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

2008; Elsevier BV; Volume: 74; Issue: 10 Linguagem: Inglês

10.1038/ki.2008.378

ISSN

1523-1755

Autores

Ihor V. Yosypiv, Mary K. Boh, Melissa A. Spera, Samir S. El‐Dahr,

Tópico(s)

Urological Disorders and Treatments

Resumo

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. 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. The metanephros develops by reciprocal inductive interactions among the ureteric bud (UB), the metanephric mesenchyme (MM), and the stroma.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: 599-608Crossref PubMed Scopus (213) Google Scholar,2.Ekblom P. Developmentally regulated conversion of mesenchyme to epithelium.FASEB J. 1989; 3: 2141-2150Crossref PubMed Scopus (227) Google Scholar,3.Hatini A. Huh S.O. 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: 1467-1478Crossref PubMed Scopus (397) Google Scholar Branching morphogenesis involves UB outgrowth from the nephric duct followed by repetitive branching to form the renal collecting system (ureters, pelvis, calyces, and collecting ducts). In turn, emerging UB tips induce surrounding mesenchymal cells to form nephrons (from the glomerulus to the distal tubule). Even subtle decreases in the efficiency of UB branching result in a profound decrease in nephron endowment.4.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: 14-26Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Decreased nephron endowment is linked to hypertension and eventual progression to chronic renal failure.5.Brenner B.M. Garcia D.L. Anderson S. Glomeruli and blood pressure. Less of one, more the other?.Am J Hypertens. 1988; 1: 335-347Crossref PubMed Scopus (1004) Google Scholar,6.Lisle S.J. Lewis R.M. Petry C.J. et al.Effect of maternal iron restriction during pregnancy on renal morphology in the adult rat offspring.Br J Nutr. 2003; 90: 33-39Crossref PubMed Scopus (113) Google Scholar In addition, aberrant UB branching morphogenesis causes renal dysplasia, a leading cause of chronic renal failure in infants.7.North American Pediatric Renal Trials and Collaborative Studies NAPRTCS Annual report. 2006Google Scholar The GDNF/c-Ret/Wnt11 signaling pathway is a major positive regulator of UB branching in the metanephros.8.Brophy P.D. Ostrom L. Lang K.M. et al.Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.Development. 2001; 128: 4747-4756PubMed Google Scholar Glial-derived neurotrophic factor (GDNF) is released from the MM and interacts with the c-Ret tyrosine kinase receptor expressed in the UB tip cells to induce branching.9.Pachnis V. Mankoo B. Costantini F. Expression of the c-ret proto-oncogene during mouse embryogenesis.Development. 1993; 119: 1005-1017Crossref PubMed Google ScholarGDNF/c-Ret and Wnt11 cooperate genetically to induce branching morphogenesis.10.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: 3175-3185Crossref PubMed Scopus (357) Google Scholar 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.Basson M.A. Akbulut S. Watson-Johnson J. et al.Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction.Dev Cell. 2005; 8: 229-239Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar Genetic inactivation of Spry1 in mice results in ectopic UB outgrowth from the Wolffian duct, increased number of UB branches, and expanded GDNF, c-Ret, and Wnt-11 expression domains,11.Basson M.A. Akbulut S. Watson-Johnson J. et al.Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction.Dev Cell. 2005; 8: 229-239Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,12.Basson M.A. 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: 466-477Crossref PubMed Scopus (117) Google Scholar 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 (AT1R) antagonists during fetal life, as well as mutations in the genes encoding angiotensinogen, renin, ACE, or AT1R in humans are associated with renal tubular dysgenesis.13.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: 964-968Crossref PubMed Scopus (172) Google Scholar Inactivation of the renin–angiotensin system genes in mice causes abnormalities in the development of the ureter, renal pelvis, and papilla.14.Nagata M. Tanimoto K. Fukamizu A. et al.Nephrogenesis and renovascular development in angiotensinogen-deficient mice.Lab Invest. 1996; 75: 745-753PubMed Google Scholar,15.Takahashi N. Lopez M.L. Cowhig Jr, J.E. et al.Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type.J Am Soc Nephrol. 2005; 16: 125-132Crossref PubMed Scopus (109) Google Scholar,16.Esther Jr, C.R. Howard T.E. Marino E.M. et al.Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 7: 953-965Google Scholar,17.Oliverio M.I. Kim H.S. Ito M. et al.Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.Proc Natl Acad Sci USA. 1998; 95: 15496-15501Crossref PubMed Scopus (268) Google Scholar,18.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: 755-760Crossref PubMed Scopus (278) Google Scholar Angiotensinogen-, renin-, ACE-, or AT1R-deficient mice exhibit pelvic dilation (hydronephrosis) and a small papilla. Mutations in the AT2R gene are associated with increased incidence of lower urinary tract anomalies including double ureters and vesicoureteral reflux.19.Oshima K. Miyazaki Y. Brock J.W. et al.Angiotensin type II receptor expression and ureteral budding.J Urol. 2001; 166: 1848-1852Abstract Full Text Full Text PDF PubMed Google Scholar These findings indicate that UB growth and development is a major target for angiotensin (Ang) II actions. We have recently reported that Ang II, acting via the AT1R, stimulates UB 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 AT1R leading to UB branching.20.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar 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 UB tip cells. Furthermore, inhibition of AT1R signaling induces apoptosis preferentially in UB tip cells. The GDNF/c-Ret/Wnt11 signaling pathway is a major positive regulator of UB branching morphogenesis program.8.Brophy P.D. Ostrom L. Lang K.M. et al.Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.Development. 2001; 128: 4747-4756PubMed Google Scholar,21.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: 380-383Crossref PubMed Scopus (1348) Google Scholar,22.Moore M.W. Klein R.D. Farinas I. et al.Renal and neuronal abnormalities in mice lacking GDNF.Nature. 1996; 382: 76-79Crossref PubMed Scopus (1048) Google Scholar,23.Costantini F. Shakya R. GDNF/Ret signaling and the development of the kidney.Bioessays. 2006; 28: 117-127Crossref PubMed Scopus (222) Google Scholar In a previous study, we demonstrated that Ang II stimulates UB branching morphogenesis in E12.5 metanephric kidneys grown ex vivo.20.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar In the present study, we examined whether Ang II-induced UB 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 UB 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 UB branching.20.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar Spry proteins function as intracellular inhibitors of RTK signaling. Genetic inactivation of Spry1 in mice results in increased number of UB 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.Basson M.A. Akbulut S. Watson-Johnson J. et al.Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction.Dev Cell. 2005; 8: 229-239Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,12.Basson M.A. 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: 466-477Crossref PubMed Scopus (117) Google Scholar 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.Basson M.A. Akbulut S. Watson-Johnson J. et al.Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction.Dev Cell. 2005; 8: 229-239Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,12.Basson M.A. 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: 466-477Crossref PubMed Scopus (117) Google ScholarSpry1 mRNA was expressed in UB branches and to a lesser extent in condensing mesenchyme (Figure 2). Ang II treatment downregulated Spry1 expression in the UB and surrounding mesenchyme (Figure 2). These findings indicate that Ang II is an important regulator of Spry1 in the intact metanephros. 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 UB cells by quantitative real-time reverse-transcription polymerase chain reaction. Treatment of UB 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, P<0.01; n=3 per treatment group). We recently demonstrated that cultured UB cells maintain expression of Ang II AT1R mRNA.24.Iosipiv I.V. Schroeder M. A role for angiotensin II AT1 receptors in ureteric bud cell branching.Am J Physiol. 2003; 285: F199-F207PubMed Google Scholar Our present findings that Ang II downregulates Spry1 mRNA expression in the UB cell lineage indicate that Ang II-mediated inhibition of Spry1 gene expression may be involved in Ang II-induced UB branching. To examine the role of endogenous Ang II in the regulation of Spry1, we utilized the AT1R 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 AT1R. As candesartan treatment decreases UB branching,20.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar AT1R-mediated effect on Spry1 is physiologically important. To begin to understand the cellular events leading to stimulation of UB branching by Ang II, we examined the direct effects of Ang II on proliferation of the UB 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 UB tip cells (28.5±2.4 vs 9.7±1.2, P<0.001) but not in UB stalks (11.3±1.9 vs 9.3±1.2, P=0.4) as compared to control (Figure 3). These results demonstrate a preferential stimulatory effect of Ang II on UB cell proliferation in the tip cells and are consistent with the notion that growth factor-induced stimulation of UB branching is initiated by focal bursts of tip cell proliferation. Aberrant apoptosis is a cardinal feature of renal dysplasia and hypoplasia.25.Woolf A.S. Price K.L. Scambler P.J. et al.Evolving concepts in human renal dysplasia.J Am Soc Nephrol. 2004; 15: 998-1007Crossref PubMed Scopus (133) Google Scholar Genetic inactivation of angiotensinogen, renin, ACE, or AT1R in mice causes hypoplasia of the medulla and papilla,14.Nagata M. Tanimoto K. Fukamizu A. et al.Nephrogenesis and renovascular development in angiotensinogen-deficient mice.Lab Invest. 1996; 75: 745-753PubMed Google Scholar,15.Takahashi N. Lopez M.L. Cowhig Jr, J.E. et al.Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type.J Am Soc Nephrol. 2005; 16: 125-132Crossref PubMed Scopus (109) Google Scholar,16.Esther Jr, C.R. Howard T.E. Marino E.M. et al.Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 7: 953-965Google Scholar,17.Oliverio M.I. Kim H.S. Ito M. et al.Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.Proc Natl Acad Sci USA. 1998; 95: 15496-15501Crossref PubMed Scopus (268) Google Scholar,18.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: 755-760Crossref PubMed Scopus (278) Google Scholar which may be a result of excessive cell death in the UB derivatives. Accordingly, we examined the effect of AT1R antagonism on UB 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 UB tips but not in the stalks (tips: 0.12±0.08 vs 0.65±0.2, P<0.05; stalks: 0.88±0.19 vs 1.0±0.25, P=0.72; Figure 4). These findings indicate a preferential inhibitory role of endogenous Ang II and its AT1R on apoptosis in UB tip cells, suggesting a role for endogenous Ang II in epithelial cell survival during UB branching morphogenesis. 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 AT1R. In addition, Ang II induces preferential proliferation and provides a survival signal to UB tip cells. We recently reported that Ang II, acting via the AT1R, stimulates UB branching morphogenesis in the metanephric kidney cultured in vitro.20.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar Furthermore, we found that activation of the EGF RTK activity is a critical step in the signal transduction pathway downstream of the AT1R leading to UB branching.20.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar These results indicated that Ang II can directly stimulate UB branching, and that cross-talk between AT1R 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 UB morphogenesis in the developing kidney. GDNF is expressed in the MM,26.Hellmich H.L. Kos L. Cho E.S. 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: 95-105Crossref PubMed Scopus (198) Google Scholar whereas c-Ret is expressed along the nephric duct and subsequently in the UB tip cells.9.Pachnis V. Mankoo B. Costantini F. Expression of the c-ret proto-oncogene during mouse embryogenesis.Development. 1993; 119: 1005-1017Crossref PubMed Google Scholar Genetic inactivation of c-Ret or GDNF in mice leads to a complete absence of the UB or significant impairment of UB morphogenesis.21.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: 380-383Crossref PubMed Scopus (1348) Google Scholar,22.Moore M.W. Klein R.D. Farinas I. et al.Renal and neuronal abnormalities in mice lacking GDNF.Nature. 1996; 382: 76-79Crossref PubMed Scopus (1048) Google Scholar Like c-Ret, Wnt11 is expressed in the UB tip cells and interacts genetically with GDNF/c-Ret pathway to induce UB branching.10.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: 3175-3185Crossref PubMed Scopus (357) Google ScholarWnt11 expression is reduced in c-Ret−/− metanephroi, indicating that Wnt11 is a downstream target gene of c-Ret. UB branching and GDNF expression is decreased in Wnt11−/− metanephroi, indicating that both mesenchymal GDNF expression and UB tree morphogenesis are dependent on Wnt11 signal from UB tip cells.10.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: 3175-3185Crossref PubMed Scopus (357) Google Scholar Thus, the GDNF/c-Ret/Wnt11 pathway is a positive feedback loop that acts to stimulate proliferation of UB tip cells and thus promote further UB 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 UB tree morphogenesis. RTK signaling is tightly controlled by positive and negative regulators. Spry proteins function as intracellular inhibitors of RTK signaling.27.Mason J.M. Morrison D.J. Basson M.A. et al.Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling.Trends Cell Biol. 2006; 16: 45-54Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar Genetic inactivation of Spry1 in mice results in increased number of UB branches, and expansion of GDNF, c-Ret, and Wnt-11 expression domains.11.Basson M.A. Akbulut S. Watson-Johnson J. et al.Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction.Dev Cell. 2005; 8: 229-239Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,12.Basson M.A. 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: 466-477Crossref PubMed Scopus (117) Google Scholar Therefore, Spry1 is a physiological negative regulator of the GDNF/c-Ret/Wnt11 pathway. In the present 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 AT1R antagonism. On the basis of these findings, we propose that AT1R 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 AT1R-Spry1 interactions may involve clustering in lipid rafts. Caveolae/lipid rafts are essential for Ang II-induced transactivation of EGF receptor.28.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: 48269-48275Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar We have demonstrated that activation of AT1R by Ang II induces tyrosine phosphorylation of EGF receptor in UB cells.20.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar As Spry proteins rapidly translocate to lipid rafts following stimulation with EGF,29.Lim J. Wong E.S.M. Ong S.H. 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: 32837-32845Crossref PubMed Scopus (84) Google Scholar AT1R activation by Ang II may hinder association of Spry1 with RTK (EGF receptor, c-Ret) to prevent inhibition and stimulate UB branching. The balance of cell proliferation and apoptosis is important in UB branching and nephron endowment.30.Pepicelli C.V. Kispert A. Rowitch D.H. et al.GDNF induces branching and increased cell proliferation in the ureter of the mouse.Dev Biol. 1997; 192: 193-198Crossref PubMed Scopus (137) Google Scholar,31.Michael L. Davies J.A. Pattern and regulation of cell proliferation during murine ureteric bud development.J Anat. 2004; 204: 241-255Crossref PubMed Scopus (107) Google Scholar,32.Meyer T.N. Schwesinger C. Bush K.T. 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: 44-67Crossref PubMed Scopus (95) Google Scholar,33.Carev D. Krnić D. Saraga M. et al.Role of mitotic, proapoptotic and anti-apoptotic factors in human kidney development.Pediatr Nephrol. 2006; 21: 627-636Crossref PubMed Scopus (33) Google Scholar,34.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: 1568-1575Crossref PubMed Scopus (54) Google Scholar,35.Sheibani N. Scheef E.A. Dimaio T.A. et al.Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells.J Cell Physiol. 2007; 210: 616-625Crossref PubMed Scopus (24) Google Scholar Derangements of the regulatory mechanisms that control these events are implicated in the pathogenesis of renal hypodysplasia,25.Woolf A.S. Price K.L. Scambler P.J. et al.Evolving concepts in human renal dysplasia.J Am Soc Nephrol. 2004; 15: 998-1007Crossref PubMed Scopus (133) Google Scholar,34.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: 1568-1575Crossref PubMed Scopus (54) Google Scholar a leading cause of pediatric end-stage renal disease.7.North American Pediatric Renal Trials and Collaborative Studies NAPRTCS Annual report. 2006Google Scholar The present study demonstrates that Ang II causes preferential proliferation of UB tip cells, whereas inhibition of endogenous AT1R signaling inhibits UB tip cell apoptosis. As Ang II stimulates UB tip cell proliferation and both c-Ret and Wnt11 are expressed in the UB tip cells, it is likely that observed increase in Ret and Wnt11 expression by Ang II is due to in part enhanced UB tip cell proliferation. We speculate that Ang II induces focal bursts of proliferation of UB tip cells, and together with decreased apoptosis, plays an important role in the expansion of the ampulla, subsequent branching, and directional bud elongation. The mechanisms by which Ang II regulates UB 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 UB branching is observed in Bcl-2−/− mice.35.Sheibani N. Scheef E.A. Dimaio T.A. et al.Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells.J Cell Physiol. 2007; 210: 616-625Crossref PubMed Scopus (24) Google Scholar Moreover, Bcl-2 overexpression in the UB suppresses UB cell apoptosis, stimulates branching of the UB tree, and increases nephron endowment.34.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: 1568-1575Crossref PubMed Scopus (54) Google Scholar The finding that p53 or bax inactivation rescues both aberrant apoptosis and UB branching in salt-stressed bradykinin B2 receptor-null mice36.Fan H. Stefkova J. El-Dahr S.S. Susceptibility to metanephric apoptosis in bradykinin B2 receptor null mice via the p53–Bax pathway.Am J Physiol Renal Physiol. 2006; 291: F670-F682Crossref PubMed Scopus (11) Google Scholar provides further evidence that G-protein-coupled receptor signaling is intimately linked to cell survival in the metanephric kidney. Stimulation of AT1R by Ang II increases intracellular calcium and activates protein kinase C.37.Berry C. Touyz R. Dominiczak A.F. et al.Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide.Am J Physiol. 2001; 281: H2337-H2365PubMed Google Scholar 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-1.38.Watanabe G. Lee R.J. Albanese C. et al.Angiotensin II activation of cyclin D1-dependent kinase activity.J Biol Chem. 1996; 271: 22570-22577Crossref PubMed Scopus (123) Google Scholar Ang II may regulate these pathways directly or may favor the release of a mesenchymal factor, such as GDNF, which, in turn, stimulates UB tip cell proliferation30.Pepicelli C.V. Kispert A. Rowitch D.H. et al.GDNF induces branching and increased cell proliferation in the ureter of the mouse.Dev Biol. 1997; 192: 193-198Crossref PubMed Scopus (137) Google Scholar,31.Michael L. Davies J.A. Pattern and regulation of cell proliferation during murine ureteric bud development.J Anat. 2004; 204: 241-255Crossref PubMed Scopus (107) Google Scholar and migration.39.Tang M.J. Cai Y. Tsai S.J. et al.Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase.Dev Biol. 2002; 243: 128-136Crossref PubMed Scopus (112) Google Scholar,40.Kim D. Dressler G.R. PTEN modulates GDNF/RET mediated chemotaxis and branching morphogenesis in the developing kidney.Dev Biol. 2007; 307: 290-299Crossref PubMed Scopus (50) Google Scholar 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.Tang M.J. Cai Y. Tsai S.J. et al.Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase.Dev Biol. 2002; 243: 128-136Crossref PubMed Scopus (112) Google Scholar,40.Kim D. Dressler G.R. PTEN modulates GDNF/RET mediated chemotaxis and branching morphogenesis in the developing kidney.Dev Biol. 2007; 307: 290-299Crossref PubMed Scopus (50) Google Scholar We propose a model in which stimulation of GDNF and c-Ret by Ang II induces preferential proliferation and survival of UB tip cells leading to UB growth and branching (Figure 5). In summary, the present study demonstrates that Ang II, acting via the AT1R, 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 UB tip cells. These results support the hypothesis that abnormal collecting system development in angiotensinogen-, renin-, ACE- or AT1R-deficient mice is at least partly due to dysregulation of the UB branching morphogenesis program as well as aberrant UB cell proliferation and apoptosis. 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 μm) 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 AT1 receptor (AT1R) antagonist candesartan (10−6 M; Sigma, St Louis, MO, USA) for 24 h at 37 °C and 5% CO220.Yosypiv I.V. Schroeder M. El-Dahr S.S. Angiotensin II type 1 receptor-EGF receptor cross-talk regulates ureteric bud branching morphogenesis.J Am Soc Nephrol. 2006; 17: 1005-1014Crossref PubMed Scopus (43) Google Scholar 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). The effect of Ang II on the GDNF/c-Ret/Wnt11 pathway during UB 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 (http://www.hhmi.ucla.edu/derobertis/protocol_page/mouse.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, and digital images were captured using Adobe Photoshop software. Quantitative real-time reverse-transcription polymerase chain reaction was utilized to determine whether Ang II alters c-Ret, Wnt11, and Spry1 mRNA expression in UB cells (generously provided by Dr Jonathan Barasch, Columbia University). We have previously demonstrated that these cells express AT1R mRNA (Iosipiv, 2003). UB cells were grown in MEM medium (Gibco BRL) that contained 10% fetal bovine serum at 37 °C in an incubator with 5% CO2. Cells were starved overnight and treated with media (control, n=3) or Ang II (10−6 M, n=3) for 24 h at 37 °C and 5% CO2. The cells were used at passages 5–8. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA (3 μg) was reverse-transcribed in the presence of 100 ng random hexamers, 0.001 ml of 10 mM dNTP, 0.002 ml of 10 × RT buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2), and 200 U of Superscript II reverse transcriptase (Invitrogen) as previously described.24.Iosipiv I.V. Schroeder M. A role for angiotensin II AT1 receptors in ureteric bud cell branching.Am J Physiol. 2003; 285: F199-F207PubMed Google Scholar 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 μl with the 12.5 μl SYBR Green ER qPCR SuperMix (Invitrogen), 1 μl first-strand cDNA template and 1 μl primer set (10 μM each). The program conditions were: 95 °C, 10 min followed by 40 cycles of 95 °C, 15 s and 60 °C, 1 min. The quantity of each target mRNA expression was normalized by that of glyceraldehyde-3-phosphate dehydrogenase mRNA expression. Three UB cell RNA samples per treatment group were analyzed in duplicates in each run. PCR reaction was performed twice. To examine the role of cell proliferation in Ang II-induced UB 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=4) or Dulbecco's modified Eagle's medium/F12 medium alone (control, n=4) for 48 h at 37 °C. 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 °C, processed for paraffin embedding, and 4-μm-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% H2O2 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 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 UBs 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 AT1R in UB 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=10) or with AT1R antagonist candesartan (10−6 M, n=10) for 24 h at 37 °C. Apoptosis was assessed by TUNEL (TACS TdT Kit; R&D Systems, Minneapolis, MN). Following digestion with 20 μg/ml proteinase K for 15 min at room temperature, the sections were peroxidase quenched with 30% H2O2, 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 °C. 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 UB tip or stalk was determined in each kidney section (n=10 kidneys per group; three sections per kidney) and the mean number of TUNEL-positive cells per UB tip or stalk was calculated. 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 <0.05 was considered statistically significant. All the authors declared no competing interests. This work was supported by NIH grants P20 RR17659 and DK-71699 to I.V.Y. and DK-56264 and DK-62250 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.

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