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Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis Sayaka Fuseya a, b, Riku Suzuki a, c, Risa Okada a, Kozue Hagiwara d, Takashi Sato d, Hisashi Narimatsu d, Hideki Yokoi e, Masato Kasahara f, Toshiaki Usui a, g, Naoki Morito g, Kunihiro Yamagata g, Takashi Kudo a, *, Satoru Takahashi a, * a
Laboratory Animal Resource Center in Transborder Medical Research Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Japan Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan c Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Japan d Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology, Japan e Department of Nephrology, Graduate School of Medicine, Kyoto University, Japan f Department of Clinical Research, Nara Medical University Hospital, Japan g Department of Nephrology, Faculty of Medicine, University of Tsukuba, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 December 2019 Accepted 5 January 2020 Available online xxx
The glomerular filtration barrier is composed of podocytes, glomerular basement membrane, and endothelial cells. Disruption of these structures causes several glomerular injuries, such as focal segmental glomerulosclerosis (FSGS). The surface of podocyte apical membranes is coated by negatively charged sialic acids on core 1-derived mucin-type O-glycans. Here, we aimed to investigate the physiological role of core 1-derived O-glycans in the podocytes using adult mice lacking podocyte-specific core 1-derived O-glycans (iPod-Cos). iPod-Cos mice exhibited early and transient proteinuria with foot process effacements and developed typical FSGS-like disease symptoms. To identify the key molecules responsible for the FSGS-like phenotype, we focused on podocalyxin and podoplanin, which possess mucin-type O-glycans. Expression and localization of podocalyxin did not change in iPod-Cos glomeruli. Besides, western blot analysis revealed significantly lower levels of intact podocalyxin in isolated glomeruli of iPod-Cos mice, and high levels of processed forms in iPod-Cos glomeruli, as compared to that in control glomeruli. Conversely, podoplanin mRNA, and protein levels were lower in iPod-Cos mice than in control mice. These results demonstrated that core 1-derived O-glycan on podocytes is required for normal glomerular filtration and may contribute to the stable expression of podocalyxin and podoplanin. © 2020 Elsevier Inc. All rights reserved.
Keywords: Mucin-type O-glycan Proteinuria FSGS Podocalyxin Podoplanin
1. Introduction Mucin-type O-glycans post-translationally modify membrane and secretory proteins. First, the N-acetylgalactosamine (GalNAc)
Abbreviations: C1galt1, core 1 b1,3-galactosyltransferase; FP, foot process; FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; GBM, glomerular basement membrane; PODXL, podocalyxin; PDPN, podoplanin. * Corresponding authors. Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan. E-mail addresses: [email protected] (T. Kudo), [email protected]. jp (S. Takahashi).
sugar is added on serine or threonine (Ser/Thr) residues of proteins by polypeptide N-acetylgalactosaminyltransferases. Subsequently, the galactose (Gal)-b1,3-GalNAc-a-Ser/Thr (core 1) structure is synthesized by core 1 b1,3-galactosyltransferase (C1galt1), which is further elongated by multiple glycosyltransferases (Fig. 1A). The C1galt1-specific molecular chaperone (Cosmc) is essential for the activity of C1galt1 [1]. Furthermore, C1galt1 knockout (KO) mice and Cosmc KO mice showed embryonic lethality due to abnormal angiogenesis and hemorrhage [2,3], indicating that core 1-derived O-glycan is critical for embryonic development. Large-scale mutagenesis screening was performed in mice using the chemical mutagen N-ethyl-N-nitrosourea [4]. plt1 mice generated in this
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Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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study harbor a point mutation in C1galt1 and exhibit low residual C1galt1 activity, thrombocytopenia, and proteinuria. We have also reported that C1galt1 is required for megakaryocyte development and proplatelet formation [5]. The glomerular filtration barrier consists of podocytes, glomerular basement membrane (GBM), and fenestrated endothelial cells, which are constitutively negatively charged. Glomerular injury causes due to the disruption of these structures, resulting in nephrotic syndromes, such as minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). FSGS is characterized by the presence of sclerotic glomeruli and progressive proteinuria, whereas MCD is accompanied by normal glomeruli, observable under a light microscope, and transient proteinuria. These diseases show similar initial symptoms such as massive proteinuria and podocyte foot process (FP) effacement, observed under an electron microscope. Therefore, a tool which distinguishes these diseases was needed for selecting more effective treatment options. The apical surface of podocytes is coated with negatively charged sialoglycoproteins, such as podocalyxin (PODXL) and podoplanin (PDPN). The mucin region of PODXL contains more than 50 putative O-linked glycosylation sites, half of which is potentially modified with sialic acids. Podxl KO mice died due to anuria within 24 h after birth [6]. Thus, PODXL and glomerular functions may be closely related. Indeed, exome sequencing led to the identification of the transmembrane domain variant, PODXL p.L442R, in patients with FSGS [7]. PDPN also contains Ser/Thr residues in the ectodomain, which can be potentially modified with core 1-derived O-glycans.
PODXL is linked to ezrin and the actin cytoskeleton via Naþ/Hþexchanger regulatory factor 2 (NHERF2) [8], while PDPN is linked to ezrin and the actin cytoskeleton, not via NHERF2 [9], which contribute to FP formation for glomerular filtration. In the present study, we aimed to investigate whether the loss of core 1-derived O-glycans affected glomerular filtration. Toward this, we generated inducible and podocyte-specific conditional Cosmc KO mice (iPod-Cos).
2. Materials and methods 2.1. Animals Mice were maintained in specific pathogen-free conditions in a Laboratory Animal Resource Center at the University of Tsukuba. All experiments were performed in compliance with the relevant Japanese and institutional laws and guidelines, and were approved by the University of Tsukuba Animal Ethics Committee (Authorization Number: 19e135). The generation of mice harboring floxed Cosmc has been described in our previous study [10]. Cosmcflox/flox mice were crossed with NPHS2-CreERT2 transgenic mice [11] to generate podocyte-specific conditional Cosmc KO mice (NPHS2CreERT2::Cosmcflox/flox; iPod-Cos). Conditional excision of the floxed allele was achieved by intraperitoneal injection of 75 mg/kg body weight of tamoxifen (TAM) once daily for a total of 5 consecutive days. The final TAM injection was defined as day 0.
Fig. 1. The onset of proteinuria in iPod-Cos mice. (A) Schematic showing the biosynthetic pathway of core 1-derived mucin-type O-glycan. (B) Quantitative analysis of urinary proteins using SDS-PAGE. (C) Urine total proteins/creatinine ratio (TP/CRE) in control and iPod-Cos mice (n ¼ 6). (D) Immunohistochemical analysis of HPA (green) lectin, MALII (red) lectin, and DAPI (blue) in glomeruli on day 20 and day 80. Scale bar: 25 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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2.2. Antibodies The details regarding antibodies and lectins used have been described in Supplementary Table 1. 2.3. Urinary analysis Murine urine was collected via spontaneous micturition. Urinary proteins were evaluated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie Brilliant Blue staining. Total urinary protein to creatinine ratio was measured by the Oriental Yeast Corporation.
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treated with 2 mM Benzyl (Bz) -a-GalNAc (Sigma) in dimethyl sulfoxide (DMSO) for 3 days from the 5th day after removing IFN-g. C1GALT1 KO HEK293 (DC1) cells were generated as described previously [10]. The mouse Podxl expression vector (pcDNA3.1) was transfected using the Lipofectamine™ 2000 transfection reagent (Invitrogen) into HEK293 parent and DC1 cells. HEK293 parent (mPodPar) and DC1 (mPodDC1) cells stably expressing mouse Podxl were selected in medium containing 450 mg/ml geneticin, and the expression of PODXL was confirmed using flow cytometric analysis with CytoFLEX (Beckman Coulter). Both cells were harvested via trypsin treatment.
2.4. Histological analysis
2.9. Western blot analysis
The kidneys were fixed in Mildform 10 N (Wako) overnight at 4 C. The sections were assessed using hematoxylin-eosin (HE) and periodic acid-schiff base (PAS) staining under light microscopy. For immunofluorescence analysis, after blocking with Carbo-free blocking solution (Vector Laboratories), paraffinized or frozen sections were stained with primary antibodies and lectins. Immunodetection was performed using Alexa Flour fluoresceinconjugated secondary antibodies (Invitrogen). Nuclei were counterstained with DAPI. Images were captured using the BIOREVO BZX800 microscope system (Keyence).
Cells were resuspended in lysis buffer (20 mM HEPES (pH7.4), 1% TritonX-100) containing cOmplete™ protease inhibitor cocktail (Roche). The cell extracts were separated on 7.5% or 10% SDS polyacrylamide gel. Signals were developed using Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore) and visualized using the iBright imaging system (Thermo Fisher Scientific).
2.5. Transmission electron microscopy
All data are shown as mean ± SEM. A two-tailed t-test and Tukey test were performed using Microsoft EXCEL at 0.05% significance levels.
Minced tissue of the renal cortex was pre-fixed in 2.5% glutaraldehyde/0.1 M phosphate buffer overnight at 4 C, and then postfixed in 1% osmium tetroxide for 30 min at 4 C. After dehydration in ethanol, the samples were embedded in Epon. The thin sections were double-stained with uranyl acetate and lead citrate and examined under a JEM1400 transmission electron microscope (Jeol). The quantitative examination of FP width and GBM thickness was performed using the Image J software (freeware; National Institute of Health) as described previously [12]. 2.6. Glomeruli isolation Glomeruli were isolated from the kidneys of control and iPodCos mice, as described previously [13]. Sixteen milligrams of iron particles (ø10 mm; Millipore) in 10 ml phosphate-buffered saline (PBS) were infused in the left ventricle over 5 min. The pieces of the renal cortex were digested with collagenase solution (1 mg/ml collagenase A (Roche), 0.1 mg/ml DNase I (Roche) in Hank’s balanced salt solution) at 37 C for 40 min with gentle shaking, and then the glomeruli were collected using a magnetic particle concentrator (Thermo Fisher Scientific). 2.7. Quantitative analysis of transcripts using real-time reverse transcription-polymerase chain reaction (RT-PCR) Real-time RT-PCR was performed as described previously [10]. The primer sequences are mentioned in Supplementary Table 2. 2.8. Cell lines The mouse podocyte cell line, SVI, was obtained from the CLC cell line service (Germany) and RPMI-1640 medium containing 10% fetal bovine serum, penicillin (100 U/l), streptomycin (100 mg/l), and 10 U/ml mouse recombinant interferon-g (IFN-g, R&D System, 485-MI) at 33 C in collagen-coated dish. To induce podocyte differentiation, SVI was cultured in medium without IFN-g at 37 C for 10 days. For depletion of core 1-derived O-glycans, the SVI was
2.10. Statistical analysis
3. Results and discussion 3.1. Early proteinuria was caused by defects of core 1-derived Oglycan in podocytes To elucidate the roles of core 1-derived O-glycan in podocytes, we generated iPod-Cos mice. The appearances of iPod-Cos mice were normal, and their body weights did not differ from those of control mice (Supplementary Fig. 1A). Analysis of the kidney functions of iPod-Cos mice revealed proteinuria on day 7, which peaked on day 10 (D10) after TAM injection (Fig. 1B) and then decreased with time. These symptoms did not differ with gender (Fig. 1C). To confirm changes in the O-glycan structures of iPod-Cos podocytes, the kidney sections were stained using terminal aGalNAc-reactive lectin, Helix pomatia agglutinin (HPA), and Maackia amurensis lectin II (MALII), an a2,3-linked sialic acid-reactive lectin (Fig. 1D). Most iPod-Cos glomeruli exhibited HPA-reactive carbohydrates on day 20 (D20), whereas control glomeruli did not. The HPA-reactive signals in iPod-Cos mice colocalized with nephrinexpressing cells (Supplementary Fig. 1B), indicating that HPA signals were specifically observed in podocytes. In contrast, MALIIreactive signals in podocytes disappeared completely in iPod-Cos glomeruli. These results suggested that a2,3-linked sialic acids predominantly existed on core 1-derived O-glycan in podocytes. Furthermore, HPA signals were expressed in iPod-Cos mice until day 80 (D80) (Fig. 1D). To confirm whether the deletions of the Cosmc gene was maintained, we determined the mRNA levels in isolated glomeruli samples using quantitative real-time RT-PCR (Supplementary Fig. 1C). Compared to that in the control, Cosmc mRNA level was significantly reduced in iPod-Cos mice, while C1galt1 mRNA level did not change on D20 and D80. These results suggested that core 1-derived O-glycan is required for normal permselectivity in podocytes and that proteinuria spontaneously recovers without any change in O-glycan structures.
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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3.2. iPod-Cos mice showed FSGS-like pathogenesis with FP effacements Next, we evaluated the histological pattern of renal injury using PAS staining. The glomeruli of iPod-Cos mice showed protein casts in tubules on D20 and some global or segmental glomerulosclerosis on D80 (Fig. 2A and Supplementary Fig. 2A). The ratio of sclerotic glomeruli to all glomeruli increased sequentially in iPod-Cos mice despite no change in the control (D10; 3.7 ± 0.6%, D20; 7.2 ± 1.7%, D80; 18.3 ± 4.6% in iPod-Cos mice) (Fig. 2B). To confirm whether the expressions of filtration-related genes changed, the transcription levels of nephrin (Nphs1) and podocin (Nphs2) were evaluated in the glomeruli. The expression levels of Nphs1 and Nphs2 in iPodCos mice were similar to those in the control (Supplementary Fig. 2B). Furthermore, ultrastructural studies showed morphological changes in podocytes, including FP effacements (Fig. 3A). The GBM thickness did not differ between the control and iPod-Cos mice on D20, whereas the average FP width was larger in iPodCos mice than that in the control on D20 (Fig. 3B and C). Furthermore, the FP effacements recovered partially on D80. Ultrastructural images revealed that the ratio of regular FPs decreased to onefifth of the control value in iPod-Cos mice on D20 (Fig. 3D). However, they were repaired on D80 (control; 37.1 ± 2.1%, iPod-Cos D20; 7.4 ± 1.7%, iPod-Cos D80; 19.8 ± 4.7% in regular FPs). These results indicated that core 1-derived O-glycan maintains the structures of FPs and that the recovery from proteinuria in iPod-Cos mice might be due to the reconstitution of FP structures.
3.3. Characterization of PODXL and PDPN in iPod-Cos glomeruli To elucidate the molecular mechanism underlying the abnormal filtration barrier of iPod-Cos podocytes, we focused on mucin-type O-glycosylated PODXL and PDPN glycoproteins. Relative to the control, immunohistochemical staining of iPod-Cos kidney showed no change in the expression level and localization of PODXL on D20 (Fig. 4A). Western blotting using glomerular extracts showed that the PODXL antibody detected a major band at approximately 150 kDa in both control and iPod-Cos samples, indicating that it was a completely glycosylated PODXL protein (Fig. 4B). However, the signal intensity in iPod-Cos samples was lower than that in the control. The remaining 150 kDa bands in iPod-Cos are possibly due to PODXL expression in endothelial cells. Furthermore, the iPod-Cos sample showed four new low molecular weight bands on both D20 and D80, which might represent the degraded components (Fig. 4B). Song et al. reported that the interaction of PODXL with NHERF2 decreases in mice systemically lacking C1galt1 after birth, which might consequently affect cytoskeleton formation via phosphorylated ezrin in podocytes [14]. However, they did not observe the degradation products of PODXL in their KO mice. We presumed that PODXL expression in podocytes could not be observed due to high PODXL expression in endothelial cells, as whole kidney lysates were used for western blotting. The expression level of the Podxl transcript in glomerular extracts was comparable to that in the control glomeruli (Fig. 4C). These data suggested that the reduction in the intact PODXL level in iPod-Cos glomeruli might be because of proteolysis in podocytes.
Fig. 2. Histological analysis of kidney in iPod-Cos mice. (A) PAS staining of kidney in control and iPod-Cos mice. Protein casts (arrows), segmental (red arrowhead) and global (arrowhead) sclerosis, and inflammatory cells (asterisk) were observed in iPod-Cos mice. Scale bar: 100 mm. (B) Ratio of glomerular sclerosis to total glomeruli using PAS staining (day 10: each n ¼ 6, day 20: each n ¼ 3, day 80: control, n ¼ 5, iPod-Cos, n ¼ 4). Two-tailed t-test; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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Fig. 3. Renal glomerulus ultrastructure changes in iPod-Cos mice. (A) Transmission electron microscopic images of glomeruli in control and iPod-Cos mice on day 20. Scale bar: 10 mm (upper panel), 2 mm (lower panel). (B, C) Average of GBM thickness (B) and FP width (C) in control and iPod-Cos mice (n ¼ 3). (D) Rate of formation of three types of foot processes (FP), namely, regular FP, irregular FP, and cell body, in control and iPod-Cos mice (n ¼ 3).
In contrast, immunohistochemical analysis showed that PDPN expression was drastically lower in iPod-Cos podocytes than in the control (Fig. 4D). However, the positive signals persisted in the parietal epithelial cells of the Bowman’s capsule of iPod-Cos mice. In western blotting, the intact PDPN was observed at around 48 kDa in iPod-Cos glomeruli on D20, as well as in the control; however, its intensity was slightly lower, and a smaller specific band appeared at 20 kDa in iPod-Cos glomeruli (Fig. 4E). The remaining 45 kDa bands are considered to be PDPN derived from the parietal epithelial cells of the Bowman’s capsule that were contaminated when the glomeruli were isolated using iron particles. Furthermore, the mRNA expression levels of Pdpn were also lower in iPod-Cos on D20 than in the control glomeruli (Fig. 4F). These data indicated the level of the Pdpn transcript was transiently reduced, and simultaneously, PDPN was degraded in the glomeruli of iPod-Cos mice on D20. 3.4. PODXL is presumed to be degraded on podocyte surface in iPodCos mice Next, we investigated whether hypoglycosylated PODXL is degraded on the cell surface or within the cells using in vitro assays. To analyze the expression level of hypoglycosylated PODXL on the cell surface, we generated some transfectants stably overexpressing mouse Podxl in HEK293 cells and C1galt1-deficient HEK293 cells, which were named mPodPar and mPodDC1, respectively. Flow cytometric analysis revealed that the expression levels of PODXL on the cell surface were similar between mPodPar and mPodDC1
clones (Fig. 4G). Compared to that in mPodPar, a major band of PODXL shifted to a slightly lower position in mPodDC1 owing to loss of core 1-derived O-glycans, as observed using western blotting (Fig. 4H). Furthermore, an additional signal was also observed in mPodDC1 after trypsin treatment for detaching cells from culture dishes or after collagenase A treatment, under the same condition used for glomeruli isolation (Fig. 4H and I). To confirm the degradation of hypoglycosylated PODXL and PDPN in podocytes, the effect of an O-glycosylation inhibitor in SVI podocyte cell lines, which were harvested by scraping, was analyzed using western blotting (Supplementary Fig. 3, left). Although a major band of endogenous PODXL shifted to a higher molecular weight range after Bz-a-GalNAc treatment owing to loss of core 1-derived O-glycans, as a previously reported [8], the expected appearance of new bands (representing degraded protein) and reduction in band intensities were not observed. These results suggested that hypoglycosylated PODXL is degraded on the cell surface, but not inside the cells, and is also experimentally susceptible to proteases. However, these results could not conclusively explain the appearance of processed forms of PODXL in iPos-Cos as observed in Fig. 4B. Previous studies have shown that metalloproteinases (MMPs) and some cysteine proteinases are expressed in podocytes and other glomerular cells for maintaining GBM turnover and promoting glomerulosclerosis [15,16]. Furthermore, human PODXL was sensitive to specific matrix metalloproteinases such as MMP-1, 2, 9, and 14 in CHO cells [17]. Therefore, our results suggested that PODXL might be degraded by specific proteinases on podocytes in iPod-Cos mice.
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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Fig. 4. Characterization of PODXL and PDPN in iPod-Cos glomeruli. (A) Immunohistochemical analysis of PODXL in glomeruli on day 20. Scale bar: 20 mm. (B) Western blotting of PODXL in isolated glomeruli of day 20 and day 80. C; control, iP-C; iPod-Cos. (C) Relative transcript levels of Podxl in the isolated glomeruli of control and iPod-Cos on day 20 and day 80 (n ¼ 3). Hprt was used for normalization. (D) Immunohistochemical analysis of PDPN in glomeruli on day 20. Scale bar: 20 mm. (E) Western blotting of PDPN in glomeruli isolated on day 20 and day 80. C; control, iP-C; iPod-Cos. (F) Relative transcript levels of Pdpn in the isolated glomeruli of control and iPod-Cos on day 20 and day 80 (n ¼ 3). Hprt was used for normalization. Two-tailed t-test; *P < 0.05. (G) Flow cytometric analysis of mPodPar (#12, 14) and mPodDC1 (#3, 8, 17) clones. (H) Western blot analysis of mPodPar (#14) and mPodDC1 (#17) using anti-PODXL antibody. Cells were harvested using trypsin treatment or cell scraper. (I) Western blotting of PODXL using mPodPar and mPodDC1 cells treated with collagenase A. mPodPar and mPodDC1 cells were detached using trypsin from the culture dish and processed using collagenase A for 40 min at 37 C. Asterisk; Same concentration, that used for glomeruli isolation (1 mg/ml), S; solvent.
Compared to that observed after DMSO treatment, PDPN shifted to a slightly lower molecular weight range after treatment with Bza-GalNAc due to truncated core 1-derived O-glycosylation (Supplementary Fig. 3, right). According to previous investigations, core 1-derived O-glycan-deficient PDPN has cleavage sites in the extracellular domain and was susceptible to several proteinases, especially MMP-2, 9 [18]. Furthermore, PDPN binds MMP-14 at the surface of cancer cells [19]. These results suggested that the 20 kDa band of PDPN in iPod-Cos glomeruli (Fig. 4E) might represent the truncated protein that degraded on the cell surface. Some reports have shown that mice lacking heparan sulfate
from GBM did not develop proteinuria, suggesting that the negative charges of the glycocalyx in endothelial cells and sialoglycoproteins in podocytes are more critical for permselectivity than GBM [20,21]. In addition, Niculovic et al. analyzed the effects of sialic acids on Nglycan, O-glycan, and glycolipid, and observed that sialylation is vital for the functioning of the glomerular filtration barrier in the postnatal period [22]. Our results, in agreement with those from Niculovic et al., indicated that sialic acids of core 1-derived O-glycan (and/or N-glycan and glycolipid) in podocytes were essential for glomerular function. iPod-Cos mice recovered from proteinuria, and the urinary
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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protein levels of iPod-Cos were similar to that of the control on D80. Although the ratio of irregular FP to total FP decreased on D80 (Fig. 3D), the cause of these phenotypes remains unclear. However, several studies have demonstrated that podocyte has the ability to recover after kidney injury [23e25]. Recently, it has been proposed that FSGS and MCD might be different phases of the same disease [26]. As experimental research, a short period-activation of Rho GTPase Rac1 is linked to FSGS pathophysiology despite reversible proteinuria, which demonstrated that the Rac1 pathway could induce a spectrum of phenotypes that range from MCD to FSGS [27]. iPod-Cos mice also showed pathogenesis of FSGS on D80 despite transient proteinuria. Furthermore, the reduced PDPN expression in iPod-Cos on D20 was recovered with improvement in proteinuria. A mouse model for MCD showed a reduction in PDPN expression and recovery with improvement in proteinuria [9]. Renal biopsies from MCD patients also showed a reduction in PDPN level, whereas its expression increased in remission [9]. The phenotypes of iPod-Cos mice may also indicate the transition from MCD to FSGS as hypothesized [26,27]. In conclusion, iPod-Cos mice showed early proteinuria with FP effacements, and its symptoms dwindled over time. Furthermore, hypoglycosylated PODXL and PDPN were suggested to be degraded in iPod-Cos podocytes. These results suggested that core 1-derived O-glycan plays a vital role in maintaining stable PODXL and PDPN expression. However, we could not conclusively prove that PODXL or PDPN is directly responsible for FSGS. The possibility that other glycoproteins carrying the core 1-derived O-glycan are expressed in podocytes cannot be excluded. Future work will aim for a comprehensive comparison of phenotypes between iPod-Cos and mice lacking its glycoproteins to shed further insight into the underlying biological implications.
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Declaration of competing interest The authors declare that they have no conflicts of interest associated with the content of this article.
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Acknowledgments This work was performed as a part of the “Medical Glycomics (MG) Project” supported by the New Energy and Industrial Technology Development Organization (NEDO). This work was supported by Grants-in-Aid for Scientific Research, JSPS KAKENHI grant number, 17590236, 22500383, 24300152, and 16H06276, and AMED grant number JP19ae0101028. We would like to thank Editage (www.editage.com) for English language editing. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.033.
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Ishidoh, et al., Selective modulation of the secretion of proteinases and their inhibitors by growth factors in cultured differentiated podocytes, Kidney Int. 62 (2002) 822e831, https://doi.org/10.1046/j.15231755.2002.00539.x. D. Fernandez, S. Larrucea, A. Nowakowski, et al., Release of podocalyxin into the extracellular space. Role of metalloproteinases, Biochim. Biophys. Acta 1813 (2011) 1504e1510, https://doi.org/10.1016/j.bbamcr.2011.05.009. Y. Pan, T. Yago, J. Fu, et al., Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets, Blood 124 (2014) 3656e3665, https://doi.org/10.1182/blood-2014-04572107. M. Quintanilla, L. Montero-Montero, J. Renart, et al., Podoplanin in inflammation and cancer, Int. J. Mol. Sci. 20 (2019), https://doi.org/10.3390/ ijms20030707. A. Singh, S.C. Satchell, C.R. Neal, et al., Glomerular endothelial glycocalyx constitutes a barrier to protein permeability, J. Am. Soc. Nephrol. 18 (2007) 2885e2893, https://doi.org/10.1681/ASN.2007010119. S. Goldberg, S.J. Harvey, J. Cunningham, et al., Glomerular filtration is normal in the absence of both agrin and perlecan-heparan sulfate from the glomerular basement membrane, Nephrol. Dial. Transplant. 24 (2009) 2044e2051, https://doi.org/10.1093/ndt/gfn758. K.M. Niculovic, L. Blume, H. Wedekind, et al., Podocyte-specific sialylationdeficient mice serve as a model for human FSGS, J. Am. Soc. Nephrol. 30 (2019) 1021e1035, https://doi.org/10.1681/ASN.2018090951. M. Nagata, K. Nakayama, Y. Terada, et al., Cell cycle regulation and differentiation in the human podocyte lineage, Am. J. Pathol. 153 (1998) 1511e1520, https://doi.org/10.1016/s0002-9440(10)65739-2. B.L. Wharram, M. Goyal, J.E. Wiggins, et al., Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene, J. Am. Soc. 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Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis Sayaka Fuseya a, b, Riku Suzuki a, c, Risa Okada a, Kozue Hagiwara d, Takashi Sato d, Hisashi Narimatsu d, Hideki Yokoi e, Masato Kasahara f, Toshiaki Usui a, g, Naoki Morito g, Kunihiro Yamagata g, Takashi Kudo a, *, Satoru Takahashi a, * a
Laboratory Animal Resource Center in Transborder Medical Research Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Japan Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan c Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Japan d Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology, Japan e Department of Nephrology, Graduate School of Medicine, Kyoto University, Japan f Department of Clinical Research, Nara Medical University Hospital, Japan g Department of Nephrology, Faculty of Medicine, University of Tsukuba, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 December 2019 Accepted 5 January 2020 Available online xxx
The glomerular filtration barrier is composed of podocytes, glomerular basement membrane, and endothelial cells. Disruption of these structures causes several glomerular injuries, such as focal segmental glomerulosclerosis (FSGS). The surface of podocyte apical membranes is coated by negatively charged sialic acids on core 1-derived mucin-type O-glycans. Here, we aimed to investigate the physiological role of core 1-derived O-glycans in the podocytes using adult mice lacking podocyte-specific core 1-derived O-glycans (iPod-Cos). iPod-Cos mice exhibited early and transient proteinuria with foot process effacements and developed typical FSGS-like disease symptoms. To identify the key molecules responsible for the FSGS-like phenotype, we focused on podocalyxin and podoplanin, which possess mucin-type O-glycans. Expression and localization of podocalyxin did not change in iPod-Cos glomeruli. Besides, western blot analysis revealed significantly lower levels of intact podocalyxin in isolated glomeruli of iPod-Cos mice, and high levels of processed forms in iPod-Cos glomeruli, as compared to that in control glomeruli. Conversely, podoplanin mRNA, and protein levels were lower in iPod-Cos mice than in control mice. These results demonstrated that core 1-derived O-glycan on podocytes is required for normal glomerular filtration and may contribute to the stable expression of podocalyxin and podoplanin. © 2020 Elsevier Inc. All rights reserved.
Keywords: Mucin-type O-glycan Proteinuria FSGS Podocalyxin Podoplanin
1. Introduction Mucin-type O-glycans post-translationally modify membrane and secretory proteins. First, the N-acetylgalactosamine (GalNAc)
Abbreviations: C1galt1, core 1 b1,3-galactosyltransferase; FP, foot process; FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; GBM, glomerular basement membrane; PODXL, podocalyxin; PDPN, podoplanin. * Corresponding authors. Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan. E-mail addresses: [email protected] (T. Kudo), [email protected]. jp (S. Takahashi).
sugar is added on serine or threonine (Ser/Thr) residues of proteins by polypeptide N-acetylgalactosaminyltransferases. Subsequently, the galactose (Gal)-b1,3-GalNAc-a-Ser/Thr (core 1) structure is synthesized by core 1 b1,3-galactosyltransferase (C1galt1), which is further elongated by multiple glycosyltransferases (Fig. 1A). The C1galt1-specific molecular chaperone (Cosmc) is essential for the activity of C1galt1 [1]. Furthermore, C1galt1 knockout (KO) mice and Cosmc KO mice showed embryonic lethality due to abnormal angiogenesis and hemorrhage [2,3], indicating that core 1-derived O-glycan is critical for embryonic development. Large-scale mutagenesis screening was performed in mice using the chemical mutagen N-ethyl-N-nitrosourea [4]. plt1 mice generated in this
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Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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study harbor a point mutation in C1galt1 and exhibit low residual C1galt1 activity, thrombocytopenia, and proteinuria. We have also reported that C1galt1 is required for megakaryocyte development and proplatelet formation [5]. The glomerular filtration barrier consists of podocytes, glomerular basement membrane (GBM), and fenestrated endothelial cells, which are constitutively negatively charged. Glomerular injury causes due to the disruption of these structures, resulting in nephrotic syndromes, such as minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). FSGS is characterized by the presence of sclerotic glomeruli and progressive proteinuria, whereas MCD is accompanied by normal glomeruli, observable under a light microscope, and transient proteinuria. These diseases show similar initial symptoms such as massive proteinuria and podocyte foot process (FP) effacement, observed under an electron microscope. Therefore, a tool which distinguishes these diseases was needed for selecting more effective treatment options. The apical surface of podocytes is coated with negatively charged sialoglycoproteins, such as podocalyxin (PODXL) and podoplanin (PDPN). The mucin region of PODXL contains more than 50 putative O-linked glycosylation sites, half of which is potentially modified with sialic acids. Podxl KO mice died due to anuria within 24 h after birth [6]. Thus, PODXL and glomerular functions may be closely related. Indeed, exome sequencing led to the identification of the transmembrane domain variant, PODXL p.L442R, in patients with FSGS [7]. PDPN also contains Ser/Thr residues in the ectodomain, which can be potentially modified with core 1-derived O-glycans.
PODXL is linked to ezrin and the actin cytoskeleton via Naþ/Hþexchanger regulatory factor 2 (NHERF2) [8], while PDPN is linked to ezrin and the actin cytoskeleton, not via NHERF2 [9], which contribute to FP formation for glomerular filtration. In the present study, we aimed to investigate whether the loss of core 1-derived O-glycans affected glomerular filtration. Toward this, we generated inducible and podocyte-specific conditional Cosmc KO mice (iPod-Cos).
2. Materials and methods 2.1. Animals Mice were maintained in specific pathogen-free conditions in a Laboratory Animal Resource Center at the University of Tsukuba. All experiments were performed in compliance with the relevant Japanese and institutional laws and guidelines, and were approved by the University of Tsukuba Animal Ethics Committee (Authorization Number: 19e135). The generation of mice harboring floxed Cosmc has been described in our previous study [10]. Cosmcflox/flox mice were crossed with NPHS2-CreERT2 transgenic mice [11] to generate podocyte-specific conditional Cosmc KO mice (NPHS2CreERT2::Cosmcflox/flox; iPod-Cos). Conditional excision of the floxed allele was achieved by intraperitoneal injection of 75 mg/kg body weight of tamoxifen (TAM) once daily for a total of 5 consecutive days. The final TAM injection was defined as day 0.
Fig. 1. The onset of proteinuria in iPod-Cos mice. (A) Schematic showing the biosynthetic pathway of core 1-derived mucin-type O-glycan. (B) Quantitative analysis of urinary proteins using SDS-PAGE. (C) Urine total proteins/creatinine ratio (TP/CRE) in control and iPod-Cos mice (n ¼ 6). (D) Immunohistochemical analysis of HPA (green) lectin, MALII (red) lectin, and DAPI (blue) in glomeruli on day 20 and day 80. Scale bar: 25 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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2.2. Antibodies The details regarding antibodies and lectins used have been described in Supplementary Table 1. 2.3. Urinary analysis Murine urine was collected via spontaneous micturition. Urinary proteins were evaluated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie Brilliant Blue staining. Total urinary protein to creatinine ratio was measured by the Oriental Yeast Corporation.
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treated with 2 mM Benzyl (Bz) -a-GalNAc (Sigma) in dimethyl sulfoxide (DMSO) for 3 days from the 5th day after removing IFN-g. C1GALT1 KO HEK293 (DC1) cells were generated as described previously [10]. The mouse Podxl expression vector (pcDNA3.1) was transfected using the Lipofectamine™ 2000 transfection reagent (Invitrogen) into HEK293 parent and DC1 cells. HEK293 parent (mPodPar) and DC1 (mPodDC1) cells stably expressing mouse Podxl were selected in medium containing 450 mg/ml geneticin, and the expression of PODXL was confirmed using flow cytometric analysis with CytoFLEX (Beckman Coulter). Both cells were harvested via trypsin treatment.
2.4. Histological analysis
2.9. Western blot analysis
The kidneys were fixed in Mildform 10 N (Wako) overnight at 4 C. The sections were assessed using hematoxylin-eosin (HE) and periodic acid-schiff base (PAS) staining under light microscopy. For immunofluorescence analysis, after blocking with Carbo-free blocking solution (Vector Laboratories), paraffinized or frozen sections were stained with primary antibodies and lectins. Immunodetection was performed using Alexa Flour fluoresceinconjugated secondary antibodies (Invitrogen). Nuclei were counterstained with DAPI. Images were captured using the BIOREVO BZX800 microscope system (Keyence).
Cells were resuspended in lysis buffer (20 mM HEPES (pH7.4), 1% TritonX-100) containing cOmplete™ protease inhibitor cocktail (Roche). The cell extracts were separated on 7.5% or 10% SDS polyacrylamide gel. Signals were developed using Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore) and visualized using the iBright imaging system (Thermo Fisher Scientific).
2.5. Transmission electron microscopy
All data are shown as mean ± SEM. A two-tailed t-test and Tukey test were performed using Microsoft EXCEL at 0.05% significance levels.
Minced tissue of the renal cortex was pre-fixed in 2.5% glutaraldehyde/0.1 M phosphate buffer overnight at 4 C, and then postfixed in 1% osmium tetroxide for 30 min at 4 C. After dehydration in ethanol, the samples were embedded in Epon. The thin sections were double-stained with uranyl acetate and lead citrate and examined under a JEM1400 transmission electron microscope (Jeol). The quantitative examination of FP width and GBM thickness was performed using the Image J software (freeware; National Institute of Health) as described previously [12]. 2.6. Glomeruli isolation Glomeruli were isolated from the kidneys of control and iPodCos mice, as described previously [13]. Sixteen milligrams of iron particles (ø10 mm; Millipore) in 10 ml phosphate-buffered saline (PBS) were infused in the left ventricle over 5 min. The pieces of the renal cortex were digested with collagenase solution (1 mg/ml collagenase A (Roche), 0.1 mg/ml DNase I (Roche) in Hank’s balanced salt solution) at 37 C for 40 min with gentle shaking, and then the glomeruli were collected using a magnetic particle concentrator (Thermo Fisher Scientific). 2.7. Quantitative analysis of transcripts using real-time reverse transcription-polymerase chain reaction (RT-PCR) Real-time RT-PCR was performed as described previously [10]. The primer sequences are mentioned in Supplementary Table 2. 2.8. Cell lines The mouse podocyte cell line, SVI, was obtained from the CLC cell line service (Germany) and RPMI-1640 medium containing 10% fetal bovine serum, penicillin (100 U/l), streptomycin (100 mg/l), and 10 U/ml mouse recombinant interferon-g (IFN-g, R&D System, 485-MI) at 33 C in collagen-coated dish. To induce podocyte differentiation, SVI was cultured in medium without IFN-g at 37 C for 10 days. For depletion of core 1-derived O-glycans, the SVI was
2.10. Statistical analysis
3. Results and discussion 3.1. Early proteinuria was caused by defects of core 1-derived Oglycan in podocytes To elucidate the roles of core 1-derived O-glycan in podocytes, we generated iPod-Cos mice. The appearances of iPod-Cos mice were normal, and their body weights did not differ from those of control mice (Supplementary Fig. 1A). Analysis of the kidney functions of iPod-Cos mice revealed proteinuria on day 7, which peaked on day 10 (D10) after TAM injection (Fig. 1B) and then decreased with time. These symptoms did not differ with gender (Fig. 1C). To confirm changes in the O-glycan structures of iPod-Cos podocytes, the kidney sections were stained using terminal aGalNAc-reactive lectin, Helix pomatia agglutinin (HPA), and Maackia amurensis lectin II (MALII), an a2,3-linked sialic acid-reactive lectin (Fig. 1D). Most iPod-Cos glomeruli exhibited HPA-reactive carbohydrates on day 20 (D20), whereas control glomeruli did not. The HPA-reactive signals in iPod-Cos mice colocalized with nephrinexpressing cells (Supplementary Fig. 1B), indicating that HPA signals were specifically observed in podocytes. In contrast, MALIIreactive signals in podocytes disappeared completely in iPod-Cos glomeruli. These results suggested that a2,3-linked sialic acids predominantly existed on core 1-derived O-glycan in podocytes. Furthermore, HPA signals were expressed in iPod-Cos mice until day 80 (D80) (Fig. 1D). To confirm whether the deletions of the Cosmc gene was maintained, we determined the mRNA levels in isolated glomeruli samples using quantitative real-time RT-PCR (Supplementary Fig. 1C). Compared to that in the control, Cosmc mRNA level was significantly reduced in iPod-Cos mice, while C1galt1 mRNA level did not change on D20 and D80. These results suggested that core 1-derived O-glycan is required for normal permselectivity in podocytes and that proteinuria spontaneously recovers without any change in O-glycan structures.
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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3.2. iPod-Cos mice showed FSGS-like pathogenesis with FP effacements Next, we evaluated the histological pattern of renal injury using PAS staining. The glomeruli of iPod-Cos mice showed protein casts in tubules on D20 and some global or segmental glomerulosclerosis on D80 (Fig. 2A and Supplementary Fig. 2A). The ratio of sclerotic glomeruli to all glomeruli increased sequentially in iPod-Cos mice despite no change in the control (D10; 3.7 ± 0.6%, D20; 7.2 ± 1.7%, D80; 18.3 ± 4.6% in iPod-Cos mice) (Fig. 2B). To confirm whether the expressions of filtration-related genes changed, the transcription levels of nephrin (Nphs1) and podocin (Nphs2) were evaluated in the glomeruli. The expression levels of Nphs1 and Nphs2 in iPodCos mice were similar to those in the control (Supplementary Fig. 2B). Furthermore, ultrastructural studies showed morphological changes in podocytes, including FP effacements (Fig. 3A). The GBM thickness did not differ between the control and iPod-Cos mice on D20, whereas the average FP width was larger in iPodCos mice than that in the control on D20 (Fig. 3B and C). Furthermore, the FP effacements recovered partially on D80. Ultrastructural images revealed that the ratio of regular FPs decreased to onefifth of the control value in iPod-Cos mice on D20 (Fig. 3D). However, they were repaired on D80 (control; 37.1 ± 2.1%, iPod-Cos D20; 7.4 ± 1.7%, iPod-Cos D80; 19.8 ± 4.7% in regular FPs). These results indicated that core 1-derived O-glycan maintains the structures of FPs and that the recovery from proteinuria in iPod-Cos mice might be due to the reconstitution of FP structures.
3.3. Characterization of PODXL and PDPN in iPod-Cos glomeruli To elucidate the molecular mechanism underlying the abnormal filtration barrier of iPod-Cos podocytes, we focused on mucin-type O-glycosylated PODXL and PDPN glycoproteins. Relative to the control, immunohistochemical staining of iPod-Cos kidney showed no change in the expression level and localization of PODXL on D20 (Fig. 4A). Western blotting using glomerular extracts showed that the PODXL antibody detected a major band at approximately 150 kDa in both control and iPod-Cos samples, indicating that it was a completely glycosylated PODXL protein (Fig. 4B). However, the signal intensity in iPod-Cos samples was lower than that in the control. The remaining 150 kDa bands in iPod-Cos are possibly due to PODXL expression in endothelial cells. Furthermore, the iPod-Cos sample showed four new low molecular weight bands on both D20 and D80, which might represent the degraded components (Fig. 4B). Song et al. reported that the interaction of PODXL with NHERF2 decreases in mice systemically lacking C1galt1 after birth, which might consequently affect cytoskeleton formation via phosphorylated ezrin in podocytes [14]. However, they did not observe the degradation products of PODXL in their KO mice. We presumed that PODXL expression in podocytes could not be observed due to high PODXL expression in endothelial cells, as whole kidney lysates were used for western blotting. The expression level of the Podxl transcript in glomerular extracts was comparable to that in the control glomeruli (Fig. 4C). These data suggested that the reduction in the intact PODXL level in iPod-Cos glomeruli might be because of proteolysis in podocytes.
Fig. 2. Histological analysis of kidney in iPod-Cos mice. (A) PAS staining of kidney in control and iPod-Cos mice. Protein casts (arrows), segmental (red arrowhead) and global (arrowhead) sclerosis, and inflammatory cells (asterisk) were observed in iPod-Cos mice. Scale bar: 100 mm. (B) Ratio of glomerular sclerosis to total glomeruli using PAS staining (day 10: each n ¼ 6, day 20: each n ¼ 3, day 80: control, n ¼ 5, iPod-Cos, n ¼ 4). Two-tailed t-test; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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Fig. 3. Renal glomerulus ultrastructure changes in iPod-Cos mice. (A) Transmission electron microscopic images of glomeruli in control and iPod-Cos mice on day 20. Scale bar: 10 mm (upper panel), 2 mm (lower panel). (B, C) Average of GBM thickness (B) and FP width (C) in control and iPod-Cos mice (n ¼ 3). (D) Rate of formation of three types of foot processes (FP), namely, regular FP, irregular FP, and cell body, in control and iPod-Cos mice (n ¼ 3).
In contrast, immunohistochemical analysis showed that PDPN expression was drastically lower in iPod-Cos podocytes than in the control (Fig. 4D). However, the positive signals persisted in the parietal epithelial cells of the Bowman’s capsule of iPod-Cos mice. In western blotting, the intact PDPN was observed at around 48 kDa in iPod-Cos glomeruli on D20, as well as in the control; however, its intensity was slightly lower, and a smaller specific band appeared at 20 kDa in iPod-Cos glomeruli (Fig. 4E). The remaining 45 kDa bands are considered to be PDPN derived from the parietal epithelial cells of the Bowman’s capsule that were contaminated when the glomeruli were isolated using iron particles. Furthermore, the mRNA expression levels of Pdpn were also lower in iPod-Cos on D20 than in the control glomeruli (Fig. 4F). These data indicated the level of the Pdpn transcript was transiently reduced, and simultaneously, PDPN was degraded in the glomeruli of iPod-Cos mice on D20. 3.4. PODXL is presumed to be degraded on podocyte surface in iPodCos mice Next, we investigated whether hypoglycosylated PODXL is degraded on the cell surface or within the cells using in vitro assays. To analyze the expression level of hypoglycosylated PODXL on the cell surface, we generated some transfectants stably overexpressing mouse Podxl in HEK293 cells and C1galt1-deficient HEK293 cells, which were named mPodPar and mPodDC1, respectively. Flow cytometric analysis revealed that the expression levels of PODXL on the cell surface were similar between mPodPar and mPodDC1
clones (Fig. 4G). Compared to that in mPodPar, a major band of PODXL shifted to a slightly lower position in mPodDC1 owing to loss of core 1-derived O-glycans, as observed using western blotting (Fig. 4H). Furthermore, an additional signal was also observed in mPodDC1 after trypsin treatment for detaching cells from culture dishes or after collagenase A treatment, under the same condition used for glomeruli isolation (Fig. 4H and I). To confirm the degradation of hypoglycosylated PODXL and PDPN in podocytes, the effect of an O-glycosylation inhibitor in SVI podocyte cell lines, which were harvested by scraping, was analyzed using western blotting (Supplementary Fig. 3, left). Although a major band of endogenous PODXL shifted to a higher molecular weight range after Bz-a-GalNAc treatment owing to loss of core 1-derived O-glycans, as a previously reported [8], the expected appearance of new bands (representing degraded protein) and reduction in band intensities were not observed. These results suggested that hypoglycosylated PODXL is degraded on the cell surface, but not inside the cells, and is also experimentally susceptible to proteases. However, these results could not conclusively explain the appearance of processed forms of PODXL in iPos-Cos as observed in Fig. 4B. Previous studies have shown that metalloproteinases (MMPs) and some cysteine proteinases are expressed in podocytes and other glomerular cells for maintaining GBM turnover and promoting glomerulosclerosis [15,16]. Furthermore, human PODXL was sensitive to specific matrix metalloproteinases such as MMP-1, 2, 9, and 14 in CHO cells [17]. Therefore, our results suggested that PODXL might be degraded by specific proteinases on podocytes in iPod-Cos mice.
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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Fig. 4. Characterization of PODXL and PDPN in iPod-Cos glomeruli. (A) Immunohistochemical analysis of PODXL in glomeruli on day 20. Scale bar: 20 mm. (B) Western blotting of PODXL in isolated glomeruli of day 20 and day 80. C; control, iP-C; iPod-Cos. (C) Relative transcript levels of Podxl in the isolated glomeruli of control and iPod-Cos on day 20 and day 80 (n ¼ 3). Hprt was used for normalization. (D) Immunohistochemical analysis of PDPN in glomeruli on day 20. Scale bar: 20 mm. (E) Western blotting of PDPN in glomeruli isolated on day 20 and day 80. C; control, iP-C; iPod-Cos. (F) Relative transcript levels of Pdpn in the isolated glomeruli of control and iPod-Cos on day 20 and day 80 (n ¼ 3). Hprt was used for normalization. Two-tailed t-test; *P < 0.05. (G) Flow cytometric analysis of mPodPar (#12, 14) and mPodDC1 (#3, 8, 17) clones. (H) Western blot analysis of mPodPar (#14) and mPodDC1 (#17) using anti-PODXL antibody. Cells were harvested using trypsin treatment or cell scraper. (I) Western blotting of PODXL using mPodPar and mPodDC1 cells treated with collagenase A. mPodPar and mPodDC1 cells were detached using trypsin from the culture dish and processed using collagenase A for 40 min at 37 C. Asterisk; Same concentration, that used for glomeruli isolation (1 mg/ml), S; solvent.
Compared to that observed after DMSO treatment, PDPN shifted to a slightly lower molecular weight range after treatment with Bza-GalNAc due to truncated core 1-derived O-glycosylation (Supplementary Fig. 3, right). According to previous investigations, core 1-derived O-glycan-deficient PDPN has cleavage sites in the extracellular domain and was susceptible to several proteinases, especially MMP-2, 9 [18]. Furthermore, PDPN binds MMP-14 at the surface of cancer cells [19]. These results suggested that the 20 kDa band of PDPN in iPod-Cos glomeruli (Fig. 4E) might represent the truncated protein that degraded on the cell surface. Some reports have shown that mice lacking heparan sulfate
from GBM did not develop proteinuria, suggesting that the negative charges of the glycocalyx in endothelial cells and sialoglycoproteins in podocytes are more critical for permselectivity than GBM [20,21]. In addition, Niculovic et al. analyzed the effects of sialic acids on Nglycan, O-glycan, and glycolipid, and observed that sialylation is vital for the functioning of the glomerular filtration barrier in the postnatal period [22]. Our results, in agreement with those from Niculovic et al., indicated that sialic acids of core 1-derived O-glycan (and/or N-glycan and glycolipid) in podocytes were essential for glomerular function. iPod-Cos mice recovered from proteinuria, and the urinary
Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033
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protein levels of iPod-Cos were similar to that of the control on D80. Although the ratio of irregular FP to total FP decreased on D80 (Fig. 3D), the cause of these phenotypes remains unclear. However, several studies have demonstrated that podocyte has the ability to recover after kidney injury [23e25]. Recently, it has been proposed that FSGS and MCD might be different phases of the same disease [26]. As experimental research, a short period-activation of Rho GTPase Rac1 is linked to FSGS pathophysiology despite reversible proteinuria, which demonstrated that the Rac1 pathway could induce a spectrum of phenotypes that range from MCD to FSGS [27]. iPod-Cos mice also showed pathogenesis of FSGS on D80 despite transient proteinuria. Furthermore, the reduced PDPN expression in iPod-Cos on D20 was recovered with improvement in proteinuria. A mouse model for MCD showed a reduction in PDPN expression and recovery with improvement in proteinuria [9]. Renal biopsies from MCD patients also showed a reduction in PDPN level, whereas its expression increased in remission [9]. The phenotypes of iPod-Cos mice may also indicate the transition from MCD to FSGS as hypothesized [26,27]. In conclusion, iPod-Cos mice showed early proteinuria with FP effacements, and its symptoms dwindled over time. Furthermore, hypoglycosylated PODXL and PDPN were suggested to be degraded in iPod-Cos podocytes. These results suggested that core 1-derived O-glycan plays a vital role in maintaining stable PODXL and PDPN expression. However, we could not conclusively prove that PODXL or PDPN is directly responsible for FSGS. The possibility that other glycoproteins carrying the core 1-derived O-glycan are expressed in podocytes cannot be excluded. Future work will aim for a comprehensive comparison of phenotypes between iPod-Cos and mice lacking its glycoproteins to shed further insight into the underlying biological implications.
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Declaration of competing interest The authors declare that they have no conflicts of interest associated with the content of this article.
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Acknowledgments This work was performed as a part of the “Medical Glycomics (MG) Project” supported by the New Energy and Industrial Technology Development Organization (NEDO). This work was supported by Grants-in-Aid for Scientific Research, JSPS KAKENHI grant number, 17590236, 22500383, 24300152, and 16H06276, and AMED grant number JP19ae0101028. We would like to thank Editage (www.editage.com) for English language editing. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.033.
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Please cite this article as: S. Fuseya et al., Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.033