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Cathepsin S Cleavage of Protease-ActivatedReceptor-2 on Endothelial Cells PromotesMicrovascular Diabetes Complications

Santhosh Kumar VR,* Murthy N. Darisipudi,* Stefanie Steiger,* Satish Kumar Devarapu,*Maia Tato,* Onkar P. Kukarni,* Shrikant R. Mulay,* Dana Thomasova,* Bastian Popper,†

Jana Demleitner,‡ Gabriele Zuchtriegel,§| Christoph Reichel,§| Clemens D. Cohen,¶**Maja T. Lindenmeyer, Helen Liapis,†† Solange Moll,‡‡ Emma Reid,§§ Alan W. Stitt,§§

Brigitte Schott,|| Sabine Gruner,|| Wolfgang Haap,|| Martin Ebeling,|| Guido Hartmann,|| andHans-Joachim Anders*

*Medizinische Klinik and Poliklinik IV, Klinikum der Universität München, Munich, Germany; †Department of Anatomyand Cell Biology, Ludwig-Maximilians Universität, Munich, Germany; ‡Walther-Straub-Institut for Pharmakologie undToxikologie, §Walter Brendel Centre of Experimental Medicine, and |Department of Otorhinolaryngology, Head andNeck Surgery, University of Munich, Munich, Germany; ¶Division of Nephrology, Krankenhaus Harlaching, Munich,Germany; **Division of Nephrology and Institute of Physiology, University Hospital and University of Zurich, Zurich,Switzerland; ††Department of Pathology and Immunology, Washington University School of Medicine, St. Louis,Missouri; ‡‡Institute of Clinical Pathology, University Hospital Geneva, Geneva, Switzerland; §§Centre for ExperimentalMedicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, Ireland; and||Cardiovascular and Metabolism, Pharma Research and Early Development, Hoffmann La Roche, Basel, Switzerland

ABSTRACTEndothelial dysfunction is a central pathomechanism in diabetes-associated complications. Wehypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease thatdegrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. Wefound that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cellinjury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 inischemia–induced microvascular permeability. In vitro transcriptome analysis and experiments usingsiRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrityand barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type2 diabetic nephropathy, only CD68+ intrarenalmonocytes expressedCat-SmRNA,whereas Cat-S proteinwas present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, thecysteine protease inhibitor cystatin Cwas expressed only in tubules. Delayed treatment of type 2 diabeticdb/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators ofdiabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers ofdiabetic retinopathy. These data identify Cat-S as a monocyte/macrophage–derived circulating PAR2 agonistand mediator of endothelial dysfunction–related microvascular diabetes complications. Thus, Cat-S or PAR2inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

J Am Soc Nephrol 27: 1635–1649, 2016. doi: 10.1681/ASN.2015020208

Received February 25, 2015. Accepted August 25, 2015.

S.K.V., M.N.D., and S.S. contributed equally to this work.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Hans-Joachim Anders, Medizinische Klinikand Poliklinik IV, Klinikum der Universität München–Innenstadt,Ziemssenstrasse 1, 80336 Munich, Germany. Email: hjanders@med.uni-muenchen.de

Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 1635–1649, 2016 ISSN : 1046-6673/2706-1635 1635

Figure 1. Extrinsic and intrinsic Cat-S triggers endothelial cell injury and microvascular permeability through PAR2 in vivo. (A) C57BL/6mice were injected with a single dose of 10 mg Cat-S 6 pretreatment with two different Cat-S antagonists. Urinary albumin-to-creatinine (A/C) ratio was quantified at several time points as indicated. Data are means6SEMs; P,0.05 versus baseline. (B) At 24 hours,transmission EM of glomeruli showed massive endothelial cell swelling with vacuolization in kidneys from Cat-S–treated mice

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Morbidity and mortality in diabetes are largely caused bymacro- and microvascular complications. The metabolicchanges of diabetes induce endothelial dysfunction (ED),which is critical to the initiation and progression of vascularcomplications.1 ED is associated with decreased endothelialnitric oxide synthase (eNOS) expression and increased endo-thelial permeability to albumin, which for example, becomesevident as microalbuminuria or retinal edema.1,2 Therefore,microalbuminuria, formerly considered to be a biomarker ofdiabetic nephropathy (DN), is now considered to be rather abiomarker of systemic ED1 and a strong predictor of cardio-vascular (CV) morbidity and mortality as well in the generalpopulation.3,4 Because DN is one of the microvascular compli-cations of diabetes, its pathogenesis is strongly related to eNOS-mediated ED, because suppression or deletion of this enzyme issufficient to cause podocyte loss, leading to macroalbuminuriaand global glomerulosclerosis.2,5 Novel strategies targeting thevicious cycle of ED-kidney-CV disease are needed.6

Diabetes and CV diseases are associated with increasedplasma levels of the cysteine protease cathepsin S (Cat-S), and itselastolytic properties contribute to CV mortality.7–12 Cystatin Climits the extracellular proteolytic activity of Cat-S to approxi-mately 1%.13 Cat-S induction or cystatin C suppression tilts thisbalance toward degeneration of the vascular wall,14 which drivesprogressive CV disease and all-cause mortality in patients withCKD.15 So far, the functional role of Cat-S has been related to itselastolytic protease activity in macrovascular diseases,7–11 butCat-S was also recognized as a protease-activated receptor-2(PAR2) agonist.16 Hence, we speculated on functional roles ofCat-S also on microvascular disease. Our data confirm thishypothesis and identify Cat-S as a circulating trigger of EDby activating PAR2 on endothelial cells, a process promotingmicrovascular complications in diabetes (e.g., DN and diabeticretinopathy [DR]).

RESULTS

Cat-S Induces Endothelial Cell Injury and MicrovascularPermeability through PAR2 In VivoTo test our hypothesis, we injected C57BL/6 mice with a singleintravenous dose of 10 mg recombinant Cat-S. Cat-S injectioninduced immediate and transient albuminuria at 90 minutes,which was blunted by pretreatment with two different Cat-Santagonists (Figure 1A).17 Transmission electron microscopy(EM) at 24 hours revealed that both drugs prevented the Cat-S–induced massive cytoplasmic swelling and vacuolization ofglomerular endothelial cells (GEnCs), whereas podocyte foot

processes remained unaffected by Cat-S (Figure 1B). Cat-S–induced albuminuria and endothelial cell injury were absentin Par2-deficient mice or on PAR2 antagonism (Figure 1, Cand D). To evaluate the role of intrinsic Cat-S in ED, we visual-ized the postischemic microvascular permeability of cremastermuscles by intravital microscopy. Both Cat-S antagonists aswell as Par2 deficiency completely diminished the extravasationof FITC-labeled dextran from the microvasculature (Figure 1,E and F) without affecting hemodynamic parameters or systemicleukocyte counts (Supplemental Figure 1). Together, extrinsicand intrinsic Cat-S promotes endothelial cell injury and micro-vascular permeability through PAR2 in vivo.

Cat-S Specifically Induces GEnC Dysfunction throughPAR2 In VitroTo better understand Cat-S–induced ED, we used the electriccell impedance sensing (ECIS) assay system on culturedGEnCs, podocytes, and tubular epithelial cells in vitro.18 In-creasing doses of recombinant Cat-S had no effect on podo-cytes or tubular epithelial cells (Supplemental Figure 2, Aand B), whereas GEnCs showed a significant increase in trans-cellular capacitance as a marker of monolayer permeability(Figure 2A). Pharmacologic Cat-S inhibition reversed thiseffect (Figure 2B). Scanning EM of such GEnC monolayersrevealed Cat-S–induced monolayer disruption, which wasprevented by Cat-S or PAR2 inhibition (Figure 2C). PAR2 in-hibition also prevented Cat-S–induced production of reactiveoxygen species or Cat-S–induced FITC-albumin permeabilityof cultured GEnCs (Figure 2, D and E). Furthermore, the Cat-S–induced death and detachment of cultured GEnCs wereprevented by siRNAs specific for PAR2 but not by siRNA spe-cific for PAR1, PAR3, or PAR4 (Figure 2, F and G). In contrast,Cat-S did not affect plastic adherence of podocytes, tubularepithelial cells, and mesangial cells with or without PAR2siRNA (Supplemental Figure 2, C–E). Transcriptome analysisof GEnC 6 hours after Cat-S exposure revealed that Cat-Ssuppressed the mRNA expression levels of mitosis-relatedgenes, whereas only a few gene groups were induced (Supple-mental Figure 3). This effect could be reversed by PAR2inhibition. Together, Cat-S specifically impairs endothelialbarrier function by inducing PAR2–dependent endothelialcell injury and dysfunction in vitro.

Cat-S and Cystatin C Expression in Human DiabeticKidney DiseaseED and increased microvascular permeability are centralpathogenic mechanisms in diabetes complications,2 but a po-tential role of Cat-S in this context is speculative. Hence, we

comparedwithhealthy kidneys,whichwaspreventedbypretreatmentwithbothCat-S inhibitors. (CandD)Also, PAR2 inhibition and lackofthe Par2 gene had the same protective effect on albuminuria and glomerular ultrastructure. (E and F) FITC dextran leakage observed byintravital microscopy was used as a marker of microvascular permeability in the postischemic (ischemia-reperfusion) cremaster muscle ofwild-type and Par2-deficient mice. Data are means6SEMs of four mice in each group. Representative images of FITC dextran leakage frompostcapillary venules are shown. Original magnification,340. *P,0.05 versus PBS/vehicle group; **P,0.01 versus PBS/vehicle group.

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Figure 2. Cat-S specifically induces ED through PAR2 in vitro. (A and B) In vitro ECIS studies with GEnCs. (A) GEnC monolayers wereexposed to increasing doses of Cat-S, and cell capacitance at 40 kHz was determined over a period of 9 hours. Note the dose-dependent

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first assessed the expression of Cat-S and its endogenous in-hibitor cystatin C in humans. The protein atlas online systemfor human tissues reports Cat-S and cystatin C expression onlyin the monocytic phagocyte system of the skin, lung, and liverand predominant Cat-S expression within the red (not white)pulp of the spleen and in epithelia of the intestinal tract, gallbladder, and renal proximal tubules (www.proteinatlas.org).Cat-S immunostaining of healthy human kidney biopsies lo-calized strong Cat-S positivity to proximal tubular epithelialcells and weak staining at GEnCs and parietal epithelial cells(Figure 3A). Human DN revealed additional Cat-S positivityinside glomerular capillaries (Figure 3B) and in infiltratingCD68+ macrophages on single and double immunostaining(Figure 3, C and D). Interestingly, in situ hybridization con-firmed Cat-S mRNA expression only in CD68+ intrarenalmacrophages and not in parenchymal cells (Figure 3E), a find-ing consistent with our recently reported data on kidney, lung,and spleen of MRLlpr mice.17 In contrast, cystatin C immu-nostaining of healthy kidneys or DN localized to tubular ep-ithelial cells only (Supplemental Figure 4). Microarray data ofmicrodissected glomerular and tubulointerstitial tissue sam-ples from human DN revealed 2- to 3-fold higher mRNA ex-pression levels for Cat-S but not cystatin C in DN versushealthy control kidneys, which implies an increased Cat-S/cystatin C ratio in DN (Supplemental Figure 5A). Real–timeRT-PCR confirmed a 2-fold induction of Cat-S mRNA in glo-meruli and a 2.5-fold induction in tubulointerstitial samplesfrom diabetic kidneys (Supplemental Figure 5B). Together,Cat-S and cystatin C protein colocalize in renal tubules. Be-cause renal nonimmune cells do not express Cat-S mRNA,circulating and filtered Cat-S protein is probably taken uppassively into tubular cells. Infiltrating CD68+ macrophagesproduce Cat-S (but no cystatin C) in DN.

Cat-S and Cystatin C Expression in Kidney Disease ofType 2 Diabetic db/db MiceIn solid organs of mice, Cat-S mRNA was consistently ex-pressed, albeit at a comparatively lower level compared withCat-A, -B, -D, -K, and -L, apattern thatwas especially evident inthe kidney (Supplemental Figure 6A). Cat-S mRNA and pro-tein (and Cat-A/K) were induced in kidneys of 6-month-oldmale type 2 diabetic (T2D) db/db mice versus nondiabeticmice, especially when early nephrectomy (1K) was used to

accelerate glomerulosclerosis (Figure 4, A and B, Supplemen-tal Figure 6B). The results of Cat-S immunostaining and in situhybridization results as well as cystatin C immunostaining in6-month-old db/db mice were identical to those in humans(Figure 4, C–E, Supplemental Figure 7). Thus, Cat-S expressionis induced among other Cats in diabetic kidney disease, butmRNA expression is restricted to mononuclear phagocytes.

RO5461111 Inhibits Cat-S Activity and ReducesPodocyte Loss, Proteinuria, Glomerulosclerosis, andRenal Inflammation in T2D db/db MiceTo test the functional contributionofCat-S indiabetes,weusedoral administration of RO5461111 (87.5 mg/kg in chow atapproximately 10 mg/kg) in 1K male db/db mice. This doseproduced stable plasma levels of RO5461111 at 400–600 ng/mlover the entire treatment period from months 4 to 6 of life.In vivo bioactivity of this dose was confirmed by a robust Lip10fragment accumulation in the spleen at month 6 (Figure 5A)as well as by measuring the plasma Cat-S activity onZ-VVR-AFC substrate at the beginning and the end of the study(Figure 5B). RO5461111 treatment did not affect food intake,body weight, blood glucose levels, or circulating leukocytecounts (Supplemental Figures 1 and 8, A and B) but signifi-cantly reduced renal Cat-S mRNA and protein expression(Figure 5, C–E). Regarding diabetes complications,RO5461111 treatment for 2 months significantly reduced in-jury of CD31+ GEnCs and glomerulosclerosis (Figure 5, F–J,Supplemental Figure 8C). Cat-S blockade reduced albumin-uria by 60% compared with the control diet–fed db/db mice(Figure 6A). This was associated with a significant increasein renal mRNA expression levels of slit diaphragm–relatedproteins nephrin and podocin (Figure 6B) and podocyte num-bers (Figure 6, C and D). Transmission EM revealed GEnCvacuolization, swelling, and loss of fenestrations in untreateddb/db mice, which was effectively reversed by RO5461111treatment (Figure 6E). The recovery of eNOS expression andthe suppression of VCAM were consistent with an improvedED (Figure 6F). As a consequence, RO5461111 significantlyreduced intrarenal CD45+ leukocytes (i.e., neutrophils andLy6C+ mononuclear phagocytes) and glomerular as well asinterstitial macrophages (Supplemental Figure 9, A–D). RenalmRNA expression levels of IL-6, TNF-a, iNOS, and CCL2 wereconsistently reduced (Supplemental Figure 9E). Taken together,

increase that occurs very quickly on Cat-S exposure. (B) Cat-S–induced increase of cell capacitance was reversed by RO5461111. Graphsare readings of single experiments representative of at least three experiments for each condition. (C) GEnCmonolayers were imaged byscanning EM after treatment as indicated. Representative images are shown. Note that either Cat-S (RO5461111) or PAR2 inhibitionprotects GEnCs from theCat-S–inducedmonolayer disintegration. (D) Cat-S–induced reactive oxygen species (ROS) production inGEnCswas determined by electron spin resonance. A PAR2-activating peptide (AP) served as a positive control. (E) Transwell endothelial cellmonolayer permeability assays with FITC albumin. Data represent FITC fluorescence in the lower well 1 hour after stimulation with Cat-Sand/or PAR2 inhibitor. Note that the Cat-S effects are reversed by a PAR2 inhibitor. *P,0.05 versus control; #P,0.05 versus PAR2-AP orCat-S. (F and G) Cell culture supernatant of GEnCs exposed to two different concentrations of Cat-S was quantified for (F) floating cells or(G) annexin positivity by flow cytometry as indicated. The cells had been pretreatedwith siRNAwith either scrambled sequence or specificsequences for PAR1, PAR2, PAR3, and PAR4. Data are means6SEMs of three identical experiments. *P,0.05 versus medium.

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oral RO5461111 treatment can effectively inhibit Cat-S bioac-tivity in db/db mice, which attenuates albuminuria, GEnC in-jury, podocyte loss, glomerulosclerosis, and renal inflammationas markers of advanced DN in db/db mice with T2D.

RO5461111 Attenuates DR in db/dbMiceDR is another clinically important micro-vascular complication of diabetes. Cat-Sblockade with RO5461111 significantly re-duced microvascular albumin leakage intothe inner plexiform layer of the retina com-pared with vehicle-treated mice (Figure 7).Cat-S blockade also restored eNOS proteinexpression in the retinal vasculature, a robustindicator of improved ED (Figure 7). Simi-larly, Cat-S blockade reduced the amount ofglial fibrillary acidic protein expression inMuller glial end feet at the internal limitingmembrane, a marker for retinal cell degen-eration and gliosis (Figure 7). Thus, Cat-Sblockade improves key components ofmicrovascular damage and retinal degener-ation in db/db mice.

PAR2 and Cat-S Inhibition Are bothEffective in AttenuatingGlomerulosclerosis and DR in db/dbMiceIf Cat-S signals through PAR2, then PAR2inhibition should also attenuate DN andDR.Also, PAR2 inhibitionoveronly 4weekssignificantly maintained GEnC CD31 pos-itivity and prevented global glomeruloscle-rosis and glomerular silver positivity(Figure 8, A–E). Concomitant Cat-S inhi-bition with RO5461111 had additive effectson CD31 positivity but not on glomerulo-sclerosis (Figure 8, A–E). PAR2 inhibitionand dual blockade were also equipotent inpreventing albumin leakage into the retina(Figure 8, F andG). Thus, late onset of Cat-SandPAR2 inhibition are both effective in atten-uating DN and DR in db/db mice with T2D.

DISCUSSION

ItwasknownthatplasmaCat-S levelscorrelatewith age, CV disease, and diabetes7–11,14

and that the proteolytic activity of Cat-Scan degrade elastic fibers (e.g., during theprogression of macrovascular diseases).19

As a new finding, the results of our ex-periments identify a previously unknownbiologic function of Cat-S (i.e., its agonis-

tic activity of PAR2 on the surface of endothelial cells). Cat-S–mediated PAR2 activation induces ED in vitro and in vivo.In mice with T2D, Cat-S–mediated PAR2 activation increa-ses the permeability of the microvasculature, which accounts

Figure 3. Cathepsin S is expressed by macrophages infiltrating the human kidney.Cat-S immunostaining in human DN. Archived kidney biopsies were stained for Cat-S.Representative images are shown at original magnifications of 3100, 3200, and31000. (A) A nondiabetic control kidney shows strong Cat-S positivity in proximaltubules. At a magnification of 31000, some positivity is noted in parietal epithelialcells as well as in podocytes in a cytoplasmic staining pattern. (B) In a patient with DN,Cat-S positivity localizes to infiltrating leukocytes inside the glomerulus. At a magnifica-tion of 31000, positivity is noted in leukocytes within capillary lumen and mesangium aswell as in GEnCs. (C) In a patient with advanced DN, Cat-S positivity localizes to interstitialcell infiltrates. (D) Dual staining for Cat-S (brown) and CD68 (red) identifies CD68+macrophages as a source of intrarenal Cat-S expression. (E) In situ hybridization does notdisplay any Cat-S mRNA in normal (panel 1) and diabetic glomeruli. In advanced DN, Cat-SmRNA was detected in interstitial cells that show a positive signal for CD68 (arrows).Original magnification, 3400.

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Figure 4. Cathepsin S is expressed by macrophages infiltrating the mouse kidney. Cat expression in kidneys of db/db mice. (A) mRNAexpression levels of cysteine Cats in kidneys from 6-month-old nondiabetic and diabetic db/db mice; db/db mice were either shamoperated (2K) or uninephrectomized (1K) at 6 weeks of age. Data are means6SEMs of 5–12 mice in each group, and the values given

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for albuminuria or retinal edema as two typical crovascular di-abetes complications. Vice versa, inhibitors for either Cat-S orPAR2 reversed this previously unknown pathomechanism andprotected T2D db/dbmice fromDNand DR.We conclude thatCat-S and PAR2 represent novel molecular targets for the pro-phylaxis or therapy of microvascular (and macrovascular)complications in diabetes.

ED is evident indiabetes andmanifests by increased vascularpermeability and associated transudates in the retinal fundusandalbumin leakage fromglomerular capillaries into theurine.ED is not only a central pathophysiologic event in diabeticvascular disease but also, serves as a predictive biomarker ofdisease progression1,20 and CV complications in patients withdiabetes like in nondiabetic populations.21–24 In fact, we foundCat-S to mediate ED and microvascular permeability not onlyin diabetes but also, in postischemic microvascular injury andon injection of recombinant Cat-S into mice. In diabetic mice,Cat-S inhibition reversed albuminuria in db/db mice alongwith a restoration of fenestrations and other ultrastructuralabnormalities of GEnC. Our in vitro studies document thatCat-S specifically and dose dependently increases endothelialcell monolayer permeability by inducing endothelial cell de-tachment and death, an effect that was not found in podocytesor other epithelial cells. Cat-S toxicity on endothelial cells invivo is less severe than in vitro, potentially because of the pres-ence of protease inhibitors,13,14 although cystatin C levels aredecreased in progressive CV disease, which should enhancethe protease activity of cysteine proteases.25 Cat-S–inducedED involves other functional endothelial cell changes, includ-ing increased oxidative stress together with reduced eNOSexpression,1,26 which is now thought to be an importantpathophysiologic link between ED and the functional and his-topathologic alterations in DN.1,2 In fact, db/db mice showimpaired eNOS activation along the progression of DN, andrestoring activity of this enzyme reduced albuminuria as amarker of improved ED.27 Cat-S inhibition markedly in-creased renal eNOS expression, which was associated withless proteinuria and protection from glomerulosclerosis inT2D db/db mice. Similarly, Cat-S inhibition also restoredeNOS expression in retinas of db/db mice, which was associ-ated with less retinal glial fibrillary acidic protein expressionas a marker for retina gliosis. Our mouse model did not showproliferative DR, but neoangiogenesis, another ED–relatedmicrovascular complications in DR, is also driven by Cat-S.28

Tissue remodeling in DN involves cytokine and chemokineproduction and the recruitment of proinflammatory and

profibrotic macrophages that amplify the inflammatory re-sponse and produce profibrotic mediators, respectively.5,29,30

ED is manifested by endothelial cell activation and luminalexpression of adhesion molecules and chemokines known formacrophage recruitment.29 Cat-S has the potential to modu-late this inflammatory component of vascular disease, be-cause activated macrophages produce Cat-S and contributeto vascular remodeling in atherosclerosis and vascular inflam-mation.9,31,32

Cat-S consistently induced ED in vitro and in vivo throughPAR2, which is in line with a recent report on Cat-S cleavage ofthe extracellular domain of PAR2 required for activation.16

Madhusudhan et al.33 reported human kidney to stain positivefor PAR2 in podocytes. We got similar results with severaldifferent antibodies inmouse kidney, but PAR2–deficient kidneysections unraveled this staining as unspecific (data not shown).We also did not detect any functional effects of PAR2 stimulationon podocytes. However, it is likely that Cat-S blockade also af-fects PAR2 signaling on other cell types in vivo. PARs are knownmodulators of vascular pathologies.34 Thrombin activates PAR1,PAR3, and PAR4.34 Activated protein C cleaves PAR1 and PAR2,and trypsin is a known activator of PAR2.34 The significance ofCat-S–mediated activation of PAR2 is supported by the protec-tive effects of PAR2 and concomitant Cat-S inhibition on DNand DR in db/dbmice, which did not exceed the effects of PAR2inhibition alone. These data are not unequivocal but consistentwith the concept that Cat-S–mediated PAR2 activation is a pre-viously unknownpathogenicmechanism inmicrovascular com-plications of diabetes.

In situ hybridization, immunolocalization studies in hu-man DN, and a relevant murine model of DN were consistentin terms of a lack of Cat-S mRNA expression in renal paren-chymal cells, whereas Cat-S protein was abundantly found intubular epithelial cells. This implies that circulating Cat-S pro-tein is filtered and passively reabsorbed in the renal tubules.Advanced DN was associated with a significant increase inrenal Cat-S mRNA expression, which we could localize to in-filtrating mononuclear phagocytes. In addition, Cat-S proteinlocalized to endothelial cells along the glomerular filtrationbarrier in association with significant ultrastructural injurysimilar to what has been described in human DN.35

Together, blocking Cat-S/PAR2–mediated ED preventsDN and DR in db/db mice. Therefore, therapeutic Cat-S orPAR2 inhibition may help to prevent and improve outcomesof microvascular (and macrovascular) complications indiabetes.

are normalized to 18S rRNA. (B) Western blot for Cat-S from kidney tissue obtained from the same mice. b-Actin is shown as a proteinloading control. (C) Cat-S immunostaining on the same kidneys localizes Cat-S protein mostly to parietal and proximal tubular epithelialcells. (D and E) In situ hybridization of mouse kidneys for Cat-S colocalizes with EMR1 mRNA expression, indicating Cat-S expression ininfiltratingmononuclear phagocytes. In contrast, no Cat-S signal is detectable in renal parenchymal cells. EMR1, epidermal growth factor-like module containing mucin-like hormone receptor 1; KO, knockout; WT, wild type. Original magnification,3400. #P,0.05 versus wildtype; *P,0.05 versus sham group.

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Figure 5. RO5461111 selectively inhibits Cat-S in vivo and prevents glomerulosclerosis in db/db mice. (A) db/db Mice were treatedfrom months 4 to 6 of age with RO5461111, which significantly increased the spleen levels of the Cat-S substrate Lip10 compared withvehicle-treated mice of the same age. The graph represents the ratio of Western blot signals for p10 and the invariant chain Li. Theeffect is indicative of target inhibition. (B) Cat-S enzymatic activity in plasma was determined by a substrate assay in samples obtained

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CONCISE METHODS

Human StudiesHuman renal biopsies from consented patients and controls were

collected.36 Microarray data from patients with DN and healthy con-

trols of the same age and sex distribution were retrieved from the

Woroniecka Database in Nephromine (http://www.nephromine.

org). For confirmatory real–time RT-PCR human renal biopsy speci-

mens from an international multicenter study, the European Renal

cDNA Bank—Kröner–Fresenius Biopsy Bank (participating centers

are in Supplemental Appendix) was used. Biopsies were obtained

from patients after informed consent and with approval of the local

ethics committees.37 Real–time RT-PCR was performed on micro-

dissected specimens from clinically indicated biopsies from patients

with diabetic kidney disease (n=12) and pretransplant kidney biop-

sies from living donors as controls (n=9) as previously described.38

Predeveloped TaqMan reagents were used for Cat-S (CTSS;

NM_004079) and the housekeeper gene 18S rRNA (Applied Biosys-

tems). The mRNA expression was analyzed by standard curve quan-

tification. Data shown are normalized to GAPDH, and target gene

expression in the control cohort is set as one.

RO5461111, a Novel Potent and Selective Inhibitor ofCat-SRO5461111 (CAS 1252637–46–9) was provided by Hoffmann La

Roche (Basel, Switzerland). It is a potent competitive inhibitor of

the active site of Cat-S, and its nitril function allows covalent revers-

ible inhibition of Cat-Swith an IC50 of 0.4 nM andmurine Cat-S with

an IC50 of 0.5 nM. The synthesis and drug development of

RO5461111 have been described in patent no. WO 2010121918.

Recombinant Cats were produced in house or purchased from com-

mercial vendors. Enzymatic assays were performed using the fluoro-

genic substrate (Z-Val-Val-Arg-AMC for human and mouse Cat-S,

human Cat-L and Cat-B, and Z-Leu-Arg-AMC for human Cat-K and

Cat-V). The generation of fluorescence over 10 minutes was mea-

sured using 10 mM substrate and 1 nM enzyme in the presence of

rising concentrations of RO5461111 to determine IC50 values. The

cellular activity of RO5461111 was tested in B cell lines of human

(RAJI) or mouse (A20) origin. Rising concentrations of RO5461111

were incubated overnight, and protein extracts were prepared for

detection of p10 upregulation by Cat-S inhibition on Western blots.

For the determination of in vivo enzyme inhibition activity of Cat-S

by RO5461111, BALBc mice were used. After oral dosing of 0.1–100

mg/kg RO5461111, mice were euthanized after 7 hours, and spleens

were harvested. Protein extracts were separated by SDS-gel electro-

phoresis for the determination of p10 upregulation by Cat-S inhibi-

tion as described below. In some experiments, we used 3mg/kg of the

previously described Cat-S inhibitor LY3000328 as a reference.39

Animal StudiesSix-week-old male wild–type or Par2–deficient C57BL/6 mice (The

Jackson Laboratory, Bar Harbor, ME) were injected intravenously

with 10 mg Cat-S; some of them were pretreated orally with two

different Cat-S inhibitors, RO5461111 (for 1 week with food) and

LY3000328 (10 mg/kg orally two times per day; provided by Fa.

Grünenthal, Aachen, Germany), or PAR2 inhibitor (10 mg/kg intra-

peritoneally, GB-83, CAS. 1252806–86–2; Axon Medchem, NL).

Urine was collected at different time intervals, analyzed for albumin

by ELISA (Bethyl Labs,Montgomery, TX), and reported as albumin-to-

creatinine ratio. After 24 hours, mice were euthanized, and kidneys

were fixed in EM fixative buffer. Five-week-old male diabetic

C57BLKS db/db or nondiabetic C57BL/6 mice (Taconic, Ry, Denmark)

underwent uninephrectomy (1Kmice) or sham surgery (2Kmice) at 6

weeks of age as described.40 After 4 months, 1K db/db mice were

divided into groups fed with either food-drug mix containing

RO5461111 (87.5 mg/kg delivering a daily dose of 10 mg/kg body

wt) or standard food until tissue harvest at 6 months. In another

experiment, db/db mice received the PAR2 inhibitor FSLLRY-amide

(Cayman Chemicals, Ann Arbor, MI) at 3 mg/kg daily intraperitone-

ally. Urinary albumin and creatinine were determined by ELISA

(Bethyl Labs, DiaSys Diagnostic Systems, Holzheim, Germany).

Spleens were used to estimate the Lip10 levels. DR was assessed by

staining for extravascular albumin (1:100; Abcam, Cambridge, United

Kingdom), glialfibrillary acidic protein (1:500;Dako,Dostrup,Denmark),

and eNOS expression (1:100; Abcam), and they were quantified in five

retina sections from each mouse at 340 using ImageJ software. Only

albumin not colocalizing with lectin+ blood vessels was measured.

The surgical procedure and the technical setup for in vivomicroscopy

on the mouse cremaster muscle and how to analyze postischemic

FITC-albumin (Sigma-Aldrich, Steinheim, Germany) leakage have

been previously described in detail.41 Briefly, in Par2-deficient or

wild-type mice treated with RO5461111, LY3000328, or vehicle, base-

line measurements were performed before ischemia of the cremaster

muscle was induced for 30 minutes, and postischemic microvascular

at 16 and 24 weeks as indicated from vehicle–treated 1K db/dbmice (black bars) and RO5461111–treated 1K db/dbmice (white bar). (C)KidneyCat-S protein levels were determined byWestern blot. The respectiveb-actin expression served as protein loading control. (D) Therespective quantification of the Western blot results is shown as means6SDs. (E) The spleen Cat-S mRNA expression levels were quan-tified by real–time RT-PCR and are expressed as the ratio to the respective 18s rRNA expression levels. Data in A, B, D, and E are means6SEMs from 8 to 12mice in each group. Kidney sections fromboth treated and untreatedmicewere stainedwith (F andG) anti-CD31 to seeGEnCs and quantification of CD31 signal in each glomeruli and (H) periodic acid–Schiff (PAS) reagent to (I) quantify glomerulosclerosis.Representative glomeruli are shown from each group at an original magnification of 3400; 50 glomeruli were scored by using a semi-quantitative PAS scores ranging from zero to four. (J) Silver-stained sections were digitally scored as a marker of fibrous extracellularmatrix. The graphs inG, I, and J illustrate themean percentage of each score6SEM from all mice in each group.Note that uninephrectomywas associated with a shift toward higher scores as seen in vehicle-treated mice, but with RO5461111 treatment, the overall scores weresignificantly reduced. WT, wild type. *P,0.05 versus vehicle-treated group; **P,0.01 versus vehicle-treated group; ***P,0.001 versusvehicle-treated group; #P,0.05 versus the earlier time point.

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Figure 6. Cat-S inhibition protects the glomerular filtration barrier in db/db mice. (A) Urinary albumin-to-creatinine ratios were de-termined as a functional marker of the glomerular filtration barrier in 6-month-old 1K db/db mice with or without treatment. (B) RNAisolates from kidneys of both treated and untreated 6-month-old 1K db/db mice underwent quantitative real–time PCR for the podocyte–specific genes nephrin and podocin as indicated. (C) Renal sections from mice of both treated and untreated groups were stained for Wilms

J Am Soc Nephrol 27: 1635–1649, 2016 Cat-S/Par2 Mediate Endothelial Dysfunction 1645

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permeability (after 160 minutes of reperfusion)

was studied. All experiments were performed ac-

cording to German animal protection laws and

had been approved by the local government

authorities.

Histopathologic EvaluationKidneys werefixed in 4% formalin, embedded in

paraffin, and stained with periodic acid–Schiff

reagent. Sclerotic lesions were scored on 50 glo-

meruli as follows: 0, no lesion; 1, ,25%; 2,

25%–49%; 3, 50%–74%; 4, 75%–100% scle-

rotic. Immunostaining was performed as de-

scribed42 using the following primary antibodies:

Cat-S (polyclonal goat anti–human Cat-S;

1:1000; Abcam), cystatin C (polyclonal rabbit

anti–human; 1:5000; EMD Millipore), CD45

(leukocytes; 1:10; BD Biosciences Pharmingen,

San Diego, CA), Wilms Tumor-1 (podocytes;

1:200; Santa Cruz Biotechnology, Santa Cruz,

CA), Mac2 (1:5000; Cederlane), and terminal de-

oxynucleotidyl transferase–mediated digoxigenin–

deoxyuridine nick–end labeling (apoptotic cells;

Roche, Mannheim, Germany). For EM, the kid-

ney cortex was cut into 131-mm cubes and im-

mediately immersed in fixative containing 3%

glutaraldehyde and 1% paraformaldehyde in

PBS. Transmission or scanning EM was per-

formed as described.43 In situ hybridization was

performed using the Quantigene ViewRNA ISH

Tissue Assay Kit (Affymetrix/Panomics Solutions)

and the following Affymetrix probe sets: human

CTSS, NM_004079-NM_001199739; mouse Ctss,

NM_001267695- NM_021281; human CD68,

NM_001251-NM_001040059; mouse Cd68,

NM_001291058-NM_009853; and mouse Emr1,

NM_010130.

Western BlottingProtein isolation and Western blotting were per-

formed as described using goat anti–mouse Cat-S

(R&D Systems, Minneapolis, MN) and detected

with a peroxidase–conjugated donkey anti–goat

(Dianova, Hamburg, Germany).44 Invariant chain

was quantified by Western blot from mouse

Tumor-1 (WT-1) and nephrin. Original magnification,3400. (D) The graph shows the mean number of WT-1–positive cells in 15 glomeruli6SEM in sections from 6-month-old 1K db/db mice with or without treatment. Note the potent effect of RO5461111 on the number ofpodocytes. Data in A–D are means6SEMs of 5–12 mice in each group. (E) Transmission EM of glomeruli from 1K db/db mice at 6 monthsdisplayed morphologic signs of glomerular endothelial damage, such as cytoplasmic swelling with whorling cytoplasmic inclusions andloss of fenestrations (arrows) in vehicle–treated 1K db/dbmice (left panel). In addition, podocytes showed partial foot process effacementas a sign of podocyte injury (asterisks). Treatment with RO5461111 prevented these abnormalities (right panel). (F) Real–time RT-PCR forVCAM and eNOS on total renal mRNA as markers of ED. Data in B and E are expressed as means of the ratio of the specific mRNA versusthose of 18S rRNA 6SEMs. VCAM, vascular cell adhesion molecule. *P,0.05 versus vehicle group; **P,0.01 versus vehicle group.

Figure 7. Cat-S inhibition attenuates DR of db/db mice. Vascular leakage of serumalbumin from isolectinB4–labeled blood vessels is reduced in (C) RO5461111–treateddb/db mice compared with (A) vehicle-treated mice. B and D show albumin leakage ata higher magnification. Scale bar, 50 mm. (E and F) Vehicle–treated 1K db/db micedisplayed increased glial fibrillary acidic protein (GFAP) expression in retinal astro-cytes, which was reduced by RO5461111. (G and H) RO5461111 also restored retinaleNOS expression. (I–K) The respective quantitative analysis is shown below. Datarepresent means6SDs of five retina cross-sections per mouse and five mice per group.GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL,outer nuclear layer; OPL, outer plexiform layer. Scale bars, 100 mm. ***P,0.001 versusvehicle.

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splenocyte supernatants loaded onto 10% SDS-

PAGE gel. On transfer onto a nitrocellulose

membrane, a rabbit anti-CD74 (BD Biosciences,

San Jose, CA) was used.

Flow CytometryFlow cytometry of the kidney cell suspensions was

performed as described45 using the following

conjugated antibodies: PE anti-CD45, APC anti-

CD11b, anti-Gr-1, FITC anti-Ly6C, and anti-

Ly6G (BD Biosciences). Cultured mouse GEnCs

that detached from the plate were stained with

either FITC antiannexinV or propidium iodide

(Becton, Dickinson & Company (BD), Franklin

Lakes, NJ).

Real–Time RT-PCRTotal mRNA fromwhole kidneys, cultured mouse

tubular cells, GEnCs, and podocytes was tran-

scribed into cDNA using Superscript II and sub-

jected to real-time PCRon a Light Cycler 480 (Real

Time PCRDetection Systems; Roche) using SYBR

green (SABiosciences) as described38 using the

genes listed in Supplemental Table 1. The mRNA

expression values for all genes were normalized to

18s rRNA.

In Vitro StudiesPrimary mouse GEnCs, tubular epithelial cells,

mesangial cells, and podocytes were generated

and cultured as described,46–48 preferentially in

serum-freemedia. Cat-S–induced changes in re-

sistance and capacitance of all cells types were

analyzed using an ECIS device (Applied Bio-

physics) at a density of 100,000 cells per well

in a volume of 400 ml media placed in ECIS–

compatible microwell slides as previously de-

scribed.49 Confluent monolayers were then

stimulated with different doses of Cat-S with

or without RO5461111. Capacitance was ana-

lyzed for indicated time points at 4 kHz. The

transwell permeability of FITC-conjugated al-

bumin in GEnC monolayer was performed as

described.50 Reactive oxygen species production

in cultured GEnCs was determined using the

electron spin resonance method. In brief, con-

fluent GEnCs were stimulated with Cat-S with

or without inhibitors in serum–free RPMI me-

dia. The cells were then washed two times with

2 ml Krebs-Hepes Buffer and incubated with

spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-

tetramethylpyrrolidine in buffer for 1 hour at

37°C. Full transcriptome sequencing on an

IonTorrentProtonSystemwasused forGEnCtran-

scriptome analysis.51 For PAR2 siRNA transfection,

Figure 8. PAR2 inhibition attenuates glomerulosclerosis and retinopathy in db/db mice. Maleuninephrectomized db/dbmice were treated with either vehicle or PAR2 inhibitor with or withoutRO5461111 from months 4 to 6 of age. (A) Kidney sections from untreated (left panel), PAR2inhibitor (center panel), and PAR2 and Cat-S inhibitor–treated (right panel) mice were stained withanti-CD31 to see GEnCs, and (B) periodic acid–Schiff (PAS) -stained sections shown at an originalmagnification of 3400 were used for scoring of glomerulosclerosis (0, no lesion; 1, #25%; 2,#50%; 3, #75%; 4, #75% sclerosis). Data are mean scores of 50 glomeruli per mouse6SEM.(C–E) quantification of CD31 fluorescence intensity, average PAS score, and mean silver area inthe kidneys of the above groups. (F and G) Retinas were prepared form the same mice, andalbumin leakage was assessed by immunostaining. Data represent means6SDs of five retinacross-sections per mouse and five mice per group. GCL, ganglion cell layer; INL, inner nuclearlayer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars,100 mm. *P,0.05 versus vehicle; **P,0.01; ***P,0.001. DAPI, 49,6-diamidino-2-phenylindole.

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2 million GEnCs were suspended in 100 ml 1 M nucleofection buffer,

siRNA and PAR2 (catalog no. AM16708 and ID no. 157431, respectively;

Ambion) were added at concentrations of 600 and 2000 ng, and scram-

bled siRNA (catalog no. 4390843; Ambion) controlswere alsomaintained

for the comparison. Lonza electroporation was used for nucleofection

using the preloaded program V-001 (compatible for endothelial cells);

then, the cells were immediately mixed with normal RPMI media con-

taining 1% FCS and 1% PS. The efficacy of transfectionwas measured by

qRT-PCR.GEnCswere cultured overnight in 12-well plates at a density of

1 million cells per well before serum fasting for 2 hours and stimulation

with different concentrations of Cat-S (1 and 2 mg/ml) for 20 hours.

Detached cells were counted using a scepter handheld automated cell

counter (catalog no. PHCC00000; EMD Millipore) and then stained

with Annexin/propidium iodide for flow cytometric analysis. Similarly,

siRNA for PAR1, PAR3, and PAR4 (catalog no. AM16708; gene ID nos.

157455, 158458, and 157434;Ambion) at a concentrationof 2000nMwas

used before analyzing GEnC detachment. Mouse podocyte, tubular epi-

thelial, and mesangial cells lines were seeded at densities of 50,000 and

250,000 cells, in 6- and 12-well plates, respectively. After allowing them

to adhere overnight the cells were stimulated with 2 mg/ml Cat-S for

20 hours and the detached cells were counted as before.

Statistical AnalysesData are presented as means6SEMs. Comparison of groups was

performed using ANOVA, and post hoc Bonferroni correction was

used for multiple comparisons. A value of P,0.05 was considered

to indicate statistical significance. Transcriptome data processing

and gene-level analysis were performed using DEseq package

(http://genomebiology.com/2010/11/10/R106).

ACKNOWLEDGMENTS

We thankDanDraganovic and JanaMandelbaum for expert technical

assistance andMarc Weidenbusch for help preparing figures. We also

thank Thomas Reinheckel (University of Freiburg) for providing

tissue from Cat-S–deficient mice.

This study was supported by Deutsche Forschungsgemeinschaft

Grants SFB 914 Project B3 to C.R. and AN372/22-1 to H.-J.A.

Parts of this project were prepared as a doctoral thesis at the Faculty

of Medicine, University of Munich by M.N.D.

DISCLOSURESS.G., W.H., and G.H. are employees from Hoffmann La Roche. All other

authors declare no duality of interest associated with this manuscript.

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See related editorial, “Cathepsin S–Dependent Protease–Activated Receptor-2 Activation: A New Mechanism of Endothelial Dysfunction,” on pages 1577–1579.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2015020208/-/DCSupplemental.

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