Angiopoietin-like protein 3 regulates the motility and permeability of podocytes by altering nephrin expression in vitro

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Abstract

It is well known that podocyte injury plays a vital role in massive proteinuria. The increase of podocyte motility results in podocyte foot process (FP) effacement, a typical form of podocyte injury. Our previous studies demonstrated that glomerular podocytes can express angiopoietin-like protein 3 (ANGPTL3) and that the increase of ANGPTL3 in dysfunctional glomerulus is correlated with podocyte FP effacement. Little is known, however, about the role of ANGPTL3 in podocyte injury. In this study, we investigated ANGPTL3’s effect on the motility and permeability of podocytes and on the expression of nephrin, a key molecule in podocytes. By scrape-wound and transwell migration assay, we found that ANGPTL3 over-expression significantly increased podocyte motility, whereas after ANGPTL3 knockdown by RNA interference, motility remained the same as that of the control group. Adriamycin (ADR) treatment significantly promoted podocyte motility. However, the same dose of ADR treatment could not promote motility after the knockdown of ANGPTL3. In addition, we assayed the diffusion of FITC-BSA across the podocytes’ monolayer to investigate whether ANGPTL3 could promote protein loss by means of an increase in podocyte motility. The results showed that the changes in the FITC-BSA permeability of the podocytes corresponded to changes in motility. Furthermore, we found that ANGPTL3 over-expression dramatically increased the expression of nephrin but that the up-regulation of nephrin induced by ADR was significantly inhibited when ANGPTL3 was diminished by RNAi. In conclusion, we found ANGPTL3 to be capable of regulating the motility and permeability of podocytes and that the mechanism of ANGPTL3’s regulation could be associated with the altered expression of nephrin.

Research highlights

ANGPTL3 can promote the motility and permeability of podocytes. ► ANGPTL3 could play a vital role in the podocyte motility promoted by adriamycin. ► Nephrin is a potential signaling molecule in this mechanism.

Introduction

Podocytes cover the outer aspect of the glomerular basement membrane (GBM). Thus, they form the final barrier to protein loss, which explains why podocyte injury is typically associated with marked proteinuria [1]. The mechanism of podocyte injury, however, is not well understood. Recent studies have reported that the kidney glomerulus could express angiopoietins (Angs) such as angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). Further studies demonstrated that Ang1 and Ang2 were involved in podocyte injury [2], [3].

Recently, a family of proteins structurally similar to the angiopoietins was identified and designated “angiopoietin-like proteins” (ANGPTLs). As a new member of this family, angiopoietin-like protein 3 (ANGPTL3) has the same structure as the C-terminal fibrinogen (FBN)-like domain (FLD) and N-terminal coiled-coil domain (CLD), among others. According to previous reports, ANGPTL3 was mainly expressed in liver cells and was only weakly expressed in the kidney [4]. In different regions, ANGPTL3 exhibited varying functions, including C-terminal FLD-induced angiogenesis when bound to integrin αVβ3 and increased plasma triglyceride levels in mice when bound to the N-terminal CLD [5], [6]. Because of its powerful inhibition of lipoprotein lipase activity, ANGPTL3 is thought to play an important role in lipid metabolism [4], [5], [7], [8]. To date, however, the relationship between ANGPTL3 and proteinuria has not been well clarified.

It is known that podocytes are highly specialized epithelial cells with a complex cellular organization consisting of a cell body, major processes, and foot processes (FPs). Podocyte FPs form a characteristic interdigitating pattern with FPs of neighboring podocytes, creating filtration slits that are bridged by the glomerular slit diaphragm (SD). Proteinuria kidney diseases are typically associated with FP effacement. FP effacement is considered to be a motile event, a characteristic that explains the spread of podocyte FPs on the GBM [9].

Nephrin, which belongs to the Ig super family, plays a key role in the SD and binds adjacent podocyte FPs. In addition to its structural role, nephrin acts as a signaling receptor molecule. Thus, it could influence podocyte motility by interacting through cytoskeleton protein signal molecules [10], [11].

In our previous studies, we observed that ANGPTL3 was up-regulated in nephrotic kidney tissues using a gene chip technique [12], [13]. Through laser micro-cutting techniques, we further detected that ANGPTL3 was only expressed in the kidney glomerulus and not in the kidney tubules [12]. In addition, by using immunohistochemistry, we found that ANGPTL3 was concentrated in the glomerular podocytes of humans and rats [13]. Furthermore, the altered expression of ANGPTL3 in the glomerulus was associated with proteinuria and FP effacement in kidney diseases [14]. These results suggested that ANGPTL3 could be involved in proteinuria development and in podocyte injury.

To better understand ANGPTL3’s function in podocytes, we investigated ANGPTL3’s effect on podocyte motility as well as the role of nephrin in podocyte motility changes regulated by ANGPTL3. In this study, we found that ANGPTL3 over-expression may contribute to the motility of the podocytes. No effect was observed in those podocytes in which there had been knockdown of ANGPTL3. In addition, adriamycin (ADR) treatment failed to promote the podocyte-directed motility of those cells in which the knockdown of ANGPTL3 had occurred. Our data suggested that nephrin was involved in the signaling mechanism for ANGPTL3-mediated motility in the podocytes.

Section snippets

Antibodies and reagents

The antibodies and reagents used in this study are listed with their sources in parentheses as follows: monoclonal antibody to glyceraldehydes-phosphate dehydrogenase (GAPDH); rabbit polyclonal antibody to nephrin (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal antibody to ANGPTL3 (R&D Systems, Minneapolis, USA); adriamycin (Pfizer Inc., USA); fluorescein-isothiocyanate-labeled bovine serum albumin (FITC-BSA, Sigma–Aldrich, St. Louis, USA).

Podocyte culture and treatment

The cultured, immortal mouse podocytes

In vitro synthesis of ANGPTL3 by cultured podocytes

After being cultured in vitro at 37 °C for 10–14 days, the immortalized mice podocytes were well differentiated and matured. The differentiated podocytes were divided into six groups according to treatment: ANGPTL3 over-expressed via transient gene transfection (ANGPTL3-cDNA); ANGPTL3 knocked down via RNAi (RNAi); ADR stimulation after ANGPTL3 knock down (RNAi + ADR); transfected negative controls (MOCK); MOCK treated with ADR at 0.5 μmol/l for 24 h (MOCK + ADR) and wild type.

To test the expression of

Discussion

Our data demonstrated that ANGPTL3 could be expressed by cultured podocytes in vitro and that its expression was significantly increased by the ADR treatment. Further, the over-expression of ANGPTL3 significantly promoted the motility and permeability of podocytes to an extent similar to ADR treatment. The knockdown of ANGPTL3 powerfully inhibited the increased motility and permeability in the podocytes treated by ADR.

In this study, we confirmed the effect of ANGPTL3 on the motility and

Conclusion

ANGPTL3 could promote the motility and permeability of podocytes. ANGPTL3 played a vital role in ADR-treated podocyte injury. As a candidate signaling molecule, nephrin was involved in ANGPTL3’s mechanism.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (Grant No. 30971375). The study was also is sponsored by the Program of Shanghai Subject Chief Scientist (Grant No. 10XD1400700). We thank all the staff in the Department of Nephrology at the Children’s Hospital of Fudan University for their technical assistance, and we are also grateful to Prof. Xiliang Zha for his helpful discussions. This study also received assistance from the Fudan University Outstanding Doctoral

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