Netrin-1 works with UNC5B to regulate angiogenesis in diabetic kidney disease

Abstract

Netrin-1, an axon guidance factor, and its receptor UNC5B play important roles in axonal development and angiogenesis. This study examined netrin-1 and UNC5B expression in kidneys with diabetic kidney disease (DKD) and investigated their roles in angiogenesis. Netrin-1 and UNC5B were upregulated in streptozotocin-induced DKD Wistar rats, and their expression was compared with that in healthy controls. However, exogenous netrin-1 in UNC5B-depleted human renal glomerular endothelial cells (HRGECs) inhibited cell migration and tubulogenesis. This effect was likely associated with SRC pathway deactivation. Netrin-1 treatment also eliminated the pro-angiogenic effects of exogenous VEGF-165 on UNC5B-silenced HRGECs. These results indicate that UNC5B antagonizes netrin-1 and that UNC5B upregulation contributes partly to enhancing angiogenesis in DKD. Therefore, introducing exogenous netrin-1 and depleting endogenous UNC5B are potential strategies for reducing the incidence of early angiogenesis and mitigating kidney injury in DKD.

Keywords

netrin-1 / VEGF-165 / UNC5B / angiogenesis / diabetic kidney disease

Introduction

Diabetes mellitus (DM) is a major public health challenge worldwide; its global prevalence has affected 347 million people, and its incidence is still rising [¹]. Diabetic kidney disease (DKD), a common microvascular complication of DM, occurs in 25%–40% of patients [²]. In many Western countries, DKD is the primary cause of end-stage renal disease [³,⁴], while type 2 diabetes is an increasing epidemic in Asia [⁵].

Abnormal angiogenesis largely contributes to DKD progression. In 1987, Osterby and Nyberg first observed abnormal angiogenesis in the glomeruli of type 1 diabetes patients [⁶]. Since then, other research groups have confirmed the existence of this effect in type 2 diabetes patients [⁷,⁸]. Similarly, the abnormal formation of glomerular capillaries was observed in streptozotocin (STZ)-induced diabetic rats [⁹,¹⁰]. Abnormal angiogenesis includes an increased number of structurally and functionally premature blood vessels with thin basal membranes and swollen vascular endothelial cells; this condition enhances permeability [⁶,¹¹]. Increased capillary formation and elongation cause glomerular hypertrophy [¹⁰,¹²], and the enhanced permeability of these capillaries allows plasma albumin extravasation. This process causes kidney damage with evident pathological changes, including small artery hyalinosis, interstitial fibrosis [¹³], and atubular glomeruli [¹⁴].

The axon guidance factor, netrin-1, is a secreted protein that regulates the directional growth of neurons and their axons [¹⁵]. Netrin-1 is widely expressed in the cells of the vascular endothelium, liver, lungs, colon, and heart and is highly present in the kidneys [¹⁶,¹⁷]. Netrin-1 primarily regulates axon growth in the nervous system and vascular cell growth in other systems [¹⁸]. Netrin-1 receptors are classified into the colorectal cancer (i.e., the DCC) family and the UNC5 family. DCC family members include DCC and neogenin, while UNC5 family members include UNC5A, UNC5B, UNC5C, and UNC5D [¹⁹,²⁰]. Axon growth is stimulated when netrin-1 forms a complex with the DCC family; it is inhibited when netrin-1 forms a complex with the UNC5 or DCC-UNC5 family receptors [¹⁹,²¹]. UNC5B is the only netrin-1 receptor that is primarily expressed in the vascular system [²⁰]. The netrin-1-induced inhibition of angiogenesis is dependent on UNC5B [²⁰].

Evidence suggests that angiogenesis regulated by either netrin-1 or UNC5B closely involves vascular endothelial growth factor (VEGF) [²²]; this growth factor, particularly VEGF-A, strongly promotes angiogenesis [²³,²⁴]. During the early stage of DKD, VEGF-A promotes abnormal angiogenesis in kidney glomeruli and interstitial tissues. VEGF-A binds its tyrosine kinase receptors, i.e., VEGFR1 and VEGFR2; VEGFR2 is mostly expressed in the glomerular and peritubular capillaries [²⁵,²⁶]. During the regulation of the vascular endothelium, VEGF-A binds VEGFR2 and activates multiple downstream signaling pathways, such as SRC, RAS, PI3K-AKT, and RAF-MEK-ERK [²⁷,²⁸]. RAF-MEK-ERK signaling primarily regulates DNA synthesis, PI3K-AKT members regulate cell survival, and the SRC pathway regulates cell migration [²⁷,²⁸].

Previous studies have shown that treatment with VEGF antibodies can attenuate pathological changes resulting from abnormal angiogenesis in DKD rat models [²⁹,³⁰]. However, VEGF antibodies, such as Eremina, may adversely affect the kidneys [³¹]. Therefore, a partial blockade of VEGF-A downstream signaling or the activities of other bypass pathway molecules (e.g., SRC, AKT, and ERK) can inhibit abnormal angiogenesis in the early stage of DKD. The current study explored the action mechanisms of netrin-1 and UNC5B in cultured human renal glomerular endothelial cells (HRGECs) and investigated angiogenesis-related pathways that potentially involved netrin-1 and UNC5B in DKD. Clarifying the molecular mechanism of angiogenesis may provide new, effective, and safe methods for inhibiting glomerular angiogenesis in the early stages of DKD.

Materials and methods

Cell culture

Primary HRGECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Cells were cultured in endothelial cell culture medium (ECM) (5% fetal bovine serum, 100 U/mL Pen/Strep, 1% endothelial cell growth supplement; ScienCell) at 37 °C in 5% CO₂ atmosphere. Cells were used for experiments after passage to P4.

Human renal biopsy tissue and urine processing

Spot urine samples were collected from healthy volunteers and patients with diabetes and albuminuria after receiving their consent. The netrin-1 level in urine was quantified via enzyme-linked immunosorbent assay (ELISA), and the data were analyzed to determine whether urinary netrin-1 levels were significantly correlated with disease progression. Renal biopsy tissues obtained from patients were fixed in 10% paraformaldehyde (PFA) at 4 °C overnight. Specimens were paraffin-embedded following standard protocols and sectioned to a thickness of 5 mm. All the participants signed written consent forms. Experiments that involved human samples were approved by The Medical Ethics Committee of the People’s Liberation Army General Hospital (No. 2014-012-01).

Establishment of a DKD animal model

Male Wistar rats (160–180 g) were selected for the experiments. Specific-pathogen-free rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in the experimental animal center of the General Hospital of the People’s Liberation Army of China (GB14925-2010). The ethics audit number was 2012-X6-27. We modeled DKD in rats via intraperitoneal injection with STZ (50 mg/kg) at 4 weeks after single nephrectomy. Blood sugar was detected 3 days after the model was established. The rats with blood sugar level greater than 16.7 mmol/L were considered successful diabetic models. The rats were divided into the DKD (n = 10) and control (n = 8) groups. The urinary albumin excretion rate rose to 50 mg/24 h in the 7th month among the experimental rats. This value was 2–3 times higher than that in the nondiabetic control rats.

The serum creatinine levels were 92.43±23.61 µmol/L in the DKD group and 59.75±3.20 µmol/L in the control group.

Small interfering (siRNA) transfection

HRGECs cultured in a 10 cm dish with ECM were split into six-well plates (4 × 10⁵ cells/well) when they reached 70% confluence. Transfection was performed using jetPRIME® reagent (Polyplus Transfection, Illkirch, France) when the cells reached 40%–50% confluence. The UNC5B interference sequence (GenePharma, China) was 5′-GUCGGACACUGCCAACUAUTT-3′. The nonsense sequence (GenePharma, China) was 5′-UUCUCCGAACGUGUCACGUTT-3′.

Cell migration and tubulogenesis assays

HRGECs cultured in six-well plates were transfected with UNC5B siRNA (si-UNC5B) or control siRNA (si-NC) when they reached 70% confluence. At 24 h after transfection, the cells were scratched with a 1 mL sterile pipette tip to create a 1 mm-long wound, followed by immediate replenishment of the fresh medium. Netrin-1 (100 ng/mL) was then added to the cells, and the cells were incubated for 6 h and cultured with or without VEGF-165 (20 ng/mL) (Huaxingbio Science, China). To measure cell migration, images were captured at 0, 6, 12, and 24 h using an inverted microscope (Olympus, Japan). Tubulogenesis assay was performed using a kit and following the manufacturer’s instructions (Cultrex, Trevigen, Gaithersburg, MD, USA).

Quantitative polymerase chain reaction (qPCR)

RNA was extracted from HRGECs using TRIzol (Invitrogen). RNA concentrations were measured using an ultraviolet spectrophotometer. First-strand complementary DNA was synthesized using a TIANScript RT kit (Tiangen Biotech, Beijing, China). Reverse transcription PCR was performed using the 2× EasyTaq® PCR SuperMix kit (-dye) (Transgen Biotech, Beijing, China) in a total volume of 20 µL with the following program: 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 55 °C–60 °C for 30 s, 72 °C for 30 s, and 72 °C for 7 min. PCR products were loaded in 2% agarose gel for electrophoresis. Semiquantitative analysis was performed using the AlphaImager^TM 2200 gel imaging analysis system (Alpha, USA).

Immunocytochemistry

HRGECs were seeded onto fibronectin-coated coverslips in six-well plates (10⁵ cells/well). Cells were synchronized in serum-free EMC medium (ScienCell) and then treated with glucose (final concentration, 30 mmol/L; Sigma), mannitol (total sugar concentration, including glucose already present in medium, 30 mmol/L; Sigma), or an equal volume of deionized water. Cells reached 70%–80% confluence after 36–48 h. Then, cells were washed with phosphate-buffered saline (PBS), fixed with 4% PFA (Beyotime Institute of Biotechnology), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich Corporation), and then blocked with 5% bovine serum albumin at room temperature (RT). The cells were then incubated with UNC5B antibody (1:200; Abcam, rabbit) at 4 °C overnight, washed with PBS, and incubated with anti-rabbit Cy3-labeled secondary antibody (1:300; Jackson Immunoresearch) for 2 h at RT. The samples were washed with PBS and mounted with 4′,6-diamidino-2-phenylindole dye (DAPI) (Abcam) that contained the mounting medium. Several fields were imaged randomly using an Olympus laser scanning confocal microscope.

Immunofluorescent staining

Rat tissue for immunofluorescence was obtained from our previous model described in the section of “Establishment of a DKD animal model.” Kidneys were embedded with Tissue-Tek® O.C.T. complex (Sakura Finetek Europe BV, Zoeterwoude, the Netherlands). Sections were soaked in PBS and then fixed with 4% PFA (Beyotime Institute of Biotechnology) at RT for 5 min and then at 4 °C for 30 min. The tissue samples were incubated with rabbit polyclonal UNC5B (1:200; Abcam) and mouse monoclonal CD31 (1:100; Santa Cruz) antibodies overnight at 4 °C. The samples were washed and incubated with fluorescein isothiocyanate (FITC)-labeled anti-rabbit (1:50; Jackson Immunoresearch) and Cy3-labeled anti-mouse (1:300; Jackson Immunoresearch) secondary antibodies at RT for 2 h, washed with PBS, and mounted with DAPI. Several fields were randomly imaged using an Olympus laser scanning confocal microscope.

Immunohistochemical staining

Kidneys were fixed in 10% formaldehyde at 4 °C overnight and then processed for paraffin embedding by following standard procedures. Sections were prepared at 3 µm. For immunohistochemical analysis, tissue sections were subjected to antigen retrieval by microwaving or autoclaving for 10 min or 15 min in 10 mmol/L sodium citrate buffer (pH 6.0). Endogenous peroxidase was blocked via incubation with 3% hydrogen peroxide for 10 min. The sections were washed with PBS and incubated with 1.5% normal goat serum for 20 min, followed by incubation with a 1:1000 dilution of rabbit polyclonal anti-UNC5B antibody (Abcam) overnight at 4 °C. The sections were washed thrice with PBS and incubated with biotin-conjugated goat anti-rabbit IgG (Invitrogen) for 30 min at room temperature. The sections were washed again with PBS and incubated with streptavidin-conjugated peroxidase (Invitrogen) for 30 min at RT. Lastly, the sections were washed with PBS, incubated with diaminobenzidine (Invitrogen), and examined under a microscope.

Western blot

Total protein was extracted from HRGECs using radioimmunoprecipitation assay buffer, and protein concentrations were measured with a Pierce BCA assay kit (Lot # JK126465; Thermo Fisher Scientific, Rockford, IL, USA). Then, 45–60 µg of total protein was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred onto nitrocellulose membranes. The membranes were first blocked with BSA at RT for 1.5 h and then incubated overnight at 4 °C with the following primary antibodies: UNC5B (1:2000; Abcam); VEGFR2 (1:1000; Huaxingbio Science, China); p-VEGFR2 (1:1000; Huaxingbio Science, China); p-AKT (1:1000; Cell Signaling Technology); AKT (1:1000; Cell Signaling Technology); p-SRC (1:1000; Cell Signaling Technology); SRC (1:1000; Cell Signaling Technology); p-ERK (1:1000; Cell Signaling Technology); ERK (1:1000; Cell Signaling Technology); and anti-b-actin (1:5000; Beyotime Institute of Biotechnology). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:1000 dilution, Beyotime Institute of Biotechnology) at RT for 2 h and then developed using an enhanced chemiluminescence reagent. b-actin was used as internal control. Band intensities were quantified using Quantity One software.

Statistical analysis

All assays were performed in triplicate. Data were obtained from three independent experiments and expressed as mean±standard deviation (SD). Statistical significance was assessed through an unpaired, two-tailed test for single comparison or ANOVA for multiple comparisons. The data were analyzed using SPSS Statistics version 21. Significance was considered at P<0.05.

Results

Abnormal glomerular angiogenesis was observed in DKD rats

CD31 is a specific vascular endothelium marker and an indicator of angiogenesis status [³²]. CD31 expression was upregulated in glomeruli from the STZ-induced Wistar rat DKD model (Fig. 1A). This result indicated an actively progressing angiogenesis.

Netrin-1 was upregulated in DKD patient urine

ELISA was used to measure netrin-1 levels in 42 DN patients and 41 normal controls. The data showed that netrin-1, a marker of early kidney injury, was upregulated in the urine of DN patients compared with that in normal controls (Table 1).

Netrin-1 receptor expression profile in human glomerular endothelial cells

qPCR was performed to measure the expression of netrin-1 receptors, including UNC5A, UNC5B, UNC5C, UNC5D, DCC, and neogenin, in HRGECs. UNC5B and neogenin were highly expressed, while UNC5C and DCC were expressed at substantially lower levels. UNC5D was barely detected, and UNC5A was undetectable (Fig. 1B and 1C).

High glucose level upregulated UNC5B in DKD kidneys and HRGECs

Immunohistochemistry was performed to detect UNC5B in human renal biopsy samples. UNC5B expression in kidney glomeruli was upregulated in DKD patients compared with that in patients with minimal change nephrosis (MCN) (Fig. 2A). Consistently, UNC5B was highly upregulated in STZ-induced DKD Wistar rat kidney glomeruli compared with that in the normal control group (Fig. 2B). UNC5B (labeled with FITC) colocalized with CD31 (labeled with Cy3) in glomeruli (Fig. 2C). UNC5B in HRGECs was upregulated in the high-glucose group compared with that in the mannitol and normal control groups (Fig. 1D).

Netrin-1 inhibited UNC5B-depleted HRGEC migration and tubulogenesis

To understand the roles of netrin-1 and UNC5B in DKD patient renal angiogenesis, we silenced UNC5B expression in HRGECs using UNC5B-specific siRNA (si-UNC5B) (Fig. 3A and 3B). Tubulogenesis and cell migration assays were performed using transfected cells treated with human recombinant netrin-1 and VEGF-165, respectively. UNC5B silencing alone did not affect HRGEC migration and tubulogenesis (Fig. 3C−3E). However, cell migration was inhibited by netrin-1 and promoted by VEGF-165 in si-UNC5B HRGECs. Netrin-1 also antagonized VEGF-165-stimulated endothelial cell migration (Fig. 3C and 3D). However, netrin-1 did not exert these effects on the siRNA control group (si-NC). A similar pattern of netrin-1 effects was observed in the tubulogenesis assay (Fig. 3E). Netrin-1 inhibited tubulogenesis and antagonized VEGF-165-stimulated tubulogenesis in UNC5B-silenced cells, but not in si-NC-transfected cells (Fig. 3E).

Netrin-1 deactivated SRC in si-UNC5B HRGECs

To understand the mechanisms that underlie netrin-1-related angiogenesis inhibition following UNC5B gene silencing, we examined VEGFR2, p-VEGFR2, p-AKT, p-SRC, and p-ERK levels in si-UNC5B HRGECs treated with netrin-1 and/or VEGF-165. p-SRC was downregulated in si-UNC5B HRGECs treated with netrin-1 in the presence or absence of VEGF-165. Other proteins, including VEGFR2, and their downstream factors, such as p-ERK and p-AKT, did not exert an effect. Exogenous netrin-1 did not affect the activation of SRC or other proteins in si-NC HRGECs (Fig. 4A). p-SRC expression in UNC5B-silenced and control cells was quantified using b-actin as an internal control, and a histogram was used to show the expression of SRC (Fig. 4B).

PP2 and netrin-1 inhibited si-UNC5B cell migration

To investigate whether netrin-1 inhibited si-UNC5B HRGEC migration and tubulogenesis through SRC signaling, we treated cells with the specific SRC inhibitor PP2. Western blot results showed that p-SRC was downregulated in PP2-treated si-UNC5B cells (Fig. 5A and 5B). si-UNC5B HRGEC migration was inhibited by PP2 or netrin-1 (Fig. 5C).

Discussion

Netrin-1 is an axon guidance factor that has been widely studied for its role in angiogenesis. Our early study showed that netrin-1 was upregulated in DKD patient urine compared with that in normal controls (Table 1), consistent with the report of Jayakumar et al. [³³]. Netrin-1 was also upregulated in STZ-induced diabetic rats [³⁴]. In addition, CD31, a specific marker for vascular endothelial cells, was upregulated in the kidneys of STZ-induced DKD Wistar rats compared with that in normal controls (Fig. 1A). These findings suggest enhanced angiogenesis in DKD kidneys.

Our qPCR results showed that UNC5B and neogenin were more abundant in HRGECs than in the other receptors of netrin-1. Previous studies have shown that UNC5B is mostly expressed in kidney proximal tubule epithelial cells [³⁵]. However, UNC5B was abundantly expressed in glomeruli and largely localized to vascular endothelial cells in rat and human kidneys. In addition, UNC5B was upregulated in the glomeruli of human and rat DKD kidneys. Glucose was used to detect the mechanisms that underlie UNC5B upregulation in DKD. The results showed that high glucose level promoted UNC5B upregulation in HRGECs. This finding indicated that high glucose level primarily caused the upregulation of UNC5B expression in DKD.

Recently, netrin-1 and UNC5B have been intensively studied for their roles in acute kidney injury that can result from ischemia, reperfusion, pyohemia, and other factors. Netrin-1 is upregulated during acute and chronic kidney injuries and excreted into the urine; it is primarily expressed in proximal tubules [³⁶,³⁷]. UNC5B may bind netrin-1 and play a protective role during kidney injury by promoting renal tubular endothelial cell proliferation and migration [²,³⁸]. UNC5B and netrin-1 also play protective roles in DKD [³⁵]. In addition to acute kidney injury, UNC5B and netrin-1 also have protective effects on DKD. UNC5B knockout in DKD rat proximal tubules upregulates urinary protein levels, while netrin-1 overexpression does otherwise and reduces mesangial expansion [³⁵].

VEGF-A promotes abnormal angiogenesis in DKD [³⁹]. To examine the effects of netrin-1 and UNC5B on glomerular capillaries, we silenced UNC5B expression in HRGECs and treated transfected cells with netrin-1 and/or VEGF-165. We then examined the levels of the VEGFA receptor, VEGFR2, and its downstream effectors. Tubulogenesis and cell migration were reduced in si-UNC5B HRGECs stimulated by recombinant netrin-1. Netrin-1 treatment also antagonized VEGF-165 stimulatory effects. Therefore, netrin-1 inhibited abnormal renal angiogenesis in UNC5B-depleted cells. However, netrin-1 did not exert these inhibitory effects on the control group in which UNC5B was highly expressed. UNC5B may directly bind netrin-1, and thus eliminate its inhibitory effects on HRGEC tubulogenesis.

Our results showed that p-SRC was downregulated in si-UNC5B HRGECs regardless of VEGF-165 treatment, while the levels of p-ERK, p-AKT, and other molecules in these pathways remained unchanged. The netrin-1-induced inhibition of cell migration and tubulogenesis may be related to the deactivation of SRC to p-SRC, which is an important regulator of endothelial cell migration in angiogenesis [²⁷,²⁸]. Other research found that SRC regulates vascular permeability in UNC5B-regulated angiogenesis [⁴⁰]. To understand the mechanisms that underlie netrin-1-regulated angiogenesis, we used the Src inhibitor PP2 [⁴¹]. The results showed that PP2 can inhibit p-SRC expression (Fig. 5A and 5B). PP2 and netrin-1 treatment inhibited si-UNC5B HRGEC migration (Fig. 5C and 5D). This finding indicated that netrin-1 can inhibit cell migration by decreasing p-SRC expression.

Netrin-1 exerted its effects on HRGECs only when UNC5B was silenced, possibly due to netrin-1’s inactivation of the VEGF downstream molecule SRC or its binding to currently unknown HRGEC surface receptors. Considering that UNC5B upregulation in DKD can prevent netrin-1 activity, we propose that netrin-1 directly or indirectly inhibits SRC signaling under normal conditions. UNC5B and neogenin were abundantly expressed in HRGECs. Neogenin may regulate angiogenesis along with netrin-1, and this phenomenon will be examined in our future studies. Under disease conditions, UNC5B is upregulated and competitively binds netrin-1, which stops the netrin-1-induced inhibition of SRC signaling and promotes angiogenesis.

Netrin-1 levels were higher in the DKD patients urine than that in the normal controls (Table 1). Angiogenesis was enhanced in early-stage DKD, suggesting that netrin-1 was insufficiently expressed during this period of the disease progression. Netrin-1 is primarily expressed in renal tubules and excreted into urine [³⁶,³⁷]. Netrin-1 hardly enters the glomerulus to function within glomerular endothelial cells due to urine flow direction. UNC5B is upregulated in glomerular endothelial cells in DKD patients and STZ-induced Wistar rat DKD models. Although netrin-1 is increased in DKD patient urine, UNC5B upregulation may antagonize netrin-1 inhibition of abnormal angiogenesis.

In treating abnormal angiogenesis, inhibiting VEGF alone does not produce satisfactory results. Netrin-1 can improve proteinuria, high glomerular filtration, and histological changes in DKD patients [³⁴,³⁵]; however, the mechanism is unclear. The removal of endogenous UNC5B with the introduction of exogenous netrin-1 may inhibit angiogenesis in glomeruli and reduce kidney damage resulting from high glomerular filtration and proteinuria. This approach can be a highly effective strategy for treating DKD. In addition, UNC5B is upregulated in the kidneys of DKD patients, and this information can be used as a diagnostic marker to evaluate DKD severity in clinical practice.

Our findings showed that netrin-1 inhibited HRGEC migration and tubulogenesis when UNC5B expression was decreased. From these observations, we hypothesize that netrin-1 inhibits angiogenesis in glomeruli, and this effect may be eliminated by UNC5B expression on endothelial cell surfaces. Therefore, introducing exogenous netrin-1 and depleting endogenous UNC5B are potential strategies for reducing the incidence of early angiogenesis and attenuating kidney injury in DKD. However, this research did not include animal experiments, and the potential for treating DKD via exogenous netrin-1 with UNC5B gene silencing or knockdown requires further study.

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