Role of the angiotensin receptor in the development of the mammalian kidney and urinary tract

Recent gene targeting and pharmacological studies have shown that the renin-angiotensin system is essential for the development of the mammalian kidney and urinary tract system. We have generated mutant mice carrying a targeted null mutation of the angiotensin type 1 receptor (AT1) or type 2 receptor (AT2) gene. Analyses of these two mutant mice revealed that both mutants have a distinct phenotype in the kidney and urinary tract system. Thus, some AT2 null mutants (Agtr2 -/Y) show anomalies that mimic human congenital anomalies of the kidney and urinary tract as a result of a defect in the in utero organogenesis of the excretory system, while AT1 null mutants (Agtr1 -/-) exhibit hydronephrosis due to a defective development of the urinary peristaltic machinery during the perinatal period. In this review, we discuss chronologically from embryos to adults how the angiotensin receptors regulate the morphogenesis of the excretory system.     

Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux

Congenital anomalies of the kidney and urinary tract (CAKUT) include vesicoureteral reflux (VUR). VUR is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter and is associated with reflux nephropathy, the cause of 15% of end-stage renal disease in children and young adults. We investigated a man with a de novo translocation, 46,X,t(Y;3)(p11;p12)dn, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, we identified two novel ROBO2 intracellular missense variants that segregate with CAKUT and VUR in two unrelated families. Adult heterozygous and mosaic mutant mice with reduced Robo2 gene dosage also exhibit striking CAKUT-VUR phenotypes. Collectively, these results implicate the SLIT-ROBO signaling pathway in the pathogenesis of a subset of human VUR.     

Congenital anomalies of the kidney and urinary tract: An embryogenetic review

Congenital anomalies of the kidney and urinary tract (CAKUT) represent a broad range of disorders that result from abnormalities of the urinary collecting system, abnormal embryonic migration of the kidneys, or abnormal renal parenchyma development. These disorders are commonly found in humans, accounting for 20–30% of all genetic malformations diagnosed during the prenatal period. It has been estimated that CAKUT are responsible for 30–50% of all children with chronic renal disease worldwide and that some anomalies can predispose to adult‐onset diseases, such as hypertension. Currently, there is much speculation regarding the pathogenesis of CAKUT. Common genetic background with variable penetrance plays a role in the development of the wide spectrum of CAKUT phenotypes. This review aims to summarize the possible mechanisms by which genes responsible for kidney and urinary tract morphogenesis might be implicated in the pathogenesis of CAKUT. Birth Defects Research (Part C) 102:374–381, 2014. © 2014 Wiley Periodicals, Inc.     

Vesicoureteral reflux and other urinary tract malformations in mice compound heterozygous for Pax2 and Emx2

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. This disease group includes a spectrum of urinary tract defects including vesicoureteral reflux, duplex kidneys and other developmental defects that can be found alone or in combination. To identify new regulators of CAKUT, we tested the genetic cooperativity between several key regulators of urogenital system development in mice. We found a high incidence of urinary tract anomalies in Pax2;Emx2 compound heterozygous mice that are not found in single heterozygous mice. Pax2^+/−;Emx2^+/− mice harbor duplex systems associated with urinary tract obstruction, bifid ureter and a high penetrance of vesicoureteral reflux. Remarkably, most compound heterozygous mice refluxed at low intravesical pressure. Early analysis of Pax2^+/−;Emx2^+/− embryos point to ureter budding defects as the primary cause of urinary tract anomalies. We additionally establish Pax2 as a direct regulator of Emx2 expression in the Wolffian duct. Together, these results identify a haploinsufficient genetic combination resulting in CAKUT-like phenotype, including a high sensitivity to vesicoureteral reflux. As both genes are located on human chromosome 10q, which is lost in a proportion of VUR patients, these findings may help understand VUR and CAKUT in humans.     

The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfra1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfra1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret''s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret''s signaling specificity and intergenic interactions that confer normal urinary system development.Key words: RET, GDNF, kidney, RTK, CAKUT, branching morphogenesis, ureter     

Renal abnormalities and their developmental origin

Congenital abnormalities of the kidney and urinary tract (CAKUT) occur in 1 out of 500 newborns, and constitute approximately 20-30% of all anomalies identified in the prenatal period. CAKUT has a major role in renal failure, and there is increasing evidence that certain abnormalities predispose to the development of hypertension and cardiovascular disease in adult life. Moreover, defects in nephron formation can predispose to Wilms tumour, the most frequent solid tumour in children. To understand the basis of human renal diseases, it is essential to consider how the kidney develops.     

The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfrα1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfrα1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret’s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret’s signaling specificity and intergenic interactions that confer normal urinary system development.     

Development of the kidney medulla

The mature renal medulla, the inner part of the kidney, consists of the medullary collecting ducts, loops of Henle, vasa recta and the interstitium. The unique spatial arrangement of these components is essential for the regulation of urine concentration and other specialized kidney functions. Thus, the proper and timely assembly of medulla constituents is a crucial morphogenetic event leading to the formation of a functioning metanephric kidney. Mechanisms that direct renal medulla formation are poorly understood. This review describes the current understanding of the key molecular and cellular mechanisms underlying morphological aspects of medulla formation. Given that hypoplasia of the renal medulla is a common manifestation of congenital obstructive nephropathy and other types of congenital anomalies of the kidney and urinary tract (CAKUT), better understanding of how disruptions in medulla formation are linked to CAKUT will enable improved diagnosis, treatment and prevention of CAKUT and their associated morbidity.     

Development of the kidney medulla

The mature renal medulla, the inner part of the kidney, consists of the medullary collecting ducts, loops of Henle, vasa recta and the interstitium. The unique spatial arrangement of these components is essential for the regulation of urine concentration and other specialized kidney functions. Thus, the proper and timely assembly of medulla constituents is a crucial morphogenetic event leading to the formation of a functioning metanephric kidney. Mechanisms that direct renal medulla formation are poorly understood. This review describes the current understanding of the key molecular and cellular mechanisms underlying morphological aspects of medulla formation. Given that hypoplasia of the renal medulla is a common manifestation of congenital obstructive nephropathy and other types of congenital anomalies of the kidney and urinary tract (CAKUT), better understanding of how disruptions in medulla formation are linked to CAKUT will enable improved diagnosis, treatment and prevention of CAKUT and their associated morbidity.     

Genetic Basis of Ureterocele

Congenital anomalies of the kidney and urinary tract (CAKUT) form a group of heterogeneous disorders that affect the kidneys, ureters and bladder, with frequent asynchronous presentations and multiple CAKUT associations in the same individual. Urinary tract formation is a complex process, dependent of the interaction of multiple genes and their sub-product. The same genic alterations can lead to different molecular expressions and different morphological anomalies. The ureterocele is a cystic dilation of the distal intramural ureter, resulting in obstruction of urine flow, dilation of the ureter and renal pelvis and loss of renal function. Two key steps in the urinary tract ontogenesis may be related to ureterocele development: formation and migration of the ureteric bud and its incorporation in the bladder. This review aims to describe the morphological, cellular and biochemical steps, as well as the genes involved in the occurrence of this anomaly.     

F2-Isoprostane level is associated with the angiotensin II type 1 receptor -153A/G gene polymorphism

Recent studies have shown that F2-isoprostane levels-a marker for lipid peroxidation-are increased in human renovascular hypertension but not in essential hypertension. Angiotensin II specifically stimulates F2-isoprostane production through activation of the AT1 receptor. The objective was to determine whether there is a relationship between the level of oxidative stress evaluated by measuring urinary F2-isoprostanes levels and polymorphisms of genes involved in the renine angiotensin aldosterone system (RAAS) regulation. The population studied included 100 subjects, 65 of whom were healthy normotensives; the other 35 were suffering from untreated, essential hypertension. The polymorphisms studied concern the genes encoding angiotensin I-converting enzyme (ACE/in16del/ins), angiotensin II receptor type I (AGTR1/A+39C[A+1166C] and AGTR1/A-153G), angiotensinogen (AGT/M235T), and aldosterone synthase (CYP11B2/T344C). Oxidative stress was evaluated by measuring urinary F2-isoprostanes levels. The characteristics of the population were as follows: men/women = 46/56; age = 50 +/- 10 years; BMI = 24 +/- 3 kg/m2; SBP = 131.7 +/- 17.2 mm Hg; DBP = 84.6 +/- 10.4 mm Hg. In univariate analysis, urinary F2-isoprostane levels were significantly lower in the presence of the G allele of AGTR1/A-153G (56 +/- 17 vs 76 +/- 39 pmol/mmol creatinine; P < 0.001, and P < 0.01 after Bonferroni correction for 10 tests). In multivariate analysis, taking into account BP, age, gender, BMI, plasma glucose, and total cholesterol, the G allele of AGTR1/A-153G is linked independently to urinary F2-isoprostanes level (P < 0.01). Our data suggest that F2-isoprostane level depends at least in part on the A-153G polymorphism of the angiotensin II AT1 receptor gene. The clinical and prognostic relevance of this polymorphism requires further investigation.     

Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice

Mutations in the receptor tyrosine kinase RET are associated with congenital anomalies of kidneys or urinary tract (CAKUT). RET tyrosine Y1015 is the docking site for PLCγ, a major regulator of RET signaling. Abrogating signaling via Y1015 causes CAKUT that are markedly different than renal agenesis in Ret-null or RetY1062F mutant mice. We performed analysis of Y1015F mutant upper and lower urinary tracts in mice to delineate its molecular and developmental roles during early urinary tract formation. We found that the degeneration of the common nephric ducts (CND), the caudal-most Wolffian duct (WD) segment, depends on Y1015 signals. The CNDs in Y1015F mutants persist owing to increased proliferation and reduced apoptosis, and showed abundance of phospho-ERK-positive cells. In the upper urinary tract, the Y1015 signals are required for proper patterning of the mesonephros and metanephros. Timely regression of mesonephric mesenchyme and proper demarcation of mesonephric and metanephric mesenchyme from the WD depends on RetY1015 signaling. We show that the mechanism of de novo ectopic budding is via increased ERK activity due to abnormal mesenchymal GDNF expression. Although reduction in GDNF dosage improved CAKUT it did not affect delayed mesenchyme regression. Experiments using whole-mount immunofluorescence confocal microscopy and explants cultures of early embryos with ERK-specific inhibitors suggest an imbalance between increased proliferation, decreased apoptosis and increased ERK activity as a mechanism for WD defects in RetY1015F mice. Our work demonstrates novel inhibitory roles of RetY1015 and provides a possible mechanistic explanation for some of the confounding broad range phenotypes in individuals with CAKUT.     

Mutation in angiotensin II type 1 receptor disrupts its binding to angiotensin II leading to hypotension: An insight into hydrogen bonding patterns

To understand the role of angiotensin II type 1 receptor gene (AGTR1) gene products in relation to hypotension we have analyzed the single nucleotide polymorphisms (SNPs) associated with this gene. This can help us to understand the genetic variations that can alter the function of the gene products. In this present study, we report the polymorphic variant associated with AGTR1 and its weak interaction with angiotensin II (AngII) which leads to hypotension. Out of 1318 SNPs, six are found to be non-synonymous, of which rs1064533 shows significant damaging effect. A missense mutation (T1255G), i.e., from thymine to guanine for rs1064533 in AGTR1 gene results in amino acid substitution from cysteine (Cys) to tryptophan (Trp) in the receptor protein. A strong hydrogen bond exists between Cys289 of native AGTR1 protein and glutamine 167 of AngII. Interestingly, it is replaced by a weak hydrogen bond in the mutant protein between Trp289 (mutant residue) and serine 340. Such a substitution from small, hydrophilic to bulky, hydrophobic residue in AGTR1 protein results in reduced binding affinity of the receptor protein with AngII, leading to hypotension. The results presented from this in silico study will open up new prospect for genetic analysis of AGTR1 gene and will be beneficial to the researchers for understanding the role played by AGTR1 gene in hypotension disease.     

Identification of target tissue glycosphingolipid receptors for uropathogenic, F1C-fimbriated Escherichia coli and its role in mucosal inflammation

Bacterial adherence to mucosal cells is a key virulence trait of pathogenic bacteria. The type 1 fimbriae and the P-fimbriae of Escherichia coli have both been described to be important for the establishment of urinary tract infections. While P-fimbriae recognize kidney glycosphingolipids carrying the Galalpha4Gal determinant, type 1 fimbriae bind to the urothelial mannosylated glycoproteins uroplakin Ia and Ib. The F1C fimbriae are one additional type of fimbria correlated with uropathogenicity. Although it was identified 20 years ago its receptor has remained unidentified. Here we report that F1C-fimbriated bacteria selectively interact with two minor glycosphingolipids isolated from rat, canine, and human urinary tract. Binding-active compounds were isolated and characterized as galactosylceramide, and globotriaosylceramide, both with phytosphingosine and hydroxy fatty acids. Comparison with reference glycosphingolipids revealed that the receptor specificity is dependent on the ceramide composition. Galactosylceramide was present in the bladder, urethers, and kidney while globotriaosylceramide was present only in the kidney. Using a functional assay, we demonstrate that binding of F1C-fimbriated Escherichia coli to renal cells induces interleukin-8 production, thus suggesting a role for F1C-mediated attachment in mucosal defense against bacterial infections.     

Traditional and targeted exome sequencing reveals common, rare and novel functional deleterious variants in RET-signaling complex in a cohort of living US patients with urinary tract malformations

Signaling by the glial cell line-derived neurotrophic factor (GDNF)-RET receptor tyrosine kinase and SPRY1, a RET repressor, is essential for early urinary tract development. Individual or a combination of GDNF, RET and SPRY1 mutant alleles in mice cause renal malformations reminiscent of congenital anomalies of the kidney or urinary tract (CAKUT) in humans and distinct from renal agenesis phenotype in complete GDNF or RET-null mice. We sequenced GDNF, SPRY1 and RET in 122 unrelated living CAKUT patients to discover deleterious mutations that cause CAKUT. Novel or rare deleterious mutations in GDNF or RET were found in six unrelated patients. A family with duplicated collecting system had a novel mutation, RET-R831Q, which showed markedly decreased GDNF-dependent MAPK activity. Two patients with RET-G691S polymorphism harbored additional rare non-synonymous variants GDNF-R93W and RET-R982C. The patient with double RET-G691S/R982C genotype had multiple defects including renal dysplasia, megaureters and cryptorchidism. Presence of both mutations was necessary to affect RET activity. Targeted whole-exome and next-generation sequencing revealed a novel deleterious mutation G443D in GFRα1, the co-receptor for RET, in this patient. Pedigree analysis indicated that the GFRα1 mutation was inherited from the unaffected mother and the RET mutations from the unaffected father. Our studies indicate that 5?% of living CAKUT patients harbor deleterious rare variants or novel mutations in GDNF-GFRα1-RET pathway. We provide evidence for the coexistence of deleterious rare and common variants in genes in the same pathway as a cause of CAKUT and discovered novel phenotypes associated with the RET pathway.     

Human alpha defensin 5 expression in the human kidney and urinary tract

Background

The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects.

Methodology/Principal Findings

Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection.

Conclusions/Significance

DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility.     

Urocortins as cardiovascular peptides

Urocortins (Ucn) 1, 2 and 3, human homologues of fish urotensin I, form the corticotropin-releasing factor (CRF) family, together with CRF, urotensin I and sauvagine. Ucn 3 is a novel member of this family and is a specific ligand for CRF type 2 receptor. CRF type 2 receptor is thought to mediate the stress-coping responses, such as anxiolysis, anorexia, vasodilatation, a positive inotropic action on myocardium and dearousal. Endogenous ligands for the CRF type 2 receptor expressed in the cardiovascular tissues, such as the myocardium, have long been unknown. We have shown expression of Ucn 3 as well as Ucn 1 in the human heart. Ucn 3 is also expressed in the kidney, particularly distal tubules. Studies in various rat tissues showed that high concentrations of immunoreactive Ucn 3 were found in the pituitary gland, adrenal gland, gastrointestinal tract, ovary and spleen in addition to the brain, heart and kidney. These observations suggest that Ucn 3 is expressed in various tissues including heart and kidney, and may regulate the circulation in certain aspects of stress and diseases, such as inflammation. Ucn 1 and 3 appear to have important pathophysiological roles in some cardiovascular diseases.     

Pre-miRNA Hsa-Let-7a-2: a Novel Intracellular Partner of Angiotensin II Type 2 Receptor Negatively Regulating its Signals

G protein-coupled receptors (GPCRs) are the largest family of druggable targets, and their biological functions depend on different ligands and intracellular interactomes. Some microRNAs (miRNAs) bind as ligands to RNA-sensitive toll-like receptor 7 to regulate the inflammatory response, thereby contributing to the pathogenesis of cancer or neurodegeneration. It is unknown whether miRNAs bind to angiotensin II (Ang II) type 2 receptor (AGTR2), a critical protective GPCR in cardiovascular diseases, as ligands or intracellular interactomes. Here, screening for miRNAs that bind to AGTR2, we identified and confirmed that the pre-miRNA hsa-let-7a-2 non-competitively binds to the intracellular third loop of AGTR2. Functionally, intracellular hsa-let-7a-2 overexpression suppressed the Ang II-induced AGTR2 effects such as cAMP lowering, RhoA inhibition, and activation of Src homology 2 domain-containing protein-tyrosine phosphatase 1, whereas hsa-let-7a-2 knockdown enhanced these effects. Consistently, overexpressed hsa-let-7a-2 restrained the AGTR2-induced antiproliferation, antimigration, and proapoptosis of cells, and vasodilation of mesenteric arteries. Our findings demonstrated that hsa-let-7a-2 is a novel intracellular partner of AGTR2 that negatively regulates AGTR2-activated signals.