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Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation
Kidney International, Vol. 68 (2005), pp. 2639–2650
Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation REIKO INAGI, MASAOMI NANGAKU, HIROSHI ONOGI, HIROSHI UEYAMA, YASUKO KITAO, KIYOKAZU NAKAZATO, SATOSHI OGAWA, KIYOSHI KUROKAWA, WILLIAM G. COUSER, and TOSHIO MIYATA Molecular and Cellular Nephrology, Institute of Medical Sciences, Tokai University, Kanagawa, Japan; Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan; Department of Neuroanatomy, Kanazawa University Medical School, Kanazawa, Japan; and Division of Nephrology, University of Washington, Seattle, Washington
Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation. Background. An imbalance between protein load and folding capacity is referred to as endoplasmic reticulum (ER) stress. As a defense mechanism, cells express ER stress inducible chaperons, such as oxygen-regulated proteins 150 (ORP150) and glucose-regulated proteins (GRPs). While ER stress is important in various diseases, a pathophysiologic role for ER stress in kidney disease remains elusive. Here we investigate expression of ER stress proteins in cultured rat podocytes as well as in our recently developed animal model of abnormal protein retention within the ER of podocytes (i.e., megsin transgenic rat). Methods. The expression of ER stress inducible proteins (ORP150, GRP78, or GRP94) in cultured podocytes treated with tunicamycin, A23187, SNAP, hypoxia, or hyperglycemia, and the renal tissues or isolated glomeruli from megsin transgenic rats was analyzed by Western blotting analysis, immunohistochemistry, or confocal microscopy. Results. Cultured podocytes demonstrated that treatment with tunicamycin, A23187, and SNAP, but not hypoxia or hyperglycemia, up-regulate expression of ER stress proteins. Extracts of isolated glomeruli from megsin transgenic rats reveal marked up-regulation of ER stress chaperones in podocytes, which was supported by immunohistochemical analysis. Confocal microscopy revealed that ER stress in podocytes was associated with cellular injury. Podocytes of transgenic rats overexpressing a mutant megsin, without the capacity for polymerization within the ER, do not exhibit ER stress or podocyte damage, suggesting a pathogenic role of ER retention of polymerized megsin. Conclusion. This paper implicates a crucial role for the accumulation of excessive proteins in the podocyte ER in the induction of ER stress and associated podocyte injury.
Key words: serpin, megsin, protein malfolding, podocyte, proteinuria, oxygen/glucose-regulated protein (ORP/GRP). Received for publication December 10, 2004 and in revised form June 6, 2005 Accepted for publication July 20, 2005 C
2005 by the International Society of Nephrology
The endoplasmic reticulum (ER) is an intracellular compartment that plays a critical role in the processing, folding, and exporting of newly synthesized proteins into the secretory pathway [1]. All cells regulate the capacity of their ER to process client proteins and adapt to an imbalance between client protein load and folding capacity, recently referred to as ER stress [2, 3]. An impairment of ER membrane integrity due to stress can be injurious by causing leakage of calcium ions and other ER luminal components. ER stress is triggered by various stimuli, including an increase in synthesis of client proteins, and also occurs in the course of various pathophysiologic states [4, 5]. The ER stress known as the unfolded protein response (UPR) is currently the best understood model of ER stress signaling, a response conserved from yeast to human [6]. The UPR involves transient attenuation of new protein synthesis, degradation of misfolded proteins, onset of apoptosis, and expression of a variety of ER stress proteins, such as oxygen-regulated protein (ORP) 150 and glucose-regulated proteins (GRPs). Under normal conditions, these ER stress proteins serve as protein chaperones, complex with defective proteins, and target them for degradation. During stress, the induction of ER stress proteins may limit accumulation of abnormal proteins in cells [6, 7], allowing cells to tolerate an accumulation of unfolded proteins. Previous studies have shown that ER stress is closely associated with the neuronal cell injury caused by ischemia [8], epileptic seizures [8], Alzheimer’s disease [9], Parkinson’s disease [10], and polyglutamine diseases [11]. ER stress is also involved in muscle cell injury in sporadic inclusion body myositis [12]. Homocysteine also induces ER stress in vascular endothelial cells [13], suggesting its potential role in thrombotic vascular events. Furthermore, ER stress induces apoptosis of pancreatic cells and subsequent development of diabetes mellitus [14, 15]. On
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the other hand, a pathophysiologic role of ER stress in kidney disease has not been defined. Accumulating evidence emphasizes an essential role of podocyte injury in proteinuria and progression of kidney failure [16, 17]. Podocytes contain a well-developed Golgi system, prominent lysosomes, many mitochondria, and abundant rough and smooth endoplasmic reticulum [18]. The density of these organelles in the cell body indicates a high level of anabolic as well as catabolic activity of podocytes, stimulating us to study susceptibility of podocytes to ER stress. Adaptive responses of podocytes to ER stress are crucial because podocytes are terminally differentiated cells that are unable to proliferate, and loss of these cells can lead to glomerulosclerosis and end-stage kidney failure [19, 20]. In the present study, we investigated expression of ER stress proteins in cultured rat podocytes as a defensive mechanism in vitro. We also employed our recently developed megsin transgenic rat to study expression of ER stress proteins in vivo. Megsin is a new member of the serpin (serine protease inhibitor) superfamily cloned from a human mesangial cDNA library [21–26]. An increased synthesis of megsin led to flooding of the protein handling system and subsequent accumulation of polymerizing megsin within the ER of podocytes [27]. It is likely that a common structure of reactive site loop and bsheet, shared by serpins, leads to the aberrant intermolecular linkage and polymerization of megsin. These animals develop periodic acid Schiff (PAS)–positive, diastaseresistant megsin inclusions within the ER of podocytes in association with proteinuria and deterioration of renal function [27]. METHODS In vitro rat podocyte experiments Primary cultured rat podocytes were isolated according to methods described elsewhere [28] In brief, the cells were initially established on Matrigel-coated tissue culture dishes (BD Biosciences, Bedford, MA, USA) and the cells were maintained onto a collagen type Icoated tissue culture dishes in K-1 medium supplemented with 2% NuSerum (Collaborative Biomedical Products, Bedford, MA, USA), 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL selenium (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) under humidified atmosphere containing 5% CO 2 . Rat podocytes used in this study showed a cobblestone-like appearance and were positive for megalin, cytokeratin, and podocalyxin, but not factor VIII or Thy-1.1, suggesting that the cells we used in this study retained the properties for podocytes. To evaluate the exposure to several stimuli including abnormal protein retention, calcium store depletion, ni-
trosative stress, hypoxia, and hyperglycemia, podocytes were treated with 5 lg/mL tunicamycin (Sigma-Aldrich), a reagent to cause an accumulation of unfolded proteins in the ER, 10 lg/mL A23187, a calcium ionophore, 110 lg/mL S-nitroso-N-acetyl-D,L-penicillamine (SNAP; Dojin, Kumamoto, Japan), a NO donor, or hypoxia utilizing Anaerocult A (<0.1% oxygen; Merck, Darmstadt, Germany) for 24 hours. For exposure to glycemic stress, podocytes were cultured in the presence of 4.5 mg/mL D- or L-glucose for two weeks. In glycemic stress we employed a long exposure time (2 weeks) to mimic chronic derangement of carbohydrate metabolism in diabetic patients. Western blot analysis Proteins (10 mg) from cell lysates or glomeruli isolated according to a conventional sieving method [29] were homogenized in 100 lL of 0.35 mol/L Tris-HCl (pH 6.8) containing 10% sodium dodecyl sulfate (SDS), 36% glycerol, 5% b-mercaptoethanol, and 0.012% bromophenol blue and centrifuged at 5000 g for 15 minutes. Three to five ll of the obtained test samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membrane. To detect ER stress inducible proteins, rabbit polyclonal antibodies to ORP150 (purified IgG fraction, 0.5 lg/mL) [30] or GRP94 (0.4 lg/mL; Stressgen Biotechnologies, Victoria, BC, Canada), or goat anti-GRP78 polyclonal antibody (20 lg/mL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used as the first antibody and horseradish peroxidase–conjugated goat anti-rabbit antibodies (1:5000 dilution; Bio-Rad, Hercules, CA, USA) or alkaline phosphatase-conjugated rabbit anti-goat IgG (H+L) (1:5000 dilution; Jackson ImmunoResearch Lab, Inc., West Grove, PA, USA) as the secondary antibody. Rabbit anti-actin polyclonal antibody (3.5 lg/mL; Sigma-Aldrich) was also used as the first antibody. The detection for ORP150, GRP94, or actin was performed by the enhanced chemiluminescence (ECL) system (Amersham Biosciences, Piscataway, NJ, USA). For detection of GRP78, p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate solution (Bio-Rad) was used. In some experiments, quantification of the intensity of each band was performed by densitometry utilizing ATTO lane analyzer software (ATTO, Tokyo, Japan) and normalized by the band intensity of actin. The experiment was repeated three times. Megsin transgenic rats To generate the human megsin transgene construct, the entire coding sequence of megsin cDNA was subcloned in the sense orientation into the pBsCAG-2 [24].
Inagi et al: ER stress in podocyte injury of megsin Tg rat
For production of mutant megsin (megsinT334R) transgenic rats, the site-directed mutagenesis at the position of P14 (substitution of Thr for Arg at 334 residue) in the reactive site loop of human megsin gene was performed utilizing a standard two-step megaprimer polymerase chain reaction (PCR) with mutated oligonucleotides, and megsinT334R cDNA was subcloned into the pBsCAG2 [27]. The megsin transgenes isolated by digestion of pBsCAG-2 containing megsin cDNA were microinjected into one pronucleus of fertilized Wistar rat eggs, followed by transfer into the oviducts of pseudopregnant rats. Rat genomic DNA extracted from the tail was used to identify megsin transgenic rats by PCR as described previously [24]. Kidneys from homozygote megsin and megsinT334R transgenic rats at the age of 8 weeks were used for PAS staining, immunohistochemistry, and Western blot analysis. Animals were treated in accordance with the guidelines of the Committee on Ethical Animal Care and Use of Tokai University. Histology For light microscopic analysis, kidneys were fixed in 4% neutral buffered formaldehyde, embedded in paraffin, and sectioned at 4 lm thickness, followed by PAS staining. Immunohistochemistry For detection of ORP150, GRP78, or desmin as a marker of podocyte injury [31], tissue sections (2 lm) of kidneys fixed with methyl Carnoy’s were used. The sections were first incubated with rabbit polyclonal antiORP150 antibodies (5 lg/mL purified IgG fraction) [30] or goat polyclonal anti-GRP78 antibodies (1:100 dilution; Santa Cruz Biotechnology, Inc.) followed by biotinylated goat anti-rabbit IgG (1:200 dilution; Zymed Laboratories, Inc, South San Francisco, CA, USA) or rabbit anti-goat IgG (1:800 dilution; DakoCytomation, Kyoto, Japan), or incubated with mouse monoclonal antibodies to desmin (11.5 lg/mL; DakoCytomation) followed by reaction with biotinylated horse anti-mouse IgG polyclonal antibody (1:400 dilution; Vector Laboratories, Inc., Burlingame, CA, USA). Development was performed with peroxidase-conjugated avidine (Vector Laboratories, Inc.) and 3,3 -diaminobenzidine tetrahydrochloride (Wako, Osaka, Japan). Finally, the tissues were counterstained with hematoxylin. In some experiments, normal rabbit IgG, goat IgG, or mouse IgG purchased from DakoCytomation were used as controls. Computer-assisted morphometry The sections stained immunohistochemically with antiORP150 or GRP78 antibodies were used for computer-
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assisted morphometry. Each staining picture was scanned using a 3CCD camera (Olympus Optical Co., Tokyo, Japan), and positive area for ORP150 or GRP78 expression per glomerulus was measured in a blinded manner using the software NIH ImageJ (NIH, Bethesda, MD, USA). Twenty consecutive glomeruli in the midcortex were measured. Glomerular cross sections containing only a minor portion of the glomerular tuft (<20 discrete capillary segments per cross-section) were not used. In order to avoid examiner’s bias, both the largest and the smallest glomerulus in the 20 glomeruli were excluded. Eighteen glomeruli from each section were examined and averages were expressed as mean ± SD. Immunofluorescence studies Rats were perfused with 4% paraformaldehyde under deep anesthesia, and the kidneys were cryostat sectioned (5 lm). Immunofluorescence studies were performed using either purified rabbit IgG to human ORP150 (5 lg/mL) [30] or human megsin (P 2 : 5 lg/mL) [23], goat polyclonal anti-GRP78 antibodies (1:100 dilution; Santa Cruz Biotechnology Inc.), or mouse monoclonal antibody to anti-synaptopodin (Progen Biotechnik GmbH, Heidelberg, Germany) followed by visualization of the binding sites of primary antibodies by the immunofluorescence method [30]. Images were then digitally overlapped. To investigate localization of megsin/ORP150 in desmin-positive injured podocytes, immunofluorescence studies with antibodies to human ORP150 and desmin followed by confocal microscopy were performed. The 4lm frozen sections were fixed with ethanol:ether (1:1), blocked with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), and incubated with purified rabbit IgG to human ORP150 (5 lg/mL) and mouse monoclonal antibody to desmin (11.5 lg/mL; DakoCytomation), followed by incubation with FITC-conjugated sheep anti-mouse IgG (1:50 dilution; DakoCytomation) and TRITC-conjugated swine anti-rabbit IgG (1:50 dilution; DAKO). The nuclei were counterstained with Hoechst Dye 33342 (Calbiochem-Novabiochem, La Jolla, CA, USA), and then the optical tomographic imaging (Z-images) for three-dimensional reconstructed images of glomeruli was performed utilizing 510 META (Carl Zeiss, Jena, Germany) [32]. Statistical analysis Data were expressed as mean ± SD. Analysis of variance (ANOVA) was utilized to evaluate the statistical significance of various differences. If the analysis detected a significant difference, the Scheffe’s t test was used to compare results obtained from the experimental animals. Values are considered significant at P < 0.05.
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Fig. 1. Expression of ER stress inducible proteins in podocytes. Podocytes were cultured in the presence of various stimuli including 5 lg/mL tunicamycin to cause an accumulation of unfolded proteins in the ER, 10 lg/mL A23187 as a calcium ionophore, 110 lg/mL SNAP as a NO donor, <0.1% oxygen as hypoxia, or 4.5 mg/mL glucose to induce hyperglycemia. Expression levels of ER stress inducible chaperone proteins, ORP150 and GRP94, were assessed by Western blot analysis followed by densitometry. (A) Representative data from three experiments are shown. (B) Quantification of the intensity of the bands was performed by densitometry and normalized to the intensity of actin bands. The mean ratio of ORP150 or GRP94 expression in the presence of various stimuli over that in the absence of stimuli was calculated for each experiment, and the average of the three experiments is expressed. Treatment with tunicamycin or A23187 markedly up-regulated expression of ORP150 and slightly of GRP94. SNAP treatment slightly up-regulated expression of both ORP150 and GRP94. By contrast, neither high glucose nor hypoxia influenced expression of ORP150 and GRP94. ∗ P < 0.05 vs. control; ∗∗ P < 0.01 vs. control. Dose and time dependency of up-regulation of OPR150 or GRP94 expression in podocytes treated with tunicamycin (C) or A23187 (D). Quantification of the intensity of the bands was performed by densitometry and normalized to the intensity of actin bands. The mean ratio of ORP150 or GRP94 expression in the presence of tunicamycin or A23187 over that in the absence of stimuli was calculated for each experiment, and the average of the three experiments is expressed. Treatment with tunicamycin or A23187 markedly up-regulated expression levels of ORP150 or GRP94 in a doseand time-dependent manner. ∗ P < 0.05 vs. control, ∗∗ P < 0.01 vs. control.
Inagi et al: ER stress in podocyte injury of megsin Tg rat
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RESULTS Expression of ER stress inducible proteins in podocytes To examine induction of ER stress in podocytes, we subjected cultured cells to various stimuli, including abnormal protein retention, calcium store depletion, nitrosative stress, hypoxia, and hyperglycemia. Expression levels of ER stress inducible chaperone proteins, ORP150 and GRP94, were assessed by Western blot analysis followed by densitometry. Representative data of Western blot analysis and densitometric quantification are shown in Figure 1A and B. Treatment with tunicamycin, a reagent to cause an accumulation of unfolded proteins in the ER, or A23187, a calcium ionophore, markedly up-regulated expression of ORP150 (tunicamycin, 5.03- ± 1.56-fold; A23187, 5.26 ± 0.97-fold, P < 0.01) and less of GRP94 (tunicamycin, 1.40 ± 0.37-fold, P < 0.05; A23187, 2.24 ± 0.53-fold, P < 0.01) in podocytes. We detected a lower molecular weight band of ORP150 in addition to a normal molecular weight band of ORP150 in podocyte treated with
24 Fig. 1. (continued)
tunicamycin. It may be due to abnormal glycosylation by tunicamycin. Up-regulation of ORP150 and GRP94 expression was observed in a time- and dose-dependent manner (Figs. 1C and D). Up-regulation of ORP150 and GRP94 in the podocytes treated with SNAP, a NO donor, was mild but statistically significant (2.32 ± 0.34-fold and 1.45 ± 0.1-fold, P < 0.05, respectively). By contrast, neither high D-glucose nor hypoxia influenced expression of ORP150 (high glucose, 0.89 ± 0.12-fold; hypoxia, 1.16 ± 0.19-fold) and GRP94 (high glucose, 0.97 ± 0.03-fold; hypoxia, 1.17 ± 0.20-fold) in podocytes. High concentration of L-glucose, which serves as a control of high osmolarity, did not change expression levels of ORP150 (0.92 ± 0.17-fold) or GRP94 (1.06 ± 0.13-fold) in podocytes, either. Megsin transgenic rats suffered from ER stress As tunicamycin blocks N-linked glycosylation and causes retention of unfolded proteins in the ER, and turned out to be a powerful inducer of ER stress proteins
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in cultured podocytes in vitro, we investigated an animal model of protein retention in the ER of podocytes in vivo. Our recently established megsin transgenic rats were utilized [27]. PAS staining (Fig. 2B) and immunoelectron microscopic studies [27] of renal tissues from these rats revealed accumulation of megsin within the ER of podocytes. Homozygote rats eventually developed proteinuria and impaired renal function [27]. In order to evaluate ER stress imposed by abnormal megsin retention, we performed immunohistochemical analysis utilizing various markers of ER stress, including ORP150 and GRP78. We employed GRP78 instead of GRP94 in immunohistochemical analysis because antiGRP94 did not work well for immunohistochemistry. Staining of ORP150 was negative in glomeruli of wildtype rats (Fig. 2E). In contrast, we observed marked up-regulation of ORP150 expression in glomeruli of homozygous megsin transgenic rats (Fig. 2F). GRP78 expression was undetectable in glomeruli of wild-type rats (Fig. 2J), and markedly up-regulated in glomeruli of homozygous megsin transgenic rats (Fig. 2K). In hetetozy-
24 Fig. 1. (continued)
gotes, up-regulation of ORP150 and GRP78 was less than in homozygotes (Figs. 2G and L). A pattern of localization of these proteins suggested that they were expressed in podocytes. These observations were also confirmed by computer-assisted morphometry of immunostained tissues: positive areas for ORP150 or GRP78 expression were increased with statistical significance in homozygotes as compared with those in heterozygotes (P < 0.001) (Fig. 2O). No immunoreactivity was observed with normal rabbit or goat IgG used as the first antibody (Figs. 2I and N). Increased expression of ORP150 and GRP78 was confirmed by Western blot analysis with isolated glomeruli from megsin transgenic rats followed by densitometric analysis (Fig. 3). We observed weak expression of both ORP150 and GRP78 in wild-type rats, although those expressions were undetectable in immunohistochemistry (Figs. 2E and J). This discrepancy may be due to the difference of sensitivity of each method. GRP94 expression was also up-regulated in the isolated glomeruli from megsin transgenic rats (Fig. 3).
Inagi et al: ER stress in podocyte injury of megsin Tg rat
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ER stress induced in podocytes with excessive megsin accumulation We previously demonstrated that accumulation of the transgene product, megsin, was localized exclusively in
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Polymerization of megsin and subsequent ER retention play a pathogenic role in development of ER stress in podocytes. Podocytes of heterozygote transgenic rats overexpressing mutant megsin (megsinT334R, a mutation at the position of P14 corresponding to the proximal hinge region of the reactive site loop), which lacks serine protease inhibitory activity as well as the capacity for polymerization within the ER, did not have megsin droplets within the cytoplasm (Fig. 2D) [27]. In addition, they did not show increased expression of ER stress markers, ORP150, GRP94, or GRP78 by immunohistochemistry (Figs. 2H and M) and Western blot analysis for isolated glomeruli (Fig. 3).
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Fig. 2. Induction of ER stress inducible proteins in the kidneys of megsin transgenic rats. PAS staining of a wild-type (A), a megsin transgenic (homozygote, B, and heterozygote, C), and a mutated-megsin (megsinT334R) transgenic rat (D) at the age of 8 weeks. PASpositive deposits (indicated by arrows) were observed within the cytoplasm of glomerular epithelial cells in a megsin transgenic rat but not in a wild-type rat or a megsinT334R transgenic rat. Immunohistochemical detection of ORP150 (E–I) and GRP78 (J–N) in the kidney of a wild-type rat (E and J), a megsin homozygote (F and K), a megsin heterozygote (G and L), and a megsinT334R transgenic rat (H and M). Expression of ORP150 and GRP78 is markedly augmented in glomeruli of megsin transgenic rats, as compared with those in wild-type and megsinT334R transgenic rats. No immunoreactivity was observed with normal rabbit or goat IgG as the first antibody in a megsin homozygote rat (I and N). The bar indicates 10 lm. (O) Computerassisted morphometry of the section stained immunohistochemically with anti-ORP150 or GRP78. Each section of three rats derived from each group was examined, and averages were expressed as mean ± SD. Positive areas for ORP150 or GRP78 expression were increased with statistical significance in homozygotes as compared with those in heterozygotes. ∗ P < 0.001 vs. wild-type or T334Rmegsin transgenic rats. #P < 0.001.
podocytes in glomeruli by immunoelectron microscopic analysis [27]. Of note, OPR150-expressed podocytes of megsin transgenic rats showed accumulation of megsin transgene product (Fig. 4). Podocyte damage by ER stress in megsin transgenic rats We further performed immunohistochemical analysis utilizing desmin as a marker of podocyte injury [31] in order to clarify whether ER stress in podocytes of megsin transgenic rats was associated with cellular injury. Glomeruli of megsin transgenic rats showed expression of desmin in podocytes and the signal intensity was higher in homozygotes than in heterozygotes (Fig. 5A, panels b and c). Whereas staining for desmin was absent in glomeruli of wild-type (Fig. 5A, panel a). No immunoreactivity was observed with normal mouse IgG used as the first antibody (Fig. 5A, panel e). Confocal microscopic analysis of double staining of desmin and ORP150 localized ORP150
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Inagi et al: ER stress in podocyte injury of megsin Tg rat
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(red) in podocytes with the cytoplasm positive for desmin (green). It suggests that podocyte injury was associated with ER stress (Fig. 5B). We further analyzed podocyte damage by double staining of synaptododin, a marker for podocytes, and GRP78 in megsin homozygote transgenic rats. The signal of synaptopodin expression adjacent to GRP78 positive signal was relatively faint as compared with that in wild-type rats, which was negative for GRP78 staining (Fig. 5C). By contrast, in megsinT334R transgenic rats, the desmin staining was extremely weak (Fig. 5A, panel d). These results, together with the lack of PAS-positive materials composed of megsin and ORP150 in the glomeruli of megsinT334R transgenic rats, clearly suggested that podocyte injury was not induced in this model. DISCUSSION ER stress in glomerular injury was recently emphasized by Cybulsky et al [33]. They reported that comple-
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Fig. 3. Western blot analysis of ER stress inducible proteins. Proteins from isolated glomeruli derived from wild-type, megsin homozygote transgenic, and mutated megsin (T334Rmegsin) transgenic rats (8-week-old) were analyzed by Western blot analysis using antibodies to ER stress inducible proteins such as ORP150, GRP78, and GRP-94. Polyclonal antibodies to b-actin were also used as an internal control. (A) Representative data from three independent experiments are shown. (B) Quantification of the intensity of the bands was performed by densitometry and normalized to the intensity of actin bands. The mean ratio of ORP150, GRP78, or GRP94 expression in megsin or T334Rmegsin transgenic rats over that in wild-type rats was calculated for each experiment, and the average of the three experiments is expressed. Increased protein expression of ORP 150, GRP78, and GRP94 was observed in isolated glomeruli from megsin transgenic rats, as compared with those from age-matched wild-type or T334Rmegsin transgenic rats. ∗ P < 0.05 vs. wild-type rats.
Fig. 4. ER stress is induced in podocytes with excessive megsin accumulation. Localization of ER stress inducible proteins in glomeruli from megsin homozygote transgenic rats (8week-old) was assessed by double staining using antibodies to ORP150 and megsin. Frozen kidney sections (5 lm) were incubated with rabbit polyclonal IgG to human ORP150 or human megsin followed by reaction with FITC- or TRITC-conjugated antirabbit IgG. OPR150-expressing podocytes of megsin transgenic rats showed accumulation of megsin transgene products. Representative positively stained cells were indicated by white arrowheads. The bar indicates 10 lm.
ment attack against podocytes induced ER injury, which was associated with up-regulation of ER stress proteins such as GPR78 (bip) and GRP94 both in vitro and in vivo. Our results confirm and extend their findings beyond complement-induced podocyte injury. Tunicamycin, which causes retention of unfolded proteins in the ER, induced ER stress in podocytes. In contrast, neither hypoxia nor high glucose resulted in ER stress in these cells. Factors inducing ER stress may be cell type–specific because hypoxia can lead to up-regulation of ER stress proteins in astrocytes [34], vascular smooth muscle cells [35], monocytes [35], ovary cells [36], and renal tubular cells [37]. Glomerular cells are relatively spared in ischemic kidney diseases compared with tubular cells [38], and hypoxia may not be as noxious to podocytes as it is to some other types of cells. In agreement with our in vitro findings, immunohistochemical analysis conducted in a rat ischemia-reperfusion of model showed that ORP150 staining is more prominent in tubular cells than in glomeruli [39].
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Fig. 5. Podocyte damage by ER stress in megsin transgenic rats. (A) Immunohistochemical detection of desmin, a marker of podocyte injury, in the kidney of a wild-type (panel a), a megsin transgenic rat (homozygote, panel b, and heterozygote, panel c), and a megsinT334R transgenic rat (panel d) at the age of 8 weeks. The sections (2 lm) were first incubated with anti-desmin monoclonal antibody, followed by reaction with biotinylated anti-mouse IgG. Expression of desmin was detected in glomeruli of a megsin transgenic rat but not in wild-type or megsinT334R transgenic rats. No immunoreactivity was observed with normal mouse IgG as the first antibody in a megsin homozygote rat (panel e). The bar indicates 10 lm.
Inagi et al: ER stress in podocyte injury of megsin Tg rat
It was not surprising that high glucose similar to what is seen in diabetes mellitus did not induce expression of ER stress proteins because ER stress proteins are known to be up-regulated by energy depletion under low glucose conditions [40, 41]. Here we demonstrated a crucial role of accumulation of excessive proteins in the ER in induction of ER stress utilizing our recently established megsin transgenic rats [27]. Megsin is a novel member of the serpin superfamily, and serpins are known to form aberrant intermolecular linkage in hepatocytes and neurons [42, 43]. To our knowledge, this is the first report to show that ER stress is associated with excessive protein accumulation in renal cells. It should be noted that the transgene of our megsin transgenic rats coded an intact molecule, in contrast to some human genetic disorders in which mutations in the coding region of highly expressed ER client proteins directly perturb their folding [2, 44]. However, previous studies also showed that unfolding or misfolding of proteins can occur under several circumstances, including macromolecular crowding, oxidative stress, exposure to toxins, and aging, suggesting that ER stress can play an important role in a wide range of diseases [8, 45]. Western blotting analysis in this study demonstrated up-regulation of a variety of ER-stress proteins in glomeruli isolated from megsin transgenic rats. We previously described predominant expression of megsin in mesangial cells of normal animals [26, 46], and cannot exclude a possible involvement of mesangial cells, endothelial cells, or infiltrating cells in induction of the ER stress proteins. However, immunohistochemical analysis as well as immunoelectron microscopic analysis [27] revealed that megsin transgenic rats unexpectedly showed accumulation of megsin in ER of podocytes. Expression patterns of the transgene revealed by in situ hybridization were ubiquitous in resident glomerular cells of the transgenic rats [27]. Accumulation of megsin also varied in different organs in spite of similar expression at the mRNA level [27]. These observations led us to speculate that individual cellular susceptibility must influence accumulation of the transgene product in ER, demonstrating predominant accumulation of megsin in podocytes of the transgenic rats. However, this requires further investigation.
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The ER stress in podocytes of megsin transgenic rats was associated with up-regulation of ER stress proteins such as ORP150 and GRP78. ORP150 was first purified and cloned as a novel stress protein induced by oxygen deprivation in cultured rat astrocytes [34]. Glucoseregulated proteins, GRP78 and GRP94, are also major constituents of the ER of mammalian cells and serve as a protective mechanism to adapt to stress on the ER [41, 47, 48]. While induction of ER stress proteins may be an important mechanism of protection of podocytes, further studies are warranted to conclude whether the ER stress response is protective or is responsible for some pathologic findings. CONCLUSION We reported induction of ER stress by various stimuli in cultured glomerular cells. Furthermore, our transgenic rats with accumulation of megsin in the ER of podocytes demonstrated ER stress-induced podocyte injury. Our findings implicated a crucial role for the accumulation of excessive proteins in the podocyte ER in the induction of ER stress and associated podocyte injury. ACKNOWLEDGMENTS We thank Dr. Johbu Itoh (Laboratories for Structure and Functional Research, Tokai University School of Medicine, Japan) for his technical support with the confocal microscopic analyses. This study was supported by grants from the New Energy and Industrial Technology Development Organization to T.M., from the Ministry of Education, Culture Sports, Science and Technology of Japan (15590861) to R.I., and from the Research and Study Program of Tokai University Educational System General Research Organization to R.I. and T.M. Reprint requests to Toshio Miyata, M.D., Ph.D., Molecular and Cellular Nephrology, Institute of Medical Sciences, Tokai University, Kanagawa 259-1193, Japan. E-mail: [email protected]
REFERENCES 1. ELLGAARD L, HELENIUS A: ER quality control: Towards an understanding at the molecular level. Curr Opin Cell Biol 13:431–437, 2001 2. LEE AS: The glucose-regulated proteins: Stress induction and clinical applications. Trends Biochem Sci 26:504–510, 2001 3. LEE AS: Mammalian stress response: Induction of the glucoseregulated protein family. Curr Opin Cell Biol 4:267–273, 1992
(B) Localization of ORP150 within injured podocytes. Three-dimensional colored intensity orthogonal section imaging of glomeruli were carried out after immunofluorescence studies using antibodies to ORP150 (red) and desmin (green), a marker of podocyte injury. Nuclei were poststained with Hoechst Dye 33342. X-Y cross-section plane image (blue line square), X-Z cross-section plane image (green line square), Y-Z cross-section plane image (red line square). ORP150 (red) was localized in podocytes with the cytoplasm positive for desmin (green), suggesting that podocyte injury was associated with ER stress. The bar indicates 5lm. (C) Double immunostaining of an ER stress inducible protein, GRP78, and synaptopodin, a marker of podocytes, in glomeruli from megsin homozygote transgenic rats (8-week-old). Frozen kidney sections (4 lm) from age-matched wild-type (upper panel) or megsin transgenic rats (lower panels) were incubated with mouse monoclonal antibodies to synaptopodin and goat polyclonal IgG to GRP78, which were detected by TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-goat IgG, respectively. The signal of synaptopodin expression adjacent to GRP78 positive signal was relatively faint in megsin transgenic rats compared with that in wild-type rats, which was negative for GRP78 expression, suggesting podocyte damage by ER stress. The bar indicates 10 lm.
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4. ARIDOR M, BALCH WE: Integration of endoplasmic reticulum signaling in health and disease. Nat Med 5:745–751, 1999 5. RON D: Proteotoxicity in the endoplasmic reticulum: Lessons from the Akita diabetic mouse. J Clin Invest 109:443–445, 2002 6. KAUFMANN RJ: Stress signaling from the lumen of the endoplasmic reticulum: Coordination of gene transcriptional and translational controls. Genes Dev 13:1211–1233, 1999 7. PAHL HL: Signal transduction from the endoplasmic reticulum to the cell nucleus. Physiol Rev 79:683–701, 1999 8. PASCHEN W, FRANDSEN A: Endoplasmic reticulum dysfunction—A common denominator for cell injury in acute and degenerative diseases of the brain? J Neurochem 79:719–725, 2001 9. KATAYAMA T, IMAIZUMI K, SATO N, et al: Presenilin-1 mutations downregulate the signaling pathway of the unfolded-protein response. Nat Cell Biol 1:479–485, 1999 10. IMAI Y, SODA M, INOUE H, et al: An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell 105:891–902, 2001 11. NISHITOH H, MATSUZAWA A, TOBIUME K, et al: ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16:1345–1355, 2002 12. VATTEMI G, ENGEL WK, MCFERRIN J, et al: Endoplasmic reticulum stress and unfolded protein response in inclusion body myositis muscle. Am J Pathol 164:1–7, 2004 13. OUTINEN PA, SOOD SK, PFEIFER SI, et al: Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells. Blood 94:959–967, 1999 14. SOCHA L, SILVA D, LESAGE S, et al: The role of endoplasmic reticulum stress in nonimmune diabetes: NOD.k iHEL, a novel model of beta cell death. Ann NY Acad Sci 1005:178–183, 2003 15. HARDING HP, RON D: Endoplasmic reticulum stress and the development of diabetes: A review. Diabetes 51(Suppl 3):S455–461, 2002 16. MUNDEL P, SHANKLAND SJ: Podocyte biology and response to injury. J Am Soc Nephrol 13:3005–3015, 2002 17. KRIZ W: Podocyte is the major culprit accounting for the progression of chronic renal disease. Microsc Res Tech 57:189–195, 2002 18. PAVENSTADT H, KRIZ W, KRETZLER M: Cell biology of the glomerular podocyte. Physiol Rev 83:253–307, 2003 19. KRIZ W: Progressive renal failure—Inability of podocytes to replicate and the consequences for development of glomerulosclerosis. Nephrol Dial Transplant 11:1738–1742, 1996 20. FRIES JW, SANDSTROM DJ, MEYER TW, et al: Glomerular hypertrophy and epithelial cell injury modulate progressive glomerulosclerosis in the rat. Lab Invest 60:205–218, 1989 21. YASUDA K, MIYATA T, NANGAKU M, et al: Functional quantitative analysis of the genome in cultured human mesangial cells. Kidney Int 53:154–158, 1998 22. MIYATA T, NANGAKU M, SUZUKI D, et al: A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy. J Clin Invest 102:828–836, 1998 23. INAGI R, MIYATA T, SUZUKI D, et al: Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy. Biochem Biophys Res Commun 286:1098–1106, 2001 24. MIYATA T, INAGI R, NANGAKU M, et al: Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion. J Clin Invest 109:585–593, 2002 25. INAGI R, MIYATA T, NANGAKU M, et al: Transcriptional regulation of a mesangium-predominant gene, megsin. J Am Soc Nephrol 13:2715–2722, 2002 26. INAGI R, NANGAKU M, MIYATA T, et al: Mesangial cell-predominant functional gene, megsin. Clin Exp Nephrol 7:87–92, 2003 27. INAGI R, NANGAKU M, USUDA N, et al: Novel serpinopathy in rat kidney and pancreas induced by overexpression of megsin. J Am Soc Nephrol 16:1339–1349, 2005 28. NANGAKU M, PIPPIN J, RICHARDSON CA, et al: Beneficial effects of systemic immunoglobulin in experimental membranous nephropathy. Kidney Int 50:2054–2062, 1996
29. SALANT DJ, DARBY C, COUSER WG: Experimental membranous glomerulonephritis in rats. J Clin Invest 66:71–81, 1980 30. KITAO Y, OZAWA K, MIYAZAKI M, et al: Expression of 150 kDa oxygen regulated protein (ORP150), a molecular chaperone in the endoplasmic reticulum, rescues hippocampal neurons from glutamate toxicity. J Clin Invest 108:1439–145, 2001 31. FLOEGE J, ALPERS CE, SAGE EH, et al: Markers of complementdependent and complement-independent glomerular visceral epithelial cell injury in vivo. Expression of antiadhesive proteins and cytoskeletal changes. Lab Invest 67:486–97, 1992 32. ITOH J, KAWAI K, SERIZAWA A, et al: Three-dimensional imaging of hormone-secreting cells and their microvessel environment in estrogen-induced prolactinoma of the rat pituitary gland by confocal laser scanning microscopy. Appl Immunohistochem Mol Morphol 9:364–370, 2001 33. CYBULSKY AV, TAKANO T, PAPILLON J, et al: Complement C5b9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells. J Biol Chem 277:41342–41351, 2002 34. KUWABARA K, MATSUMOTO M, IKEDA J, et al: Purification and characterization of a novel stress protein, the 150-kDa oxygenregulated protein (ORP150), from cultured rat astrocytes and its expression in ischemic mouse brain. J Biol Chem 271:5025–5032, 1996 35. TSUKAMOTO Y, KUWABARA K, HIROTA S, et al: 150-kD oxygenregulated protein is expressed in human atherosclerotic plaques and allows mononuclear phagocytes to withstand cellular stress on exposure to hypoxia and modified low density lipoprotein. J Clin Invest 98:1930–1941, 1996 36. ROLL DE, MURPHY BJ, LADEROUTE KR, et al: Oxygen regulated 80 kDa protein and glucose regulated 78 kDa protein are identical. Mol Cell Biochem 103:141–148, 1991 37. BANDO Y, OGAWA S, YAMAUCHI A, et al: 150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells. Am J Physiol Cell Physiol 278:C1172–1182, 2000 38. TRUONG LD, FARHOOD A, TASBY J, et al: Experimental chronic renal ischemia: Morphologic and immunologic studies. Kidney Int 41:1676–1689, 1992 39. BANDO Y, TSUKAMOTO Y, KATAYAMA T, et al: ORP150/HSP12A protects renal tubular epithelium from ischemia-induced cell death. FASEB J 18:1401–1403, 2004 40. MASSA SM, LONGO FM, ZUO J, et al: Cloning of rat grp75, an hsp70family member, and its expression in normal and ischemic brain. J Neuro Res 40:807–819, 1995 41. LITTLE E, RAMAKRISHNAN M, ROY B, et al: The glucose-regulated proteins (GRP78 and GRP94): Functions, gene regulation, and applications. Crit Rev Eukaryot Gene Expr 4:1–18, 1994 42. PERLMUTTER DH: Liver injury in alpha1-antitrypsin deficiency: An aggregated protein induces mitochondrial injury. J Clin Invest 110:1579–1583, 2002 43. DAVIS RL, SHRIMPTON AE, HOLOHAN PD, et al: Familial dementia caused by polymerization of mutant neuroserpin. Nature 401:376– 379, 1999 44. WANG J, TAKEUCHI T, TANAKA S, et al: A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse. J Clin Invest 103:27–37, 1999 45. KAUFMAN RJ: Orchestrating the unfolded protein response in health and disease. J Clin Invest 110:1389–1398, 2002 46. NANGAKU M, MIYATA T, SUZUKI D, et al: Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis. Kidney Int 60:641–652, 2001 47. KOZUTSUMI Y, SEGAL M, NORMINGTON K, et al: The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332:462–464, 1988 48. SHIU RP, POUYSSEGUR J, PASTAN I: Glucose depletion accounts for the induction of two transformation-sensitive membrane proteins in Rous Sarcoma virus-transformed chick embryo fibroblasts. Proc Natl Acad Sci USA 74:3840–3844, 1977
Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation REIKO INAGI, MASAOMI NANGAKU, HIROSHI ONOGI, HIROSHI UEYAMA, YASUKO KITAO, KIYOKAZU NAKAZATO, SATOSHI OGAWA, KIYOSHI KUROKAWA, WILLIAM G. COUSER, and TOSHIO MIYATA Molecular and Cellular Nephrology, Institute of Medical Sciences, Tokai University, Kanagawa, Japan; Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan; Department of Neuroanatomy, Kanazawa University Medical School, Kanazawa, Japan; and Division of Nephrology, University of Washington, Seattle, Washington
Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation. Background. An imbalance between protein load and folding capacity is referred to as endoplasmic reticulum (ER) stress. As a defense mechanism, cells express ER stress inducible chaperons, such as oxygen-regulated proteins 150 (ORP150) and glucose-regulated proteins (GRPs). While ER stress is important in various diseases, a pathophysiologic role for ER stress in kidney disease remains elusive. Here we investigate expression of ER stress proteins in cultured rat podocytes as well as in our recently developed animal model of abnormal protein retention within the ER of podocytes (i.e., megsin transgenic rat). Methods. The expression of ER stress inducible proteins (ORP150, GRP78, or GRP94) in cultured podocytes treated with tunicamycin, A23187, SNAP, hypoxia, or hyperglycemia, and the renal tissues or isolated glomeruli from megsin transgenic rats was analyzed by Western blotting analysis, immunohistochemistry, or confocal microscopy. Results. Cultured podocytes demonstrated that treatment with tunicamycin, A23187, and SNAP, but not hypoxia or hyperglycemia, up-regulate expression of ER stress proteins. Extracts of isolated glomeruli from megsin transgenic rats reveal marked up-regulation of ER stress chaperones in podocytes, which was supported by immunohistochemical analysis. Confocal microscopy revealed that ER stress in podocytes was associated with cellular injury. Podocytes of transgenic rats overexpressing a mutant megsin, without the capacity for polymerization within the ER, do not exhibit ER stress or podocyte damage, suggesting a pathogenic role of ER retention of polymerized megsin. Conclusion. This paper implicates a crucial role for the accumulation of excessive proteins in the podocyte ER in the induction of ER stress and associated podocyte injury.
Key words: serpin, megsin, protein malfolding, podocyte, proteinuria, oxygen/glucose-regulated protein (ORP/GRP). Received for publication December 10, 2004 and in revised form June 6, 2005 Accepted for publication July 20, 2005 C
2005 by the International Society of Nephrology
The endoplasmic reticulum (ER) is an intracellular compartment that plays a critical role in the processing, folding, and exporting of newly synthesized proteins into the secretory pathway [1]. All cells regulate the capacity of their ER to process client proteins and adapt to an imbalance between client protein load and folding capacity, recently referred to as ER stress [2, 3]. An impairment of ER membrane integrity due to stress can be injurious by causing leakage of calcium ions and other ER luminal components. ER stress is triggered by various stimuli, including an increase in synthesis of client proteins, and also occurs in the course of various pathophysiologic states [4, 5]. The ER stress known as the unfolded protein response (UPR) is currently the best understood model of ER stress signaling, a response conserved from yeast to human [6]. The UPR involves transient attenuation of new protein synthesis, degradation of misfolded proteins, onset of apoptosis, and expression of a variety of ER stress proteins, such as oxygen-regulated protein (ORP) 150 and glucose-regulated proteins (GRPs). Under normal conditions, these ER stress proteins serve as protein chaperones, complex with defective proteins, and target them for degradation. During stress, the induction of ER stress proteins may limit accumulation of abnormal proteins in cells [6, 7], allowing cells to tolerate an accumulation of unfolded proteins. Previous studies have shown that ER stress is closely associated with the neuronal cell injury caused by ischemia [8], epileptic seizures [8], Alzheimer’s disease [9], Parkinson’s disease [10], and polyglutamine diseases [11]. ER stress is also involved in muscle cell injury in sporadic inclusion body myositis [12]. Homocysteine also induces ER stress in vascular endothelial cells [13], suggesting its potential role in thrombotic vascular events. Furthermore, ER stress induces apoptosis of pancreatic cells and subsequent development of diabetes mellitus [14, 15]. On
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the other hand, a pathophysiologic role of ER stress in kidney disease has not been defined. Accumulating evidence emphasizes an essential role of podocyte injury in proteinuria and progression of kidney failure [16, 17]. Podocytes contain a well-developed Golgi system, prominent lysosomes, many mitochondria, and abundant rough and smooth endoplasmic reticulum [18]. The density of these organelles in the cell body indicates a high level of anabolic as well as catabolic activity of podocytes, stimulating us to study susceptibility of podocytes to ER stress. Adaptive responses of podocytes to ER stress are crucial because podocytes are terminally differentiated cells that are unable to proliferate, and loss of these cells can lead to glomerulosclerosis and end-stage kidney failure [19, 20]. In the present study, we investigated expression of ER stress proteins in cultured rat podocytes as a defensive mechanism in vitro. We also employed our recently developed megsin transgenic rat to study expression of ER stress proteins in vivo. Megsin is a new member of the serpin (serine protease inhibitor) superfamily cloned from a human mesangial cDNA library [21–26]. An increased synthesis of megsin led to flooding of the protein handling system and subsequent accumulation of polymerizing megsin within the ER of podocytes [27]. It is likely that a common structure of reactive site loop and bsheet, shared by serpins, leads to the aberrant intermolecular linkage and polymerization of megsin. These animals develop periodic acid Schiff (PAS)–positive, diastaseresistant megsin inclusions within the ER of podocytes in association with proteinuria and deterioration of renal function [27]. METHODS In vitro rat podocyte experiments Primary cultured rat podocytes were isolated according to methods described elsewhere [28] In brief, the cells were initially established on Matrigel-coated tissue culture dishes (BD Biosciences, Bedford, MA, USA) and the cells were maintained onto a collagen type Icoated tissue culture dishes in K-1 medium supplemented with 2% NuSerum (Collaborative Biomedical Products, Bedford, MA, USA), 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL selenium (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) under humidified atmosphere containing 5% CO 2 . Rat podocytes used in this study showed a cobblestone-like appearance and were positive for megalin, cytokeratin, and podocalyxin, but not factor VIII or Thy-1.1, suggesting that the cells we used in this study retained the properties for podocytes. To evaluate the exposure to several stimuli including abnormal protein retention, calcium store depletion, ni-
trosative stress, hypoxia, and hyperglycemia, podocytes were treated with 5 lg/mL tunicamycin (Sigma-Aldrich), a reagent to cause an accumulation of unfolded proteins in the ER, 10 lg/mL A23187, a calcium ionophore, 110 lg/mL S-nitroso-N-acetyl-D,L-penicillamine (SNAP; Dojin, Kumamoto, Japan), a NO donor, or hypoxia utilizing Anaerocult A (<0.1% oxygen; Merck, Darmstadt, Germany) for 24 hours. For exposure to glycemic stress, podocytes were cultured in the presence of 4.5 mg/mL D- or L-glucose for two weeks. In glycemic stress we employed a long exposure time (2 weeks) to mimic chronic derangement of carbohydrate metabolism in diabetic patients. Western blot analysis Proteins (10 mg) from cell lysates or glomeruli isolated according to a conventional sieving method [29] were homogenized in 100 lL of 0.35 mol/L Tris-HCl (pH 6.8) containing 10% sodium dodecyl sulfate (SDS), 36% glycerol, 5% b-mercaptoethanol, and 0.012% bromophenol blue and centrifuged at 5000 g for 15 minutes. Three to five ll of the obtained test samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membrane. To detect ER stress inducible proteins, rabbit polyclonal antibodies to ORP150 (purified IgG fraction, 0.5 lg/mL) [30] or GRP94 (0.4 lg/mL; Stressgen Biotechnologies, Victoria, BC, Canada), or goat anti-GRP78 polyclonal antibody (20 lg/mL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used as the first antibody and horseradish peroxidase–conjugated goat anti-rabbit antibodies (1:5000 dilution; Bio-Rad, Hercules, CA, USA) or alkaline phosphatase-conjugated rabbit anti-goat IgG (H+L) (1:5000 dilution; Jackson ImmunoResearch Lab, Inc., West Grove, PA, USA) as the secondary antibody. Rabbit anti-actin polyclonal antibody (3.5 lg/mL; Sigma-Aldrich) was also used as the first antibody. The detection for ORP150, GRP94, or actin was performed by the enhanced chemiluminescence (ECL) system (Amersham Biosciences, Piscataway, NJ, USA). For detection of GRP78, p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate solution (Bio-Rad) was used. In some experiments, quantification of the intensity of each band was performed by densitometry utilizing ATTO lane analyzer software (ATTO, Tokyo, Japan) and normalized by the band intensity of actin. The experiment was repeated three times. Megsin transgenic rats To generate the human megsin transgene construct, the entire coding sequence of megsin cDNA was subcloned in the sense orientation into the pBsCAG-2 [24].
Inagi et al: ER stress in podocyte injury of megsin Tg rat
For production of mutant megsin (megsinT334R) transgenic rats, the site-directed mutagenesis at the position of P14 (substitution of Thr for Arg at 334 residue) in the reactive site loop of human megsin gene was performed utilizing a standard two-step megaprimer polymerase chain reaction (PCR) with mutated oligonucleotides, and megsinT334R cDNA was subcloned into the pBsCAG2 [27]. The megsin transgenes isolated by digestion of pBsCAG-2 containing megsin cDNA were microinjected into one pronucleus of fertilized Wistar rat eggs, followed by transfer into the oviducts of pseudopregnant rats. Rat genomic DNA extracted from the tail was used to identify megsin transgenic rats by PCR as described previously [24]. Kidneys from homozygote megsin and megsinT334R transgenic rats at the age of 8 weeks were used for PAS staining, immunohistochemistry, and Western blot analysis. Animals were treated in accordance with the guidelines of the Committee on Ethical Animal Care and Use of Tokai University. Histology For light microscopic analysis, kidneys were fixed in 4% neutral buffered formaldehyde, embedded in paraffin, and sectioned at 4 lm thickness, followed by PAS staining. Immunohistochemistry For detection of ORP150, GRP78, or desmin as a marker of podocyte injury [31], tissue sections (2 lm) of kidneys fixed with methyl Carnoy’s were used. The sections were first incubated with rabbit polyclonal antiORP150 antibodies (5 lg/mL purified IgG fraction) [30] or goat polyclonal anti-GRP78 antibodies (1:100 dilution; Santa Cruz Biotechnology, Inc.) followed by biotinylated goat anti-rabbit IgG (1:200 dilution; Zymed Laboratories, Inc, South San Francisco, CA, USA) or rabbit anti-goat IgG (1:800 dilution; DakoCytomation, Kyoto, Japan), or incubated with mouse monoclonal antibodies to desmin (11.5 lg/mL; DakoCytomation) followed by reaction with biotinylated horse anti-mouse IgG polyclonal antibody (1:400 dilution; Vector Laboratories, Inc., Burlingame, CA, USA). Development was performed with peroxidase-conjugated avidine (Vector Laboratories, Inc.) and 3,3 -diaminobenzidine tetrahydrochloride (Wako, Osaka, Japan). Finally, the tissues were counterstained with hematoxylin. In some experiments, normal rabbit IgG, goat IgG, or mouse IgG purchased from DakoCytomation were used as controls. Computer-assisted morphometry The sections stained immunohistochemically with antiORP150 or GRP78 antibodies were used for computer-
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assisted morphometry. Each staining picture was scanned using a 3CCD camera (Olympus Optical Co., Tokyo, Japan), and positive area for ORP150 or GRP78 expression per glomerulus was measured in a blinded manner using the software NIH ImageJ (NIH, Bethesda, MD, USA). Twenty consecutive glomeruli in the midcortex were measured. Glomerular cross sections containing only a minor portion of the glomerular tuft (<20 discrete capillary segments per cross-section) were not used. In order to avoid examiner’s bias, both the largest and the smallest glomerulus in the 20 glomeruli were excluded. Eighteen glomeruli from each section were examined and averages were expressed as mean ± SD. Immunofluorescence studies Rats were perfused with 4% paraformaldehyde under deep anesthesia, and the kidneys were cryostat sectioned (5 lm). Immunofluorescence studies were performed using either purified rabbit IgG to human ORP150 (5 lg/mL) [30] or human megsin (P 2 : 5 lg/mL) [23], goat polyclonal anti-GRP78 antibodies (1:100 dilution; Santa Cruz Biotechnology Inc.), or mouse monoclonal antibody to anti-synaptopodin (Progen Biotechnik GmbH, Heidelberg, Germany) followed by visualization of the binding sites of primary antibodies by the immunofluorescence method [30]. Images were then digitally overlapped. To investigate localization of megsin/ORP150 in desmin-positive injured podocytes, immunofluorescence studies with antibodies to human ORP150 and desmin followed by confocal microscopy were performed. The 4lm frozen sections were fixed with ethanol:ether (1:1), blocked with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), and incubated with purified rabbit IgG to human ORP150 (5 lg/mL) and mouse monoclonal antibody to desmin (11.5 lg/mL; DakoCytomation), followed by incubation with FITC-conjugated sheep anti-mouse IgG (1:50 dilution; DakoCytomation) and TRITC-conjugated swine anti-rabbit IgG (1:50 dilution; DAKO). The nuclei were counterstained with Hoechst Dye 33342 (Calbiochem-Novabiochem, La Jolla, CA, USA), and then the optical tomographic imaging (Z-images) for three-dimensional reconstructed images of glomeruli was performed utilizing 510 META (Carl Zeiss, Jena, Germany) [32]. Statistical analysis Data were expressed as mean ± SD. Analysis of variance (ANOVA) was utilized to evaluate the statistical significance of various differences. If the analysis detected a significant difference, the Scheffe’s t test was used to compare results obtained from the experimental animals. Values are considered significant at P < 0.05.
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Fig. 1. Expression of ER stress inducible proteins in podocytes. Podocytes were cultured in the presence of various stimuli including 5 lg/mL tunicamycin to cause an accumulation of unfolded proteins in the ER, 10 lg/mL A23187 as a calcium ionophore, 110 lg/mL SNAP as a NO donor, <0.1% oxygen as hypoxia, or 4.5 mg/mL glucose to induce hyperglycemia. Expression levels of ER stress inducible chaperone proteins, ORP150 and GRP94, were assessed by Western blot analysis followed by densitometry. (A) Representative data from three experiments are shown. (B) Quantification of the intensity of the bands was performed by densitometry and normalized to the intensity of actin bands. The mean ratio of ORP150 or GRP94 expression in the presence of various stimuli over that in the absence of stimuli was calculated for each experiment, and the average of the three experiments is expressed. Treatment with tunicamycin or A23187 markedly up-regulated expression of ORP150 and slightly of GRP94. SNAP treatment slightly up-regulated expression of both ORP150 and GRP94. By contrast, neither high glucose nor hypoxia influenced expression of ORP150 and GRP94. ∗ P < 0.05 vs. control; ∗∗ P < 0.01 vs. control. Dose and time dependency of up-regulation of OPR150 or GRP94 expression in podocytes treated with tunicamycin (C) or A23187 (D). Quantification of the intensity of the bands was performed by densitometry and normalized to the intensity of actin bands. The mean ratio of ORP150 or GRP94 expression in the presence of tunicamycin or A23187 over that in the absence of stimuli was calculated for each experiment, and the average of the three experiments is expressed. Treatment with tunicamycin or A23187 markedly up-regulated expression levels of ORP150 or GRP94 in a doseand time-dependent manner. ∗ P < 0.05 vs. control, ∗∗ P < 0.01 vs. control.
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RESULTS Expression of ER stress inducible proteins in podocytes To examine induction of ER stress in podocytes, we subjected cultured cells to various stimuli, including abnormal protein retention, calcium store depletion, nitrosative stress, hypoxia, and hyperglycemia. Expression levels of ER stress inducible chaperone proteins, ORP150 and GRP94, were assessed by Western blot analysis followed by densitometry. Representative data of Western blot analysis and densitometric quantification are shown in Figure 1A and B. Treatment with tunicamycin, a reagent to cause an accumulation of unfolded proteins in the ER, or A23187, a calcium ionophore, markedly up-regulated expression of ORP150 (tunicamycin, 5.03- ± 1.56-fold; A23187, 5.26 ± 0.97-fold, P < 0.01) and less of GRP94 (tunicamycin, 1.40 ± 0.37-fold, P < 0.05; A23187, 2.24 ± 0.53-fold, P < 0.01) in podocytes. We detected a lower molecular weight band of ORP150 in addition to a normal molecular weight band of ORP150 in podocyte treated with
24 Fig. 1. (continued)
tunicamycin. It may be due to abnormal glycosylation by tunicamycin. Up-regulation of ORP150 and GRP94 expression was observed in a time- and dose-dependent manner (Figs. 1C and D). Up-regulation of ORP150 and GRP94 in the podocytes treated with SNAP, a NO donor, was mild but statistically significant (2.32 ± 0.34-fold and 1.45 ± 0.1-fold, P < 0.05, respectively). By contrast, neither high D-glucose nor hypoxia influenced expression of ORP150 (high glucose, 0.89 ± 0.12-fold; hypoxia, 1.16 ± 0.19-fold) and GRP94 (high glucose, 0.97 ± 0.03-fold; hypoxia, 1.17 ± 0.20-fold) in podocytes. High concentration of L-glucose, which serves as a control of high osmolarity, did not change expression levels of ORP150 (0.92 ± 0.17-fold) or GRP94 (1.06 ± 0.13-fold) in podocytes, either. Megsin transgenic rats suffered from ER stress As tunicamycin blocks N-linked glycosylation and causes retention of unfolded proteins in the ER, and turned out to be a powerful inducer of ER stress proteins
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in cultured podocytes in vitro, we investigated an animal model of protein retention in the ER of podocytes in vivo. Our recently established megsin transgenic rats were utilized [27]. PAS staining (Fig. 2B) and immunoelectron microscopic studies [27] of renal tissues from these rats revealed accumulation of megsin within the ER of podocytes. Homozygote rats eventually developed proteinuria and impaired renal function [27]. In order to evaluate ER stress imposed by abnormal megsin retention, we performed immunohistochemical analysis utilizing various markers of ER stress, including ORP150 and GRP78. We employed GRP78 instead of GRP94 in immunohistochemical analysis because antiGRP94 did not work well for immunohistochemistry. Staining of ORP150 was negative in glomeruli of wildtype rats (Fig. 2E). In contrast, we observed marked up-regulation of ORP150 expression in glomeruli of homozygous megsin transgenic rats (Fig. 2F). GRP78 expression was undetectable in glomeruli of wild-type rats (Fig. 2J), and markedly up-regulated in glomeruli of homozygous megsin transgenic rats (Fig. 2K). In hetetozy-
24 Fig. 1. (continued)
gotes, up-regulation of ORP150 and GRP78 was less than in homozygotes (Figs. 2G and L). A pattern of localization of these proteins suggested that they were expressed in podocytes. These observations were also confirmed by computer-assisted morphometry of immunostained tissues: positive areas for ORP150 or GRP78 expression were increased with statistical significance in homozygotes as compared with those in heterozygotes (P < 0.001) (Fig. 2O). No immunoreactivity was observed with normal rabbit or goat IgG used as the first antibody (Figs. 2I and N). Increased expression of ORP150 and GRP78 was confirmed by Western blot analysis with isolated glomeruli from megsin transgenic rats followed by densitometric analysis (Fig. 3). We observed weak expression of both ORP150 and GRP78 in wild-type rats, although those expressions were undetectable in immunohistochemistry (Figs. 2E and J). This discrepancy may be due to the difference of sensitivity of each method. GRP94 expression was also up-regulated in the isolated glomeruli from megsin transgenic rats (Fig. 3).
Inagi et al: ER stress in podocyte injury of megsin Tg rat
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ER stress induced in podocytes with excessive megsin accumulation We previously demonstrated that accumulation of the transgene product, megsin, was localized exclusively in
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Polymerization of megsin and subsequent ER retention play a pathogenic role in development of ER stress in podocytes. Podocytes of heterozygote transgenic rats overexpressing mutant megsin (megsinT334R, a mutation at the position of P14 corresponding to the proximal hinge region of the reactive site loop), which lacks serine protease inhibitory activity as well as the capacity for polymerization within the ER, did not have megsin droplets within the cytoplasm (Fig. 2D) [27]. In addition, they did not show increased expression of ER stress markers, ORP150, GRP94, or GRP78 by immunohistochemistry (Figs. 2H and M) and Western blot analysis for isolated glomeruli (Fig. 3).
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Fig. 2. Induction of ER stress inducible proteins in the kidneys of megsin transgenic rats. PAS staining of a wild-type (A), a megsin transgenic (homozygote, B, and heterozygote, C), and a mutated-megsin (megsinT334R) transgenic rat (D) at the age of 8 weeks. PASpositive deposits (indicated by arrows) were observed within the cytoplasm of glomerular epithelial cells in a megsin transgenic rat but not in a wild-type rat or a megsinT334R transgenic rat. Immunohistochemical detection of ORP150 (E–I) and GRP78 (J–N) in the kidney of a wild-type rat (E and J), a megsin homozygote (F and K), a megsin heterozygote (G and L), and a megsinT334R transgenic rat (H and M). Expression of ORP150 and GRP78 is markedly augmented in glomeruli of megsin transgenic rats, as compared with those in wild-type and megsinT334R transgenic rats. No immunoreactivity was observed with normal rabbit or goat IgG as the first antibody in a megsin homozygote rat (I and N). The bar indicates 10 lm. (O) Computerassisted morphometry of the section stained immunohistochemically with anti-ORP150 or GRP78. Each section of three rats derived from each group was examined, and averages were expressed as mean ± SD. Positive areas for ORP150 or GRP78 expression were increased with statistical significance in homozygotes as compared with those in heterozygotes. ∗ P < 0.001 vs. wild-type or T334Rmegsin transgenic rats. #P < 0.001.
podocytes in glomeruli by immunoelectron microscopic analysis [27]. Of note, OPR150-expressed podocytes of megsin transgenic rats showed accumulation of megsin transgene product (Fig. 4). Podocyte damage by ER stress in megsin transgenic rats We further performed immunohistochemical analysis utilizing desmin as a marker of podocyte injury [31] in order to clarify whether ER stress in podocytes of megsin transgenic rats was associated with cellular injury. Glomeruli of megsin transgenic rats showed expression of desmin in podocytes and the signal intensity was higher in homozygotes than in heterozygotes (Fig. 5A, panels b and c). Whereas staining for desmin was absent in glomeruli of wild-type (Fig. 5A, panel a). No immunoreactivity was observed with normal mouse IgG used as the first antibody (Fig. 5A, panel e). Confocal microscopic analysis of double staining of desmin and ORP150 localized ORP150
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(red) in podocytes with the cytoplasm positive for desmin (green). It suggests that podocyte injury was associated with ER stress (Fig. 5B). We further analyzed podocyte damage by double staining of synaptododin, a marker for podocytes, and GRP78 in megsin homozygote transgenic rats. The signal of synaptopodin expression adjacent to GRP78 positive signal was relatively faint as compared with that in wild-type rats, which was negative for GRP78 staining (Fig. 5C). By contrast, in megsinT334R transgenic rats, the desmin staining was extremely weak (Fig. 5A, panel d). These results, together with the lack of PAS-positive materials composed of megsin and ORP150 in the glomeruli of megsinT334R transgenic rats, clearly suggested that podocyte injury was not induced in this model. DISCUSSION ER stress in glomerular injury was recently emphasized by Cybulsky et al [33]. They reported that comple-
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Fig. 3. Western blot analysis of ER stress inducible proteins. Proteins from isolated glomeruli derived from wild-type, megsin homozygote transgenic, and mutated megsin (T334Rmegsin) transgenic rats (8-week-old) were analyzed by Western blot analysis using antibodies to ER stress inducible proteins such as ORP150, GRP78, and GRP-94. Polyclonal antibodies to b-actin were also used as an internal control. (A) Representative data from three independent experiments are shown. (B) Quantification of the intensity of the bands was performed by densitometry and normalized to the intensity of actin bands. The mean ratio of ORP150, GRP78, or GRP94 expression in megsin or T334Rmegsin transgenic rats over that in wild-type rats was calculated for each experiment, and the average of the three experiments is expressed. Increased protein expression of ORP 150, GRP78, and GRP94 was observed in isolated glomeruli from megsin transgenic rats, as compared with those from age-matched wild-type or T334Rmegsin transgenic rats. ∗ P < 0.05 vs. wild-type rats.
Fig. 4. ER stress is induced in podocytes with excessive megsin accumulation. Localization of ER stress inducible proteins in glomeruli from megsin homozygote transgenic rats (8week-old) was assessed by double staining using antibodies to ORP150 and megsin. Frozen kidney sections (5 lm) were incubated with rabbit polyclonal IgG to human ORP150 or human megsin followed by reaction with FITC- or TRITC-conjugated antirabbit IgG. OPR150-expressing podocytes of megsin transgenic rats showed accumulation of megsin transgene products. Representative positively stained cells were indicated by white arrowheads. The bar indicates 10 lm.
ment attack against podocytes induced ER injury, which was associated with up-regulation of ER stress proteins such as GPR78 (bip) and GRP94 both in vitro and in vivo. Our results confirm and extend their findings beyond complement-induced podocyte injury. Tunicamycin, which causes retention of unfolded proteins in the ER, induced ER stress in podocytes. In contrast, neither hypoxia nor high glucose resulted in ER stress in these cells. Factors inducing ER stress may be cell type–specific because hypoxia can lead to up-regulation of ER stress proteins in astrocytes [34], vascular smooth muscle cells [35], monocytes [35], ovary cells [36], and renal tubular cells [37]. Glomerular cells are relatively spared in ischemic kidney diseases compared with tubular cells [38], and hypoxia may not be as noxious to podocytes as it is to some other types of cells. In agreement with our in vitro findings, immunohistochemical analysis conducted in a rat ischemia-reperfusion of model showed that ORP150 staining is more prominent in tubular cells than in glomeruli [39].
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Fig. 5. Podocyte damage by ER stress in megsin transgenic rats. (A) Immunohistochemical detection of desmin, a marker of podocyte injury, in the kidney of a wild-type (panel a), a megsin transgenic rat (homozygote, panel b, and heterozygote, panel c), and a megsinT334R transgenic rat (panel d) at the age of 8 weeks. The sections (2 lm) were first incubated with anti-desmin monoclonal antibody, followed by reaction with biotinylated anti-mouse IgG. Expression of desmin was detected in glomeruli of a megsin transgenic rat but not in wild-type or megsinT334R transgenic rats. No immunoreactivity was observed with normal mouse IgG as the first antibody in a megsin homozygote rat (panel e). The bar indicates 10 lm.
Inagi et al: ER stress in podocyte injury of megsin Tg rat
It was not surprising that high glucose similar to what is seen in diabetes mellitus did not induce expression of ER stress proteins because ER stress proteins are known to be up-regulated by energy depletion under low glucose conditions [40, 41]. Here we demonstrated a crucial role of accumulation of excessive proteins in the ER in induction of ER stress utilizing our recently established megsin transgenic rats [27]. Megsin is a novel member of the serpin superfamily, and serpins are known to form aberrant intermolecular linkage in hepatocytes and neurons [42, 43]. To our knowledge, this is the first report to show that ER stress is associated with excessive protein accumulation in renal cells. It should be noted that the transgene of our megsin transgenic rats coded an intact molecule, in contrast to some human genetic disorders in which mutations in the coding region of highly expressed ER client proteins directly perturb their folding [2, 44]. However, previous studies also showed that unfolding or misfolding of proteins can occur under several circumstances, including macromolecular crowding, oxidative stress, exposure to toxins, and aging, suggesting that ER stress can play an important role in a wide range of diseases [8, 45]. Western blotting analysis in this study demonstrated up-regulation of a variety of ER-stress proteins in glomeruli isolated from megsin transgenic rats. We previously described predominant expression of megsin in mesangial cells of normal animals [26, 46], and cannot exclude a possible involvement of mesangial cells, endothelial cells, or infiltrating cells in induction of the ER stress proteins. However, immunohistochemical analysis as well as immunoelectron microscopic analysis [27] revealed that megsin transgenic rats unexpectedly showed accumulation of megsin in ER of podocytes. Expression patterns of the transgene revealed by in situ hybridization were ubiquitous in resident glomerular cells of the transgenic rats [27]. Accumulation of megsin also varied in different organs in spite of similar expression at the mRNA level [27]. These observations led us to speculate that individual cellular susceptibility must influence accumulation of the transgene product in ER, demonstrating predominant accumulation of megsin in podocytes of the transgenic rats. However, this requires further investigation.
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The ER stress in podocytes of megsin transgenic rats was associated with up-regulation of ER stress proteins such as ORP150 and GRP78. ORP150 was first purified and cloned as a novel stress protein induced by oxygen deprivation in cultured rat astrocytes [34]. Glucoseregulated proteins, GRP78 and GRP94, are also major constituents of the ER of mammalian cells and serve as a protective mechanism to adapt to stress on the ER [41, 47, 48]. While induction of ER stress proteins may be an important mechanism of protection of podocytes, further studies are warranted to conclude whether the ER stress response is protective or is responsible for some pathologic findings. CONCLUSION We reported induction of ER stress by various stimuli in cultured glomerular cells. Furthermore, our transgenic rats with accumulation of megsin in the ER of podocytes demonstrated ER stress-induced podocyte injury. Our findings implicated a crucial role for the accumulation of excessive proteins in the podocyte ER in the induction of ER stress and associated podocyte injury. ACKNOWLEDGMENTS We thank Dr. Johbu Itoh (Laboratories for Structure and Functional Research, Tokai University School of Medicine, Japan) for his technical support with the confocal microscopic analyses. This study was supported by grants from the New Energy and Industrial Technology Development Organization to T.M., from the Ministry of Education, Culture Sports, Science and Technology of Japan (15590861) to R.I., and from the Research and Study Program of Tokai University Educational System General Research Organization to R.I. and T.M. Reprint requests to Toshio Miyata, M.D., Ph.D., Molecular and Cellular Nephrology, Institute of Medical Sciences, Tokai University, Kanagawa 259-1193, Japan. E-mail: [email protected]
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(B) Localization of ORP150 within injured podocytes. Three-dimensional colored intensity orthogonal section imaging of glomeruli were carried out after immunofluorescence studies using antibodies to ORP150 (red) and desmin (green), a marker of podocyte injury. Nuclei were poststained with Hoechst Dye 33342. X-Y cross-section plane image (blue line square), X-Z cross-section plane image (green line square), Y-Z cross-section plane image (red line square). ORP150 (red) was localized in podocytes with the cytoplasm positive for desmin (green), suggesting that podocyte injury was associated with ER stress. The bar indicates 5lm. (C) Double immunostaining of an ER stress inducible protein, GRP78, and synaptopodin, a marker of podocytes, in glomeruli from megsin homozygote transgenic rats (8-week-old). Frozen kidney sections (4 lm) from age-matched wild-type (upper panel) or megsin transgenic rats (lower panels) were incubated with mouse monoclonal antibodies to synaptopodin and goat polyclonal IgG to GRP78, which were detected by TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-goat IgG, respectively. The signal of synaptopodin expression adjacent to GRP78 positive signal was relatively faint in megsin transgenic rats compared with that in wild-type rats, which was negative for GRP78 expression, suggesting podocyte damage by ER stress. The bar indicates 10 lm.
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