Introduction

Diabetic nephropathy (DN), is microvascular complication of diabetes causes to kidney dysfunction and end-stage renal disease (ESRD). DN progression is characterized by renal fibrosis, which is attributed to the excessive accumulation of the extracellular matrix (ECM) [1]. Renal fibrosis is generally considered to result from maladaptive repair processes, stimulate the formation of myofibroblasts through the differentiation from tissue-resident fibroblasts (FBs) and bone marrow-derived mesenchymal stem cells (MSCs), epithelial-to-mesenchymal transition (EMT), and endothelial-to-mesenchymal transition (EndoMT). The accumulated myofibroblasts induces ECM production and fibrosis progression [2]. In particular EndoMT affects vascular dysfunction [3, 4], and contributes to the early development of renal fibrosis in diabetes [5].

Advanced glycation end products (AGEs) are metabolic consequence of hyperglycemia, and play an important roles in the progression of DN [6]. The generation of AGEs is facilitated through nonenzymatic glycation reactions under the hyperglycemic conditions, and AGEs activate several intracellular signaling pathways throught receptor for AGEs (RAGE), and induce fibroblast activation, ECM production, EndoMT, and renal fibrosis [5, 7].

Alpha2-antiplasmin (α2AP) rapidly inhibits plasmin, and results in the formation of a stable inactive complex, plasmin-α2AP complex (PAP) [4, 8, 9]. α2AP is synthesized in various tissues including kidney [10], and has various functions, such as cytokine production, cell growth, cell differentiation, and regulates angiogenesis, inflammatory responses, tissue remodeling [11,12,13]. In our previous studies, we showed that α2AP expression is elevated in fibrotic tissue [14,15,16]. In addition, α2AP is associated with vascular injury, EMT, myofibroblast differentiation from tissue-resident fibroblatsts and bone marrow-derived mesenchymal stem cells in the process of fibrosis progression [15, 17, 18], and the blockade of α2AP alleviates fibrosis [16]. Furthermore, the increased circulating levels of α2AP and PAP have been reported in patients with diabetes and DN [19, 20]. In the present study, we investigated the roles of α2AP in the pathogenesis of DN using a streptozotocin (STZ)-induced DN mouse model, and demonstrated that α2AP is associated with the progression of fibrosis in DN.

Materials and methods

Animals

The α2AP deficient (α2AP−/−) mice were generated by homologous recombination using embryonic stem cells, as described previously [21]. Wild type (α2AP+/+) and α2AP−/− mice littermates were housed in groups of two to five in filter-top cages with a fixed 12 h light and 12 h dark cycle.

Mice experiments

The mice experiments in this study were approved by the Animal Research Committee of Doshisha Women’s College of Liberal Arts (Approval ID: Y17-015). The saline or STZ (50 mg/kg) were administrated intraperitoneally for five consecutive days in 10-week-old male α2AP+/+ and α2AP−/− mice. Mice were euthanized at 3 months after administration of saline or STZ, and tissues were dissected for studies. Blood glucose levels were checked by tail vein puncture using One Touch Ultra glucose meter (Johnson and Johnson, NJ, USA). Mice with blood glucose levels of ≥ 400 mg/dL were considered diabetes. The samples of kidney were placed immediately in liquid nitrogen, and stored at –80 °C until use.

Measurement of serum insulin

The collected blood samples were centrifuged to separate serum, and the concentration of serum insulin was measured by ELISA (mouse insulin ELISA kit (Fujifilm Wako Shibayagi corporation, Gunma, Japan).

Glomerular cell proliferation and mesangial matrix expansion

The glomerular cell proliferation and mesangial matrix expansion were assessed using periodic acid Schiff (PAS) staining as described previously [12]. The stained images obtained from separate fields on the specimens were analyzed by using ImageJ.

Collagen content in kidney

The collagen content was measured as previously described [22]. The collagen content was assessed using Sirius red staining. The stained images obtained from separate fields on the specimens were analyzed by using ImageJ. Sirius red positive area was expressed as a percent of the observed with sham mice.

Western blot analysis

We performed western blot analysis as previously described [23]. The kidney samples from mice were homogenized on ice, and the homogenized samples were centrifuged at 15,000 rpm for 30 min at 4 °C and the supernatant was collected. The protein concentration in each lysate was measured using a BCA protein assay kit (Pierce, IL, USA). Proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to a PVDF membrane. We detected each protein by incubation with each antibodies followed by incubation with horseradish peroxidase-conjugated antibodies to IgG (Santa Cruz Biotechnology, CA, USA).

Cell culture

NIH3T3 FBs (RIKEN BioResource Center, Tsukuba, Japan) and UV♀2 vascular endothelial cells (ECs) (mouse cell line derived from UV-induced angioendothelioma-like tumor)(RIKEN BioResource Center, Tsukuba, Japan) were seeded into the 60-mm diameter dishes and maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS) at 37 °C in a humidified atmosphere with 5% CO2/95% air. Then, the cells were used for experiments. AGE-BSA was purchased from Sigma-Aldrich (MO, USA).

siRNAs study

We performed western blot analysis as previously described [24, 25]. NIH3T3 FBs were transfected with RAGE siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, sc-36375) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. A nonspecific siRNA (Santa Cruz Biotechnology, CA, sc-37007) was employed as the control.

Co-culture

Co-culture of NIH3T3 FBs and UV♀2 vascular ECs were was performed using a Transwell cell culture insert (1.0 μm pore size) (Corning, NY, USA). UV♀2 vascular ECs were seeded in 6-well plates, and NIH3T3 FBs with inserts were added to the upper chamber of the Transwell system. Both cells were maintained in DMEM containing 10% FCS at 37 °C in a humidified atmosphere with 5% CO2/95% air. Then, the cells were used for experiments.

Statistical analysis

All data were expressed as mean±SEM. The statistical analysis was conducted with unpaired t-test for two-group comparison, with one‐way ANOVA followed by Tukey test for multiple comparison. Statistical significance was defined as a P value of <0.05.

Results

The expression of α2AP was elevated in the STZ-induced diabetic nephropathy model mice

Diabetic nephropathy (DN) is a major complication of diabetes. To clarify the role of α2AP on the pathogenesis of DN, we examined the expression of α2AP in the kidney of STZ-induced DN model mice. The expression of α2AP in the kidney of STZ-treated mice was 2.2-fold higher than that in saline-treated mice (Fig. 1).

Fig. 1
figure 1

The expression of α2AP in the STZ-induced DN model mice. The expression of α2AP in kidney from the saline or STZ-treated mice was examined by a western blot analysis. The histogram shows quantitative representations of α2AP (n = 3). The data represent the mean ± SEM. ***P<0.05

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α2AP deficiency attenuated the progression of renal fibrosis in the STZ-induced diabetic nephropathy model mice

To clarify the effect of α2AP on the progression of renal fibrosis in STZ-induced DN model mice, we compared the pathologic changes in the kidney of STZ-treated α2AP+/+ and α2AP−/− mice. First, we confirmed that there was no difference in the blood glucose and insulin concentration in the α2AP+/+ and α2AP−/− mice in the fed state and that α2AP did not affect glucose metabolism (data not shown). Glomerular cell proliferation (Fig. 2a, b), mesangial matrix expansion (Fig. 2a, c) and collagen deposition (Fig. 2d, e) were elevated in the kidney of STZ-treated mice, and their levels in STZ-treated α2AP+/+ mice were significantly higher than those in STZ-treated α2AP−/− mice. In addition, the expression of myofibroblast and mesenchymal cell phenotype including α-smooth muscle actin (α-SMA) and vimentin, and type I collagen were elevated in the kidney of STZ-treated mice, and their levels in STZ-treated α2AP+/+ mice was significantly higher than those in the kidneys of STZ-treated α2AP−/− mice (Fig. 2f). Furthermore, the expression of vascular endothelial cell phenotype, VE-cadherin was decreased in the kidney of STZ-treated α2AP+/+ mice. In contrast, the treatment of STZ did not affect VE-cadherin expression in the kidney of STZ-treated α2AP−/− mice (Fig. 2f).

Fig. 2
figure 2

α2AP deficiency attenuated renal fibrosis progression in the STZ-induced DN model mice. a Representative kidney sections from saline or STZ-treated α2AP+/+ and α2AP−/− mice (PAS stain). b The number of Nuclei per glomerulus in the saline or STZ-treated α2AP+/+ and α2AP−/− mice (n = 6). c Mesangial matrix was measured by PAS stain in the kidney from STZ-treated α2AP+/+ and α2AP−/− mice (n = 6). Mesangial matrix area was expressed as a percent of the observed with control in the STZ-treated α2AP+/+ mice. d Representative kidney sections from the saline or STZ-treated α2AP+/+ and α2AP−/− mice (Sirius red stain). e The collagen content in the kidney from saline or STZ-treated α2AP+/+ and α2AP−/− mice (n = 4). f The expression of each protein in kidney from the saline or pristane-treated α2AP+/+ and α2AP−/− mice was examined by a western blot analysis. The histogram on the right panels shows quantitative representations of each protein obtained from densitometry analysis (n = 3). The data represent the mean ± SEM. NS not significant. *P<0.001. **P<0.01. ***P<0.05

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α2AP is associated with the high glucose condition/AGEs-induced pro-fibrotic changes in fibroblasts

We showed that the treatment of high glucose induced the expression of α2AP, α-SMA and type I collagen in FBs (Fig. 3a). We also showed that AGEs induced the expression of α2AP, α-SMA and type I collagen in FBs (Fig. 3b). These data suggest that the high glucose condition/AGEs induced α2AP production and pro-fibrotic changes in FBs. AGEs has various functions through RAGE [7]. We examined the effect of RAGE reduction in FBs by using siRNA (Fig. 3c). The reduction of RAGE attenuated the AGEs-induced α2AP production in FBs (Fig. 3d). In our previous study, we demonstrated that the ERK1/2 and JNK pathways regulate the induction of α2AP expression [14]. AGEs induced the activation of ERK1/2 and JNK pathways (Fig. 3e). In addition, we examined whether the ERK1/2 or JNK pathway is associated with the AGEs-induced α2AP production by using the MEK specific inhibitors (PD98059) or JNK inhihitor (SP600125). PD98059 and SP600125 attenuated the AGEs-induced α2AP production in FBs (Fig. 3f). We also examined the effect of the RAGE-specific inhibitor FPS-ZM1 [26] on the high glucose condition-induced α2AP production in FBs. FPS-ZM1 attenuated the high glucose-induced α2AP production in FBs (Fig. 3 g). Furthermore, the neutralization of α2AP attenuated the high glucose-induced pro-fibrotic changes in FBs (Fig. 3 h).

Fig. 3
figure 3

α2AP is associated with the high glucose condition/AGEs-induced pro-fibrotic changes in FBs. a NIH3T3 fibroblasts (FBs) were stimulated by glucose (50 mM) for the indicated periods. The expression of each protein was measured by a western blot analysis. b FBs were stimulated by BSA (10 µg/ml) or AGE-BSA (10 µg/ml) for 24 h. The expression of each protein was measured by a western blot analysis. The histogram on the right panels shows quantitative representations of each proteins expression obtained from densitometry analysis (n = 3). c FBs were transfected with control or RAGE siRNA. At 24 h after transfection, the cells were used for experiments. d The siRNA-transfected FBs stimulated by BSA (10 µg/ml) or AGE-BSA (10 µg/ml) for 24 h. The expression of α2AP was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n = 3). e FBs were stimulated by AGE-BSA (10 µg/ml) for the indicated periods. The expression of each protein was measured by a western blot analysis. f FBs were cultured for 24 h in the absence or presence of BSA (10 µg/ml), AGE-BSA (10 µg/ml), PD98059 (30 µM) or SP600125 (30 µM) as indicated. The histogram on the bottom panels shows quantitative representations of α2AP expression obtained from densitometry analysis (n = 3). g FBs were cultured for 24 h in the absence or presence of FPS-ZM1 as indicated in the high glucose condition (50 mM). The expression of α2AP was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n = 3). h FBs were cultured for 7 days in the absence or presence of control IgG (1 µg/ml), anti-α2AP antibody (1 µg/ml). The histogram on the right panels shows quantitative representations of each proteins expression obtained from densitometry analysis (n = 3). The data represent the mean ± SEM. *P<0.001. **P<0.01. ***P<0.05

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The effect of α2AP on the high glucose condition/AGEs-induced EndoMT

The treatment of high glucose induced EndoMT (the increase in a-SMA and vimentin, and the decrease in VE-cadherin) in vascular ECs (Fig. 4a). We also showed that AGEs induced EndoMT in ECs (Fig. 4b). AGEs is known to induce EndoMT through smad pathway [5]. We examined the effect of α2AP on the AGEs-induced smad activation, and showed that α2AP promoted the AGEs-induced smad2/3 activation in ECs (Fig. 4c). It has been reported that the inhibition of SHP2 suppresses smad2/3 activation [27], and we previously reported that α2AP activates SHP2 in ECs [18]. Therefore, we exmined the effect of SHP2 on the α2AP-promoted EndoMT induced by AGEs. α2AP promoted the AGEs-induced EndoMT, and the SHP2 inhibitor (NSC87877) reversed the α2AP-promoted EndoMT induced by AGEs (Fig. 4d).

Fig. 4
figure 4

The effect of α2AP on the high glucose condition/AGEs-induced EndoMT in ECs. a UV♀2 vascular endothelial cells (ECs) were stimulated by glucose (50 mM) for the indicated periods. The expression of each protein was measured by a western blot analysis. b ECs were stimulated by BSA (10 µg/ml) or AGE-BSA (10 µg/ml) for 24 h. The expression of each protein was measured by a western blot analysis. The histogram on the right panels shows quantitative representations of each proteins expression obtained from densitometry analysis (n = 3). c ECs were pretreated by α2AP (4 nM) for 30 min and then stimulated by AGE-BSA (10 µg/ml) for the indicated periods. d ECs were cultured for 24 h in the absence or presence of AGE-BSA (10 µg/ml), α2AP (4 nM), or NSC87877 (100 µM) as indicated. The histogram on the bottom panels shows quantitative representations of each protein expression obtained from densitometry analysis (n = 3). The data represent the mean ± SEM. *P<0.001. **P<0.01. ***P<0.05

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The effect of α2AP on the high glucose condition-induced EndoMT in fibroblast/vascular endothelial cell co-culture

We showed that the high glucose condition/AGEs induced α2AP production in FBs, but not in ECs (Figs. 3 and 4a). Therefore, we co-cultured FBs and ECs to determine whether the FB-produced α2AP affects EndoMT progression. The FB/EC co-culture promoted the high glucose-induced EndoMT compared to ECs mono-culture (Fig. 5a). In addition, we showed that α2AP neutralizaiton attenuated the FBs/ECs co-culture-promoted EndoMT under the high glucose condition (Fig. 5b).

Fig. 5
figure 5

The effect of α2AP on the high glucose condition-induced EndoMT in FBs and ECs co-culture. a ECs (mono-culture) or FBs/ECs (co-culture) were cultured in the high-glucose condition (50 mM) for 7 days. The expression of each protein was measured by a western blot analysis. b FBs/ECs (co-culture) were cultured in the high-glucose condition (50 mM) for 7 days in the absence or presence of control IgG (1 µg/ml), anti-α2AP antibody (1 µg/ml). The expression of each protein was measured by a western blot analysis. The histogram on the bottom panels shows quantitative representations of each protein expression obtained from densitometry analysis (n = 3). The data represent the mean ± SEM. *P<0.001. ***P<0.05

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Discussion

In the present study, we examined the role of α2AP on the progression of renal fibrosis in DN using a STZ-induced DN mouse model. The expression of α2AP was elevated in the STZ-induced DN model mice (Fig. 1). In addition, α2AP deficiency attenuated the pro-fibrotic changes, such as myofibroblast and collagen deposition and EndoMT in the DN model mice (Fig. 2). These data suggest that the expression of α2AP is induced on the process of DN onset, and the increased α2AP expression may play a pivotal role in the progression of fibrosis in DN.

The persistent of hyperglycemic conditions in diabetes accerates AGEs production and exacerbates DN, and then accelerates fibrosis progression [5, 7]. The high glucose conditions and AGEs caused the pro-fibrotic changes and α2AP production in FBs (Fig. 3a, b), and the blockade of α2AP attenuated the high glucose condition-induced pro-fibrotic chanegs in FBs (Fig. 3h). AGEs can bind RAGE, and activate several intracellular signaling pathways [7]. The reduction of RAGE by siRNA attenuated the AGEs-induced α2AP production in FBs (Fig. 3d). In addition, the activation of ERK1/2 and JNK pathways is associated with the induction of α2AP expression [14]. AGEs activated the ERK1/2 and JNK pathways, and the inhibition of ERK1/2 and JNK pathways attenuated the AGEs-induced α2AP production in FBs (Fig. 3e, f). Furthermore, we showed that the RAGE specific inhibitor attenuated the high glucose condition-induced α2AP production in FBs (Fig. 3d). On the other hand, the hyperglycemia is also known to induce oxidative stress and diacyglycerol (DAG) production, and oxidative stress and DAG can lead the production of cytokines [28, 29]. We confirmed that the effect of oxidative stress and DAG on the α2AP production in FBs. The oxidative stress inducer, H2O2 and DAG analog did not induce the α2AP production in FBs (data not shown). These data suggest that the high glucose condition/AGEs induced α2AP production in FBs, and RAGE is associated with the production of α2AP induced by high glucose condition/AGEs.

EndoMT contributes to the progression of vascular dysfunction and fibrosis [3, 4]. EndoMT is regulated by various inflammatory cytokines [1]. α2AP is a serine protease inhibitor that rapidly inactivates plasmin, and regulates thrombolysis. We recently reported that α2AP induces cytokine production, cell differntiation and cell growth indpendent of plasmin [13, 17, 30]. These findings suggest that α2AP has biological effects other than the regulation of thrombolysis. In addition, α2AP is associated with EMT, which exhibits features similar to those of EndoMT [15]. In the present study, we showed that α2AP deficiency attenuated EC injury and EndoMT in the DN model mice (Fig. 2). AGEs is known to induce EndoMT through smad signaling [5]. α2AP did not induce smad2/3 activation (data not shown), but α2AP promoted the AGEs-induced smad2/3 activation and EndoMT (Fig. 5b, c). It has been reported that SHP2 mediates smad2/3 activation [27], and we previously demonstrated that α2AP activates SHP2 in ECs [18]. We herein showed that the inhibition of SHP2 reversed the α2AP-promoted EndoMT induced by AGEs (Fig. 5c). These data suggest that the α2AP-related SHP2 activation is associated with EndoMT induced by high glucose condition/AGEs.

Furthermore, we showed that the high glucose conditions/AGEs induced α2AP production in FBs, but not in ECs (Figs. 3 and 4a). In addition, the FBs/ECs co-culture promoted the high glucose condition-induced EndoMT (Fig. 5a), and the blockade of α2AP attenuated the FBs/ECs co-culture-promoted EndoMT under the high glucose condition (Fig. 5b). These data suggest that the high conditions/AGEs induced α2AP production in FBs, and α2AP affected the pro-fibrotic changes in FBs. On the other hand, the FB-derived α2AP promoted the high glucose conditions-induced EC injury and EndoMT. α2AP may play a pivotal rolte on the progression of fibrosis in DN (Fig. 6).

Fig. 6
figure 6

The role of α2AP on the progression of renal fibrosis in diabetes. The hyperglycemic conditions induces AGEs overproduction, and AGEs stimulates α2AP production in FBs. And then, AGEs and α2AP induce the pro-fibrotic changes in FBs. On the other hand, the FB-derived α2AP promotes the high glucose/AGEs-induced EC injury and EndoMT, and exacerbates fibrosis progression

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It has been reported that the patients with diabetes had a hypercoagulable state by increased coagulability and decreased fibrinolysis, and the hypercoagulable contributes to the increased risk of vascular disease and the development of microvascular complications in diabetes [31, 32]. The increased circulating levels of α2AP and PAP have been reported in patients with diabetes and DN [19, 20]. On the other hand, plasmin reduces ECM deposition in kidney with DN [33]. In addition, plasmin activates the AMPK pathway [8], and the activation of AMPK improves renal function in DN model rat [34]. The increase in α2AP expression in diabetes may also affect the onset and progression of DN through plasmin inactivation.

In conclusion, the high glucose conditions induced α2AP production, and α2AP is associated with EndoMT and fibrosis progression in DN. The regulation of α2AP expression in diabetes may be useful for DN and provide new insight into the development of clinical therapies for DN.