Basic Research

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular 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 and Hans-Joachim Anders
JASN June 2016, 27 (6) 1635-1649; DOI: https://doi.org/10.1681/ASN.2015020208

Abstract

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia–induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68+ intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage–derived circulating PAR2 agonist and mediator of endothelial dysfunction–related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

Morbidity and mortality in diabetes are largely caused by macro- and microvascular complications. The metabolic changes of diabetes induce endothelial dysfunction (ED), which is critical to the initiation and progression of vascular complications.1 ED is associated with decreased endothelial nitric oxide synthase (eNOS) expression and increased endothelial permeability to albumin, which for example, becomes evident as microalbuminuria or retinal edema.1,2 Therefore, microalbuminuria, formerly considered to be a biomarker of diabetic nephropathy (DN), is now considered to be rather a biomarker of systemic ED1 and a strong predictor of cardiovascular (CV) morbidity and mortality as well in the general population.3,4 Because DN is one of the microvascular complications of diabetes, its pathogenesis is strongly related to eNOS-mediated ED, because suppression or deletion of this enzyme is sufficient to cause podocyte loss, leading to macroalbuminuria and global glomerulosclerosis.2,5 Novel strategies targeting the vicious cycle of ED-kidney-CV disease are needed.6

Diabetes and CV diseases are associated with increased plasma levels of the cysteine protease cathepsin S (Cat-S), and its elastolytic properties contribute to CV mortality.7–12 Cystatin C limits the extracellular proteolytic activity of Cat-S to approximately 1%.13 Cat-S induction or cystatin C suppression tilts this balance toward degeneration of the vascular wall,14 which drives progressive CV disease and all-cause mortality in patients with CKD.15 So far, the functional role of Cat-S has been related to its elastolytic protease activity in macrovascular diseases,7–11 but Cat-S was also recognized as a protease-activated receptor-2 (PAR2) agonist.16 Hence, we speculated on functional roles of Cat-S also on microvascular disease. Our data confirm this hypothesis and identify Cat-S as a circulating trigger of ED by activating PAR2 on endothelial cells, a process promoting microvascular complications in diabetes (e.g., DN and diabetic retinopathy [DR]).

Results

Cat-S Induces Endothelial Cell Injury and Microvascular Permeability through PAR2 In Vivo

To test our hypothesis, we injected C57BL/6 mice with a single intravenous dose of 10 µg recombinant Cat-S. Cat-S injection induced immediate and transient albuminuria at 90 minutes, which was blunted by pretreatment with two different Cat-S antagonists (Figure 1A).17 Transmission electron microscopy (EM) at 24 hours revealed that both drugs prevented the Cat-S–induced massive cytoplasmic swelling and vacuolization of glomerular endothelial cells (GEnCs), whereas podocyte foot processes remained unaffected by Cat-S (Figure 1B). Cat-S–induced albuminuria and endothelial cell injury were absent in Par2-deficient mice or on PAR2 antagonism (Figure 1, C and D). To evaluate the role of intrinsic Cat-S in ED, we visualized the postischemic microvascular permeability of cremaster muscles by intravital microscopy. Both Cat-S antagonists as well as Par2 deficiency completely diminished the extravasation of FITC-labeled dextran from the microvasculature (Figure 1, E and F) without affecting hemodynamic parameters or systemic leukocyte counts (Supplemental Figure 1). Together, extrinsic and intrinsic Cat-S promotes endothelial cell injury and microvascular permeability through PAR2 in vivo.

Figure 1.
Figure 1.

Extrinsic and intrinsic Cat-S triggers endothelial cell injury and microvascular permeability through PAR2 in vivo. (A) C57BL/6 mice were injected with a single dose of 10 µg Cat-S ± pretreatment with two different Cat-S antagonists. Urinary albumin-to-creatinine (A/C) ratio was quantified at several time points as indicated. Data are means±SEMs; 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 compared with healthy kidneys, which was prevented by pretreatment with both Cat-S inhibitors. (C and D) Also, PAR2 inhibition and lack of the Par2 gene had the same protective effect on albuminuria and glomerular ultrastructure. (E and F) FITC dextran leakage observed by intravital microscopy was used as a marker of microvascular permeability in the postischemic (ischemia-reperfusion) cremaster muscle of wild-type and Par2-deficient mice. Data are means±SEMs of four mice in each group. Representative images of FITC dextran leakage from postcapillary venules are shown. Original magnification, ×40. *P<0.05 versus PBS/vehicle group; **P<0.01 versus PBS/vehicle group.

Cat-S Specifically Induces GEnC Dysfunction through PAR2 In Vitro

To better understand Cat-S–induced ED, we used the electric cell impedance sensing (ECIS) assay system on cultured GEnCs, podocytes, and tubular epithelial cells in vitro.18 Increasing doses of recombinant Cat-S had no effect on podocytes or tubular epithelial cells (Supplemental Figure 2, A and B), whereas GEnCs showed a significant increase in transcellular capacitance as a marker of monolayer permeability (Figure 2A). Pharmacologic Cat-S inhibition reversed this effect (Figure 2B). Scanning EM of such GEnC monolayers revealed Cat-S–induced monolayer disruption, which was prevented by Cat-S or PAR2 inhibition (Figure 2C). PAR2 inhibition also prevented Cat-S–induced production of reactive oxygen species or Cat-S–induced FITC-albumin permeability of cultured GEnCs (Figure 2, D and E). Furthermore, the Cat-S–induced death and detachment of cultured GEnCs were prevented by siRNAs specific for PAR2 but not by siRNA specific for PAR1, PAR3, or PAR4 (Figure 2, F and G). In contrast, Cat-S did not affect plastic adherence of podocytes, tubular epithelial cells, and mesangial cells with or without PAR2 siRNA (Supplemental Figure 2, C–E). Transcriptome analysis of GEnC 6 hours after Cat-S exposure revealed that Cat-S suppressed the mRNA expression levels of mitosis-related genes, whereas only a few gene groups were induced (Supplemental Figure 3). This effect could be reversed by PAR2 inhibition. Together, Cat-S specifically impairs endothelial barrier function by inducing PAR2–dependent endothelial cell injury and dysfunction in vitro.

Figure 2.
Figure 2.

Cat-S specifically induces ED through PAR2 in vitro. (A and B) In vitro ECIS studies with GEnCs. (A) GEnC monolayers were exposed to increasing doses of Cat-S, and cell capacitance at 40 kHz was determined over a period of 9 hours. Note the dose-dependent increase that occurs very quickly on Cat-S exposure. (B) Cat-S–induced increase of cell capacitance was reversed by RO5461111. Graphs are readings of single experiments representative of at least three experiments for each condition. (C) GEnC monolayers were imaged by scanning EM after treatment as indicated. Representative images are shown. Note that either Cat-S (RO5461111) or PAR2 inhibition protects GEnCs from the Cat-S–induced monolayer disintegration. (D) Cat-S–induced reactive oxygen species (ROS) production in GEnCs was determined by electron spin resonance. A PAR2-activating peptide (AP) served as a positive control. (E) Transwell endothelial cell monolayer permeability assays with FITC albumin. Data represent FITC fluorescence in the lower well 1 hour after stimulation with Cat-S and/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 or Cat-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 pretreated with siRNA with either scrambled sequence or specific sequences for PAR1, PAR2, PAR3, and PAR4. Data are means±SEMs of three identical experiments. *P<0.05 versus medium.

Cat-S and Cystatin C Expression in Human Diabetic Kidney Disease

ED and increased microvascular permeability are central pathogenic mechanisms in diabetes complications,2 but a potential role of Cat-S in this context is speculative. Hence, we first assessed the expression of Cat-S and its endogenous inhibitor cystatin C in humans. The protein atlas online system for human tissues reports Cat-S and cystatin C expression only in the monocytic phagocyte system of the skin, lung, and liver and predominant Cat-S expression within the red (not white) pulp of the spleen and in epithelia of the intestinal tract, gall bladder, and renal proximal tubules (www.proteinatlas.org). Cat-S immunostaining of healthy human kidney biopsies localized strong Cat-S positivity to proximal tubular epithelial cells and weak staining at GEnCs and parietal epithelial cells (Figure 3A). Human DN revealed additional Cat-S positivity inside glomerular capillaries (Figure 3B) and in infiltrating CD68+ macrophages on single and double immunostaining (Figure 3, C and D). Interestingly, in situ hybridization confirmed Cat-S mRNA expression only in CD68+ intrarenal macrophages and not in parenchymal cells (Figure 3E), a finding consistent with our recently reported data on kidney, lung, and spleen of MRLlpr mice.17 In contrast, cystatin C immunostaining of healthy kidneys or DN localized to tubular epithelial cells only (Supplemental Figure 4). Microarray data of microdissected glomerular and tubulointerstitial tissue samples from human DN revealed 2- to 3-fold higher mRNA expression levels for Cat-S but not cystatin C in DN versus healthy control kidneys, which implies an increased Cat-S/cystatin C ratio in DN (Supplemental Figure 5A). Real–time RT-PCR confirmed a 2-fold induction of Cat-S mRNA in glomeruli and a 2.5-fold induction in tubulointerstitial samples from diabetic kidneys (Supplemental Figure 5B). Together, Cat-S and cystatin C protein colocalize in renal tubules. Because renal nonimmune cells do not express Cat-S mRNA, circulating and filtered Cat-S protein is probably taken up passively into tubular cells. Infiltrating CD68+ macrophages produce Cat-S (but no cystatin C) in DN.

Figure 3.
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 ×100, ×200, and ×1000. (A) A nondiabetic control kidney shows strong Cat-S positivity in proximal tubules. At a magnification of ×1000, some positivity is noted in parietal epithelial cells 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 magnification of ×1000, positivity is noted in leukocytes within capillary lumen and mesangium as well as in GEnCs. (C) In a patient with advanced DN, Cat-S positivity localizes to interstitial cell 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 not display any Cat-S mRNA in normal (panel 1) and diabetic glomeruli. In advanced DN, Cat-S mRNA was detected in interstitial cells that show a positive signal for CD68 (arrows). Original magnification, ×400.

Cat-S and Cystatin C Expression in Kidney Disease of Type 2 Diabetic db/db Mice

In solid organs of mice, Cat-S mRNA was consistently expressed, albeit at a comparatively lower level compared with Cat-A, -B, -D, -K, and -L, a pattern that was especially evident in the kidney (Supplemental Figure 6A). Cat-S mRNA and protein (and Cat-A/K) were induced in kidneys of 6-month-old male type 2 diabetic (T2D) db/db mice versus nondiabetic mice, especially when early nephrectomy (1K) was used to accelerate glomerulosclerosis (Figure 4, A and B, Supplemental Figure 6B). The results of Cat-S immunostaining and in situ hybridization results as well as cystatin C immunostaining in 6-month-old db/db mice were identical to those in humans (Figure 4, C–E, Supplemental Figure 7). Thus, Cat-S expression is induced among other Cats in diabetic kidney disease, but mRNA expression is restricted to mononuclear phagocytes.

Figure 4.
Figure 4.

Cathepsin S is expressed by macrophages infiltrating the mouse kidney. Cat expression in kidneys of db/db mice. (A) mRNA expression levels of cysteine Cats in kidneys from 6-month-old nondiabetic and diabetic db/db mice; db/db mice were either sham operated (2K) or uninephrectomized (1K) at 6 weeks of age. Data are means±SEMs of 5–12 mice in each group, and the values given are normalized to 18S rRNA. (B) Western blot for Cat-S from kidney tissue obtained from the same mice. β-Actin is shown as a protein loading control. (C) Cat-S immunostaining on the same kidneys localizes Cat-S protein mostly to parietal and proximal tubular epithelial cells. (D and E) In situ hybridization of mouse kidneys for Cat-S colocalizes with EMR1 mRNA expression, indicating Cat-S expression in infiltrating mononuclear 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, ×400. #P<0.05 versus wild type; *P<0.05 versus sham group.

RO5461111 Inhibits Cat-S Activity and Reduces Podocyte Loss, Proteinuria, Glomerulosclerosis, and Renal Inflammation in T2D db/db Mice

To test the functional contribution of Cat-S in diabetes, we used oral administration of RO5461111 (87.5 mg/kg in chow at approximately 10 mg/kg) in 1K male db/db mice. This dose produced stable plasma levels of RO5461111 at 400–600 ng/ml over the entire treatment period from months 4 to 6 of life. In vivo bioactivity of this dose was confirmed by a robust Lip10 fragment accumulation in the spleen at month 6 (Figure 5A) as well as by measuring the plasma Cat-S activity on Z-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 leukocyte counts (Supplemental Figures 1 and 8, A and B) but significantly reduced renal Cat-S mRNA and protein expression (Figure 5, C–E). Regarding diabetes complications, RO5461111 treatment for 2 months significantly reduced injury of CD31+ GEnCs and glomerulosclerosis (Figure 5, F–J, Supplemental Figure 8C). Cat-S blockade reduced albuminuria by 60% compared with the control diet–fed db/db mice (Figure 6A). This was associated with a significant increase in renal mRNA expression levels of slit diaphragm–related proteins nephrin and podocin (Figure 6B) and podocyte numbers (Figure 6, C and D). Transmission EM revealed GEnC vacuolization, swelling, and loss of fenestrations in untreated db/db mice, which was effectively reversed by RO5461111 treatment (Figure 6E). The recovery of eNOS expression and the suppression of VCAM were consistent with an improved ED (Figure 6F). As a consequence, RO5461111 significantly reduced intrarenal CD45+ leukocytes (i.e., neutrophils and Ly6C+ mononuclear phagocytes) and glomerular as well as interstitial macrophages (Supplemental Figure 9, A–D). Renal mRNA expression levels of IL-6, TNF-α, iNOS, and CCL2 were consistently reduced (Supplemental Figure 9E). Taken together, oral RO5461111 treatment can effectively inhibit Cat-S bioactivity in db/db mice, which attenuates albuminuria, GEnC injury, podocyte loss, glomerulosclerosis, and renal inflammation as markers of advanced DN in db/db mice with T2D.

Figure 5.
Figure 5.

RO5461111 selectively inhibits Cat-S in vivo and prevents glomerulosclerosis in db/db mice. (A) db/db Mice were treated from months 4 to 6 of age with RO5461111, which significantly increased the spleen levels of the Cat-S substrate Lip10 compared with vehicle-treated mice of the same age. The graph represents the ratio of Western blot signals for p10 and the invariant chain Li. The effect is indicative of target inhibition. (B) Cat-S enzymatic activity in plasma was determined by a substrate assay in samples obtained at 16 and 24 weeks as indicated from vehicle–treated 1K db/db mice (black bars) and RO5461111–treated 1K db/db mice (white bar). (C) Kidney Cat-S protein levels were determined by Western blot. The respective β-actin expression served as protein loading control. (D) The respective quantification of the Western blot results is shown as means±SDs. (E) The spleen Cat-S mRNA expression levels were quantified 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 means±SEMs from 8 to 12 mice in each group. Kidney sections from both treated and untreated mice were stained with (F and G) anti-CD31 to see GEnCs 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 ×400; 50 glomeruli were scored by using a semiquantitative PAS scores ranging from zero to four. (J) Silver-stained sections were digitally scored as a marker of fibrous extracellular matrix. The graphs in G, I, and J illustrate the mean percentage of each score±SEM from all mice in each group. Note that uninephrectomy was associated with a shift toward higher scores as seen in vehicle-treated mice, but with RO5461111 treatment, the overall scores were significantly reduced. WT, wild type. *P<0.05 versus vehicle-treated group; **P<0.01 versus vehicle-treated group; ***P<0.001 versus vehicle-treated group; #P<0.05 versus the earlier time point.

Figure 6.
Figure 6.

Cat-S inhibition protects the glomerular filtration barrier in db/db mice. (A) Urinary albumin-to-creatinine ratios were determined as a functional marker of the glomerular filtration barrier in 6-month-old 1K db/db mice with or without treatment. (B) RNA isolates 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 Tumor-1 (WT-1) and nephrin. Original magnification, ×400. (D) The graph shows the mean number of WT-1–positive cells in 15 glomeruli ±SEM in sections from 6-month-old 1K db/db mice with or without treatment. Note the potent effect of RO5461111 on the number of podocytes. Data in A–D are means±SEMs of 5–12 mice in each group. (E) Transmission EM of glomeruli from 1K db/db mice at 6 months displayed morphologic signs of glomerular endothelial damage, such as cytoplasmic swelling with whorling cytoplasmic inclusions and loss of fenestrations (arrows) in vehicle–treated 1K db/db mice (left panel). In addition, podocytes showed partial foot process effacement as a sign of podocyte injury (asterisks). Treatment with RO5461111 prevented these abnormalities (right panel). (F) Real–time RT-PCR for VCAM 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 versus those of 18S rRNA ±SEMs. VCAM, vascular cell adhesion molecule. *P<0.05 versus vehicle group; **P<0.01 versus vehicle group.

RO5461111 Attenuates DR in db/db Mice

DR is another clinically important microvascular complication of diabetes. Cat-S blockade with RO5461111 significantly reduced microvascular albumin leakage into the inner plexiform layer of the retina compared with vehicle-treated mice (Figure 7). Cat-S blockade also restored eNOS protein expression in the retinal vasculature, a robust indicator of improved ED (Figure 7). Similarly, Cat-S blockade reduced the amount of glial fibrillary acidic protein expression in Muller glial end feet at the internal limiting membrane, a marker for retinal cell degeneration and gliosis (Figure 7). Thus, Cat-S blockade improves key components of microvascular damage and retinal degeneration in db/db mice.

Figure 7.
Figure 7.

Cat-S inhibition attenuates DR of db/db mice. Vascular leakage of serum albumin from isolectinB4–labeled blood vessels is reduced in (C) RO5461111–treated db/db mice compared with (A) vehicle-treated mice. B and D show albumin leakage at a higher magnification. Scale bar, 50 μm. (E and F) Vehicle–treated 1K db/db mice displayed increased glial fibrillary acidic protein (GFAP) expression in retinal astrocytes, which was reduced by RO5461111. (G and H) RO5461111 also restored retinal eNOS expression. (I–K) The respective quantitative analysis is shown below. Data represent means±SDs 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 μm. ***P<0.001 versus vehicle.

PAR2 and Cat-S Inhibition Are both Effective in Attenuating Glomerulosclerosis and DR in db/db Mice

If Cat-S signals through PAR2, then PAR2 inhibition should also attenuate DN and DR. Also, PAR2 inhibition over only 4 weeks significantly maintained GEnC CD31 positivity and prevented global glomerulosclerosis and glomerular silver positivity (Figure 8, A–E). Concomitant Cat-S inhibition with RO5461111 had additive effects on CD31 positivity but not on glomerulosclerosis (Figure 8, A–E). PAR2 inhibition and dual blockade were also equipotent in preventing albumin leakage into the retina (Figure 8, F and G). Thus, late onset of Cat-S and PAR2 inhibition are both effective in attenuating DN and DR in db/db mice with T2D.

Figure 8.
Figure 8.

PAR2 inhibition attenuates glomerulosclerosis and retinopathy in db/db mice. Male uninephrectomized db/db mice were treated with either vehicle or PAR2 inhibitor with or without RO5461111 from months 4 to 6 of age. (A) Kidney sections from untreated (left panel), PAR2 inhibitor (center panel), and PAR2 and Cat-S inhibitor–treated (right panel) mice were stained with anti-CD31 to see GEnCs, and (B) periodic acid–Schiff (PAS) -stained sections shown at an original magnification of ×400 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 mouse±SEM. (C–E) quantification of CD31 fluorescence intensity, average PAS score, and mean silver area in the kidneys of the above groups. (F and G) Retinas were prepared form the same mice, and albumin leakage was assessed by immunostaining. Data represent means±SDs 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 μm. *P<0.05 versus vehicle; **P<0.01; ***P<0.001. DAPI, 4′,6-diamidino-2-phenylindole.

Discussion

It was known that plasma Cat-S levels correlate with age, CV disease, and diabetes7–11,14 and that the proteolytic activity of Cat-S can degrade elastic fibers (e.g., during the progression of macrovascular diseases).19 As a new finding, the results of our experiments identify a previously unknown biologic function of Cat-S (i.e., its agonistic 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 increases the permeability of the microvasculature, which accounts for albuminuria or retinal edema as two typical crovascular diabetes complications. Vice versa, inhibitors for either Cat-S or PAR2 reversed this previously unknown pathomechanism and protected T2D db/db mice from DN and DR. We conclude that Cat-S and PAR2 represent novel molecular targets for the prophylaxis or therapy of microvascular (and macrovascular) complications in diabetes.

ED is evident in diabetes and manifests by increased vascular permeability and associated transudates in the retinal fundus and albumin leakage from glomerular capillaries into the urine. ED is not only a central pathophysiologic event in diabetic vascular disease but also, serves as a predictive biomarker of disease progression1,20 and CV complications in patients with diabetes like in nondiabetic populations.21–24 In fact, we found Cat-S to mediate ED and microvascular permeability not only in diabetes but also, in postischemic microvascular injury and on injection of recombinant Cat-S into mice. In diabetic mice, Cat-S inhibition reversed albuminuria in db/db mice along with a restoration of fenestrations and other ultrastructural abnormalities of GEnC. Our in vitro studies document that Cat-S specifically and dose dependently increases endothelial cell monolayer permeability by inducing endothelial cell detachment and death, an effect that was not found in podocytes or other epithelial cells. Cat-S toxicity on endothelial cells in vivo is less severe than in vitro, potentially because of the presence of protease inhibitors,13,14 although cystatin C levels are decreased in progressive CV disease, which should enhance the protease activity of cysteine proteases.25 Cat-S–induced ED involves other functional endothelial cell changes, including increased oxidative stress together with reduced eNOS expression,1,26 which is now thought to be an important pathophysiologic link between ED and the functional and histopathologic alterations in DN.1,2 In fact, db/db mice show impaired eNOS activation along the progression of DN, and restoring activity of this enzyme reduced albuminuria as a marker of improved ED.27 Cat-S inhibition markedly increased renal eNOS expression, which was associated with less proteinuria and protection from glomerulosclerosis in T2D db/db mice. Similarly, Cat-S inhibition also restored eNOS expression in retinas of db/db mice, which was associated with less retinal glial fibrillary acidic protein expression as a marker for retina gliosis. Our mouse model did not show proliferative DR, but neoangiogenesis, another ED–related microvascular complications in DR, is also driven by Cat-S.28

Tissue remodeling in DN involves cytokine and chemokine production and the recruitment of proinflammatory and profibrotic macrophages that amplify the inflammatory response and produce profibrotic mediators, respectively.5,29,30 ED is manifested by endothelial cell activation and luminal expression of adhesion molecules and chemokines known for macrophage recruitment.29 Cat-S has the potential to modulate this inflammatory component of vascular disease, because activated macrophages produce Cat-S and contribute to vascular remodeling in atherosclerosis and vascular inflammation.9,31,32

Cat-S consistently induced ED in vitro and in vivo through PAR2, which is in line with a recent report on Cat-S cleavage of the extracellular domain of PAR2 required for activation.16 Madhusudhan et al.33 reported human kidney to stain positive for PAR2 in podocytes. We got similar results with several different antibodies in mouse kidney, but PAR2–deficient kidney sections unraveled this staining as unspecific (data not shown). We also did not detect any functional effects of PAR2 stimulation on podocytes. However, it is likely that Cat-S blockade also affects PAR2 signaling on other cell types in vivo. PARs are known modulators 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 of Cat-S–mediated activation of PAR2 is supported by the protective effects of PAR2 and concomitant Cat-S inhibition on DN and DR in db/db mice, which did not exceed the effects of PAR2 inhibition alone. These data are not unequivocal but consistent with the concept that Cat-S–mediated PAR2 activation is a previously unknown pathogenic mechanism in microvascular complications of diabetes.

In situ hybridization, immunolocalization studies in human DN, and a relevant murine model of DN were consistent in terms of a lack of Cat-S mRNA expression in renal parenchymal cells, whereas Cat-S protein was abundantly found in tubular epithelial cells. This implies that circulating Cat-S protein is filtered and passively reabsorbed in the renal tubules. Advanced DN was associated with a significant increase in renal Cat-S mRNA expression, which we could localize to infiltrating mononuclear phagocytes. In addition, Cat-S protein localized to endothelial cells along the glomerular filtration barrier in association with significant ultrastructural injury similar to what has been described in human DN.35

Together, blocking Cat-S/PAR2–mediated ED prevents DN and DR in db/db mice. Therefore, therapeutic Cat-S or PAR2 inhibition may help to prevent and improve outcomes of microvascular (and macrovascular) complications in diabetes.

Concise Methods

Human Studies

Human renal biopsies from consented patients and controls were collected.36 Microarray data from patients with DN and healthy controls 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 specimens 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 microdissected specimens from clinically indicated biopsies from patients with diabetic kidney disease (n=12) and pretransplant kidney biopsies 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 Biosystems). The mRNA expression was analyzed by standard curve quantification. 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 of Cat-S

RO5461111 (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 reversible inhibition of Cat-S with an IC50 of 0.4 nM and murine 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 commercial vendors. Enzymatic assays were performed using the fluorogenic 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 measured using 10 µM 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 electrophoresis for the determination of p10 upregulation by Cat-S inhibition as described below. In some experiments, we used 3 mg/kg of the previously described Cat-S inhibitor LY3000328 as a reference.39

Animal Studies

Six-week-old male wild–type or Par2–deficient C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were injected intravenously with 10 µg 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 intraperitoneally, 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 (1K mice) or sham surgery (2K mice) 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 intraperitoneally. 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), glial fibrillary 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 ×40 using ImageJ software. Only albumin not colocalizing with lectin+ blood vessels was measured. The surgical procedure and the technical setup for in vivo microscopy 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, baseline measurements were performed before ischemia of the cremaster muscle was induced for 30 minutes, and postischemic microvascular permeability (after 160 minutes of reperfusion) was studied. All experiments were performed according to German animal protection laws and had been approved by the local government authorities.

Histopathologic Evaluation

Kidneys were fixed in 4% formalin, embedded in paraffin, and stained with periodic acid–Schiff reagent. Sclerotic lesions were scored on 50 glomeruli as follows: 0, no lesion; 1, <25%; 2, 25%–49%; 3, 50%–74%; 4, 75%–100% sclerotic. Immunostaining was performed as described42 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 deoxynucleotidyl transferase–mediated digoxigenin–deoxyuridine nick–end labeling (apoptotic cells; Roche, Mannheim, Germany). For EM, the kidney cortex was cut into 1×1-mm cubes and immediately immersed in fixative containing 3% glutaraldehyde and 1% paraformaldehyde in PBS. Transmission or scanning EM was performed 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 Blotting

Protein isolation and Western blotting were performed 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 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 Cytometry

Flow 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-PCR

Total mRNA from whole kidneys, cultured mouse tubular cells, GEnCs, and podocytes was transcribed into cDNA using Superscript II and subjected to real-time PCR on a Light Cycler 480 (Real Time PCR Detection 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 Studies

Primary mouse GEnCs, tubular epithelial cells, mesangial cells, and podocytes were generated and cultured as described,46–48 preferentially in serum-free media. Cat-S–induced changes in resistance and capacitance of all cells types were analyzed using an ECIS device (Applied Biophysics) at a density of 100,000 cells per well in a volume of 400 µl media placed in ECIS–compatible microwell slides as previously described.49 Confluent monolayers were then stimulated with different doses of Cat-S with or without RO5461111. Capacitance was analyzed for indicated time points at 4 kHz. The transwell permeability of FITC-conjugated albumin 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, confluent GEnCs were stimulated with Cat-S with or without inhibitors in serum–free RPMI media. 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 IonTorrent Proton System was used for GEnC transcriptome analysis.51 For PAR2 siRNA transfection, 2 million GEnCs were suspended in 100 µl 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 scrambled siRNA (catalog no. 4390843; Ambion) controls were also maintained 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 containing 1% FCS and 1% PS. The efficacy of transfection was measured by qRT-PCR. GEnCs were 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 µg/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 concentration of 2000 nM was used before analyzing GEnC detachment. Mouse podocyte, tubular epithelial, 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 µg/ml Cat-S for 20 hours and the detached cells were counted as before.

Statistical Analyses

Data are presented as means±SEMs. 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).

Disclosures

S.G., W.H., and G.H. are employees from Hoffmann La Roche. All other authors declare no duality of interest associated with this manuscript.

Acknowledgments

We thank Dan Draganovic and Jana Mandelbaum for expert technical assistance and Marc 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.

Footnotes

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