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Role of angiotensin in renal vascular development
Kidney International, Vol. 54, Suppl. 67 (1998), pp. S-12–S-16
Role of angiotensin in renal vascular development R. ARIEL GOMEZ Department of Pediatrics, University of Virginia, School of Medicine, Charlottesville, Virginia, USA
Role of angiotensin in renal vascular development. All components of the renin-angiotensin system (RAS) are expressed in the developing kidney in a temporospatial pattern that suggests a role for this system in kidney morphogenesis. Pharmacological blockade of angiotensin actions in fetal and newborn animals results in striking alterations in kidney architecture, including immature glomeruli and papillae, dilated tubuli, and arrested vascular development. Inactivation of angiotensinogen or angiotensin converting enzyme genes in mice results in similar anomalies that begin as subtle alterations in early life and become more pronounced as extrauterine life progresses. However, inactivation of each angiotensin receptor subtype does not result in obvious morphological abnormalities, suggesting functional redundancy at the receptor level. Crossing of mice lacking the various receptor subtypes should be revealing. Overall, the available information suggests that the RAS is necessary for the normal morphological and functional development of the kidney and the preservation of kidney architecture in adult life.
The definitive mammalian kidney, the metanephros, results from coinductive interaction between the primitive ureter and the metanephric blastema. The differentiating ureter arising from the mesonephric (Wolffian) duct induces the transformation of the metanephric mesenchyma into tubular and glomerular epithelium [1]. In turn, the induced mesenchyma stimulates the ureter to continue to differentiate and branch within the renal mesenchyma. As the ureter branches, the mesenchymal cells condense around each ureteric tip. The condensate develops into a vesicle, followed by a comma-shaped body. The creation of a lower cleft into the latter structure results in the formation of the S-shaped glomerulus. The formation of the S-shaped glomerulus seems to be critical for glomerular vascularization, because at this stage, endothelial cell precursors invade the lower cleft and form the glomerular capillaries. At this stage, proximal and distal tubules can be distinguished readily. Further tubular and glomerular growth results in the formation of a nephron with adult characteristics. In humans, nephrogenesis starts around the
Key words: angiotensin converting enzyme, extrauterine nephrogenesis, glomerulus, metanephros, receptors, renin-angiotensin system, Wolffian duct.
© 1998 by the International Society of Nephrology
fifth week of gestation and culminates around weeks 32 to 36. Thus, full-term infants are born with a full complement of nephrons. In mice and rats, the metanephros is visible around the embryonic (E) days 11 to 12 and 13 to 14 (term: 21 to 22 days), respectively, and continues in postnatal life. Thus, the neonatal mice and rat kidneys are useful models to study extrauterine nephrogenesis. VASCULAR DEVELOPMENT Precise and timely assembly of the kidney vasculature in concert with the developing nephron is fundamental for the development of a kidney capable of sustaining independent, extrauterine life. Kidney vascularization and nephrogenesis are simultaneous and coordinated processes [2]. The embryonic origin and mechanism underlying blood vessel formation in the kidney are still under debate. Endothelial cells may differentiate in situ from local mesenchymal cell precursors (vasculogenesis), or they may originate and sprout from pre-existing extrarenal or intrarenal vessel (angiogenesis) and migrate into the developing nephron [3, 4]. Both paradigms are not mutually exclusive and are supported by available experimental evidence. Precursors for smooth muscle, endothelial, and reninexpressing cells are present in the embryonic rat kidney well before vessel formation can be detected [5–7]. These precursor cells have the potential to differentiate and participate in the assembly of the arteriolar vessel wall. Once the arteriolar vessels are formed, the anatomical arrangement of the kidney vasculature is as follows: In the rat, a single renal artery divides at the level of the hilum into three main lobar arteries. Each lobar artery is continued by corticomedullary (arcuate) arteries that course through the corticomedullary junction parallel to the kidney cortex. From the arcuate arteries, numerous corticoradial (interlobular) arteries originate that in turn give off the afferent arterioles of the glomeruli. As expected, this basic blueprint of arteriolar development is already established in the fetal rat by E20. As maturation continues, the arterial tree grows in complexity and surface area. This is achieved mainly by a progressive and continued increase in the number of small arterial branches, especially interlobular arteries and afferent arterioles [7]. In fact, in the developing rat, arteriolar branching is very rapid up to day
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Gomez: Angiotensin and renal development
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Fig. 1. Expression of angiotensin receptor subtypes AT1 and AT2 during nephrogenesis. AT1 is represented by the hatched areas and AT2 by the gray areas. The density of pattern indicates the intensity of expression. (A) Embryonic day 14 (E14) metanephros showing intense expression of AT2 (right side) and sparse expression of AT1 throughout the renal mesenchyma. (B) E15 to E20 metanephros showing persistence of AT2 in mesenchyma and termination in epithelial structures while AT1 expression increases in more mature nephrons. (C) Postnatal days 7 to 15 showing the gradient of maturing nephrons. AT2 predominates in the differentiating areas, whereas AT1 predominates in more mature juxtamedullary nephrons. (D) Adult kidney showing the persistence of AT1 in mature glomeruli and tubules. AT2 is not detectable at this stage. Taken from [16] with permission from the American Society of Physiology.
10 postnatum (P10), while nephrogenesis is still occurring. The mechanisms that control arteriolar branching are not well understood and currently are being investigated. As discussed later here, components of the renin-angiotensin system (RAS) seem to play a role in this process. RAS AND VASCULAR DEVELOPMENT IN THE KIDNEY All the components of the RAS are expressed in the developing kidney [8]. In the rat metanephros, reninexpressing cells are found as early as day E14, before vessel or nephron formation has taken place. At E19, these cells are found along arcuate and interlobular arteries [9, 10]. As the kidney continues its development through fetal and postnatal life, renin distribution shifts from the larger arteries in the fetus to its mature juxtaglomerular localization in the adult animal [9]. These developmental patterns of renin expression have been observed in every species examined thus far [8]. In addition, we have recently observed an association between renin-expressing cells and branching of renal arterioles, suggesting a role for these cells in renal angiogenesis [10]. Angiotensinogen (Atg), the only known precursor of angiotensin (Ang) peptides, is detected in the fetal rat kidney by E17 to E18 [8, 11, 12]. However, the renal expression of Atg is highest around the
time of birth. In newborn rats, Atg is expressed in proximal tubules, mesangial cells, and vascular bundles. Ang-converting enzyme (ACE) is seen in endothelial precursors invading the lower cleft of the S-shaped body, in glomerular capillaries, corticomedullary arteries, proximal tubules, and peritubular capillaries [13, 14]. In addition to ACE, other proteases are capable of generating Ang in the developing kidney [15]. The high expression of components of the RAS in the developing kidney is reflected functionally by its higher levels of ANG compared with the adult rat kidney [15]. ANG receptors are also expressed in the maturing kidney in a characteristic developmental pattern [16, 17]. In the rat, both Ang receptor subtypes (AT1 and AT2) are expressed throughout metanephric development, albeit in differing temporal and spatial patterns (Fig. 1). At E14, when the ureteric bud is beginning to branch and nephrons and vessels have not yet been formed, AT2 receptors are more abundant and are present in the undifferentiated metanephric blastema. AT1 receptors are also present in the mesenchyma, although in lesser numbers (Fig. 1). At E17, nephrogenesis and vascularization are both occurring actively. A sagittal section of the kidney at this stage shows numerous glomeruli and arterioles at various stages of maturation, with the more mature glomeruli localized deeper within the cortex. At this stage, AT1
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Gomez: Angiotensin and renal development
Fig. 2. Effect of deletion of the gene for angiotensin converting enzyme (ACE) on the renal arterial tree. Left: wild type (ACE1/1); right knock-out (ACE2/2) mice. Both vascular trees are stained with renin antibody (dark brown color). The arterioles in the ACE2/2 mice are thickened and sparse compared with the delicate arterioles in the wild-type. Renin staining is limited to the end of afferent arterioles in ACE1/1 but is distributed broadly along the vascular tree in ACE2/2 mice. Taken from [8] with permission.
receptors are found in the more mature glomeruli, whereas AT2 receptors are present in condensed mesenchyme and (transiently) in newly formed nephrons (Fig. 1). At E20, the kidney architecture is better defined, showing distinct medullary and cortical regions; there is still an outer nephrogenic zone. AT1 and AT2 are equally prevalent within the outer medullary stripe, the more developed glomeruli, large arteries, and the medullary rays. AT2 is abundant in the medullary rays, the nephrogenic cortex, and the columns of Bertin. After birth, AT1 predominates in more mature juxtamedullary nephrons, and AT2 continues to be expressed in differentiating structures. As nephron maturation is achieved, AT2 expression is down-regulated until it disappears after the second postnatal week (Fig. 1). In contrast, as nephrons differentiate, AT1 expression increases and becomes maximal around the first postnatal week. By the end of 1 month of postnatal life, AT1 expression is similar to that of the adult (Fig. 1). The pattern of receptor expression shown previously here has been documented in several animal species, suggesting unique and perhaps opposite functions of the two receptors. Overall, the expression pattern of RAS components during ontogeny suggests a role for this system in kidney development, as discussed later here. Blockade of the AT1 receptor in newborn rats up to P12 results in striking alterations in the developing kidney architecture [18]. In addition to a reduced number of immature glomeruli and dilated tubules, the renal arterioles are thick, short, and markedly reduced in numbers. Papillary atrophy that correlates functionally with a decreased urinary concentrating capacity and inability to excrete an acute volume load properly is found also [19, 20]. Interestingly, these functional abnormalities persist into adulthood, even if the treatment is stopped after P23.
Pigs treated neonatally with ACE inhibitors develop similar morphological and functional abnormalities [21]. Fetal anuria and renal abnormalities have also been described in humans treated with ACE inhibitors [22]. More severe renal-vascular abnormalities have also been noted in frog tadpoles receiving ACE inhibitors during their metamorphosis [18], suggesting that the mesonephric kidney of frogs is more susceptible to blockers of the RAS. Contrary to the findings with ACE and AT1 inhibitors, treatment of neonatal animals with AT2 blockers does not seem to induce any developmental abnormalities in vivo. Treatment of weaning rats with losartan does not induce renal abnormalities [23]. These findings suggest that the developing rat kidney is susceptible to RAS blockade mainly during the last five days of pregnancy and during the first two to three postnatal weeks [24]. As discussed earlier here, this period is associated with high expression of renin, Atg, and AT1 receptors. Presumably, the human kidney is more susceptible to the deleterious effects of RAS blockers during the last weeks of pregnancy. Contrary to the results obtained with pharmacological inhibitors of the RAS, targeted inactivation of each of the AT1 receptors (AT1A, AT1B) and AT2 receptors does not result in alterations in morphological kidney development [25–30], suggesting that the contribution of each individual receptor to kidney morphogenesis is limited or that it can be compensated by the action of the other Ang receptor subtypes. It would be interesting to determine whether mice with inactivation of both receptor subtypes (AT1A and AT1B) develop renal and/or vascular abnormalities. Targeted inactivation of Atg and ACE genes, however, produces remarkably similar renal abnormalities in adult mice, consisting of diminished numbers of thickened, hypercellular renal arterioles (Fig. 2), interstitial fibrosis,
Gomez: Angiotensin and renal development
papillary atrophy, and tubular dilatation [31–33]. In both knockout models, the abnormalities are obvious by P7 to P10 and become more pronounced with increasing age. There is disagreement as to whether abnormalities are seen at earlier stages in life: Nagata et al [33] found no effect of lack of Atg on nephrogenesis, whereas Niimura et al [32] reported delay in glomerular maturation and smaller glomeruli in Atg2/2 mice. Consistent with the latter, we find subtle alterations in interstitial architecture at E18. Arrest in vascular and glomerular development is already obvious by P7. These studies clearly demonstrate that both the ACE and Atg genes are necessary for the preservation of normal kidney structure during development and in adult life. Whereas in rats treated with RAS blockers and in mutant mice, there is disagreement as to whether glomerular development is affected [18, 32, 34] or not [19, 24, 33], and there is agreement that the renal arterioles and tubules are most severely affected by perturbations of the RAS during development. The similarities of findings between Atg- and ACEmutant mice suggest that lack of angiotensin II is responsible for the kidney pathology. It is also possible that other Ang fragments may be involved. In either case, the identity of the receptor(s) mediating the phenomenon is less clear and remains to be established. In summary, the aforementioned studies indicate that the RAS is an important regulator of renal-vascular development, a function that seems to be conserved across the phylogenetic scale. ACKNOWLEDGMENTS This study was supported by the Child Health Research Center (P30-HD28810), the National Institutes of Health Center of Excellence in Pediatric Nephrology (DK 52612), and the O’Brien Research Center (DK 45179). The authors thank Karen Trentham for her excellent secretarial assistance. Reprint requests R. Ariel Gomez, M.D., MR4 –2001, Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: Ang, angiotensin; ACE, angiotensin converting enzyme; Atg, angiotensinogen; E, embryonic; RAS, renin-angiotensin system.
NOTE ADDED IN PROOF Recently, Tsuchida et al [35] reported that mice deficient in both AT1A and AT1B receptors have kidney vascular abnormalities similar to those found in Atg2/2 mice.
REFERENCES 1. SAXEN L: Organogenesis of the Kidney (Developmental and Cell Biology Series). New York, Cambridge University Press, 1987 2. SARIOLA H: Mechanisms and regulation of the vascular growth during kidney differentiation. Issues Biomed 14:69 – 80, 1991 3. RISAU W, SARIOLA H, ZERWES H, SASSE J, EKBLOM P, KEMLER R, DOETSCHMAN T: Vasculogenesis and angiogenesis in embryonic-stemcell-derived embryoid bodies. Development 102:471– 478, 1988
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4. RISAU W, EKBLOM P: Production of a heparin-binding angiogenesis factor by the embryonic kidney. J Cell Biol 103:1101–1107, 1986 5. CAREY AV, CAREY RM, GOMEZ RA: Expression of a-smooth muscle actin in the developing kidney vasculature. Hypertension 19(Suppl 2):168 –175, 1992 6. TUFRO-MCREDDIE A, NORWOOD VF, GOMEZ RA: Vascular endothelial growth factor (VEGF) induces nephrogenesis and endothelial cell differentiation during kidney development. (abstract) J Am Soc Nephrol 6:779, 1995 7. GOMEZ RA, NORWOOD VF, TUFRO-MCREDDIE A: Development of the kidney vasculature. Microsc Res Tech 39:254, 1997 8. HILGERS KF, NORWOOD VF, GOMEZ RA: Angiotensin’s role in renal development. Semin Nephrol 17:492–501, 1997 9. GOMEZ RA, LYNCH KR, STURGILL BC, ELWOOD JP, CHEVALIER RL, CAREY RM, PEACH MJ: Distribution of renin mRNA and its protein in the developing kidney. Am J Physiol 257:F850 –F858, 1989 10. REDDI V, PENTZ ES, GOMEZ RA: Renin expressing cells are associated with arterial branching in the developing kidney. (abstract) J Am Soc Nephrol 7:1640, 1996 11. GOMEZ RA, CASSIS L, LYNCH KR, CHEVALIER RL, WILFONG N, CAREY RM, PEACH MJ: Fetal expression of the angiotensinogen gene. Endocrinology 123:2298 –2302, 1988 12. DARBY IA, SERNIA C: In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys. Cell Tissue Res 281:197–206, 1995 13. WIGGER HJ, STALCUP SA: Distribution and development of angiotensin converting enzyme in the fetal and newborn rabbit. Lab Invest 38:581–585, 1978 14. MOUNIER F, HINGLAIS N, SICH M, GROAS F, LACOSTE M, DERIS Y, ALHENC-GELAS F, GUBLER MC: Ontogenesis of angiotensin-I converting enzyme in human kidney. Kidney Int 32:684 – 690, 1987 15. YOSIPIV IV, EL-DAHR SS: Activation of angiotensin-generating systems in the developing rat kidney. Hypertension 27:281–286, 1996 16. NORWOOD VF, CRAIG MR, HARRIS JM, GOMEZ RA: Differential expression of angiotensin II receptors during early renal morphogenesis. Am J Physiol 272:R662–R667, 1997 17. HOBART PM, FOGLIANO M, O’CONNOR BA: Human renin gene: Structure and sequence analysis. Proc Natl Acad Sci USA 81:5026 – 5030, 1984 18. TUFRO-MCREDDIE A, ROMANO LM, HARRIS JM, FERDER L, GOMEZ RA: Angiotensin II regulates nephrogenesis and renal vascular development. Am J Physiol 269:F110 –F115, 1995 19. FRIBERG P, SUNDELIN B, BOHMAN S-O, BOBIK A, NILSSON H, WICKMAN A, GUSTAFSSON H, PETERSEN J, ADAMS MA: Renin-angiotensin system in neonatal rats: Induction of a renal abnormality in response to ACE inhibition or angiotensin II antagonism. Kidney Int 45:485– 492, 1994 20. NILSSON A, ADAMS M, GURON G, SUNDELIN B, FRIBERG P: Reduced ability to excrete an acute volume load after neonatal ACE inhibition in the rat. (abstract) J Am Soc Nephrol 7:1586, 1996 21. FRIBERG P, GURON G, SUNDELIN B, WICKMAN A, ADAMS MA: Neonatal ACE inhibition in the pig causes irreversible abnormalities in the kidney. J Hypertens 14(Suppl 1):S45, 1996 22. PRYDE PG, SEDMAN AB, NUGENT CE, BARR M: Angiotensin-converting enzyme inhibitor fetopathy. J Am Soc Nephrol 3:1575–1582, 1993 23. TUFRO-MCREDDIE A, JOHNS DW, GEARY KM, DAGLI H, EVERETT AD, CHEVALIER RL, CAREY RM, GOMEZ RA: Angiotensin II type 1 receptor: Role in renal growth and gene expression during normal development. Am J Physiol 266:F911–F918, 1994 24. SPENCE SG, ALLEN HL, CUKIERSKI MA, MANSON JM, ROBERTSON RT, EYDELLOTH RS: Defining the susceptible period of developmental toxicity for the At1-selective angiotensin II receptor antagonist losartan in rats. Teratology 51:367–382, 1995 25. ITO M, OLIVERIO MI, MANNON PJ, BEST CF, MAEDA N, SMITHIES O, COFFMAN TM: Regulation of blood pressure by the type IA angiotensin II receptor gene. Proc Natl Acad Sci USA 92:3521–3525, 1995 26. SUGAYA T, SHIN-ICHIRO N, TANIMOTO K, TAKIMOTO E, YAMAGISHI T, IMAMURA K, GOTO S, IMAIZUMI K, HISADA Y, OTSUKA A, UCHIDA H, SAGIURA M, FUKUTA K, FUKAMIZU A, MURAKAMI K: Angiotensin II Type 1a receptor-deficient mice with hypotension and hyperreninemia. J Biol Chem 270:18719 –18722, 1995 27. CHEN X, LI W, YOSHIDA H, TSUCHIDA S, TAKEMOTO F, OKUBO S, FOGO A, MATSUSAKA T, ICHIKAWA I: Gene targeting of angiotensin
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type 1A or 1B receptor alone does not produce phenotype comparable to angiotensinogen gene targeting. (abstract) J Am Soc Nephrol 7:1630, 1996 HEIN L, BARSH GS, PRATT RE, DZAU VJ, KOBILKA BK: Behavioural and cardiovascular effects of disrupting the angiotensin II type 2 receptor gene in mice. Nature 377:744 –747, 1995 ICHIKI T, LABOSKY PA, SHIOTA C, OKUYAMA S, IMAGAWA Y, FOGO A, NIIMURA F, ICHIKAWA I, HOGAN BLM, INAGAMI T: Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type 2 receptor. Nature 377:748 –750, 1995 NISHIMURA H, MATSUSAKA T, UTSUNOMIYA H, FOGO A, KON V, INAGAMI T, ICHIKAWA I: Gene targeting reveals that angiotensin type 2 receptor is not essential for embryonic development of the kidney. (abstract) J Am Soc Nephrol 7:1602, 1996 KIM H-S, KREGE JH, KLUCKMAN KD, HAGAMAN JR, HODGIN JB, BEST CF, JENNETTE JC, COFFMAN TM, MAEDA N, SMITHIES O: Genetic control of blood pressure and the angiotensinogen locus. Proc Natl Acad Sci USA 92:2735–2739, 1995
32. NIIMURA F, LABOSKY PA, KAKUCHI J, OKUBO S, YOSHIDA H, OIKAWA T, ICHIKI T, NAFTILAN A, FOGO A, INAGAMI T, HOGAN B, ICHIKAWA L: Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation. J Clin Invest 96:2947–2954, 1995 33. NAGATA M, TANIMOTO K, FUKAMIZU A, KON Y, SUGIYAMA F, YAGAMI K, MURAKAMI K, WATANABE T: Nephrogenesis and renovascular development in angiotensinogen-deficient mice. Lab Invest 75:745–753, 1996 34. ESTHER CR JR, HOWARD TE, MARINO EM, GODDARD JM, CAPECCHI MR, BERNSTEIN KE: Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 74:953–965, 1996 35. TSUCHIDA T, MATSUSAKA T, CHEN X, OKUBO S, NIIMURA F, NISHIMURA H, FOGO A, UTSUNOMIYA H, INAGAMI T, ICHIKAWA I: Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes. J Clin Invest 101:755–760, 1998
Role of angiotensin in renal vascular development R. ARIEL GOMEZ Department of Pediatrics, University of Virginia, School of Medicine, Charlottesville, Virginia, USA
Role of angiotensin in renal vascular development. All components of the renin-angiotensin system (RAS) are expressed in the developing kidney in a temporospatial pattern that suggests a role for this system in kidney morphogenesis. Pharmacological blockade of angiotensin actions in fetal and newborn animals results in striking alterations in kidney architecture, including immature glomeruli and papillae, dilated tubuli, and arrested vascular development. Inactivation of angiotensinogen or angiotensin converting enzyme genes in mice results in similar anomalies that begin as subtle alterations in early life and become more pronounced as extrauterine life progresses. However, inactivation of each angiotensin receptor subtype does not result in obvious morphological abnormalities, suggesting functional redundancy at the receptor level. Crossing of mice lacking the various receptor subtypes should be revealing. Overall, the available information suggests that the RAS is necessary for the normal morphological and functional development of the kidney and the preservation of kidney architecture in adult life.
The definitive mammalian kidney, the metanephros, results from coinductive interaction between the primitive ureter and the metanephric blastema. The differentiating ureter arising from the mesonephric (Wolffian) duct induces the transformation of the metanephric mesenchyma into tubular and glomerular epithelium [1]. In turn, the induced mesenchyma stimulates the ureter to continue to differentiate and branch within the renal mesenchyma. As the ureter branches, the mesenchymal cells condense around each ureteric tip. The condensate develops into a vesicle, followed by a comma-shaped body. The creation of a lower cleft into the latter structure results in the formation of the S-shaped glomerulus. The formation of the S-shaped glomerulus seems to be critical for glomerular vascularization, because at this stage, endothelial cell precursors invade the lower cleft and form the glomerular capillaries. At this stage, proximal and distal tubules can be distinguished readily. Further tubular and glomerular growth results in the formation of a nephron with adult characteristics. In humans, nephrogenesis starts around the
Key words: angiotensin converting enzyme, extrauterine nephrogenesis, glomerulus, metanephros, receptors, renin-angiotensin system, Wolffian duct.
© 1998 by the International Society of Nephrology
fifth week of gestation and culminates around weeks 32 to 36. Thus, full-term infants are born with a full complement of nephrons. In mice and rats, the metanephros is visible around the embryonic (E) days 11 to 12 and 13 to 14 (term: 21 to 22 days), respectively, and continues in postnatal life. Thus, the neonatal mice and rat kidneys are useful models to study extrauterine nephrogenesis. VASCULAR DEVELOPMENT Precise and timely assembly of the kidney vasculature in concert with the developing nephron is fundamental for the development of a kidney capable of sustaining independent, extrauterine life. Kidney vascularization and nephrogenesis are simultaneous and coordinated processes [2]. The embryonic origin and mechanism underlying blood vessel formation in the kidney are still under debate. Endothelial cells may differentiate in situ from local mesenchymal cell precursors (vasculogenesis), or they may originate and sprout from pre-existing extrarenal or intrarenal vessel (angiogenesis) and migrate into the developing nephron [3, 4]. Both paradigms are not mutually exclusive and are supported by available experimental evidence. Precursors for smooth muscle, endothelial, and reninexpressing cells are present in the embryonic rat kidney well before vessel formation can be detected [5–7]. These precursor cells have the potential to differentiate and participate in the assembly of the arteriolar vessel wall. Once the arteriolar vessels are formed, the anatomical arrangement of the kidney vasculature is as follows: In the rat, a single renal artery divides at the level of the hilum into three main lobar arteries. Each lobar artery is continued by corticomedullary (arcuate) arteries that course through the corticomedullary junction parallel to the kidney cortex. From the arcuate arteries, numerous corticoradial (interlobular) arteries originate that in turn give off the afferent arterioles of the glomeruli. As expected, this basic blueprint of arteriolar development is already established in the fetal rat by E20. As maturation continues, the arterial tree grows in complexity and surface area. This is achieved mainly by a progressive and continued increase in the number of small arterial branches, especially interlobular arteries and afferent arterioles [7]. In fact, in the developing rat, arteriolar branching is very rapid up to day
S-12
Gomez: Angiotensin and renal development
S-13
Fig. 1. Expression of angiotensin receptor subtypes AT1 and AT2 during nephrogenesis. AT1 is represented by the hatched areas and AT2 by the gray areas. The density of pattern indicates the intensity of expression. (A) Embryonic day 14 (E14) metanephros showing intense expression of AT2 (right side) and sparse expression of AT1 throughout the renal mesenchyma. (B) E15 to E20 metanephros showing persistence of AT2 in mesenchyma and termination in epithelial structures while AT1 expression increases in more mature nephrons. (C) Postnatal days 7 to 15 showing the gradient of maturing nephrons. AT2 predominates in the differentiating areas, whereas AT1 predominates in more mature juxtamedullary nephrons. (D) Adult kidney showing the persistence of AT1 in mature glomeruli and tubules. AT2 is not detectable at this stage. Taken from [16] with permission from the American Society of Physiology.
10 postnatum (P10), while nephrogenesis is still occurring. The mechanisms that control arteriolar branching are not well understood and currently are being investigated. As discussed later here, components of the renin-angiotensin system (RAS) seem to play a role in this process. RAS AND VASCULAR DEVELOPMENT IN THE KIDNEY All the components of the RAS are expressed in the developing kidney [8]. In the rat metanephros, reninexpressing cells are found as early as day E14, before vessel or nephron formation has taken place. At E19, these cells are found along arcuate and interlobular arteries [9, 10]. As the kidney continues its development through fetal and postnatal life, renin distribution shifts from the larger arteries in the fetus to its mature juxtaglomerular localization in the adult animal [9]. These developmental patterns of renin expression have been observed in every species examined thus far [8]. In addition, we have recently observed an association between renin-expressing cells and branching of renal arterioles, suggesting a role for these cells in renal angiogenesis [10]. Angiotensinogen (Atg), the only known precursor of angiotensin (Ang) peptides, is detected in the fetal rat kidney by E17 to E18 [8, 11, 12]. However, the renal expression of Atg is highest around the
time of birth. In newborn rats, Atg is expressed in proximal tubules, mesangial cells, and vascular bundles. Ang-converting enzyme (ACE) is seen in endothelial precursors invading the lower cleft of the S-shaped body, in glomerular capillaries, corticomedullary arteries, proximal tubules, and peritubular capillaries [13, 14]. In addition to ACE, other proteases are capable of generating Ang in the developing kidney [15]. The high expression of components of the RAS in the developing kidney is reflected functionally by its higher levels of ANG compared with the adult rat kidney [15]. ANG receptors are also expressed in the maturing kidney in a characteristic developmental pattern [16, 17]. In the rat, both Ang receptor subtypes (AT1 and AT2) are expressed throughout metanephric development, albeit in differing temporal and spatial patterns (Fig. 1). At E14, when the ureteric bud is beginning to branch and nephrons and vessels have not yet been formed, AT2 receptors are more abundant and are present in the undifferentiated metanephric blastema. AT1 receptors are also present in the mesenchyma, although in lesser numbers (Fig. 1). At E17, nephrogenesis and vascularization are both occurring actively. A sagittal section of the kidney at this stage shows numerous glomeruli and arterioles at various stages of maturation, with the more mature glomeruli localized deeper within the cortex. At this stage, AT1
S-14
Gomez: Angiotensin and renal development
Fig. 2. Effect of deletion of the gene for angiotensin converting enzyme (ACE) on the renal arterial tree. Left: wild type (ACE1/1); right knock-out (ACE2/2) mice. Both vascular trees are stained with renin antibody (dark brown color). The arterioles in the ACE2/2 mice are thickened and sparse compared with the delicate arterioles in the wild-type. Renin staining is limited to the end of afferent arterioles in ACE1/1 but is distributed broadly along the vascular tree in ACE2/2 mice. Taken from [8] with permission.
receptors are found in the more mature glomeruli, whereas AT2 receptors are present in condensed mesenchyme and (transiently) in newly formed nephrons (Fig. 1). At E20, the kidney architecture is better defined, showing distinct medullary and cortical regions; there is still an outer nephrogenic zone. AT1 and AT2 are equally prevalent within the outer medullary stripe, the more developed glomeruli, large arteries, and the medullary rays. AT2 is abundant in the medullary rays, the nephrogenic cortex, and the columns of Bertin. After birth, AT1 predominates in more mature juxtamedullary nephrons, and AT2 continues to be expressed in differentiating structures. As nephron maturation is achieved, AT2 expression is down-regulated until it disappears after the second postnatal week (Fig. 1). In contrast, as nephrons differentiate, AT1 expression increases and becomes maximal around the first postnatal week. By the end of 1 month of postnatal life, AT1 expression is similar to that of the adult (Fig. 1). The pattern of receptor expression shown previously here has been documented in several animal species, suggesting unique and perhaps opposite functions of the two receptors. Overall, the expression pattern of RAS components during ontogeny suggests a role for this system in kidney development, as discussed later here. Blockade of the AT1 receptor in newborn rats up to P12 results in striking alterations in the developing kidney architecture [18]. In addition to a reduced number of immature glomeruli and dilated tubules, the renal arterioles are thick, short, and markedly reduced in numbers. Papillary atrophy that correlates functionally with a decreased urinary concentrating capacity and inability to excrete an acute volume load properly is found also [19, 20]. Interestingly, these functional abnormalities persist into adulthood, even if the treatment is stopped after P23.
Pigs treated neonatally with ACE inhibitors develop similar morphological and functional abnormalities [21]. Fetal anuria and renal abnormalities have also been described in humans treated with ACE inhibitors [22]. More severe renal-vascular abnormalities have also been noted in frog tadpoles receiving ACE inhibitors during their metamorphosis [18], suggesting that the mesonephric kidney of frogs is more susceptible to blockers of the RAS. Contrary to the findings with ACE and AT1 inhibitors, treatment of neonatal animals with AT2 blockers does not seem to induce any developmental abnormalities in vivo. Treatment of weaning rats with losartan does not induce renal abnormalities [23]. These findings suggest that the developing rat kidney is susceptible to RAS blockade mainly during the last five days of pregnancy and during the first two to three postnatal weeks [24]. As discussed earlier here, this period is associated with high expression of renin, Atg, and AT1 receptors. Presumably, the human kidney is more susceptible to the deleterious effects of RAS blockers during the last weeks of pregnancy. Contrary to the results obtained with pharmacological inhibitors of the RAS, targeted inactivation of each of the AT1 receptors (AT1A, AT1B) and AT2 receptors does not result in alterations in morphological kidney development [25–30], suggesting that the contribution of each individual receptor to kidney morphogenesis is limited or that it can be compensated by the action of the other Ang receptor subtypes. It would be interesting to determine whether mice with inactivation of both receptor subtypes (AT1A and AT1B) develop renal and/or vascular abnormalities. Targeted inactivation of Atg and ACE genes, however, produces remarkably similar renal abnormalities in adult mice, consisting of diminished numbers of thickened, hypercellular renal arterioles (Fig. 2), interstitial fibrosis,
Gomez: Angiotensin and renal development
papillary atrophy, and tubular dilatation [31–33]. In both knockout models, the abnormalities are obvious by P7 to P10 and become more pronounced with increasing age. There is disagreement as to whether abnormalities are seen at earlier stages in life: Nagata et al [33] found no effect of lack of Atg on nephrogenesis, whereas Niimura et al [32] reported delay in glomerular maturation and smaller glomeruli in Atg2/2 mice. Consistent with the latter, we find subtle alterations in interstitial architecture at E18. Arrest in vascular and glomerular development is already obvious by P7. These studies clearly demonstrate that both the ACE and Atg genes are necessary for the preservation of normal kidney structure during development and in adult life. Whereas in rats treated with RAS blockers and in mutant mice, there is disagreement as to whether glomerular development is affected [18, 32, 34] or not [19, 24, 33], and there is agreement that the renal arterioles and tubules are most severely affected by perturbations of the RAS during development. The similarities of findings between Atg- and ACEmutant mice suggest that lack of angiotensin II is responsible for the kidney pathology. It is also possible that other Ang fragments may be involved. In either case, the identity of the receptor(s) mediating the phenomenon is less clear and remains to be established. In summary, the aforementioned studies indicate that the RAS is an important regulator of renal-vascular development, a function that seems to be conserved across the phylogenetic scale. ACKNOWLEDGMENTS This study was supported by the Child Health Research Center (P30-HD28810), the National Institutes of Health Center of Excellence in Pediatric Nephrology (DK 52612), and the O’Brien Research Center (DK 45179). The authors thank Karen Trentham for her excellent secretarial assistance. Reprint requests R. Ariel Gomez, M.D., MR4 –2001, Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: Ang, angiotensin; ACE, angiotensin converting enzyme; Atg, angiotensinogen; E, embryonic; RAS, renin-angiotensin system.
NOTE ADDED IN PROOF Recently, Tsuchida et al [35] reported that mice deficient in both AT1A and AT1B receptors have kidney vascular abnormalities similar to those found in Atg2/2 mice.
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