Departments of * Nephrology and Colorectal Surgery, Chiayi Chang Gung Memorial Hospital, Graduate Institute of Clinical Medical Sciences, Chang Gung University, College of Medicine, Chia-Yi School, Chang Gung Institute of Technology, and || Department of Medical Research, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan; and ¶ Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri
Address correspondence to: Dr. Feng-Sheng Wang, Department of Medical Research, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung 833, Taiwan. Phone: +886-7-731-7123, ext. 8876; Fax: +886-7-7338456; E-mail: linchunliang{at}adm.cgmh.org.tw
Received for publication December 20, 2005.
Accepted for publication July 24, 2006.
Glomerulosclerosis and diabetic nephropathy are attributableto high glucose induction of mesangial cell apoptosis. WhereasWnt signaling has been found to regulate renal morphogenesisand pathogenesis, the biologic role of Wnt/-catenin signalingin controlling high glucose–induced mesangial cell apoptosisis not well defined. Herein is reported that Wnt/-catenin signalingis required for protecting glomerular mesangial cells from highglucose–mediated cell apoptosis. High glucose downregulatedWnt4 and Wnt5a expression and the subsequent nuclear translocationof -catenin, whereas it increased glycogen synthase kinase-3(GSK-3) and caspase-3 activities and apoptosis of glomerularmesangial cells. Suppression of GSK-3 activation or increasein nuclear -catenin by transfection of Wnt4 or Wnt5a or stable-catenin (S33Y) reversed Akt activation and reduced the highglucose–mediated caspase-3 cleavage and cell apoptosis.Pharmacologic inhibition of GSK-3 by recombinant Wnt5a or bromoindirubin-3'-oximeor LiCl increased Akt phosphorylation and -catenin translocationand abrogated high glucose–mediated proapoptotic activities.Exogenous bromoindirubin-3'-oxime treatment reduced phospho-Ser9-GSK-3and -catenin expression and apoptosis of cells adjacent to glomeruliin diabetic kidneys and attenuated urinary protein secretionin diabetic rats. Taken together, mesangial cells respondedto high glucose by impairing that canonical Wnt pathway to increaseproapoptotic activities. Sustaining Wnt/-catenin signaling isbeneficial for promoting survival of mesangial cells that areexposed to high glucose stress.
Advanced stages of glomerulosclerosis are characterized by lossof cellular components (1) and accumulation of extracellularmatrix around glomerular mesangial cells (2,3). Increased mesangialcell apoptosis has been found to mediate the pathogenesis ofglomerular sclerosis and crescentic glomerulonephritis (4,5)and correlate with proteinuria in remnant kidney (6,7). Hyperglycemiahas been shown to induce renal glomerulosclerosis in patientswith diabetes (8). Increased mesangial cell apoptosis correlateswith extracellular matrix accumulation in hyperglycemia-inducedsegmentation of glomerular tuft (9). High glucose through provokingproapoptotic signaling or sensitivity to TGF-1 increases apoptosisof mesangial cells (10,11). Whereas high glucose–inducedmesangial cell apoptosis contributes to the development of glomerulosclerosisand diabetic nephropathy, the molecular mechanism by which highglucose promotes mesangial cell apoptosis is not well defined.
Canonical Wnt proteins via inactivation of glycogen synthasekinase-3 (GSK-3) and -catenin translocation into the nucleusincrease Wnt responsive gene transcription (12). Wnt signalingmolecules act as potent regulators for renal tissue morphogenesisand pathogenesis. Wnt4 regulates mesenchymal-to-epithelial transitionduring nephrogenesis (13) and controls cell-cycle progressionduring tissue regeneration after acute renal failure (14). Transplantationof fibroblasts that express Wnt4 proteins under the renal capsuleinduces lesions with tubular epithelial destruction in mice(15). Elevated -catenin expression correlates with renal dysplasiaand increased collecting duct cysts in ALK3 transgenic mice(16). Transgenic mice with overexpression of -catenin displaysevere polycystic lesions in glomeruli, proximal and distaltubules, and collecting ducts (17). Decreased -catenin is associatedwith excess TGF-1 synthesis and dysfunction of peritoneal mesothelialcells in the presence of high glucose (18). We recently foundthat TGF-1 was involved in reactive oxygen radical–mediatedfibronectin accumulation of high glucose–stressed mesangialcells and early renal injuries in diabetic rats (19).
GSK-3 signaling regulates many biologic processes, includingcell death, cell survival, and transcriptional regulation ofseveral cell types (20,21). Modulation of GSK-3 activity hasbeen reported to control glucose metabolism of renal cells (22)and hypertonic-induced apoptosis of renal medullary interstitialcells (23). Whereas previous studies have suggested that Wntsignaling molecules are important for modulating renal cellfunction, the biologic role of Wnt/-catenin signaling pathwayin high glucose–stressed glomerular mesangial cells isnot well defined. We hypothesized that Wnt signaling may beinvolved in regulating the fate of mesangial cells that areexposed to high-glucose conditions. The purposes of this studywere to investigate whether Wnt/-catenin signaling in mesangialcells is altered in the presence of high glucose and whethermodulation of Wnt/-catenin signaling controls high glucose–inducedapoptosis in mesangial cells and in a rodent model of diabeticnephropathy.
Cell Cultures
Rat mesangial cells and mouse SV40 MES-13 glomerular mesangialcells (American Type Culture Collection, Manassas, VA) werecultured, respectively, in DMEM and 10% FBS (Life Technologies,Gaithersburg, MD) or a mixture of DMEM and Ham’s F12 medium(3:1; vol/vol), 5% FBS, and 14 mM HEPES in a 5% CO2, 37°Cincubator for 6 d and harvested by trypsinization for furtherstudies. Cell viability was determined using trypan blue exclusion.
High Glucose Treatment
Cells (1 x 106 cells/well, six-well plate) were cultured inbasal medium (5 mM d-glucose) with or without 35 mM d-glucosefor 96 h. Cell cultures that were exposed to 35 mM mannitolwere used as osmolar control. In some experiments, cells wereco-cultured in high glucose with 250 ng/ml recombinant Wnt5a(R&D Systems, Minneapolis, MN) or pretreated with 10 mMLiCl or 10 µM (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO)or 10 µM caspase-3 inhibitor Z-DEVD-FMK (Calbiochem, LaJolla, CA).
Cell Growth
Cell proliferation was measured using a Cell Proliferation Kit(Boehringer Mannheim GmbH, Mannheim, Germany). Briefly, cells(2 x 104 cells/well, 96-well plate) with or without high glucosein the presence or absence of Wnt signaling modulators werecultured for 24, 48, or 96 h before addition of 10 µl/well3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromidefor an additional 4-h culture. Formazan synthesis in each wellwas resolved by 10% SDS–0.01 M HCl and colorimetricallymeasured at 550 nm. In some experiments, cells (5 x 104 cells/well,24-well plate) that were cultured with or without high glucoseor Wnt signaling modulators were trypsinized and counted usinga hemacytometer.
Real-Time PCR
Total RNA was extracted and purified from 106 cells using QIAzolreagent (Qiagen, Valencia, CA). Total RNA (1 µg) was reverse-transcribedinto cDNA. Twenty-five microliters of PCR mixture that containedcDNA template equivalent to 20 ng of total RNA, 2.5 µMeach forward and reverse primer, and 2x iQ SYBR Green Supermixwas amplified using the iCycler iQ Real-time PCR Detection System(Bio-Rad Laboratories, Hercules, CA) with an initial melt at95°C for 5 min followed by 40 cycles at 94°C for 15s, 52°C for 20 s, and 72°C for 30 s using the followingprimer oligonucleotide sequences followed by PCR amplification:Wnt1 (forward 5'-ATA GCC TCC TCC ACG AAC CT-3', reverse 5'-GGAATT GCC ACT TGC ACT CT-3', 175 bp expected), Wnt3a (forward5'-ACC TGG AGA AGG CTG GAA AT-3', reverse 5'-ATG TGA TCC AGGATG GTC GT-3'; 162 bp expected), Wnt4 (forward 5'-GCC ACG CACTAA AGG AGA AG-3, reverse 5'-GGC CTT AGA CGT CTT GTT GC-3';215 bp expected), Wnt5a (forward 5'-AGC CGA GAG ACA GCC TTCAC-3', reverse 5'-TCC TGC GAC CTG CTTCATTG-3'; 289 bp expected),and -actin (forward 5'-CGC CAA CCG CGA GAA GAT-3', reverse 5'-CGTCAC CGG AGT CCA TCA-3'; 168 bp expected). The number of amplificationsteps required to reach an arbitrary intensity threshold (Ct)was computed. The relative gene expression levels were presented2–GCt, where GCt = Cttarget – Ct-actin. Fold changefor the treatment was defined as the relative expression, comparedwith the vehicle and was calculated as 2–Ct, where Ct= Cttreatment – Ctvehicle (19).
Western Blotting
Membrane, cytosolic, and nuclear extracts of cell cultures wereprepared as described previously (19). Aliquots of cytosolicor nuclear extracts (100 µg) were subjected to Westernblot assay. The designated proteins on the blots were probedby antibodies against GSK-3, phospho-Ser9-GSK-3, phospho-Ser473-Akt,-catenin, caspase-3, cleaved caspase-3, and phospho–poly(ADP-ribose)polymerase (phospho-PARP) (Cell Signaling Technology, Beverly,MA), followed by horseradish peroxidase–conjugated IgGas the secondary antibody and visualized with chemiluminescenceagents. Protein band intensity on each blot from three repeatedexperiments was quantified by scan densitometry. The fold ofincrease was calculated by dividing the band intensity fromthe high glucose–stressed sample by that of the respectivecontrol sample.
Wnt4, Wnt5, and -Catenin cDNA Transfection
cDNA encoding Wnt4 or Wnt5a or stable (S33Y) -catenin or wild-type-catenin (24) were ligated and cloned, respectively, into pUSE(Upstate Biotechnology, Lake Placid, NY) and pC1-neo vectors.Cells (5 x 105 cells/well, in six-well plate) were plated toreach 60 to 80% confluence and transfected using FuGENE 6 transfectionreagent (Roche Diagnostic Corp., Indianapolis, IN). Cells thatwere stably transfected with the plasmids were selected in mediumthat contained 600 µg/ml G418 (Life Technologies, Gaithersburg,MD).
Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridine Triphosphate-Biotin Nick End-Labeling
Trypsinized and floating cells that were cultured in high glucosewith or without Wnt signaling modulators were pooled, spun (1x 104 cells) onto glass slides, and fixed in 70% methanol forinvestigation of cell apoptosis using in situ cell death detectionkits (Roche Diagnostics, Mannheim, Germany). Specimens thatwere pretreated with 50 U/ml DNAse I (Sigma Chemical, St. Louis,MO) or incubated in reaction buffer without terminal deoxynucleotidyltransferase were used as positive or negative controls. Terminaldeoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotinnick end-labeling (TUNEL)-stained cells were recognized usingfast red as substrates.
Streptozotocin-Induced Diabetes
Four-month-old male Wistar rats were caged in pairs and maintainedon rodent diet and water ad libitum. Diabetes in rats was inducedas described previously (19). Briefly, diabetes was inducedby a single intraperitoneal injection of 50 mg/kg streptozotocin(Sigma Chemical). One week after injection, blood glucose wasmeasured from tails. Rats with blood glucose >300 mg/dl,defined as successful induction of diabetes, were used for succeedingexperiments. For equalization of blood glucose levels in alldiabetic rats, intermittent-acting insulin was administeredsubcutaneously once a day until the rats were killed. Bloodglucose levels were measured every day just before insulin injections.The dose of insulin was adjusted to reach the target blood glucoselevel of 200 to 250 mg/dl. All studies were approved by theInstitutional Animal Care and Use Committee of the hospital.
Exogenous BIO Treatment
Diabetic rats were given BIO subcutaneously (n = 6; 200 µg/kgper d) or vehicle (n = 6; 200 µl of corn oil) for 28 consecutivedays. Six rats without streptozotocin injections were used asnormal controls. At day 28, urine was collected using metaboliccage systems, and urinary protein and creatinine levels weremeasured using respective assay kits (Sigma-Aldrich, St. Louis,MO). Rats were killed with an overdose of pentobarbital sodium,and kidneys were harvested for immunohistochemical analysis.After perfusion with PBS, fresh kidney tissues were ground witha mortar and pestle under liquid nitrogen; lysed with ice-coldPBS that contained 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate,100 µg/ml PMSF, and 30 µg/ml aprotinin; and homogenizedby ultrasonication. Aliquots of kidney tissue homogenate (50µg) were subjected to assessment of Wnt4 and Wnt5a, phospho-Ser9-GSK-3,and -catenin expression using immunoblotting.
Immunohistochemistry
Kidneys were fixed in 4% PBS-buffered formaldehyde, embeddedin paraffin, sliced longitudinally into 5-µm-thick sections,and subjected to immunohistochemical or TUNEL staining. Antibodiesagainst Wnt4, Wnt5a, phospho-Ser9-GSK-3 and -catenin were usedfor immunohistochemistry. Immunoreactivity in sections was demonstratedusing a horseradish peroxidase–3'-,3'-diaminobenzidinekit (R&D Systems), followed by counterstaining with hematoxylin,dehydration, and mounting. Sections without primary antibodieswere enrolled as negative controls for immunostaining. Six regionswithin renal glomeruli from three sections that were obtainedfrom four rats were studied. Each region that contained positiveimmunostained cells were analyzed microscopically and quantitatively(Carl Zeiss, Gottingen, Germany). Three random images from eachselected region then were taken, captured, and analyzed underx400 magnification using image analysis software (Media Cybernetics,Silver Spring, MD). The percentage of positive immunolabeledcells and total cells in each area was counted. Renal mesangialand tubular cells were identified morphologically. Apoptosisof mesangial cell cultures was counted as the ratio of TUNEL-positivestained mesangial cell number and total cell number under x200magnification.
Statistical Analyses
All values were expressed as means ± SE calculated fromat least three repeated experiments. Wilcoxon test was usedto evaluate differences between the sample of interest and itsrespective control. For analysis of time course, a multiplerange of ANOVA and post hoc tests were used. P < 0.05 wasconsidered significant.
High Glucose–Induced Caspase-3 Activation and Cell Apoptosis
We examined whether high glucose altered proliferation or apoptosisof mesangial cells. In comparison with the control groups, highglucose significantly reduced cell proliferation (Figure 1A)and promoted cell apoptosis (Figure 1B) by 48 h. We furtherexamined whether high glucose–mediated cell apoptosiswas linked to caspase-3 activation. Immunoblotting showed thathigh glucose significantly increased the levels of cleaved caspase-3and activated PARP (Figure 1C). Inhibition of caspase-3 activityby Z-DEVD-FMK reduced the high glucose–mediated activationof caspase-3, PARP (Figure 1D), and cell apoptosis (Figure 1B).Furthermore, TUNEL staining indicated that high glucose inducesDNA fragmentation, which was abrogated with caspase-3 inhibitorpretreatment (Figure 1E).
Figure 1. High glucose (HG) significantly reduced proliferation (A) and increased apoptosis (B) of mesangial cells in 48 h. (C) HG increased caspase-3 and poly(ADP-ribose) polymerase (PARP) activation. (D) Inhibition of caspase-3 activity by Z-PEVD-FMK evidently reduced the promoting effect of HG on caspase-3 and PARP activation. (E) Apoptotic mesangial cells with or without HG stress or 10 µM caspase-3 inhibitor pretreatment. Cells (2 x 104 cells/well, 96-well plate) were cultured in medium that contained 35 mM d-glucose for 4 d. Cell growth was determined using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide kits and cell number counting. Cells that were positive for terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) exhibited red staining in nucleus. Immunoblotting of total caspase-3 showed equal loadings and transfer for all lanes. P < 0.05, *vehicle- and #HG-treated groups. Magnification, x200.
High Glucose Downregulated Wnt/-Catenin Signaling
We investigated whether high-glucose stress could alter Wnt/-cateninsignaling. Real-time PCR showed that high glucose altered Wnt1,Wnt3a, Wnt4, and Wnt5a mRNA expression (Figure 2A). Of the Wntexpression, high glucose reduced Wnt4 and Wnt5a expression by>50% throughout the study period and was analyzed further.A reduction in phosphorylation of Ser9 in GSK-3 is known toincrease GSK-3 activity and attenuate -catenin translocationinto the nucleus. An antibody against phospho-Ser9-GSK-3 wasused to determine the level of GSK-3 activation (25). Immunoblottingshowed that high glucose reduced Wnt4 and Wnt5a expression,which correlated with a reduction in phospho-Ser9-GSK-3 levelsand nuclear -catenin expression (Figure 2B). These findingsindicate that mesangial cells respond to high glucose by promotingGSK-3 activation and suppressing Wnt/-catenin signaling.
Figure 2. (A) HG significantly reduced Wnt4 and Wnt5a mRNA gene expression throughout the study period. (B) HG reduced Wnt4, Wnt5a, inhibitory phosphorylated Ser9–glycogen synthase kinase-3 (phospho-Ser9-GSK-3), and nuclear -catenin expression. The experimental results graphed represent the relative abundance of the Wnt gene by real-time PCR normalized to housekeeping gene -actin. Immunoblotting of total GSK-3 showed equal loadings and transfer for all lanes.
Wnt4, Wnt5a, and -Catenin Signaling Is Required for Cell Survival
We examined the biologic role of Wnt4, Wnt5a, and -catenin inhigh glucose–mediated mesangial cell apoptosis. Transfectionof mesangial cells with either Wnt4 or Wnt5a (Figure 3A) suppressedhigh glucose–mediated GSK-3 activity as evidenced by increasesin phosphorylated Ser9-GSK-3 and Akt activation and nuclear-catenin accumulation (Figure 3B). Overexpression of Wnt4 orWnt5a suppressed high glucose–mediated activation of caspase-3,PARP (Figure 3C), and cell apoptosis (Figure 3D) and increasedcell growth (Figure 3E). Moreover, transfection of -cateninmutant (S33Y) increased nuclear -catenin and phosphorylatedAkt expression (Figure 4A) and reduced the high glucose–mediatedactivation of caspase-3, PARP (Figure 4B), and cell apoptosis(Figure 4C) and subsequently reversed cell proliferation (Figure 4D).This result suggests that the stability of -catenin is criticalfor mesangial cell homeostasis.
Figure 3. Transfection of Wnt4 and Wnt5a increased Wnt4 and Wnt5a expression (A) and abrogated the suppressing effect of HG on phospho-Ser9-GSK-3, nuclear -catenin, and phosphorylated Akt expression (B). Overexpression of Wnt4 or Wnt5a reduced the promoting effect of HG on caspase-3 and PARP activation (C), cell apoptosis (D), and increased cell growth (E). Mesangial cells were subjected to transfection with pUSE or Wnt4 or Wnt5a cDNA. Stably transfected cell cultures were subjected to HG stress for 48 h. Immunoblotting of total GSK-3 or nuclear caspase-3 showed equal loadings and transfer for all lanes. P < 0.05, *vehicle- and #HG-treated groups.
Figure 4. Transfection of stable -catenin (S33Y) increased nuclear -catenin and phosphorylated Akt expression (A) and reduced the promoting effect of HG on caspase-3 and PARP activation (B), cell apoptosis (C), and reversed cell proliferation (D). Mesangial cells were subjected to transfection with wild-type or stable -catenin cDNA. Stably transfected cell cultures were subjected to HG stress for 48 h. Immunoblotting of total caspase-3 and actin showed equal loadings and transfer for all lanes. P < 0.05, *vehicle and #HG-treated groups.
Wnt Signaling Modulators Alleviated High Glucose–Mediated Apoptotic Signaling
We investigated whether pharmacologic modulation of Wnt/-cateninsignaling could alter proapoptotic activities of mesangial cellsthat are exposed to high glucose. Recombinant Wnt5a or GSK-3inhibitors BIO or LiCl suppressed high glucose–mediatedactivation of GSK-3 and restored nuclear -catenin levels andphospho-Akt expression (Figure 5A). Wnt/GSK-3 modulators abrogatedhigh-glucose induction of caspase-3, PARP activation (Figure 5B),and cell apoptosis (Figure 5C) and increased cell proliferation(Figure 5D).
Figure 5. Recombinant Wnt5a and (2'Z, 3'E)-6-bromoindirubin-3'-oxime (BIO) and LiCl increased the inhibitory phospho-Ser9-GSK-3, nuclear -catenin, and phosphorylated Akt expression (A) and abrogated HG-induced caspase-3 and PARP activation (B) and cell apoptosis (C). (D) Wnt5a and GSK-3 inhibitors reversed proliferation of cell cultures. Cell cultures were co-cultured or pretreated with 250 ng/ml recombinant Wnt5a or 10 mM LiCl or 10 µM BIO for 48 h. Immunoblotting of total GSK-3 and caspase-3 showed equal loadings and transfer for all lanes. P < 0.05 from the *vehicle- and #HG-treated groups.
BIO Alleviated Urinary Protein Secretion and Mesangial Cell Apoptosis in a Model of Diabetic Nephropathy
We examined whether modulation of GSK-3 signaling by BIO couldalter diabetes-induced glomerulopathy. In comparison with thenormal group, diabetes significantly increased blood glucoseand urinary protein excretion (Table 1). BIO treatment significantlyreduced the promoting effect of diabetes on urinary proteinsecretion (Table 1). BIO treatment did not seem to alter bloodglucose in diabetic rats throughout the study period.
Table 1. Biochemical characteristics and Wnt4, Wnt5a, phospho-GSK-3, -catenin, and TUNEL expression in renal glomerular mesangial cells of diabetic rats with or without BIO treatmenta
In the diabetes group, mesangial cells in glomeruli and tubularcells surrounding glomeruli displayed intensive DNA fragmentationas demonstrated by positive TUNEL staining. Fewer mesangialcells and tubular cells adjacent to glomeruli of diabetic kidneywith BIO treatment showed TUNEL staining (Figure 6A). Immunoblottingshowed that diabetes decreased Wnt4, Wnt5a, phospho-Ser9-GSK-3,and -catenin expression in kidney tissue. BIO treatment reducedthe suppressing effect of diabetes on phosphor-Ser9-GSK-3 and-catenin expression but not Wnt4 or Wnt5a expression in thekidneys (Figure 6B).
Figure 6. Effect of BIO treatment on glomerular mesangial cell apoptosis and Wnt signaling molecule expression of renal tissue in diabetic rats with or without BIO treatment. (A) In comparison with the normal group, mesangial and tubular cells around glomeruli in diabetic kidneys expressed intensive TUNEL staining. BIO treatment attenuated the promoting effect of diabetes on TUNEL staining in diabetic kidney. Cells that were positive for TUNEL exhibited red staining in nucleus. (B) Diabetes evidently reduced Wnt4, Wnt5a, phospho-Ser9-GSK-3, and -catenin expression of kidney tissue. BIO abrogated the suppressing effect of diabetes on phosphor-Ser9-GSK-3 and -catenin expression. Immunoblotting of actin showed equal loadings and transfer for all lanes. Magnification, x400.
In the absence of primary antibodies, no immunostaining wasvisible (Figures 7 and 8). Cells that were adjacent to glomeruliand showed positive Wnt4, Wnt5a, phosphor-Ser9-GSK-3, or -cateninimmunoexpression exhibited brown color in cell periphery orcytoplasm. In the diabetes group, mesangial cells and tubularcells around glomeruli in the renal cortex expressed lower amountsof Wnt4, Wnt5a (Figure 7), phosphor-Ser9-GSK-3, and -catenin(Figure 8) when compared with the normal group. In the BIO groups,mesangial cells and tubular cells expressed evident phosphor-Ser9-GSK-3and -catenin expression (Figure 8). We found that diabetes significantlypromoted cell apoptosis that was associated with attenuatedWnt4, Wnt5a, phospho-Ser9-GSK-3, and -catenin expression inglomerular mesangial cells when compared with the control group(Table 1). BIO treatment significantly suppressed mesangialcell apoptosis and correlated with increased phospho-Ser9-GSK-3and -catenin immunoreactivities in renal glomeruli of diabeticrats. BIO treatment did not markedly affect Wnt4 and Wnt5a expressionin renal tissue (Table 1).
Figure 7. Representative photographs of Wnt4 and Wnt5a immunostaining of glomeruli in diabetic kidneys with or without BIO treatment. At low-power field, cells that were located in diabetic kidney tissue with vehicle or BIO treatment displayed weak Wnt4 and Wnt5a expression when compared with the normal group. At high-power field, mesangial and tubular cells around glomeruli in diabetic kidneys with vehicle or BIO treatment expressed weak Wnt 4 and Wnt5a expression. Immunostained cells exhibited brown color in cell periphery and cytoplasm. Magnifications: x100 and x400.
Figure 8. Representative photographs of phospho-Ser9-GSK-3 and -catenin immunostaining of glomeruli in diabetic kidneys with or without BIO treatment. At low-power field, cells that were located in diabetic kidney tissue with vehicle treatment displayed weak phospho-Ser9-GSK-3 and -catenin expression when compared with the normal group. At high-power field, mesangial and tubular cells around glomeruli in diabetic kidneys with vehicle or BIO treatment expressed weak phospho-Ser9-GSK-3 and -catenin expression. In the BIO-treated group, tubular and mesangial cells expressed evident phospho-Ser9-GSK-3 and -catenin immunoreactivities. Immunostained cells exhibited brown color in cell periphery and cytoplasm. Magnifications: x100 and x400.
In this study, increased proapoptotic activities in mesangialcells followed high-glucose stress, which downregulated Wnt/-cateninsignaling. Whereas previous studies have demonstrated that highglucose–promoted mesangial cell apoptosis is attributableto increasing oxidative stress or altering growth factor expression(26–28), little research has been done to define the biologicrole of Wnt/-catenin in regulating the homeostasis of mesangialcells that are exposed to high glucose. Our findings providethe first indication that high-glucose induction of mesangialcell apoptosis was through activation of GSK-3 and subsequentdestabilization of -catenin–responsive cell survival activities.We suggest that it is the modulation of apoptosis- or survival-regulatorymolecules by altering canonical Wnt signal transduction pathwaythat bring about high-glucose promotion of mesangial cell apoptosis.
We found that mesangial cells that were exposed to high-glucoseconditions responded by activating GSK-3 and subsequently promotedproapoptotic cascades. GSK-3 is reported to regulate apoptosisof renal medullary interstitial cells (23) and glucose intolerancein GSK-3 transgenic mice and diabetic animals (29,30). In ourstudy, inhibiting GSK-3 activation by LiCl or BIO reduced highglucose–promoted caspase-3 and PARP phosphorylation andcell apoptosis, suggesting that GSK-3–dependent signalingpathways are involved in mediating high-glucose stress to increaseapoptotic programs of mesangial cells.
We noted that high glucose suppressed nuclear -catenin translocation,Akt activation, and growth of mesangial cells. -Catenin is involvedin regulating apoptosis of renal cell carcinoma (31) and multiplekidney cysts (32). Increased phosphorylated -catenin expressionwas noted in placenta vessels of patients with diabetes (33),suggesting that diabetes may destabilize -catenin signalingto alter tissue function. We provide evidence that -cateninsignaling acts as a survival-stimulatory molecule for mesangialcells that are exposed to high glucose. These are based on thefindings that increasing nuclear -catenin accumulation reducedhigh glucose–promoted DNA fragmentation and reversed Aktphosphorylation and proliferation of stable -catenin–transfectedcell cultures. To our knowledge, this is the first report thathigh glucose raised mesangial cell apoptosis that is regulatedby attenuation of -catenin signaling. Stabilization of -cateninis required for sustaining survival of mesangial cells thatare exposed to high-glucose stress.
We previously showed that alteration of Wnt signaling correlateswith renal injury (34,35). In this study, we found that mesangialcells that were exposed to high-glucose conditions reduced Wntexpression. Restoring Wnt4 or Wnt5a expression by gene transfectionor recombinant protein reduced high glucose–induced cellapoptosis. These findings suggest that Wnt4 and Wnt5a moleculesare beneficial for promoting mesangial cell survival. Previousstudies demonstrated that Wnt5a signals through a -catenin–independentpathway or by inhibition of the canonical Wnt signaling pathwayto regulate chondrocyte dedifferentiation (36,37). In our study,cell cultures that overexpressed Wnt4 or Wnt5a raised nuclear-catenin accumulation to promote mesangial cell survival, suggestingthat Wnt4 and Wnt5a initialized a -catenin–responsivemechanism. We speculate the discrepancy that Wnt4 and Wnt5aregulation of -catenin signaling depends on cell type and stimulationused.
To our knowledge, control of GSK-3 and -catenin signaling inglomerular mesangial cells of the diabetic kidney in vivo hasnot been reported previously. This study provides the firstevidence that glomerular mesangial cells and tubular cells indiabetic kidneys displayed weak Wnt level, phospho-Ser9-GSK-3,and -catenin expression. Impairing GSK-3 activation by exogenousBIO administration increased -catenin signaling and alleviateddiabetes-induced glomerular mesangial cell death and urinaryprotein secretion in diabetic rats. These phenomena in vivoare in line with those of cell culture models. We cannot excludethe possibility that high glucose may alter Wnt/-catenin signalingin tubular cells. The role of Wnt signaling in regulating diabetes-stressedtubular cells needs to be explored in the future. We suggestedthat renal mesangial cells actively respond to high-glucosestress by altering Wnt/-catenin signaling and subsequently promotedcell apoptosis.
The role of mesangial cell apoptosis in proteinuria and diabeticglomerulosclerosis remains controversial. Previous studies suggestedthat apoptosis of resident glomerular mesangial cells is theearliest cellular lesion in the development diabetic nephropathy(38,39), and several bioactive molecules are involved in regulatingapoptotic activities of high glucose–stressed mesangialcells (40,41). Our observation revealed that high glucose perturbedWnt/-catenin signaling and induced glomerular mesangial cellapoptosis and proteinuria. GSK-3 and -catenin had a distinctrole in modulating mesangial cell fate. We cannot exclude thepossibilities that other Wnt-signaling molecules may be linkedto high glucose–induced mesangial cell apoptosis. Furtherstudies are needed to define the biologic role of these moleculesin diabetic nephropathy.
Taken together, we have provided evidence that canonical Wntsignaling is involved in controlling high glucose–mediatedmesangial cell death. Modulation of canonical Wnt/GSK-3/-cateninsignal transduction is beneficial for enhancing mesangial cellsurvival in diabetic kidney.
Acknowledgments
This work was supported in part by grants NSC94-2314-B-182A-127(to C.L.L.) from the National Science Council, Taiwan, and ChangGung Memorial Hospital (CMRPG630042 to C.L.L.), Taiwan.
We thank Dr. Bert Vogelstein (John Hopkins Medical Instituteand Howard Hughes Medical Institutes) for the generous giftof mutant -catenin cDNA construct.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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[Abstract][Full Text][PDF]
-Catenin Signaling Modulates Survival of High Glucose–Stressed Mesangial Cells
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Abstract
Introduction
-catenin signaling in controlling high glucose–induced mesangial cell apoptosis is not well defined. Herein is reported that Wnt/
GCt, where 
, and 
