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.     

Pain threshold, learning and formation of brain edema in mice lacking the angiotensin II type 2 receptor

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the functional role of the AT2 receptor in the central nervous system (CNS). We have previously shown that AT2-deficient mice displayed anxiety-like behavior in comparisons with wild-type mice. In the present study, we analyzed the pain threshold, learning behavior and brain edema formation using the tail-flick test, the tail-pinch test, the passive avoidance task and cold injury, respectively. In the passive avoidance task and cold injury, no differences were found between wild-type mice and AT2-deficient mice. In contrast, the pain threshold was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of beta-endorphin in the brain was analyzed quantitatively in AT2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of beta-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. We found that the AT2 receptor does not influence learning behavior and brain edema formation. As AT2-deficient mice have increased sensitivity to pain and decreased levels of brain beta-endorphin, AT2 receptors may perhaps mediate regulation of the pain threshold.     

Angiotensin II type 1 receptor blockade attenuates renal fibrogenesis in an immune-mediated nephritic kidney through counter-activation of angiotensin II type 2 receptor

The relative roles of angiotensin II (Ang II) type 1 receptor (AT(1)R) and Ang II type 2 receptor (AT(2)R) in immune-mediated nephritis are unknown, and the effect of the blockade of AT(1)R and its indirect counter-activation of AT(2)R relative to the anti-fibrotic action in this disease is unclear. To address this question, we studied the role of AT(1)R and AT(2)R in anti-glomerular basement membrane nephritis in SJL mice. Groups of mice were treated with either an AT(1)R antagonist (CGP-48933; CGP group), an AT(2)R antagonist (PD-123319; PD group), both (CGP/PD group), or a vehicle (PCt group) from Day 29 to 56. At Day 56 post-treatment, fibrosis-related parameters such as interstitial matrix deposition, and the expression of genes of TGF-beta1, plasminogen activator inhibitor-1, and type I collagen were significantly reduced in the kidney in the CGP group. There were no significant effects on these parameters in the PD group. However, this anti-fibrotic action by CGP-48933 was totally abolished by co-treatment with PD-123319 in the CGP/PD group. The gene expression of renin was significantly increased in the kidneys in the CGP and CGP/PD groups, suggesting that CGP-48933 had increased Ang II generation in those groups. In conclusion, counter-activation of AT(2)R by increased Ang II under AT(1)R blockade likely conferred an anti-fibrotic protection in this model.     

Reciprocal regulation of cholesterol excretion in apolipoprotein E-null mice by angiotensin II type 1 and type 2 receptor deficiency

AimsThe effects of AT₁ and AT₂ receptor deficiency on the intake and excretion of cholesterol were examined using atherosclerotic apolipoprotein E-null (ApoEKO) mice.Main methodsApoEKO, AT₁a/ApoEKO and AT₂/ApoEKO mice received a high-cholesterol diet (HCD: 1.25% cholesterol) for 10 days before sampling.Key findingsPlasma total cholesterol level was lower in AT₁a/ApoEKO mice and higher in AT₂/ApoEKO mice than in ApoEKO mice with a high cholesterol intake. In these mice, cholesterol content in feces was higher in AT₁a/ApoEKO mice and lower in AT₂/ApoEKO mice than in ApoEKO mice. Moreover, cholesterol content in bile tended to be higher in AT₁a/ApoEKO mice and lower in AT₂/ApoEKO mice than in ApoEKO mice, while a significant difference was observed only between AT₁a/ApoEKO and AT₂/ApoEKO mice. Cholesterol content and expression of HMG-CoA reductase and LDL receptor in liver were not different among the groups. Similar but weaker changes were also observed with a normal standard diet. Treatment with an AT₁ receptor blocker, irbesartan, increased cholesterol content in bile and tended to increase cholesterol excretion into feces in ApoEKO mice with HCD.SignificanceThese results suggest that AT₁ and AT₂ receptor stimulation was involved in the regulation of cholesterol excretion into bile and feces, and that the regulation acted reciprocally in a cholesterol overload condition with HCD.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Role of transmembrane helix IV in G-protein specificity of the angiotensin II type 1 receptor

G-protein activation by G-protein coupled receptors (GPCRs) is accomplished through proper interaction with the cytoplasmic loops rather than through sequence-specific interactions. However, the mechanism by which a specific G-protein is selected by a GPCR is not known. In the current model of GPCR activation, agonist binding modulates helix-helix interactions, which is necessary for fully determining G-protein specificity and stimulation of GDP/GTP exchange. In this study, we report that a single-residue deletion in transmembrane helix IV leads the angiotensin II type 1 (AT(1)) receptor chimera CR17 to retain GTP-sensitive high affinity for the agonist angiotensin II but results in complete inactivation of intracellular inositol phosphate production. The agonist dissociation profile of CR17 in the presence of guanosine 5'-3-O-(thio)triphosphate suggests that the activation-induced conformational changes of the chimeric receptor itself remain intact. Insertion of an alanine at position 149 (CR17triangle down149A) in this chimera rescued the inactive phenotype, restoring intracellular inositol phosphate production by the chimera. This finding suggests that in the wild-type AT(1) receptor the orientation of transmembrane helix IV-residues following Cys(149) is a key determinant for effectively distinguishing among various structurally similar G-proteins. The results emphasize that the contacts within the membrane-embedded portion of transmembrane helix IV in the AT(1) receptor is important for specific G-protein selection.     

Cloning, expression, and characterization of a gene encoding the human angiotensin II type 1A receptor.

The human angiotensin II (AII) type 1a receptor gene and its upstream control sequence has been cloned from a human leukocyte genomic library. The promoter element CAAT and TATA sequences were found at -602 and -538, respectively, upstream from the translational initiation site. The deduced protein sequence is homologous to rat and bovine AT1a receptors (94.7% and 95.3% identity). The expressed gene exhibited high-affinity AII and Dup753 binding and was functionally coupled to inositol phosphate turnover. Northern analysis of human tissues showed AT1 receptor mRNA expression in placenta, lung, heart, liver, and kidney. Using 5' untranslated and coding sequence as probes in a Southern blot analysis, it was established that another AT1 subtype exists in the human genome.     

Malformations of kidney and urinary tract in common chromosomal aberrations

Summary A review of urinary tract malformations in chromosomal aberrations is presented. This study is based on a careful evaluation of published cases as well as of our own patients and autopsy material.In trisomy D and E and in Turner's syndrome the incidence of gross urinary system malformations is more than 60%, in the cat-eye syndrome up to 100%. In trisomy 21, 4p-, 18q- und 18r syndrome malformations of the renal system are less frequent.However, in patients with the cri-du-chat syndrome (5p-) and in 13q-, 13r, 18p- and Klnefelter's syndrome, gross malformations of the urinary tract seem to occur only occasionally.There is no direct correlation of urinary tract malformation with concomitant malformations of other organs. Moreover, no specific renal malformations can be correlated to any particular chromosomal anomaly.

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Zusammenfassung Die vorliegende Arbeit über Mißbildungen des Harntraktes bei Chromosomenaberrationen beruht auf einem sorgfältigen und systematischen Studium publizierter Fälle und eigener beobachteter Patienten sowie von Autopsiebefunden. Bei fehlender Angabe über den Nierenstatus wurde versucht, durch persönliche Anfrage beim Autor der Arbeit zusätzliche Angaben zu erhalten.Viele Chromosomenaberrationen gehen mit einer deutlich erhöhten Häufigkeit schwerer Mißbildungen der Nieren und Harnwege einher. Bei der Trisomie D und E sowie beim Turner-Syndrom finden sich Mißbildungen des Urinsystems in über 60%, beim Cat-eye-Syndrom sogar in 100% der Fälle.Bei der Trisomie 21, bei Deletion des kurzen Armes von Chromosom 4 (4p-) sowie bei Deletion des langen Armes von Chromosom 18 (18q-) und bei Ringchromosom 18 (18r) finden sich Mißbildungen der Nieren- und Harnwege seltener, aber dennoch signifikant häufiger als bei einer Normalbevökerung.Bei Patienten mit dem Cri-du-chat-Syndrom (5p-), mit Deletion des langen Armes des Chromosoms 13 (13q-), bei 13 Ringchromosom (13r) sowie Deletion des kurzen Armes des Chromosoms 18 (18p-) und beim Klinefelter-Syndrom dagegen scheinen gröbere Nierenmißbildungen nur gelegentlich vorzukommen.Es besteht keine direkte Korrelation zwischen Mißbildungen der Nieren oder Harnwege und Mißbildungen anderer Organe oder dysplastischen Zeichen.Zudem kann den einzelnen Trisomien und Chromosomenaberrationen keine spezifische Mißbildung des Harntraktes zugeordnet werden. Es ist deshalb unmöglich, irgendwelche Nierenanomalie mit einem speziellen Chromosom oder einer bestimmten Chromosomenregion zu korrelieren.

     

Role of 12-lipoxygenase in decreasing P-cadherin and increasing angiotensin II type 1 receptor expression according to glomerular size in type 2 diabetic rats

12-lipoxygenase (12-LO) was implicated in the development of diabetic nephropathy (DN), in which the proteinuria was thought to be associated with a decreased expression of glomerular P-cadherin. Therefore, we investigated the role of 12-LO in the glomerular P-cadherin expression in type 2 diabetic rats according to the glomerular sizes. Rats fed with high-fat diet for 6 wk were treated with low-dose streptozotocin. Once diabetes onset, diabetic rats were treated with 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) for 8 wk. Then glomeruli were isolated from diabetic and control rats with a sieving method. RT-PCR, Western blotting, and immunofluorescent staining were used for mRNA and protein expressions of P-cadherin and angiotensin II (Ang II) type 1 receptor (AT1). We found that CDC did not affect the glucose levels but completely attenuated diabetic increases in glomerular volume and proteinuria. Diabetes significantly decreased the P-cadherin mRNA and protein expressions and increased the AT1 mRNA and protein expressions in the glomeruli. These changes were significantly prevented by CDC and recaptured by direct infusion of 12-LO product [12(S)-HETE] to normal rats for 7 days. The decreased P-cadherin expression was similar between large and small glomeruli, but the increased AT1 expression was significantly higher in the large than in the small glomeruli from diabetic and 12(S)-HETE-treated rats. Direct infusion of normal rats with Ang II for 14 days also significantly decreased the glomerular P-cadherin expression. These results suggest that diabetic proteinuria is mediated by the activation of 12-LO pathway that is partially attributed to the decreased glomerular P-cadherin expression.     

Augmented renal vascular nNOS and renin protein expression in angiotensin type 1 receptor null mice

The present study was performed to determine the influence of absence of angiotensin type 1A (AT(1A)) and/or AT(1B) receptor feedback regulation of kidney neuronal nitric oxide synthase (nNOS) and renin protein expression. Kidneys were harvested from wild-type (WT), AT(1A)(-/-), AT(1B)(-/-), and AT(1A)(-/-)AT(1B)(-/-) mice and immunostained for nNOS and renin protein localization. AT(1A)(-/-) and AT(1A)(-/-)AT(1B)(-/-) kidneys demonstrated an increase in the percentage of glomeruli with nNOS-positive afferent and interlobular arterioles compared with WT mice. Density of vascular nNOS immunostaining was 20-fold higher in kidneys of AT(1A)(-/-) and AT(1A)(-/-)AT(1B)(-/-) compared with WT mice. Density of macula densa nNOS immunostaining was 7-fold higher in AT(1A)(-/-)AT(1B)(-/-) than in WT mice. Percent of glomeruli positive for juxtaglomerular (JG) cell renin was 3-fold higher, whereas the density of JG cell renin immunostaining was 15-fold higher in kidneys of AT(1A)(-/-) and AT(1A)(-/-)AT(1B)(-/-) compared with WT mice. Kidneys of AT(1A)(-/-) and AT(1A)(-/-)AT(1B)(-/-) mice displayed recruitment of renin protein expression along afferent and interlobular arterioles. Absence of AT(1) receptor signaling resulted in enhanced nNOS protein expression in both microvascular and tubular structures. Enhanced NO generation may contribute to the reduced renal vascular tone and blood pressure observed with blockade of the renin-angiotensin system.     

Upregulation of angiotensin II type 2 receptor expression in estrogen-induced pituitary hyperplasia

Recent evidence shows that reexpression and upregulation of angiotensin II (ANG II) type 2 (AT2) receptor in adult tissues occur during pathological conditions such as tissue hyperplasia, inflammation, and remodeling. In particular, expression of functional AT2 receptors in the pituitary and their physiological significance and regulation have not been described. In this study, we demonstrate that chronic in vivo estrogen treatment, which induces pituitary hyperplasia, enhances local AT2 expression (measured by Western blot and RT-PCR) concomitantly with downregulation of ANG II type 1 (AT1) receptors. In vivo progesterone treatment of estrogen-induced pituitary hyperplasia did not modify either the ANG II receptor subtype expression pattern or octapeptide-induced and AT1-mediated calcium signaling. Nevertheless, an unexpected potentiation of the ANG II prolactin-releasing effect was observed in this group, and this response was sensitive to both AT1 and AT2 receptor antagonists. These data are the first to document that ANG II can act at the pituitary level through the AT2 receptor subtype and that estrogens display a differential regulation of AT1 and AT2 receptors at this level.     

Localization of angiotensin II receptor subtypes in the rabbit adrenal and kidney

The localization of subtypes of the angiotensin II receptor has been determined by autoradiographic techniques using iodinated angiotensin II and two nonpeptide antagonists that exhibit selective affinities. DuP 753 specifically displaces type 1 sites (AII-1) and PD123177 inhibits only type 2 sites (AII-2). The rabbit adrenal cortex contains predominately AII-1 sites and the few AII-2 sites that are present are nonuniformly distributed. In the rabbit kidney, the fibrous outer sheath contains exclusively AII-2 sites whereas the glomeruli of the renal cortex and the renal medulla exhibit only AII-1 sites.     

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.