Netrin-1 alleviates high-glucose-induced renal proximal tubule injury by activating AKT/mTOR-mediated autophagy

doi:10.21203/rs.3.rs-2217868/v1

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Netrin-1 alleviates high-glucose-induced renal proximal tubule injury by activating AKT/mTOR-mediated autophagy

https://doi.org/10.21203/rs.3.rs-2217868/v1

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The kidneys have a high level of netrin-1 expression, which protects against some acute & chronic kidney disorders. However, it is yet unknown how Netrin-1 affects renal proximal tubule cells in diabetic nephropathy (DN) under pathological circumstances. Research has shown that autophagy protects the kidneys in animal models of renal disease. In this study, we looked at the probable autophagy regulation mechanism of Netrin-1 and its function in the pathogenesis of DN. We proved that high blood sugar levels caused Netrin-1 to be down-regulated, which then triggered the AKT/mTOR signalling pathway, enhanced HK-2 cell death, and actin cytoskeleton disruption. By deleting Netrin-1 or adding an autophagy activator in vitro, these pathogenic alterations were reverted. Our results indicate that Netrin-1 stimulates autophagy by blocking the AKT/mTOR signalling pathway, which underlies high glucose-induced malfunction of the renal proximal tubules. This study reveals that targeting Netrin-1 related signalling has therapeutic potential for DN and advances our knowledge of the processes operating in renal proximal tubules in DN.

Cell & Tissue Engineering

diabetic nephropathy

Netrin-1

autophagy

AKT

mTOR

One of the leading causes of end-stage renal disease & all death in diabetic people is diabetic nephropathy (DN).
Netrin-1 is promising for ameliorating apoptosis and actin cytoskeleton derangement.
Netrin-1 promoted autophagy & blocked the AKT/mTOR pathway.
The impact of Netrin-1 on proteinuria & albuminuria in diabetic kidney disease need more investigation.

End-stage renal disease (ESRD), which is a consequence of diabetes, is largely brought on by diabetic nephropathy (DN). Nephrologists have shown a great deal of interest in the glomerular cell damage brought on by high glucose (HG) levels in DN; nevertheless, growing evidence suggests that renal tubule damage, specifically to the proximal tubule, occurs in DN [1]. In the aetiology of DN, progressive renal tubulointerstitial fibrosis plays a crucial role [2]. Intense scientific research has focused on the essential functions that renal proximal tubule damage and loss play in kidney disease [3].

A member of the netrin protein family, netrin-1 is a secreted glycoprotein with a significant chemotropic role in axonal guidance [4]. Previous research has shown that the laminin-related secreted protein known as Netrin-1 is excreted in the urine of both mice and humans and is strongly increased following acute and chronic kidney injury. Furthermore, a study found that renal fibrogenesis is aided by aberrant Netrin-1 stimulation in the kidneys [5]. The mechanism underlying Netrin-1's impact on the renal proximal tubules in diseased situations is still unknown.

Animal models of kidney disorders have renoprotective effects due to autophagy, which is essential for cellular metabolism & organelle homeostasis [6]. Apoptosis results from impaired autophagy [7]. The AKT pathway is frequently abnormally activated and is linked to numerous disorders, including DN [8, 9], where it is crucial for mediating cell differentiation, cell cycle, metastasis, & apoptosis. When the AKT signalling pathway is activated, mTOR is phosphorylated, ultimately inhibiting autophagy. According to a recent study, Netrin-1 modulates autophagic activity via controlling lysosomal processes [10]. Exogenous Netrin-1 decreases autophagosome production & ultimately lowers autophagic activity, according to a different study [11]. Uncertainty exists on how the altered AKT/mTOR signalling & proximal tubule cell activity are affected by the reduced expression of Netrin-1 in DN.

In order to shed light on potential pathogenic causes and develop new therapeutic approaches for DN, the goal of this work was to examine the biological importance of Netrin-1 in high-glucose-triggered renal tubular cell dysfunction.

2.1 Cell Culture

The Shanghai FuHeng Biotechnology Co., Ltd. sold us the human renal proximal tubule (HK-2) (FH0228; Shanghai, China). 0.05 mg/mL bovine pituitary extract and 5 ng/mL epidermal growth factor were added to keratinocyte serum free media (K-SFM; Invitrogen, Carlsbad, CA, USA), which was used to subculture the cells (EGF, Invitrogen). For 10–14 days, the cells were incubated in a CO2 environment at 37°C. Depending on the rate of cell growth, the medium was changed every day or every other day. Additionally, during 24 hours, HK-2 cells received either 5.5 mM (LG group) or 30 mM glucose. Rapamycin (100 nmol/L), an inhibitor of mTOR, was bought from Selleckchem (S1039; Houston, Texas, USA). We bought rat recombinant Netrin-1 from R&D Systems, Inc. (6419-N1-025; Minneapolis, Minnesota, USA). For 24 hours, Netrin-1 (0 or 20 ng/mL) was applied to HK-2 cells.

2.2 Human Netrin-1 is downregulated by lentiviruses

HEK293T cells were transfected with the lentiviral packaging plasmid pGC-LV, envelope plasmid pHelper 1.0, and the pHelper 2.0 vectors producing small hairpin RNA (shRNA) specific to the Netrin-1 gene (shNetrin-1) and the scramble shRNA lentiviral particle. In accordance with the manufacturer's instructions, diluted DNA is combined with Lipofectamine 2000 Reagent (Invitrogen). Prior to use in all studies, HK-2 cells were transduced by being cultured with viral supernatants supplemented with 8 g/ml polybrene for 24 hours, followed by 72 hours of puromycin selection.

2.3 Western Blotting

Radioimmunoprecipitation assay lysis buffer (P0013B; Solarbio) was used to extract cell proteins, and 10% SDS-PAGE was used to separate them. Anti-Netrin-1 (20235-1-AP; Proteintech), and anti-cleaved caspase 3 were added after the membrane was transferred to PVDF (9664T; Cell Signaling Technology), anti-Bax (14796; Cell Signaling Technology), anti-Bcl-2 (AB1722; Millipore), anti-LC3 (12741S; Cell Signaling Technology), anti-P62 (5114S; Cell Signaling Technology), anti-Beclin-1 (3738S; Cell Signaling Technology), anti-p-AKT (4060S; Cell Signaling Technology), anti-AKT (4691; Cell Signaling Technology), anti-p-mTOR (ab109268; Abcam), anti-mTOR (ab134903; Abcam), anti-mouse IgG (HRP-linked, 7076; Cell Signaling Technology), anti-rabbit IgG (HRP-linked, 7074; Cell Signaling Technology), and anti-GAPDH (5174; Cell Signaling Technology) antibodies were applied. A method for enhanced chemiluminescence (ECL) detection was used to detect the membrane after PBS washing and secondary antibody incubation (Santa Cruz Biotechnology).

2.4 Analysis of cell apoptosis

After being digested with 0.25% trypsin (without EDTA) and centrifuged for five minutes at 1,000 rpm, the various groups of HK-2 cells were resuspended in PBS. After being stained with Annexin V-FITC (5 L) and PI (10 L) for 30 minutes in binding buffer, the cells were removed. Then, using flow cytometry, the apoptosis rate was discovered (40302ES20; YEASENusing).

2.5 The use of Real-Time Quantitative PCR & RNA Extraction (RT-qPCR)

TRIzol reagent (Invitrogen) was used to extract total RNA, and the Prime Script RT Reagent Kit was used to reverse-transcribe the total RNA into complementary DNA (cDNA) (Takara Bio, Kusatsu, Shiga, Japan). RT-qPCR was employed to identify changes in gene expression using SYBR Premix Ex Taq II (Takara Bio). Target gene mRNA levels were adjusted to GAPDH levels. The following primer sequences were used: Netrin-1:5′-TGCAAGCCCTTCCACTACG-3′ (F) & 5′-TGTTGTGGCGACAGTTGAGG-3′ (R); GAPDH: 5′-GGAGCGAGATCCCTCCAAAAT-3′ (F) & 5′-GGCTGTTGTCATACTTCTCATGG-3′ (R).

2.6 Adenoviral Transfection of GFP-mRFP-LC3

GFP-mRFP-LC3-containing adenovirus vector was used to infect HK-2 cells (Asia-Vector Biotechnology, Shanghai, China). On brand-new media, HK-2 cells were cultured for a whole day. Then, HK-2 cells were observed using a confocal laser scanning microscope (FV1000; OLYMPUS) to track autophagy flux and gauge the quantity of yellow and green dots.

2.7 Score for Actin Cytoskeletal Disorder

Phalloidin-TRITC (G1401, Servicebio, Wuhan, China) was used to stain F-actin filaments in HK-2 cells so that they could be seen under a fluorescence microscope (Zeiss, Oberkochen, Germany). After being put into a 24-well plate, the cells were fixed in PBS containing 4% paraformaldehyde. The PBS solution with 0.5% Triton X-100 permeated the cell membrane. For cell structure labelling, DAPI (G1012; Servicebio) & phalloidin-TRITC (100 nM; G1401; Servicebio) were utilised. Using a grading system from a prior study, a semi-quantitative analysis of actin cytoskeleton abnormality was carried out [12]. Phalloidin staining of F-actin revealed the disordered patches, which were classified as such.

2.8 Statistical Analysis

SEMs and means are used to express data. The means among more than two groups were analysed using one-way ANOVA and the Bonferroni correction. When there were two groups, unpaired t-tests were performed to examine the data. The statistical evaluations used GraphPad Prism programme. Representative results from each experiment, which were carried out at minimum three times, are displayed. The cutoff for statistical significance was P < 0.05.

3.1 Netrin-1 Overexpression Reduced Hyperglycemia Induced Apoptosis & Aberrant Actin Cytoskeleton

HG treatment was shown to decrease Netrin-1 expression (Fig. 1A). The development of DN is significantly influenced by the dysfunction of the renal proximal tubules. We found the function of Netrin-1 in apoptosis & the actin cell skeleton of HK-2 cells under HG circumstances to ascertain its impact on renal proximal tubules. Expression of pro-apoptotic proteins (cleaved caspase-3 and Bax) was noticeably upregulated in HK-2 cells but downregulated following exposure to recombinant Netrin-1 (Fig. 1B). It was discovered that Bcl-2, a crucial anti-apoptotic protein, had the opposite impact. According to flow cytometry, the number of HK-2 cells that underwent apoptosis was much higher under the HG circumstances compared to the controls, however adding Netrin-1 dramatically decreased cell death in comparison to cells under the HG circumstances only (Fig. 1C).

Using Netrin-1 recombination protein, Netrin-1 was overexpressed in the renal proximal tubule to examine whether it affects cytoskeletal reorganisation there. Treatment with HG dramatically increased the amount of disordered HK-2 cells and decreased the quantity of F-actin fibres, and Netrin-1 overexpression had the opposite effects (Fig. 1D). These findings demonstrate the critical role of Netrin-1 in renal proximal tubule dysfunction in HG.

3.2 Netrin-1 overexpression promoted autophagy & the AKT/mTOR pathway.

So, it was determined how HG circumstances affected the autophagy of HK2 cells. In order to investigate the regulatory functions of Netrin-1 in autophagy, the expression levels of Beclin-1, P62, & LC3, three biological indicators of autophagic activity, were assessed in HG-treated HK-2 (Fig. 2A). After receiving HG therapy, comparing to the control category, Beclin-1 expression levels & the ratio of LC3B-II/LC3B-I expression levels both dropped. Additionally, after HG treatment, overexpression of Netrin-1 increased the ratio of LC3B-II/LC3B-I expression and the level of Beclin-1 expression, showing that HG treatment significantly decreased LC3 fluorescence intensity, whereas overexpression of Netrin-1 restored LC3 expression in HG-treated cells (Fig. 2B). In HG circumstances, HK-2 cells showed a stable low autophagic activity. Cells that overexpressed Netrin-1, however, greatly increased autophagy (Fig. 2C). According to these results, Netrin-1 therapy could be able to increase autophagy in HG-treated HK-2 cells.

AKT activates mTOR, a crucial protein in the control of autophagy, which thus prevents autophagy from occurring. A treatment with netrin-1 resulted in lower ratios of p-AKT/AKT & p-mTOR/mTOR (Fig. 2A). This result suggested that, in HG circumstances, Netrin-1 further suppressed the AKT/mTOR signalling pathway.

3.3 Decreased Netrin-1 Expression Reduced Autophagy

To examine the relationship between autophagy & HK-2 cells treated with rapamycin after Netrin-1 knockdown, Netrin-1 in HK-2 cells in response to HG conditions, an activator of autophagy, to promote autophagosome formation. In comparison to the control group, decreased Netrin-1 expression dramatically raised the ratio of p-AKT/AKT, p-mTOR/mTOR, & the level of P62. Rapamycin and Netrin-1 silencing dramatically reduced the ratios of p-AKT/AKT, p-mTOR/mTOR, and P62 in comparison to the shNetrin-1 group while considerably increasing the ratios of LC-3II/LC-3I & Beclin-1 (Fig. 3A).

Rapamycin treatment or Netrin-1 silencing were used to produce LC-3 fluorescence. In HK-2 cells treated with HG, Netrin-1 knockdown resulted in autophagy flux; this behaviour was reversed by rapamycin administration (Fig. 3B). In HK-2 cells exposed to HG, autophagosome formation was reduced by down-regulating Netrin-1, and this effect was reversed by rapamycin treatment (Fig. 3C), confirming that Netrin-1 down-regulation attenuated autophagy through the AKT/mTOR pathway and negatively regulates its expression.

3.4 Rapamycin Decreases High-Glucose Injury in Netrin-1 Knockdown Cells

It was hypothesized that HK-2 cells secreted Netrin-1 to activate autophagy, thereby inhibiting apoptosis and actin cytoskeleton derangement. To test this hypothesis, Netrin-1 synthesis was knocked-down in differentiated HK-2 cells using lentivirus-mediated shRNA interference or by adding rapamycin. HK-2 cells incubated with shNetrin-1 under HG conditions for 24 h showed reduced apoptosis (Fig. 4A), as shown by the higher levels of Bcl-2 & lower levels of Bax and cleaved caspase-3. The inclusion of rapamycin reversed these effects (Fig. 4B), as was Netrin-1 induced cytoskeletal derangement (Fig. 4C).

Under HG circumstances, Netrin-1 expression in renal proximal tubule cells was considerably reduced. Additionally, in HG circumstances, it triggers proximal tubule cell death and actin cytoskeleton derangement. Additionally, these effects were lessened by utilising a lentivirus to suppress Netrin-1. Finally, Netrin-1-induced protection of proximal tubule cells may be due to decreased apoptosis and promotion of autophagy.

Under HG circumstances and in diabetic patients, there has been an increase in renal tubular epithelial cell apoptosis [13]. Dysfunction of the proximal tubule due to apoptosis characterizes the early stages of DN [14]. To identify new therapeutic strategies, we first examined the protective effects of Netrin-1 against proximal tubule cell apoptosis in the diabetic kidneys. Our study provides evidence that HG treatment reduced Netrin-1 expression. Apoptosis in HK-2 cells is further triggered by Netrin-1 downregulation. The concentrations of Bcl-2, cleaved caspase-3 & Bax were measured to detect proximal tubule apoptosis accurately. This process can be blocked by up-regulation of Netrin-1 expression. Conversely, the expression of the autophagy activator rapamycin reversed the effects of HG's suppression of apoptosis in the shNetrin-1 transfection group.

Cell movement depends on the rearrangement of the cytoskeleton, which is a process that has been extensively investigated [12, 15]. Related proteins to this process include actin stress fibres, microtubules, and microfilaments. Our research revealed that overexpressing Netrin-1 in HK-2 cells inhibited actin reorganisation and that Netrin-1 potentially alters proximal tubular epithelial cell morphology by activating autophagy under HG conditions.

AKT/mTOR signalling is stimulated by HG and is linked to proximal tubular injury and autophagy suppression in DN, according to earlier research [16]. In order to maintain intracellular lysosomal homeostasis in diabetes patients, autophagy is crucial [17]. Because DN is prevented in its early stages by the mTOR inhibitor rapamycin, diabetic kidney morphological and functional abnormalities are decreased [18]. These findings suggest that AKT/mTOR signaling is essential for regulating autophagy and proximal tubular injury and that targeting this signaling pathway is a promising way to ameliorate DN progression. In renal proximal tubular epithelial cells, a prior study revealed that Netrin-1 might activate the AKT/mTOR pathway [19, 20].

In neural growth cones, Netrin-1 inhibits the phosphorylated/activated versions of AKT and mTOR [21]. Similar to this, our research showed that proximal tubular epithelial cells treated with HG increased phosphorylation of AKT and mTOR. AKT & mTOR activities were, however, markedly suppressed by Netrin-1 overexpression. Incubation with rapamycin exacerbated a reduction in LC3II brought on by decreased Netrin-1 expression, indicating that Netrin-1 triggers AKT/mTOR-mediated autophagy.

In conclusion, decreased Netrin-1 expression under HG conditions results in renal proximal tubule cell apoptosis and actin cytoskeletal derangement. By activating the AKT/mTOR signalling pathway & protecting cells from apoptosis & actin cytoskeletal disorder, up-regulation of Netrin-1 led to autophagy activity in HK-2 cells. These discoveries shed new light on the processes of autophagy & apoptosis. As a result, techniques based on Netrin-1 may be used to treat DN. Additionally, the crucial role that Netrin-1 plays in DN suggests that manipulating pathways connected to Netrin-1 is likely to be successful.

Competing interests: The authors declare no competing interests.

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Netrin-1 alleviates high-glucose-induced renal proximal tubule injury by activating AKT/mTOR-mediated autophagy

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Version 1

Abstract

Figures

Highlights

1 Introduction

2 Materials & Methods

2.1 Cell Culture

2.2 Human Netrin-1 is downregulated by lentiviruses

2.3 Western Blotting

2.4 Analysis of cell apoptosis

2.5 The use of Real-Time Quantitative PCR & RNA Extraction (RT-qPCR)

2.6 Adenoviral Transfection of GFP-mRFP-LC3

2.7 Score for Actin Cytoskeletal Disorder

2.8 Statistical Analysis

3 Results

3.1 Netrin-1 Overexpression Reduced Hyperglycemia Induced Apoptosis & Aberrant Actin Cytoskeleton

3.2 Netrin-1 overexpression promoted autophagy & the AKT/mTOR pathway.

3.3 Decreased Netrin-1 Expression Reduced Autophagy

3.4 Rapamycin Decreases High-Glucose Injury in Netrin-1 Knockdown Cells

4 Discussion

Declarations

References

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