Deletion of the prorenin receptor in the ureteric bud in mice inhibits Dot1/H3K79 pathway.

BACKGROUND: The prorenin receptor (PRR) plays a critical role in ureteric bud (UB) branching morphogenesis. DOT1 Like (DOT1L), a histone methyltransferase specific for Histone 3 lysine 79 (H3K79), is important for differentiation of the UB-derived renal collecting duct cells. In this study, we tested whether DOT1L/H3 dimethyl K79 (H3m2K79) are regulated by PRR deletion in the UB and UB-derived collecting ducts in the embryonic mouse kidneys. METHODS: Mutant Hoxb7Cre+/PRRflox/flox (PRRUB-/-) and control PRRUB+/+, mice were studied on embryonic (E) day E17.5. DOT1L mRNA and protein expression in the kidney was examined by real-time qRT-PCR and immunohistochemistry, respectively. H3m2K79 protein expression was determined by immunohistochemistry and Western blot analysis. RESULTS: DOT1L mRNA levels were decreased in mutant compared to control mice (0.68 ± 0.06 vs. 1.0 ± 0.01, p < 0.01). DOT1L and H3m2K79 immunostaining was reduced in the mutant vs. control kidneys (Dot1: 0.62 ± 0.03 vs. 1.0 ± 0.01, p < 0.05; H3m2K79: 0.64 ± 0.04 vs.1.1 ± 0.01. p < 0.05.). Western blot analysis revealed decreased H3m2K79 protein levels in mutant compared to control kidneys (1.0 ± 0.06 vs. 1.5 ± 0.02, p < 0.05). CONCLUSION: Targeted deletion of the PRR in the UB and UB-derived collecting ducts results in reduced DOT1L gene/protein and H3m2K79 protein expression in the embryonic mouse metanephroi in vivo. IMPACT: The role of histone methylation in mediating the effect of the prorenin receptor on the ureteric bud branching (UB) morphogenesis and urine acidification during kidney development is unknown. We demonstrate that histone H3 lysine (K) 79 dimethylation by methyltransferase Dot1 is reduced in the embryonic kidney of mice that lack the prorenin receptor in the UB lineage.

Nephrotic Syndrome in a Child With NPHS2 Mutation.

Steroid resistant nephrotic syndrome (SRNS) accounts for 30% of all cases of nephrotic syndrome (NS) in children and frequently leads to end stage kidney disease (ESKD). About 30% of children with SRNS demonstrate causative mutations in podocyte- associated genes. Early identification of genetic forms of SRNS is critical to avoid potentially harmful immunosuppressive therapy. A 2-year-old male patient with NS and no family history of renal disease did not respond to 4-week steroid treatment. Kidney biopsy demonstrated mesangial proliferative glomerulopathy with basement membrane dysmorphism. Tacrolimus and Lisinopril were added to therapy pending results of genetic testing. Kidney Gene panel showed a NPHS2 c.413G>A (p.Arg138Gln) homozygous pathogenic variant. This missense variant is considered a common pathogenic founder mutation in European populations. A diagnosis of autosomal-recessive form of nonsyndromic SRNS due to NPHS2 causative variant was made. Immunosuppresive therapy was stopped, Lizinopril dose was increased and weekly infusions of Albumin/furosemide were initiated to manage edema. This case demonstrates that early genetic testing in children with SRNS avoids prolonged potentially harmful immunosuppressive therapy, allows for timely genetic family counseling, and allows earlier consideration for future living related donor kidney transplantation.

Cytogenomic aberrations in isolated multicystic dysplastic kidney in children.

BACKGROUND: Multicystic dysplastic kidney (MCDK) is a common form of congenital kidney anomaly. The cause of MCDK is unknown. We investigated whether MCDK in children is linked to cytogenomic aberrations. METHODS: We conducted array comparative genomic hybridization (aCGH) in ten unrelated children with MCDK. The pattern of inheritance was determined by real-time PCR in patients and their biological parents. RESULTS: Pathogenic aberrations were detected in three patients: a deletion at 7p14.3 with a size of 2.07 Mb housing 12 genes, including BBS9 (Bardet-Biedl syndrome 9) and BMPER (BMP binding endothelial regulator); a duplication at 16p13.11p12.3 with a size of 3.28 Mb that included >20 genes; and monosomy X for a female patient. The deletion at 7p14.3 was inherited from the patient's father, while the duplication at 16p13.11p12.3 was derived from the patient's mother. CONCLUSIONS: Up to 30% of patients with MCDK possess cytogenomic aberrations. BBS9 and BMPER variants have been reported to result in cystic kidney dysplasia, suggesting a possible pathogenic function for the deletion at 7p14.3 in children with MCDK. The duplication at 16p13.11p12.3 was not reported previously to associate with MCDK. Both variations were inherited from parents, indicating hereditary contributions in MCDK. Thus, aCGH is an informative tool to unravel the pathogenic mechanisms of MCDK. IMPACT: Cytogenomic aberrations are common in children with MCDK. Cytogenomic aberrations are inherited from parents, indicating hereditary contributions in MCDK. aCGH is a valuable tool to reveal pathogenic mechanisms of MCDK.

Sequence variants in the renin-angiotensin system genes are associated with isolated multicystic dysplastic kidney in children.

BACKGROUND: Multicystic dysplastic kidney (MCDK) is a common form of congenital cystic kidney disease in children. The etiology of MCDK remains unclear. Given an important role of the renin-angiotensin system in normal kidney development, we explored whether MCDK in children is associated with variants in the genes encoding renin-angiotensin system components by Sanger sequencing. METHODS: The coding regions of renin (REN), angiotensinogen (AGT), ACE, and angiotensin 1 receptor (AGTR1) genes were amplified by PCR. The effect of DNA sequence variants on protein function was predicted with PolyPhen-2 software. RESULTS: 3 novel and known AGT variants were found. 1 variant was probably damaging, 1 was possibly damaging and one was benign. Out of 7 REN variants, 4 were probably damaging and 3 were benign. Of 6 ACE variants, 3 were probably damaging and 3-benign. 3 AGTR1 variants were found. 2 variants were possibly damaging, and one was benign. CONCLUSION: We report novel associations of sequence variants in REN, AGT, ACE, or AGTR1 genes in children with isolated MCDK in the United States. Our findings suggest a recessive disease model and support the hypothesis of multiple renin-angiotensin system gene involvement in MCDK. IMPACT: Discovery of novel gene variants in renin-angiotensin genes in children with MCDK. Novel possibly damaging gene variants discovered. Multiple renin-angiotensin system gene variants are involved in MCDK.

Renin-angiotensin system in mammalian kidney development.

Mutations in the genes of the renin-angiotensin system result in congenital anomalies of the kidney and urinary tract (CAKUT), the main cause of end-stage renal disease in children. The molecular mechanisms that cause CAKUT are unclear in most cases. To improve the care of children with CAKUT, it is critical to determine the underlying mechanisms of CAKUT. In this review, we discuss recent advances that have helped to better understand how disruption of the renin-angiotensin system during kidney development contributes to CAKUT.

(Pro)renin receptor regulates lung development via the Wnt/ß-catenin signaling pathway.

The (pro)renin receptor [(P)RR] binds to prorenin to activate the renin-angiotensin system and is essential for the development of many different organ systems. Whether the (P)RR also plays a role in lung development is unknown. Immunostaining was used to determine the spatial-temporal distribution of (P)RR in the embryonic, postnatal, and adult lungs. We created a lung-specific (P)RR knockout mouse [Foxd1cre/+-(P)RRflox/flox] and assessed changes in lung morphology, cell proliferation, and apoptosis using immunohistochemistry and TUNEL staining. (P)RR function was confirmed by using siRNA to knock down (P)RR in human bronchial epithelial cells (HBECs) and then using the CCK-8 assay and flow cytometry to assess cell proliferation and apoptosis. Gene expression changes after knockdown were assessed by RT-PCR and Western blotting. (P)RR is expressed in the club cells of the bronchial epithelium, and expression increases throughout development. Lung-specific (P)RR knockout disrupted branching morphogenesis, leading to lung hypoplasia and neonatal mortality. These defects were associated with increased apoptosis and decreased proliferation of the pulmonary epithelial and mesenchymal cells and may be mediated by downregulation of Wnt11, ß-catenin, and Axin2. (P)RR regulates lung development through canonical Wnt/ß-catenin signaling and may present a new target for strategies to treat lung hypoplasia.

Stromal prorenin receptor is critical for normal kidney development.

Formation of the metanephric kidney requires coordinated interaction among the stroma, ureteric bud, and cap mesenchyme. The transcription factor Foxd1, a specific marker of renal stromal cells, is critical for normal kidney development. The prorenin receptor (PRR), a receptor for renin and prorenin, is also an accessory subunit of the vacuolar proton pump V-ATPase. Global loss of PRR is embryonically lethal in mice, indicating an essential role of the PRR in embryonic development. Here, we report that conditional deletion of the PRR in Foxd1+ stromal progenitors in mice (cKO) results in neonatal mortality. The kidneys of surviving mice show reduced expression of stromal markers Foxd1 and Meis1 and a marked decrease in arterial and arteriolar development with the subsequent decreased number of glomeruli, expansion of Six2+ nephron progenitors, and delay in nephron differentiation. Intrarenal arteries and arterioles in cKO mice were fewer and thinner and showed a marked decrease in the expression of renin, suggesting a central role for the PRR in the development of renin-expressing cells, which in turn are essential for the proper formation of the renal arterial tree. We conclude that stromal PRR is crucial for the appropriate differentiation of the renal arterial tree, which in turn may restrict excessive expansion of nephron progenitors to promote a coordinated and proper morphogenesis of the nephrovascular structures of the mammalian kidney.

Conditional ablation of the prorenin receptor in nephron progenitor cells results in developmental programming of hypertension.

Nephron induction during kidney development is driven by reciprocal interactions between progenitor cells (NPCs) of the cap mesenchyme (CM) and the ureteric bud (UB). The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump V-ATPase. Previously, we demonstrated that conditional ablation of the PRR in Six2+ NPCs in mice (Six2PRR-/- ) causes early neonatal death. Here, we identified genes that are regulated by PRR in Six2+ NPCs FACS-isolated from Six2PRR-/- and control kidneys on embryonic day E15.5 using whole-genome expression analysis. Seven genes with expression in CM cells previously shown to direct kidney development, including Notch1, ß-catenin, Lef1, Lhx1, Jag1, and p53, were downregulated. The functional groups within the downregulated gene set included genes involved in embryonic and cellular development, renal regeneration, cellular assembly and organization, cell morphology, death and survival. Double-transgenic Six2PRR-/- /BatGal+ mice, a reporter strain for ß-catenin transcriptional activity, showed decreased ß-catenin activity in the UB in vivo. Reduced PRR gene dosage in heterozygous Six2PRR+/- mice was associated with decreased glomerular number, segmental thickening of the glomerular basement membrane with focal podocyte foot process effacement, development of hypertension and increased soluble PRR (sPRR) levels in the urine at 2 months of age. Together, these data demonstrate that NPC PRR performs essential functions during nephrogenesis via control of hierarchy of genes that regulate critical cellular processes. Both reduced nephron endowment and augmented urine sPRR likely contribute to programming of hypertension in Six2PRR+/- mice.

Foxd1 is an upstream regulator of the renin-angiotensin system during metanephric kidney development.

BackgroundWe tested the hypothesis that Foxd1, a transcription factor essential for normal kidney development, is an upstream regulator of the renin-angiotensin system (RAS) during ureteric bud (UB)-branching morphogenesis.MethodsUB branching, RAS gene, and protein expression were studied in embryonic mouse kidneys. RAS mRNA expression was studied in mesenchymal MK4 cells.ResultsThe number of UB tips was reduced in Foxd1-/- compared with that in Foxd1+/+ metanephroi on embryonic day E12.5 (14±2.1 vs. 28±1.3, P<0.05). Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated that renin, angiotensin I-converting enzyme (ACE), and angiotensin (Ang) II receptor type 1 (AT1R) mRNA levels were decreased in Foxd1-/- compared with those in Foxd1+/+ E14.5 metanephroi. Western blot analysis and immunohistochemistry showed decreased expression of AGT and renin proteins in Foxd1-/- metanephroi compared with that in Foxd1+/+ metanephroi. Foxd1 overexpression in mesenchymal MK4 cells in vitro increased renin, AGT, ACE, and AT1R mRNA levels. Exogenous Ang II stimulated UB branching equally in whole intact E12.5 Foxd1-/- and Foxd1+/+ metanephroi grown ex vivo (+364±21% vs. +336±18%, P=0.42).ConclusionWe conclude that Foxd1 is an upstream positive regulator of RAS during early metanephric development and propose that the cross-talk between Foxd1 and RAS is essential in UB-branching morphogenesis.

Prorenin receptor controls renal branching morphogenesis via Wnt/ß-catenin signaling.

The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump H+-ATPase. Renal branching morphogenesis, defined as growth and branching of the ureteric bud (UB), is essential for mammalian kidney development. Previously, we demonstrated that conditional ablation of the PRR in the UB in PRRUB-/- mice causes severe defects in UB branching, resulting in marked kidney hypoplasia at birth. Here, we investigated the UB transcriptome using whole genome-based analysis of gene expression in UB cells, FACS-isolated from PRRUB-/-, and control kidneys at birth (P0) to determine the primary role of the PRR in terminal differentiation and growth of UB-derived collecting ducts. Three genes with expression in UB cells that previously shown to regulate UB branching morphogenesis, including Wnt9b, ß-catenin, and Fgfr2, were upregulated, whereas the expression of Wnt11, Bmp7, Etv4, and Gfrα1 was downregulated. We next demonstrated that infection of immortalized UB cells with shPRR in vitro or deletion of the UB PRR in double-transgenic PRRUB-/-/BatGal+ mice, a reporter strain for ß-catenin transcriptional activity, in vivo increases ß-catenin activity in the UB epithelia. In addition to UB morphogenetic genes, the functional groups of differentially expressed genes within the downregulated gene set included genes involved in molecular transport, metabolic disease, amino acid metabolism, and energy production. Together, these data demonstrate that UB PRR performs essential functions during UB branching and collecting duct morphogenesis via control of a hierarchy of genes that control UB branching and terminal differentiation of the collecting duct cells.

Prorenin receptor in kidney development.

Prorenin receptor (PRR), a receptor for renin and prorenin and an accessory subunit of the vacuolar proton pump H+-ATPase, is expressed in the developing kidney. Global loss of PRR is lethal in mice, and PRR mutations are associated with a high blood pressure, left ventricular hypertrophy and X-linked mental retardation in humans. With the advent of modern gene targeting techniques, including conditional knockout approaches, several recent studies have demonstrated critical roles for the PRR in several lineages of the developing kidney. PRR signaling has been shown to be essential for branching morphogenesis of the ureteric bud (UB), nephron progenitor survival and nephrogenesis. PRR regulates these developmental events through interactions with other transcription and growth factors. Several targeted PRR knockout animal models have structural defects mimicking congenital anomalies of the kidney and urinary tract observed in humans. The aim of this review, is to highlight new insights into the cellular and molecular mechanisms by which PRR may regulate UB branching, terminal differentiation and function of UB-derived collecting ducts, nephron progenitor maintenance, progression of nephrogenesis and normal structural kidney development and function.

Prorenin receptor is critical for nephron progenitors.

Deficient nephrogenesis is the major factor contributing to renal hypoplasia defined as abnormally small kidneys. Nephron induction during kidney development is driven by reciprocal interactions between progenitor cells of the cap mesenchyme (CM) and the ureteric bud (UB). The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump H(+)-ATPase. Global loss of PRR is lethal in mice and PRR mutations are associated with a high blood pressure, left ventricular hypertrophy and X-linked mental retardation in humans. To circumvent lethality of the ubiquitous PRR mutation in mice and to determine the potential role of the PRR in nephrogenesis, we generated a mouse model with a conditional deletion of the PRR in Six2(+) nephron progenitors and their epithelial derivatives (Six2(PRR-/-)). Targeted ablation of PRR in Six2(+) nephron progenitors caused a marked decrease in the number of developing nephrons, small cystic kidneys and podocyte foot process effacement at birth, and early postnatal death. Reduced congenital nephron endowment resulted from premature depletion of nephron progenitor cell population due to impaired progenitor cell proliferation and loss of normal molecular inductive response to canonical Wnt/ß-catenin signaling within the metanephric mesenchyme. At 2 months of age, heterozygous Six2(PRR+/-) mice exhibited focal glomerulosclerosis, decreased kidney function and massive proteinuria. Collectively, these findings demonstrate a cell-autonomous requirement for the PRR within nephron progenitors for progenitor maintenance, progression of nephrogenesis, normal kidney development and function.

Renin-angiotensin system in ureteric bud branching morphogenesis: implications for kidney disease.

Failure of normal branching morphogenesis of the ureteric bud (UB), a key ontogenic process that controls organogenesis of the metanephric kidney, leads to congenital anomalies of the kidney and urinary tract (CAKUT), the leading cause of end-stage kidney disease in children. Recent studies have revealed a central role of the renin-angiotensin system (RAS), the cardinal regulator of blood pressure and fluid/electrolyte homeostasis, in the control of normal kidney development. Mice or humans with mutations in the RAS genes exhibit a spectrum of CAKUT which includes renal medullary hypoplasia, hydronephrosis, renal hypodysplasia, duplicated renal collecting system and renal tubular dysgenesis. Emerging evidence indicates that severe hypoplasia of the inner medulla and papilla observed in angiotensinogen (Agt)- or angiotensin (Ang) II AT 1 receptor (AT 1 R)-deficient mice is due to aberrant UB branching morphogenesis resulting from disrupted RAS signaling. Lack of the prorenin receptor (PRR) in the UB in mice causes reduced UB branching, resulting in decreased nephron endowment, marked kidney hypoplasia, urinary concentrating and acidification defects. This review provides a mechanistic rational supporting the hypothesis that aberrant signaling of the intrarenal RAS during distinct stages of metanephric kidney development contributes to the pathogenesis of the broad phenotypic spectrum of CAKUT. As aberrant RAS signaling impairs normal renal development, these findings advocate caution for the use of RAS inhibitors in early infancy and further underscore a need to avoid their use during pregnancy and to identify the types of molecular processes that can be targeted for clinical intervention.

Deletion of the prorenin receptor from the ureteric bud causes renal hypodysplasia.

The role of the prorenin receptor (PRR) in the regulation of ureteric bud (UB) branching morphogenesis is unknown. Here, we investigated whether PRR acts specifically in the UB to regulate UB branching, kidney development and function. We demonstrate that embryonic (E) day E13.5 mouse metanephroi, isolated intact E11.5 UBs and cultured UB cells express PRR mRNA. To study its role in UB development, we conditionally ablated PRR in the developing UB (PRR (UB-/-)) using Hoxb7 (Cre) mice. On E12.5, PRR (UB-/-) mice had decreased UB branching and increased UB cell apoptosis. These defects were associated with decreased expression of Ret, Wnt11, Etv4/Etv5, and reduced phosphorylation of Erk1/2 in the UB. On E18.5, mutants had marked kidney hypoplasia, widespread apoptosis of medullary collecting duct cells and decreased expression of Foxi1, AE1 and H(+)-ATPase α4 mRNA. Ultimately, they developed occasional small cysts in medullary collecting ducts and had decreased nephron number. To test the functional consequences of these alterations, we determined the ability of PRR (UB-/-) mice to acidify and concentrate the urine on postnatal (P) day P30. PRR (UB-/-) mice were polyuric, had lower urine osmolality and a higher urine pH following 48 hours of acidic loading with NH4Cl. Taken together, these data show that PRR present in the UB epithelia performs essential functions during UB branching morphogenesis and collecting duct development via control of Ret/Wnt11 pathway gene expression, UB cell survival, activation of Erk1/2, terminal differentiation and function of collecting duct cells needed for maintaining adequate water and acid-base homeostasis. We propose that mutations in PRR could possibly cause renal hypodysplasia and renal tubular acidosis in humans.

Ontogeny of the (pro)renin receptor.

BACKGROUND: This study examined temporal expression of the (pro)renin receptor ((P)RR), during renal, heart, lung, and brain organogenesis in the mouse. METHODS: (P)RR expression was determined by quantitative reverse-transcription PCR, western blotting, and immunohistochemistry. RESULTS: Brain, kidney, and lung (P)RR mRNA levels increased progressively during gestation and peak on postnatal day (P)10. (P)RR protein contents were high during gestation in all organs studied and declined with maturation. Brain (P)RR was expressed most prominently in the ependymal lining of the ventricles. In the embryonic day (E)16.5 and E18.5 metanephros, (P)RR was present in the ureteric bud and ureteric bud-derived collecting ducts. In the fetal heart, (P)RR was expressed diffusely in the myocardium, whereas pulmonary (P)RR was detected at highest levels in the epithelium of branching airways. Treatment of newborn kidneys with the angiotensin (Ang) II type 1 receptor (AT1R) antagonist candesartan increased (P)RR mRNA levels. CONCLUSION: (P)RR gene and protein expressions in the brain, kidney, heart, and lung are developmentally regulated in a tissue-specific manner. Endogenous Ang II, acting via the AT1R, exerts a negative feedback on (P)RR in the newborn kidney. These findings suggest that high (P)RR protein levels observed during gestation may play a role in brain, kidney, heart, and lung organogenesis.

Developmental programming of hypertension and kidney disease.

A growing body of evidence supports the concept that changes in the intrauterine milieu during "sensitive" periods of embryonic development or in infant diet after birth affect the developing individual, resulting in general health alterations later in life. This phenomenon is referred to as "developmental programming" or "developmental origins of health and disease." The risk of developing late-onset diseases such as hypertension, chronic kidney disease (CKD), obesity or type 2 diabetes is increased in infants born prematurely at <37 weeks of gestation or in low birth weight (LBW) infants weighing <2,500 g at birth. Both genetic and environmental events contribute to the programming of subsequent risks of CKD and hypertension in premature or LBW individuals. A number of observations suggest that susceptibility to subsequent CKD and hypertension in premature or LBW infants is mediated, at least in part, by reduced nephron endowment. The major factors influencing in utero environment that are associated with a low final nephron number include uteroplacental insufficiency, maternal low-protein diet, hyperglycemia, vitamin A deficiency, exposure to or interruption of endogenous glucocorticoids, and ethanol exposure. This paper discusses the effect of premature birth, LBW, intrauterine milieu, and infant feeding on the development of hypertension and renal disease in later life as well as examines the role of the kidney in developmental programming of hypertension and CKD.

Congenital anomalies of the kidney and urinary tract: a genetic disorder?

Congenital anomalies of the kidney and urinary tract (CAKUTs) occur in 3-6 per 1000 live births, account for the most cases of pediatric end-stage kidney disease (ESKD), and predispose an individual to hypertension and cardiovascular disease throughout life. Although CAKUTs are a part of many known syndromes, only few single-candidate causative genes have been implicated so far in nonsyndromic cases of human CAKUT. Evidence from mouse models supports the hypothesis that non-syndromic human CAKUT may be caused by single-gene defects. Because increasing numbers of children with CAKUT are surviving to adulthood, better understanding of the molecular pathogenesis of CAKUT, development of new strategies aiming at prevention of CAKUT, preservation of renal function, and avoidance of associated cardiovascular morbidity are needed. In this paper, we will focus on the knowledge derived from the study of syndromic and non-syndromic forms of CAKUT in humans and mouse mutants to discuss the role of genetic, epigenetic, and in utero environmental factors in the pathogenesis of non-syndromic forms of CAKUT in children with particular emphasis on the genetic contributions to CAKUT.

Ontogeny of angiotensin-converting enzyme 2.

INTRODUCTION: This study examined the temporal expression of angiotensin (Ang)-converting enzyme 2 (ACE2) during renal, heart, lung, and brain organogenesis in the mouse. RESULTS: We demonstrate that kidney ACE2 mRNA levels are low on embryonic day (E) 12.5, increase fourfold during development, and decline in adulthood. In extrarenal tissues, ACE2 mRNA levels are also low during early gestation, increase in perinatal period, and peak in adulthood. The lung shows the highest age-related increase in ACE2 mRNA levels followed by the brain, kidney, and heart. ACE2 protein levels and enzymatic activity are high in all organs studied during gestation and decline postnatally. Ang II decreases ACE2 mRNA levels and enzymatic activity in kidneys grown ex vivo. These effects of Ang II are blocked by the specific Ang II AT(1) receptor (AT(1)R) antagonist candesartan, but not by the AT(2) receptor (AT(2)R) antagonist PD123319. DISCUSSION: We conclude that ACE2 gene and protein expression and enzymatic activity are developmentally regulated in a tissue-specific manner. Ang II, acting through AT(1)R, exerts a negative feedback on ACE2 during kidney development. We postulate that relatively high ACE2 protein levels and enzymatic activity observed during gestation may play a role in kidney, lung, brain, and heart organogenesis.