A genetic linkage map of the laboratory rat, Rattus norvegicus.

We report the construction of the first complete genetic linkage map of the laboratory rat. By testing 1171 simple sequence length polymorphisms (SSLPs), we have identified 432 markers that show polymorphisms between the SHR and BN rat strains and mapped them in a single (SHR x BN) F2 intercross. The loci define 21 large linkage groups corresponding to the 21 rat chromosomes, together with a pair of nearby markers on chromosome 9 that are not linked to the rest of the map. Because 99.5% of the markers fall into one of the 21 large linkage groups, the maps appear to cover the vast majority of the rat genome. The availability of the map should facilitate whole genome scans for genes underlying qualitative and quantitative traits relevant to mammalian physiology and pathobiology.

Molecular therapies for vascular diseases.

Vascular disease is the most common cause of death in the industrialized world. Although significant progress has been made in treating these disorders, more therapeutic agents must be developed that effectively prevent, arrest, or reverse this disease. Recent insights into the pathogenesis of vascular disease have opened up a new frontier of molecular therapies that target molecules as diverse as adhesion molecules and transcription factors. The biological rationale for these new therapies and their prospects for success are discussed.

Renin-specific antibody for study of cardiovascular homeostasis.

Antiserum specific for purified canine renal renin was used to inhibit this enzyme in trained, conscious dogs. The antiserum did not affect blood pressure in sodium-replete dogs but decreased plasma renin activity and blood pressure in sodium-depleted animals. The antiserum also reduced blood pressure to control levels concomitant with suppression of plasma renin activity in uninephrectomized dogs with acute renovascular hypertension. These observations establish the role of the renin-angiotensin system in the maintenance of blood pressure in the sodium-depleted state as well as in the initiation of renovascular hypertension.

Gene expression of the renin-angiotensin system in human tissues. Quantitative analysis by the polymerase chain reaction.

Activation of tissue-specific gene expression of the components of the renin-angiotensin system (RAS) in humans may play an important role in cardiovascular regulation and pathophysiology. Studies of human tissue RAS expression, however, have been limited by the lack of availability of sufficient amounts of fresh human tissues and a sensitive method for detecting specific mRNAs. To demonstrate the presence of components of local RASs in humans we used the polymerase chain reaction (PCR) after reverse transcription to detect renin- angiotensinogen-, and angiotensin-converting enzyme-mRNA in small quantities of human tissues. Results indicated that all components of the RAS were widely expressed in human organ samples. In order to study changes of gene expression in small tissue samples (e.g., renal biopsies) obtained from patients, we established a competitive PCR assay for quantification of renin, using a 155-basepair deletion mutant of the human renin cDNA as an internal standard. Renin-mRNA concentration was quantitated in the kidney (1.74 +/- 0.2 pg renin/micrograms total RNA), adrenal gland (1.15 +/- 0.15 pg renin/micrograms total RNA), placenta (0.7 +/- 0.1 pg renin/micrograms total RNA), and saphenous vein (0.02 +/- 0.01 pg renin/micrograms total RNA). The method described here may serve as a highly sensitive tool to quantify alterations in gene expression in man under various pathophysiologic conditions. This study should provide the methodological basis for future studies of tissue RAS in human physiology and disease.

Characterization of inactive renin from human kidney and plasma. Evidence of a renal source of circulating inactive renin.

An inactive form of renin has been isolated from human plasma. It has been suggested that this may represent renin precursor secreted from the kidney. However, early studies failed to isolate inactive renin from human renal tissue. In this investigation, rapid processing of human kidney cortex at temperatures below 4 degrees C in the presence of protease inhibitors followed by cibacron-blue affinity chromatography allowed us to extract a totally inactive form of renal renin. Furthermore, we found that in kidney inactive renin constituted from 10 to as much as 50% of the total renin concentration. Biochemical characterization of the inactive renin from plasma and from kidney indicates that they are structural homologues and, when activated, have enzymatic properties that resemble active renal renin. Renal and plasma inactive renin were found to have the following properties in common: (a) a pH optimum of activation of 3.3; (b) reversible activation by acid dialysis on return to pH 7.4, 37 degrees C; (c) pH optima of enzyme activity of 7.8 with sheep angiotensinogen and 5.5 and 6.7 (biphasic) with human angiotensinogen; (d) Michaelis-Menten constants, Km, of 0.29-0.34 microM with sheep angiotensinogen, and 0.99-1.25 microM with human angiotensinogen; (e) an antibody to human renal renin mean inhibitory titer of 1:30,000 with 1 X 10(-4) Goldblatt units of activated renal or plasma inactive renin; (f) gel filtration profiles consisting of two peaks with apparent molecular weights of 56,000 +/- 1,500 and 49,200 +/- 1,000. Activation of plasma and kidney inactive renin by acid plus renal kallikrein was not accompanied by a change in gel filtration elution patterns. To determine whether inactive renin is released by the kidney, we measured inactive renin in samples obtained simultaneously from both the renal veins and inferior vena cava below the origin of the renal veins. In eight consecutive patients, inactive renin concentration was significantly higher in renal venous blood than in inferior vena caval blood. These data indicate that human kidney contains and secretes significant quantities of inactive renin. Thus, the kidney appears to be a major source of inactive renin in human plasma.

Atrial natriuretic polypeptide inhibits hypertrophy of vascular smooth muscle cells.

Vascular remodeling is central to the pathophysiology of hypertension and atherosclerosis. Recent evidence suggests that vasoconstrictive substances, such as angiotensin II (AII), may function as a vascular smooth muscle growth promoting substance. To explore the role of the counterregulatory hormone, atrial natriuretic polypeptide (ANP) in this process, we examined the effect of ANP (alpha-rat ANP [1-28]) on the growth characteristics of cultured rat aortic smooth muscle (RASM) cells. ANP (10(-7) M) significantly suppressed the proliferative effect of 1% and 5% serum as measured by 3H-thymidine incorporation and cell number, confirming ANP as an antimitogenic factor. In quiescent RASM cells, ANP (10(-7), 10(-6) M) significantly suppressed the basal incorporations of 3H-uridine and leucine by 50 and 30%, respectively. ANP (10(-7), 10(-6) M) also suppressed AII-induced RNA and protein syntheses (by 30-40%) with the concomitant reduction of the cell size. Furthermore, ANP also significantly attenuated the increase of 3H-uridine and leucine incorporations caused by transforming growth factor-beta (4 x 10(-11), 4 x 10(-10) M), a potent hypertrophic factor. These results indicate that ANP possesses an antihypertrophic action on vascular smooth muscle cells. Down-regulation of protein kinase C by 24-h treatment with phorbol 12,13-dibutyrate did not inhibit ANP-induced suppression on 3H-uridine incorporation. Based on the observation that ANP was more potent than a ring-deleted analogue of ANP on inhibiting 3H-uridine incorporation, we conclude that the ANP's inhibitory effect is primarily mediated via the activation of a guanylate cyclase-linked ANP receptor(s). Indeed 8-bromo cGMP mimicked the antihypertrophic action of ANP. Accordingly, we speculate that in addition to its vasorelaxant and natriuretic effects, the antihypertrophic action of ANP observed in the present study may serve as an additional compensatory mechanism of ANP in hypertension.

Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells.

Recently, angiotensin II (Ang II) has been shown to cause hypertrophy of cultured quiescent rat aortic smooth muscle (RASM) cells. This observation along with the demonstration of angiotensinogen mRNA in the vessel wall has led us to postulate a role for vascular angiotensin in hypertensive blood vessel hypertrophy. To investigate further the possible molecular mechanisms, we examined the effect of Ang II on the expression of two genes known to be involved with cellular growth response. Near-confluent RASM cells were made quiescent by 48-h exposure to a defined serum-free medium. Ang II (10(-6) to 10(-11) M) resulted in an induction of the protooncogene c-myc mRNA within 30 min which persisted for 6 h. Interestingly, 6 h after the addition of Ang II, platelet-derived growth factor (PDGF) A-chain mRNA expression was elevated, peaked in 9 h, and persisted for 11 h. This was accompanied with a 15-20-fold increase in PDGF concentration in the culture medium. These effects were dose-dependent and were blocked by saralasin. Whereas the inhibition of protein synthesis by cycloheximide resulted in a stabilization of c-myc mRNA, cycloheximide abolished the elevation of the PDGF A-chain mRNA. Taken together, our data show that exposure of RASM cells to Ang II results in the sequential activation of c-myc and PDGF A-chain mRNA expressions. This sequential activation of protooncogene and growth factor gene may be an important mechanism in angiotensin-induced smooth muscle growth and hypertrophy.

Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II.

Recent observations in our laboratory suggest that angiotensin II (Ang II) is a bifunctional vascular smooth muscle cell (VSMC) growth modulator capable of inducing hypertrophy or inhibiting mitogen-stimulated DNA synthesis. Because transforming growth factor-beta 1 (TGF beta 1) has similar bifunctional effects on VSMC growth, we hypothesized that autocrine production of TGF beta 1 may mediate the growth modulatory effects of Ang II. Indeed, this study demonstrates that Ang II induces a severalfold increase in TGF beta 1 mRNA levels within 4 h that is dependent on de novo protein synthesis and appears to be mediated by activation of protein kinase C (PKC). Ang II not only stimulates the synthesis of latent TGF beta 1, but also promotes its conversion to the biologically active form as measured by bioassay. The coincubation of VSMCs with Ang II and control IgG has no significant mitogenic effect. However, the co-administration of Ang II and the anti-TGF beta 1 antibody stimulates significantly DNA synthesis and cell proliferation. We conclude that: (a) Ang II induces increased TGF beta 1 gene expression via a PKC dependent pathway involving de novo protein synthesis; (b) Ang II promotes the conversion of latent TGF beta 1 to its biologically active form; (c) Ang II modulates VSMC growth by activating both proliferative and antiproliferative pathways; and (d) Autocrine active TGF beta 1 appears to be an important determinant of VSMC growth by hypertrophy or hyperplasia.

Distinct nuclear proteins competing for an overlapping sequence of cyclic adenosine monophosphate and negative regulatory elements regulate tissue-specific mouse renin gene expression.

The mouse renin locus (Ren-1d) exhibits specific patterns of tissue expression. It is expressed in kidney but not submandibular gland (SMG). This locus contains a negative regulatory element (NRE) and a cAMP responsive element (CRE) that share an overlapping sequence. In the kidney, CRE binding proteins (CREB) and NRE binding proteins (NREB) compete for binding to this sequence, with the CREB having a greater affinity. In the SMG, CREB is inactivated by an inhibitory protein, permitting NREB to bind to the sequence, thus inhibiting Ren-1d expression. We hypothesize that the competition between NREB and CREB for this sequence governs tissue-specific expression of mouse renin. We speculate that this may be a general paradigm that determines tissue-specific gene expression.

Fluid shear stress differentially modulates expression of genes encoding basic fibroblast growth factor and platelet-derived growth factor B chain in vascular endothelium.

Fluid shear stress has been shown to be an important regulator of vascular structure and function through its effect on the endothelial cell. We have explored the effect of shear stress on the expression of the heparin-binding growth factors platelet-derived growth factor B chain (PDGF-B) and basic fibroblast growth factor (bFGF) in bovine aortic endothelial cells using a purpose-built cone-plate viscometer. Using morphometric analysis, we have mimicked the endothelial cell shape changes encountered in vivo in response to shear stress and correlated these with changes in gene expression. Steady laminar shear stress of 15 and 36 dyn/cm2 both resulted in endothelial cell shape change, but the higher shear stress induced greater and more uniform alignment in the direction of flow and nuclear protrusion after 24 h. Steady laminar shear stress of both 15 and 36 dyn/cm2 induced a significant 3.9- and 4.2-fold decrease, respectively, in PDGF-B mRNA at 9 h. In contrast, steady laminar shear of 15 dyn/cm2 induced a mild and transient 1.5-fold increase in bFGF mRNA while shear of 36 dyn/cm2 induced a significant 4.8-fold increase at 6 h of shear which remained at 2.9-fold at 9 h. Pulsatile and turbulent shear stress showed the same effect as steady laminar shear stress (all at 15 dyn/cm2 time-average magnitude) on PDGF-B and bFGF mRNA content. Cyclic stretch (20% strain, 20/min) of cells grown on silicone substrate did not significantly affect either PDGF-B or bFGF mRNA levels. These results suggest that expression of each peptide growth factor gene is differentially regulated by fluid shear stress in the vascular endothelial cell. These results may have implications on vascular structure and function in response to hemodynamic forces and present a model for the study of transduction of mechanical stimuli into altered gene expression.

Novel and effective gene transfer technique for study of vascular renin angiotensin system.

Vascular renin angiotensin system (RAS) has been reported to exist in vascular wall. However, there is no direct evidence whether the vascular RAS per se can modulate growth of vascular smooth muscle cells (VSMC), because there is no suitable method to investigate the effect of endogenously produced vasoactive substances on growth of these cells. In this study, we transferred angiotensin-converting enzyme (ACE) and/or renin cDNAs into cultured VSMC using the efficient Sendai virus (hemagglutinating virus of Japan) liposome-mediated gene transfer method, to examine their relative roles in VSMC growth in vitro. Within 35 min or 6 h, the transfection of ACE cDNA into VSMC by hemagglutinating virus of Japan method resulted in a twofold higher ACE activity than control vector, whereas a cationic liposome (Lipofectin)-mediated method failed to show any effect. This in vitro system provided us with the opportunity to investigate the influence of endogenous vascular RAS on VSMC growth. Transfection of ACE or renin cDNA resulted in increased DNA and RNA synthesis, which was inhibited with the specific angiotensin II receptor antagonist (DuP 753: 10(-6) M). Angiotensin I added to ACE-transfected VSMC increased RNA synthesis in a dose-dependent manner. Cotransfection of renin and ACE cDNAs stimulated further RNA synthesis as compared to ACE or renin cDNA alone. These results showed that transfected components of RAS can modulate VSMC growth through the endogenous production of vascular angiotensin II, and that ACE as well as renin are rate limiting in determining the VSMC RAS activity. We conclude that the hemagglutinating virus of Japan liposome-mediated gene transfer technique provides a new and useful tool for study of endogenous vascular modulators such as vascular RAS.

Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.

Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of 60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by L,N-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions of potassium, verapamil, calcium-free medium, and antagonists of the KCa channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses to other agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on micro-carrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator; the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the KCa channel) on the endothelial cell membrane that leads to the release of nitric oxide.

Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production. Modulation by potassium channel blockade.

The endothelium has the capacity to modulate vascular structure in response to hemodynamic stimuli. We tested the hypothesis that exposure of the endothelium to increased laminar shear stress induces the expression of TGF beta 1 via a signal transduction pathway modulated by K+ channel currents. Although TGF beta 1 is normally secreted in a latent, inactive form, exposure of cultured endothelial cells to steady laminar shear stress (20 dynes/cm2) induced increased generation of biologically active TGF beta 1. This increase in active TGF beta 1 was associated with a sustained increase in TGF beta 1 mRNA expression within 2 h of stimulation. TGF beta 1 mRNA levels increased in direct proportion to the intensity of the shear stress within the physiologic range. The effect of shear stress on TGF beta 1 mRNA expression was regulated at the transcriptional level as defined by nuclear run-off studies and transient transfection of a TGF beta 1 promoter-reporter gene construct. Blockade of endothelial K+ channels with tetraethylammonium significantly inhibited: activation of TGF beta 1 gene transcription; increase in steady state mRNA levels; and generation of active TGF beta 1 in response to shear stress. These data suggest that endothelial K+ channels and autocrine-paracrine TGF beta 1 may be involved in the mechanotransduction mechanisms mediating flow-induced vascular remodeling.

H19, a developmentally regulated gene, is reexpressed in rat vascular smooth muscle cells after injury.

Vascular smooth muscle cell migration, proliferation, and differentiation are central to blood vessel development. Since neointimal formation after vascular injury may require the reexpression of a smooth muscle developmental sequence, we examined the expression of H19, a developmentally regulated gene, in rat blood vessels. Expression of the H19 gene is associated with the differentiation process that takes place during development of many tissues. Consistent with this, H19 was highly expressed in the 1-d-old rat aorta but was undetectable in the adult. H19 transcripts were only minimally detected in uninjured carotid artery but were abundant at 7 and 14 d after injury and were localized by in situ hybridization, primarily to the neointima. H19 transcript were undetectable in proliferating neointimal cells in culture but became highly abundant in postconfluent, differentiated neointimal cells. H19 transcripts were only minimally expressed in adult medial smooth muscle cells grown under the identical conditions. Thus, H19 may play an important role in the normal development and differentiation of the blood vessel and in the phenotypic changes of the smooth muscle cells, which are associated with neointimal lesion formation. The vascular injury model may be a useful system to use in examining the function of H19.

Sodium regulation of angiotensinogen mRNA expression in rat kidney cortex and medulla.

Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-1D2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P less than 0.001) and medulla, 1.5-fold (P less than 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P less than 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen.

Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II.

Angiotensin (Ang) II stimulates hypertrophic growth of vascular smooth muscle cells (VSMC). Accompanying this growth is the induction of the expression of growth-related protooncogenes (c-fos, c-jun, and c-myc), as well as the synthesis of the autocrine growth factors, such as PDGF-A and TGF-beta 1. In this study, we demonstrate further that Ang II also induces the synthesis of basic fibroblast growth factor (bFGF), a potent mitogen for VSMC. To examine how these factors interact to modulate the growth response of VSMC to Ang II, we used antisense oligomers to determine the relative contribution of these three factors. Treatment of confluent, quiescent smooth muscle cells with specific antisense oligomers complementary to bFGF, PDGF-A, and TGF-beta 1 efficiently inhibited the syntheses of these factors. Our results demonstrate that in these VSMC, TGF-beta 1 affects a key antiproliferative action, modulating the mitogenic properties of bFGF. Autocrine PDGF exerts only a minimal effect on DNA synthesis. An imbalance in these signals activated by Ang II may result in abnormal VSMC growth leading to the development of vascular disease.

Vasopressin-mediated forearm vasodilation in normal humans. Evidence for a vascular vasopressin V2 receptor.

Arginine vasopressin (AVP) is a potent vasopressor and antidiuretic neurohormone. However, when administered intravenously to humans, AVP causes forearm vasodilation. This effect has been attributed to sympathetic withdrawal, secondary to AVP-induced sensitization of baroreceptors. The possibility that AVP also causes forearm vasodilation directly has not been examined. Accordingly, the direct effect of AVP was determined by studying the forearm blood flow (FBF) response to intraarterial (IA) AVP infusion (0.01-1.0 ng/kg per min). Infusion of IA AVP increased FBF (96%) in the infused arm, but not the control arm, in a dose-dependent manner. The role of specific AVP V1 receptors in mediating this FBF response was determined before and after pretreatment with a V1 antagonist (AVP-A). AVP-A alone had no effect on FBF, but coadministration of AVP and AVP-A potentiated the vasodilatory response (223%). IA infusion of the V2 agonist, 1-desamino[8-D-arginine] vasopressin, caused a dose-dependent increase in FBF. These findings suggest that AVP causes direct, dose-dependent vasodilation in the human forearm that may be mediated by V2 vasopressinergic receptors. In contrast, AVP infusion caused digital vasoconstriction that was blocked by AVP-A, whereas dDAVP did not affect digital blood flow. Thus, AVP induces regionally selective vascular effects, with concurrent forearm vasodilation and digital vasoconstriction.

Androgen regulation of rat renal angiotensinogen messenger RNA expression.

Renal angiotensinogen (ang-n) mRNA concentration in the male WKY rat increases significantly during puberty. Furthermore, renal angiotensinogen mRNA level in the adult female WKY rat is considerably lower than in the male. The present study investigates the role of androgen in differential renal ang-n mRNA expression. Northern and slot blot analyses with alpha-32P labeled ang-n cDNA (pRang 3) demonstrated that castration lowered ang-n mRNA levels in the male kidney by greater than or equal to 60% compared with control, suggesting that androgen may be involved with renal ang-n gene regulation. Moreover, male WKY rats castrated as weanlings and normal adult female WKY rats each implanted with testosterone displayed significant (P less than 0.05) increases in renal ang-n mRNA levels. Our observations, taken together with previous reports that androgen influences proximal tubule morphology and the tubular expression of transport proteins (e.g., Na+/H+ antiporter), may have important physiological implications for understanding the relationship between androgen and angiotensin in the regulation of tubular function.

Localization and differential regulation of angiotensinogen mRNA expression in the vessel wall.

Recent data demonstrate the existence of a vascular renin angiotensin system. In this study we examine the localization of angiotensinogen mRNA in the blood vessel wall of two rat strains, the Wistar and Wistar Kyoto (WKY), as well as the regulation of vascular angiotensinogen mRNA expression by dietary sodium. Northern blot analysis and in situ hybridization histochemistry demonstrate that in both strains angiotensinogen mRNA is detected in the aortic medial smooth muscle layer as well as the periaortic fat. In WKY rats fed a 1.6% sodium diet, angiotensinogen mRNA concentration is 2.6-fold higher in the periaortic fat than in the smooth muscle, as analyzed by quantitative slot blot hybridization. Angiotensinogen mRNA expression in the medial smooth muscle layer is sodium regulated. After 5 d of a low (0.02%) sodium diet, smooth muscle angiotensinogen mRNA levels increase 3.2-fold (P less than 0.005) as compared with the 1.6% sodium diet. In contrast, angiotensinogen mRNA level in the periaortic fat is not influenced by sodium diet. In summary, our data demonstrate regional (smooth muscle vs. periaortic fat) differential regulation of angiotensinogen mRNA levels in the blood vessel wall by sodium. This regional differential regulation by sodium may have important physiological implications.

In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer.

DBA/2J mouse contains two renin gene loci (Ren1d and Ren2d). Ren2d but not Ren1d is expressed in submandibular gland (SMG) while both are expressed in the kidney. Based on vitro studies, we have postulated that a negative regulatory element (NRE) in the renin gene promoter is involved in its tissue-specific expression. In this study, we examined the molecular mechanism at the in vivo level using direct gene transfer. Fragments of the Ren1d or Ren2d promoter were fused to a chloramphenicol acetyltransferase (CAT) gene expression vector. These constructs complexed in fusogenic liposomes were injected directly into the mouse SMG or intraarterially into the mouse kidney via the renal artery. The vector containing the CAT exhibited readily detectable in vivo expressions in both SMG and kidney. In the SMG, Ren1d fragment containing the NRE abolished CAT expression while deletion of the NRE restored CAT expression. The homologous fragment from the Ren2d promoter did not inhibit CAT expression while deletion of the 150-bp insertion resulted in the inhibition. Cotransfection of Ren1d construct with Ren1d-NRE oligonucleotides as transcriptional factor decoy restored CAT expression. Contrary to the SMG, transfection with Ren1d fragment-CAT construct or Ren2d fragment-CAT construct into the kidney resulted in similar levels of CAT expression. Interestingly, human c-myc NRE oligonucleotides which share homology with Ren1d-NRE competed effectively with these oligonucleotides for the regulation of Ren1d gene expression in vivo. This NRE sequence is also homologous to silencer elements found in multiple mammalian genes, suggesting the presence of a family of NRE/NRE binding proteins regulating expression of diverse genes.