Cell culture

Conditionally immortalized mouse podocytes (MPs), a gift from Dr. Zhihong Liu, Nanjing University (Nanjing, China), were culture, maintained or differentiated as described previously. In brief, MPs were maintained in RPMI 1640 medium (Gibco, Life Technologies, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Life Technologies). To propagate MPs, they were cultured at 33°C in permissive conditions in culture medium supplemented with 10 U/ml mouse recombinant γ-interferon (Life Technologies) to enhance the expression of temperature-sensitive large T-antigen. To induce differentiation, MPs were maintained at 37°C without γ-interferon in non-permissive conditions for 2 weeks.

Establishment of adenovirus vectors of RNAi, wild-type (WT) and mutant (MU) mouse FAM40A gene expressions

Three pairs of miRNA oligos were designed for the target gene FAM40A and connected with the pcDNA6.2-GW/EmGFP expression vectors to construct stable RNAi plasmids, from which the most optimal was pAd-EGFP-FAM40A-miR with an interference efficiency of 69% for knocking down the target gene FAM40A. Likewise, the second-generation adenovirus expression system pAd/CMV/V5-DEST was used to establish adenovirus that over-expressed FAM40A-WT/MU. Finally, silenced FAM40A, FAM40A-WT and FAM40A-MU over-expression models were established successfully (Supplementary Figure 1).

Supplementary Figure 1

Evolutionary conservation and the causing mutant (c.1562T>C, p521M>T) of FAM40A gene

Immunofluorescence microscopy and image analysis

All procedures were performed at room temperature unless otherwise specified. MPs were grown on collagen-coated glass coverslips, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, washed with PBS for 3 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, and rinsed with PBS. The nonspecific binding sites were saturated in blocking solution (3% bovine serum albumin, and 0.02% NaN₃ in PBS) for 40 min. Cells were then incubated with primary antibodies (pre-diluted in blocking solution) and stained overnight at 4°C. Secondary antibodies were diluted in blocking solution and incubated for 50 min. The primary and secondary control antibodies were stained simultaneously. F-actin was stained with 100 nM rhodamine-labeled phalloidin (Cat. # PHDR1 Lot # 053) for 30 min, and α-tubulin was stained with 1 μM paclitaxel, Oregon Green 488 conjugate for 120 min. Cells were stained with DAPI (1 μg/ml, Sigma-Aldrich, Gillingham, UK) for nuclear staining. Coverslips were mounted on glass slides using the commercial mounting medium GelTol. Specimens were visualized and photographed using a fluorescence microscope (OLYMPUS BX51, Japan).

To quantify actin assembly, transfected cells were randomly selected and F-actin fluorescence images were acquired with a fluorescence microscope. Cell membranes and membrane protrusions were outlined on the fluorescence image with a width of 5 μm (“membrane region”). The mean fluorescence intensity (mean pixel density) in the membrane region and remaining cytosol region was quantitated using Adobe Photoshop CS5. Fluorescence intensity was collected from 10–12 different cells per group.

RT-qPCR

mRNA was isolated from differentiated immortalized MPs using an RNA assay kit (Qiagen, Hilden, Germany) and transcribed in cDNA by reverse transcriptase. No reverse transcriptase was added in the negative control.

Immunoblotting

Cells were lysed in ice-cold buffer containing 1% Triton X-100, 125 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 2 mM Na₃VO₄, 10 mM sodium pyrophosphate, 25 mM NaF, and a protease inhibitor cocktail (Roche Diagnostics). After removing insoluble components by centrifugation (10,000 rpm) at 4°C for 10 min, the protein concentration of the supernatant was quantified using a commercial reagent (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE under reducing conditions. Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with 5% dry milk and incubated with primary antibodies (1 : 100 dilution) at 4°C for 12 h. After three washes, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase, and signals were detected by Pierce ECL Western Blotting Substrate.

Expression and localization of FAM40A in MPs

Characterized by marked proteinuria and podocyte injury, FSGS remains a challenge, and the alterations in structural genes of the podocyte contribute to the monogenetic forms of FSGS in the early stage [8, 9]. Podocyte-specific mutated genes, including NPHS1, NPHS2, WT-1, LAMB2, CD2AP, TRPC6, ACTN4 and INF2, have been shown to be responsible for FSGS [1, 10, 11]. However, the treatment options for those FSGS patients are limited, and steroid resistance has been an increased risk of dysfunction of the kidney [12–14]. Recently, we identified FAM40A as a contributing gene for familial FSGS by a genome-wide association study (GWAS, data not shown). To determine the role of FAM40A in podocytes, we first examined the expression of FAM40A in cultured MPs by RT-qPCR, immunofluorescence and protein immunoblotting. In immunoblotting, both undifferentiated and differentiated MPs showed the expression of FAM40A (96 kDa) (Figure 1 A). Expression of FAM40A in MPs was also confirmed by RT-qPCR. We next analyzed the localization of FAM40A in MPs by immunofluorescence staining. Based on the bioinformatic analysis of FAM40A, it was predicted to have three transmembrane domains binding to DNA [15, 16]. In undifferentiated MPs, FAM40A proteins showed nuclear localization with some enrichment on the nuclear membrane (Figure 1 B). Similarly, FAM40A had punctate localization mainly in the nucleus in differentiated podocytes (Figure 1 B). Remarkably, FAM40A was expressed and localized in podocytes, and significantly enriched in the nucleus and perinuclear zone (Figure 1 B).

Figure 1

Expression and localization of FAM40A in podocytes. A – Podocytes express FAM40A as shown by Western blotting from undifferentiated and differentiated MPs. B – Immunofluorescence staining of FAM40A in undifferentiated and differentiated MPs. FAM40A staining was concentrated in the nucleus with little cytoplasm around the protrusion. Scale bars = 10 μm

Altered expression of FAM40A in MPs disrupts the regular cytoskeletal organization and reduces the e expression of nephrin

FAM40A has been identified previously in a novel large multiprotein assembly, together with FAM40B [7]. Moreover, FAM40A negatively regulates the MST3 and MST4 kinases and determines cancer cell migration and metastasis [17]. To explore the role of FAM40A in MPs, FAM40A-siRNA and FAM40A-overexpressed plasmids were transfected to fully differentiated mature MPs. Compared with the control group, MP morphology changed, and the normal foot-process organization disappeared when knocking down FAM40A. In addition, the intracellular expression of evenly distributed cytoskeletal protein F-actin was decreased (Figure 2 A), and the expression of F-actin was also decreased at both mRNA and protein levels (Figures 2 B, C). Moreover, the functional protein nephrin that was supported and fixed on the slit diaphragm by F-actin decreased (Figure 2 C). In contrast, MPs became larger and round, without the normal foot process organization, and the typical pseudopodial extension of foot processes disappeared (Figure 2 A). When the expression of F-actin protein was increased, its distribution was disrupted with obvious marginalization (Figure 2 A). Taken together, change in FAM40A expression altered the normal morphology and cytoskeletal architecture of MPs, mainly represented by disruption of F-actin distribution and disappearance of foot-process organization.

Figure 2

Altered expression of FAM40A in podocytes disrupts the regular cytoskeletal organization and decreases the expression of nephrin. A – Immunofluorescence staining of FAM40A depletion and over-expression on cytoskeletal organization in podocytes. Podocytes were transfected with the pAd-EGFP- FAM40A-miR or pAd-FAM40A-WT-EGFP in thin-bottomed multi-well plates, fixed for 72 h and stained for FAM40A, F-actin and nephrin. B – Detection of F-actin and nephrin mRNA by RT-PCR. C – Immunoblotting analysis of differentiated MPs

NC – negative control, MiR – interference group, FAM40A-up – FAM40A over-expression group. *Compared with NC group, p < 0.05; **compared with NC group, p < 0.01. Scale bars: 20 μm.

Mutation of FAM40A in podocytes disrupts F-actin and reduces the expression of nephrin

The mechanism by which FAM40A is involved in the progression of FSGS is largely unknown. To further explore the effect of FAM40A-MU on MPs, a plasmid harboring FAM40A-MU was transfected to fully differentiate mature MPs via the adenovirus vector. Fluorescence imaging showed that FAM40A-MU protein was expressed in MPs, and it changed the cell morphology of MPs, as represented by the wave-like irregular change of the cell periphery, and loss or fusion of the typical pseudopodia, extension of foot processes, increased marginalization of cytoskeletal protein F-actin distribution (Figure 3 A), and up-regulation of F-actin protein expression (Figure 3 C). When FAM40A mutation occurred, both immunocytochemistry and Western blot showed that the nephrin protein level was decreased, while nephrin mRNA expression remained unchanged (Figures 3 B, C). Taken together, FAM40A mutation induced morphological change of MPs. In addition, the expression of the functional protein nephrin of the slit diaphragm was decreased. Podocytopathy resulting from FAM40A-MU over-expression was similar to that from FAM40A-MT over-expression but it was not so dramatic.

Figure 3

FAM40A mutation in MPs disrupts F-actin and reduces the expression of nephrin. A – Immunofluorescence study of mutant FAM40A on cytoskeletal organization in MPs. MPs were transfected with pAd-FAM40A-MU-EGFP in thin-bottomed multi-well plates for 72 h, fixed and stained for FAM40A, F-actin and nephrin. Scale bars: 20 μm. B – RT-PCR mRNA of F-actin and nephrin. C – immunoblotting analysis of differentiated MPs

NC – negative control, MiR – interference group, FAM40A-up – FAM40A over-expression group compared with NC group, **p < 0.01. Scale bars: 20 μm.