Treatment of renal interstitial fibrosis with an agonistic Tie2 monoclonal antibody via amelioration of peritubular capillary lesions in a mouse model of nephropathy

doi:10.21203/rs.3.rs-1784752/v1

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Treatment of renal interstitial fibrosis with an agonistic Tie2 monoclonal antibody via amelioration of peritubular capillary lesions in a mouse model of nephropathy

https://doi.org/10.21203/rs.3.rs-1784752/v1

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Background: Chronic kidney disease (CKD) eventually manifests as renal interstitial fibrosis, and loss of peritubular capillaries in the fibrotic area is the main marker of disease progression. Tie2, as the main functional receptor of angiopoietin, is characteristically expressed in vascular endothelial cells. This study aimed to evaluate the effect and mechanism of an agonistic Tie2 monoclonal antibody on folic acid (FA)-induced renal fibrosis (FAN) in mice.

Methods: Mice were randomly divided into wild-type mice, FAN model mice, and FAN model mice treated with the agonistic Tie2 antibody. Human umbilical vein endothelial cells with lipopolysaccharide-induced injury were treated with the agonistic Tie2 antibody in vitro.

Results: FA-induced renal tubulointerstitial lesions could be prevented with the agonistic Tie2 antibody. The extent of renal fibrosis induced by FA was suppressed by treatment with the agonistic Tie2 antibody. The level of fibroblast-specific protein 1 was reduced in mice that were administered the agonistic Tie2 antibody. Further, the agonistic Tie2 antibody inhibited FA‐induced vascular inflammation by downregulating vascular cell adhesion molecule-1. In addition, peritubular capillary density was upregulated by agonistic Tie2 antibody treatment, as evidenced by an increase in the level of cluster of differentiation 31. These findings were verified by the in vivo results, which showed that the levels of VCAM-1, Bax, and α-smooth muscle actin were downregulated after treatment with the agonistic Tie2 antibody.

Conclusions: Based on all these findings, it appears that the agonistic Tie2 antibody attenuated renal interstitial fibrosis in FAN model mice by promoting peritubular capillary regeneration, inhibiting inflammation, and improving peritubular capillary lesions, and therefore, it may have promise for the treatment of CKD.

Agonistic Tie2 monoclonal antibody

Peritubular capillary

Renal interstitial fibrosis

Renal tubulointerstitial fibrosis is an important factor in the progression of chronic kidney disease (CKD)[1, 2]. More and more animal and clinical studies have suggested that tubulointerstitial fibrosis is associated with peritubular capillary loss[3–6]. In one such study, Yuan et al.[7] demonstrated that peritubular capillary loss may result in tubulointerstitial fibrosis of folic acid (FA)-induced nephrotoxicity (FAN) in mice. Consequently, protecting damaged renal microvasculature is a crucial approach to ameliorating renal interstitial fibrosis. In the process, angiogenesis plays an important role[8, 9].

Tie2 is a transmembrane receptor tyrosine kinase found almost exclusively on endothelial cells. Angiopoietin-1 (Ang-1) is a endogenous ligand for Tie2 which can activate Tie2. Binding of Tie2 by Ang1 triggers pathways that promote angiogenesis and regulate vascular stability[10–14]. Previous reports have shown that Ang-1/Tie2 signaling improves endothelial survival, downregulates inflammatory effect[15, 16], and rescues apoptosis[17, 18]. Recent studies have found that dysregulation of Ang-1/Tie2 signaling is a significant feature in patients of CKD[19]. Interestingly, previous researches have shown contradictory results about the effect of Ang-1 in renal fibrosis. A soluble, stable, and potent Ang1 variant (COMP-Ang1) protects peritubular capillaries, downregulates inflammation and delays fibrotic changes in a unilateral ureteral obstruction (UUO) mice model[20]. Contrary to the findings observed in UUO models, administration of a soluble form of Ang-1 enhances fibrosis and inflammation in FAN models[21]. In previous studies, Yuan et al. have demonstrated that the agonistic Tie2 monoclonal antibody stimulates Tie2 activation[22], which maintains the integrity of recently formed interstitial vessels[23]. Further, Tie-2-expressing capillaries were found to undergo proliferation in the fibrotic interstitium between atrophic tubules in a mouse model of FAN[24]. In the present study, we have tried to expand this line of research by exploring the potential renoprotective effects of an agonistic Tie2 monoclonal antibody.

In this study, a mouse model of FAN and lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs) were used to verify that exogenous treatment with an agonistic Tie2 monoclonal antibody can improve tubulointerstitial lesions and decrease tubulointerstitial fibrosis by reducing peritubular capillary inflammation and upregulating peritubular capillary density. Our in vivo and in vitro findings indicate that agonistic Tie2 monoclonal antibodies may have therapeutic applications in the treatment of CKD.

2.1. FA-induced mouse model of renal fibrosis

Male CD1 mice (4 weeks old, 16 ± 2 g) (Suzhou Sinosure Biotechnology Co. Ltd., China) were intraperitoneally administered FA (F7876, 240 mg/kg; Sigma, USA) in vehicle (0.2 ml of 0.3 mol/L NaHCO3) or only vehicle. The FA dose used has been previously shown to reliably induce severe nephrotoxicity[24]. Nine days after FA injection, mice in the Tie2 treatment group were treated by intraperitoneal injection of 1 mg each of agonistic Tie2 monoclonal antibody (mT1002r-m, Angio-Proteomie). On day 28, the left kidneys of all the mice were resected and preserved.

2.2. Culture and treatment of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVECs) were bought from Procell Life Science and Technology Co. Ltd. (Wuhan, China) and were kept at 37°C in a 5% CO₂-containing humidified incubator. Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Chemical Company, St Louis, MO) media supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) was used as the culture medium. HUVECs were incubated in a 5% CO₂ incubator at 37°C, and the medium was replaced every 2–3 days. HUVECs were then exposed to lipopolysaccharide (LPS, 10 µg/ml; L4005, Sigma, USA) for construction of the in vitro model. Following this, HUVECs with LPS-induced injury were treated with the agonistic Tie2 monoclonal antibody (5 µg/ml; AF313, R&D).

2.3. Hematoxylin–eosin and Masson staining

Masson staining was performed using the standard procedure as described previously[25]. At 7 days after FA injection, the right kidneys of the mice were freshly dissected, fixed, processed, and embedded in paraffin. The kidney sections were cut to a thickness of 3 mm, and the sections were stained with Masson’s trichrome. At 28 days after FA injection, the left kidneys were freshly dissected, fixed, processed, and embedded in paraffin. Then, 3-mm sections were cut and stained with hematoxylin-eosin (HE) and Masson’s trichrome. To observe the pathological changes in renal tissues, the sections were subjected to HE staining and scored on a scale from 0 to 4 (0: no changes, 1: changes affecting < 25% of the section, 2: changes affecting 25–50% of the section, 3: changes affecting 50–75% of the section; and 4: changes affecting 75–100% of the section) [26]. The tissue sections were visualized and photographed under a light microscope (Olympus Corporation, Tokyo, Japan). Tubulointerstitial fibrotic areas were evaluated by Masson’s trichrome. Using the Olympus Cell Soft Imaging System, the area of tubulointerstitial fibrosis and the total area of each visual field were measured and their ratio was calculated. Then, the mean of all the values was calculated.

2.4. Immunohistochemistry Staining

At 28 days after FA injection, the left kidneys of the mice were preserved in 4% paraformaldehyde. The tissues were then embedded in paraffin, and transverse paraffin sections (thickness, 5 mm) were mounted on silane-coated slides. The sections were deparaffinized, rehydrated, and then incubated with the FSP-1 antibody (1:100, ab41532; Abcam) or CD31 antibody (1:100, ab124432; Abcam). Immunolabelled sections were visualized with 0.05% DAB plus 0.3% H2O2 in phosphate-buffered saline.

2.5. Western Blot Analysis

Protein was extracted from renal tissues and HUVECs using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing 1% phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). The Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to determine protein concentration. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were probed with primary antibodies against VCAM-1 (1:2000, ab134047; Abcam) or α-SMA (1:2500, ab32575; Abcam). Western blot analysis for specific protein expression was performed according to established procedures as described previously [26].

2.6. RNA Isolation and Quantitative PCR

RNA was isolated from HUVECs by using the standard protocol of the TRIzol manufacturer. cDNA was reverse-transcribed by using Cell Counting Kit-8 (Japan Tonin Co. Ltd., Japan). The following primers against VCAM-1 and Bax were used: VCAM-1, 5′-CCTGCTGGGATATTAGCTCCA-3′ (forward) and 5′-CAGCGGTAGGTGTCGAAGC-3′ (reverse); Bax, 5′-GTGCACCAAGGTGCCGGAAC-3′ (forward) and 5′-TCAGCCCATCTTCTTCCAGA-3′ (reverse). The thermocycling conditions were as follows: denaturation for 5 min at 95℃ followed by 40 cycles of 10 s at 94℃, 20 s at 60℃, and 30 s at 72℃. The relative mRNA levels were calculated using the 2-ΔΔCq method[27].

2.7. Statistical Analysis

GraphPad Prism Version 6 (GraphPad Prism Software Inc., San Diego, California, USA) was used for data analysis and figure preparation. Results were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used to compare the means of the three groups. P < 0.05 was considered to indicate significance.

3.1. Attenuation of renal atrophy and lesions in FAN model mice treated with an agonistic Tie2 monoclonal antibody

On day 2, urine samples from FAN model mice were collected for determining the urinary albumin-to-creatinine ratio (uACR), which is an established marker for renal dysfunction [29]. uACR was higher than normal in the model mice. On day 7, the right kidneys were resected for routine histological analysis by Masson staining. Tubular damage was detected 1 d after FA administration. Over the next few weeks, most tubules regenerated but exhibited patchy atrophy, and interstitial fibrosis was also observed [24]. Consistent with these findings, the present results revealed FA-induced renal injury, as evidenced by high uACR and renal pathological changes (Fig. S1a & b).

Next, we divided the mice into three groups: WT mice, FAN model mice, and FAN model mice treated with an agonistic Tie2 monoclonal antibody. Figure 1 shows the changes induced in FAN mice treated with the agonistic Tie2 monoclonal antibody. The mice were intraperitoneally administered 1 mg of the agonistic Tie2 monoclonal antibody on day 9 (Fig. 1a). Visual examination by autopsy of the FAN mice revealed renal shrinkage with uneven surfaces, and a lower kidney/body weight ratio than the WT group (P < 0.01). In the FAN mice treated with the agonistic Tie2 monoclonal antibody, the degree of kidney shrinkage was reduced (Fig. 1b) and the kidney/body weight ratio was substantially improved compared with the FAN mice that did not receive this treatment (Fig. 1c, Table 1). These data suggest that treatment with the agonistic Tie2 monoclonal antibody ameliorated renal atrophy in FAN mice. We further evaluated histopathological changes in kidney tissue by HE staining. A considerable number of tubular casts and severe cell infiltration, tubular atrophy, and interstitial fibrosis were detected in the FAN mice; these effects were suppressed by agonistic Tie2 monoclonal antibody administration (Fig. 1d). In addition, the tubulointerstitial injury score was decreased after agonistic Tie2 monoclonal antibody treatment in the FAN mice (P < 0.001) (Fig. 1e). These findings suggest that treatment with the agonistic Tie2 monoclonal antibody mitigated FA-induced renal lesions in mice.

Table 1

Kidney weight and body weights
Group		Kidney weight (g)	Body weight (g)	Kidney/body weight ratio (×100)
WT		0.33 ± 0.02	35.61 ± 2.00	0.93 ± 0.09
FAN		0.23 ± 0.02	34.08 ± 1.86	0.68 ± 0.06
FAN + Tie2		0.26 ± 0.05	33.35 ± 5.19	0.79 ± 0.12
F	21.160		0.299	7.538
P	0.003		0.608	0.019

3.2. Suppression of renal tubulointerstitial fibrosis in FAN mice treated with the agonistic Tie2 monoclonal antibody

Masson’s trichome staining was performed to observe collagen deposition and renal interstitial fibrosis. Significantly increased interstitial collagen deposition was observed on day 28 in the FAN mice, but this effect was suppressed by agonistic Tie2 monoclonal antibody administration in the antibody-treated FAN mice (Fig. 2a). Treatment with the agonistic Tie2 monoclonal antibody resulted in a marked reduction in the FA-induced tubulointerstitial fibrosis area (P < 0.01) (Fig. 2b). In addition, the expression of fibroblast-specific protein-1 (FSP-1) in renal tissues was evaluated by immunohistochemical staining (Fig. 2c). FSP-1 has been shown to be correlated with serum creatinine levels, creatinine clearance, and fibrosis area as confirmed by renal biopsy, and is a predictor of end-stage renal disease[30]. A significant increase in FSP-1 staining in renal tissues was observed in the FAN mice on day 28, whereas agonistic Tie2 monoclonal antibody treatment effectively inhibited FA-induced expression of FSP-1 (P < 0.05) (Fig. 2d).

3.3. Attenuation of inflammation and improved density of peritubular capillaries in FAN mice treated with the agonistic Tie2 monoclonal antibody

The expression of vascular cell adhesion molecule-1 (VCAM-1), an important mediator of vascular inflammation, was assessed by western blotting (Fig. 3a). Elevated expression of VCAM-1 was observed in the FAN mice, but this effect was diminished on treatment with the agonistic Tie2 monoclonal antibody (P < 0.01) (Fig. 3b). The full-length blots were presented in Supplementary Fig. 2 (Fig. S2).This indicates that the agonistic Tie2 monoclonal antibody had an anti-inflammatory effect. Furthermore, immunohistochemical staining of CD31, a typical marker of endothelial cells (Fig. 3c), showed that peritubular capillaries could be easily observed by CD31 immunostaining. In the FAN mice, a decrease in the number of CD31-positive capillaries was observed in renal tissues, but this effect was mitigated on administration of the agonistic Tie2 monoclonal antibody (P < 0.001) (Fig. 3d). These results suggest that treatment with the agonistic Tie2 monoclonal antibody prevented decrease in the density of peritubular capillaries in FAN mice.

3.4. In vitro protective effect of the agonistic Tie2 monoclonal antibody on vascular endothelial cells

Based on the ameliorative effect of the agonistic Tie2 monoclonal antibody on peritubular capillary lesions and renal interstitial fibrosis in the mouse model of FAN, we used HUVECs with LPS-induced injury as an in vitro model to visualize and quantify the effect of the agonistic Tie2 monoclonal antibody on vascular endothelial cells. The mRNA expression levels of the inflammatory factor VCAM-1 (Fig. 4a) and the apoptosis gene Bax (Fig. 4b) in the LPS group were significantly higher than those in the WT group (P < 0.05), while those in the agonistic Tie2 monoclonal antibody treatment group were significantly lower than those in the LPS group (P < 0.05). This suggests that the agonistic Tie2 monoclonal antibody had anti-inflammatory and anti-apoptotic effects on injured HUVEC cells. Thus, the agonistic Tie2 monoclonal antibody promoted endothelial cell regeneration.

Next, the in vitro effect of the agonistic Tie2 monoclonal antibody on the expression of the pro-fibrotic factor α-smooth muscle actin (α-SMA) was examined in HUVECs. As shown in Fig. 4c and d, the protein expression level of α-SMA was significantly increased on stimulation with LPS in HUVECs. However, this increase in α-SMA levels was significantly suppressed by treatment with the agonistic Tie2 monoclonal antibody (P < 0.05). The full-length blot was presented in Supplementary Fig. 3 (Fig. S3).These results demonstrate that agonistic Tie2 monoclonal antibody treatment resulted in a weakened fibrotic response in endothelial cells.

Our data demonstrate for the first time that administration of an agonistic Tie2 monoclonal antibody ameliorates renal tubulointerstitial fibrosis, dampens renal inflammation, and promotes the growth of peritubular capillaries after FA-induced renal injury in a mouse model. The potential protective effect of this antibody on endothelial cells was confirmed through in vitro experiments on HUVECs with LPS-induced injury, and the findings were in line with previous studies that have reported the protective effect of Ang-1/Tie2 activation on endothelial cells[15–18].

Recently, Carota et al.[31] suggested that activation of Tie2 by inhibition of vascular endothelial protein tyrosine phosphatase reduces the expression of pro-inflammatory and pro-fibrotic gene targets in diabetic nephropathy. Rübig et al.[32] also provided evidence that the in vivo activation of Tie2 by PEGylated VT, a drug-like Tie2 receptor agonist, can counteract microvascular endothelial barrier dysfunction, improve renal recovery, and reduce mortality in ischemic acute kidney injury. Here, we used a reliable model of CKD to study the impact of agonistic Tie2 monoclonal antibody administration on kidney fibrosis. In accordance with these previous findings, we found that agonistic Tie2 monoclonal antibody treatment prevented FA-induced renal vascular inflammation, improved the density of peritubular capillaries, and significantly attenuated interstitial fibrosis.

In the present study, we observed that the accumulation of FSP-1-positive cells and the interstitial fibrotic area were significantly decreased by agonistic Tie2 monoclonal antibody treatment. The findings indicate that agonistic Tie2 monoclonal antibodies have an anti-fibrotic effect. Consistent with these findings, the in vitro protein expression level of α-SMA, a putative marker of myofibroblasts[33], was increased in HUVECs with LPS-induced injury, while it was markedly repressed by treatment with the agonistic Tie2 monoclonal antibody. In addition, the anti-inflammatory effect of the agonistic Tie2 monoclonal antibody was demonstrated by downregulation of VCAM-1 expression. In accordance with these findings, there is increasing evidence that Tie2 activation is associated with anti-inflammatory effects in endothelial cells[34–36], and that Tie2 agonists inhibit endothelial VCAM-1 expression[1]. In addition, in the present study, we examined LPS-induced apoptosis in endothelial cells as evidenced by elevated levels of the proapoptotic gene Bax[37]. Interestingly, agonistic Tie2 monoclonal antibody administration enhanced endothelial cell survival in response to apoptotic injuries[2.38]. Hence, agonistic Tie2 monoclonal antibody inhibited inflammation, rescued apoptosis, and suppressed fibrosis. The preservation of peritubular capillaries by agonistic Tie2 monoclonal antibody treatment is a potential future research direction to further explore the mechanism of Tie2 monoclonal antibodies in the treatment of CKD or improving renal fibrosis.

There is increasing evidence to show that peritubular capillaries play an important role in CKD and are a key regulator of CKD progression[39]. Peritubular capillary rarefaction is found not only in diabetic nephropathy[40] and hypertensive nephropathy[41], but also in IgA nephropathy[42], congenital nephrotic syndrome[43], lupus nephritis[44], and polycystic kidney disease[45]. Thus, protecting peritubular capillaries is a crucial approach to alleviating renal fibrosis[46–49]. In the present study, we observed that agonistic Tie2 monoclonal antibody administration in FAN mice is associated with an increase in the density of peritubular capillaries. This effect might be due to a Tie2 activation-induced elevation in endothelial cell number that directly enhances endothelial cell proliferation[50] or prevents endothelial cells apoptosis [51]. We were unable to explore the mechanisms in detail in the present study, so more research is needed to explore the signaling pathway involved in renal tubulointerstitial fibrosis and how it is affected by the administration of an agonistic Tie2 monoclonal antibody.

In summary, agonistic Tie2 monoclonal antibody administration has a protective effect on kidneys via amelioration of peritubular capillary lesions and interstitial fibrosis. These results, along with the in vitro results in HUVECs, suggest that agonistic Tie2 treatment may be a novel, endothelium-specific therapeutic modality in CKD.

CKD

Chronic kidney disease

folic acid

FAN

folic acid-induced nephropathy

FSP-1

fibroblast-specific protein 1

VCAM-1

vascular cell adhesion molecule-1

CD31

cluster of differentiation 31

αSMA

α-smooth muscle actin

Ang-1

Angiopoietin-1

LPS

lipopolysaccharide

HUVEC

human umbilical vein endothelial cell

hematoxylin-eosin

uACR

urinary albumin-to-creatinine ratio

UUO

unilateral ureteral obstruction.

Acknowledgements

Not applicable.

Authors’ contributions

L Jiang and XH Hu contributed to the design of the study, data collection and drafting of this paper. L Jiang, YJ Feng, Z Wang and HY Tang participated in performing the study. Q Lin, YY Shen, Y Zhu and QY Xu participated in data analysis. XZ Li contributed to the design, organization of the work and prepared the manuscript. The authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant number: 81370787).

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval

The current study was conducted with the approval of the Animal Ethics Committee of Soochow University (SUDA20201110A03). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.

No human participants were involved in the study. It was not a clinical trial.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

^a Department of Nephrology and Immunology, Children's Hospital of Soochow University, Suzhou 215003, China. ^b Institute of Pediatrics, Children’s Hospital of Soochow University, Suzhou 215003, China. ^c Department of Pediatrics, Jiangyin People's Hospital, Jiangyin 214400, China. ^d Department of Pediatrics, Zibo Maternal and Child Health Care Hospital, Zibo 255000, China.

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No competing interests reported.

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Treatment of renal interstitial fibrosis with an agonistic Tie2 monoclonal antibody via amelioration of peritubular capillary lesions in a mouse model of nephropathy

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Version 1

Abstract

Figures

1. Background

2. Methods

2.1. FA-induced mouse model of renal fibrosis

2.2. Culture and treatment of human umbilical vein endothelial cells

2.3. Hematoxylin–eosin and Masson staining

2.4. Immunohistochemistry Staining

2.5. Western Blot Analysis

2.6. RNA Isolation and Quantitative PCR

2.7. Statistical Analysis

3. Results

3.1. Attenuation of renal atrophy and lesions in FAN model mice treated with an agonistic Tie2 monoclonal antibody

3.2. Suppression of renal tubulointerstitial fibrosis in FAN mice treated with the agonistic Tie2 monoclonal antibody

3.3. Attenuation of inflammation and improved density of peritubular capillaries in FAN mice treated with the agonistic Tie2 monoclonal antibody

3.4. In vitro protective effect of the agonistic Tie2 monoclonal antibody on vascular endothelial cells

4. Discussion

5. Conclusions

Abbreviations

Declarations

Authors’ contributions

Funding

Competing interests

References

Competing Interests

Supplementary Files

Status:

Version 1