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