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Endocrinology, doi:10.1210/en.2007-1372
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Endocrinology Vol. 149, No. 6 2934-2942
Copyright © 2008 by The Endocrine Society

Superoxide Destabilization of β-Catenin Augments Apoptosis of High-Glucose-Stressed Mesangial Cells

Chun-Liang Lin, Jeng-Yi Wang, Jih-Yang Ko, Kameswaran Surendran, Yu-Ting Huang, Yu-Hsia Kuo and Feng-Sheng Wang

Departments of Nephrology (C.-L.L.) and Colorectal Surgery (J.-Y.W.), Chiayi Chang Gung Memorial Hospital and Graduate Institute of Clinical Medical Sciences (C.-L.L., Y.-Y.K., F.-S.W.), Chang Gung University, College of Medicine, Taoyuan 333, Taiwan; Chia-Yi School (C.L.-L.), Chang Gung Institute of Technology; Departments of Medical Research (J.-Y.W.) and Orthopedic Surgery (Y.-T.H., Y.-H.K.), Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung 833, Taiwan; and Department of Molecular Biology and Pharmacology (K.S.), Washington University School of Medicine, St. Louis, Missouri 63310

Address all correspondence and requests for reprints to: Feng-Sheng Wang, Ph.D., Department of Medical Research, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung 833, Taiwan. E-mail: linchunliang{at}adm.cgmh.org.tw or wangfs{at}ms33.hinet.net.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intense mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Although reactive oxygen radicals and Wnt signaling components are potent regulators that modulate renal tissue remodeling and morphogenesis, cross-talk between oxidative stress and Wnt/β-catenin signaling in controlling high-glucose-impaired mesangial cell survival and renal function have not been tested. In this study, high glucose induced Ras and Rac1 activation, superoxide burst, and Wnt5a/β-catenin destabilization and subsequently promoted caspase-3 and poly (ADP-ribose) polymerase cleavage and apoptosis in mesangial cell cultures. The pharmacological and genetic suppression of superoxide synthesis by superoxide dismutase and diphenyloniodium, dominant-negative Ras (S17N), and dominant-negative Rac1 (T17N) abrogated high-glucose-induced glycogen synthase kinase (GSK-3β) activation and caspase-3 and poly (ADP-ribose) polymerase degradation. Inactivation of Ras and Racl also reversed Wnt/β-catenin expression and survival of mesangial cells. Stabilization of β-catenin by the transfection of stable β-catenin ({Delta}45) and kinase-inactive GSK-3β attenuated high-glucose-mediated mesangial cell apoptosis. Exogenous superoxide dismutase administration attenuated urinary protein secretion in diabetic rats and abrogated diabetes-mediated reactive oxygen radical synthesis in renal glomeruli. Immunohistological observation revealed that superoxide dismutase treatment abrogated diabetes-induced caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) and increased Wnt5a/β-catenin expression in renal glomeruli. Taken together, high glucose induced oxidative stress and apoptosis in mesangial cells. The Ras and Rac1 regulation of superoxide appeared to raise apoptotic activity by activating GSK-3β and inhibiting Wnt5a/β-catenin signaling. Controlling oxidative stress and Wnt/β-catenin signaling has potential for protecting renal tissue against the deleterious effect of high glucose.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RENAL MESANGIAL CELLS in the presence of high-glucose stress undergo cascades of deleterious reactions including cell injury, necrosis, and extracellular matrix deposition, leading to glomeruli dysfunction (1, 2). Promoted mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy (3, 4), indicating that mesangial cells respond to extracellular high-glucose stress by altering intracellular biochemical reactions and subsequently disturbing mesangial cell survival.

Oxidative stress mediates the deleterious effects of diabetes on renal tissue function (5). The high-glucose induction of reactive oxygen radicals interrupts microvascularity, impairs the sodium ion transportation of tubular cells, and promotes the collagen IV accumulation of mesangial cells (6, 7, 8). The suppression of oxidative stress by modulating oxidase activity and antioxidant capacity alleviates high-glucose-mediated mesangial fibrosis (9, 10) and renal cell apoptosis (11, 12).

The G protein family Ras and Rac-1 are responsible for regulating the proliferation, apoptosis, and differentiation of several cell types that can adapt to deleterious stress (13, 14). These membrane-bound molecules modulate the high-glucose- and glycooxidative-product-induced oxidative damage of mesangial cells and diabetic nephropathy (15, 16). Inactivation of Rac1 prevents high-glucose-mediated oxidative stress in vascular endothelial cells (17). We earlier demonstrated that the Ras induction of oxidative stress mediated fibrosis matrix accumulation in the early stage of high-glucose-induced renal injuries (15).

Canonical Wnt signaling through GSK-3β inactivation and subsequent β-catenin stabilization raises Wnt protein-responsive biological activities (18, 19, 20). Wnt signaling is essential to the mesenchymal-epithelial transition of the medullar region in nephrogenesis (21). The modulation of β-catenin signaling increases nephron formation (20) and regulates the development of kidney vesicles (22). Wnt/β-catenin was recently found to protect renal tissue against the deleterious actions of high glucose (23). Although Wnt/β-catenin signaling was recently found to modulate reactive oxygen radical-mediated senescence, apoptosis, and proliferation in several cell cultures (24, 25, 26), the function of Wnt/β-catenin signaling in regulating the survival of mesangial cells that are exposed to high-glucose-induced oxidative stress has not been determined.

This study investigates whether the high-glucose induction of reactive oxygen radicals is linked to the alteration of glycogen synthase kinase-3β (GSK-3β) or β-catenin signaling and to examine whether modulating the activation of Ras or Rac1 can alter the high-glucose-induced loss of β-catenin signaling and the apoptosis of mesangial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Rat mesangial cells and human embryonic kidney 293T cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM with 10% fetal bovine serum (Life Technologies, Gaithersburg, MD). Mouse SV40 MES-13 glomerular mesangial cells (American Type Culture Collection) were maintained in a mixture of DMEM and Ham’s F12 medium (3:1; vol/vol), 5% fetal bovine serum, and 14 mM HEPES. Cells were cultured in a 5% CO2, 37 C incubator until subconfluent and then harvested by trypsinization for subsequent study. Cell viability was determined using trypan blue exclusion.

High-glucose treatment
Cells (1 x 106 cells per well, six-well plate) were cultured in basal medium (5 mM D-glucose) with and without 35 mM D-glucose for 2, 4, 6, 12, 24, and 48 h. Cell cultures exposed to 35 mM mannitol were used as osmolar controls. To investigate the role of superoxide in the promotion of cell apoptosis by high glucose, subconfluent cell cultures were pretreated with and without 500 U/ml bovine erythrocyte superoxide dismutase-polyethylene glycol (SOD-PEG), 20 µM diphenyloniodium (DPI; Sigma-Aldrich, St. Louis, MO) and 10 µM PD98059 (Calbiochem, La Jolla, CA). In some experiments, trypsinized and floating cells were pooled, spun (1 x 104 cells) onto glass slides, and fixed in ice-cold 70% methanol to detect apoptotic cells.

Superoxide production
The production of superoxide by mesangial cell cultures (1 x 105 cells per well, in 96-well plate) with and without high-glucose treatment was determined using horse heart cytochrome c reduction assay in the absence and presence of SOD and calculated using the molar extinction coefficient of 0.0282 µM–1·cm–1 as previously described (15).

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL)
Apoptotic cells in cell cultures and kidney tissue were detected using in situ cell death detection kits (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Specimens incubated in reaction buffer without terminal deoxynucleotidyl transferase were used as negative controls. TUNEL-stained cell cultures were recognized using fast red as a substrate. Cyrosections (8 µm) from OCT-embedded snap-frozen tissues were fixed in ice-cold methanol and then the apoptotic cells were immunofluorescence stained and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Dako, Carpinteria, CA). Six random images from each section, obtained from 12 sections from six repeated experiments or renal tissue, were randomly selected and observed under x400 magnification using an Axiovert 200 inverted microscope with an A-plan x10/0.25 ph1 (matching condenser position, phase 1) objective lens. Images were taken using an Axiocam HRM cool charge-coupled device camera and AxioVision 4 image-analysis software (Carl Zeiss, Gottingen, Germany). The TUNEL-positive-stained mesangial cells and total cells per high-power field in each section were counted, and the percentage of positively labeled cells was calculated.

cDNA transfection
The cDNAs that encoded wild-type Ras, dominant-negative Ras (S17N), wild-type Rac1 and dominant-negative Rac1 (T17N; Upstate Biotechnology, Lake Placid, NY), kinase-inactive GSK-3β (27), and stable β-catenin ({Delta}45) (28) were ligated and cloned into pUSE (Upstate Biotechnology), pcDNA3.1 (Invitrogen, Carlsbad, CA) and pNeo vectors (Invitrogen), respectively. Stable transfection and selection were performed as previously described (23).

Real-time PCR
Total RNA was extracted and purified from 106 cells using QIAzol reagent (QIAGEN Inc., Valencia, CA). Total RNA (1 µg) was reverse transcribed into cDNA, and 25 µl PCR mixture containing cDNA template equivalent to 20 ng total RNA, 2.5 µM each forward and reverse primer, and 2x iQ SYBR Green Supermix was amplified using the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) with an initial melt at 95 C for 5 min followed by 40 cycles at 94 C for 15 sec, 52 C for 20 sec, and 72 C for 30 sec using the following primer oligonucleotide sequences: rat Wnt5a forward, 5'-AGC CGA GAG ACA GCC TTC AC-3', and reverse, 5'-TCC TGC GAC CTG CTTCATTG-3' (289-bp expected); mouse Wnt5a forward, 5'-AGG AGT TCG TGG ACG CTA GA-3', and reverse, 5'-GCC GCG CTA TCA TAC TTC TC-3' (241-bp expected); human Wnt5a forward, 5'-TGG CTT TGG CCA TAT TTT TC-3', and reverse, 5'-CCG ATG TAC TGC ATT TGG AT-3' (199-bp expected); human SOD1 forward, 5'-GGG CAA AGG TGG AAA TGA AG-3', and reverse, 5'-TTA GCA GGA CAG CAG ATG AG-3' (133-bp expected); and β-actin forward, 5'-CGC CAA CCG CGA GAA GAT-3', and reverse, 5'-CGT CAC CGG AGT CCA TCA-3' (168-bp expected). The number of amplification steps required to reach an arbitrary intensity threshold (Ct) was computed. The relative gene expression levels were presented as 2–{Delta}Ct, where {Delta}Ct = CtWnt5a – Ctβ-actin. The fold change in the treatment was defined as the relative expression, which was compared with the vehicle and was calculated as 2–{Delta}{Delta}Ct, where {Delta}{Delta}Ct = {Delta}Cttreatment – {Delta}Ctvehicle (23).

Western blotting
Membrane, cytosolic, and nuclear extracts of cell cultures were prepared as previously described (29), and aliquots of cell lysates (75 µg) were subjected to Western blot. The designated proteins on the blots were probed by antibodies against ERK, phosphorylated ERK, GSK-3β, phospho-Ser9-GSK-3β, β-catenin, caspase-3, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology Inc., Beverly, MA), followed by horseradish peroxidase-conjugated IgG as the secondary antibodies that were visualized with chemiluminescence agents. Ras and Rac1 activation was assessed using Ras and Rac1 activation assay kits (Upstate Biotechnology, Charlottesville, VA) according to the manufacturer’s instructions. The protein band intensity on each blot from three repeated experiments was quantified by scan densitometry. The relative fold change was expressed as treatment/vehicle x 100%.

Exogenous SOD-PEG administration for diabetic rats
All studies were approved by the Institutional Animal Care and Use Committee of the hospital. Diabetes was induced in 4-month-old male Wistar rats by a single ip injection of 50 mg/kg streptozotocin (Sigma-Aldrich) as previously described (23). Twelve diabetic rats randomly divided into two groups received an ip injection with 50 U/kg·d SOD-PEG (n = 6) and normal saline (n = 6) for 28 consecutive days. Six rats without streptozotocin injection were used as normal controls. At d 28, urine was collected using metabolic cage systems and urinary protein and creatinine levels were measured using respective assay kits (Sigma-Aldrich). Rats were killed with an overdose of pentobarbital sodium, and their kidneys were dissected. After perfusion with PBS, fresh kidney tissues were subjected to OCT embedding in liquid nitrogen or fixation in 4% PBS-buffered formaldehyde and paraffin embedding.

In situ detection of reactive oxygen radicals
Cyrostat sections (8 µm) from OCT-embedded snap-frozen tissues were fixed with ice-cold methanol. The formation of reactive oxygen radicals in renal tissue was detected as previously described (30). Briefly, sections were incubated in 100 mM Tris-maleate buffer (pH 8.0) that contained 10% polyvinyl alcohol (wt/vol) and 5 mM sodium azide to inhibit endogenous myeloperoxidase activity. Then 12.5 mM 3,3'-diaminobenzidine (DAB; Sigma-Aldrich) was added, and the sections were incubated at 37 C for 2 h. Sections were washed and mounted in glycerol for microscopic observation. Sections without DAB were used as negative controls. The DAB-accumulated area in six random images from 12 sections obtained from four rats were taken, captured, and analyzed under x400 magnification using a using a Zeiss Axioskop 2 plus microscope (Carl Zeiss) with a cool charge-coupled device camera and Image-Pro Plus image analysis software (SNAP-Pro Digital Kit; Media Cybernetics, Silver Spring, MD). The percentage of the area within renal mesangium in which DAB accumulated was calculated.

Immunohistochemistry
Active Raf-1, cleaved caspase-3, Wnt5a, phospho-Ser9-GSK-3β, and β-catenin immunoreactivities in 5-µm-thick paraffin-embedded sections were demonstrated using specific primary antibodies and a nonbiotin horseradish peroxidase detection system (BioGenex, San Ramon, CA), followed by counterstaining with hematoxylin, dehydration, and mounting. Sections without primary antibodies and rat IgG were used as negative controls and isotype control for immunostaining. Six random images from 12 sections obtained from four rats were captured and analyzed under x400 magnification. The percentage of the positively immunolabeled cells and of the total cells in each area within the glomeruli was calculated. All cells including renal mesangial cells were identified morphologically.

Statistical analysis
All values are expressed as mean + SE calculated from at least three repeated experiments. A Wilcoxon test was conducted to evaluate the differences between each sample of interest and its respective control. To analyze the time course, multiple-range ANOVA and post hoc tests were conducted. A P value of <0.05 was treated as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Superoxide-mediated high-glucose promotion of mesangial cell apoptosis
We examined whether superoxide was involved in high-glucose-induced mesangial cell apoptosis. Rat mesangial cells were pretreated with and without SOD and DPI and then cultured under high-glucose conditions. High glucose significantly increased superoxide synthesis by 2 h (Fig. 1AGo), promoted the cleavage of caspase-3 and PARP (Fig. 1BGo), and increased the number of apoptotic cells (Fig. 1CGo). Pretreatments with SOD and DPI significantly abrogated high-glucose-induced superoxide burst, the cleavage of caspase-3, and PARP and cell apoptosis (Fig. 1Go). TUNEL staining indicated that high glucose induced cell apoptosis, which was abrogated by pretreatments with SOD and DPI (Fig. 1DGo). Osmolar control 35 mM mannitol did not significantly alter superoxide synthesis and the apoptotic activity of cell cultures throughout the study period (Fig. 1Go).


Figure 1
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  		FIG. 1. A–C, SOD and DPI pretreatments reduced high-glucose-induced superoxide burst (A), the cleavage of nuclear caspase-3 and PARP (B), and apoptosis of mesangial cells (C). D, Representative photographs of apoptotic mesangial cells with or without high-glucose stress or pretreated with SOD or DPI. Cells positive for TUNEL exhibited red staining in nucleus. Cell cultures (1 x 106 cells per well in six-well plates) were pretreated with 500 U/ml bovine erythrocyte SOD-PEG or 20 µM DPI and subjected to 35 mM D-glucose or osmolar control (35 mM mannitol) for 2 d. Superoxide production was measured by SOD-inhibitable cytochrome c reduction assays. Immunoblotting of total cytosolic caspase-3 showed equal loading and transfer for all lanes. Apoptotic cells were detected by TUNEL staining. * and #, Differences (P < 0.05) from the vehicle- and high-glucose-treated groups, respectively. HG, High glucose.

 
High-glucose induction of superoxide-attenuated Wnt/β-catenin signaling
We further investigated whether high-glucose induction of superoxide could alter Wnt5a/β-catenin expression. Compared with the control group, high glucose significantly reduced Wnt5a mRNA (Fig. 2AGo) and nuclear β-catenin expression and increased GSK-3β activation as demonstrated by the impairment of phospho-Ser9-GSK-3β expression (Fig. 2BGo). Pretreatments with SOD and DPI significantly reversed Wnt5a mRNA expression (Fig. 2AGo) and phospho-Ser9-GSK-3β and nuclear β-catenin expression (Fig. 2BGo). High glucose or pretreatments with either SOD or DPI did not significantly alter the expression of GSK-3β of cell cultures throughout the study period (Fig. 2BGo).


Figure 2
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  		FIG. 2. SOD and DPI pretreatments reversed the high-glucose (HG)-mediated loss of Wnt5a mRNA expression (A) and the expression of cytosolic phospho-Ser9-GSK-3 and nuclear β-catenin (B). Cell cultures (1 x 106 cell per well, in six-well plates) were pretreated with 500 U/ml SOD-PEG or 20 µM DPI and subjected to 35 mM D-glucose treatment for 2 d. The Wnt5a mRNA expression in the representative electrophoretogram was assessed by reversed transcribing 1 µg total RNA into cDNA templates followed by 30 cycles of PCR amplification. The plotted experimental results represent the relative abundance of the Wnt5a gene normalized to housekeeping gene β-actin by quantitative RT-PCR within logarithmic amplification. Immunoblotting of total cytosolic GSK-3β and actin showed equal loading and transfer for all lanes. * and #, Differences (P < 0.05) from the vehicle- and high-glucose-treated groups, respectively.

 
Ras and Rac1 induction of superoxide-controlled Wnt signaling and cell apoptosis
High glucose increased Ras and Racl activation by 2 h (Fig. 3AGo). SOD pretreatment did not significantly alter high-glucose-mediated Ras or Rac-1 activation throughout the study period. Whether Ras or Rac1 mediated high-glucose-inhibited Wnt5a/β-catenin expression of mesangial cells was investigated. Mouse SV40 MES-13 glomerular mesangial cells were transfected with dominant-negative Ras (S17N) and Rac1 (T17N) cDNAs to abrogate high-glucose-induced Ras and Rac1 activation, ERK activation (Fig. 3BGo), and superoxide synthesis (Fig. 3CGo). Inactivating Ras and Rac1 significantly reversed Wnt5a mRNA expression (Fig. 4AGo) and phospho-Ser9-GSK-3β and nuclear β-catenin expression (Fig. 4AGo) and abrogated the high-glucose-induced caspase-3 and PARP cleavage (Fig. 4BGo) and reduced the number of apoptotic cells (Fig 4CGo). Inactivating ERK by PD98059, a Ras downstream effector that is known to mediate high-glucose-induced nephropathy (15), abrogated high-glucose-inhibited Wnt5a mRNA expression and increased phosphor-Ser9-GSK-3β and nuclear β-catenin accumulation (Fig. 4Go).


Figure 3
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  		FIG. 3. A, High glucose (HG) increased Ras and Racl activation. B and C, Transfection of dominant-negative (DN) Ras and dominant-negative Rac1 reduced Ras and Rac1 activation and ERK phosphorylation (B) and attenuated high glucose-induced production of superoxide by mesangial cells (C). Immunoblotting of total membrane Ras and Rac1 showed equal loading and transfer for all lanes. * and #, Differences (P < 0.05) from the vehicle- and high-glucose-treated groups, respectively. WT, Wild-type.

 

Figure 4
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  		FIG. 4. Transfection of dominant-negative (DN) Ras and Rac1 mutants increased Wnt5a mRNA expression (A) and cytosolic phospho-Ser9-GSK-3β and nuclear β-catenin expression (B) and reduced high-glucose (HG)-induced nuclear cleaved caspase-3 and PARP expression (C) and apoptosis (D) of mesangial cells. Inactivating ERK by PD98059 abrogated high-glucose-inhibited Wnt5a mRNA expression and increased phosphor-Ser9-GSK-3β and nuclear β-catenin accumulation. Wnt5a mRNA expression in the representative electrophoretogram was assessed by reverse transcribing 1 µg total RNA into cDNA templates followed by 30 cycles of PCR amplification. The plotted experimental results revealed the relative abundance of the Wnt5a gene normalized to housekeeping gene β-actin by quantitative RT-PCR within logarithmic amplification. Immunoblotting of cytosolic actin and caspase-3 showed equal loading and transfer for all lanes.* and #, Differences (P < 0.05) from the vehicle and high glucose-treated groups, respectively. WT, Wild-type.

 
In addition to mouse and rat mesangial cells, high glucose significantly increased Ras activation and suppressed Wnt5a mRNA expression and phospho-Ser9-GSK-3β and nuclear β-catenin expression in HEK 293 cells. The inactivation of Ras by transfecting dominant-negative Ras (S17N) cDNA reversed Wnt5a mRNA expression and phospho-Ser9-GSK-3β and nuclear β-catenin accumulation in HEK 293 cells (supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

β-Catenin alleviated high-glucose-induced cell apoptosis
We investigated whether GSK-3β or β-catenin modulated high-glucose-induced cell apoptosis. Mouse SV40 MES-13 glomerular mesangial cells were transfected with kinase-inactive GSK-3β and stable β-catenin ({Delta}45) cDNAs to inactivate GSK-3β and stabilize nuclear β-catenin expression. Transfection of kinase-inactive GSK-3β mutant increased phospho-Ser9-GSK-3β and nuclear β-catenin expression (Fig. 5AGo), alleviated high-glucose-promoted caspase-3 and PARP cleavage (Fig. 5BGo), and reduced the number of apoptotic cells (Fig. 5CGo). Transfection of stable β-catenin ({Delta}45) mutant increased nuclear β-catenin expression (Fig. 6AGo) and abrogated high-glucose-induced caspase-3 and PARP cleavage (Fig. 6BGo) and the number of apoptotic mesangial cells (Fig. 6CGo). Moreover, stabilization of β-catenin significantly alleviated the suppressing effect of high glucose on SOD1 mRNA expression in cell cultures (Fig. 6DGo).


Figure 5
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  		FIG. 5. A and B, Transfection of kinase-inactive (KI) GSK-3β increased cytosolic phospho-Ser9-GSK-3β and nuclear β-catenin expression (A) and reduced high-glucose (HG)-induced nuclear caspase-3 and PARP cleavage (B). C, Inactivating GSK-3β alleviated high-glucose-induced apoptosis of mesangial cells. Wild-type (WT) or kinase-inactive GSK-3β mutant-transfected mesangial cells were cultured under high-glucose conditions for 2 d. Immunoblotting of cytosolic actin and caspase-3 showed equal loading and transfer for all lanes. * and #, Differences (P < 0.05) from the vehicle- and high-glucose-treated groups, respectively.

 

Figure 6
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  		FIG. 6. A and B, Transfection of stable β-catenin ({Delta}45) mutant increased nuclear β-catenin expression (A) and reduced cleavage of high-glucose (HG)-induced nuclear caspase-3 and PARP (B). C, Stabilization of β-catenin alleviated high-glucose-induced apoptosis of mesangial cells. D, High glucose reduced SOD1 mRNA expression of mesangial cells. Transfection of stable β-catenin ({Delta}45) mutant restored high-glucose-depressed SOD1 mRNA expression. Wild-type or stable β-catenin mutant-transfected mesangial cells were cultured under high-glucose conditions for 2 d. The electrophoretogram of nuclear extracts indicated equal loading for all lanes. Immunoblotting of cytosolic actin and caspase-3 showed equal loading and transfer for all lanes. * and #, Differences (P < 0.05) from the vehicle- and high-glucose-treated groups, respectively.

 
Oxidative stress-modulated Wnt/β-catenin signaling in diabetic rat kidneys
We verified whether in vivo modulation of superoxide regulated diabetes-induced mesangial cell apoptosis. Compared with the normal group, diabetes significantly increased blood glucose, glycated hemoglobin (HbA1c), and urinary protein excretion (Table 1Go). Exogenous SOD treatment significantly reduced diabetes-induced urinary protein secretion. SOD treatment did not significantly affect blood glucose and HbA1c throughout the study period.


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  		TABLE 1. Biochemical characteristics and immunohistology in renal glomerular mesangial cells of diabetic rats with or without SOD treatment

 
DAB in the presence of reactive oxygen radicals (reactive oxygen species) forms insoluble and brown oxidized DAB precipitate. It is employed to detect in situ reactive oxygen radicals in tissue (30). In the diabetes group, cells in glomeruli displayed intense reactive oxygen radical synthesis, active Raf-1 and cleaved caspase-3 expression (Fig. 7Go), and TUNEL staining (Fig. 8Go). Few cells in the glomeruli of diabetic rats with SOD treatment showed slight reactive oxygen radical formation, cleaved caspase-3 expression (Fig. 7Go), and TUNEL staining (Fig. 8Go).


Figure 7
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  		FIG. 7. Representative photographs of production of reactive oxygen species (ROS), active Raf-1, and cleaved caspase-3 of glomeruli in diabetic kidneys with or without SOD treatment. Cells located at diabetic glomerular tissue displayed intensive reactive oxygen radical production, active Raf-1, and cleaved caspase-3 expression when compared with the normal cells. Cells in mesangium expressed weak reactive oxygen radical production and cleaved caspase-3 expression after SOD treatment. Cells within glomeruli in diabetic kidneys with oxidized DAB precipitate and immunostained cells were brown in cell periphery and cytoplasm. Specimens were observed under x400 magnification. DM, Diabetes mellitus.

 

Figure 8
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  		FIG. 8. Representative photographs of TUNEL immunofluorescence staining of glomeruli in diabetic kidneys with or without SOD treatment. Cells within diabetic glomerular tissue displayed intensive TUNEL staining when compared with the normal group. Few mesangial cells expressed weak TUNEL staining after SOD treatment. Cells within glomeruli in diabetic kidneys with TUNEL-positive stained cells exhibited green fluorescence in nuclei. Specimens were counterstained with 4',6-diamidino-2-phenylindole (blue fluorescence) and observed under magnification x400. Glomeruli (dotted line area) in renal tissue were localized using a light microscope, and then fluorescence microscopy was used to observe the staining of the cells. Dotted lines were manually plotted on the images using Image-Pro Plus image analysis software. DM, Diabetes mellitus.

 
In the diabetes group, cells in glomeruli weakly expressed Wnt5a, phospho-Ser9-GSK-3β, and β-catenin immunoreactivities. Mesangial cells displayed evident Wnt5a, phospho-Ser9-GSK-3β, and β-catenin expression after SOD treatment (Fig. 9Go). Histomorphometry results demonstrated that diabetes significantly promoted the accumulation of reactive oxygen radical, the cleavage of caspase-3, and apoptosis associated with attenuated Wnt5a and phospho-Ser9-GSK-3β as well as β-catenin expression in glomerular mesangial cells when compared with the control group (Table 1Go). Exogenous SOD treatment significantly reduced diabetes-mediated reactive oxygen radical burst and mesangial cell apoptosis along with increased Wnt5a, phospho-Ser9-GSK-3β, and β-catenin expression in the renal glomeruli of diabetic rats (Table 1Go).


Figure 9
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  		FIG. 9. Representative photographs of Wnt5a, phospho-Ser9-GSK-3β and β-catenin immunostaining of glomeruli in diabetic kidneys with or without SOD treatment. Cells in glomeruli displayed weaker Wnt5a, phospho-Ser9-GSK-3β, and β-catenin expression than the normal controls. Mesangial cells expressed evident Wnt5a, phospho-Ser9-GSK-3β, and β-catenin immunoreactivities after SOD treatment. Immunostained cells were brown in the cell periphery and cytoplasm. Specimens were observed under x400 magnification. DM, Diabetes mellitus.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
High glucose raised oxidative stress and progressively promoted apoptotic activity in mesangial cells. Although recent studies have demonstrated the deleterious actions of reactive oxygen radicals on high-glucose-stressed renal tissue remodeling (29, 31, 32, 33), little research has been done to define the functional role of Wnt/β-catenin in responding to oxidative stress caused by high glucose. The current findings are the first indication that the Ras/Rac1 induction of the superoxide burst mediated high-glucose-impaired Wnt5a/β-catenin signaling and subsequently induced mesangial cell apoptosis and renal dysfunction. We suggest that the oxidative stress disturbed Wnt/β-catenin-dependent mesangial cell survival that accelerated renal tissue deterioration, thus providing a new insight into the molecular mechanisms underlying high-glucose-induced renal injury.

Scavenging superoxide by SOD and DPI reduced high-glucose-induced mesangial cell apoptosis. Reactive oxygen radicals produced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase control the biological reactions and apoptosis of several cell types (34, 35, 36). Superoxide-mediated oxidative stress participated in mediating high-glucose-induced renal injury, based on the finding that exogenous SOD administration alleviates urinary protein secretion and renal cell apoptosis in diabetic rat kidneys. The phenomena observed in the in vitro model are in line with those detected in the in vivo model, suggesting that sustained oxidative stress is responsible for the high-glucose disturbance of homeostasis in the renal tissue.

High-glucose-induced Ras-regulated mesangial cell apoptosis. Inactivating Ras attenuated oxidative stress and improved mesangial cell survival. Ras signaling modulates high-glucose- and glycooxidative-product-induced oxidative damage of mesangial cells (37, 38). The molecular mechanism by which high glucose activates Ras signaling is unclear. Previous studies demonstrate that the membrane-bound receptor for advanced glycation end products (RAGE) mediates high-glucose- and glycooxidative-product-induced oxidative stress and tissue injury (37). We speculate that high glucose may perturb membrane-bound signaling molecules that trigger Ras signaling and induce apoptosis activity of mesangial cells.

Inactivation of Rac1 abrogated high-glucose-induced superoxide and mesangial cell apoptosis. Few studies have demonstrated the biological role of Rac-1 in diabetic nephropathy. Rac-1 is one of the membrane-bound molecules that is responsible for triggering superoxide production (39). The inactivation of Ras and Rac1 prevents mesangial cells from the deleterious effect of high glucose. Our findings support previous findings that inactivating Ras attenuates Rac-1 activity in several cell types under pathological conditions (40, 41). Dominant-negative Rac1 abrogates cell apoptosis that is induced by the overexpression of Ras (42). However, the mechanism by which Ras regulates Rac-1 is unclear. The current findings indicate that Rac-l acts, at least in part, as a high-glucose- and Ras-responsive molecule that raises production of reactive oxygen radical and progressively promotes the proapoptosis activity of mesangial cells.

Inactivating GSK-3β and stabilizing β-catenin improved high-glucose-suppressed mesangial cell survival. These findings agree with our and others’ previous studies that have shown that GSK-3β and β-catenin have a distinct role in controlling apoptotic and proliferative activity in several cell types (24, 43, 44). This study provides the first evidence that inactivating Ras and Rac1 by dominant-negative Ras and Rac1 and scavenging superoxide by SOD and DPI reversed Wnt5a/β-catenin expression and impaired GSK-3β activation in high-glucose-stressed mesangial cells, suggesting that GSK-3β and β-catenin acted as potent superoxide-responsive molecules that are able to modulate apoptotic programs of high-glucose-stressed mesangial cells. Ras/ERK cascades modulate the growth factor-mediated proliferation of cell cultures (45, 46) and activate Wnt/β-catenin signaling components in cancer cells (47, 48). In this study, inactivating Ras and ERK signaling abrogated high-glucose-impaired Wnt5a/β-catenin signaling and mesangial cell apoptosis. We speculate that the discrepant action of Ras/ERK on cell fate may depend on the type of cell and the extracellular stress. Inactivating Ras alleviated high-glucose-suppressed β-catenin signaling in HEK 293T cell cultures as another explanation of the diverse effects of Ras/ERK on cell growth and apoptosis. We suggest that Ras and Rac1 may directly or indirectly mediate the deleterious action of high glucose on hindering Wnt/β-catenin signaling cascades and survival of mesangial cells.

Interestingly, stabilization of β-catenin reversed high-glucose-suppressed SOD1 mRNA expression by mesangial cells. Reciprocal regulation between β-catenin and antioxidant capacity occurs in oxidative stress-induced cell senescence (24). SOD1 knockout mice with diabetes display accelerated diabetic nephropathy (49). We suggest that multiple pathways are responsible for controlling homeostasis between oxidative stress and antioxidative activity of mesangial cells that are exposed to high-glucose stress. β-Catenin restoration of SOD1 is proposed as another explanation of the protection afforded by administering exogenous SOD against the diabetes-induced loss of mesangial cells and renal function in diabetic animals in vivo.

Antioxidant modulation of in situ reactive oxygen radical synthesis and Wnt/β-catenin signaling in glomerular mesangial cells of the diabetic kidney in vivo has not, to our knowledge, been previously reported. This study demonstrates that glomeruli in diabetic kidneys intensively accumulated reactive oxygen radicals as evidenced by oxidized DAB accumulation. Mesangial cells in diabetic kidneys weakly expressed Wnt5a, phospho-Ser9-GSK-3β, and β-catenin. The suppression of oxidative stress by exogenous SOD increased Wnt5a/β-catenin levels and alleviated glomerular mesangial cell apoptosis and urinary protein secretion in diabetic rats, suggesting that diabetic renal mesangial cells actively respond to SOD treatment. These in vivo phenomena are consistent with those of cell culture models. To our knowledge, this work is the first to report that attenuation of oxidative stress by SOD treatment restores the survival-promoting signaling of Wnt5a/β-catenin in glomerular mesangial cells in diabetic kidneys.

Taken together, we have provided evidence that Wnt5a/β-catenin is essential to mesangial cell survival (Fig. 10AGo). High glucose activated Ras/Rac1/ERK and provoked the superoxide disturbance of β-catenin signaling and increased GSK-3β-dependent proapoptotic activity, leading to mesangial cell apoptosis (Fig. 10BGo). Modulation of redox reactions merits further study as an alternative strategy for protecting renal tissue from the deleterious effects of high-glucose stress.


Figure 10
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  		FIG. 10. The scheme of Ras- and Rac1-induced superoxide mediation of apoptotic program in high-glucose-stressed mesangial cells. A, Wnt5a and β-catenin are critical to mesangial cell survival. B, High glucose induces Ras/Rac-1/ERK-dependent superoxide and activated GSK-3β leading to destabilization of β-catenin, loss of Wnt5a expression, and apoptosis of mesangial cell.

 


   Acknowledgments
 
We thank Dr. D. C. Seldin (Molecular Medicine Program, Department of Medicine, Boston University Medical Center, Boston, MA) and Dr. B. Vogelstein (John Hopkins Medical Institute and Howard Hughes Medical Institutes) for the generous gifts of kinase-inactive GSK-3β cDNA and stable β-catenin ({Delta}45) constructs. Ted Knoy is also appreciated for his editorial assistance.


   Footnotes
 
This work was supported in part by grants (NSC 95-2314-B-182A-182-MY3 to C.-L.L.) from the National Science Council, Taiwan, and Genomic and Proteomic Facilities (CMRPG83038) and grants (CMRPG630043 to C.-L.L.) from Chang Gung Memorial Hospital, Taiwan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 13, 2008

Abbreviations: DAB, 3,3'-Diaminobenzidine; DPI, diphenyloniodium; GSK-3β, glycogen synthase kinase-3β; HbA1c, glycosylated hemoglobin; PARP, poly (ADP-ribose) polymerase; PEG, polyethylene glycol; SOD, superoxide dismutase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling.

Received October 5, 2007.

Accepted for publication March 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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