Downregulation of Spry-1, an inhibitor of GDNF/Ret, causes angiotensin II-induced ureteric bud branching

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1 & 2008 International Society of Nephrology original article Downregulation of Spry-1, an inhibitor of GDNF/Ret, causes angiotensin II-induced ureteric bud branching Ihor V. Yosypiv 1, Mary K. Boh 1, Melissa A. Spera 1 and Samir S. El-Dahr 1 1 Section of Pediatric Nephrology, Department of Pediatrics, Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center, New Orleans, Louisiana, USA Mutations of genes in the renin-angiotensin system are associated with congenital abnormalities of the kidney and urinary tract. The major signaling pathway for branching morphogenesis during kidney development is the c-ret receptor tyrosine kinase whose ligand is GDNF and whose downstream target is Wnt11. We determined whether angiotensin II, an inducer of ureteric bud branching in vitro, influences the GDNF/c-Ret/Wnt11 pathway. Mouse metanephroi were grown in the presence or absence of angiotensin II or an angiotensin type 1 receptor (AT1R) antagonist and gene expression was measured by whole mount in situ hybridization. Angiotensin II induced the expression of c-ret and Wnt11 in ureteric bud tip cells. GDNF, a Wnt11-regulated gene expressed in the mesenchyme, was also upregulated by angiotensin II but this downregulated Spry1, an endogenous inhibitor of Ret tyrosine kinase activity in an AT1R-dependent manner. Angiotensin II also decreased Spry1 mrna levels in cultured ureteric bud cells. Exogenous angiotensin II preferentially stimulated ureteric bud tip cell proliferation in vivo while AT1R blockade increased cell apoptosis. Our findings suggest AT1R-mediated inhibition of the Spry1 gene increases c-ret tyrosine kinase activity leading to upregulation of its downstream target Wnt11. Enhanced Wnt11 expression induces GDNF in adjacent mesenchyme causing focal bursts of ureteric bud tip cell proliferation, decreased tip cell apoptosis and branching. Kidney International (2008) 74, ; doi: /ki ; published online 23 July 2008 KEYWORDS: ureteric bud; renin angiotensin; GDNF; c-ret; Spry1; branching morphogenesis Correspondence: Ihor V. Yosypiv, Section of Pediatric Nephrology, Department of Pediatrics, SL-37, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. iiosipi@tulane.edu Received 20 August 2007; revised 17 April 2008; accepted 20 May 2008; published online 23 July 2008 The metanephros develops by reciprocal inductive interactions among the ureteric bud (), the metanephric mesenchyme (MM), and the stroma. 1 3 Branching morphogenesis involves outgrowth from the nephric duct followed by repetitive branching to form the renal collecting system (ureters, pelvis, calyces, and collecting ducts). In turn, emerging tips induce surrounding mesenchymal cells to form nephrons (from the glomerulus to the distal tubule). Even subtle decreases in the efficiency of branching result in a profound decrease in nephron endowment. 4 Decreased nephron endowment is linked to hypertension and eventual progression to chronic renal failure. 5,6 In addition, aberrant branching morphogenesis causes renal dysplasia, a leading cause of chronic renal failure in infants. 7 The GDNF/c-Ret/Wnt11 signaling pathway is a major positive regulator of branching in the metanephros. 8 Glial-derived neurotrophic factor (GDNF) is released from the MM and interacts with the c-ret tyrosine kinase receptor expressed in the tip cells to induce branching. 9 GDNF/ c-ret and Wnt11 cooperate genetically to induce branching morphogenesis. 10 Uncontrolled activation of the GDNF/ c-ret/wnt11 pathway is prevented by Sprouty (Spry) proteins that function as intracellular inhibitors of receptor tyrosine kinase (RTK) signaling. 11 Genetic inactivation of Spry1 in mice results in ectopic outgrowth from the Wolffian duct, increased number of branches, and expanded GDNF, c-ret, and Wnt-11 expression domains, 11,12 indicating that Spry1 is a negative regulator of the GDNF/c-Ret/Wnt11 pathway. Exposure to angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT 1 R) antagonists during fetal life, as well as mutations in the genes encoding angiotensinogen, renin, ACE, or AT 1 R in humans are associated with renal tubular dysgenesis. 13 Inactivation of the renin angiotensin system genes in mice causes abnormalities in the development of the ureter, renal pelvis, and papilla Angiotensinogen-, renin-, ACE-, or AT 1 R- deficient mice exhibit pelvic dilation (hydronephrosis) and a small papilla. Mutations in the AT 2 R gene are associated with increased incidence of lower urinary tract anomalies including double ureters and vesicoureteral reflux. 19 These findings indicate that growth and development is a major target for angiotensin (Ang) II actions. Kidney International (2008) 74,

2 o r i g i n a l a r t i c l e IV Yosypiv et al.: Angiotensin II in kidney development We have recently reported that Ang II, acting via the AT 1 R, stimulates branching morphogenesis in the intact metanephric kidney cultured in vitro, and that activation of the epidermal growth factor (EGF) RTK activity is a critical step in the signal transduction pathway downstream of the AT 1 R leading to branching. 20 We report here that Ang II stimulates the GDNF/Ret/Wnt11 pathway indirectly by repression of Spry1. This effect is accompanied by increased proliferation of tip cells. Furthermore, inhibition of AT 1 R signaling induces apoptosis preferentially in tip cells. RESULTS Effect of Ang II on c-ret, Wnt11, and GDNF gene expression in the ex vivo cultured metanephric kidney The GDNF/c-Ret/Wnt11 signaling pathway is a major positive regulator of branching morphogenesis program. 8,21 23 In a previous study, we demonstrated that Ang II stimulates branching morphogenesis in E12.5 metanephric kidneys grown ex vivo. 20 In the present study, we examined whether Ang II-induced branching is accompanied by activation of the GDNF/c-Ret/Wnt11 pathway. Treatment with Ang II (10 5 M) for 24 h increased c-ret and Wnt11 mrna expression in the tip cells compared to control as determined by in situ hybridization (ISH; Figure 1). GDNF expression in the mesenchyme was also upregulated by Ang II. These data suggest that activation of this signaling pathway is critical in Ang II-mediated branching. 20 Effect of Ang II on Spry1 gene expression in the ex vivo cultured metanephric kidney and cells Spry proteins function as intracellular inhibitors of RTK signaling. Genetic inactivation of Spry1 in mice results in increased number of branches, and expanded GDNF, c-ret, and Wnt-11 expression domains, indicating that Spry1 is a negative regulator of the GDNF/c-Ret/Wnt11 pathway. 11,12 We therefore examined whether Ang II stimulates the GDNF/c-Ret/Wnt11 pathway indirectly by repression of Spry1. E12.5 wild-type metanephroi were treated with media or Ang II (10 5 M) for 24 h and processed for whole-mount ISH. As previously reported, 11,12 Spry1 mrna was expressed in branches and to a lesser extent in condensing mesenchyme (Figure 2). Ang II treatment downregulated Spry1 expression in the and surrounding mesenchyme (Figure 2). These findings indicate that Ang II is an important regulator of Spry1 in the intact metanephros. a c Figure 2 Angiotensin (Ang) II downregulates Spry1 mrna expression in E12.5 mouse metanephroi that were grown ex vivo for 24 h. After 24 h in culture, kidney explants were processed for whole-mount in situ hybridization. Representative images demonstrate that treatment with Ang II (10 5 M, b) decreases Spry1 mrna expression in the and in the mesenchyme compared to control (media, a). AT 1 R antagonist candesartan (d) abrogates Ang II-induced decrease in Spry1 gene expression (c, d). (c) Ang II alone (10 5 M); (d) Ang II (10 5 M) þ candesartan (10 5 M). b d c-ret Wnt11 GDNF Media Ang II Figure 1 Angiotensin (Ang) II upregulates c-ret, Wnt11, and GDNF mrna expression in E12.5 mouse metanephroi that were grown ex vivo for 24 h. After 24 h in culture, kidney explants were processed for whole-mount in situ hybridization. Representative images demonstrate that treatment with Ang II increased c-ret and Wnt11 expression in the tips and GDNF expression in the mesenchyme Kidney International (2008) 74,

3 IV Yosypiv et al.: Angiotensin II in kidney development o r i g i n a l a r t i c l e To confirm the observed effect of Ang II on Spry1 and to allow a more quantitative analysis of changes in Spry1 gene expression, we examined the effect of Ang II on Spry1 mrna levels in cultured cells by quantitative real-time reversetranscription polymerase chain reaction. Treatment of cells with Ang II (10 5 M) for 24 h resulted in a decrease of Spry1 mrna levels compared to control (0.66±0.03 vs 1.0±0.01, Po0.01; n ¼ 3 per treatment group). We recently demonstrated that cultured cells maintain expression of Ang II AT 1 R mrna. 24 Our present findings that Ang II downregulates Spry1 mrna expression in the cell lineage indicate that Ang II-mediated inhibition of Spry1 gene expression may be involved in Ang II-induced branching. To examine the role of endogenous Ang II in the regulation of Spry1, we utilized the AT 1 R antagonist, candesartan. Treatment of E12.5 metanephroi with candesartan (10 6 M) for 24 h abrogated Ang II-induced downregulation of Spry1 gene expression (Figure 2). The inhibitory effects of endogenous Ang II on Spry1 gene expression are therefore mediated by AT 1 R. As candesartan treatment decreases branching, 20 AT 1 R-mediated effect on Spry1 is physiologically important. Effect of Ang II on cell proliferation in the ex vivo cultured embryonic kidney To begin to understand the cellular events leading to stimulation of branching by Ang II, we examined the direct effects of Ang II on proliferation of the epithelium utilizing 5-bromo-2-deoxyuridine (BrdU) incorporation as an index of DNA synthesis in vivo. Treatment with Ang II (10 5 M) for 48 h increased cell proliferation index in the tip cells (28.5±2.4 vs 9.7±1.2, Po0.001) but not in stalks (11.3±1.9 vs 9.3±1.2, P ¼ 0.4) as compared to control (Figure 3). These results demonstrate a preferential stimulatory effect of Ang II on cell proliferation in the tip cells and are consistent with the notion that growth factorinduced stimulation of branching is initiated by focal bursts of tip cell proliferation. AT 1 R blockade promotes apoptosis in metanephric kidneys Aberrant apoptosis is a cardinal feature of renal dysplasia and hypoplasia. 25 Genetic inactivation of angiotensinogen, renin, ACE, or AT 1 R in mice causes hypoplasia of the medulla and papilla, which may be a result of excessive cell death in the derivatives. Accordingly, we examined the effect of AT 1 R antagonism on cell apoptosis in metanephroi grown ex vivo. Treatment of E13.5 metanephroi with candesartan (10 6 M) for 24 h significantly increased the number of terminal uridine triphosphate (UTP) end-labeling (TUNEL)-positive cells in the tips but not in the stalks (tips: 0.12±0.08 vs 0.65±0.2, Po0.05; stalks: 0.88±0.19 vs 1.0±0.25, P ¼ 0.72; Figure 4). These findings indicate a preferential inhibitory role of endogenous Ang II and its AT 1 R on apoptosis in tip cells, suggesting a role for endogenous Ang II in epithelial cell survival during branching morphogenesis. DISCUSSION The present study demonstrates that Ang II stimulates GDNF, c-ret, Wnt-11 gene expression, while inhibiting expression of Spry1 in the metanephric kidney cultured in vitro. This effect of Ang II on Spry1 is mediated by the AT 1 R. In addition, Ang II induces preferential proliferation and provides a survival signal to tip cells. We recently reported that Ang II, acting via the AT 1 R, stimulates branching morphogenesis in the metanephric kidney cultured in vitro. 20 Furthermore, we found that activation of the EGF RTK activity is a critical step in the signal transduction pathway downstream of the AT 1 R leading to branching. 20 These results indicated that Ang II can a b 40 c 40 d e Cell proliferation index P<0.001 Tips Ang II (10 5 M) Media P=0.4 Stalks Figure 3 Angiotensin (Ang) II stimulates proliferation of the ureteric bud () tip cells. Ang II-treated metanephroi (b, d) have more BrdU-positive cells (brown staining, arrows) in tips compared to control (media, a, c). (e) Ang II increases cell proliferation index in tip but not in stalk cells. Kidney International (2008) 74,

4 o r i g i n a l a r t i c l e IV Yosypiv et al.: Angiotensin II in kidney development a G b AT 1 R Ang II c-ret c 10 Number of TUNEL-positive cells per structure 1.4 Media ND Candesartan P< tip stalk Figure 4 The AT 1 R antagonist, candesartan, stimulates apoptosis in tip cells. Apoptotic cells were identified by TUNEL (brown staining). (a) Media, (b) candesartan (10 6 M), (c) kidney treated with TACS-nuclease to generate DNA breaks in every cell (positive control); (d) bar graph shows the effect of media or candesartan on the number of TUNEL-positive cells in the tip and stalk cells. G, glomerulus;, ureteric bud; ND, not different. (b) TUNEL-positive cells in are indicated by arrows. directly stimulate branching, and that cross-talk between AT 1 R and RTK signaling is important in the development of the renal collecting system. c-ret is an RTK that is activated by GDNF and is crucial in morphogenesis in the developing kidney. GDNF is expressed in the MM, 26 whereas c-ret is expressed along the nephric duct and subsequently in the tip cells. 9 Genetic inactivation of c-ret or GDNF in mice leads to a complete absence of the or significant impairment of morphogenesis. 21,22 Like c-ret, Wnt11 is expressed in the tip cells and interacts genetically with GDNF/c-Ret pathway to induce branching. 10 Wnt11 expression is reduced in c- Ret / metanephroi, indicating that Wnt11 is a downstream target gene of c-ret. branching and GDNF expression is decreased in Wnt11 / metanephroi, indicating that both mesenchymal GDNF expression and tree morphogenesis are dependent on Wnt11 signal from tip cells. 10 Thus, the GDNF/c-Ret/Wnt11 pathway is a positive feedback loop that acts to stimulate proliferation of tip cells and thus promote further growth and branching. Our present findings that Ang II enhances GDNF, c-ret, and Wnt11 expression indicate that activation of this pathway by Ang II is important in Ang II-mediated signaling to stimulate tree morphogenesis. RTK signaling is tightly controlled by positive and negative regulators. Spry proteins function as intracellular inhibitors of RTK signaling. 27 Genetic inactivation of Spry1 in mice results in increased number of branches, and expansion of GDNF, c-ret, and Wnt-11 expression domains. 11,12 Therefore, Spry1 is a physiological negative regulator of the GDNF/c-Ret/Wnt11 pathway. In the present d Branching GDNF Increased tip cell proliferation Decreased tip cell apoptosis Ureteric bud Spry1 Erk1/2 PI3K PLC Wnt11 c-ret Figure 5 A proposed model for Ang II-induced branching morphogenesis. Ang II AT 1 R-mediated inhibition of Spry1 gene expression releases c-ret tyrosine kinase activity leading to upregulation of c-ret and its downstream target gene, Wnt-11. Enhanced Wnt-11 expression, in turn, induces GDNF expression in the adjacent mesenchyme. This causes focal bursts of tip cell proliferation and branching. Decreased tip cell apoptosis may also contribute to Ang II-induced branching. The mechanisms of Spry1 inhibition by Ang II remain to be determined. study, we found that exogenous Ang II suppresses Spry1 gene expression in cultured embryonic kidneys. Thus, Ang II may stimulate the GDNF/c-Ret/Wnt11 pathway indirectly by repression of Spry1. Moreover, Ang II-induced downregulation of Spry1 expression is abrogated by AT 1 R antagonism. On the basis of these findings, we propose that AT 1 R signaling negatively regulates Spry1 gene expression. This in turn facilitates c-ret RTK signaling leading to activation of the GDNF/Ret/Wnt11 positive feedback loop (Figure 5). The mechanism of AT 1 R-Spry1 interactions may involve clustering in lipid rafts. Caveolae/lipid rafts are essential for Ang IIinduced transactivation of EGF receptor. 28 We have demonstrated that activation of AT 1 R by Ang II induces tyrosine phosphorylation of EGF receptor in cells. 20 As Spry proteins rapidly translocate to lipid rafts following stimulation with EGF, 29 AT 1 R activation by Ang II may hinder association of Spry1 with RTK (EGF receptor, c-ret) to prevent inhibition and stimulate branching. The balance of cell proliferation and apoptosis is important in branching and nephron endowment Derangements of the regulatory mechanisms that control these events are implicated in the pathogenesis of renal hypodysplasia, 25,34 a leading cause of pediatric end-stage renal disease. 7 The present study demonstrates that Ang II causes preferential proliferation of tip cells, whereas inhibition of endogenous AT 1 R signaling inhibits tip cell apoptosis. As Ang II stimulates tip cell proliferation and both c-ret and Wnt11 are expressed in the tip cells, it is likely that observed increase in Ret and Wnt11 expression by Ang II is due to in part enhanced tip cell proliferation. We speculate that Ang II induces focal bursts of proliferation of tip cells, and together with decreased apoptosis, plays an important role in the expansion of the ampulla, subsequent branching, and directional bud elongation Kidney International (2008) 74,

5 IV Yosypiv et al.: Angiotensin II in kidney development o r i g i n a l a r t i c l e The mechanisms by which Ang II regulates tip cell proliferation and apoptosis are not known. Potential mechanisms include upregulation of antiapoptotic (Bcl-2) and downregulation of proapoptotic (bax, p53) factors. In this regard, decreased branching is observed in Bcl-2 / mice. 35 Moreover, Bcl-2 overexpression in the suppresses cell apoptosis, stimulates branching of the tree, and increases nephron endowment. 34 The finding that p53 or bax inactivation rescues both aberrant apoptosis and branching in salt-stressed bradykinin B2 receptor-null mice 36 provides further evidence that G-protein-coupled receptor signaling is intimately linked to cell survival in the metanephric kidney. Stimulation of AT 1 R by Ang II increases intracellular calcium and activates protein kinase C. 37 Activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway by protein kinase C stimulates transcription of cell-cycle progression genes, such as cyclin D1, through activation of the transcription factor AP Ang II may regulate these pathways directly or may favor the release of a mesenchymal factor, such as GDNF, which, in turn, stimulates tip cell proliferation 30,31 and migration. 39,40 Recent data indicate that GDNF-induced migration of Ret-transfected MDCK cells is critically dependent on Ret and its downstream signaling via the PI3 kinase pathway. 39,40 We propose a model in which stimulation of GDNF and c-ret by Ang II induces preferential proliferation and survival of tip cells leading to growth and branching (Figure 5). In summary, the present study demonstrates that Ang II, acting via the AT 1 R, downregulates Spry1 and upregulates GDNF/Ret/Wnt11 gene expression in the metanephros. The stimulatory effects of Ang II on the GDNF/Ret/Wnt11 pathway are accompanied by preferential proliferation and survival of tip cells. These results support the hypothesis that abnormal collecting system development in angiotensinogen-, renin-, ACE- or AT 1 R-deficient mice is at least partly due to dysregulation of the branching morphogenesis program as well as aberrant cell proliferation and apoptosis. MATERIALS AND METHODS Metanephric organ culture Wild-type CD1 mice embryos (Charles River Laboratories, New York, NY) were dissected aseptically from the surrounding tissues on E12. 5 and the metanephroi were isolated. The day when the vaginal plug was observed was considered to be E0.5. Metanephroi were grown on air fluid interface on polycarbonate transwell filters (Corning Costar, 0.5 mm) inserted into six-well plates containing Dulbecco s modified Eagle s medium/f12 medium (Gibco BRL, Carlsbad, CA, USA) alone or in the presence of Ang II (10 5 M) alone or combined with the AT 1 receptor (AT 1 R) antagonist candesartan (10 6 M; Sigma, St Louis, MO, USA) for 24 h at 37 1C and 5% CO 2 20 and then processed for the whole-mount ISH. The effect of drug treatment was studied in paired kidneys obtained from the same fetus (that is, left kidney was incubated with media and right kidney with Ang II or left kidney with media and right kidney with candesartan). In situ hybridization The effect of Ang II on the GDNF/c-Ret/Wnt11 pathway during branching was examined by whole-mount ISH. c-ret, GDNF, Wnt11, and Spry1 cdnas were kind gifts from Dr F. Costantini, Dr A. McMahon, and Dr J.D. Licht, respectively. Preparation of RNA probes and whole-mount ISH were performed according to protocols ( PDF) established in the De Robertis Laboratory. Five embryonic kidneys per treatment group per probe were examined. All experiments were done at least twice. The metanephroi were photographed using an Olympus model SC35 camera mounted on an Olympus model BH-2 microscope,anddigitalimageswerecapturedusingadobephotoshop software. Quantitative real-time reverse-transcription polymerase chain reaction Quantitative real-time reverse-transcription polymerase chain reaction was utilized to determine whether Ang II alters c-ret, Wnt11, and Spry1 mrna expression in cells (generously provided by Dr Jonathan Barasch, Columbia University). We have previously demonstrated that these cells express AT 1 R mrna (Iosipiv, 2003). cells were grown in MEM medium (Gibco BRL) that contained 10% fetal bovine serum at 37 1C in an incubator with 5% CO 2. Cells were starved overnight and treated with media (control, n ¼ 3) or Ang II (10 6 M, n ¼ 3) for 24 h at 37 1C and 5% CO 2. The cells were used at passages 5 8. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA (3 mg) was reversetranscribed in the presence of 100 ng random hexamers, ml of 10 mm dntp, ml of 10 RT buffer (200 mm Tris-HCl (ph 8.4), 500 mm KCl, 15 mm MgCl 2 ), and 200 U of Superscript II reverse transcriptase (Invitrogen) as previously described. 24 SYBR Green quantitative real-time reverse-transcription polymerase chain reaction was conducted in the Mx3000P equipment (Stratagene, La Jolla, CA) using MxPro QPCR software (Stratagene). Mouse Spry1 gene-specific primers obtained from SuperArray (Frederick, MD). Each PCR reaction was run in 25 ml with the 12.5 ml SYBR Green ER qpcr SuperMix (Invitrogen), 1 ml firststrand cdna template and 1 ml primer set (10 mm each). The program conditions were: 95 1C, 10 min followed by 40 cycles of 95 1C, 15 s and 60 1C, 1 min. The quantity of each target mrna expression was normalized by that of glyceraldehyde-3-phosphate dehydrogenase mrna expression. Three cell RNA samples per treatment group were analyzed in duplicates in each run. PCR reaction was performed twice. Cell proliferation and apoptosis assays To examine the role of cell proliferation in Ang II-induced branching, we examined the effect of exogenous Ang II on in vivo incorporation of BrdU. CD1 mice metanephroi isolated on E11.5 were grown on filters in the presence of Ang II (10 5 M, n ¼ 4) or Dulbecco s modified Eagle s medium/f12 medium alone (control, n ¼ 4) for 48 h at 37 1C. BrdU (10 4 M; Sigma) was added to the media during the last 6 h of incubation. The kidneys were fixed in 10% neutral buffered formalin overnight at 4 1C, processed for paraffin embedding, and 4-mm-thick sections were cut. Slides were deparaffinized in two exchanges of xylene and rehydrated in a series of graded ethanol. After quenching of endogenous peroxidase with 30% H 2 O 2 and trypsin digestion, the sections were treated with blocking solution and sequentially incubated with biotinylated mouse anti-brdu antibody (Sigma; 1:50), streptavidin-peroxidase substrate, and stained with diaminobenzidine (Zymed, San Kidney International (2008) 74,

6 o r i g i n a l a r t i c l e IV Yosypiv et al.: Angiotensin II in kidney development Francisco, CA). The slides were counterstained with hematoxylin, mounted, and coverslipped. The number of BrdU-positive (brown) and -negative (blue) cells was determined in four randomly selected s of each kidney section by light microscopy. Cell proliferation index (percentage of BrdU-positive cells) was calculated from the ratio of BrdU-positive to total nuclei. To investigate the role of endogenous Ang II and AT 1 R in cell apoptosis, E12.5 CD1 mice metanephroi were grown on filters in the presence of Dulbecco s modified Eagle s medium/f12 medium alone (n ¼ 10) or with AT 1 R antagonist candesartan (10 6 M, n ¼ 10) for 24 h at 37 1C. Apoptosis was assessed by TUNEL (TACS TdT Kit; R&D Systems, Minneapolis, MN). Following digestion with 20 mg/ml proteinase K for 15 min at room temperature, the sections were peroxidase quenched with 30% H 2 O 2, and the TUNEL labeling reaction mixture was added to cover each section. The slides were then incubated in a humidified chamber for 60 min at 37 1C. The reaction was stopped by a stop buffer. The slides were counterstained with 0.5% methyl green and examined by light microscopy. The number of TUNEL-positive cells per tip or stalk was determined in each kidney section (n ¼ 10 kidneys per group; three sections per kidney) and the mean number of TUNEL-positive cells per tip or stalk was calculated. Statistical analysis Differences among the treatment groups in Spry1 mrna levels and the number of BrdU- and TUNEL-positive cells in media vs Ang II or candesartan vs Ang II were analyzed by Student s t-test. A P-value of o0.05 was considered statistically significant. DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS This work was supported by NIH grants P20 RR17659 and DK to I.V.Y. and DK and DK to S.E.D. We thank Dr Frank Costantini (Columbia University Medical Center), Andrew P. McMahon (Harvard University) and Jonathan D. Licht (Northwestern University) for providing the probes for in situ hybridization, and Dr Renfang Song for help with qpcr. REFERENCES 1. Aufderheide E, Chiquet-Ehrismann R, Ekblom P. Epithelial mesenchymal interactions in the developing kidney lead to expression of tenascin in the mesenchyme. J Cell Biol 1987; 105: Ekblom P. Developmentally regulated conversion of mesenchyme to epithelium. FASEB J 1989; 3: Hatini A, Huh SO, Herzlinger D et al. Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes Dev 1996; 10: Sakurai H, Nigam S. In vitro branching tubulogenesis: implications for developmental and cystic disorders, nephron number, renal repair, and nephron engineering. Kidney Int 1998; 54: Brenner BM, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens 1988; 1: Lisle SJ, Lewis RM, Petry CJ et al. Effect of maternal iron restriction during pregnancy on renal morphology in the adult rat offspring. Br J Nutr 2003; 90: North American Pediatric Renal Trials and Collaborative Studies. NAPRTCS Annual report Brophy PD, Ostrom L, Lang KM et al. Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene. Development 2001; 128: Pachnis V, Mankoo B, Costantini F. Expression of the c-ret protooncogene during mouse embryogenesis. Development 1993; 119: Majumdar A, Vainio S, Kispert A et al. Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development. Development 2003; 130: Basson MA, Akbulut S, Watson-Johnson J et al. Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction. Dev Cell 2005; 8: Basson MA, Watson-Johnson J, Shakya R et al. Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1. Dev Biol 2006; 299: Gribouval O, Gonzales M, Neuhaus T et al. Mutations in genes in the renin angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Nat Genet 2005; 37: Nagata M, Tanimoto K, Fukamizu A et al. Nephrogenesis and renovascular development in angiotensinogen-deficient mice. Lab Invest 1996; 75: Takahashi N, Lopez ML, Cowhig Jr JE et al. Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type. J Am Soc Nephrol 2005; 16: Esther Jr CR, Howard TE, Marino EM et al. Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 1996; 7: Oliverio MI, Kim HS, Ito M et al. Reduced growth, abnormal kidney structure, and type 2 (AT 2 ) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT 1A and AT 1B receptors for angiotensin II. Proc Natl Acad Sci USA 1998; 95: Tsuchida S, Matsusaka T, Chen X et al. Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes. J Clin Invest 1998; 101: Oshima K, Miyazaki Y, Brock JW et al. Angiotensin type II receptor expression and ureteral budding. J Urol 2001; 166: Yosypiv IV, Schroeder M, El-Dahr SS. Angiotensin II type 1 receptor-egf receptor cross-talk regulates ureteric bud branching morphogenesis. J Am Soc Nephrol 2006; 17: Schuchardt A, D Agati V, Larsson-Blomberg L et al. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 1994; 367: Moore MW, Klein RD, Farinas I et al. Renal and neuronal abnormalities in mice lacking GDNF. Nature 1996; 382: Costantini F, Shakya R. GDNF/Ret signaling and the development of the kidney. Bioessays 2006; 28: Iosipiv IV, Schroeder M. A role for angiotensin II AT1 receptors in ureteric bud cell branching. Am J Physiol 2003; 285: F199 F Woolf AS, Price KL, Scambler PJ et al. Evolving concepts in human renal dysplasia. J Am Soc Nephrol 2004; 15: Hellmich HL, Kos L, Cho ES et al. Embryonic expression of glial cell-line derived neurotrophic factor (GDNF) suggests multiple developmental roles in neural differentiation and epithelial mesenchymal interactions. Mech Dev 1996; 54: Mason JM, Morrison DJ, Basson MA et al. Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling. Trends Cell Biol 2006; 16: Ushio-Fukai M, Hilenski L, Santanam N et al. Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling. J Biol Chem 2001; 276: Lim J, Wong ESM, Ong SH et al. Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain. J Biol Chem 2000; 275: Pepicelli CV, Kispert A, Rowitch DH et al. GDNF induces branching and increased cell proliferation in the ureter of the mouse. Dev Biol 1997; 192: Michael L, Davies JA. Pattern and regulation of cell proliferation during murine ureteric bud development. J Anat 2004; 204: Meyer TN, Schwesinger C, Bush KT et al. Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney. Dev Biol 2004; 275: Carev D, Krnić D, Saraga M et al. Role of mitotic, proapoptotic and antiapoptotic factors in human kidney development. Pediatr Nephrol 2006; 21: Dziarmaga A, Eccles M, Goodyer P. Suppression of ureteric bud apoptosis rescues nephron endowment and adult renal function in Pax2 mutant mice. J Am Soc Nephrol 2006; 17: Kidney International (2008) 74,

7 IV Yosypiv et al.: Angiotensin II in kidney development o r i g i n a l a r t i c l e 35. Sheibani N, Scheef EA, Dimaio TA et al. Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells. J Cell Physiol 2007; 210: Fan H, Stefkova J, El-Dahr SS. Susceptibility to metanephric apoptosis in bradykinin B2 receptor null mice via the p53 Bax pathway. Am J Physiol Renal Physiol 2006; 291: F670 F Berry C, Touyz R, Dominiczak AF et al. Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide. Am J Physiol 2001; 281: H2337 H Watanabe G, Lee RJ, Albanese C et al. Angiotensin II activation of cyclin D1-dependent kinase activity. J Biol Chem 1996; 271: Tang MJ, Cai Y, Tsai SJ et al. Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase. Dev Biol 2002; 243: Kim D, Dressler GR. PTEN modulates GDNF/RET mediated chemotaxis and branching morphogenesis in the developing kidney. Dev Biol 2007; 307: Kidney International (2008) 74,