Ihor V. Yosypiv

Genetics of congenital anomalies of the kidney and urinary tract

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in 1 in 500 births and are a major cause of morbidity in children. Notably, CAKUT account for the most cases of pediatric end-stage renal disease and predispose the individual to hypertension and cardiovascular disease throughout life. Although some forms of CAKUT are a part of a syndrome or are associated with a positive family history, most cases of renal system anomalies are sporadic and isolated to the urinary tract. Broad phenotypic spectrum of CAKUT and variability in genotype–phenotype correlation indicate that pathogenesis of CAKUT is a complex process that depends on interplay of many factors. This review focuses on the genetic mechanisms (single-gene mutations, modifier genes) leading to renal system anomalies in humans and discusses emerging insights into the role of epigenetics, in utero environmental factors, and micro-RNAs (miRNAs) in the pathogenesis of CAKUT. Common gene networks that function in defined temporospatial fashion to orchestrate renal system morphogenesis are highlighted. Derangements in cellular, molecular, and morphogenetic mechanisms that direct normal renal system development are emphasized as a major cause of CAKUT. Integrated understanding of how morphogenetic process disruptions are linked to CAKUT will enable improved diagnosis, treatment, and prevention of congenital renal system anomalies and their consequences.

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.

Angiotensin II Type 1 Receptor–EGF Receptor Cross-Talk Regulates Ureteric Bud Branching Morphogenesis

Angiotensinogen-, angiotensin-converting enzyme-, and angiotensin II (Ang II) type 1 receptor (AT(1)R)-deficient mice exhibit a dilated renal pelvis (hydronephrosis) and a small papilla. These abnormalities have been attributed to impaired development of the ureteral and pelvic smooth muscle. Defects in the growth and branching of the ureteric bud (UB), which gives rise to the collecting system, have not been examined carefully. This study tested the hypothesis that Ang II stimulates UB growth and branching in the intact metanephros. Immunohistochemistry demonstrated that embryonic mouse kidneys express AT(1)R in the UB and its branches. Embryonic day 11.5 metanephroi were microdissected from Hoxb7-green fluorescence protein mice and grown for 48 h in serum-free medium in the presence or absence of Ang II. The number of green fluorescence protein-positive UB branch points (BP) and tips was monitored in each explant at 24 and 48 h. Ang II increased the number of UB tips and BP at 24 h (tips: 24.3 +/- 1.1 versus 18.3 +/- 0.7, P < 0.01; BP: 14.4 +/- 0.6 versus 11.7 +/- 0.6, P < 0.01) and 48 h (tips: 30.2 +/- 1.3 versus 22.9 +/- 0.8, P < 0.01; BP: 21.3 +/- 0.9 versus 15.7 +/- 0.6, P < 0.01) compared with control. In contrast, treatment of metanephroi with the AT(1)R antagonist candesartan inhibited UB branching, decreasing the number of UB tips and BP. Similarly, inhibition of EGF receptor (EGFR) tyrosine kinase activity abrogated Ang II-stimulated UB branching. A cross-talk between the renin-angiotensin system and EGFR signaling was elicited at the cellular level by the ability of Ang II to induce tyrosine phosphorylation of EGFR in UB cells and through abrogation of Ang II-induced UB cell branching using an EGFR tyrosine kinase inhibitor. These data demonstrate that Ang II, acting via the AT(1)R, stimulates UB branching morphogenesis. This process depends on tyrosine phosphorylation of the EGFR. Cooperation of AT(1)R and EGFR signaling therefore is important in the development of the renal collecting system.

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

Angiotensin II AT2 receptor regulates ureteric bud morphogenesis.

ANG II AT2 receptor (AT2R)-deficient mice exhibit abnormal ureteric bud (UB) budding, increased incidence of double ureters, and vesicoureteral reflux. However, the role of the AT2R during UB morphogenesis and the mechanisms by which aberrant AT2R signaling disrupts renal collecting system development have not been fully defined. In this study, we mapped the expression of the AT2R during mouse metanephric development, examined the impact of disrupted AT2R signaling on UB branching, cell proliferation, and survival, and investigated the cross talk of the AT2R with the glial-derived neurotrophic factor (GDNF)/c-Ret/Wnt11 signaling pathway. Embryonic mouse kidneys express AT2R in the branching UB and the mesenchyme. Treatment of embryonic day 12.5 (E12.5) metanephroi with the AT2R antagonist PD123319 or genetic inactivation of the AT2R in mice inhibits UB branching, decreasing the number of UB tips compared with control (34 +/- 1.0 vs. 43 +/- 0.6, P < 0.01; 36 +/- 1.8 vs. 48 +/- 1.3, P < 0.01, respectively). In contrast, treatment of metanephroi with the AT2R agonist CGP42112 increases the number of UB tips compared with control (48 +/- 1.8 vs. 39 +/- 12.3, P < 0.05). Using real-time quantitative RT-PCR and whole mount in situ hybridization, we demonstrate that PD123319 downregulates the expression of GDNF, c-Ret, Wnt11, and Spry1 mRNA levels in E12.5 metanephroi grown ex vivo. AT(2)R blockade or genetic inactivation of AT2R stimulates apoptosis and inhibits proliferation of the UB cells in vivo. We conclude that AT2R performs essential functions during UB branching morphogenesis via control of the GDNF/c-Ret/Wnt11 signaling pathway, UB cell proliferation, and survival.

Histone deacetylases are critical regulators of the renin-angiotensin system during ureteric bud branching morphogenesis.

Mutations in the genes encoding components of the renin-angiotensin system (RAS) in mice or humans cause congenital abnormalities of the kidney and urinary tract. We hypothesized that absence of angiotensin (Ang) II in angiotensinogen (AGT)-deficient mice leads to defects in ureteric bud (UB) branching and that RAS genes are critically dependent on histone deacetylase (HDAC) activity. The number of UB tips was lower in AGT−/− compared with AGT+/+ embryonic (E) day E13.5 metanephroi (24 ± 1.5 versus 36 ± 3.7, p < 0.05). Real-time RT-PCR demonstrated that pharmacological inhibition of HDAC activity with Scriptaid increases AGT, renin, angiotensin-converting enzyme (ACE), and AT1 receptor (AT1R) mRNA levels in E12.5 mouse metanephroi and early mesenchymal (MK3) cells. Furthermore, Scriptaid enhanced Ang II-induced decrease in Sprouty (Spry) 1 gene expression in cultured UB cells. Treatment of intact E12.5 mouse metanephroi grown ex vivo with Ang II (10−5 M, 24 h) increased HDAC-1 and decreased total acetylated histone H3 protein levels. These findings indicate that lack of endogenous Ang II in AGT-deficient mice inhibits UB branching. We conclude that intact RAS is critical in structural integrity of the renal collecting system and that UB morphogenetic program genes, such as AGT, renin, ACE, AT1R, or Spry1, are epigenetically controlled via HDACs.

Renin–angiotensin system in ureteric bud branching morphogenesis: insights into the mechanisms

Branching morphogenesis of the ureteric bud (UB) is a key developmental process that controls organogenesis of the entire metanephros. Notably, aberrant UB branching may result in a spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). Genetic, biochemical and physiological studies have demonstrated that the renin–angiotensin system (RAS), a key regulator of the blood pressure and fluid/electrolyte homeostasis, also plays a critical role in kidney development. All the components of the RAS are expressed in the metanephros. Moreover, mutations in the genes encoding components of the RAS in mice or humans cause diverse types of CAKUT which include renal papillary hypoplasia, hydronephrosis, duplicated collecting system, renal tubular dysgenesis, renal vascular abnormalities, abnormal glomerulogenesis and urinary concentrating defect. Despite widely accepted role of the RAS in metanephric kidney and renal collecting system (ureter, pelvis, calyces and collecting ducts) development, the mechanisms by which an intact RAS exerts its morphogenetic actions are incompletely defined. Emerging evidence indicates that defects in UB branching morphogenesis may be causally linked to the pathogenesis of renal collecting system anomalies observed under conditions of aberrant RAS signaling. This review describes the role of the RAS in UB branching morphogenesis and highlights emerging insights into the cellular and molecular mechanisms whereby RAS regulates this critical morphogenetic process.

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.

Role of the renin-angiotensin system in the development of the ureteric bud and renal collecting system.

Genetic, biochemical and physiological studies have demonstrated that the renin-angiotensin system (RAS) plays a fundamental role in kidney development. All of the components of the RAS are expressed in the metanephros. Mutations in the genes encoding components of the RAS in mice or pharmacological inhibition of RAS in animals or humans cause diverse congenital abnormalities of the kidney and lower urinary tract. The latter include renal vascular abnormalities, abnormal glomerulogenesis, renal papillary hypoplasia, hydronephrosis, aberrant UB budding, duplicated collecting system, and urinary concentrating defect. Thus, the actions of angiotensin (ANG) II during kidney development are pleiotropic both spatially and temporally. Whereas the role of ANG II in renovascular and glomerular development has received much attention, little is known about the potential role of ANG II and its receptors in the morphogenesis of the collecting system. In this review, we discuss recent genetic and functional evidence gathered from transgenic knockout mice and in vitro organ and cell culture implicating the RAS in the development of the ureteric bud and collecting ducts. A novel conceptual framework has emerged from this body of work which states that stroma-derived ANG II elicits activation of AT1/AT2 receptors expressed on the ureteric bud to stimulate branching morphogenesis as well as collecting duct elongation and papillogenesis.

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.