Reactive Oxygen Species in the Presence of High Glucose Alter Ureteric Bud Morphogenesis
Shao-Ling Zhang*,
Yun-Wen Chen*,
Stella Tran*,
Isabelle Chenier*,
Marie-Josée Hébert* and
Julie R. Ingelfinger
* University of Montreal, Centre hospitalier de l'Université de Montréal-Hôtel-Dieu, Montreal, Quebec, Canada; and Pediatric Nephrology Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Correspondence: Dr. Shao-Ling Zhang, University of Montreal, Centre hospitalier de l'Université de Montréal-Hôtel-Dieu, Pavillon Masson, 3850 Saint Urbain Street, Montreal, Quebec H2W 1T7, Canada. Phone: 514-890-8000, ext. 15633; Fax: 514-412-7204; E-mail: shao.ling.zhang{at}umontreal.ca
Received for publication October 17, 2006.
Accepted for publication April 11, 2007.
Renal malformations are a major cause of childhood renal failure.During the development of the kidney, ureteric bud (UB) branchingmorphogenesis is critical for normal nephrogenesis. These studiesinvestigated whether renal UB branching morphogenesis is alteredby a high ambient glucose environment and studied underlyingmechanism(s). Kidney explants that were isolated from differentperiods of gestation (embryonic days 12 to 18) from Hoxb7–greenfluorescence protein mice were cultured for 24 h in either normalD-glucose (5 mM) or high D-glucose (25 mM) medium with or withoutvarious inhibitors. Alterations in renal morphogenesis wereassessed by fluorescence microscopy. Paired-homeobox 2 (Pax-2)gene expression was determined by real-time quantitative PCR,Western blotting, and immunohistology. The results revealedthat high D-glucose (25 mM) specifically stimulates UB branchingmorphogenesis via Pax-2 gene expression, whereas other glucoseanalogs, such as d-mannitol, L-glucose, and 2-deoxy-D-glucose,had no effect. The stimulatory effect of high glucose on UBbranching was blocked in the presence of catalase and inhibitorsof NADPH oxidase, mitochondrial electron transport chain complexI, and Akt signaling. Moreover, in in vivo studies, it seemsthat high glucose induces, via Pax-2 (mainly localized in UB),acceleration of UB branching but not nephron formation. Takentogether, these data demonstrate that high glucose alters UBbranching morphogenesis. This occurs, at least in part, viareactive oxygen species generation, activation of Akt signaling,and upregulation of Pax-2 gene expression.
Maternal diabetes constitutes a major risk factor for congenitalmalformations in the offspring. When the fetus is exposed tosustained levels of high ambient glucose, widespread fetal damagemay develop, affecting multiple systems, including cardiovascular,nervous, skeletal and urogenital systems—a condition calleddiabetic embryopathy.1,2 Infants who are born to women withpregestational diabetes have a 10-fold risk of congenital malformations,and those who are born to women with gestational diabetes havea five-fold relative risk of congenital malformations. Boththe mother with diabetes and her fetus are at risk for significantmorbidity and mortality, even in the 21st century.3,4 Of thosecongenital malformations that are seen in offspring of pregestationalmaternal diabetes, renal malformations such as renal agenesisand congenital abnormalities of kidney and urinary tract aremost prevalent.5
Renal morphogenesis involves complex events in which many genesinteract to coordinate the formation of the final kidney. Abnormalitiesoccur when the normal pattern of nephrogenesis is interrupted.In humans, the fetal kidneys begin to develop at 5 weeks gestation,glomeruli at 8 to 9 weeks, and tubular function after the 14thweek. The full complement of nephrons (between 200,000 and wellover 1 million) has formed by approximately 36 weeks of gestation;no further nephrons are formed after this time.6,7 However,rodents, which have a gestation period in the range of 19 to21 days, continue nephrogenesis postnatally—until approximately10 days after birth. Diabetes constitutes an adverse in uteroenvironment that may impair nephrogenesis in both human andexperimental animal models, resulting in renal agenesis, dysplasiaor aplasia, and hypoplasia.8–11 We have initiated investigationsconcerning the interaction between high glucose and paired-homeobox2 (Pax-2) gene expression in renal development. As a "kidney-specific"master gene, Pax-2 is expressed in both ureteric bud (UB) andmetanephric mesenchyme (MM) lineages, normally optimizing UBbranching and mesenchymal-to-epithelial transformation in kidneydevelopment.12–14 Mutations in the Pax-2 gene cause increasedapoptosis,15–17 associated with renal hypoplasia.15,18,19We recently reported that high D(+) glucose (25 mM), as comparedwith normal glucose (5 mM), specifically induced Pax-2 geneexpression in both in vitro (mouse metanephric mesenchymal cells[MK4]) and ex vivo (kidney explant from Hoxb7–green fluorescenceprotein [Hox7-GFP] mice) models.20,21 High glucose–inducedPax-2 gene expression is mediated, at least in part, via reactiveoxygen species (ROS) generation and activation of the NF-B signalingpathway but not via protein kinase C, p38 mitogen-activatedprotein kinase and p44/42 mitogen-activated protein kinase signaling.22
This work is designed to demonstrate the influence of a high-glucosemilieu on UB branching morphogenesis and its underlying mechanism(s)using real time in an ex vivo model. Additional in vivo studiesthat complement the ex vivo studies are also included. Our resultsindicate that high glucose stimulates UB branching via ROS generationand Pax-2 gene expression. We conclude that the stimulatoryeffect of high glucose is mediated, at least to some extent,via activation of NADPH oxidase and mitochondrial oxidativemetabolism and stimulation of Akt signaling pathway.
High D-Glucose Stimulates UB Branching in a Time-Dependent Manner
Kidney explants that were isolated from timed-pregnant miceat embryonic day 13 (E13) were cultured either in normal (5mM) D-glucose DMEM plus 20 mM d-mannitol (left kidney) or high(25 mM) D-glucose DMEM (right kidney) supplemented with 1% depletedFBS (dFBS) up to 96 h with fresh medium changed every 24 h.As seen in fluorescence microscopic sequential images, as comparedwith 5 mM glucose (Figure 1, A and B), 25 mM D-glucose (Figure 1,C and D) stimulates UB branching morphogenesis in a time-dependentmanner. Because a stimulatory effect of high glucose is presentafter 24 h of incubation, we used 24 h of stimulation for subsequentstudies. By carefully measuring the diameter of metanephroithat were cultured from 0 to 96 h (Figure 1E), we found thathigh glucose reduced the size of metanephroi in a time-dependentmanner.
Figure 1. High D-glucose stimulates ureteric bud (UB) branching in a time-dependent manner. Kidney explants that were isolated from time-pregnant mice at embryonic day 13 (E13) were cultured in either normal (5 mM) D-glucose DMEM (left kidney) or high (25 mM) D-glucose DMEM (right kidney) supplied with 1% dFBS up to 96 h with fresh medium changed every 24 h. The UB branching morphogenesis sequential images were recorded by fluorescence microscope. (A and B) Representative of the kidney explants that were cultured in 5 mM glucose supplemented with 20 mM d-mannitol DMEM to maintain constant isotonicity or osmolality. (C and D) Representative of the kidney explants that were cultured in 25 mM glucose DMEM. The sequential images was recorded by every 24 h. (E) High ambient glucose reduced the surface area of cultured metanephroi in a time-dependent manner. Magnifications: x2 in A and C; x10 in B and D.
High D-Glucose Specificity on UB Branching Morphogenesis
E13 kidney explants were incubated in medium that contained1% dFBS and 25 mM different glucose analogues such as d-mannitol,L-glucose, 2-deoxy-D-glucose, and D-glucose. After incubationfor 24 h, high D-glucose (Figure 2A-a) specifically stimulatesUB branching morphogenesis by fluorescence microscopy, whereasother glucose analogs, such as d-mannitol (Figure 2A-b), L-glucose(Figure 2A-c), and 2-deoxy-D-glucose (Figure 2A-d), had no effect.Moreover, high D-glucose stimulates UB branching in a dosage-dependentmanner (Figure 2B). For maintaining constant isotonicity orosmolality, 5 mM glucose medium was supplemented with d-mannitol(20 mM final concentration) in additional studies. High glucosestimulated a greater than two-fold increase of UB tip numbersas compared with normal glucose (Figure 3). This increase inUB branching is induced by high D-glucose (25 mM) but not byother glucose analogs.
Figure 2. High D-glucose on UB branching morphogenesis. (A) Specificity of the effect of glucose analogs on UB branching. E13 kidney explants were incubated in medium that contained 1% dFBS and 25 mM different glucose analogues, such as D-glucose (a), d-mannitol (b), L-glucose (c), and 2-deoxy-D-glucose (d), for 24 h. The images were recorded by fluorescence microscope (2X and 10X magnification). (B) The D-glucose dosage-dependent effect on UB branching from 5 to 30 mM in E13 kidney explant. Magnifications: x2 in A and B; x10 in A.
Figure 3. High D-glucose stimulates UB branching morphogenesis. (A) E13 kidney explants were incubated in either 5 (left kidney) or 25 mM glucose DMEM (right kidney) that contained 1% dFBS for 24 h. The images were recorded by fluorescence microscope. (B) Quantification of UB numbers. Kidney explants that were incubated in 5 mM glucose were considered the control (100%). Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005. Magnifications: x2, x4, and x10.
High Glucose Stimulated Pax-2 Gene Expression
High glucose increased Pax-2 gene and protein expression—mRNA(real-time quantitative PCR [RT-qPCR]; Figure 4A) and protein(Western blot; Figure 4B)—as compared with normal glucosein E18 kidney explants. Immunohistologic staining indicatedthat the upregulation of Pax-2 is localized mainly to the UB(Figure 4C). These data indicate that high glucose stimulationof UB branching morphogenesis is mediated via Pax-2 gene expression.
Figure 4. High glucose upregulates Pax-2 expression in E18 kidney explant as analyzed by real-time quantitative PCR (RT-qPCR; A) Western blot (B), and immunohistologic staining (C). The normalized Pax-2 level in explants that were incubated in 5 mM glucose was considered the control (100%). Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005. Magnifications: x20 and x32.
Inhibitors of NADPH Oxidase and Mitochondrial Electron Transport Chain Complex I Block the Stimulatory Effect of High Glucose on Pax-2 Gene Expression and UB Branching Ex Vivo
It is apparent that inhibitors of NADPH oxidase (diphenyleneiodinium [DPI]; 10–6 M) and mitochondrial electron transportchain complex I (rotenone, 10–6 M) block the stimulatoryaction of high glucose on Pax-2 expression (Figure 5) and UBbranching morphogenesis (Figure 6) in E14 kidney explants. Thesedata indicate that the stimulatory effect of high glucose ismediated, at least in part, via NADPH oxidase activation andROS generation.
Figure 5. Inhibitory effect of diphenylene iodinium (DPI) and rotenone on Pax-2 gene expression in E16 kidney explants. E16 kidney explants were cultured in either 5 or 25 mM glucose DMEM with or without DPI (10–6 M) and rotenone (10–6 M) for 24 h. The Pax-2 gene expression was analyzed by either RT-qPCR (A) or Western blot (B). The relative densities of Pax-2 were compared with -actin. The normalized Pax-2 level in kidney explants that were incubated in 5 mM glucose was considered the control (100%). Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005.
Figure 6. Inhibitory effect of DPI and rotenone on UB branching morphogenesis stimulated by high glucose. E14 kidney explants were incubated in either 5 (A) or 25 mM glucose DMEM (B) in the absence or presence of DPI (10–6 M; C) and rotenone (10–6 M; D) for 24 h. The images were recorded by fluorescence microscope. (E) Quantification of UB numbers. Kidney explants that were incubated in 5 mM glucose were considered to be controls (100%). Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005. Magnifications: x2 and x10.
High Glucose–-Induced ROS Generation on UB Branching and Pax-2 Gene Expression
We previously reported that H2O2 modestly increases Pax-2 geneexpression. However, this stimulatory effect of H2O2 was significantlyenhanced in the presence of high glucose in E16 kidney explants.22The question has been raised about whether H2O2 also directlymodulates UB branching. Indeed, exogenous H2O2 at 10–5M could trigger UB branching morphogenesis, particularly inthe presence of high glucose in E13 kidney explants (Figure 7,A through E). Our data also indicate that high glucose activatesthe total ROS generation in E18 kidney explant (Figure 7F).Moreover, high glucose–induced UB branching in E12 kidneyexplants could be partially blocked by catalase (250 U) andcompletely abolished by Akt inhibitor IV (a cell-permeable benzimidazolecompound that inhibits Akt phosphorylation/activation by targetingthe ATP binding site of a kinase upstream of Akt but downstreamof phosphatidylinositol-3-kinase [PI3-K]) at a concentrationof 10–6 M (Figure 8). Similar results were also foundin Pax-2 mRNA and protein expression (Figure 9). These dataindicate that high glucose evokes ROS generation and upregulatesPax-2 gene expression via Akt signaling and subsequently stimulatesUB branching morphogenesis.
Figure 7. H2O2 effect on UB branching morphogenesis in E13 kidney explants. E13 kidney explants were incubated in either 5 (A and B) or 25 mM glucose DMEM (C and D) with or without H2O2 (10–5 M) for 24 h. The images were recorded by fluorescence microscope. (E) Quantification of UB numbers. Kidney explants that were incubated in 5 mM glucose were considered as controls (100%). (F) reactive oxygen species (ROS) generation was assessed by lucigenin method, and the final value of ROS generation was normalized by the protein concentration of sample. The normalized ROS generation in cells that were incubated in 5 mM glucose was considered as the control (100%). Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005. Magnification, x10.
Figure 8. Inhibitory effect of catalase and AKT inhibitor on UB branching morphogenesis in E12 kidney explants. E12 kidney explants were incubated in either 5 (A) or 25 mM glucose DMEM (B) in the absence or presence of catalase (250 U; C) and AKT inhibitor (10–6 M; (D) for 24 h. The images were recorded by fluorescence microscope. (E) Quantification of UB numbers. Kidney explants that were incubated in 5 mM glucose were considered the control (100%). Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005. Magnifications: x2 and x10.
Figure 9. Inhibitory effect of catalase and AKT inhibitor on Pax-2 gene expression in E17 kidney explants. E17 kidney explants were cultured in either 5 or 25 mM glucose DMEM with or without catalase (250 U) and AKT inhibitor (10–6 M) for 24 h. The Pax-2 gene expression was analyzed by either RT-qPCR (A) or Western blot (B). The relative densities of Pax-2 were compared with -actin. The normalized Pax-2 level in kidney explants that were incubated in 5 mM glucose was considered the control (100%). Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005.
Effect of Gestational Diabetes on Offspring Neonatal Kidney
We have used streptozotocin (STZ) to induce gestational diabetesin the pregnant mother at E13.23–29Figure 10A shows ourexperimental protocol in detail. As may be seen, the newbornoffspring of STZ-induced diabetic dams remained significantlysmaller and lighter (average 20% less body weight) as comparedwith offspring of control dams (control versus STZ: 1.414 ±0.11 versus 1.03 ± 0.07 g; Figure 10B). Most impressive,kidneys of the diabetic offspring were significantly smallerand growth retarded as compared with the kidneys of controloffspring (Figure 10C).
Figure 10.In vivo studies on the effect of maternal diabetes on the neonatal kidney of the offspring. (A) Representative mouse in our experimental protocol in detail. Streptozotocin (STZ)-induced diabetic newborn offspring remained significantly smaller and lighter (average 20% less) as compared with control animals, as shown in B (control versus STZ: 1.414 ± 0.11 versus 1.03 ± 0.07 g). Kidney of diabetic offspring was significantly smaller and growth retarded (hematoxylin/eosin staining and Dolichos Biflorus Agglutinin staining) compared with control (C).
Pax-2 Expression in Neonatal Kidney In Vivo
We evaluated the Pax-2 gene expression as analyzed by immunohistologicstaining (Figure 11, A and B), qRT-PCR (Figure 11C), and Westernblot (Figure 11D) in neonatal kidney of both control and diabeticdams. Our data indicated that maternal hyperglycemia via Pax-2(mainly localized in UB) seems to accelerate UB branching butnot nephron formation in neonatal kidney of diabetic dams.
Figure 11.In vivo Pax-2 expression in neonate kidney. Pax-2 immunohistologic staining in newborn kidney of offspring: Control (A) and STZ (B). Pax-2 gene expression in neonatal kidney was analyzed by qRT-PCR (C) and Western blotting (D). The normalized Pax-2 level in control neonate kidney was considered as 100%. Each point represents the mean ± SD of three independent experiments. *P 0.05; **P 0.01; ***P 0.005. Magnifications: x2 and x10.
In this study, we observed that high glucose alters UB branchingmorphogenesis via Pax2 gene expression. The high glucose effectseems to be mediated, at least in part, via ROS generation andactivation of the PI3-K–AKT pathway.
Maternal diabetes creates a high-risk intrauterine environmentthat has been directly linked to the development of congenitalrenal abnormalities, including caudal regression syndrome, whichis highly associated with renal agenesis and abnormalities ofthe kidney and urinary tract.8–11 These anomalies havebeen noted as either isolated events or part of multiple malformationsyndromes that are more common in offspring of mothers withdiabetes. The incidence seems to be proportional to the degreeof maternal hyperglycemia. For example, in the human, a high-glucoseambient environment throughout pregnancy (pregestational diabetes)is present before embryonic development, which may result ina fetus with markedly teratogenic features that may includethe caudal regression syndrome. In experimental Hoxb7-GFP micein our study, when the pregnant dams were exposed to STZ beforethe budding process, in which the Wolffian duct becomes UB,the dams were barely able to deliver. We observed the same consequences:Teratogenic embryos including caudal regression syndrome withrenal agenesis under those conditions. On the basis of the timingfound in the literature26,27 and our own experiences, we inducedgestational diabetes by STZ injection at E13, and we successfullymanaged to obtain small litters of offspring from diabetic dams.Because the budding process is already completed before E13,from day E13 to birth, it seems likely that the high-glucoseambient environment may impair the nephrogenesis.
To date, most human and experimental studies on gestationaldiabetes have focused on the phenotype but in only a few instanceson the mechanism(s). Most maternal diabetes–related kidneyexplant studies, however, based on long culture time (average4 to 6 d) revealed that hyperglycemia dosage-dependently reducesthe size of the metanephros, UB branching dysmorphogenesis,and the population of nascent nephrons.26,27,30 This observationhas been questioned, because the ideal observation time of therecognized architectural "pattern" of UB tree is between 18and 48 h.31 In this study, experiments were designed to resolvepotential ambiguities that arise from specific protocols orto examine UB morphogenesis under conditions that allow directcomparison with previous studies.32,33
To understand the effect of high glucose per se on renal UBdevelopment and its underlying molecular mechanisms, we usedHoxb7-GFP–transgenic mice20,21 as a model with which wewould be able to monitor UB branching under normal- or high-glucosecondition ex vivo. By using sequential images, we observed thatE13 kidney explant cultured in high D(+) glucose (25 mM) conditiondisplay more UB branching as compared with normaL-glucose medium(5 mM). A stimulatory effect was obvious at 24 h after exposure.Moreover, high D(+) glucose specifically triggers UB branchingmorphogenesis and increased UB tip numbers in a dosage-dependentmanner, whereas other glucose analogs, such as d-mannitol, L-glucose,and 2-deoxy-D-glucose, had no effect. We previously reportedthat high glucose specifically induces Pax-2 gene expressionvia ROS generation in E16 kidney explants from Hoxb7-GFP mice.22In this study, we observed that high glucose elevated totalROS generation and triggered UB branching morphogenesis. Itseems that the stimulatory effect of high glucose could be blockedby ROS inhibitors such as DPI and rotenone. Although the Pax-2gene is expressed in both UB and MM lineages,12,34–39our immunohistologic staining data have clearly revealed thatthe greatest upregulation of Pax-2 by high glucose is in thearea of the UB. Meanwhile, to clarify the direct functionalimpact of ROS on UB branching morphogenesis, we also testedH2O2, an important source of superoxide, and observed that exogenousH2O2 at 10–5 M stimulates UB branching morphogenesis;however, in combination with high glucose, the H2O2 stimulatoryeffect is enhanced substantially, the similar response patternas Pax-2 gene expression.22 Taken together, our data suggestthat high glucose–induced ROS generation has a functionalimpact on UB lineage ex vivo.
Evidence indicates that the high glucose–ROS–PI3-K/Akt–NF-Bpathway seems to be a major signaling pathway that almost coversall major renal cell types.40–45 This scenario has beenpostulated as that by which high glucose leads to kidney damage.40–45Indeed, one study46 suggested that downregulation of Pax-2 expressioncorrelates with a decreased Akt phosphorylation and an enhancedsensitivity to renal endothelial cell apoptosis both in vivoand in vitro, suggesting that Pax-2 promotes angiogenesis, likelyvia survival, proliferation, invasion, and cell organizationvia the PI3-K/Akt-dependent pathway. Therefore, we hypothesizedthat the pathway high glucoseROSAktNF-BPax-2 is involved inimpairment in the UB lineage induced by high glucose. Indeed,our data suggest that high glucose action on UB branching morphogenesisas well as on Pax-2 gene expression could be completely abolishedby Akt inhibitors but partially blocked by catalase. The reasonfor this partial blocking effect may be because catalase couldonly convert H2O2 into H2O but has no effect on other species,such peroxynitrites, hydroxyl radicals, etc. Indeed, more studiesare needed to elucidate the action of other ROS.
We used STZ to induce gestational diabetes in an in vivo modelto generate a high-glucose ambient environment during pregnancy.We observed that this impaired UB branching morphogenesis inE13 pregnant Hoxb7-GFP mice. This in vivo strategy avoids thelimitations that are imposed by using ex vivo studies. Our dataindicate that the body weight (in g) of neonate offspring fromthe diabetic mother remained significantly lower and smaller(average 20% less) than that in control animals. Renal morphologyrevealed that kidneys of diabetic offspring showed growth retardation.However, it seems that high glucose via Pax-2 (mainly localizedin UB) could accelerate UB branching but not nephron formation,but at the same time, high glucose also triggers cell apoptosisin both UB and nephron, which we believe is the major mechanismby which renal function is ultimately affected in diabetic offspringover time (our long-term follow-up study).
Our data demonstrate that high glucose alters UB branching morphogenesisvia Pax-2 gene and protein expression. The stimulatory effectof high glucose seems to be mediated via ROS generation andactivation of the Akt signaling pathway.
Reagents
Normal-glucose medium (5 mM D-glucose DMEM [cat. no. 12320])was purchased from Invitrogen (Burlington, ON, Canada). D(+)-glucose,L-glucose, d-mannitol, 2-deoxy-D-glucose, DPI, rotenone, H2O2,catalase, and 5-(2-benzothiazolyl)-3-ethyl-2-[2-(methylphenylamine)ethenyl]-1-phenyl-1H-benzimidazoliumiodide (Akt Inhibitor IV) were purchased from Sigma-AldrichCanada Ltd. (Oakville, ON, Canada). Mouse anti–-actinmAb (clone AC-15) and rabbit polyclonal anti–Pax-2 antibodywere purchased from Sigma-Aldrich Canada Ltd. and Covance (Richmond,CA), respectively.
Animals
We used the murine Hoxb7-GFP model20,21 (obtained from Dr. FrankCostantini, Department of Genetics and Development, ColumbiaUniversity Medical Center, New York, NY), which is useful forstudying alterations in UB branching morphogenesis because theGFP permits direct observation of the branching process. Thisis especially useful for studying adverse in utero developmentas in diabetes. Hoxb7-GFP mice express GFP driven by the Hoxb7promoter throughout the Wolffian duct and UB epithelium butnot in the surrounding MM or its epithelial derivatives, allowingUB branching morphogenesis to be visualized in real time duringgrowth of the kidney, either in organ culture or in fixed tissue.20,21We used these features for the direct study of UB branchingmorphogenesis pattern under nondiabetic and diabetic conditionsex vivo.
Animal care in these experiments met the standards set forthby the Canadian Council on Animal Care, and the procedures usedwere approved by Institutional Animal Care Committee of theCentre hospitalier de l'Université de Montréal.Hoxb7-GFP mice were housed under standard humidity and lightingconditions (12-h light-dark cycles) and were allowed free accessto standard mouse diet and water ad libitum. Timed-pregnantmice aged 8 to 10 wk were used in all experiments. Vaginal wetmounts were made to determine the estrous cycles of the mice.On the evening before estrus, female mice were housed overnightwith male mice; the presence of spermatozoa in a vaginal smearthe next morning was defined as day 1 of pregnancy.
Metanephric Organ Culture
Embryos (E12 to E18) were dissected aseptically from timed-pregnantmice, and the metanephroi were isolated under sterile conditions.22GFP-positive metanephroi were photographed immediately afterisolation (time 0) and were individually cultured in 1 ml ofeither normal-glucose (5 mM) or high-glucose (25 mM) DMEM suppliedwith 1% dFBS, 100 U/ml penicillin, and 100 µg/ml streptomycinin 95% air and 5% CO2 at 37°C in separate wells of a 24-wellplate for different time periods, depending on the experiment.For example, we constantly monitored and recorded every 8 hthe sequential images of UB branching in metanephroi that werecultured either in normal-glucose or high-glucose DMEM suppliedwith 1% dFBS condition up to 96 h with fresh medium changedevery 24 h. On the basis of initial results, a 24-h incubationperiod was subsequently used for the rest of our experiments.The surface area of cultured metanephroi in either 5- or 25-mMglucose DMEM from time 0 to time 96 h was measured by QCapturePro 5.1 image analysis program provided in Olympus 1x 71 Microscope(Carsen, Ontario, Canada).
To address the variability in embryonic kidney size and in UBbranching patterns among conceptuses, we studied the effectof various treatments on UB branching in kidneys from the samefetus; for example, the left kidney was incubated with normalglucose and the right kidney with high D-glucose or L-glucose,or the left kidney was incubated with high D-glucose and theright kidney with high D-glucose in the presence or absenceof DPI (10–6 M), rotenone (10–6 M), catalase (250U), and Akt inhibitor (10–6 M).
Sequential images of branching UB were recorded with a Olympus1x 71 Microscope. Quantitative assessment of UB branching ineach treatment group was performed by manually counting thenumber of UB tips at time 0 and at 24 h.
Western Blotting
Western blots were performed as in previous studies.22,47,48Briefly, small aliquots (20 to 50 µl) of homogenized kidneyexplant sample were subjected to 10% SDS-PAGE and then transferredonto a polyvinylidene difluoride membrane (Hybond-P; GE HealthcareBiosciences, Baie d'Urfe, Quebec, Canada). The membrane wasfirst blotted for anti–Pax-2 and then reblotted for -actin.The relative densities of the Pax-2 versus-actin bands weremeasured by computerized laser densitometry.
RT-qPCR
RT-qPCR was performed as reported previously.22,47 In brief,first-strand cDNA was produced from 2 µg of random hexamerprimed total RNA using Super-Script preamplification system(Invitrogen, Burlington, Ontario, Canada). Relative quantificationby real-time PCR was carried out using iQ SYBR Green SupermixKit (Bio-Rad Laboratories, Mississauga, ON, Canada) and MiniOpticonReal-Time PCR Detection System (Bio-Rad), following the protocoldescribed by the supplier. PCR reactions in triplicate underwent40 cycles of 95°C for 20 s, 60°C for 20 s, 72°Cfor 20 s, and 79°C for 5 s in the thermal cycler. The parameterthreshold cycle value was measured to determine starting copynumber of target genes using the standard curve. Lower valueof threshold cycle indicates a higher amount of PCR products.We used the following forward and reverse primers: Forward 5'-ACATCAAATCAGAACAGGGGAAC-3'and reverse 5'-CATGTCACGACCAGTCACAAC-3'; these correspond tothe nucleotide sequences N + 1319 to N + 1341 and N + 1453 toN + 1473 of Pax-2 cDNA (NM_003990). For internal control, wedeployed primers specific for mouse -actin (forward and reverseprimers 5'-CGTGCGTGACATCAAAGAGAA-3' and 5'-GCTCGTTGCCAATAGTGATGA-3',corresponding to nucleotide sequences N + 704 to N + 724 andN + 820 to N + 840 of mouse -actin cDNA [NM_007393]).47
Immunohistochemistry
Kidney explants were fixed in 4% paraformaldehyde in PBS (FisherScientific, Nepean, ON, Canada) after 24 h in culture and thenparaffin embedded. Kidney sections of 5 µm were deparaffinizedin xylene and rehydrated. Immunohistochemical examination wasperformed by the standard avidin-biotin-peroxidase complex method(ABC Staining System; Santa Cruz Biotechnologies, Santa Cruz,CA). Endogenous peroxidase was inhibited in 1% hydrogen peroxide–methanolfor 10 min at room temperature and followed by trypsin treatmentfor 10 min in a moist chamber at 37°C. After serum blocking,the sections were incubated with primary anti–Pax-2 polyclonalantibody diluted 1:100 overnight at 4°C humidity chamber,then biotinylated secondary antibody was added, followed bythe addition of preformed ABC reagents supplied by the ABC kit.The Pax-2 protein was visualized by color development with 3,3'-diaminobenzidinetetrahydrochloride. All sections were counterstained with hematoxylin,dehydrated, and covered with glass coverslips.
ROS Generation
ROS production was monitored by the lucigenin method with minormodifications.22,49,50 ROS that were generated in E18 kidneyexplant were normalized with protein concentration and expressedas relative light units per milligram of protein.
In Vivo Study
We induced gestational diabetes in pregnant Hoxb7-GFP mice withan intraperitoneal injection of STZ (150 mg/kg body wt) at E13.23–29Maternal glucose concentration (mM) was carefully monitoredby Accu-Chek Compact Plus Blood Glucose Meter (Roche Diagnostics,Laval, QC, Canada; Figure 10 A). Newborn birth weight was carefullyrecorded, as shown in Figure 10B. Hematoxylin/eosin stainingwas used to review renal morphology, whereas Dolichos BiflorusAgglutinin-FITC (Vector Laboratories) staining was used forUB identification15 in 5 µm of paraformaldehyde (4%)-fixed,paraffin-embedded kidney sections under a light microscope (Figure 10C).Pax-2 expression was analyzed by immunohistologic staining,qRT-PCR, and Western blot as mentioned previously (Figure 11).
Statistical Analyses
Statistical significance between experimental groups was analyzedinitially by t test or by one-way ANOVA followed by the Bonferronitest as appropriate. Three to four separate experiments wereperformed for each protocol. Data are expressed as means ±SD. P 0.05 was considered statistically significant.
This research was supported by KRESCENT and Kidney Foundationof Canada and a New Investigator Award from KRESCENT of KidneyFoundation of Canada for Shao-Ling Zhang.
We acknowledge the kind gifts of Hoxb7-GFP mice from Dr. FrankCostantini (Columbia University, New York, NY). Thanks are alsodue to Dr. John S.D. Chan (CHUM-Hôtel-Dieu, Montreal,QC, Canada) for unconditional support and discussion for theproject. The editorial assistance of Ovid Da Silva (ResearchSupport Office, Research Centre, CHUM) is acknowledged.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
B signaling pathway but not via protein kinase C, p38 mitogen-activated protein kinase and p44/42 mitogen-activated protein kinase signaling.22 
ROS
-actin mAb (clone AC-15) and rabbit polyclonal anti–Pax-2 antibody were purchased from Sigma-Aldrich Canada Ltd. and Covance (Richmond, CA), respectively. 
0.05 was considered statistically significant. 
