2-(8-Hydroxy-6-Methoxy-1-Oxo-1H-2-Benzopyran-3-yl) Propionic Acid, an Inhibitor of Angiogenesis, Ameliorates Renal Alterations in Obese Type
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糖尿病学杂志 2006年第5期
1 Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2 Department of Medicine and Clinical Science, Genzyme, Framingham, Massachusetts
Ang, angiopoietin; CCr, creatinine clearance; ESRD, end-stage renal disease; GBM, glomerular basement membrane; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; NM-3, 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid; TGF, transforming growth factor; TNF-, tumor necrosis factor-; UACR, urinary albumin-to-creatinine ratio; VEGF, vascular endothelial growth factor
ABSTRACT
One of the mechanisms involved in the progression of diabetic nephropathy, the most common cause of end-stage renal failure, is angiogenic phenomenon associated with the increase of angiogenic factors such as vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-2, an antagonist of Ang-1. In the present study, we examined the therapeutic efficacy of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3), a small molecule isocoumarin with antiangiogenic activity, using diabetic db/db mice, a model of obese type 2 diabetes. Increases in kidney weight, glomerular volume, creatinine clearance, urinary albumin excretion, total mesangial fraction, glomerular type IV collagen, glomerular endothelial area (CD31+), and monocyte/macrophage accumulation (F4/80+) observed in control db/db mice were significantly suppressed by daily intraperitoneal injection of NM-3 (100 mg/kg, for 8 weeks). Increases in renal expression of VEGF-A, Ang-2, fibrogenic factor transforming growth factor (TGF)-1, and chemokine monocyte chemoattractant protein-1 but not tumor necrosis factor- were also inhibited by NM-3 in db/db mice. Furthermore, decreases of nephrin mRNA and protein levels in db/db mice were recovered by NM-3. In addition, treatment of db/db mice with NM-3 did not affect body weight, blood glucose, serum insulin, or food consumption. NM-3 significantly suppressed the increase of VEGF induced by high glucose in cultured podocytes and also suppressed the increase of VEGF and TGF- induced by high glucose in cultured mesangial cells. Taken together, these results demonstrate the potential use of NM-3 as a novel therapeutic agent for renal alterations in type 2 diabetes.
Diabetic nephropathy is complicated in 30eC40% of patients with type 2 diabetes and is the most common pathological disorder predisposing end-stage renal disease (ESRD) in Japan and the Western world. Early alterations in diabetic nephropathy include glomerular hyperfiltration, glomerular and tubular epithelial hypertrophy, glomerular basement membrane (GBM) thickening, and the development of microalbuminuria, followed by the development of the accumulation of mesangial matrix and overt proteinuria, eventually leading to glomerulosclerosis and ESRD (1). The involvement of various growth factors and cytokines, including angiotensin II, IGF-I, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and transforming growth factor (TGF)-1, in the development of diabetic nephropathy have been reported (2).
Although angiogenesis, the development of new blood vessels from preexisting ones, plays a crucial role in physiological events, such as wound repair, uncontrolled neovascularization is associated with a number of pathological disorders, including tumor growth, rheumatoid arthritis, and diabetic retinopathy (3). Vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, promotes endothelial cell proliferation, migration, and endothelial cell tube formation (4). VEGF-A also induces vascular permeability (5) in association with inflammatory lesions. The protein and mRNA level of VEGF-A and its receptor flk-1/KDR were upregulated in experimental diabetic nephropathy (6eC8). Studies using neutralizing antieCVEGF-A antibody further demonstrated the involvement of VEGF-A in early glomerular hypertrophy in type 1 diabetic model and mesangial matrix accumulation in the progressive stage of type 2 diabetic model (9,10).
Angiopoietin (Ang)-1, a major physiological ligand for endothelial receptor Tie2, induces the recruitment and stable attachment of pericytes leading to the maturation of newly formed blood vessels (11). In contrast, Ang-2 competitively inhibits the binding of Ang-1 to Tie2 and renders blood vessels "unstable" (12). Ang-2 loosens the attachment of pericytes and synergizes with VEGF-A to promote angiogenesis (12,13). However, when insufficient angiogenic stimuli are present, Ang-2 causes endothelial cell apoptosis and vessel regression (12). During kidney development, Ang-1, Ang-2, and Tie2 are highly expressed and play pivotal roles in the maturation of glomeruli and renal blood vessels (14). We previously reported the upregulation of Ang-2 in the type 1 mouse diabetic nephropathy model, although the expression of Ang-1 was not altered (15,16).
Nephrin, a glomerular podocyte protein, is crucial for maintaining the integrity of the interpodocyte slit membrane structure and an intact filtration barrier. In diabetic nephropathy, the protein level of nephrin decreases possibly via the loss of nephrin into urine due to synthesis of the splice variant isoform of the nephrin lacking a transmembrane domain (17,18).
A previous study demonstrated that the increased glomerular filtration surface in diabetic nephropathy resulted from the formation of new glomerular capillaries in accordance with a slight elongation of the preexisting capillaries (19), analogous to the changes observed in pathological diabetic retinopathy.
2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3) is a novel synthetic derivative of cytogenin, a natural compound isolated from culture filtrate of Streptoverticillium eurocidicum (20,21). NM-3 potently inhibits endothelial cell proliferation, migration, sprouting, tube formation in vitro, and tumor growth in vivo (22). Moreover, NM-3 suppresses endothelial cell migration induced by VEGF and exerts inhibitory effects on angiogenesis in VEGF-secreting malignant tumors (21,22). A phase I clinical study of NM-3 in patients with cancer has demonstrated that NM-3 is a highly orally bioavailable and well-tolerated drug in humans (23). NM-3 is currently entering a phase II clinical trial in patients with multiple myeloma (24).
In the present study, to examine whether NM-3 could provide beneficial effects on type 2 diabetic nephropathy, we applied NM-3 to type 2 diabetic db/db mice. Here, we demonstrate that treatment with NM-3 markedly suppresses glomerular hypertrophy, hyperfiltration, and urinary albumin excretion as well as the expansion of mesangial matrix and the accumulation of monocytes/macrophages. These therapeutic effects are possibly mediated through the downregulation of proangiogenic signals induced by VEGF-A and Ang-2 and through the suppression of TGF-1 and the recovery of nephrin.
RESEARCH DESIGN AND METHODS
The experimental protocol was approved by the Animal Ethics Review Committee of Okayama University. Adult female db/db mice (C57BL/KsJ-db/db Jcl; Clea Japan, Osaka, Japan) and their age-matched nondiabetic db/m littermates (C57BL/KsJ-db/+m Jcl; Clea Japan) were used in the present study. Mice were fed a standard pellet laboratory chow and were provided with water ad libitum. The db/db mice were included in this study at the age of 8 weeks because they develop diabetes (hyperglycemia) at the age of week 7eC8 (25). At the age of 8 weeks, blood glucose of db/db mice was in the range of 20.3eC22.1 mmol/l. Mice were divided into three subgroups (n = 6 for each subgroup): 1) db/db mice treated with daily intraperitoneal injection of NM-3 (Genzyme, Cambridge, MA) at 100 mg · kg body wteC1 · dayeC1 for 8 weeks, 2) db/db mice treated with daily intraperitoneal injection of vehicle buffer (PBS) for the same interval, and 3) nondiabetic, control db/m mice receiving vehicle buffer.
Blood glucose was monitored every week. No mice died, and no signs of apparent exhaustion were observed during the experimental period. At 0 and 8 weeks after initiating the intraperitoneal injection of NM-3, the body weight and the individual 24-h food consumption were measured. At 8 weeks after initiating treatment, individual 24-h urine sample collection was performed using metabolic cages. Nonfasting blood samples were drawn from the retro-orbital venous plexus using heparinized capillary tubes under anesthesia at the time of death. Kidney, liver, and heart weights were measured just after death. NM-3 was dissolved in PBS and filtered to be used for the animal experiments. The dosage of NM-3 used in the present study was determined according to previous reports using NM-3 (22,26). NM-3 has a serum half-life of 3eC10 h in preclinical models, a low toxicity profile, and thus a potentially wide therapeutic window (22). According to the manufacturer’s information, oral dosage of NM-3 at 1 g/kg was well tolerated in animals without acute toxicity.
Blood and urine examination.
Blood glucose was measured in tail-vein blood, and urine was tested for ketone bodies and glucose by SRL (Okayama, Japan). Serum and urinary creatinine levels were measured by the enzymatic colorimetric method as described previously (15). Urinary albumin concentration was measured by nephelometry (Organon Teknika-Cappel, Durham, NC) using anti-mouse albumin antibody (ICN Pharmaceuticals, Aurora, OH) as previously described (15). Results were normalized to the urinary creatinine levels and expressed as urinary albumin-to-creatinine ratio (UACR). The creatinine clearance (CCr) was calculated and expressed as milliliters per minute. Serum levels of mouse insulin were determined by enzyme-linked immunosorbent assay using an ultrasensitive rat insulin enzyme-linked immunosorbent assay kit and mouse insulin standard (Morinaga, Yokohama, Japan) following the manufacturer’s instructions. According to the manufacturer’s technical information, mouse insulin can be measured in combination with mouse insulin standard because of a high homology among mammalian animals. All samples were examined in duplicate, and mean values of individual sera were used for statistical analysis. The intra- and interassay coefficients of variation for the insulin assays were <5 and 10%, respectively.
Histological analysis.
At 8 weeks after starting treatment, kidneys were removed, fixed in 10% buffered formalin, and embedded in paraffin. Sections (4-e蘭) were stained with periodic acid Schiff for light microscopic observation to determine glomerular volume and mesangial matrix index as described previously (15,16) (online appendix [available at http://diabetes.diabetesjournals.org]).
Immunohistochemistry.
Immunohistochemistry of CD31, type IV collagen, F4/80, and VEGF was performed using frozen sections as previously described (15,16,27,28) (online appendix).
RNA extraction and quantitative real-time PCR.
RNA extraction and real-time PCR were performed to determine the mRNA levels of TGF-1, IL-6, MCP-1, and nephrin in renal cortex as previously described (15,16) (online appendix).
Immunoblot.
Immunoblot assay was performed to determine the protein levels of VEGF-A, Ang-1, Ang-2, nephrin, TGF-, MCP-1, and tumor necrosis factor- (TNF-) in renal cortex as previously described (15,16,29eC31) (online appendix).
Cell culture.
Conditionally immortalized mouse podocytes (provided by Dr. Peter Mundel, Mount Sinai School of Medicine, New York, NY) and primary murine mesangial cells (MES13) were used to determine the direct effect of NM-3 on high glucoseeCinduced increase of the protein levels of VEGF and/or TGF- as previously described (32,33) (online appendix).
Statistical analysis.
All values are expressed as means ± SE. A Kruskal-Wallis test with post hoc comparisons using the Scheffe’s test was used for intergroup comparisons of multiple variables. Statistical analysis was performed by StatView software (Abacus Concepts, Berkeley, CA). A level of P < 0.05 was considered statistically significant.
RESULTS
Changes in blood glucose, body weight, food consumption, mean serum insulin, liver weight, heart weight, and kidney weight.
Body weight, blood glucose, food consumption, serum levels of insulin, liver weight, heart weight, and kidney weight were significantly increased in db/db mice compared with db/m mice at the age of 16 weeks. Administration of NM-3 did not alter plasma glucose concentrations, food consumption, and mean serum insulin in db/db mice after 8 weeks of treatment (Tables 1 and 2). There was no significant difference in liver weight, heart weight, and kidney weight between treatments with NM-3 and vehicle. Kidney weight and liver weight tended to be lower in the NM-3eCtreated group compared with the vehicle-treated group (Table 2). Mean systolic blood pressure measured at the age of 16 weeks by a tail cuff method did not significantly differ among the experimental groups (db/m mice, 118 ± 5 mmHg; db/db mice treated with vehicle, 114 ± 3 mmHg; and db/db mice treated with NM-3, 112 ± 5 mmHg).
Changes in CCr and urinary albumin excretion.
Serum creatinine levels did not significantly differ among the experimental groups. To evaluate the effect of NM-3 on preventing hyperfiltration associated with diabetic nephropathy, CCr and UACR were determined (Fig. 1). Although db/db mice treated with vehicle showed a marked elevation of CCr and UACR, NM-3 treatment resulted in the suppression of diabetes-induced increase of CCr (db/m mice, 0.18 ± 0.03 ml/min; db/db mice treated with vehicle, 0.45 ± 0.06 ml/min; and db/db mice treated with NM-3, 0.27 ± 0.03 ml/min) and UACR (db/m mice, 50.0 ± 1.1 e蘥 albumin/mg creatinine; db/db mice treated with vehicle, 470.3 ± 20.6 e蘥 albumin/mg creatinine; and db/db mice treated with NM-3, 170.4 ± 20.8 e蘥 albumin/mg creatinine, at 8 weeks after initiating treatment). The development of glomerular hyperfiltration and albuminuria in db/db mice was significantly suppressed by treatment with NM-3.
Histology and morphometric analysis.
Histological examination of the kidneys revealed glomerular hypertrophy and expansion of the mesangial area in vehicle-treated db/db mice. After 8 weeks of treatment with NM-3, glomerular hypertrophy and mesangial matrix expansion were significantly inhibited compared with vehicle treatment in db/db mice (Fig. 2). Systemic administration of NM-3 did not cause any pathological alterations in liver, lung, or heart of diabetic mice or of nondiabetic mice (data not shown).
Immunohistochemical analysis of glomerular type IV collagen expression.
To further evaluate the therapeutic effect of NM-3 in the diabetic nephropathy model, the expression level of type IV collagen was examined by immunofluorescence staining (Fig. 3). The amount of type IV collagen in glomeruli was increased in the vehicle-treated db/db mice (Fig. 3B) compared with nondiabetic db/m mice (Fig. 3A). Enhanced immunoreactivity in db/db mice was observed mainly in the GBM and mesangial area. Treatment with NM-3 significantly decreased the accumulation of type IV collagen in db/db mice (Fig. 3C and D).
Expression of TGF-1 mRNA and protein.
TGF-1 is known as a profibrotic growth factor involved in the expansion of mesangial matrix and renal hypertrophy in diabetic nephropathy (34). Vehicle-treated db/db mice exhibited increased expression of TGF-1 mRNA and protein compared with db/m mice in the renal cortex. Treatment with NM-3 resulted in the suppression of TGF-1 mRNA expression in db/db mice (Fig. 3E). In addition, treatment with NM-3 resulted in the suppression of the increase of renal TGF- protein levels in db/db mice as detected by immunoblot analysis (Fig. 3F). These results suggest that NM-3 suppresses the increase of glomerular type IV collagen, mesangial matrix expansion, and renal hypertrophy via, at least in part, downregulating TGF-1.
Immunohistochemical analysis of glomerular CD31+ endothelial area.
We next examined the expression of endothelial cell marker CD31 in glomeruli. In db/m mice, CD31 was detected in glomerular capillaries (Fig. 4A), and increased expression of CD31 in glomerular capillary area was observed in the vehicle-treated db/db mice (Fig. 4B). Treatment with NM-3 significantly suppressed the increase of the glomerular CD31+ area in db/db mice (Fig. 4C and E). These results demonstrate that treatment with NM-3 leads to inhibition of increase in the glomerular CD31+ endothelial area in db/db mice, possibly via its anti-angiogenic efficacy.
Immunohistochemical analysis of monocyte/macrophage accumulation.
We next examined the expression of mouse monocyte/macrophage surface marker F4/80 in glomeruli. In the vehicle-treated db/db mice (Fig. 5B), the number of F4/80+ cells was significantly increased compared with the db/m mice (Fig. 5A). Treatment with NM-3 decreased the accumulation of monocyte/macrophage in glomeruli (Fig. 5C), suggesting the anti-inflammatory action of NM-3 in this model.
Expression of IL-6 and MCP-1 mRNA.
IL-6 and MCP-1 are involved in leukocyte infiltration in association with the development of inflammatory lesions. There were no significant differences in the mRNA levels of these cytokines in renal cortex between the vehicle-treated db/db mice and db/m mice, as detected by real-time PCR. Treatment with NM-3 failed to affect renal mRNA expression of IL-6 and MCP-1 in db/db mice (Fig. 5E and F), suggesting that the inhibitory effect of NM-3 on monocyte/macrophage recruitment is possibly independent of alterations in the level of these chemokines.
Protein levels of MCP-1 and TNF-.
The effect of NM-3 on the protein levels of inflammation-associated factors MCP-1 and TNF- in the renal cortex was studied by immunoblot assay. The level of MCP-1 was significantly increased in vehicle-treated db/db mice compared with db/m mice (Fig. 5G and H). Treatment with NM-3 mildly suppressed the increase of MCP-1 in db/db mice with statistical significance. The level of TNF- was increased in db/db mice, but there was no difference between NM-3 and vehicle treatment (Fig. 5G and I).
Expression of VEGF-A, Ang-1, and Ang-2.
The effect of NM-3 on the expression of angiogenesis-associated factors VEGF-A, Ang-1, and Ang-2 in the renal cortex was studied by immunoblot assay. The levels of VEGF-A and Ang-2 were significantly increased in vehicle-treated db/db mice compared with db/m mice (Fig. 6B and D). Treatment with NM-3 significantly suppressed the increase of VEGF-A and Ang-2 in db/db mice. The level of Ang-1 was detected in db/m mice but was not significantly altered in vehicle- or NM-3eCtreated db/db mice (Fig. 6C).
Immunohistochemical analysis of VEGF-A.
We next examined the expression of VEGF-A in glomeruli by indirect immunohistochemistry. In vehicle-treated db/db mice, immunoreactivity for VEGF-A was increased compared with db/m mice, mainly localized to podocytes (Fig. 6EeCG). Treatment with NM-3 resulted in decreased immunoreactivity for VEGF-A in glomeruli compared with vehicle-treated db/db mice.
Expression of nephrin mRNA and protein.
The expression of nephrin, an essential component of the slit-diaphragm involved in the regulation of the glomerular filtration barrier, was studied using real-time PCR and immunoblot assay. Vehicle buffereCtreated db/db mice exhibited reduced renal expression levels of nephrin mRNA and protein compared with db/m mice. Treatment with NM-3 resulted in the recovery of nephrin mRNA and protein expression (Fig. 7). These results suggest the supportive role of NM-3 in maintaining nephrin and thus the glomerular filtration barrier possibly associated with the suppression of albuminuria in db/db mice.
Protein expression of TGF- in cultured mouse mesangial cells.
To further clarify the mechanism of action of NM-3 in suppressing the increase of TGF- in diabetic mice, we performed cell culture analysis using primary mouse mesangial cells. In a preliminary study, the protein level of TGF- in mesangial cells was significantly increased at 48 h after incubation in medium supplemented with 25 mmol/l glucose compared with incubation for the same time interval under normal glucose conditions (immunoblot). Therefore, mesangial cells were incubated for 48 h under normal or high glucose conditions in the following experiments. Addition of mannitol to culture condition under normal glucose did not lead to the increase of TGF-, thus excluding the potential effect by elevated osmotic pressure. Treatment with NM-3 resulted in the suppression of the increase of TGF- protein induced by high glucose in a dose-dependent manner (Fig. 8A and C). These results demonstrate the direct action of NM-3 on mesangial cells in suppressing upregulation of TGF- induced by high glucose.
Expression of VEGF-A protein in cultured mouse mesangial cells and podocytes.
To further clarify the mechanism of action of NM-3 in suppressing the increase of VEGF-A in diabetic mice, we performed cell culture analysis using primary mouse mesangial cells and mouse podocytes. In a preliminary study, the protein level of VEGF-A in mesangial cells and podocytes was significantly increased at 48 h after incubating in medium supplemented with 25 mmol/l glucose compared with incubation for the same time interval under normal glucose conditions (immunoblot). Therefore, cells were incubated for 48 h under normal or high glucose conditions in the following experiments. We did not observe apparent morphological alterations suggestive of cytotoxicity by treatment with NM-3 under different concentrations (1eC100 mg/ml) in mesangial cells and podocytes. Incubation of cells in medium containing normal glucose concentration supplemented with mannitol failed to present any differences in the levels of TGF- or VEGF compared with normal glucose alone, indicating the induction of these proteins by high glucose independent of hyperosmolarity. Treatment with NM-3 resulted in the suppression of the increase of VEGF protein induced by high glucose in a dose-dependent manner in both mesangial cells and podocytes (Fig. 8A, B, D, and E). These results demonstrate the direct action of NM-3 on mesangial cells and podocytes in suppressing upregulation of VEGF-A induced by high glucose condition.
DISCUSSION
Diabetic nephropathy is the major underlying cause of ESRD, and appropriate management of blood glucose and blood pressure is known to be important in preventing the progression. Although clinical trials revealed the beneficial effect of angiotensin type 1 receptor blockade in preventing the progression of diabetic nephropathy, novel and distinct therapeutic approaches are required, considering the vast and increasing population of diabetic nephropathy.
Angiogenesis is regulated by the balance between pro-angiogenic factors and antiangiogenic factors (3,35,36). The involvement of VEGF-A in progressing diabetic nephropathy has been demonstrated in a number of previous reports suggesting the therapeutic potential of the inhibition of VEGF-A signaling (6,7,9,10,37). We recently reported that the renal expression of VEGF and Ang-2 was increased in a streptozotocin-induced type 1 diabetic mouse model. Antiangiogenic tumstatin peptide and endostatin peptide inhibited the increase of VEGF and Ang-2, potentially associated with the observed therapeutic effects in early diabetic nephropathy (15,16), suggesting the involvement of these angiogenesis-associated factors in the progression of diabetic nephropathy.
Cytogenin is a natural product of microbial origin possessing the potent antiarthritic effects against type II collageneCinduced arthritis (38) and antiangiogenic activity in a mouse dorsal air sac assay model (20). Whereas cytogenin is biologically instable with limited therapeutic effects, NM-3, a derivative of cytogenin, is considerably orally bioavailable and biologically stable (39). NM-3 was also revealed to possess potent therapeutic effects on the type II collageneCinduced arthritis (40) and antiangiogenic activity in the mouse dorsal air sac model (21). Furthermore, in vitro proliferation of endothelial cells was inhibited by NM-3 at concentrations 10-fold less than those required to inhibit normal fibroblasts or tumor cells (26), suggesting its potent direct antiangiogenic action on endothelial cells.
In the present study, we used db/db mice, a model of obese type 2 diabetes, to demonstrate the potential therapeutic efficacy of NM-3 in diabetic nephropathy. The db/db mouse is characterized by obesity, sustained hyperglycemia, hyperinsulinemia, and lack of ketonuria due to the defect of leptin receptor in the hypothalamus accompanied by the typical diabetic renal alterations characterized by renal/glomerular hypertrophy, thickening of GBM, albuminuria, and mesangial matrix accumulation within 2 months of onset of diabetes (25,41,42).
Administration of NM-3 did not affect blood glucose, body weight, food consumption, and serum insulin concentration in db/db mice. In addition, treatment with NM-3 did not give rise to any pathological alterations in heart or liver and failed to affect wound repair (data not shown). Previous reports described inconsistent results on CCr within 2 months after the onset of diabetes in db/db mice (10,25). The discrepant outcomes may be attributed to a variable susceptibility to diabetes in subbreedings of the db/db mouse strain. In the present study, we observed increased CCr and albuminuria at this time point in vehicle-treated db/db mice. Renal alterations such as increased urinary albumin excretion, glomerular hypertrophy, glomerular hyperfiltration, and the increase of mesangial matrix were significantly inhibited by NM-3 at 8 weeks after starting treatment (16 weeks of age). Renal hypertrophy observed in db/db mice tended to be slightly decreased by NM-3. Considering the dominant contribution of the tubular compartment in organizing renal mass, we speculate that NM-3 might have mainly regulated glomerular hypertrophy observed in diabetic nephropathy, thus not leading to remarkable effects on renal hypertrophy.
The accumulation of type IV collagen in glomeruli in db/db mice was also inhibited by NM-3, potentially associated with the suppression of renal TGF-1 mRNA levels. Connective tissue growth factor mediates the effect of TGF- in inducing mesangial matrix expansion and mesangial cell hypertrophy in diabetic nephropathy (43). The role of VEGF-A in inducing the expression of connective tissue growth factor in retinal capillary cells in association with the development of retinal neovascular diseases has been reported (44). In addition, podocyte-derived VEGF-A induced by TGF-1 was reported to stimulate the production of 3(IV) collagen, a component of -chains in GBM (45). Therefore, the inhibitory effect of NM-3 on the increase of glomerular type IV collagen and mesangial matrix expansion may, at least in part, be mediated via downregulation of VEGF-A, which presumably is involved in mediating the profibrotic effect of TGF-1 in diabetic nephropathy. It has been reported that expression of TGF- is increased in mesangial cells cultured under high glucose conditions (46). To determine the potential direct effect of NM-3 on the synthesis of TGF- in mesangial cells induced by hyperglycemia, we performed in vitro experiments and observed inhibitory effects of NM-3 on high glucoseeCinduced increase of TGF- in cultured mouse mesangial cells.
Treatment with NM-3 suppressed the increase of the CD31+ glomerular endothelial area in db/db mice, potentially due to the antiangiogenic activity of NM-3 in association with the suppression of VEGF. Increased accumulation of monocytes/macrophages in glomeruli has been reported in type 1 diabetic (15) and db/db mouse models (47). IL-6 and MCP-1 induce the recruitment of monocyte/macrophage, and MCP-1 especially is involved in the development of diabetic nephropathy (48). In the present study, the mRNA expression of IL-6 and MCP-1 did not alter in db/db mice, and NM-3 did not affect either. The protein level of MCP-1 was partially suppressed by treatment with NM-3 with statistical significance as detected by immunoblot, suggesting its regulatory role on MCP-1 via posttranscriptional regulation. The protein level of TNF-, another inflammatory cytokine involved in various inflammation processes and the development of diabetic nephropathy, was not altered by treatment with NM-3. VEGF-A promotes vascular permeability and also mediates recruitment of monocyte/macrophage in a receptor-dependent fashion (49). Therefore, the observed increase of monocyte/macrophage accumulation in db/db mice was possibly mediated via stimulation of VEGF-A, and the inhibitory effect of NM-3 on the recruitment of monocytes/macrophages may result from interference with the VEGF-A signaling by NM-3, in addition to its inhibitory effect on the protein level of MCP-1.
We next examined the effect of NM-3 on the renal level of angiogenesis-associated factors. A previous report demonstrated the increased VEGF expression mainly on podocytes in db/db mice by 13 weeks and even at 27 weeks of age (8). Consistent with this previous report, the level of VEGF-A was increased in the renal cortex of db/db mice in the present study at 16 weeks of age. Treatment with NM-3 significantly suppressed the increase of VEGF-A in the renal cortex of db/db mice. The therapeutic effect of NM-3 in diabetic nephropathy may be attributed, at least in part, to the inhibition of the VEGF-A pathway in analogy with previous studies using neutralizing antieCVEGF-A antibodies (9,10). A previous report described podocyte-specific localization of VEGF in db/db mice (8), which is consistent with our immunohistochemical findings for VEGF. Inhibitory effect by NM-3 on immunoreactivity for VEGF in podocytes was also observed. In addition to production of VEGF by cultured podocytes in response to high glucose (50), the increase of VEGF protein synthesis was also observed in mesangial cells cultured under a high ambient glucose conditions (51). To examine the potential direct effect of NM-3 on the synthesis of VEGF induced by high glucose in mesangial cells and podocytes, we next performed cell culture experiments. Inhibitory effect of NM-3 on high glucoseeCinduced upregulation of VEGF was observed in both cultured mouse mesangial cells and podocytes in a dose-dependent manner. Treatment with high concentrations of NM-3 did not exhibit apparent morphological changes suggestive of cytotoxicity in both cell types. Our results demonstrate the direct suppressive effect of NM-3 on VEGF expression in mesangial cells and podocytes under high glucose condition, in addition to its known effects on endothelial cells.
In regard to Angs, the expression of Ang-1 in the renal cortex was not altered in db/db mice, in line with our previous observation in type 1 diabetic mouse model (15,16). Treatment with NM-3 failed to affect the level of renal Ang-1. In contrast, the expression of Ang-2 was markedly increased in db/db mice, which is similar to our previous results in type 1 diabetic mouse model (15,16). Treatment with NM-3 significantly suppressed the increase of Ang-2 in renal cortex of db/db mice. The marked increase of Ang-2 over Ang-1 in db/db mice suggests the proangiogenic milieu considering simultaneous upregulation of VEGF-A and the "leaky " condition of capillaries in association with increased monocyte recruitment.
Among various podocyte proteins composing slit diaphragm, we investigated alteration in the levels of nephrin, a functional molecule of the interpodocyte filtration slit diaphragm. Altered expression of nephrin has been reported in the setting of diabetic nephropathy (17,18,37,52). Reduction in both mRNA and protein expression of nephrin was associated with proteinuria in diabetic spontaneously hypertensive rats (52). In human diabetic nephropathy, reduced mRNA and protein expression of nephrin was demonstrated (17,18). A recent report describes reduced expression of nephrin in db/db mouse kidney (53). In the present study, we observed decreased mRNA and protein expression of nephrin in db/db mice, which is consistent with previous observations from our laboratory and others (15,16,53). Administration of NM-3 resulted in significant recovery of nephrin in db/db mice. Considering the pivotal role of nephrin in the maintenance of the glomerular filtration barrier, the recovery of nephrin by treatment with NM-3 may be associated with decreased albuminuria. The indirect influence of glomerular endothelial cells toward podocytes possibly mediated via secreted factors or alteration on a matrix microenvironment may explain the observed effect of NM-3 on nephrin levels. A genetic study using mice with podocyte-specific overexpression or deletion of VEGF-A suggested the importance of an appropriate level of VEGF-A in maintaining a proper glomerular filtration barrier (54). The role of Ang-1 in maintaining glomerular endothelium and regulating the action of VEGF-A on glomerular permselectivity (55) further demonstrates that the interaction between glomerular endothelial cells and podocytes is involved in maintaining proper glomerular structures as well as biological functions. The direct effect of NM-3 on podocytes leading to the restoration of nephrin needs further investigation. A recent report describes lower mRNA expression of other podocyte proteins such as CD2AP and actinin-4 in microalbuminuric patients compared with normoalbuminuric patients of type 2 diabetes (56), and alteration of the expression of these other podocyte proteins in the present experimental groups also need to be investigated in future.
In the present study, we treated db/db mice with systemic administration of antiangiogenic NM-3. Although no adverse effects were observed in NM-3eCtreated diabetic mice, which is consistent with the results from a recent phase I clinical trial of NM-3 showing no dose-limiting toxicities (24,57), it is possible that systemic administration of antiangiogenic reagents may suppress angiogenic response in a setting requiring angiogenic neovessel formation, such as peripheral neuropathy, nonhealing ulcers, and limb ischemia, pathological conditions complicated in patients with advanced diabetes. Because we attempted to observe the therapeutic effects of NM-3 in a relatively early stage of diabetic nephropathy before the incidence of apparent renal dysfunction, further assessment by the administration of NM-3 for an extended period of time may address these issues.
In conclusion, we demonstrated here that the administration of antiangiogenic NM-3 significantly ameliorated renal alterations in obese type 2 diabetic mice, in addition to its known therapeutic effects on solid tumors (22,23,26) and rheumatoid arthritis (40). Considering its high oral bioavailability and excellent safety profile demonstrated by the phase I clinical trials in patients with cancers, our results convincingly indicate the potential of NM-3 for application into clinic to prevent the progression of diabetic nephropathy alone or in combination with known therapeutic approaches.
ACKNOWLEDGMENTS
Y.M. has received a research grant from a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and grants-in-aid from the Tokyo Biochemical Research Foundation, the Inamori Foundation, and the Suzuken Memorial Foundation. Y.M. is a recipient of the 2003 Research Award from the Kobayashi Magobei Memorial Foundation for Medical Science, the 2003 Research Award from the Ryobi Teien Memorial Foundation, the 2004 Research Award from the Sanyo Broadcast Academic and Cultural Foundation, and the 2005 Oshima Award (Young Investigator Award) from the Japanese Society of Nephrology.
We appreciate Dr. Peter Mundel and Dr. Jun Wada (Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan) for providing cultured podocytes and mesangial cells. We also appreciate Dr. Tatsuo Okada (Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan) for technical assistance in cell culture experiments.
FOOTNOTES
K.I., Y.M., and Y.Yamam. contributed equally to this work.
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2 Department of Medicine and Clinical Science, Genzyme, Framingham, Massachusetts
Ang, angiopoietin; CCr, creatinine clearance; ESRD, end-stage renal disease; GBM, glomerular basement membrane; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; NM-3, 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid; TGF, transforming growth factor; TNF-, tumor necrosis factor-; UACR, urinary albumin-to-creatinine ratio; VEGF, vascular endothelial growth factor
ABSTRACT
One of the mechanisms involved in the progression of diabetic nephropathy, the most common cause of end-stage renal failure, is angiogenic phenomenon associated with the increase of angiogenic factors such as vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-2, an antagonist of Ang-1. In the present study, we examined the therapeutic efficacy of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3), a small molecule isocoumarin with antiangiogenic activity, using diabetic db/db mice, a model of obese type 2 diabetes. Increases in kidney weight, glomerular volume, creatinine clearance, urinary albumin excretion, total mesangial fraction, glomerular type IV collagen, glomerular endothelial area (CD31+), and monocyte/macrophage accumulation (F4/80+) observed in control db/db mice were significantly suppressed by daily intraperitoneal injection of NM-3 (100 mg/kg, for 8 weeks). Increases in renal expression of VEGF-A, Ang-2, fibrogenic factor transforming growth factor (TGF)-1, and chemokine monocyte chemoattractant protein-1 but not tumor necrosis factor- were also inhibited by NM-3 in db/db mice. Furthermore, decreases of nephrin mRNA and protein levels in db/db mice were recovered by NM-3. In addition, treatment of db/db mice with NM-3 did not affect body weight, blood glucose, serum insulin, or food consumption. NM-3 significantly suppressed the increase of VEGF induced by high glucose in cultured podocytes and also suppressed the increase of VEGF and TGF- induced by high glucose in cultured mesangial cells. Taken together, these results demonstrate the potential use of NM-3 as a novel therapeutic agent for renal alterations in type 2 diabetes.
Diabetic nephropathy is complicated in 30eC40% of patients with type 2 diabetes and is the most common pathological disorder predisposing end-stage renal disease (ESRD) in Japan and the Western world. Early alterations in diabetic nephropathy include glomerular hyperfiltration, glomerular and tubular epithelial hypertrophy, glomerular basement membrane (GBM) thickening, and the development of microalbuminuria, followed by the development of the accumulation of mesangial matrix and overt proteinuria, eventually leading to glomerulosclerosis and ESRD (1). The involvement of various growth factors and cytokines, including angiotensin II, IGF-I, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and transforming growth factor (TGF)-1, in the development of diabetic nephropathy have been reported (2).
Although angiogenesis, the development of new blood vessels from preexisting ones, plays a crucial role in physiological events, such as wound repair, uncontrolled neovascularization is associated with a number of pathological disorders, including tumor growth, rheumatoid arthritis, and diabetic retinopathy (3). Vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, promotes endothelial cell proliferation, migration, and endothelial cell tube formation (4). VEGF-A also induces vascular permeability (5) in association with inflammatory lesions. The protein and mRNA level of VEGF-A and its receptor flk-1/KDR were upregulated in experimental diabetic nephropathy (6eC8). Studies using neutralizing antieCVEGF-A antibody further demonstrated the involvement of VEGF-A in early glomerular hypertrophy in type 1 diabetic model and mesangial matrix accumulation in the progressive stage of type 2 diabetic model (9,10).
Angiopoietin (Ang)-1, a major physiological ligand for endothelial receptor Tie2, induces the recruitment and stable attachment of pericytes leading to the maturation of newly formed blood vessels (11). In contrast, Ang-2 competitively inhibits the binding of Ang-1 to Tie2 and renders blood vessels "unstable" (12). Ang-2 loosens the attachment of pericytes and synergizes with VEGF-A to promote angiogenesis (12,13). However, when insufficient angiogenic stimuli are present, Ang-2 causes endothelial cell apoptosis and vessel regression (12). During kidney development, Ang-1, Ang-2, and Tie2 are highly expressed and play pivotal roles in the maturation of glomeruli and renal blood vessels (14). We previously reported the upregulation of Ang-2 in the type 1 mouse diabetic nephropathy model, although the expression of Ang-1 was not altered (15,16).
Nephrin, a glomerular podocyte protein, is crucial for maintaining the integrity of the interpodocyte slit membrane structure and an intact filtration barrier. In diabetic nephropathy, the protein level of nephrin decreases possibly via the loss of nephrin into urine due to synthesis of the splice variant isoform of the nephrin lacking a transmembrane domain (17,18).
A previous study demonstrated that the increased glomerular filtration surface in diabetic nephropathy resulted from the formation of new glomerular capillaries in accordance with a slight elongation of the preexisting capillaries (19), analogous to the changes observed in pathological diabetic retinopathy.
2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3) is a novel synthetic derivative of cytogenin, a natural compound isolated from culture filtrate of Streptoverticillium eurocidicum (20,21). NM-3 potently inhibits endothelial cell proliferation, migration, sprouting, tube formation in vitro, and tumor growth in vivo (22). Moreover, NM-3 suppresses endothelial cell migration induced by VEGF and exerts inhibitory effects on angiogenesis in VEGF-secreting malignant tumors (21,22). A phase I clinical study of NM-3 in patients with cancer has demonstrated that NM-3 is a highly orally bioavailable and well-tolerated drug in humans (23). NM-3 is currently entering a phase II clinical trial in patients with multiple myeloma (24).
In the present study, to examine whether NM-3 could provide beneficial effects on type 2 diabetic nephropathy, we applied NM-3 to type 2 diabetic db/db mice. Here, we demonstrate that treatment with NM-3 markedly suppresses glomerular hypertrophy, hyperfiltration, and urinary albumin excretion as well as the expansion of mesangial matrix and the accumulation of monocytes/macrophages. These therapeutic effects are possibly mediated through the downregulation of proangiogenic signals induced by VEGF-A and Ang-2 and through the suppression of TGF-1 and the recovery of nephrin.
RESEARCH DESIGN AND METHODS
The experimental protocol was approved by the Animal Ethics Review Committee of Okayama University. Adult female db/db mice (C57BL/KsJ-db/db Jcl; Clea Japan, Osaka, Japan) and their age-matched nondiabetic db/m littermates (C57BL/KsJ-db/+m Jcl; Clea Japan) were used in the present study. Mice were fed a standard pellet laboratory chow and were provided with water ad libitum. The db/db mice were included in this study at the age of 8 weeks because they develop diabetes (hyperglycemia) at the age of week 7eC8 (25). At the age of 8 weeks, blood glucose of db/db mice was in the range of 20.3eC22.1 mmol/l. Mice were divided into three subgroups (n = 6 for each subgroup): 1) db/db mice treated with daily intraperitoneal injection of NM-3 (Genzyme, Cambridge, MA) at 100 mg · kg body wteC1 · dayeC1 for 8 weeks, 2) db/db mice treated with daily intraperitoneal injection of vehicle buffer (PBS) for the same interval, and 3) nondiabetic, control db/m mice receiving vehicle buffer.
Blood glucose was monitored every week. No mice died, and no signs of apparent exhaustion were observed during the experimental period. At 0 and 8 weeks after initiating the intraperitoneal injection of NM-3, the body weight and the individual 24-h food consumption were measured. At 8 weeks after initiating treatment, individual 24-h urine sample collection was performed using metabolic cages. Nonfasting blood samples were drawn from the retro-orbital venous plexus using heparinized capillary tubes under anesthesia at the time of death. Kidney, liver, and heart weights were measured just after death. NM-3 was dissolved in PBS and filtered to be used for the animal experiments. The dosage of NM-3 used in the present study was determined according to previous reports using NM-3 (22,26). NM-3 has a serum half-life of 3eC10 h in preclinical models, a low toxicity profile, and thus a potentially wide therapeutic window (22). According to the manufacturer’s information, oral dosage of NM-3 at 1 g/kg was well tolerated in animals without acute toxicity.
Blood and urine examination.
Blood glucose was measured in tail-vein blood, and urine was tested for ketone bodies and glucose by SRL (Okayama, Japan). Serum and urinary creatinine levels were measured by the enzymatic colorimetric method as described previously (15). Urinary albumin concentration was measured by nephelometry (Organon Teknika-Cappel, Durham, NC) using anti-mouse albumin antibody (ICN Pharmaceuticals, Aurora, OH) as previously described (15). Results were normalized to the urinary creatinine levels and expressed as urinary albumin-to-creatinine ratio (UACR). The creatinine clearance (CCr) was calculated and expressed as milliliters per minute. Serum levels of mouse insulin were determined by enzyme-linked immunosorbent assay using an ultrasensitive rat insulin enzyme-linked immunosorbent assay kit and mouse insulin standard (Morinaga, Yokohama, Japan) following the manufacturer’s instructions. According to the manufacturer’s technical information, mouse insulin can be measured in combination with mouse insulin standard because of a high homology among mammalian animals. All samples were examined in duplicate, and mean values of individual sera were used for statistical analysis. The intra- and interassay coefficients of variation for the insulin assays were <5 and 10%, respectively.
Histological analysis.
At 8 weeks after starting treatment, kidneys were removed, fixed in 10% buffered formalin, and embedded in paraffin. Sections (4-e蘭) were stained with periodic acid Schiff for light microscopic observation to determine glomerular volume and mesangial matrix index as described previously (15,16) (online appendix [available at http://diabetes.diabetesjournals.org]).
Immunohistochemistry.
Immunohistochemistry of CD31, type IV collagen, F4/80, and VEGF was performed using frozen sections as previously described (15,16,27,28) (online appendix).
RNA extraction and quantitative real-time PCR.
RNA extraction and real-time PCR were performed to determine the mRNA levels of TGF-1, IL-6, MCP-1, and nephrin in renal cortex as previously described (15,16) (online appendix).
Immunoblot.
Immunoblot assay was performed to determine the protein levels of VEGF-A, Ang-1, Ang-2, nephrin, TGF-, MCP-1, and tumor necrosis factor- (TNF-) in renal cortex as previously described (15,16,29eC31) (online appendix).
Cell culture.
Conditionally immortalized mouse podocytes (provided by Dr. Peter Mundel, Mount Sinai School of Medicine, New York, NY) and primary murine mesangial cells (MES13) were used to determine the direct effect of NM-3 on high glucoseeCinduced increase of the protein levels of VEGF and/or TGF- as previously described (32,33) (online appendix).
Statistical analysis.
All values are expressed as means ± SE. A Kruskal-Wallis test with post hoc comparisons using the Scheffe’s test was used for intergroup comparisons of multiple variables. Statistical analysis was performed by StatView software (Abacus Concepts, Berkeley, CA). A level of P < 0.05 was considered statistically significant.
RESULTS
Changes in blood glucose, body weight, food consumption, mean serum insulin, liver weight, heart weight, and kidney weight.
Body weight, blood glucose, food consumption, serum levels of insulin, liver weight, heart weight, and kidney weight were significantly increased in db/db mice compared with db/m mice at the age of 16 weeks. Administration of NM-3 did not alter plasma glucose concentrations, food consumption, and mean serum insulin in db/db mice after 8 weeks of treatment (Tables 1 and 2). There was no significant difference in liver weight, heart weight, and kidney weight between treatments with NM-3 and vehicle. Kidney weight and liver weight tended to be lower in the NM-3eCtreated group compared with the vehicle-treated group (Table 2). Mean systolic blood pressure measured at the age of 16 weeks by a tail cuff method did not significantly differ among the experimental groups (db/m mice, 118 ± 5 mmHg; db/db mice treated with vehicle, 114 ± 3 mmHg; and db/db mice treated with NM-3, 112 ± 5 mmHg).
Changes in CCr and urinary albumin excretion.
Serum creatinine levels did not significantly differ among the experimental groups. To evaluate the effect of NM-3 on preventing hyperfiltration associated with diabetic nephropathy, CCr and UACR were determined (Fig. 1). Although db/db mice treated with vehicle showed a marked elevation of CCr and UACR, NM-3 treatment resulted in the suppression of diabetes-induced increase of CCr (db/m mice, 0.18 ± 0.03 ml/min; db/db mice treated with vehicle, 0.45 ± 0.06 ml/min; and db/db mice treated with NM-3, 0.27 ± 0.03 ml/min) and UACR (db/m mice, 50.0 ± 1.1 e蘥 albumin/mg creatinine; db/db mice treated with vehicle, 470.3 ± 20.6 e蘥 albumin/mg creatinine; and db/db mice treated with NM-3, 170.4 ± 20.8 e蘥 albumin/mg creatinine, at 8 weeks after initiating treatment). The development of glomerular hyperfiltration and albuminuria in db/db mice was significantly suppressed by treatment with NM-3.
Histology and morphometric analysis.
Histological examination of the kidneys revealed glomerular hypertrophy and expansion of the mesangial area in vehicle-treated db/db mice. After 8 weeks of treatment with NM-3, glomerular hypertrophy and mesangial matrix expansion were significantly inhibited compared with vehicle treatment in db/db mice (Fig. 2). Systemic administration of NM-3 did not cause any pathological alterations in liver, lung, or heart of diabetic mice or of nondiabetic mice (data not shown).
Immunohistochemical analysis of glomerular type IV collagen expression.
To further evaluate the therapeutic effect of NM-3 in the diabetic nephropathy model, the expression level of type IV collagen was examined by immunofluorescence staining (Fig. 3). The amount of type IV collagen in glomeruli was increased in the vehicle-treated db/db mice (Fig. 3B) compared with nondiabetic db/m mice (Fig. 3A). Enhanced immunoreactivity in db/db mice was observed mainly in the GBM and mesangial area. Treatment with NM-3 significantly decreased the accumulation of type IV collagen in db/db mice (Fig. 3C and D).
Expression of TGF-1 mRNA and protein.
TGF-1 is known as a profibrotic growth factor involved in the expansion of mesangial matrix and renal hypertrophy in diabetic nephropathy (34). Vehicle-treated db/db mice exhibited increased expression of TGF-1 mRNA and protein compared with db/m mice in the renal cortex. Treatment with NM-3 resulted in the suppression of TGF-1 mRNA expression in db/db mice (Fig. 3E). In addition, treatment with NM-3 resulted in the suppression of the increase of renal TGF- protein levels in db/db mice as detected by immunoblot analysis (Fig. 3F). These results suggest that NM-3 suppresses the increase of glomerular type IV collagen, mesangial matrix expansion, and renal hypertrophy via, at least in part, downregulating TGF-1.
Immunohistochemical analysis of glomerular CD31+ endothelial area.
We next examined the expression of endothelial cell marker CD31 in glomeruli. In db/m mice, CD31 was detected in glomerular capillaries (Fig. 4A), and increased expression of CD31 in glomerular capillary area was observed in the vehicle-treated db/db mice (Fig. 4B). Treatment with NM-3 significantly suppressed the increase of the glomerular CD31+ area in db/db mice (Fig. 4C and E). These results demonstrate that treatment with NM-3 leads to inhibition of increase in the glomerular CD31+ endothelial area in db/db mice, possibly via its anti-angiogenic efficacy.
Immunohistochemical analysis of monocyte/macrophage accumulation.
We next examined the expression of mouse monocyte/macrophage surface marker F4/80 in glomeruli. In the vehicle-treated db/db mice (Fig. 5B), the number of F4/80+ cells was significantly increased compared with the db/m mice (Fig. 5A). Treatment with NM-3 decreased the accumulation of monocyte/macrophage in glomeruli (Fig. 5C), suggesting the anti-inflammatory action of NM-3 in this model.
Expression of IL-6 and MCP-1 mRNA.
IL-6 and MCP-1 are involved in leukocyte infiltration in association with the development of inflammatory lesions. There were no significant differences in the mRNA levels of these cytokines in renal cortex between the vehicle-treated db/db mice and db/m mice, as detected by real-time PCR. Treatment with NM-3 failed to affect renal mRNA expression of IL-6 and MCP-1 in db/db mice (Fig. 5E and F), suggesting that the inhibitory effect of NM-3 on monocyte/macrophage recruitment is possibly independent of alterations in the level of these chemokines.
Protein levels of MCP-1 and TNF-.
The effect of NM-3 on the protein levels of inflammation-associated factors MCP-1 and TNF- in the renal cortex was studied by immunoblot assay. The level of MCP-1 was significantly increased in vehicle-treated db/db mice compared with db/m mice (Fig. 5G and H). Treatment with NM-3 mildly suppressed the increase of MCP-1 in db/db mice with statistical significance. The level of TNF- was increased in db/db mice, but there was no difference between NM-3 and vehicle treatment (Fig. 5G and I).
Expression of VEGF-A, Ang-1, and Ang-2.
The effect of NM-3 on the expression of angiogenesis-associated factors VEGF-A, Ang-1, and Ang-2 in the renal cortex was studied by immunoblot assay. The levels of VEGF-A and Ang-2 were significantly increased in vehicle-treated db/db mice compared with db/m mice (Fig. 6B and D). Treatment with NM-3 significantly suppressed the increase of VEGF-A and Ang-2 in db/db mice. The level of Ang-1 was detected in db/m mice but was not significantly altered in vehicle- or NM-3eCtreated db/db mice (Fig. 6C).
Immunohistochemical analysis of VEGF-A.
We next examined the expression of VEGF-A in glomeruli by indirect immunohistochemistry. In vehicle-treated db/db mice, immunoreactivity for VEGF-A was increased compared with db/m mice, mainly localized to podocytes (Fig. 6EeCG). Treatment with NM-3 resulted in decreased immunoreactivity for VEGF-A in glomeruli compared with vehicle-treated db/db mice.
Expression of nephrin mRNA and protein.
The expression of nephrin, an essential component of the slit-diaphragm involved in the regulation of the glomerular filtration barrier, was studied using real-time PCR and immunoblot assay. Vehicle buffereCtreated db/db mice exhibited reduced renal expression levels of nephrin mRNA and protein compared with db/m mice. Treatment with NM-3 resulted in the recovery of nephrin mRNA and protein expression (Fig. 7). These results suggest the supportive role of NM-3 in maintaining nephrin and thus the glomerular filtration barrier possibly associated with the suppression of albuminuria in db/db mice.
Protein expression of TGF- in cultured mouse mesangial cells.
To further clarify the mechanism of action of NM-3 in suppressing the increase of TGF- in diabetic mice, we performed cell culture analysis using primary mouse mesangial cells. In a preliminary study, the protein level of TGF- in mesangial cells was significantly increased at 48 h after incubation in medium supplemented with 25 mmol/l glucose compared with incubation for the same time interval under normal glucose conditions (immunoblot). Therefore, mesangial cells were incubated for 48 h under normal or high glucose conditions in the following experiments. Addition of mannitol to culture condition under normal glucose did not lead to the increase of TGF-, thus excluding the potential effect by elevated osmotic pressure. Treatment with NM-3 resulted in the suppression of the increase of TGF- protein induced by high glucose in a dose-dependent manner (Fig. 8A and C). These results demonstrate the direct action of NM-3 on mesangial cells in suppressing upregulation of TGF- induced by high glucose.
Expression of VEGF-A protein in cultured mouse mesangial cells and podocytes.
To further clarify the mechanism of action of NM-3 in suppressing the increase of VEGF-A in diabetic mice, we performed cell culture analysis using primary mouse mesangial cells and mouse podocytes. In a preliminary study, the protein level of VEGF-A in mesangial cells and podocytes was significantly increased at 48 h after incubating in medium supplemented with 25 mmol/l glucose compared with incubation for the same time interval under normal glucose conditions (immunoblot). Therefore, cells were incubated for 48 h under normal or high glucose conditions in the following experiments. We did not observe apparent morphological alterations suggestive of cytotoxicity by treatment with NM-3 under different concentrations (1eC100 mg/ml) in mesangial cells and podocytes. Incubation of cells in medium containing normal glucose concentration supplemented with mannitol failed to present any differences in the levels of TGF- or VEGF compared with normal glucose alone, indicating the induction of these proteins by high glucose independent of hyperosmolarity. Treatment with NM-3 resulted in the suppression of the increase of VEGF protein induced by high glucose in a dose-dependent manner in both mesangial cells and podocytes (Fig. 8A, B, D, and E). These results demonstrate the direct action of NM-3 on mesangial cells and podocytes in suppressing upregulation of VEGF-A induced by high glucose condition.
DISCUSSION
Diabetic nephropathy is the major underlying cause of ESRD, and appropriate management of blood glucose and blood pressure is known to be important in preventing the progression. Although clinical trials revealed the beneficial effect of angiotensin type 1 receptor blockade in preventing the progression of diabetic nephropathy, novel and distinct therapeutic approaches are required, considering the vast and increasing population of diabetic nephropathy.
Angiogenesis is regulated by the balance between pro-angiogenic factors and antiangiogenic factors (3,35,36). The involvement of VEGF-A in progressing diabetic nephropathy has been demonstrated in a number of previous reports suggesting the therapeutic potential of the inhibition of VEGF-A signaling (6,7,9,10,37). We recently reported that the renal expression of VEGF and Ang-2 was increased in a streptozotocin-induced type 1 diabetic mouse model. Antiangiogenic tumstatin peptide and endostatin peptide inhibited the increase of VEGF and Ang-2, potentially associated with the observed therapeutic effects in early diabetic nephropathy (15,16), suggesting the involvement of these angiogenesis-associated factors in the progression of diabetic nephropathy.
Cytogenin is a natural product of microbial origin possessing the potent antiarthritic effects against type II collageneCinduced arthritis (38) and antiangiogenic activity in a mouse dorsal air sac assay model (20). Whereas cytogenin is biologically instable with limited therapeutic effects, NM-3, a derivative of cytogenin, is considerably orally bioavailable and biologically stable (39). NM-3 was also revealed to possess potent therapeutic effects on the type II collageneCinduced arthritis (40) and antiangiogenic activity in the mouse dorsal air sac model (21). Furthermore, in vitro proliferation of endothelial cells was inhibited by NM-3 at concentrations 10-fold less than those required to inhibit normal fibroblasts or tumor cells (26), suggesting its potent direct antiangiogenic action on endothelial cells.
In the present study, we used db/db mice, a model of obese type 2 diabetes, to demonstrate the potential therapeutic efficacy of NM-3 in diabetic nephropathy. The db/db mouse is characterized by obesity, sustained hyperglycemia, hyperinsulinemia, and lack of ketonuria due to the defect of leptin receptor in the hypothalamus accompanied by the typical diabetic renal alterations characterized by renal/glomerular hypertrophy, thickening of GBM, albuminuria, and mesangial matrix accumulation within 2 months of onset of diabetes (25,41,42).
Administration of NM-3 did not affect blood glucose, body weight, food consumption, and serum insulin concentration in db/db mice. In addition, treatment with NM-3 did not give rise to any pathological alterations in heart or liver and failed to affect wound repair (data not shown). Previous reports described inconsistent results on CCr within 2 months after the onset of diabetes in db/db mice (10,25). The discrepant outcomes may be attributed to a variable susceptibility to diabetes in subbreedings of the db/db mouse strain. In the present study, we observed increased CCr and albuminuria at this time point in vehicle-treated db/db mice. Renal alterations such as increased urinary albumin excretion, glomerular hypertrophy, glomerular hyperfiltration, and the increase of mesangial matrix were significantly inhibited by NM-3 at 8 weeks after starting treatment (16 weeks of age). Renal hypertrophy observed in db/db mice tended to be slightly decreased by NM-3. Considering the dominant contribution of the tubular compartment in organizing renal mass, we speculate that NM-3 might have mainly regulated glomerular hypertrophy observed in diabetic nephropathy, thus not leading to remarkable effects on renal hypertrophy.
The accumulation of type IV collagen in glomeruli in db/db mice was also inhibited by NM-3, potentially associated with the suppression of renal TGF-1 mRNA levels. Connective tissue growth factor mediates the effect of TGF- in inducing mesangial matrix expansion and mesangial cell hypertrophy in diabetic nephropathy (43). The role of VEGF-A in inducing the expression of connective tissue growth factor in retinal capillary cells in association with the development of retinal neovascular diseases has been reported (44). In addition, podocyte-derived VEGF-A induced by TGF-1 was reported to stimulate the production of 3(IV) collagen, a component of -chains in GBM (45). Therefore, the inhibitory effect of NM-3 on the increase of glomerular type IV collagen and mesangial matrix expansion may, at least in part, be mediated via downregulation of VEGF-A, which presumably is involved in mediating the profibrotic effect of TGF-1 in diabetic nephropathy. It has been reported that expression of TGF- is increased in mesangial cells cultured under high glucose conditions (46). To determine the potential direct effect of NM-3 on the synthesis of TGF- in mesangial cells induced by hyperglycemia, we performed in vitro experiments and observed inhibitory effects of NM-3 on high glucoseeCinduced increase of TGF- in cultured mouse mesangial cells.
Treatment with NM-3 suppressed the increase of the CD31+ glomerular endothelial area in db/db mice, potentially due to the antiangiogenic activity of NM-3 in association with the suppression of VEGF. Increased accumulation of monocytes/macrophages in glomeruli has been reported in type 1 diabetic (15) and db/db mouse models (47). IL-6 and MCP-1 induce the recruitment of monocyte/macrophage, and MCP-1 especially is involved in the development of diabetic nephropathy (48). In the present study, the mRNA expression of IL-6 and MCP-1 did not alter in db/db mice, and NM-3 did not affect either. The protein level of MCP-1 was partially suppressed by treatment with NM-3 with statistical significance as detected by immunoblot, suggesting its regulatory role on MCP-1 via posttranscriptional regulation. The protein level of TNF-, another inflammatory cytokine involved in various inflammation processes and the development of diabetic nephropathy, was not altered by treatment with NM-3. VEGF-A promotes vascular permeability and also mediates recruitment of monocyte/macrophage in a receptor-dependent fashion (49). Therefore, the observed increase of monocyte/macrophage accumulation in db/db mice was possibly mediated via stimulation of VEGF-A, and the inhibitory effect of NM-3 on the recruitment of monocytes/macrophages may result from interference with the VEGF-A signaling by NM-3, in addition to its inhibitory effect on the protein level of MCP-1.
We next examined the effect of NM-3 on the renal level of angiogenesis-associated factors. A previous report demonstrated the increased VEGF expression mainly on podocytes in db/db mice by 13 weeks and even at 27 weeks of age (8). Consistent with this previous report, the level of VEGF-A was increased in the renal cortex of db/db mice in the present study at 16 weeks of age. Treatment with NM-3 significantly suppressed the increase of VEGF-A in the renal cortex of db/db mice. The therapeutic effect of NM-3 in diabetic nephropathy may be attributed, at least in part, to the inhibition of the VEGF-A pathway in analogy with previous studies using neutralizing antieCVEGF-A antibodies (9,10). A previous report described podocyte-specific localization of VEGF in db/db mice (8), which is consistent with our immunohistochemical findings for VEGF. Inhibitory effect by NM-3 on immunoreactivity for VEGF in podocytes was also observed. In addition to production of VEGF by cultured podocytes in response to high glucose (50), the increase of VEGF protein synthesis was also observed in mesangial cells cultured under a high ambient glucose conditions (51). To examine the potential direct effect of NM-3 on the synthesis of VEGF induced by high glucose in mesangial cells and podocytes, we next performed cell culture experiments. Inhibitory effect of NM-3 on high glucoseeCinduced upregulation of VEGF was observed in both cultured mouse mesangial cells and podocytes in a dose-dependent manner. Treatment with high concentrations of NM-3 did not exhibit apparent morphological changes suggestive of cytotoxicity in both cell types. Our results demonstrate the direct suppressive effect of NM-3 on VEGF expression in mesangial cells and podocytes under high glucose condition, in addition to its known effects on endothelial cells.
In regard to Angs, the expression of Ang-1 in the renal cortex was not altered in db/db mice, in line with our previous observation in type 1 diabetic mouse model (15,16). Treatment with NM-3 failed to affect the level of renal Ang-1. In contrast, the expression of Ang-2 was markedly increased in db/db mice, which is similar to our previous results in type 1 diabetic mouse model (15,16). Treatment with NM-3 significantly suppressed the increase of Ang-2 in renal cortex of db/db mice. The marked increase of Ang-2 over Ang-1 in db/db mice suggests the proangiogenic milieu considering simultaneous upregulation of VEGF-A and the "leaky " condition of capillaries in association with increased monocyte recruitment.
Among various podocyte proteins composing slit diaphragm, we investigated alteration in the levels of nephrin, a functional molecule of the interpodocyte filtration slit diaphragm. Altered expression of nephrin has been reported in the setting of diabetic nephropathy (17,18,37,52). Reduction in both mRNA and protein expression of nephrin was associated with proteinuria in diabetic spontaneously hypertensive rats (52). In human diabetic nephropathy, reduced mRNA and protein expression of nephrin was demonstrated (17,18). A recent report describes reduced expression of nephrin in db/db mouse kidney (53). In the present study, we observed decreased mRNA and protein expression of nephrin in db/db mice, which is consistent with previous observations from our laboratory and others (15,16,53). Administration of NM-3 resulted in significant recovery of nephrin in db/db mice. Considering the pivotal role of nephrin in the maintenance of the glomerular filtration barrier, the recovery of nephrin by treatment with NM-3 may be associated with decreased albuminuria. The indirect influence of glomerular endothelial cells toward podocytes possibly mediated via secreted factors or alteration on a matrix microenvironment may explain the observed effect of NM-3 on nephrin levels. A genetic study using mice with podocyte-specific overexpression or deletion of VEGF-A suggested the importance of an appropriate level of VEGF-A in maintaining a proper glomerular filtration barrier (54). The role of Ang-1 in maintaining glomerular endothelium and regulating the action of VEGF-A on glomerular permselectivity (55) further demonstrates that the interaction between glomerular endothelial cells and podocytes is involved in maintaining proper glomerular structures as well as biological functions. The direct effect of NM-3 on podocytes leading to the restoration of nephrin needs further investigation. A recent report describes lower mRNA expression of other podocyte proteins such as CD2AP and actinin-4 in microalbuminuric patients compared with normoalbuminuric patients of type 2 diabetes (56), and alteration of the expression of these other podocyte proteins in the present experimental groups also need to be investigated in future.
In the present study, we treated db/db mice with systemic administration of antiangiogenic NM-3. Although no adverse effects were observed in NM-3eCtreated diabetic mice, which is consistent with the results from a recent phase I clinical trial of NM-3 showing no dose-limiting toxicities (24,57), it is possible that systemic administration of antiangiogenic reagents may suppress angiogenic response in a setting requiring angiogenic neovessel formation, such as peripheral neuropathy, nonhealing ulcers, and limb ischemia, pathological conditions complicated in patients with advanced diabetes. Because we attempted to observe the therapeutic effects of NM-3 in a relatively early stage of diabetic nephropathy before the incidence of apparent renal dysfunction, further assessment by the administration of NM-3 for an extended period of time may address these issues.
In conclusion, we demonstrated here that the administration of antiangiogenic NM-3 significantly ameliorated renal alterations in obese type 2 diabetic mice, in addition to its known therapeutic effects on solid tumors (22,23,26) and rheumatoid arthritis (40). Considering its high oral bioavailability and excellent safety profile demonstrated by the phase I clinical trials in patients with cancers, our results convincingly indicate the potential of NM-3 for application into clinic to prevent the progression of diabetic nephropathy alone or in combination with known therapeutic approaches.
ACKNOWLEDGMENTS
Y.M. has received a research grant from a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and grants-in-aid from the Tokyo Biochemical Research Foundation, the Inamori Foundation, and the Suzuken Memorial Foundation. Y.M. is a recipient of the 2003 Research Award from the Kobayashi Magobei Memorial Foundation for Medical Science, the 2003 Research Award from the Ryobi Teien Memorial Foundation, the 2004 Research Award from the Sanyo Broadcast Academic and Cultural Foundation, and the 2005 Oshima Award (Young Investigator Award) from the Japanese Society of Nephrology.
We appreciate Dr. Peter Mundel and Dr. Jun Wada (Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan) for providing cultured podocytes and mesangial cells. We also appreciate Dr. Tatsuo Okada (Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan) for technical assistance in cell culture experiments.
FOOTNOTES
K.I., Y.M., and Y.Yamam. contributed equally to this work.
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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