Ecology explains anhydrobiotic performance across tardigrades, but the shared evolutionary history matters more.

Desiccation stress is lethal to most animals. However, some microinvertebrate groups have evolved coping strategies, such as the ability to undergo anhydrobiosis (i.e. survival despite the loss of almost all body water). Tardigrades are one such group, where the molecular mechanisms of anhydrobiosis have been more thoroughly studied. Despite the ecological, evolutionary and biotechnological importance of anhydrobiosis, little is known about its inter- and intra-specific variability nor its relationship with natural habitat conditions or phylogenetic history. We developed a new index-anhydrobiotic recovery index (ARI)-to evaluate the anhydrobiotic performance of tardigrade populations from the family Macrobiotidae. Moreover, we compared the explanatory role of habitat humidity and phylogenetic history on this trait using a variance partitioning approach. We found that ARI is correlated with both microhabitat humidity and yearly rainfall, but it is mostly driven by phylogenetic niche conservatism (i.e. a high portion of ARI variation is explained by phylogeny alone). Finally, we showed that anhydrobiotic performance is highly variable, even between closely related species, and that their response to local ecological conditions is tightly linked to their phylogenetic history. This study not only presents key insights into an emerging model system, but also provides a new methodological approach for wider scale studies of the ecological and evolutionary implications of anhydrobiosis.

Chronic carbon monoxide treatment attenuates development of obesity and remodels adipocytes in mice fed a high-fat diet.

OBJECTIVE: Induction of heme oxygenase-1 (HO-1) has been demonstrated to result in chronic weight loss in several rodent models of obesity. However, the specific contribution of the HO metabolite, carbon monoxide (CO) to this response remains unknown. In this study, we determined the effect of chronic low level administration of a specific CO donor on the progression of obesity and its effects on metabolism and adipocyte biology in mice fed a high-fat diet. DESIGN: Experiments were performed on C57BL/6J mice fed a high-fat diet (60%) from 4 weeks until 30 weeks of age. Mice were administered either the CO donor, carbon monoxide releasing molecules (CORM)-A1 (5 mg kg(-1), intraperitoneally every other day) or the inactive form of the drug (iCORM-A1). Body weights were measured weekly and fasted blood glucose, insulin as well as body composition were measured every 6 weeks. Food intake, O2 consumption, CO2 production, activity and body heat production were measured at 28 weeks after start of the experimental protocol. RESULTS: Chronic CORM-A1 attenuated the development of high fat induced obesity from 18 weeks until the end of the study. Chronic CORM-A1 treatment in mice fed a high-fat diet resulted in significant decreases in fasted blood glucose, insulin and body fat and increased O2 consumption and heat production as compared with mice treated with iCORM-A1. Chronic CORM-A1 treatment also resulted in a significant decrease in adipocyte size and an increase in adipocyte number and in NRF-1, PGC-1α and UCP1 protein levels in epidydmal fat. CONCLUSION: Our results demonstrate that chronic CO treatment prevents the development of high-fat diet induced obesity via stimulation of metabolism and remodeling of adipocytes.

Chronic HO-1 induction with cobalt protoporphyrin (CoPP) treatment increases oxygen consumption, activity, heat production and lowers body weight in obese melanocortin-4 receptor-deficient mice.

OBJECTIVE: Heme oxygenase-1 induction (HO-1) elicits chronic weight loss in several rodent models of obesity. Despite these findings, the mechanism by which HO-1 induction reduces body weight is unclear. Chronic HO-1 induction does not alter food intake, suggesting other mechanisms such as increases in metabolism and activity may be responsible for the observed reduction of body weight. In this study, we investigated the mechanism of weight loss elicited by chronic HO-1 induction in a model of genetic obesity due to melanocortin-4 receptor (MC4R) deficiency. DESIGN: Experiments were performed on loxTB MC4R-deficient mice as well as lean controls. Mice were administered cobalt protoporphyrin (CoPP, 5 mg kg(-1)), an inducer of HO-1, once weekly, from 4 to 23 weeks of age. Body weights were measured weekly and fasted blood glucose and insulin, as well as food intake were determined at 18 weeks of age. Oxygen consumption (VO(2)), CO(2) production (VCO(2)), activity and body heat production were measured at 20 weeks of age. RESULTS: Chronic CoPP treatment resulted in a significant decrease in body weight from 5 weeks on in loxTB mice. Chronic CoPP treatment resulted in a significant decrease in fasted blood glucose levels, plasma insulin and a significant increase in plasma adiponectin levels in MC4R-deficient mice. Chronic CoPP treatment increased VO(2) (47 ± 4 vs 38 ± 3 ml kg(-1) per min, P<0.05) and VCO(2) (44 ± 7 vs 34 ± 4 ml kg(-1) per min, P<0.05) in treated vs non-treated, MC4R-deficient mice (n=4). Heat production (10%) and activity (18%) were also significantly (P<0.05) increased in CoPP-treated MC4R-deficient mice. CONCLUSION: Our results suggest that chronic HO-1 induction with CoPP induction elicits weight loss by increasing metabolism and activity by an MC4R-independent pathway.

Effects of renin gene transfer on blood pressure and renin gene expression in a congenic strain of Dahl salt-resistant rats.

To investigate whether a BP-regulatory locus exists in the vicinity of the renin locus on rat chromosome 13, we transferred this chromosome segment from the Dahl salt-sensitive (S) rat onto the genetic background of the Dahl salt-resistant (R) rat. In congenic Dahl R rats carrying the S renin gene and fed an 8% salt diet, systolic BP was significantly lower than in progenitor Dahl R rats: 127 +/- 1 mmHg versus 138 +/- 4 mmHg, respectively (P < 0.05). Moreover, the decreased BP in the congenic Dahl R strain was associated with decreased kidney renin mRNA and decreased plasma renin concentration. These findings demonstrate that the Dahl S strain carries alleles in or near the renin locus that confer lower plasma renin concentration and lower BP than the corresponding alleles in the Dahl R strain, at least when studied on the genetic background of the Dahl R rat and in the environment of a high salt diet. The occurrence of coincident reductions in kidney renin mRNA, plasma renin concentration, and BP after interstrain transfer of naturally occurring renin gene variants strongly suggests that genetically determined variation in renin gene expression can affect BP.

Overexpression of protein kinase C-zeta (PKC-zeta) inhibits invasive and metastatic abilities of Dunning R-3327 MAT-LyLu rat prostate cancer cells.

Previously, we reported that protein kinase C (PKC)-zeta mRNA levels are reduced markedly in metastatic Dunning R-3327 rat prostate tumors relative to the nonmetastatic Dunning H tumor and normal rat prostate (C.T. Powell et al., Cell Growth & Differ., 5: 143-149, 1994). To examine the effect of PKC-zeta on metastatic and invasive abilities of an aggressive Dunning R-3327 cell line, we generated stably transfected clones of MAT-LyLu cells that overexpress active PKC-zeta. PKC-zeta-overexpressing MAT-LyLu cells exhibited tumorigenicity and growth rates in syngeneic rats similar to those of MAT-LyLu cells transfected with vector alone or untransfected MAT-LyLu. However, nine independent clones of PKC-zeta-expressing cells exhibited an average 2-fold lower tendency to metastasize to lungs relative to vector-transfected MAT-LyLu cell clones, with about 2-fold and 4.5-fold fewer metastases per rat in two separate protocols. In addition, the ability of four PKC-zeta overexpressing MAT-LyLu clones to invade through Matrigel in a Boyden chamber assay was reduced an average of 12-fold relative to three vector-transfected clones. These results indicate that increased PKC-zeta expression can substantially suppress invasion and metastasis by an aggressive rat prostate tumor.

Transgenesis and gene targeting in the mouse; tools for studying genetic determinants of hypertension.

As more effort is made to identify genes responsible for hypertension in human populations and genetically hypertensive animal models, the need for experimental systems in which the functional significance of genes, gene variants, and quantitative trait loci (QTL) can be determined is becoming increasingly important. Over the past five years, transgenic and gene-targeting technology has been utilized to study the cardiovascular effects of over-expression or ablation of genes which have been considered candidates in the genetic basis of hypertension. This review focuses on the most recent major advances in this area, and how this technology aids in our understanding of the molecular mechanisms by which newly discovered genes or gene variants affect blood pressure in the whole organism. We also discuss the potential use of transgenic models in refining the location of a QTL, and discuss some of the limitations and potential pitfalls in the application of these tools to the field of hypertension research. The coupling of genetic manipulations afforded by transgenesis and gene targeting, along with advances in our ability to assess the cardiovascular phenotype in the mouse, provides us with a powerful system for examining the genes responsible for causing essential hypertension.

Novel mechanism of hypertension revealed by cell-specific targeting of human angiotensinogen in transgenic mice.

We tested the hypothesis that the tissue-specific intrarenal renin-angiotensin system (RAS) can participate in the regulation of blood pressure independently of its endocrine counterpart, by generating two transgenic models that differ in their tissue-specific expression of human angiotensinogen (AGT). Human AGT expression was driven by its endogenous promoter in the systemic model and by the kidney androgen-regulated protein promoter in the kidney-specific model. Using molecular, biochemical, and physiological measurements, we demonstrate that human AGT mRNA and protein are restricted to the kidney in the kidney-specific model. Plasma ANG II was elevated in the systemic model but not in the kidney-specific model. Nevertheless, blood pressure was markedly elevated in both the systemic and kidney-specific transgenic mice. Acute administration of the selective ANG II AT-1 receptor antagonist losartan lowered blood pressure in the systemic model but not in the kidney-specific model. These results provide evidence for the potential importance of the intrarenal RAS in blood pressure regulation by showing that expression of AGT specifically in the kidney leads to chronic hypertension independently of the endocrine RAS.

Inhibition of renal outer medullary 20-HETE production produces hypertension in Lewis rats.

Recent studies have indicated that a deficiency in the production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the outer medulla of the kidney may contribute to the abnormalities in the renal handling of sodium and the development of hypertension in Dahl salt-sensitive rats. To determine whether a reduction in 20-HETE production in the outer medulla is sufficient to induce hypertension, an inhibitor of the renal metabolism of arachidonic acid by P450 enzymes, 17-octadecenoic acid (17-ODYA), was chronically infused directly into the outer medulla of the left kidney of uninephrectomized Lewis rats fed a high salt diet. Renal medullary interstitial infusion of 17-ODYA (400 pmol/min) reduced the formation of 20-HETE in the outer medulla of the infused kidney by 70% compared with values seen in the right kidney collected when the rat was uninephrectomized, but it had no effect on the production of 20-HETE in the renal cortex. After 5 days, mean arterial pressure rose from 115 +/- 2 to 142 +/- 2 mm Hg (n = 6) in the rats infused with 17-ODYA, while mean arterial pressure was not significantly altered in the rats infused with vehicle alone (116 +/- 1 versus 117 +/- 2 mm Hg, n = 6). These results suggest that inhibition of the renal metabolism of arachidonic acid by P450 enzymes in the outer medulla of the kidney is sufficient to induce the development of hypertension in Lewis rats fed a high salt diet and support the view that P450 metabolites of arachidonic acid play an important role in the regulation of renal function and the long-term control of arterial pressure.

Cytochrome P4504A genotype cosegregates with hypertension in Dahl S rats.

Recent studies indicate that the production of 20-HETE by a P4504A2 enzyme in the outer medulla of the kidney is reduced in Dahl salt-sensitive (SS/Jr) rats, but the contribution of this abnormality to the elevation in loop Cl- transport and development of hypertension in this model is unknown. THe present study found that alleles at the locus for the P4504A2 gene cosegregate with blood pressure in an F2 population (n=151) derived from a cross between SS/Jr and Lewis rats (P < .0001). The P4504A2 locus is located in a region on rat chromosome 5 where a blood pressure quantitative trait locus was previously detected. Systolic blood pressure averaged 201 +/- 6 mm Hg in rats with the SS genotype (n=36), 192 +/- 4 mm Hg in SL genotype rats (n=77), and 169 +/- 3 mm Hg in LL genotype rats (n=38). In further studies, we confirmed that there are phenotypic differences in the expression of the P4504A2 gene in the kidneys of SS/Jr and Lewis rats. Although the production of 20-HETE from 14C-arachidonic acid was similar in microsomes prepared from the renal cortex of SS/Jr and Lewis rats (54 +/- 3 versus 55 +/- 3 pmol/min/mg protein), the production of 20-HETE in microsomes prepared from the outer medulla (OM) was markedly reduced in SS/Jr rats (2.8 +/- 0.8 versus 6.7 +/- 1 pmol/min/mg protein). The diminished production of 20-HETE in the OM was due to a threefold reduction in the level of P4504A2 protein. These results suggest that an altered expression of the P4504A2 enzyme in the OM may contribute to the development of hypertension in SS/Jr rats.

Renal cytochrome P4504A activity and salt sensitivity in spontaneously hypertensive rats.

Differences in the renal metabolism of arachidonic acid by cytochrome P450 have been reported in the spontaneously hypertensive rat (SHR) and Wistar-Kyoto rats, but the contribution of this system to the development of hypertension is unclear. The present study compared renal P450 activity and blood pressure in SHR and Brown-Norway rats (BN) under control conditions and in response to an elevation in sodium intake; genetic linkage analysis was performed in an F2 population (n=219) derived from these strains. Basal renal P4504A enzyme activity measured by conversion of [C(14)]arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) was significantly greater in the kidneys of adult SHR (n=7) than of BN (n=8) (82 +/- 7 versus 60 +/- 5 pmol/min per milligram protein). Renal 20-HETE production fell 45 percent in SHR and 22 percent in BN in which salt intake was elevated by drinking of saline instead of water for 2 weeks. Mean arterial pressure averaged 157 +/- 3mm Hg in SHR (n = 9) and 100 +/- 2 mm Hg in BN fed a normal salt diet, and it rose to 170 +/- 7 mm Hg (P<.05) in SHR and fell to 90 +/- 3 mm Hg (P<.05) in BN (n=8) after sodium intake was elevated. A polymorphic marker, D5Rjr1, that spanned a repeated element in the P4504A gene on chromosome 5, where all three P4504A isoforms are located, was used for genotyping of the F2 population. The P4504A genotype did not cosegregate with baseline mean arterial pressure in the F2 population; however, significant linkage was observed with the change in mean arterial pressure after sodium intake of the rats was elevated. The degree of linkage differed in male and female rats, and the highest LOD score (3.6) was observed in male F2 rats with a BN grandfather. These findings suggest that the difference in renal P450 activity in SHR and BN does not contribute to the development of hypertension in this F2 population, but it may play some role in determining the blood pressure response to an elevation in salt intake.

Transfer of a salt-resistant renin allele raises blood pressure in Dahl salt-sensitive rats.

To evaluate the role of the renin gene in the development of hypertension in Dahl salt-sensitive rats (SS/Jr/Hsd), we derived a congenic strain of rats homozygous for the salt-resistant renin allele (S/renrr) and compared them with a control strain homozygous for the salt-sensitive renin allele (S/ren(ss). Mean arterial pressure was significantly higher in 12-week-old S/renrr rats fed a high salt (8.0%) diet for 3 weeks than in S/ren(ss) rats or in SS/Jr/Hsd rats rederived from the foundation colony we used to generate the cogenic strain (195 +/- 3 [n = 49] versus 168 +/- 3 [n = 17] or 161 +/- 3 [n = 16] mm Hg). Mean arterial pressure was also higher in S/renrr rats than in S/ren(ss) rats raised from birth on either a very low salt (0.1%) diet (119 +/- 9 [n = 6] versus 100 +/- 7 [n = 7] mm Hg) or a low salt (0.4%) diet (143 +/- 1 [n = 22] versus 117 +/- 3 [n = 10] mm Hg). Plasma renin activity of S/renrr rats was significantly higher than that of S/ren(ss) rats fed a very low salt diet (5.7 +/- 2.0 versus 1.8 +/- 0.3) ng angiotensin l/mL per hour), a low salt diet (4.4 +/- 1.0 versus 1.1 +/- 0.3), or a high salt diet (1.5 +/- 0.2 versus 0.9 +/- 0.1). Urinary protein excretion was greater in S/renrr rats than in S/ren(ss) rats fed a high salt diet (244.2 +/- 48.5 versus 43.6 +/- 19.5 mg/24 h), and this was associated with significant reductions in renal blood flow (3.3 +/- 0.6 versus 4.6 +/- 0.5 mL/min per gram kidney weight) and glomerular filtration rate (0.49 +/- 0.11 versus 0.82 +/- 0.08 mL/min per gram kidney weight). Captopril (20 mg/kg i.v.) had no effect on blood pressure in S/ren(ss) rats fed a low salt diet, but it lowered blood pressure by 20 mm Hg in S/ren(rr) rats to the same level seen in untreated S/ren(ss) rats. Chronic administration of captopril (5 mg/100 mL drinking water) reduced blood pressure in S/renrr rats fed a high salt diet (170 +/- 5 mm Hg) to the same level seen in untreated S/ren(ss) rats, whereas it had no significant effect on blood pressure in S/ren(ss) rats. These results indicate that transfer of a salt-resistant renin allele to SS/Jr/Hsd rats raises plasma renin activity and augments the severity of hypertension and renal disease.

Genetic contamination of Dahl SS/Jr rats. Impact on studies of salt-sensitive hypertension.

The Dahl salt-sensitive rat (SS/Jr) is a widely used animal model of salt-sensitive hypertension. SS/Jr rats are believed to be highly inbred and uniformly sensitive to the hypertensinogenic effects of sodium chloride, but we have recently observed that SS/Jr rats from Harlan Sprague Dawley, Inc, exhibit considerable variability in their blood pressure response to supplemental dietary salt. To test the possibility that commercially available SS/Jr rats are genetically contaminated and therefore no longer fully inbred, we performed molecular genetic studies and blood pressure measurements in several groups of SS/Jr rats purchased from Harlan Sprague Dawley. We found molecular evidence of heterozygosity and/or atypical allelic variants involving loci on at least five different chromosomes. Many of the rats also failed to exhibit a salt-sensitive blood pressure phenotype. We conclude that SS/Jr rats being sold by the only commercial vendor of Dahl rats in the United States are genetically contaminated and resistant to the hypertensinogenic effects of salt. These findings raise serious questions about the interpretation of research conducted with SS/Jr rats obtained from Harlan Sprague Dawley.

Genetic Manipulation of the Renin-Angiotensin System Using Cre-loxP-Recombinase.

The advent of gene-targeting technology in embryonic stem cells has provided an important tool for the dissection of complex biological systems by allowing investigators to generate germ line mutations in selected genes. Since the introduction of this technology in the early 1980s, hundreds of genes have been targeted for systemic deletion (knocked out), including each gene of the renin-angiotensin system (RAS). Although the technique is very powerful, there are weaknesses that limit its usefulness for studying the RAS. For example, systemic deletion of several of the RAS genes leads to a phenotype, of varying severity depending on the gene in question, in which the mice suc5cumb to severe renal lesions and ultimately die before the age of weaning. This is observed in angiotensinogen (Agt-/-), angiotensin-converting enzyme (ACE-/-), and combined angiotensin receptor subtype 1A and 1B (At1a-/-, At1b-/-) deficient "knockout" mice (1-5). Mice deficient in At1a, but wild type at the At1b locus, are phenotypically normal on mixed genetic backgrounds, but exhibit the same renal lesions and reduced mortality when bred onto "pure" genetic backgrounds, suggesting that renal morphology in response to Ang-II may be under some complex genetic control (6). Presumably, Ang-II is required during the early neonatal period for the continued development of the kidney, and the mice die if they are unable to either generate or utilize An-II during this period.

Heme oxygenase-1 protects against radiocontrast-induced acute kidney injury by regulating anti-apoptotic proteins.

Radiocontrast agents are thought to induce acute kidney injury in part through increased production of reactive oxygen species and increased cellular apoptosis. In this study we determined whether heme oxygenase-1 could prevent or reduce radiocontrast-induced acute kidney injury and, if so, what were the mechanisms by which this can occur. Sodium iothalamate was administered to uninephrectomized, salt-depleted male Sabra rats to initiate acute kidney injury. Heme oxygenase-1 was induced with cobalt protoporphyrin or inhibited with stannous mesoporphyrin. Inhibition of heme oxygenase exacerbated kidney injury as measured by an increase in plasma creatinine and in superoxide production. Heme oxygenase-1 induction prevented the increase in plasma creatinine and in superoxide in both the cortex and medulla compared to untreated rats with acute kidney injury. This protective effect of heme oxygenase-1 was associated with increased anti-apoptotic proteins Bcl-2 and Bcl-xl and a decrease of pro-apoptotic caspase-3 and caspase-9 along with increased expression of inactive BAX. Our study suggests that increased levels of heme oxygenase-1 are protective against acute kidney injury due to radiocontrast exposure.

Physiological insights from genetic manipulation of the renin-angiotensin system.

The renin-angiotensin system is one of the most widely studied endocrine systems. It has an important role in the regulation of normal homeostasis, and disturbances in this system may be important in numerous pathological states. This review will focus on the major insights and important questions raised from gene targeting of this system.

Modifiable gene expression in mice: kidney-specific deletion of a target gene via the cre-loxP system.

With the advent of gene-targeting in mouse embryonic stem (ES) cells, the use of knockout mice to study the physiological effects of loss of gene function has become increasingly prevalent. However, there are several drawbacks with conventional gene-targeting approaches which may make phenotyping of the resultant mice difficult, if not, impossible. Conventional gene-targeting results in the loss of function of the targeted gene in all cells and tissues, which can be problematic for genes which are required developmentally, which exhibit a wide tissue-specific expression pattern, or are part of complex paracrine systems. As with mice that lack the angiotensinogen or endothelin-1 gene, loss of gene function may lead to a lethal phenotype which can be manifested during embryonic development, at birth or postnatally. These limitations could potentially be circumvented by using a system in which the loss of gene function is placed under spatial and/or temporal control. We will discuss how the cre-loxP recombinase system can be applied to delete a gene in a tissue- and developmentally regulated fashion.

Sequence analysis of the polymerase L gene of human respiratory syncytial virus and predicted phylogeny of nonsegmented negative-strand viruses.

The complete nucleotide sequence of the large (L) polymerase gene of human respiratory syncytial virus (RSV) strain A2 was determined by analysis of cloned-cDNAs representing the entire gene and confirmed in part by dideoxy sequencing of genomic RNA. The RSV L gene is 6578 nucleotides in length and contains a single major open reading frame that encodes a protein of 2165 amino acids. The molecular weight (250,226) and amino acid composition of the deduced RSV L protein are similar to those of other negative-strand RNA viruses. Regions of statistically significant amino acid sequence similarity were identified in pairwise global alignments of the RSV L protein with its counterparts in four paramyxoviruses (parainfluenza virus type 3, Sendai virus, measles virus, Newcastle disease virus) and two rhabdoviruses (rabies virus, vesicular stomatitis virus). In addition, amino acid sequence alignments showed that the RSV L protein has a 70-amino acid amino-terminal extension relative to the others. This is suggested to be due to the acquisition of gene overlap of the RSV L gene with its upstream neighbor, the 22K (M2) gene and the use of a new translational start site. The most highly related region among these seven proteins is located within the amino-terminal half, representing approximately 20% of each protein sequences. This region contains six discrete segments that are colinear and highly conserved in each paramyxovirus and rhabdovirus L protein, and three of these overlapped with sequence motifs found previously in other RNA-dependent RNA and DNA polymerases. A phylogenetic tree was constructed from the paramyxovirus and rhabdovirus L protein sequences to further define their relationships. The branching order indicates that RSV represents a lineage within the paramyxovirus family which is relatively distinct from the others, which in turn are more closely interrelated. Among these other members of the family Paramyxoviridae, the branching order does not entirely conform to their current taxonomic organization, providing support for its reevaluation.

Genetic evidence that lethality in angiotensinogen-deficient mice is due to loss of systemic but not renal angiotensinogen.

Angiotensinogen (AGT)-deficient mice die shortly after birth presumably due to renal dysfunction caused by the presence of severe vascular and tubular lesions in the kidney. Because AGT is expressed in renal proximal tubule cells, we hypothesized that its loss may be the primary mediator of the lethal phenotype. We generated two models to test this hypothesis by breeding transgenic mice expressing human renin with mice expressing human AGT (hAGT) either systemically or kidney-specifically. We then bred double transgenic mice with AGT+/- mice, intercrossed the compound heterozygotes, and examined the offspring. We previously reported that the presence of the human renin and systemically expressed hAGT transgene complemented the lethality observed in AGT-/- mice. On the contrary, we show herein that the presence of the human renin and kidney-specific hAGT transgene cannot rescue lethality in AGT-/- mice. An analysis of newborns indicated that AGT-/- mice were born in normal numbers, and collection of dead 10-day old pups revealed an enrichment in AGT-/-. Importantly, we demonstrated that angiotensinogen protein and functional angiotensin II was generated in the kidney, and the kidney-specific transgene was temporally expressed during renal development similar to the endogenous AGT gene. These data strongly support the notion that the loss of systemic AGT, but not intrarenal AGT, is responsible for death in the AGT-/- mouse model. Taken together with our previous studies, we conclude that the intrarenal renin-angiotensin system located in the proximal tubule plays an important role in blood pressure regulation and may cause hypertension if overexpressed, but may not be required for continued development of the kidney after birth.