Jang KY, Sung MJ, WC: Tissue inhibitor of metalloproteinases 3 regulates resolution of in- Park SK, Kim W: Peritubular capillary preservation with COMP- flammation following acute lung injury. Am J Pathol 176: 64– 73, 2010 angiopoietin-1 decreases ischemia-reperfusion-induced acute kid- 32. Cai X, Lin Y, Friedrich CC, Neville C, Pomerantseva I, Sundback CA, ney injury. Am J Physiol Renal Physiol 297: F952–F960, 2009 Zhang Z, Vacanti JP, Hauschka PV, Grottkau BE: Bone marrow derived 15. Rabelink TJ, Wijewickrama DC, de Koning EJ: Peritubular endothelium: pluripotent cells are pericytes which contribute to vascularization. Stem The Achilles heel of the kidney? Kidney Int 72: 926–930, 2007 Cell Rev 5: 437–445, 2009 16. Davis GE, Saunders WB: Molecular balance of capillary tube formation 33. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, versus regression in wound repair: Role of matrix metalloproteinases Sun B, Zheng B, Zhang L, Norotte C, Teng PN, Traas J, Schugar R, and their inhibitors. J Investig Dermatol Symp Proc 11: 44–56, 2006 Deasy BM, Badylak S, Buhring HJ, Giacobino JP, Lazzari L, Huard J, 17. Bausch D, Pausch T, Krauss T, Hopt UT, Fernandez-del-Castillo C, Péault B: A perivascular origin for mesenchymal stem cells in multiple Warshaw AL, Thayer SP, Keck T: Neutrophil granulocyte derived human organs. Cell Stem Cell 3: 301–313, 2008 MMP-9 is a VEGF independent functional component of the angio- 34. Hayden MR, Karuparthi PR, Habibi J, Lastra G, Patel K, Wasekar C, genic switch in pancreatic ductal adenocarcinoma. Angiogenesis 14: Manrique CM, Ozerdem U, Stas S, Sowers JR: Ultrastructure of islet 235–243, 2011 microcirculation, pericytes and the islet exocrine interface in the HIP rat 18. Darmanin S, Chen J, Zhao S, Cui H, Shirkoohi R, Kubo N, Kuge Y, model of diabetes. Exp Biol Med (Maywood) 233: 1109–1123, 2008 Tamaki N, Nakagawa K, Hamada J, Moriuchi T, Kobayashi M: All-trans 35. Caruso RA, Fedele F, Finocchiaro G, Pizzi G, Nunnari M, Gitto G, retinoic acid enhances murine dendritic cell migration to draining Fabiano V, Parisi A, Venuti A: Ultrastructural descriptions of pericyte/ lymph nodes via the balance of matrix metalloproteinases and their endothelium peg-socket interdigitations in the microvasculature of inhibitors. JImmunol179: 4616–4625, 2007 human gastric carcinomas. Anticancer Res 29: 449–453, 2009 19. Ries C, Egea V, Karow M, Kolb H, Jochum M, Neth P: MMP-2, MT1-MMP, 36. Suzuki K, Masawa N, Sakata N, Takatama M: Pathologic evidence of and TIMP-2 are essential for the invasive capacity of human mesenchymal microvascular rarefaction in the brain of renal hypertensive rats. J stem cells: Differential regulation by inflammatory cytokines. Blood 109: Stroke Cerebrovasc Dis 12: 8–16, 2003 4055–4063, 2007 37. Saunders WB, Bayless KJ, Davis GE: MMP-1 activation by serine pro- 20. Vigetti D, Moretto P, Viola M, Genasetti A, Rizzi M, Karousou E, Pallotti teases and MMP-10 induces human capillary tubular network collapse F, De Luca G, Passi A: Matrix metalloproteinase 2 and tissue inhibitors and regression in 3D collagen matrices. JCellSci118: 2325–2340, of metalloproteinases regulate human aortic smooth muscle cell mi- 2005 gration during in vitro aging. FASEB J 20: 1118–1130, 2006 38. Davis GE, Pintar Allen KA, Salazar R, Maxwell SA: Matrix metalloproteinase- 21. Hartwell LH, Hopfield JJ, Leibler S, Murray AW: From molecular to 1 and -9 activation by plasmin regulates a novel endothelial cell- modular cell biology. Nature 402[Suppl]: C47–C52, 1999 mediated mechanism of collagen gel contraction and capillary tube 22. Lin SL, Li B, Rao S, Yeo EJ, Hudson TE, Nowlin BT, Pei H, Chen L, Zheng regression in three-dimensional collagen matrices. JCellSci114: 917– JJ, Carroll TJ, Pollard JW, McMahon AP, Lang RA, Duffield JS: Mac- 930, 2001 rophage Wnt7b is critical for kidney repair and regeneration. Proc Natl 39. Schrimpf C, Duffield JS: Mechanisms of fibrosis: The role of the peri- Acad Sci USA 107: 4194–4199, 2010 cyte. Curr Opin Nephrol Hypertens 20: 297–305, 2011

882 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 868–883, 2012 www.jasn.org BASIC RESEARCH

40. Li B, Cohen A, Hudson TE, Motlagh D, Amrani DL, Duffield JS: Mobilized 53. Mittaz L, Russell DL, Wilson T, Brasted M, Tkalcevic J, Salamonsen LA, human hematopoietic stem/progenitor cells promote kidney repair after Hertzog PJ, Pritchard MA: Adamts-1 is essential for the development ischemia/reperfusion injury. Circulation 121: 2211–2220, 2010 and function of the urogenital system. Biol Reprod 70: 1096–1105, 41. Lim NH, Kashiwagi M, Visse R, Jones J, Enghild JJ, Brew K, Nagase H: 2004 Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 54. Ozerdem U, Grako KA, Dahlin-Huppe K, Monosov E, Stallcup WB: NG2 and ADAMTS-5: Biological and structural implications. Biochem J 431: proteoglycan is expressed exclusively by mural cells during vascular 113–122, 2010 morphogenesis. Dev Dyn 222: 218–227, 2001 42. Kassiri Z, Oudit GY, Kandalam V, Awad A, Wang X, Ziou X, Maeda N, 55. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Herzenberg AM, Scholey JW: Loss of TIMP3 enhances interstitial Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov nephritis and fibrosis. J Am Soc Nephrol 20: 1223–1235, 2009 A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J: 43. Janssen A, Hoellenriegel J, Fogarasi M, Schrewe H, Seeliger M, Tamm TM4: A free, open-source system for microarray data management and E, Ohlmann A, May CA, Weber BH, Stöhr H: Abnormal vessel formation analysis. Biotechniques 34: 374–378, 2003 in the choroid of mice lacking tissue inhibitor of metalloprotease-3. 56. Huang W, Sherman BT, Lempicki RA: Systematic and integrative anal- Invest Ophthalmol Vis Sci 49: 2812–2822, 2008 ysis of large gene lists using DAVID bioinformatics resources. Nat 44. Basile DP, Fredrich K, Chelladurai B, Leonard EC, Parrish AR: Renal Protoc 4: 44–57, 2009 ischemia reperfusion inhibits VEGF expression and induces ADAMTS-1, 57. Baldi P, Long AD: A Bayesian framework for the analysis of microarray a novel VEGF inhibitor. Am J Physiol Renal Physiol 294: F928–F936, 2008 expression data: Regularized t -test and statistical inferences of gene 45. Ronco C, House AA, Haapio M: Cardiorenal syndrome: Refining the changes. Bioinformatics 17: 509–519, 2001 definition of a complex symbiosis gone wrong. Intensive Care Med 34: 58. Storey JD, Tibshirani R: Statistical significance for genomewide studies. 957–962, 2008 Proc Natl Acad Sci USA 100: 9440–9445, 2003 46. Anania MC, Sensi M, Radaelli E, Miranda C, Vizioli MG, Pagliardini S, 59. Lin SL, Chen RH, Chen YM, Chiang WC, Lai CF, Wu KD, Tsai TJ: Pen- Favini E, Cleris L, Supino R, Formelli F, Borrello MG, Pierotti MA, Greco toxifylline attenuates tubulointerstitial fibrosis by blocking Smad3/4- A: TIMP3 regulates migration, invasion and in vivo tumorigenicity of activated transcription and profibrogenic effects of connective tissue thyroid tumor cells. Oncogene 30: 3011–3023, 2011 growth factor. J Am Soc Nephrol 16: 2702–2713, 2005 47. Kornfeld JW, Meder S, Wohlberg M, Friedrich RE, Rau T, Riethdorf L, 60. McMahon AP, Aronow BJ, Davidson DR, Davies JA, Gaido KW, Grimmond Löning T, Pantel K, Riethdorf S: Overexpression of TACE and TIMP3 S, Lessard JL, Little MH, Potter SS, Wilder EL, Zhang P GUDMAP project: mRNA in head and neck cancer: Association with tumour development GUDMAP: The genitourinary developmental molecular anatomy project. and progression. Br J Cancer 104: 138–145, 2011 J Am Soc Nephrol 19: 667–671, 2008 48. Song B, Wang C, Liu J, Wang X, Lv L, Wei L, Xie L, Zheng Y, Song X: 61. Brenes F, Harris S, Paz MO, Petrovic LM, Scheuer PJ: PLP fixation for MicroRNA-21 regulates breast cancer invasion partly by targeting tis- combined routine histology and immunocytochemistry of liver bi- sue inhibitor of metalloproteinase 3 expression. J Exp Clin Cancer Res opsies. JClinPathol39: 459–463, 1986 29: 29, 2010 62. David S, Ghosh CC, Kümpers P, Shushakova N, Van Slyke P, Khankin 49. Qi JH, Dai G, Luthert P, Chaurasia S, Hollyfield J, Weber BH, Stöhr H, EV, Karumanchi SA, Dumont D, Parikh SM: Effects of a synthetic PEG- Anand-Apte B: S156C mutation in tissue inhibitor of metalloproteinases-3 ylated Tie-2 agonist peptide on endotoxemic lung injury and mortality. induces increased angiogenesis. JBiolChem284: 19927–19936, 2009 Am J Physiol Lung Cell Mol Physiol 300: L851–L862, 2011 50. Nakamura A, Sakai Y, Ohata C, Komurasaki T: Expression and significance 63. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori of a disintegrin and metalloproteinase with thrombospondin motifs S, Wu S, Lang R, Iredale JP: Selective depletion of macrophages reveals (ADAMTS)-1 in an animal model of renal interstitial fibrosis induced by distinct, opposing roles during liver injury and repair. JClinInvest115: unilateral ureteral obstruction. Exp Toxicol Pathol 59: 1–7, 2007 56–65, 2005 51. Luque A, Carpizo DR, Iruela-Arispe ML: ADAMTS1/METH1 inhibits endothelial cell proliferation by direct binding and sequestration of VEGF165. JBiolChem278: 23656–23665, 2003 See related editorial, “Activated Pericytes and the Inhibition of Renal Vascular 52. Mittaz L, Ricardo S, Martinez G, Kola I, Kelly DJ, Little MH, Hertzog PJ, Stability: Obstacles for Kidney Repair,” on pages 767–769. Pritchard MA: Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental This article contains supplemental material online at http://jasn.asnjournals. dysgenesis. Nephrol Dial Transplant 20: 419–423, 2005 org/lookup/suppl/doi:10.1681/ASN.2011080851/-/DCSupplemental.

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