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Keywords:

  • activated protein C;
  • diabetes;
  • glomerulosclerosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

Summary.  Background:  Activated protein C (APC) can regulate immune and inflammatory responses and apoptosis. Protein C transgenic mice develop less diabetic nephropathy but whether exogenous administration of APC suppresses established diabetic nephropathy is unknown.

Objectives:  We investigated the therapeutic potential of APC in mice with streptozotocin-induced diabetic nephropathy.

Methods:  Diabetes was induced in unilaterally nephrectomized C57/Bl6 mice using intraperitoneal (i.p.) injection of streptozotocin. Four weeks later, the mice were treated with i.p. exogenous APC every other day for 1 month.

Results:  APC-treated mice had a significantly improved blood nitrogen urea-to-creatinine ratio, urine total protein to creatinine ratio and proteinuria, and had significantly less renal fibrosis as measured by the levels of collagen and hydroxyproline. The renal tissue concentration of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and the RNA expression of platelet-derived growth factor (PDGF), transforming growth factor-β1 and connective tissue growth factor (CTGF) were significantly lower in APC-treated mice than in untreated animals. The percentage of apoptotic cells was reduced and the expression of podocin, nephrin and WT-1 in the glomeruli was significantly improved in mice treated with APC compared with untreated mice. The levels of coagulation markers were not affected by APC treatment.

Conclusion:  Exogenous APC improves renal function and mitigates pathological changes in mice with diabetic nephropathy by suppressing the expression of fibrogenic cytokines, growth factors and apoptosis, suggesting its potential usefulness for the therapy of this disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

The incidence and prevalence of end-stage renal disease have increased over the past 30 years up to 2010 and are expected to continue increasing [1]. Diabetic nephropathy is the single most frequent cause of end-stage renal disease [2]. Angiotensin I-converting enzyme inhibition and angiotensin II receptor antagonists, the standard treatment for diabetic nephropathy, only delay the late stage symptoms of the disease, and thus the patient evolves with progressive renal damage, often requiring dialysis or kidney transplantation [2]. Consequently, diabetic nephropathy has become a public health burden and new therapeutic strategies that target mechanistic pathways underlying diabetes-associated renal injury are needed [2].

The precise pathogenic mechanisms of diabetic nephropathy are still unclear. Diabetic nephropathy has been traditionally considered to be caused by metabolic and hemodynamic alterations [3], but recent studies suggest that inflammatory processes and aberrant immune responses are also involved in the development and progression of the disease [4]. Hyperglycemia stimulates renal cells to produce humoral mediators, cytokines and growth factors that are responsible in part for the structural and functional alterations seen in the diabetic kidney, such as increased extracellular matrix deposition and increased permeability of the glomerular basement membrane [5].

Activated protein C (APC), the effector enzyme of the protein C pathway, plays a fundamental role not only as an inhibitor of the coagulation system but also as a modulator of inflammatory responses, tissue remodeling and apoptosis. These effects are mediated mostly through its receptors, the endothelial protein C receptor and protease-activated receptor-1 [6]. The cytoprotective effects of APC include protection of the endothelial barrier functions, suppression of the expression of inflammatory cytokines, growth factors and adhesion molecules from a variety of cells, blockade of the activation and the extravasation of leukocytes at sites of tissue injury, and inhibition of apoptosis through the tumor suppressor protein p53 [7].

The beneficial and protective effects of APC administration have been demonstrated in animal models including sepsis, ischemic stroke and lung disorders as well as in human diseases [6]. The APC system may also regulate disorders in the microcirculation, such as those seen in the diabetic kidney. We have previously reported that circulating APC levels inversely correlated with progression of diabetic nephropathy [8]. A recent study has showed that APC can prevent the development of diabetic nephropathy in mice with high circulating levels of protein C [9]. In the present study, we hypothesized that exogenous administration of APC would inhibit the progression of diabetic nephropathy. To investigate this hypothesis we induced accelerated diabetes with streptozotocin (STZ) in mice unilaterally nephrectomized and treated them with intraperitoneal (i.p.) injections of APC.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

Animals

Male 6- to 8-week-old C57/Bl6 mice weighting 20–25 g were maintained in a pathogen-free environment on a constant 12-h light/12-h dark cycle in a temperature- and humidity-controlled room and were given ad libitum access to food and water. The experimental protocol for the animal studies conformed to NIH guidelines: Principles of Laboratory Animal Care (NIH publication no. 85–23, revised 1985; http://grants1.nih.gov/grants/olaw/references/phspol.htm) and was reviewed and approved by Mie University’s Committee for Animal Investigation.

Nephrectomy

All mice used in the present study underwent a unilateral nephrectomy performed with the purpose of accelerating the progression of hyperglycemia-induced nephropathy. The mice were anesthetized using a low dose of pentobarbital (1 mg kg−1) administered by i.p. injection. An incision of approximately 1 cm was made in the right dorso-lumbar area. The ureter and the renal artery and vein were located and subsequently ligated using silk thread before cutting them proximal to the right renal hilium. The kidney was removed and the incision was then closed with silk sutures.

Induction of diabetes and treatment with APC

After complete recovery from nephrectomy (1 month after surgery), a group of mice received i.p. injections of STZ (40 mg kg−1 body weight) for five consecutive days; control mice received 200 μL of saline solution intraperitoneally for five consecutive days. After 4 weeks, the STZ-treated mice reached a non-fasting blood-glucose level of over 300 mg dL−1 (> 15 mmol L−1) and were randomized to either STZ/SAL or STZ/APC groups. The STZ/APC group received 1 mg kg−1 of human APC dissolved in 200 μL of saline every other day i.p. and the STZ/SAL mice received 200 μL of saline solution intraperitoneally every other day (Fig. S1a). Human APC was provided by Kaketsuken (Kumamoto, Japan) [10]. Mice that did not receive STZ injections (SAL/SAL group) received 200 μL of saline (SAL/SAL) or 1 mg kg−1 of human APC dissolved in a 200-μL solution (SAL/APC) i.p. every other day. Thus, there were four experimental groups of animals: negative control mice treated with saline (SAL/SAL, n = 8), APC (SAL/APC, n = 5), mice with diabetic nephropathy treated with saline (STZ/SAL, n = 11) and mice with diabetic nephropathy treated with APC (STZ/APC, n = 13). Mice were weighed, urine and plasma collected and glucose determined at three time points: before STZ injections, before the initiation of APC treatment and at sacrifice. Systolic, diastolic and mean blood pressures were determined non-invasively in conscious mice using the tail-cuff method 1 day before sacrifice (BA-98A System; Softron Co., Tokyo, Japan). Animals were habituated to the device when measurements were conducted. Mice were sacrificed on day 29 after the start of APC or saline injections. The time interval between the last APC treatment and sampling at sacrifice was 48 h.

Biochemical analysis

Blood glucose was measured using the glucose-oxidase method. Plasma creatinine, blood urea nitrogen and albumin in urine were measured by the KINKIGOKEN Corp. (Tokyo, Japan). The concentration of total protein was measured using a dye-binding assay (BCA™ protein assay kit; Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Immunoassays were used for measuring monocyte chemoattractant protein-1 (MCP-1; BD Biosciences Pharmingen, San Diego, CA, USA) and vascular endothelial growth factor (VEGF; R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. Mouse-specific thrombin–antithrombin complex (TAT), a marker of coagulation activation, was measured using EIA kits from Cusabio Biotech (Wuhan, China) according to the manufacturer’s instructions. Mouse endogenous activated protein C (APC) and mouse D-dimer were measured using mouse-specific EIA kits from Uscn Life Science Inc. (Wuhan, China) according to the manufacturer’s instructions [11].

Statistical analysis

Statistical analyzes were done using the StatView 4.1 package software for the Macintosh (Abacus Concepts, Berkeley, CA, USA). Data are expressed as the mean ± standard error (SE). The statistical difference between variables was calculated by analysis of variance with post hoc analysis using the Turkey–Kramer test and when appropriate the Student’s t-test or Mann–Whitney U-test was used. Statistical significance was considered as P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

Induction of diabetes and changes in body weight

Diabetes was induced in all mice that received i.p. injections of STZ with blood glucose levels of over 300 mg dL−1 (> 15 mmol L−1) at both 4 weeks after the injections and at sacrifice (Fig. S1a–c). Moreover, the normal increase in body weight was impeded in mice receiving STZ injections (about 14% ± 8% in controls) (Fig. S1d,e). No significant differences were observed in body weight between groups during the APC therapy (Fig. S1f,g).

Renal function

Renal function was evaluated in nephrectomized mice treated with saline or STZ on the 4th week before APC administration. Albuminuria and the ratio of urine albumin to creatinine were significantly elevated in mice treated with STZ (STZ/SAL) compared with those treated with saline (SAL/SAL) (Fig. S2a,b). Although a toxic effect of STZ cannot be ruled out, these observations suggest that the development of diabetic nephropathy occurs early in unilaterally nephrectomized mice treated with STZ (Table S2); this is consistent with the results reported in a previous study [12].

Four weeks after APC treatment, the results showed that the ratio of blood urea nitrogen to plasma creatinine and proteinuria were significantly elevated in the STZ/SAL group compared with the SAL/SAL and SAL/APC groups (Fig. 1A,B). The plasma concentration of albumin was significantly decreased and the ratio of urine total protein to creatinine was significantly increased in STZ/SAL compared with both SAL/SAL and SAL/APC control groups (Fig. 1C,D). These observations confirm that STZ/SAL mice had diabetic nephropathy. Diabetic mice treated with APC had a decreased blood nitrogen urea-to-creatinine ratio, proteinuria, ratio of urine total protein to creatinine and elevated plasma albumin compared with the STZ/SAL group (Fig. 1A–D). No significant difference was found in any of these parameters between the SAL/SAL and SAL/APC groups.

image

Figure 1.  Renal functional parameters. The blood nitrogen urea (BUN)-to-creatinine ratio (A) and the concentration of total protein (B) in urine were significantly different between the SAL/SAL and STZ/SAL groups and between the STZ/SAL and STZ/APC groups. The plasma level of albumin (C) and the urine total protein-to-creatinine ratio (D) were significantly different between the STZ/SAL and control groups and between the STZ/SAL and STZ/APC groups. Bars indicate mean ± standard error of mean (SEM). Statistical difference was analyzed using anova with post-hoc analysis. *P < 0.001 vs. the SAL/SAL and SAL/APC groups. ‡P < 0.05 vs. STZ/SAL.

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Kidney weight and blood arterial pressure

The kidney weight in diabetic mice (STZ/SAL and STZ/APC groups) significantly increased compared with the saline control group but no difference was found between them (Table S2). Arterial hypertension can also cause nephropathy. Systolic, diastolic and media blood pressures were measured 1 day before sacrifice but no differences were found between the SAL/SAL, STZ/SAL and STZ/APC groups (Table S2).

Mesangial expansion

Increased deposition of the extracellular matrix with concurrent mesangial expansion occurs in the glomeruli of patients with diabetes mellitus [13]. In agreement with this, enhanced mesangial expansion was observed in glomeruli from mice in the STZ/SAL group compared with the SAL/SAL and SAL/APC control groups. Treatment with APC significantly blocked this mesangial expansion (Fig. 2A,B).

image

Figure 2.  Mesangial expansion. Compared with the control groups and STZ/APC there is increased deposition of periodic acid-Schiff (PAS) positive substances in glomeruli from the STZ/SAL group (A). Quantification of PAS (+) areas disclosed a significant deposition of extracellular matrix in the STZ/SAL group compared with both the SAL/SAL and STZ/APC groups (B). Bars indicate mean ± SEM. The scale bars indicate 50 μm. Statistical difference was analyzed using anova with post-hoc analysis. *P < 0.0001 vs. the SAL/SAL and SAL/APC groups. ‡P < 0.0001 vs. STZ/SAL.

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Renal sclerosis

Deposition of collagen in glomeruli and renal interstitium was assessed using Masson’s trichrome staining and by measuring the contents of collagen and hydroxyproline in the kidney [14]. There was intense staining for collagen in the glomerular and renal interstitial areas in mice in the STZ/SAL group compared with the SAL/SAL and SAL/APC groups (Fig. 3A,B). Quantification of positively stained areas showed significant deposition of collagen in the STZ/SAL group compared with the SAL/SAL group, but collagen staining was significantly lower in mice treated with APC (Fig. 3A,B). Measurement of collagen and hydroxyproline content confirmed these results as the contents of collagen and hydroxyproline were significantly different between the STZ/SAL group and the SAL/SAL and SAL/APC groups, and between the STZ/SAL and STZ/APC groups (Fig. 3B).

image

Figure 3.  Renal fibrosis. Masson staining demonstrated increased collagen staining in STZ/SAL mice compared with the SAL/SAL, SAL/APC and STZ/APC groups (A). Quantification of Masson-stained areas showed enhanced deposition of collagen in the STZ/SAL group compared with the control and STZ/APC groups (B). There was increased renal content of collagen and hydroxyproline in the STZ/SAL group compared with the control and STZ/APC groups (B). Bars indicate mean ± standard error of mean (SEM). The scale bars indicate 50 μm. Statistical difference was analyzed using anova with post-hoc analysis. *P < 0.0002 vs. the SAL/SAL and SAL/APC groups. ‡P < 0.001 vs. STZ/SAL.

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The coagulation and fibrinolysis system

The plasma concentration of endogenous APC was significantly increased in both STZ/SAL and STZ/APC groups compared with the SAL/SAL group but there was no difference between other groups (Table 1). Human APC was undetectable in plasma in all groups at the time of sacrifice. APC exerts its anticoagulant activity by inhibiting the coagulation factors (F)Va and VIIIa [6]. The effect of APC treatment on the coagulation system was evaluated by measuring the plasma concentration of TAT. The plasma concentration of TAT tended to decrease in the STZ/SAL group compared with both SAL/SAL and SAL/APC groups but did not reach significance (Table 1).

Table 1.  Coagulation parameters
ParameterSAL/SALSAL/APCSTZ/SALSTZ/APC
  1. Samples were taken at sacrifice from each group of mice. Statistics by anova and the Turkey–Kramer test. TAT, thrombin–antithrombin complex; APC, activated protein C; D-D, D-dimer. *P = 0.1 vs. the SAL/SAL and STZ/APC groups. P < 0.002 vs. the SAL/SAL group. §P = 0.03 vs. the SAL/SAL group.

APC
 Plasma (ng mL−1)0.93 ± 0.181.53 ± 0.422.99 ± 0.203.59 ± 0.39
 Tissue (ng per kidney)5.58 ± 1.695.97 ± 2.101.90 ± 0.284.91 ± 0.34
TAT
 Plasma (ng mL−1)9.46 ± 0.957.76 ± 1.445.68 ± 1.64*2.88 ± 0.39
 Tissue (ng per kidney)19.77 ± 0.5320.37 ± 0.6117.87 ± 0.9217.93 ± 0.42
D-D
 Plasma (ng mL−1)426.53 ± 27.19601.53 ± 82.73517.94 ± 65.32692.16 ± 73.11§
 Tissue (ng per kidney)545.28 ± 104.49566.57 ± 139.25562.09 ± 114.88541.42 ± 19.36

The plasma concentration of D-dimer was significantly elevated in STZ/APC mice compared with SAL/SAL mice but there was no significant difference between other groups (Table 1). Plasminogen activator inhibitor-1 (PAI-1), a component of the fibrinolysis system, has been also associated with increased deposition of the extracellular matrix in diabetic nephropathy [15]. The RNA expression of PAI-1 was significantly increased in STZ/SAL mice compared with both SAL/SAL and SAL/APC groups. The RNA expression of PAI-1 was significantly decreased in the group treated with APC (STZ/APC) compared with the untreated group (STZ/SAL; Table 2).

Table 2.  Semiquantitative RT-PCR
Gene/gapdhSAL/SALSAL/APCSTZ/SALSTZ/APC
  1. ctgf, connective tissue factor growth factor; pdgf-α, platelet-derived growth factor; pai-1, plasminogen activator inhibitor-1; vegf, vascular endothelial growth factor; mcp-1, monocyte-chemoattractant protein-1; wt-1, Wilms tumor-1. *P < 0.001 vs. the SAL/SAL and SAL/APC groups. P < 0.05 vs. the STZ/SAL group. §P < 0.02 vs. the SAL/SAL and SAL/APC groups.

pai-1/gapdh 0.136 ± 0.0010.124 ± 0.0040.401 ± 0.047*0.253 ± 0.055
mcp-1/gapdh 0.226 ± 0.010.203 ± 0.0190.970 ± 0.09*0.570 ± 0.103
vegf/gapdh 0.144 ± 0.0060.142 ± 0.010.778 ± 0.145*0.217 ± 0.05
ctgf/gapdh 0.131 ± 0.0030.115 ± 0.0070.540 ± 0.049*0.403 ± 0.051
pdfg-α/gapdh 0.078 ± 0.0020.075 ± 0.0050.617 ± 0.054*0.346 ± 0.067
podocin/gapdh 0.749 ± 0.0460.711 ± 0.0140.284 ± 0.081§0.395 ± 0.145
nephrin/gapdh 0.717 ± 0.0350.689 ± 0.0420.096 ± 0.026*0.213 ± 0.030
wt-1/gapdh 0.547 ± 0.0700.730 ± 0.1010.597 ± 0.0490.947 ± 0.060

No significant differences were detected in the tissue concentrations of TAT, D-dimer or endogenous APC between groups (Table 1).

Chemokines and growth factors

The inflammatory response plays an important role in the pathogenesis of diabetic nephropathy via MCP-1, which is a key factor in the recruitment of inflammatory cells and VEGF which enhances vascular permeability at sites of tissue injury [3]. The protein and RNA expression of MCP-1 and VEGF in renal tissue homogenates were significantly increased in STZ/SAL mice compared with mice of the SAL/SAL and SAL/APC groups; but they were significantly decreased in mice treated with APC (STZ/APC) compared with STZ/SAL mice (Fig. 4A,B; Table 2).

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Figure 4.  Profibrogenic cytokines, VEGF and αSMA. The renal tissue concentrations of MCP-1 (A) and VEGF (B) were significantly increased in the STZ/SAL group compared with the control and STZ/APC groups. Reverse transcription-PCR analysis of TGF-β1 (C) and αSMA (D) disclosed significant differences between the control and STZ/SAL groups and between the STZ/SAL and STZ/APC groups. Bars indicate mean ± standard error of the mean (SEM). A statistical difference was analyzed using anova with post-hoc analysis. *P < 0.05 vs. the SAL/SAL and SAL/APC groups. ‡P < 0.05 vs. STZ/SAL.

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TGF-β1 and connective tissue growth factor (CTGF) play critical roles in the secretion of extracellular matrix deposition from myofibroblasts [16]. The RNA expressions of TGF-β1 (Fig. 4C) and CTGF (Table 2) were significantly enhanced in STZ/SAL compared with SAL/SAL and SAL/APC mice but they were significantly decreased by APC therapy (STZ/APC). The expression of α-smooth muscle actin, a marker of myofibroblast activation, was significantly enhanced in STZ/SAL compared with control mice but it was significantly decreased in APC-treated mice (Fig. 4D). Proliferation of myofibroblasts occurs during renal fibrotic processes [17]. Platelet-derived growth factor (PDGF) is one of the main stimulators of myofibroblast proliferation [17]. We found increased RNA expression of PDGF in STZ/APC mice compared with control non-diabetic mice but the PDGF concentration was reduced in mice treated with APC compared with the STZ/SAL group (Table 2).

Apoptosis

Apoptosis of structural glomerular cells, particularly podocytes, has been implicated in the pathogenesis of diabetic nephropathy [18]. Apoptosis in glomeruli was evaluated using the TUNEL method in our mouse model of diabetic nephropathy and the results showed a significantly increased number of apoptotic cells in the glomerular areas in mice from the STZ/SAL group compared with the SAL/SAL and SAL/APC groups. The STZ/APC group has a reduced number of apoptotic cells compared with the STZ/SAL group (Fig. 5A,B).

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Figure 5.  Apoptosis in diabetic nephropathy. The TUNEL method was used to assess apoptosis. The area of apoptotic cells was counted in the glomeruli and it was expressed as the percentage of the total glomerular area. Apoptotic areas were prominent in the STZ/SAL group compared with the control and STZ/APC groups (A). Quantification disclosed a significant increased number of apoptotic cells in the STZ/SAL group compared with the control and STZ/APC groups (B). Bars indicate mean ± standard error of the mean (SEM). The scale bars indicate 50 μm. Statistical difference was analyzed using anova with post-hoc analysis. *P < 0.001 vs. the SAL/SAL and SAL/APC groups. ‡P < 0.001 vs. STZ/SAL.

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To clarify the mechanism of APC-mediated inhibition of apoptosis, we assessed the renal tissue expression of pro- and anti-apoptotic factors in each group of mice by semi-quantitative PCR. The renal expression of the pro-apoptotic factors Apaf-1 and caspase-3 were significantly decreased whereas the expression of the anti-apoptotic XIAP was significantly elevated in diabetic mice treated with APC (STZ/APC) compared with the diabetic STZ/SAL group treated with saline (Fig. S3a,b). In addition, the renal expression of Apaf-1 was significantly decreased and XIAP was significantly increased in the SAL/APC group compared with the SAL/SAL group. The anti-apoptotic factors Bcl-2 and Bcl-xL also tended to increase after APC treatment (Fig. S3a,b).

Podocytes-specific markers

Podocytes and their slit diaphragm play a key role in the prevention of proteinuria [19,20]. Nephrin and podocin are structural components of the slit diaphragm. We found decreased RNA expression of nephrin and podocin in STZ/SAL compared with the SAL/SAL and SAL/APC groups (Table 2). There was a significant difference in the RNA expression level of nephrin but not that of podocin between the STZ/SAL and STZ/APC groups (Table 2); however, the protein expression of podocin as evaluated by immunohistochemistry significantly improved after APC treatment (Fig. 6A,B).

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Figure 6.  Staining of podocin and WT-1. The number of podocin (+) cells (A, B) and WT-1 (+) cells (C, D) was significantly reduced in STZ/SAL mice compared with the control and STZ/PC groups. Bars indicate mean ± standard error of the mean (SEM). The scale bars indicate 20 μm. Statistical difference was analyzed by anova with post-hoc analysis. *P < 0.0001 vs. the SAL/SAL and SAL/APC groups. ‡P < 0.0001 vs. STZ/SAL.

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The expression of WT-1, a transcription factor specifically expressed in podocytes, was also evaluated by PCR and immunohistochemistry. The RNA expression of WT-1 was significantly higher in the STZ/APC group than in the STZ/SAL group but no significant difference was observed between other groups (Table 1). The protein expression of WT-1 was decreased in the STZ/SAL group compared with both control (SAL/SAL and SAL/APC) groups but it was significantly improved by APC treatment (Fig. 6C,D).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

The present study shows that treatment with APC improves renal functional parameters and retards the development of diabetes-associated renal fibrosis by decreasing apoptosis in a mouse model of diabetic nephropathy.

Unilateral renal ablation and diabetic nephropathy

In the present study, in order to accelerate the development of diabetes nephropathy, we injected STZ to previously unilaterally nephrectomized mice. Previously the occurrence of mild segmental expansion of the mesangial areas was demonstrated in nephrectomized mice after 4 weeks of STZ injection [12]; further, the results of the present study showed that these diabetic mice already have renal dysfunction. However, it should be noted that the microvascular changes of diabetic nephropathy observed in these mice with short-term hyperglycemia are milder than in mice with prolonged hyperglycemia shown earlier [9]. The limitations of this short-term model may explain the lack of a difference in the kidney weights between the untreated and APC-treated diabetic mice. Another limitation of the present study is that it is mainly characterized by persistent hyperglycaemia and hence captures only one aspect of diabetes mellitus. Further, we have not ruled out the possibility that the early renal dysfunction detected in the present study is because of STZ-induced toxicity.

Coagulation activation and nephropathy

Patients with diabetes are in a hypercoagulable state because they have increased circulating levels of coagulation factors (fibrinogen, FX and FVIII), impaired fibrinolysis as a consequence of an increased plasma level of PAI-1, thrombin-activatable fibrinolysis inhibitor and a decreased activity of the anticoagulant system [21]. However, in the present study, we found no significant changes in the plasma levels of TAT or D-dimer in mice with diabetic nephropathy compared with control mice suggesting that diabetic nephropathy does not affect the systemic coagulation and fibrinolysis systems, at least in the present study. Interestingly, the plasma level of endogenous APC was significantly elevated in diabetic mice treated without (STZ/SAL) or with APC (STZ/APC) compared with control mice, suggesting that there is a compensatory increase in APC generation that occurs in our diabetic model. APC is both an anti-coagulant via its inactivation of coagulation FVa and VIIIa and an anti-inflammatory via its receptors. As there was no change in coagulation markers in animals treated with APC compared with diabetic mice, it suggests that the protective effects of APC were as a result of its other activities.

Another common morbid association of diabetes is arterial hypertension, which may also worsen microangiopathy in diabetes [22]. To rule out this possibility, arterial pressure was measured in all groups of mice but none of them showed abnormal values, suggesting that arterial pressure was not involved in our model of nephropathy. However, it is worth noting that the absence of an arterial complication may be because of the short duration (8 weeks) of diabetes.

Chemokines and nephropathy

Long-term low-grade inflammation plays a critical role in the mechanism of pathological conditions that cause diabetic nephropathy including insulin resistance and endothelial dysfunction [23]. MCP-1 is involved in chronic inflammation and diabetic nephropathy. Increased levels of MCP-1 are correlated with the number of infiltrating macrophages in the renal interstitium in humans and in animal models [24,25]. The fact that the increased urinary MCP-1 concentration in patients or animals with renal disease (including diabetic nephropathy) correlates with the degree of proteinuria and renal damage further supports the role of MCP-1 in the development of diabetic glomerulosclerosis [26]. In the kidneys, MCP-1 can be produced by cortical tubules, glomerular podocytes and mesangial cells [25]. In agreement with these previous observations, in the present study, the protein and RNA expression of MCP-1 were significantly increased in the kidneys of diabetic mice compared with control mice. Therapy with APC reduced the concentration of MCP-1 in the kidneys of mice with established diabetes. The inhibitory effect of APC on MCP-1 has been reported in several types of cells and it is mediated by both protease activated receptor-1 and endothelial protein C receptor [6]. This is the first report showing the inhibitory effect of APC on renal expression of MCP-1 in diabetic nephropathy. The inhibitory effect of APC on MCP-1 may also explain, at least in part, the beneficial effect of APC on diabetic nephropathy.

Podocytopathy and renal dysfunction

Podocyte injury plays a critical role in the mechanism of albuminuria in diabetic kidney disease. Previous studies suggested that in the early stages of diabetic nephropathy, the expression of components of the slit diaphragm is decreased in podocytes and that this results in foot effacement from the glomerular basement membrane, apoptosis and alteration of the renal filtration barrier to protein passage [27]. Nephrin, an 185-kDa transmembrane protein, is a major component of the slit diaphragm that contains a large extracellular portion with eight immunoglobulin-like domains [20,27]. Podocin is another protein expressed by podocytes in the slit diaphragm; podocin interacts with nephrin and this interaction is required for efficient signaling through nephrin [28]. In agreement with the results of previous studies, in the present study we found an increased number of apoptotic cells and decreased expression of both nephrin and podocin in mice with untreated STZ-induced diabetes compared with mice of the control groups; the expression of WT-1, a transcription factor specifically expressed in podocytes, was also reduced in diabetic mice compared with controls. APC treatment increased the expression of nephrin, podocin and WT-1 and significantly inhibited apoptosis. Further investigation into the mechanism of the changes in apoptosis showed that diabetic mice treated with APC have reduced expression of pro-apoptotic factors and increased expression of anti-apoptotic factors compared with untreated mice. Overall, these findings agree with the result of a previous study showing that APC can prevent diabetic nephropathy by inhibiting apoptosis in a transgenic mouse overexpressing protein C [9]. However, this is the first study to show that administration of exogenous APC effectively inhibits apoptosis and inflammation in diabetic nephropathy.

VEGF and nephropathy

Diabetes is associated with enhanced glomerular expression of VEGF, which is believed to have a detrimental effect at least in late stages of the disease [29]. VEGF may act as a chemoattractant for macrophages as well as activating them [30]. Stimulation of VEGF production by podocytes also increases macromolecular permeability of the glomerular capillary [30]. Consistent with previous reports, we found enhanced expression of VEGF in untreated STZ/SAL mice compared with control mice. We also evaluated the effect of therapy with APC on VEGF expression in diabetic nephropathy; APC has been previously shown to increase the expression of VEGF from several cell types [31]; surprisingly, mice treated with APC had decreased levels of VEGF in the kidneys compared with untreated mice. This observation suggests that APC may also be involved in the maintenance of optimal renal expression of VEGF in the kidneys.

Growth factors and glomerulosclerosis

The hallmark of diabetic nephropathy is the extensive deposition of extracellular matrix protein in the kidneys with thickening of the glomerular basement membrane, mesangial expansion, mesangiolysis and nodular sclerosis [32]. Excessive collagen deposition in the kidneys may result from disruption of the balance between processes of synthesis and degradation which is regulated by several growth factors and chemokines including TGF-β1, CTGF, PDGF and MCP-1 [32]. PDGF favors glomerular sclerosis by promoting the proliferation of fibroblasts, MCP-1, TGF-β1 and CTGF by stimulating the secretion of extracellular matrix proteins including collagens. TGF-β1 may also promote collagen deposition in the kidneys by stimulating the secretion of tissue-type metalloproteinase inhibitors [32]. Consistent with previous observations, in the present study, diabetes caused enhanced fibrosis of the renal interstitium and glomeruli and this was associated with increased renal expressions of PDGF, MCP-1, CTGF and TGF-β1 compared with non-diabetic mice; these pathological alterations and the expression levels of PDGF, MCP-1, CTGF and TGF-β1 were significantly inhibited by APC treatment, demonstrating the protective effect of APC on established diabetic glomerulosclerosis. A previous study has demonstrated the anti-fibrotic effect of APC but this is the first time that it was shown in a model of diabetic nephropathy [10].

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

A previous study has shown that high generation of APC in a mouse overexpressing protein C can prevent the development of diabetic nephropathy by inhibiting apoptosis. The results of the present study demonstrated for the first time that in addition to inhibit apoptosis and to improve renal function, exogenous administration of APC can also inhibit pro-fibrogenic cytokines, growth factors and renal fibrosis in a model of diabetic nephropathy, suggesting the potential beneficial effect of APC treatment for diabetic nephropathy.

Recombinant human APC has been developed and approval has been obtained for its use in patients with sepsis; recombinant human APC was found to be the first drug showing a survival benefit in patients with sepsis [33]; variants of recombinant APC with reduced anticoagulant activity have been also developed to avoid the risk of a bleeding complication [34]. Therefore, further investigations should be also carried out to evaluate the applicability of recombinant APC as a novel tool for treating diabetic nephropathy in humans.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

P. Gil-Bernabe, D. Boveda Ruiz, and C. N. D'Alessandro-Gabazza prepared the mouse model of disease and carried out tissue staining. M. Toda and Y. Miyake measured several markers, cytokines and growth factors in plasma and renal homogenates. T. Suzuki, Y. Onishi and Y. Yano measured renal functional parameters. J. Morser made corrections in the manuscript and important intellectual contributions. E.C. Gabazza wrote the first draft of the manuscript and contributed intellectually for the completion of the study. Y. Takei contributed to the intellectual content of the manuscript.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

This research was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Mie Medical Research Foundation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Addendum
  9. Acknowledgements
  10. Disclosure of Conflict of Interests
  11. References
  12. Supporting Information

Data S1. Materials and Methods.

Figure S1. Experimental design, glycemia and body weight.

Figure S2. Early diabetic nephropathy in unilaterally nephrectomized mice after STZ administration.

Figure S3. Changes in the expression of transcription and apoptosis-related factors by activated protein C.

Table S1. Primers for RT-PCR.

Table S2. Renal weight at sacrifice and blood pressures.

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JTH_4621_sm_FigS1-S3-TableS1-S2.pdf1307KSupporting info item

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