In situ demonstration of angiotensin-dependent and independent pathways for hyperaldosteronism during chronic extracellular fluid volume depletion.

In wild-type mice, 2-wk administration of losartan, an angiotensin (Ang) II type 1 (AT1) receptor antagonist, along with dietary sodium restriction, resulted in an elevation of plasma aldosterone greater than that seen with sodium restriction alone (2.75 +/- 0.35 vs. 1.38 +/- 0.16 ng/ml, P < 0.01). Plasma potassium increased in sodium-restricted, losartan-treated mice (6.0 +/- 0.2 mEq/liter), while potassium remained unchanged in mice with sodium restriction alone. To study the effect of Ang II on glomerulosa cells that may operate independently of plasma potassium in situ, we used chimeric mice made of cells with or without the intact AT1A gene (Agtr1a). When animals were fed a normal diet or chronically infused with Ang II, the aldosterone synthase mRNA was detectable only in Agtr1a+/+ but not Agtr1a-/- zona glomerulosa cells. After 2 wk of sodium restriction, plasma aldosterone increased (1.51 +/- 0.27 ng/ml) and potassium remained on average at 4.5 +/- 0.2 mEq/liter, with aldosterone synthase mRNA expressed intensively in Agtr1a+/+, but not detectable in Agtr1a-/- cells. Simultaneous sodium restriction and losartan treatment caused increases in plasma potassium (5.5 +/- 0.1 mEq/liter) and aldosterone (1.84 +/- 0.38 ng/ml), with both Agtr1a-/- and Agtr1a+/+ cells intensively expressing aldosterone synthase mRNA. Thus, aldosterone production is regulated by Ang II in the adrenal gland during chronic alterations in extracellular fluid volume when plasma potassium is maintained within the normal range. In the light of a previous observation that dietary potassium restriction superimposed on sodium restriction abolished secondary hyperaldosteronism in angiotensinogen null-mutant mice, the present findings demonstrate that when the renin-Ang system is compromised, plasma potassium acts as an effective alternative mechanism for the volume homeostasis through its capacity to induce hyperaldosteronism.

Angiotensin type II receptor expression and ureteral budding.

PURPOSE: Deletion of the angiotensin type II receptor gene (Agtr2) in mice results in a spectrum of urinary tract anomalies similar to that in humans. The mechanism behind this anomalous development is poorly understood. We evaluated Agtr2 expression as it relates to normal and abnormal ureteral budding. MATERIALS AND METHODS: A total of 400 wild type mice were inspected at birth for gross evidence of a urinary tract anomaly. In addition, the urinary tracts of 30 wild type embryos were evaluated at 11.0/11.5 and 13.5 weeks of gestation. These embryos were examined for ureteral budding site via section and whole mount in situ hybridization with c-ret probe and Agtr2 expression via in situ hybridization with Agtr2 riboprobe. There were 740 newborn mice homozygous for the null mutation of Agtr2 also evaluated along with 55 angiotensin type II knockout embryos at the aforementioned gestational ages. RESULTS: All wild type newborn animals were grossly normal. Of the angiotensin type II knockout newborns 23 (3.1%) had gross abnormalities of the urinary tract at birth. The predominant finding was a duplicated collecting system associated with a hydronephrotic upper pole moiety. These duplicated collecting systems fulfilled the Meyer-Weigert law. Interestingly, 25 (59.5%) of the knockout embryos showed abnormal ureteral budding. However, in wild type embryos Agtr2 was expressed at this "ectopic" cranial site between the wolffian duct and metanephric mesenchyme. CONCLUSIONS: Although not the sole regulator, angiotensin type II receptor expression may have a role in the embryological development of the urinary tract by inhibiting aberrant ureteral budding. A defect in this inhibitory process appears to cause ectopic ureteral budding, and may subsequently lead to renal dysplasia and other congenital anomalies of the kidney and urinary tract.

Moyamoya disease complicated with renal artery stenosis and nephrotic syndrome: reversal of nephrotic syndrome after nephrectomy.

A 7-year-old boy with moyamoya disease developed sustained hypertension, nephrotic syndrome, hyperreninemia, and occlusion of the right renal artery. After right nephrectomy, hyperreninemia and hypertension improved. Proteinuria was resolved after nephrectomy, in parallel with the decrease in plasma renin activity. Moyamoya disease can cause nephrotic-range proteinuria, which is caused hemodynamically by hyperreninemia.

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.

Nephrotoxicity of angiotensin inhibition during the perinatal period.

As the only ex utero mechanism for the removal of nitrogenous waste, the mammalian kidney achieves some 50-fold increase in urine production during the perinatal period when the placental circulation becomes no longer available as a functional dialyzer. This urine is efficiently removed from the kidney by the renal pelvis, a smooth muscle structure unique to mammals, which develops during the perinatal period. We found that mutant mice completely devoid of angiotensinogen or its type 1 receptor, as well as wild-type neonates given an ACE inhibitor, fail to develop a renal pelvis or a ureteral peristaltic movement. These structural and functional defects in the urinary tract are followed by severe obstructive injury of the renal parenchyma. The ability of angiotensin to directly induce the pelvis is demonstrated in an organ culture system, in which treatment with angiotensin induces the characteristic smooth muscle layer in the wild type, but not in homozygous null mutants. Upregulation of both renal angiotensin content and type 1 receptor at the renal hilum are also demonstrated in the wild type during the transition from intra- to extra-uterine life. By inducing the timely development of the renal pelvis, angiotensin thus facilitates the removal from the renal parenchyma of the urine that promptly increases at birth, thereby effectively preventing a buildup of intrarenal pressure and a consequent development of dysmorphic kidney.

Congenital anomalies of the kidney and urinary tract--role of the loss of function mutation in the pluripotent angiotensin type 2 receptor gene.

PURPOSE: Recent studies of the human genome and genetic engineering experiments in mice revealed that congenital anomalies of the kidney and urinary tract commonly seen in newborns in various anatomical forms are polygenic disease, that is a disease caused by simultaneous defects in multiple genes. We discuss some possible genetic explanations of the classic theories of the formation of congenital kidney and urinary tract anomalies. MATERIALS AND METHODS: We reviewed classic and current theories regarding urinary tract development. Included in our review are recent results from our laboratory evaluating the genetic role of normal and abnormal urinary tract development. RESULTS: We observed a genetic abnormality that may explain many classic anatomical theories of congenital kidney and urinary tract anomalies. One of the genes involved in urinary tract ontogenesis is the angiotensin type 2 receptor gene, which is the "other" angiotensin receptor. While the type 1 receptor mediates essentially all known actions of angiotensin, including its hypertensive effect, relatively little is known about the angiotensin type 2 receptor. Careful dissection studies in mutant mouse embryos selectively lacking the angiotensin type 2 receptor gene revealed that this gene is pleiotropic, that is its defect causes not only ectopic ureteral budding from the wolffian duct, but also disturbance in other subsequent ontogenic events that are critical for the normal growth of the kidney and urinary tract. CONCLUSIONS: Many congenital anomalies of the kidney and urinary tract appear to share a common genetic cause. While these anomalies are caused by various genetic hits, abnormalities in the angiotensin type 2 receptor gene are often involved in this anomalous development. This review article offers a better understanding of the genetics involved in urinary tract development and ties some of the newly emerging genetic theories with classic anatomical theories.

Production and use of chimeric mice.

The gene-targeting technology allows complete and selective inactivation of a specific gene to study the consequence of total absence of the gene product. The availability of such null-mutant mice enables investigators to identify the biological function of the gene product in physiological or pathophysiological conditions. This technology, popularly known as "gene knockout," can also induce more subtle mutations at the targeted site of the genome.

Salutary role for angiotensin in partial urinary tract obstruction.

BACKGROUND: In the neonatal period, angiotensin II (Ang II) is up-regulated and induces a timely development of the renal pelvis and ureteral peristalsis, thereby protecting the kidney from hydronephrosis. We tested the possibility that in adulthood, Ang II may act salutarily on the kidney structure during partial urinary tract obstruction by inducing adaptive changes in the peristaltic machinery. METHODS: Adult male Sprague-Dawley rats were subjected to partial unilateral ureteral obstruction (UUO) and divided into two groups, that is, those treated with (group L, N = 21) and those without (group C, N = 21) an angiotensin type 1 (AT1) receptor antagonist (losartan). Control animals were sham operated (N = 10). Rats were sacrificed either at day 7 or day 14. RESULTS: The degree of hydronephrosis determined morphometrically was significantly more severe in group L than group C at both day 7 and day 14, indicating that Ang II inhibition accentuated hydronephrosis. The measurement of upstream pressure within the partially ligated ureter in vivo revealed that losartan significantly attenuates the frequency of ureteral peristaltic activities. In in vitro studies using ureteral strips harvested from normal adult Sprague-Dawley rats (N = 10), Ang II (10(-8) mol/L) was shown to augment contraction, which was completely inhibited by losartan (10(-6) mol/L). CONCLUSIONS: Ang II has a salutary effect of protecting kidneys from hydronephrosis during partial ureteral obstruction through its ability to augment ureteral peristalsis.

Comparison of the effects of zolpidem and triazolam on nocturnal sleep and sleep latency in the morning: a cross-over study in healthy young volunteers.

1. Zolpidem (ZPD, 10 mg) was directly compared with triazolam (TRZ, 0.25 mg), a benzodiazepine hypnotic of a short action comparable to ZPD. The compounds were given to healthy young subjects for three nights, in a crossover design. 2. Polysomnographic data of three 150-min sections of the nights as well as the whole nights were analyzed, to clearly detect the proper effects of the very short acting hypnotics, which might be missed in the analysis of whole night. 3. Time courses were significantly different between the two compounds in the ratios (%) of stage wake (SW), stage 2 (S2), slow wave sleep (SWS) and stage REM (SR). 4. Compared to the baseline, SWS was increased by ZPD on the first night, not by TRZ. The separate analysis of the three 150-min sections revealed an increase of SWS during the first 150-min of the ZPD night, suggesting a proper action of ZPD to augment SWS. An increase of S2 and a decrease of SR were caused by TRZ, not by ZPD. However, the separate analysis indicated that ZPD might reduce SR during the first 150-min, which was cancelled by a subsequent rebound increase in the whole night analysis. 5. During the withdrawal period, TRZ, not ZPD, increased SW and SR with worsening of mood in the morning. ZPD did not affect sleep latency in the morning, while TRZ caused a trend of the reduction.

Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter.

In the normal mouse embryo, Bmp4 is expressed in mesenchymal cells surrounding the Wolffian duct (WD) and ureter stalk, whereas bone morphogenetic protein (BMP) type I receptor genes are transcribed either ubiquitously (Alk3) or exclusively in the WD and ureter epithelium (Alk6). Bmp4 heterozygous null mutant mice display, with high penetrance, abnormalities that mimic human congenital anomalies of the kidney and urinary tract (CAKUT), including hypo/dysplastic kidneys, hydroureter, ectopic ureterovesical (UV) junction, and double collecting system. Analysis of mutant embryos suggests that the kidney hypo/dysplasia results from reduced branching of the ureter, whereas the ectopic UV junction and double collecting system are due to ectopic ureteral budding from the WD and accessory budding from the main ureter, respectively. In the cultured metanephros deprived of sulfated glycosaminoglycans (S-GAGs), BMP4-loaded beads partially rescue growth and elongation of the ureter. By contrast, when S-GAGs synthesis is not inhibited, BMP4 beads inhibit ureter branching and expression of Wnt 11, a target of glial cell-derived neurotrophic factor signaling. Thus, Bmp4 has 2 functions in the early morphogenesis of the kidney and urinary tract. One is to inhibit ectopic budding from the WD or the ureter stalk by antagonizing inductive signals from the metanephric mesenchyme to the illegitimate sites on the WD. The other is to promote the elongation of the branching ureter within the metanephros, thereby promoting kidney morphogenesis.

Dual renin gene targeting by Cre-mediated interchromosomal recombination.

This study describes a new approach to targeting clustered genes. Our study began with the establishment of two lines of mice carrying different mutations in either Ren1 or Ren2. These two genes, both encoding renin, span over 40 kb in tandem on chromosome 1. Each gene was mutated by gene targeting to contain loxP sites. These two mutants and Cre transgenic mice were mated to produce offspring carrying the mutant Ren1 and Ren2 genes, as well as the Cre transgene concurrently. Initially, two mutant Ren genes were located on separate chromosomes. Southern analysis of mice from the second generation revealed that the mutant Ren1 and Ren2 were interchromosomally recombined at the loxP sites to produce a new dually mutated allele on the chromosome at the rate of 9.6% (7/73). Thus, interchromosomal recombination can be efficiently programmed by mating as designed using the Cre-loxP system.

Renal physiology of the mouse.

As the transgenic and gene-targeting technology has become an invaluable experimental approach to study the function of gene products, the need has been expanded to assess the physiology in the mouse, which is virtually the only animal species to which that new genetic technology can apply. In this regard, renal physiologists have also received fruits of success from modern technology in several key areas, and areas are expanding in both depth and scope.

How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT.

CAKUT are problems that often require surgical intervention or, in the worst case, lead to renal failure and the need for dialysis and/or renal transplantation. It is believed that these anomalies share a common genetic cause and to date there has been no good animal model with which to study these abnormalities. Although the abnormal interaction between the ureteral bud and metanephric blastema leads to renal hypodysplasia, vesicoureteral reflux, and ectopic ureters to name a few, the genetic and biochemical modulation of urinary tract development is not understood. Studies using the mouse strain mutant for angiotensin type 2 (AT2) receptors have given new insight into this mystery. The animals show defective apoptosis of undifferentiated mesenchymal cells in the area surrounding the developing kidney and urinary tract. This abnormal apoptosis may well interfere with the normal interaction between the ureteral bud and metanephric blastema resulting in CAKUT. This abnormal interaction would theoretically lead to preexisting intrinsic abnormalities of the kidney, which are programmed and take effect early in embryonic development. In the worst cases, the renal abnormalities would lead to progressive deterioration of renal function. Undoubtedly, there are more genes and biochemical modulators involved in this process other than the RAS and AT2 receptors. Our current animal model gives new and unique possibilities with which to study development of the kidney and urinary tract and ultimately seek ways of preventing an often debilitating disease process.

Potent antihypertrophic effect of the bradykinin B2 receptor system on the renal vasculature.

BACKGROUND: Angiotensin type 1 (AT1) receptor-deficient mice (Agtr1-/-), which selectively lack both AT1A and AT1B receptor genes, are characterized by marked intrarenal vascular thickening. In the present study, we explored the possible involvement of the kinin-kallikrein system in the development of this renal vascular hypertrophy. METHODS: Wild-type and Agtr1-/- mice were examined for the developmental regulation pattern of the kinin-kallikrein system and treated with aprotinin (a kallikrein inhibitor), AcLys [D-b Nal7, Ile8] des-Arg9-bradykinin (a bradykinin B1 receptor antagonist), or Hoe-140 (a bradykinin B2 receptor antagonist) from 3 to 14 days of age. RESULTS: The normal postnatal up-regulation of kininase II was organ-specifically suppressed in Agtr1-/- kidneys at 2 and 3 weeks of age. Immunohistochemical staining in Agtr1-/- mice revealed tissue kallikrein staining along the nephron from connecting tubules to cortical collecting tubules in proximity to the hypertrophic vasculature, whereas tissue kallikrein staining was confined to connecting tubules in wild-type mice. Aprotinin and Hoe-140 accelerated the vascular hypertrophy significantly as determined by wall thickness ratio, whereas B1 receptor antagonism had no effect. CONCLUSION: The kinin-kallikrein system in the Agtr1-/- mouse kidney is functionally activated by local suppression of kininase II and extensive redistribution of kallikrein to perivascular areas. This activation, specific to the kidney, serves to dampen a development of the marked vascular hypertrophy. These results demonstrate, to our knowledge for the first time, the antihypertrophic effect of the bradykinin B2 receptor system on the renal vasculature in vivo.

What have we learned from gene targeting studies for the renin angiotensin system of the kidney?

Over the last decade, gene targeting technologies have provided investigators with powerful new tools to study the physiology and pathophysiology of the kidney. In that, the renin-angiotensin system (RAS) has been a subject of intense investigation. Detailed analyses of mutant mice have not only confirmed notions already suggested by other studies, but also shed a new light on previously unrecognized functions of RAS. In this review, we will focus on what we have learned from these gene targeted animals in particular relevance to nephrology.

The renal lesions that develop in neonatal mice during angiotensin inhibition mimic obstructive nephropathy.

BACKGROUND: Inhibition of angiotensin action, pharmacologically or genetically, during the neonatal period leads to renal anomalies involving hypoplastic papilla and dilated calyx. Recently, we documented that angiotensinogen (Agt -/-) or angiotensin type 1 receptor nullizygotes (Agtr1 -/-) do not develop renal pelvis nor ureteral peristaltic movement, both of which are essential for isolating the kidney from the high downstream ureteral pressure. We therefore examined whether these renal anomalies could be characterized as "obstructive" nephropathy. METHODS: Agtr1 -/- neonatal mice were compared with wild-type neonates, the latter subjected to surgical complete unilateral ureteral ligation (UUO), by analyzing morphometrical, immunohistochemical, and molecular indices. Agtr1 -/- mice were also subjected to a complete UUO and were compared with wild-type UUO mice by quantitative analysis. To assess the function of the urinary tract, baseline pelvic and ureteral pressures were measured. RESULTS: The structural anomalies were qualitatively indistinguishable between the Agtr1 -/- without surgical obstruction versus the wild type with complete UUO. Thus, in both kidneys, the calyx was enlarged, whereas the papilla was atrophic; tubulointerstitial cells underwent proliferation and also apoptosis. Both were also characterized by interstitial macrophage infiltration and fibrosis, and within the local lesion, transforming growth factor-beta 1, platelet-derived growth factor-A and insulin-like growth factor-1 were up-regulated, whereas epidermal growth factor was down-regulated. Moreover, quantitative differences that exist between mutant kidneys without surgical obstruction and wild-type kidneys with surgical UUO were abolished when both underwent the same complete surgical UUO. The hydraulic baseline pressure was always lower in the pelvis than that in the ureter in the wild type, whereas this pressure gradient was reversed in the mutant. CONCLUSION: The abnormal kidney structure that develops in neonates during angiotensin inhibition is attributed largely to "functional obstruction" of the urinary tract caused by the defective development of peristaltic machinery.