Clin Exp Nephrol DOI 10.1007/s10157-014-1003-0
ORIGINAL ARTICLE
Histochemical and immunoelectron microscopic analysis of ganglioside GM3 in human kidney Tetsuya Kaneko • Yoshiharu Tsubakihara • Hiroaki Fushimi Seiji Yamaguchi • Yoshitsugu Takabatake • Hiromi Rakugi • Hayato Kawakami • Yoshitaka Isaka
•
Received: 19 March 2014 / Accepted: 15 June 2014 Ó Japanese Society of Nephrology 2014
Abstract Background Gangliosides are amphipathic lipids ubiquitously expressed in all vertebrate cells. They have been reported to play pivotal roles in cell morphology, cell adhesion, signal transduction, and modulation of immune reaction. Although human kidney contains various kinds of ganglioside, their physiological and pathophysiological roles have not been elucidated yet. As ganglioside GM3 is the most abundant ganglioside in human kidney, we tried to reveal the distribution of GM3 using histological analysis. Methods Macroscopically normal parts of operatively resected kidney from renal cell carcinoma patients were
used for analyses. Immunohistochemical and immunoelectron microscopic analyses were performed with antiGM3 antibody. Results Immunohistochemical analyses showed that GM3 was observed in glomeruli and renal proximal tubules. Immunoelectron microscopy demonstrated that GM3 was localized on the foot process of podocyte and also in Golgi region of renal proximal tubule cells. Conclusions Ganglioside GM3 might take a part of the negative electric charge on the surface of podocyte and its multiple physiological actions may play pivotal roles for maintaining glomerular function. Keywords
T. Kaneko (&) Department of Nephrology, NTT West Japan Osaka Hospital, Osaka, Japan e-mail: [email protected] Y. Tsubakihara Department of Comprehensive Kidney Disease Research, Osaka University Graduate School of Medicine, Osaka, Japan H. Fushimi The Department of Pathology, Osaka General Medical Center, Osaka, Japan S. Yamaguchi The Department of Urology, Osaka General Medical Center, Osaka, Japan Y. Takabatake H. Rakugi Y. Isaka Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Osaka, Japan H. Kawakami Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
Ganglioside GM3 Lipid raft Podocyte
Introduction Gangliosides are sialic acid-containing glycosphingolipid that are ubiquitous in neural and extraneural organs [1–3]. They were identified first from nerve tissue in 1942 by Ernst Klenk. But to date, various kinds of ganglioside have been detected in outer leaflet of plasma membranes and endoplasmic reticulum in many tissues. In neuron, ganglioside plays pivotal roles in neural differentiation and dendrite formation [4, 5]. Recently, ganglioside in extraneural cells has been also reported to play important roles in cell differentiation, adhesion, signal transduction, and oncogenic transformation, and modulation of immune reaction [1, 6–9]. Various kinds of ganglioside have been reported to be located in ‘‘lipid raft’’. Hakomori reported about the importance of lipid raft in signal transduction and cell adhesion [2]. Inokuchi reported that the signal transduction of insulin receptor was interfered by the increase of GM3 in lipid raft [10].
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Although kidney contains various kinds of ganglioside, the physiological role has not been totally elucidated yet [11, 12]. Investigation for the role of ganglioside in renal tubule might help understanding of renal physiology. Concerning about glomerulus, Jing reported that GM3 on mouse podocyte surface functioned as a receptor for sFlt1(soluble fms-like tyrosine kinase-1) and composed lipid raft that was crucial for podocyte–podocyte adhesion by nephrin and intracellular actin reorganization [13]. GM3 has the simplest structure and was regarded as a prototype of a series of ganglioside [14]. Histochemical analysis on human glomeruli might be helpful for understanding the role of GM3. Experiments on animals should be performed, but we know that the ganglioside distribution in rat kidney should be different from human [15]. Moreover, in vertebrate, only human tissue lacks n-glycolyl neuraminic acid, suggesting that analysis of human tissue is indispensable [16]. Therefore, we investigated the distribution of GM3 in human kidney tissue using immunohistochemical and immunoelectron microscopic analyses.
Materials and methods The present study titled ‘‘Microscopic analysis of operatively resected kidney or biopsied renal tissue by anti glycolipid antibody staining.’’ was approved by the ethical committee of clinical medicine in Osaka General Medical Center on May 21st in 2008. Renal tissues were obtained from resected kidneys diagnosed as renal cell carcinoma from June 2008 to March 2009. Immunohistochemical and immunoelectron microscopic analyses Two kidney tissues were obtained from fifty-year-old male person and seventy-year-old female person. Before surgical operations, informed consents were accomplished according to the prepared printed format approved by the clinical medicine ethical committee in Osaka General Medical Center. After resection, the kidneys were cut sagittally into two parts as usual. Kidney tissues that looked macroscopically normal were cut out for analyses. For immunofluorescent analysis, kidney tissues were embedded in OCT compound (Miles; Elkhart, IL) and frozen with liquid nitrogen. Sections (8 lm thick) were made with a cryostat, placed on egg albumin-coated slides, and fixed with acetone at 0 °C for 10 min. After washing with PBS, nonspecific binding sites were blocked by incubation with 10 % goat serum and 3 % BSA for 10 min. After washing with PBS, sections were then incubated with anti-GM3 antibody (0.04 mg/ml) (M2590: COSMO BIO CO., LTD) for 12 h at 4 °C, washed with PBS, and
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subsequently incubated for 1 h with secondary antibody (goat anti-mouse IgM(l)F(ab0 )2 fragments adsorbed against human, bovine, and horse and conjugated to fluorescein: American Qualex Antibodies). After washing with PBS, the specimens were mounted in Perma Fluor (Japan Turner Corporation), and observed under a fluorescent microscope (Nikon; Tokyo, Japan). For analysis with avidin–biotin-peroxidase complex (ABC) technique and immunoelectron microscopy, sections (8 lm thick) were made with a cryostat, placed on egg albumin-coated slides. We tried various conditions for fixation according to previous reports (Table1) [17, 18]. After all, we decided to fix with 4 % formaldehyde–PBS (pH 7.4) at room temperature for 5 min. After fixation, some of the specimens were treated with 0.3 % H2O2 containing methanol for 20 min at room temperature to inactivate the endogenous peroxidase in renal proximal tubule. In the following procedure, we used VECTASTAIN ABC-PO (anti-mouse IgM kit: VECTOR). After washing with PBS, nonspecific binding sites were blocked by incubation with normal horse serum for 20 min at room temperature. After washing with PBS, sections were then incubated with anti-GM3 antibody (0.04 mg/ml) (M2590) for 12 h at 4 °C, washed with PBS, and subsequently incubated for 1 h with secondary antibody (anti-mouse IgM labeled with biotin). After washing with PBS, the specimens were incubated with VECTASTAIN ABC reagent (VECTOR) for 1 h at room temperature. Then, the specimens were washed with PBS and stained with DAB (3, 30 -Di aminobenzidine, tetrahydrochloride: DAB) kit (VECTOR). After all the procedure, they were observed with light microscope. After observation, the specimens were immersed in 1 % OsO4 for 30 min, dehydrated and embedded in Epon812. Ultrathin sections were cut and observed under a transmission electron microscope (JEOL, JEM-1011). Thin layer chromatography and western blot analysis Other parts of kidney tissues were dipped in the extraction solvent (chloroform/methanol 2/1 by volume). After extraction of tissue lipid, tissues were removed and the solvents were served for thin layer chromatography (TLC) analyses [19]. Especially, microdissection technique was performed for glomeruli isolation [20]. In brief, renal cortex tissue was placed in conditioned medium and glomeruli were divided under microscopic observation. Sixty isolated glomeruli were served for TLC. HPTLC plates (MERCK KGaA) were used for lipid separation. The developing solvent system was chloroform/methanol/0.2 % aqueous CaCl2 (60/35/5 by volume). Lipids on plates were made visible with Primuline reagent. As control samples, mouse brain ganglioside and purified GM3 were
Clin Exp Nephrol
generously offered by Dr. Takao Taki (Tokushima Institute, Otsuka Pharmaceutical Co., Ltd). To ascertain the results from histological analyses, commercially obtained premade membrane for western blot analysis (Single Tissue Blots-Single Species, Human Kidney: Geno Technology) was used to test the cross reaction of M2590 to human kidney protein.
human kidney protein was transferred, to avoid the nonspecific cross reaction of M2590 to human kidney protein. Western blot analysis with M2590 showed no signal (data not shown), indicating that M2590 did not bind nonspecifically to human renal proteins. These results confirmed the reliability of M2590 in the histochemical study. Light microscopic analyses
Results Confirmation of the reliability of M2590 antibody in vivo We first confirmed the reliability of M2590, an antibody for GM3, using TLC and Western blot analysis [14, 21, 22]. TLC analysis revealed that at least two bands of GM3, which corresponded to the different-sized ceramide portion, were observed in human kidney cortex and medulla as reported previously (Fig. 1a) [11, 15]. Glomeruli also contained GM3 (Fig. 1b). As reactivity of M2590 has been well established in vitro, immunostaining on TLC plate was not performed [21, 22]. Western blot analysis was performed using premade membrane (Single Tissue BlotsSingle Species, Human Kidney: Geno Technology), where Table 1 Fixation condition for immunohistochemical and immunoelectron microscopy
Fig. 1 Thin layer chromatography (TLC) of mouse brain ganglioside (lane 1), human kidney lipid extracted from renal cortex (lane 2) and medulla (lane 3), and ganglioside GM3 (lane 4) revealed that existence of GM3 in human kidney (a). TLC of lipid extracted from sixty glomeruli (lane 5) and mouse brain ganglioside (lane 6) also revealed the existence of GM3 in human glomeruli (b). GM1 indicated ganglioside GM1
Immunofluorescent staining of the kidney tissue (fifty-yearold male) using M2590 showed that GM3 was more intensely observed in cortex than in medulla (Fig. 2a–c). In renal cortex, GM3 was intense in renal proximal tubule and also found in glomeruli (Fig. 2b, d). Serial section of renal cortex (Fig. 2e, f) revealed that staining with secondary antibody (goat anti-mouse IgM antibody conjugated to fluorescein) only did not show any signals. Immunohistochemical staining by ABC technique also revealed that GM3 existed more intensely in cortex than in medulla (Fig. 3a). To eliminate the endogenous peroxidase activity in renal proximal tubule, inactivation by 0.3 % H2O2 containing methanol was performed. Positive staining on renal proximal tubule (Fig. 3e) disappeared in specimens treated with secondary antibody only (negative control) (Fig. 3f), although remained in M2590 treated
Fixative temperature
Fixative solution
Duration
Microstructure preservation
Antigenicity of GM3
4 °C
4 % Formaldehyde in PBS
5h
Preserved
Lost
4 °C
4 % Formaldehyde in PBS
1h
Poor
Poor study
4 °C Room (15–20 °C)
4 % Paraformaldehyde in PBS 4 % Formaldehyde in PBS
1h 5 min
Poor Preserved
Poor study Preserved
Room (15–20 °C)
4 % Formaldehyde in PBS
10 min
Preserved
Preserved
Room (15–20 °C)
4 % Formaldehyde in PBS
15 min
Preserved
Decreased
Room (15–20 °C)
10 % Formaldehyde in PBS
10 min
Preserved
Lost
a
b
] GM3 GM1
GM3
[[
]] GM1
[
1
2
3
4
5
6
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Clin Exp Nephrol Fig. 2 Immunohistochemical analyses of human kidney with anti-GM3 antibody M2590, macroscopic view of kidney tissue (a), higher magnification of cortex (b), higher magnification of medulla (c), and higher magnification of glomerulus (d). Positive staining was observed mainly in proximal tubule. In glomerulus, staining was observed on the peripheral of glomerular capillary and not remarkable in mesangial area. ‘‘Secondary’’ means negative control using only secondary antibody (fluorescent goat anti-mouse IgM antibody). Serial section of cortex revealed that staining was positive only with antiGM3 antibody (e, f)
a anti-GM3
medulla
b
anti-GM3
d
anti-GM3
f
secondary
anti-GM3
cortex
c
e
specimen (Fig. 3d). In glomerulus, staining was not remarkable in mesangial area and generally weak compared with renal proximal tubule cell at this magnification (Fig. 3b). No difference was observed in glomeruli after inactivation of endogenous peroxidase (Fig. 3b–d). Immunohistochemical analyses indicated that GM3 might be rich in cortex, especially in renal proximal tubule. Similar results were observed in the kidney of seventyyear-old female (data not shown).
anti-GM3
(Fig. 4b). GM3 was also observed on the plasma membrane of glomerular vascular endothelial cells (Fig. 4c). In proximal tubule cell, GM3 was observed in Golgi region (Fig. 4d). As specimens without endogenous peroxidase inactivation were served for analysis, positive staining in renal proximal tubule (Fig. 4d) might be enhanced by endogenous peroxidase. However, positive staining in glomerulus was not possibly affected by endogenous peroxidase of the adjacent renal proximal tubule. The results of fifty-year-old male were similar (data not shown).
Immunoelectron microscopic analyses As 0.3 % H2O2 containing methanol treatment induced degenerative change on renal tissue (Fig. 3d, f), specimens without endogenous peroxidase inactivation were served for immunoelectron microscopic analysis. Immunoelectron microscopy of the kidney specimen from seventy-year-old woman exhibited GM3 staining on the surface of podocyte (Fig. 4a), especially on the surface of foot processes
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Discussion Glycolipid is difficult to fix for histological analysis. Concerning about fixation, the present result was provided by trial and error. Kawakami et al. [17, 18] previously reported immunoelectron microscopic analyses of ganglioside. They suggested that acetone fixation might not be
Clin Exp Nephrol Fig. 3 Immunohistochemical analyses of human kidney by ABC technique with anti-GM3 antibody (M2590), low magnification (a) and high magnification of glomerulus (b). ‘‘Anti-GM3’’ means specimens stained with anti-GM3 antibody. ‘‘H2O2 (?)’’ indicates specimens after inactivation of endogenous peroxidase with H2O2. ‘‘H2O2 (-)’’ indicates specimens without inactivation. ‘‘Secondary’’ means specimens incubated with secondary antibody only (biotin labeled anti-mouse IgM) (negative control). Positive staining was observed mainly in renal proximal tubules and not remarkable in medullary collecting duct (a). In glomerulus, staining was not remarkable (b). The staining by anti-GM3 antibody was preserved after inactivation of endogenous peroxidase (c, d). Inactivation of endogenous peroxidase eliminated the staining of negative control (e, f). Arrows indicate glomeruli
cortex
anti-GM3 H202(-)
anti-GM3 H202(-)
a anti-GM3
medulla b H202(-)
e sufficient for electron microscopic analysis because lipid would disperse considerably and aldehyde might be better. As shown in Table 1, the retention of microstructure and the preservation of antigenicity were incompatible problems. We cannot tell why 4 % paraformaldehyde was not successful. Concerning about the timing of fixation, it was most desirable to fix raw tissue right after operative resection because glycolipids are labile. However, it was difficult to fix tissue immediately after resection in the clinical settings. The present data suggested that staining of GM3 was not remarkable in renal medulla, although both cortex and medulla contained GM3 (Fig. 1a). Previous report revealed that human renal cortex and renal medulla might contain 0.446 and 0.281 lmols/g of GM3, respectively [11]. In
H202(+)
d
c secondary
anti-GM3
H202(-)
secondary
H202(+)
f
immunohistochemical analysis, the difference of staining intensity among cortex, glomeruli, and medulla, might be dependent on GM3 concentration. In the present study, positive staining of renal proximal tubule cells was observed, suggesting active synthesis, glycosylation and intracellular transport of GM3 as reported in other cells [23]. GM3 was reported to be accumulated in hypertrophic kidney of diabetic rats, suggesting the active synthesis in rat renal tubules [24, 25]. GM3 might compose lipid raft on cell surface or brush border membrane and help or modulate the transporter function [25, 26]. By immunoelectron microscopy, positive staining was remarkable on the surface of podocyte and glomerular vascular endothelial cell. GM3 on vascular endothelial cell has been reported and its inhibitory effect against
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FP v
v
a
b
v
FP
c
N
d
Fig. 4 Immunoelectron microscopic analyses of GM3 with anti-GM3 antibody (M2590), glomerular capillary (a) (98000). Higher magnification of podocyte foot processes (FP indicates ‘‘foot processes’’ and V indicates ‘‘vascular lumen of glomerular capillary’’) and glomerular endothelial cells (b) (912000). Arrows indicate
glomerular vascular endothelial cells (c) (915000). Arrows indicate positive signals in cytoplasm of renal proximal tubule cell (d) (92000). N indicates nucleus. Positive staining was observed on the surface of podocyte, especially on foot process. In renal proximal tubule cell, positive staining was supposed to locate in Golgi region
angiogenesis has been documented [8, 27]. Further studies are needed about the role of ganglioside in glomerular vascular endothelial cell. The distribution of GM3 in human kidney podocyte in the present study is quite suggestive. Positive staining was observed on the surface of foot process, as if they were coated by GM3. It has been accepted that the negative electric charge on podocyte might be composed of sialic acid on podocalyxin [28, 29]. But the present results suggested that not only podocalyxin but also GM3 might take part in the electrical negative charge. In 2012, Jing reported about the binding of s-Flt1 to GM3 on mouse podocyte [13]. Their report suggested that changes of GM3 on the surface of podocyte may contribute to pathological cell transformation in human glomerular diseases. In human and experimental nephrotic syndrome, reduction of negative charge on podocyte has been reported as a result of the desialylation on podocalyxin [28, 29]. If GM3 might compose some part of the negative electric charge on human podocyte, we can speculate that desialylation or reduction of GM3 might take place in human nephrotic syndrome. What is the role of GM3 in podocyte? From morphological point of view, previous reports indicated that ganglioside was abundant in neuronal cells which possessed
cellular protrusion, neurite. But recent report also revealed that GM3 might be accumulated in leading head of T lymphocyte [30]. The negative charge of ganglioside has been reported to be beneficial for tubular formation or curved shape of plasma membrane physicochemically [31, 32]. Ganglioside may be a basic membrane structure component for cells with protrusion including neuron, lymphocyte, and podocyte. From functional point of view, ganglioside is a component of lipid raft. In lipid raft, ganglioside might play pivotal roles in adhesion between podocytes (side to side) and podocyte to glomerular basement membrane (GBM) [1, 13, 33]. It is conceivable that the qualitative and quantitative change of molecular components on lipid raft might disrupt adhesion. For example, dysfunction of tetraspanin CD151 has been reported to induce proteinuria and renal dysfunction in human kidney [34]. There are several reports that overexpression of GM3 in the lipid raft of adipocyte might have induced dissociation of insulin receptor and caveolin-1, consequently induced insulin resistance [10, 14]. We speculate that decrease (or desialylation) of GM3 might also contribute to the dysfunction of lipid raft on podocyte, resulting in the detachment from glomerular basement membrane or neighboring podocytes [13, 33].
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Clin Exp Nephrol Fig. 5 Functional role of GM3 in human podocyte (hypothesis)
Hypothesis Multiple function of ganglioside GM3 in human podocyte
3. side to side adhesion by nephrin
2. actin organization sFlt1
actin
1. foot process stabilization
4. adhesion to GBM
Glomerular basement membrane (GBM) GM3
All speculations in this discussion about podocyte are summarized in Fig. 5. Further studies are needed on human kidneys with various kinds of glomerulonephritis, minimal change nephrotic syndrome, and diabetic nephropathy.
Conclusions Ganglioside GM3 is located in renal proximal tubule and glomeruli. Its multiple physiological actions may play pivotal roles for renal tubule and glomerular function. Acknowledgments We thank Ms. Sachie Matubara (Laboratory for Electron microscopy, Kyorin University School of Medicine) for electron microscopic analyses. We also thank Dr. Takao Taki (Tokushima Institute, Otsuka Pharmaceutial Co., Ltd), for providing control samples of ganglioside, technical advice and helpful discussion. Conflict of interest Potential financial conflict of interest.Honoraria: Yoshiharu Tsubakihara (Chugai Pharm Co., Ltd, Kyowa Hakko, Kirin Co.,Ltd, Mitsubishi Tanabe Co., Ltd, Bayer Co., Ltd,), Research funding: Yoshiharu Tsubakihara (Chugai Pharm Co., Ltd, Baxter Japan Ltd, Otsuka Pharm Co., Ltd, Bayer Co., Ltd,), Endowed departments by commercial entities: Yoshiharu Tsubakihara (Chugai Pharm Co., Ltd, Baxter Japan Ltd,). The other authors have declared that no conflict of interest exists.
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ORIGINAL ARTICLE
Histochemical and immunoelectron microscopic analysis of ganglioside GM3 in human kidney Tetsuya Kaneko • Yoshiharu Tsubakihara • Hiroaki Fushimi Seiji Yamaguchi • Yoshitsugu Takabatake • Hiromi Rakugi • Hayato Kawakami • Yoshitaka Isaka
•
Received: 19 March 2014 / Accepted: 15 June 2014 Ó Japanese Society of Nephrology 2014
Abstract Background Gangliosides are amphipathic lipids ubiquitously expressed in all vertebrate cells. They have been reported to play pivotal roles in cell morphology, cell adhesion, signal transduction, and modulation of immune reaction. Although human kidney contains various kinds of ganglioside, their physiological and pathophysiological roles have not been elucidated yet. As ganglioside GM3 is the most abundant ganglioside in human kidney, we tried to reveal the distribution of GM3 using histological analysis. Methods Macroscopically normal parts of operatively resected kidney from renal cell carcinoma patients were
used for analyses. Immunohistochemical and immunoelectron microscopic analyses were performed with antiGM3 antibody. Results Immunohistochemical analyses showed that GM3 was observed in glomeruli and renal proximal tubules. Immunoelectron microscopy demonstrated that GM3 was localized on the foot process of podocyte and also in Golgi region of renal proximal tubule cells. Conclusions Ganglioside GM3 might take a part of the negative electric charge on the surface of podocyte and its multiple physiological actions may play pivotal roles for maintaining glomerular function. Keywords
T. Kaneko (&) Department of Nephrology, NTT West Japan Osaka Hospital, Osaka, Japan e-mail: [email protected] Y. Tsubakihara Department of Comprehensive Kidney Disease Research, Osaka University Graduate School of Medicine, Osaka, Japan H. Fushimi The Department of Pathology, Osaka General Medical Center, Osaka, Japan S. Yamaguchi The Department of Urology, Osaka General Medical Center, Osaka, Japan Y. Takabatake H. Rakugi Y. Isaka Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Osaka, Japan H. Kawakami Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
Ganglioside GM3 Lipid raft Podocyte
Introduction Gangliosides are sialic acid-containing glycosphingolipid that are ubiquitous in neural and extraneural organs [1–3]. They were identified first from nerve tissue in 1942 by Ernst Klenk. But to date, various kinds of ganglioside have been detected in outer leaflet of plasma membranes and endoplasmic reticulum in many tissues. In neuron, ganglioside plays pivotal roles in neural differentiation and dendrite formation [4, 5]. Recently, ganglioside in extraneural cells has been also reported to play important roles in cell differentiation, adhesion, signal transduction, and oncogenic transformation, and modulation of immune reaction [1, 6–9]. Various kinds of ganglioside have been reported to be located in ‘‘lipid raft’’. Hakomori reported about the importance of lipid raft in signal transduction and cell adhesion [2]. Inokuchi reported that the signal transduction of insulin receptor was interfered by the increase of GM3 in lipid raft [10].
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Although kidney contains various kinds of ganglioside, the physiological role has not been totally elucidated yet [11, 12]. Investigation for the role of ganglioside in renal tubule might help understanding of renal physiology. Concerning about glomerulus, Jing reported that GM3 on mouse podocyte surface functioned as a receptor for sFlt1(soluble fms-like tyrosine kinase-1) and composed lipid raft that was crucial for podocyte–podocyte adhesion by nephrin and intracellular actin reorganization [13]. GM3 has the simplest structure and was regarded as a prototype of a series of ganglioside [14]. Histochemical analysis on human glomeruli might be helpful for understanding the role of GM3. Experiments on animals should be performed, but we know that the ganglioside distribution in rat kidney should be different from human [15]. Moreover, in vertebrate, only human tissue lacks n-glycolyl neuraminic acid, suggesting that analysis of human tissue is indispensable [16]. Therefore, we investigated the distribution of GM3 in human kidney tissue using immunohistochemical and immunoelectron microscopic analyses.
Materials and methods The present study titled ‘‘Microscopic analysis of operatively resected kidney or biopsied renal tissue by anti glycolipid antibody staining.’’ was approved by the ethical committee of clinical medicine in Osaka General Medical Center on May 21st in 2008. Renal tissues were obtained from resected kidneys diagnosed as renal cell carcinoma from June 2008 to March 2009. Immunohistochemical and immunoelectron microscopic analyses Two kidney tissues were obtained from fifty-year-old male person and seventy-year-old female person. Before surgical operations, informed consents were accomplished according to the prepared printed format approved by the clinical medicine ethical committee in Osaka General Medical Center. After resection, the kidneys were cut sagittally into two parts as usual. Kidney tissues that looked macroscopically normal were cut out for analyses. For immunofluorescent analysis, kidney tissues were embedded in OCT compound (Miles; Elkhart, IL) and frozen with liquid nitrogen. Sections (8 lm thick) were made with a cryostat, placed on egg albumin-coated slides, and fixed with acetone at 0 °C for 10 min. After washing with PBS, nonspecific binding sites were blocked by incubation with 10 % goat serum and 3 % BSA for 10 min. After washing with PBS, sections were then incubated with anti-GM3 antibody (0.04 mg/ml) (M2590: COSMO BIO CO., LTD) for 12 h at 4 °C, washed with PBS, and
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subsequently incubated for 1 h with secondary antibody (goat anti-mouse IgM(l)F(ab0 )2 fragments adsorbed against human, bovine, and horse and conjugated to fluorescein: American Qualex Antibodies). After washing with PBS, the specimens were mounted in Perma Fluor (Japan Turner Corporation), and observed under a fluorescent microscope (Nikon; Tokyo, Japan). For analysis with avidin–biotin-peroxidase complex (ABC) technique and immunoelectron microscopy, sections (8 lm thick) were made with a cryostat, placed on egg albumin-coated slides. We tried various conditions for fixation according to previous reports (Table1) [17, 18]. After all, we decided to fix with 4 % formaldehyde–PBS (pH 7.4) at room temperature for 5 min. After fixation, some of the specimens were treated with 0.3 % H2O2 containing methanol for 20 min at room temperature to inactivate the endogenous peroxidase in renal proximal tubule. In the following procedure, we used VECTASTAIN ABC-PO (anti-mouse IgM kit: VECTOR). After washing with PBS, nonspecific binding sites were blocked by incubation with normal horse serum for 20 min at room temperature. After washing with PBS, sections were then incubated with anti-GM3 antibody (0.04 mg/ml) (M2590) for 12 h at 4 °C, washed with PBS, and subsequently incubated for 1 h with secondary antibody (anti-mouse IgM labeled with biotin). After washing with PBS, the specimens were incubated with VECTASTAIN ABC reagent (VECTOR) for 1 h at room temperature. Then, the specimens were washed with PBS and stained with DAB (3, 30 -Di aminobenzidine, tetrahydrochloride: DAB) kit (VECTOR). After all the procedure, they were observed with light microscope. After observation, the specimens were immersed in 1 % OsO4 for 30 min, dehydrated and embedded in Epon812. Ultrathin sections were cut and observed under a transmission electron microscope (JEOL, JEM-1011). Thin layer chromatography and western blot analysis Other parts of kidney tissues were dipped in the extraction solvent (chloroform/methanol 2/1 by volume). After extraction of tissue lipid, tissues were removed and the solvents were served for thin layer chromatography (TLC) analyses [19]. Especially, microdissection technique was performed for glomeruli isolation [20]. In brief, renal cortex tissue was placed in conditioned medium and glomeruli were divided under microscopic observation. Sixty isolated glomeruli were served for TLC. HPTLC plates (MERCK KGaA) were used for lipid separation. The developing solvent system was chloroform/methanol/0.2 % aqueous CaCl2 (60/35/5 by volume). Lipids on plates were made visible with Primuline reagent. As control samples, mouse brain ganglioside and purified GM3 were
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generously offered by Dr. Takao Taki (Tokushima Institute, Otsuka Pharmaceutical Co., Ltd). To ascertain the results from histological analyses, commercially obtained premade membrane for western blot analysis (Single Tissue Blots-Single Species, Human Kidney: Geno Technology) was used to test the cross reaction of M2590 to human kidney protein.
human kidney protein was transferred, to avoid the nonspecific cross reaction of M2590 to human kidney protein. Western blot analysis with M2590 showed no signal (data not shown), indicating that M2590 did not bind nonspecifically to human renal proteins. These results confirmed the reliability of M2590 in the histochemical study. Light microscopic analyses
Results Confirmation of the reliability of M2590 antibody in vivo We first confirmed the reliability of M2590, an antibody for GM3, using TLC and Western blot analysis [14, 21, 22]. TLC analysis revealed that at least two bands of GM3, which corresponded to the different-sized ceramide portion, were observed in human kidney cortex and medulla as reported previously (Fig. 1a) [11, 15]. Glomeruli also contained GM3 (Fig. 1b). As reactivity of M2590 has been well established in vitro, immunostaining on TLC plate was not performed [21, 22]. Western blot analysis was performed using premade membrane (Single Tissue BlotsSingle Species, Human Kidney: Geno Technology), where Table 1 Fixation condition for immunohistochemical and immunoelectron microscopy
Fig. 1 Thin layer chromatography (TLC) of mouse brain ganglioside (lane 1), human kidney lipid extracted from renal cortex (lane 2) and medulla (lane 3), and ganglioside GM3 (lane 4) revealed that existence of GM3 in human kidney (a). TLC of lipid extracted from sixty glomeruli (lane 5) and mouse brain ganglioside (lane 6) also revealed the existence of GM3 in human glomeruli (b). GM1 indicated ganglioside GM1
Immunofluorescent staining of the kidney tissue (fifty-yearold male) using M2590 showed that GM3 was more intensely observed in cortex than in medulla (Fig. 2a–c). In renal cortex, GM3 was intense in renal proximal tubule and also found in glomeruli (Fig. 2b, d). Serial section of renal cortex (Fig. 2e, f) revealed that staining with secondary antibody (goat anti-mouse IgM antibody conjugated to fluorescein) only did not show any signals. Immunohistochemical staining by ABC technique also revealed that GM3 existed more intensely in cortex than in medulla (Fig. 3a). To eliminate the endogenous peroxidase activity in renal proximal tubule, inactivation by 0.3 % H2O2 containing methanol was performed. Positive staining on renal proximal tubule (Fig. 3e) disappeared in specimens treated with secondary antibody only (negative control) (Fig. 3f), although remained in M2590 treated
Fixative temperature
Fixative solution
Duration
Microstructure preservation
Antigenicity of GM3
4 °C
4 % Formaldehyde in PBS
5h
Preserved
Lost
4 °C
4 % Formaldehyde in PBS
1h
Poor
Poor study
4 °C Room (15–20 °C)
4 % Paraformaldehyde in PBS 4 % Formaldehyde in PBS
1h 5 min
Poor Preserved
Poor study Preserved
Room (15–20 °C)
4 % Formaldehyde in PBS
10 min
Preserved
Preserved
Room (15–20 °C)
4 % Formaldehyde in PBS
15 min
Preserved
Decreased
Room (15–20 °C)
10 % Formaldehyde in PBS
10 min
Preserved
Lost
a
b
] GM3 GM1
GM3
[[
]] GM1
[
1
2
3
4
5
6
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Clin Exp Nephrol Fig. 2 Immunohistochemical analyses of human kidney with anti-GM3 antibody M2590, macroscopic view of kidney tissue (a), higher magnification of cortex (b), higher magnification of medulla (c), and higher magnification of glomerulus (d). Positive staining was observed mainly in proximal tubule. In glomerulus, staining was observed on the peripheral of glomerular capillary and not remarkable in mesangial area. ‘‘Secondary’’ means negative control using only secondary antibody (fluorescent goat anti-mouse IgM antibody). Serial section of cortex revealed that staining was positive only with antiGM3 antibody (e, f)
a anti-GM3
medulla
b
anti-GM3
d
anti-GM3
f
secondary
anti-GM3
cortex
c
e
specimen (Fig. 3d). In glomerulus, staining was not remarkable in mesangial area and generally weak compared with renal proximal tubule cell at this magnification (Fig. 3b). No difference was observed in glomeruli after inactivation of endogenous peroxidase (Fig. 3b–d). Immunohistochemical analyses indicated that GM3 might be rich in cortex, especially in renal proximal tubule. Similar results were observed in the kidney of seventyyear-old female (data not shown).
anti-GM3
(Fig. 4b). GM3 was also observed on the plasma membrane of glomerular vascular endothelial cells (Fig. 4c). In proximal tubule cell, GM3 was observed in Golgi region (Fig. 4d). As specimens without endogenous peroxidase inactivation were served for analysis, positive staining in renal proximal tubule (Fig. 4d) might be enhanced by endogenous peroxidase. However, positive staining in glomerulus was not possibly affected by endogenous peroxidase of the adjacent renal proximal tubule. The results of fifty-year-old male were similar (data not shown).
Immunoelectron microscopic analyses As 0.3 % H2O2 containing methanol treatment induced degenerative change on renal tissue (Fig. 3d, f), specimens without endogenous peroxidase inactivation were served for immunoelectron microscopic analysis. Immunoelectron microscopy of the kidney specimen from seventy-year-old woman exhibited GM3 staining on the surface of podocyte (Fig. 4a), especially on the surface of foot processes
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Discussion Glycolipid is difficult to fix for histological analysis. Concerning about fixation, the present result was provided by trial and error. Kawakami et al. [17, 18] previously reported immunoelectron microscopic analyses of ganglioside. They suggested that acetone fixation might not be
Clin Exp Nephrol Fig. 3 Immunohistochemical analyses of human kidney by ABC technique with anti-GM3 antibody (M2590), low magnification (a) and high magnification of glomerulus (b). ‘‘Anti-GM3’’ means specimens stained with anti-GM3 antibody. ‘‘H2O2 (?)’’ indicates specimens after inactivation of endogenous peroxidase with H2O2. ‘‘H2O2 (-)’’ indicates specimens without inactivation. ‘‘Secondary’’ means specimens incubated with secondary antibody only (biotin labeled anti-mouse IgM) (negative control). Positive staining was observed mainly in renal proximal tubules and not remarkable in medullary collecting duct (a). In glomerulus, staining was not remarkable (b). The staining by anti-GM3 antibody was preserved after inactivation of endogenous peroxidase (c, d). Inactivation of endogenous peroxidase eliminated the staining of negative control (e, f). Arrows indicate glomeruli
cortex
anti-GM3 H202(-)
anti-GM3 H202(-)
a anti-GM3
medulla b H202(-)
e sufficient for electron microscopic analysis because lipid would disperse considerably and aldehyde might be better. As shown in Table 1, the retention of microstructure and the preservation of antigenicity were incompatible problems. We cannot tell why 4 % paraformaldehyde was not successful. Concerning about the timing of fixation, it was most desirable to fix raw tissue right after operative resection because glycolipids are labile. However, it was difficult to fix tissue immediately after resection in the clinical settings. The present data suggested that staining of GM3 was not remarkable in renal medulla, although both cortex and medulla contained GM3 (Fig. 1a). Previous report revealed that human renal cortex and renal medulla might contain 0.446 and 0.281 lmols/g of GM3, respectively [11]. In
H202(+)
d
c secondary
anti-GM3
H202(-)
secondary
H202(+)
f
immunohistochemical analysis, the difference of staining intensity among cortex, glomeruli, and medulla, might be dependent on GM3 concentration. In the present study, positive staining of renal proximal tubule cells was observed, suggesting active synthesis, glycosylation and intracellular transport of GM3 as reported in other cells [23]. GM3 was reported to be accumulated in hypertrophic kidney of diabetic rats, suggesting the active synthesis in rat renal tubules [24, 25]. GM3 might compose lipid raft on cell surface or brush border membrane and help or modulate the transporter function [25, 26]. By immunoelectron microscopy, positive staining was remarkable on the surface of podocyte and glomerular vascular endothelial cell. GM3 on vascular endothelial cell has been reported and its inhibitory effect against
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FP v
v
a
b
v
FP
c
N
d
Fig. 4 Immunoelectron microscopic analyses of GM3 with anti-GM3 antibody (M2590), glomerular capillary (a) (98000). Higher magnification of podocyte foot processes (FP indicates ‘‘foot processes’’ and V indicates ‘‘vascular lumen of glomerular capillary’’) and glomerular endothelial cells (b) (912000). Arrows indicate
glomerular vascular endothelial cells (c) (915000). Arrows indicate positive signals in cytoplasm of renal proximal tubule cell (d) (92000). N indicates nucleus. Positive staining was observed on the surface of podocyte, especially on foot process. In renal proximal tubule cell, positive staining was supposed to locate in Golgi region
angiogenesis has been documented [8, 27]. Further studies are needed about the role of ganglioside in glomerular vascular endothelial cell. The distribution of GM3 in human kidney podocyte in the present study is quite suggestive. Positive staining was observed on the surface of foot process, as if they were coated by GM3. It has been accepted that the negative electric charge on podocyte might be composed of sialic acid on podocalyxin [28, 29]. But the present results suggested that not only podocalyxin but also GM3 might take part in the electrical negative charge. In 2012, Jing reported about the binding of s-Flt1 to GM3 on mouse podocyte [13]. Their report suggested that changes of GM3 on the surface of podocyte may contribute to pathological cell transformation in human glomerular diseases. In human and experimental nephrotic syndrome, reduction of negative charge on podocyte has been reported as a result of the desialylation on podocalyxin [28, 29]. If GM3 might compose some part of the negative electric charge on human podocyte, we can speculate that desialylation or reduction of GM3 might take place in human nephrotic syndrome. What is the role of GM3 in podocyte? From morphological point of view, previous reports indicated that ganglioside was abundant in neuronal cells which possessed
cellular protrusion, neurite. But recent report also revealed that GM3 might be accumulated in leading head of T lymphocyte [30]. The negative charge of ganglioside has been reported to be beneficial for tubular formation or curved shape of plasma membrane physicochemically [31, 32]. Ganglioside may be a basic membrane structure component for cells with protrusion including neuron, lymphocyte, and podocyte. From functional point of view, ganglioside is a component of lipid raft. In lipid raft, ganglioside might play pivotal roles in adhesion between podocytes (side to side) and podocyte to glomerular basement membrane (GBM) [1, 13, 33]. It is conceivable that the qualitative and quantitative change of molecular components on lipid raft might disrupt adhesion. For example, dysfunction of tetraspanin CD151 has been reported to induce proteinuria and renal dysfunction in human kidney [34]. There are several reports that overexpression of GM3 in the lipid raft of adipocyte might have induced dissociation of insulin receptor and caveolin-1, consequently induced insulin resistance [10, 14]. We speculate that decrease (or desialylation) of GM3 might also contribute to the dysfunction of lipid raft on podocyte, resulting in the detachment from glomerular basement membrane or neighboring podocytes [13, 33].
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Clin Exp Nephrol Fig. 5 Functional role of GM3 in human podocyte (hypothesis)
Hypothesis Multiple function of ganglioside GM3 in human podocyte
3. side to side adhesion by nephrin
2. actin organization sFlt1
actin
1. foot process stabilization
4. adhesion to GBM
Glomerular basement membrane (GBM) GM3
All speculations in this discussion about podocyte are summarized in Fig. 5. Further studies are needed on human kidneys with various kinds of glomerulonephritis, minimal change nephrotic syndrome, and diabetic nephropathy.
Conclusions Ganglioside GM3 is located in renal proximal tubule and glomeruli. Its multiple physiological actions may play pivotal roles for renal tubule and glomerular function. Acknowledgments We thank Ms. Sachie Matubara (Laboratory for Electron microscopy, Kyorin University School of Medicine) for electron microscopic analyses. We also thank Dr. Takao Taki (Tokushima Institute, Otsuka Pharmaceutial Co., Ltd), for providing control samples of ganglioside, technical advice and helpful discussion. Conflict of interest Potential financial conflict of interest.Honoraria: Yoshiharu Tsubakihara (Chugai Pharm Co., Ltd, Kyowa Hakko, Kirin Co.,Ltd, Mitsubishi Tanabe Co., Ltd, Bayer Co., Ltd,), Research funding: Yoshiharu Tsubakihara (Chugai Pharm Co., Ltd, Baxter Japan Ltd, Otsuka Pharm Co., Ltd, Bayer Co., Ltd,), Endowed departments by commercial entities: Yoshiharu Tsubakihara (Chugai Pharm Co., Ltd, Baxter Japan Ltd,). The other authors have declared that no conflict of interest exists.
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