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American Journal of Physiology - Renal Physiology logoLink to American Journal of Physiology - Renal Physiology
. 2020 Mar 23;318(5):F1067–F1073. doi: 10.1152/ajprenal.00595.2019

Dual blockade of protease-activated receptor 1 and 2 additively ameliorates diabetic kidney disease

Shohei Mitsui 1,*, Yuji Oe 2,3,*, Akiyo Sekimoto 1, Emiko Sato 1,3, Yamato Hashizume 1, Shu Yamakage 3, Satoshi Kumakura 3, Hiroshi Sato 1,3, Sadayoshi Ito 3, Nobuyuki Takahashi 1,3,✉
PMCID: PMC7294339  PMID: 32200667

Abstract

Protease-activated receptors (PARs) are coagulation protease targets, and they increase expression of inflammatory cytokines and chemokines in various diseases. Of all PARs, previous reports have shown that PAR1 or PAR2 inhibition is protective against diabetic glomerular injury. However, how PAR1 and PAR2 cooperatively contribute to diabetic kidney disease (DKD) pathogenesis and whether dual blockade of PARs is more effective in DKD remain elusive. To address this issue, male type I diabetic Akita mice heterozygous for endothelial nitric oxide synthase were used as a model of DKD. Mice (4 mo old) were divided into four treatment groups and administered vehicle, PAR1 antagonist (E5555, 60 mg·kg−1·day−1), PAR2 antagonist (FSLLRY, 3 mg·kg−1·day−1), or E5555 + FSLLRY for 4 wk. The results showed that the urinary albumin creatinine ratio was significantly reduced when both PAR1 and PAR2 were blocked with E5555 + FSLLRY compared with the vehicle-treated group. Dual blockade of PAR1 and PAR2 by E5555 + FSLLRY additively ameliorated histological injury, including mesangial expansion, glomerular macrophage infiltration, and collagen type IV deposition. Marked reduction of inflammation- and fibrosis-related gene expression in the kidney was also observed. In vitro, PAR1 and PAR2 agonists additively increased mRNA expression of macrophage chemoattractant protein 1 or plasminogen activator inhibitor-1 in human endothelial cells. Changes induced by the PAR1 agonist were blocked by a NF-κB inhibitor, whereas those of the PAR2 agonist were blocked by MAPK and/or NF-κB inhibitors. These findings suggest that PAR1 and PAR2 additively contribute to DKD pathogenesis and that dual blockade of both could be a novel therapeutic option for treatment of patients with DKD.

Keywords: coagulation, cytokine, endothelium, fibrosis

INTRODUCTION

Diabetic kidney disease (DKD) is a serious complication of both type I and II diabetes mellitus (1, 3). Although inhibitors of the renin-angiotensin system or hypoglycemic drugs are widely used, they are not able to prevent disease progression (1, 3). Because DKD is a main cause of end-stage kidney disease requiring renal replacement therapy worldwide, exploration of novel therapeutic targets is an urgent need.

The protease-activated receptor (PAR), a G protein-coupled receptor, is involved in the pathogenesis of inflammation through the production of cytokines and chemokines (19, 31). There are four PAR family members (PAR1–PAR4) that are activated by specific coagulation proteases; for example, tissue factor and factor VIIa complex activate PAR2, factor Xa activates both PAR1 and PAR2, and thrombin activates PAR1 and PAR4 (31). Specifically, PAR1 and PAR2 contribute to pathogenesis of several kidney injury models (7, 13, 24, 25, 29). In DKD, expression levels of PAR1 and PAR2 in the kidney are elevated (13). A lack or pharmacological inhibition of PAR1 or PAR2 reduces urinary albumin excretion and alleviates glomerular injury in diabetic mice (9, 13, 25, 26). However, how PAR1 and PAR2 cooperatively contribute to DKD and whether dual blockade of PARs is more effective in DKD remain elusive. To address this issue, the present study investigated the effect of PAR1 and PAR2 inhibitor cotreatment of diabetic mice. We found that distinct roles of PAR1 and PAR2 in vascular inflammation and that dual blockade of them can be a novel therapeutic option of DKD.

MATERIALS AND METHODS

Animals.

All experiments were conducted in compliance with guidelines of Tohoku University (Sendai, Japan). In this study, type I diabetic Akita (Ins2C96Y/+) mice with reduced expression of endothelial nitric oxide synthase (eNOS+/−) were used as a model of DKD. Our previous study has shown that reduced expression of eNOS (eNOS+/−), comparable to that associated with human NOS3 variants, was shown to express ~25% glomerular eNOS protein compared with diabetic eNOS+/+ mice. A modest decrease of eNOS was sufficient to enhance diabetic glomerulosclerosis and to elevate blood pressure, suggesting one of the suitable models to study human DKD (28). Herein, the male F1 progeny of 129S6/SvEvTac eNOS−/− × C57BL/6J Ins2Akita/+ mice were generated, which have a survival advantage compared with diabetic C57BL/6J eNOS+/− mice (28). To test the effect of PAR1 and PAR2 inhibition, the PAR1 inhibitor E5555 (Eisai, Tokyo, Japan) and the PAR2 inhibitor FSLLRY-NH2 (Peptide Institute, Osaka, Japan) were used. E5555 was suspended in 0.5% carboxymethylcellulose and administered by oral gavage. FSLLRY-NH2 (referred to as FSLLRY) was dissolved in PBS for intraperitoneal injection. Mice (4 mo old) were randomly divided the following four treatment groups: vehicle (carboxymethylcellulose + PBS), E5555 (60 mg·kg−1·day−1), FSLLRY (3 mg·kg−1·day−1), and E5555 + FSLLRY (Fig. 1A). eNOS+/− mice without treatment were used as a nondiabetic group. Inhibitors were administered every day for 4 wk, and samples, such as the kidney, blood, and urine, were collected. The dose of inhibitor used was determined based on previous reports (8, 9, 15).

Fig. 1.

Fig. 1.

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Dual blockade of protease-activated receptor (PAR)1 and PAR2 ameliorates urinary albumin excretion. A: experimental protocol. B6, C57BL/6J; 129, 129S6/SvEvTac; CMC, carboxymethylcellulose. B–E: systolic blood pressure (BP; B), random nonfasting blood glucose (C), plasma creatinine (D), and urinary albumin excretion (U-Alb; E) in a spot urine sample from diabetic mice before euthanasia. DKD, diabetic kidney disease; NS, not significant. *P < 0.05. The dashed line indicates the level of nondiabetic mice (n = 7–8). Data are shown as means ± SE. Two-way ANOVA was used in A, B, and D and a Kruskal-Wallis test in C for statsistical analysis.

Urinary analysis.

Spot urine samples were collected before euthanasia. An ELISA kit was used to measure urinary albumin (Exocell, Philadelphia, PA) according to the manufacturer’s protocol. Urinary creatinine was determined using liquid chromatography-mass spectrometry/mass spectrometry as previously described (22). Urinary albumin excretion was defined as the ratio of urinary albumin to creatinine.

Measurement of blood pressure.

Blood pressure was measured by a computerized tail-cuff method using the CODA system (Kent Scientific, Torrington, CT) as previously described (13).

Quantitative real-time PCR.

Total RNA was extracted from the whole kidney or cultured cells using TRI Reagent (Molecular Research Center, Cincinnati, OH). Reverse transcription and real-time PCR were performed using an iScript Advanced cDNA Synthesis and SsoAdvanced Universal Probe/SYBR Supermix kits (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. Hypoxanthine-guanine phosphoribosyltransferase was used as a reference gene. Primer sequences were provided shown in a previous report (13).

Histological evaluation.

Kidney samples were fixed in 2% paraformaldehyde and embedded in paraffin before being cut into 1.5-µm-thick sections and stained with periodic acid-Schiff (PAS). The severity of mesangial expansion was quantified on a scale of 0−2 as follows: 0 = normal, 1 = mild proliferation of the mesangial area, and 2 = severe proliferation of the mesangial area or global sclerosis. The glomeruli in each group were chosen and evaluated in a blinded manner.

Immunohistochemistry.

Rabbit anti-mouse collagen type IV antibody (1:200, Merck Millipore, Burlington, MA) and rabbit anti-human CD68 antibody (1:2,000, Abcam, Cambridge, UK) were used. Heat-induced antigen retrieval was performed using sodium citrate buffer to detect collagen type IV. Proteinase K (Dako) was used to detect CD68. Primary antibodies were incubated overnight at 4 °C. N-histofine simple stain kits (Nichirei Biosciences, Tokyo, Japan) were used as a secondary antibody according to the manufacturer’s protocol. Sections were incubated without primary antibody as a negative control. The ratio of the glomerular collagen type IV-positive area to glomerular tuft area was assessed using ImageJ (National Institutes of Health, Bethesda, MD).

Cell culture.

Human endothelial cells (EA.hy926) were cultured in Dulbecco’s modified Eagle medium-high glucose containing 10% FBS, 100 IU/mL of penicillin, 100 IU/mL of streptomycin, and 200 mM L-glutamine in a humidified incubator at 37°C with 5% CO2 in air (4). The PAR1 agonist TFLLR-NH2 (referred to as TFL) was purchased from Abcam (Cambridge, UK). The PAR2 agonist 2f-LIGRLO (referred to as 2f-LI) was purchased from Tocris Bioscience (Bristol, UK), U-0126 was obtained from Wako Pure Chemical Industries (Osaka, Japan). Bay 11-7082 was obtained from Sigma-Aldrich (St. Louis, MO). All experiments were performed after serum starvation for 24 h. Both U-0126 and Bay 11-7082 were administered 1 h before TFL or 2f-LI treatment. For quantitative PCR analysis, cells were harvested after a 3-h incubation with TFL or 2f-LI.

Statistical analysis.

Values are presented as means ± SE. Data were analyzed using JMP Pro 14 (SAS Institute, Cary, NC). Statistical analysis was only performed for diabetic groups. After normality was checked, two-way ANOVA followed by a Tukey-Kramer test was used to test the effect of PAR1 and PAR2 inhibitors and their interaction. For nonparametric values, a Kruskal-Wallis test followed by a Steel-Dwass test was used. Differences were considered statistically significant at P < 0.05.

RESULTS

Dual blockade of PAR1 and PAR2 additively reduces urinary albumin excretion in diabetic mice.

The basal characteristics of diabetic mice are shown in Fig. 1, B–E. Administration of E5555, FSLLRY, or E5555 + FSLLRY did not affect systolic blood pressure or levels of nonfasting blood glucose and plasma creatinine in diabetic mice (Fig. 1, B–D). Neither E5555 nor FSLLRY alone affected urinary albumin excretion, but their coadministration significantly reduced excretion compared with vehicle-treated mice (Fig. 1E). Body and kidney weights were similar among groups (data not shown).

Dual blockade of PAR1 and PAR2 additively reduces mesangial proliferation in DKD.

Glomerular mesangial expansion is a hallmark of diabetic kidney injury (14); therefore, the PAS-positive mesangial area in diabetic mice was measured (Fig. 2, A and B). Administration of E5555 or FSLLRY significantly reduced the mesangial expansion score compared with the vehicle-treated group (vehicle: 1.51 ± 0.06, E5555: 0.85 ± 0.07, and FSLLRY: 0.99 ± 0.06). Cotreatment with E5555 and FSLLRY further decreased mesangial expansion (0.69 ± 0.07) to the level of nondiabetic mice (0.62 ± 0.07).

Fig. 2.

Fig. 2.

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Mesangial expansion scores in diabetic mice. A: representative photomicrographs of the glomeruli from diabetic or nondiabetic mice. DKD, diabetic kidney disease; PAS, periodic acid-Schiff stain. Scale bar = 50 μm. B: comparison of mesangial expansion scores among diabetic mice treated with vehicle, E5555, FSLLRY, or both E5555 + FSLLRY. Approximately 100 glomeruli of each group were randomly chosen in blind and evaluated. **P < 0.01; ***P < 0.001. The dashed line indicates the level of nondiabetic control mice. Data are shown as means ± SE. A Kruskal-Wallis test was used for statistical analysis.

Dual blockade of PAR1 and PAR2 ameliorates inflammation in DKD.

Increase of inflammatory cell infiltration and cytokine production is important in DKD pathogenesis (11, 17). Therefore, glomerular infiltration of CD68-positive macrophages and expression of cytokines and chemokines in the kidney were quantified. The results showed that cotreatment with E5555 and FSLLRY significantly reduced the number of CD68-positive cells compared with that of E5555- or vehicle-treated diabetic groups (Fig. 3, A and B). E5555 alone and E5555 + FSLLRY cotreatment reduced EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1) mRNA (macrophage marker) compared with vehicle-treated mice (Fig. 3C). Expression levels of TNF-α (Tnfa) and macrophage chemoattractant protein (MCP)-1 (Mcp1) mRNA were significantly reduced in E5555 + FSLLRY-treated mice compared with vehicle-treated mice (Fig. 3C). Together, amelioration of inflammation was evident when PAR1 and PAR2 were dually blocked in this diabetic model.

Fig. 3.

Fig. 3.

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Macrophage infiltration and inflammatory gene expression in kidneys from diabetic mice. A: representative photomicrographs of CD68 mice (a marker of macrophages). Arrows indicate CD68-positive cells. Scale bar = 50 μm. B: numbers of CD68-positive cells in the glomeruli. Approximately 100 glomeruli in each group were blindly and randomly chosen and evaluated. C: gene expression levels of EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1), tumor necrosis factor-α (Tnfa), and macrophage chemoattractant protein-1 (Mcp1) mRNA in kidneys from diabetic mice (n = 7–8). AU, arbitrary units; DKD, diabetic kidney disease. *P < 0.05; **P < 0.01; ***P < 0.001. The dashed line indicates the level of nondiabetic control mice. Data are shown as means ± SE. A Kruskal-Wallis test was used for statistical analysis.

Dual blockade of PAR1 and PAR2 ameliorates collagen IV deposition in DKD.

Next, the fibrotic response in kidney injury of diabetic mice was characterized. Immunohistochemistry showed that E5555 or FSLLRY alone significantly reduced glomerular collagen type IV-positive areas compared with the vehicle-treated group. Cotreatment with E5555 and FSLLRY additively reduced collagen type IV positivity compared with E5555- or vehicle-treated mice (Fig. 4, A and B). Next, the expression of fibrosis-related genes, such as transforming growth factor-β (Tgfb), plasminogen activator inhibitor (PAI)-1 (Pai1), and collagen type I (Col1), was quantified. E5555 + FSLLRY cotreatment significantly reduced renal expression of Tgfb and Pai1 mRNA compared with that of vehicle-treated mice. FSLLRY alone and E5555 + FSLLRY cotreatment significantly reduced Col1 mRNA in the kidney compared with vehicle-treated mice. Collectively, dual blockade of PAR1 and PAR2 greatly ameliorated fibrotic responses in diabetic kidney injury.

Fig. 4.

Fig. 4.

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Glomerular collagen type IV deposition and fibrosis-related gene expression in kidneys from diabetic mice. A: representative photomicrographs of collagen type IV. Scale bar = 50 μm. B: comparison of glomerular collagen type IV-positive areas among diabetic mice treated with vehicle, E5555, FSLLRY, and E5555 + FSLLRY. Approximately 100 glomeruli in each group were blindly and randomly chosen and evaluated. C: gene expression levels of transforming growth factor-β (Tgfb), plasminogen activator inhibitor-1 (Pai1), and collagen type I (Col1) mRNA in kidneys from diabetic mice (n = 7–8). AU, arbitrary units; DKD, diabetic kidney disease. *P < 0.05; **P < 0.01; ***P < 0.001. The dashed line indicates the level of nondiabetic control mice. Data are shown as means ± SE. A Kruskal-Wallis test was used for statistical analysis.

Effect of PAR1 and PAR2 agonists on proinflammatory cytokine expression in human endothelial cells.

Because dual blockade of PAR1 and PAR2 additively reduced inflammation in DKD, they could contribute to kidney injury via different mechanisms. Both PAR1 and PAR2 are highly expressed in endothelial cells and because vascular inflammation is an important pathogenesis in diabetic complication (18, 21), the effect of PAR1 agonist TFL (200 µM) and PAR2 agonist 2f-LI (20 μM) on cytokine expression in human endothelial cells was tested. TFL and 2f-LI alone significantly increased MCP-1 mRNA (TFL: 1.7-fold and 2f-LI: 7.5-fold) compared with vehicle, and TFL + 2f-LI cotreatment additively increased MCP-1 levels compared with vehicle (12.5-fold). When cells were pretreated with the MAPK inhibitor U-0126, TFL still increased MCP-1 mRNA (1.8-fold), whereas 2f-LI only caused a small change (1.8-fold). After pretreatment with the NF-κB inhibitor Bay11-7082, the change in MCP-1 mRNA levels induced by TFL largely disappeared (0.8-fold), whereas 2f-LI significantly increased levels (5.3-fold). The change in MCP-1 levels by TFL + 2f-LI cotreatment under NF-κB inhibition was similar to that of 2f-LI alone, suggesting that the additive effect by TFL disappeared when the NF-κB pathway was blocked (Fig. 5A). Collectively, PAR1 largely increased MCP-1 mRNA via the NF-κB pathway, whereas PAR2 increased levels via the MAPK pathway.

Fig. 5.

Fig. 5.

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Effect of protease-activated receptor (PAR)1 and PAR2 agonists on macrophage chemoattractant protein-1 (MCP1) and plasminogen activator inhibitor-1 (PAI1) mRNA levels in human endothelial cells. A, left: effect of PAR1 agonist [TFLLR (TFL), 200 μM], PAR2 agonist [2f-LIGRLO (2f-LI), 20 μM], and their cotreatment on the expression of MCP1 mRNA. A, middle: effect of a MAPK inhibitor (U-0126, 10 μM) on the expression of MCP1 mRNA. A, right: effect of a NF-kB inhibitor (Bay11-7082, 10 μM) on the expression of MCP1 mRNA. n = 6–7. B, left: effect of TFL, 2f-LI, and their cotreatment on the expression of PAI1 mRNA. B, middle: effects of U-0126 on the expression of PAI1 mRNA. B, right: effects of Bay11-7082 on the expression of PAI1 mRNA. n = 6–7. AU, arbitrary units; NS, not significant. *P < 0.05. Data are shown as means ± SE. A Kruskal-Wallis test was used for statistical analysis.

Change in PAI-1 mRNA levels were also examined. TFL or 2f-LI alone both significantly increased PAI-1 mRNA levels (TFL: 1.8-fold; 2f-LI: 2.4-fold). Furthermore, TFL + 2f-LI cotreatment additively increased PAI-1 mRNA compared with vehicle (4.3-fold). After pretreatment with U-0126, TFL still significantly increased PAI-1 mRNA (1.8-fold), but the change induced by 2f-LI was smaller and not significant (1.7-fold). After Bay11-7092 pretreatment, the changes induced by TFL, 2f-LI, and TFL + 2f-LI all disappeared (Fig. 5B). These findings suggest that PAR1 increases PAI-1 expression via the NF-κB pathway, whereas PAR2 increases it via both MAPK and NF-κB pathways.

DISCUSSION

In the present study, both PAR1 and PAR2 inhibitors were shown to ameliorate DKD. Individually, these inhibitors reduced glomerular PAS-positive and collagen type IV-positive areas in the kidney of diabetic mice. When combined, additive therapeutic effects were obtained, including a reduction of the urinary albumin-to-creatinine ratio, marked amelioration of histological injury (mesangial expansion, glomerular macrophage infiltration, and collagen type IV deposition), and a reduction of inflammation and fibrosis-related gene expression in the kidney.

Previous reports have shown the PAR1 inhibitor vorapaxar or genetic deletion of PAR1 ameliorates streptozotocin-induced diabetic glomerulosclerosis, such as mesangial expansion and collagen type IV deposition (25, 26). Similarly, the PAR2 inhibitor FSLLRY has been shown to reduce mesangial expansion in db/db mice (9). Lack of PAR2 has also been shown to ameliorate urinary albumin excretion, mesangial expansion, and elevation of proinflammatory- or fibrosis-related markers in diabetic Akita mice with eNOS deletion (13). These changes were consistent with the present results obtained by treatment of diabetic mice with E5555 or FSLLRY alone. Furthermore, the present results demonstrated that dual blockade of PAR1 and PAR2 additively ameliorated glomerular injury. These findings suggest that PAR1 and PAR2 contribute to DKD in distinct manners.

Both PAR1 and PAR2 are known to increase production of cytokines and chemokines. Using microarray analysis, the PAR1 agonist thrombin was shown to increase expression of proinflammatory genes, such as IL-8, MCP-1, chemokine (C-X-C motif) ligand (CXCL)1, and CXCL2 mRNA, in human umbilical vein endothelial cells (5). Others have shown that thrombin upregulates expression of CXCL3 via a NF-κB-dependent mechanism (16). We have also previously demonstrated that the PAR2 agonist peptide 2f-LI increases MCP-1 and CXCL1 expression through either MAPK or phosphatidylinositol 3-kinase pathways in human endothelial cells (12). Moreover, trypsin and 2f-LI have been shown to increase IL-6 and IL-8 expression and activate the NF-κB pathway in human dermal microvascular endothelial cells (20). In the present study, PAR1 and PAR2 agonists were found to have an additive effect on expression of MCP-1 and PAI-1 mRNA in human endothelial cells. Mechanistically, PAR1 elevated MCP-1 and PAI-1 mRNA levels via the NF-κB pathway, whereas PAR2 elevated their expression via MAPK or NF-κB pathways. These findings suggest that PAR1 and PAR2 additively increase vascular inflammation via different mechanisms, which explains why dual blockade of PARs additively alleviates DKD.

Based on the present and previous studies, PAR1 and PAR2 inhibitors may be promising drugs for treatment of patients with DKD. Clinical trials of PAR1 inhibitors, such as vorapaxar or atopaxar, are ongoing for treatment of coronary heart disease (6, 23). The expansion of their use for improving glycemic control and delaying diabetic kidney injury is expected (26, 27). The development of PAR2 inhibitor compounds is also being actively studied (2, 30). More interestingly, a recent study (10) developed bivalent PAR1-PAR2 antagonists that can block both PAR1 and PAR2 pathways.

A limitation of this study is that a metabolic cage study was not performed. Some fundamental data, such as urinary volume, are lacking. How diabetes and/or PAR inhibitors affect urinary parameters are unclear.

In conclusion, the present study demonstrated that PAR1 and PAR2 contribute to vascular inflammation in DKD via different mechanisms and additively exacerbate diabetic kidney injury. These findings suggest that their dual blockade may represent a novel therapeutic option to prevent onset and progression of DKD in humans.

GRANTS

This work was supported by the DiaComp Pilot and Feasibility Program (30835-14) from National Institute of Diabetes and Digestive and Kidney Diseases Diabetic Complications Consortium Grant DK-076169, Grants-In-Aid from the Japan Society for Promotion of Science (JSPS18K15993), and the Suzuken Memorial Foundation (18-022).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.M., Y.O., A.S., E.S., and Y.H. performed experiments; S.M., Y.O., and E.S. analyzed data; S.M., Y.O., A.S, E.S., S.Y., S.K., and N.T. interpreted results of experiments; S.M. and Y.O. prepared figures; Y.O. and N.T. conceived and designed research; S.M., Y.O. and N.T. drafted manuscript; E.S., S.Y., S.K., H.S., and S.I. edited and revised manuscript; S.M., Y.O., A.S., E.S., Y.H., S.Y., S.K., H.S., S.I., and N.T. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank members of Tohoku University Faculty of Pharmaceutical Sciences for assistance and Eisai for providing E5555.

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