Biomedical subjects

Oliver Smithies

Publications and source records attributed to Oliver Smithies.

At least 37 records · Page 2Linked to original sources

PubMed2005

An improved technique for tail-cuff blood pressure measurements with dark-tailed mice.

Study of the genetics of hypertension has been facilitated greatly by the use of mice with modified genes that affect blood pressure. A current successful method for measuring blood pressure in mice relies on detection of light passing through the tail to determine the pressure in a tail-cuff necessary to stop pulsed flow. Success in obtaining reliable blood pressure measurements in light-tailed strains of mice (e.g., C57BL/6J) has been excellent. However, in our and others' experience, mice having highly pigmented tails (e.g., 129S6/SvEvTac) have yielded less consistent measurements. We report here that simple modifications to the channel containing the pulse detection sensor can greatly improve the pulse detection of dark-tailed mice. The first modification--lining the sensor channel with four layers of clear plastic wrap--increased the frequency of successful blood pressure measurements of 129S6/SvEvTac mice twofold and reduced variability by one-third. The second modification--lining the sides of the channel with reflective foil--also improved the success rate with dark-tailed mice. Mean blood pressures were unaffected by these modifications, which enhance detection of the pulse wave and likely will be helpful in diverse applications in which blood pressure is measured in rodent strains with pigmented tails.

Animals↗

PubMed2004

Lifelong genetic minipumps.

Most physiologists working with animals are familiar with osmotic minipumps. These surgically implanted devices can, for a limited period, administer a reagent at a constant predetermined rate that is unaffected by concurrent procedures. The investigator can then test the physiological effects of other treatments knowing that the animals' homeostatic responses will not be able to alter the dose of the pumped reagent. To develop the genetic equivalent of a lifelong minipump, simply inherited as an autosomal dominant, we here combine three of our previously described strategies, genetic clamping, single-copy chosen-site integration, and modification of untranslated regions (UTRs). As a test of the procedure, we have generated a series of intrinsically useful animals having genetic minipumps secreting renin ectopically from the liver at levels controlled by the investigator but not subject to homeostatic changes. To achieve the different dosage levels of these genetic minipumps, we altered the UTRs of a renin transgene driven by an albumin promoter and inserted it into the genome as a single copy at the ApoA1/ApoC3 locus, a locus that is strongly expressed in the liver. The resulting mice express plasma renin over ranges from near physiological to eightfold wild type and develop graded cardiovascular and kidney disease consequent to their different levels of ectopically secreted renin. The procedure and DNA constructs we describe can be used to generate genetic minipumps for controlling plasma levels of a wide variety of secreted protein products.

Animals↗

PubMed2004

Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type.

Mice lacking Ren1c were generated using C57BL/6-derived embryonic stem cells. Mice homozygous for Ren1c disruption (Ren1c-/-) are born at the expected ratio, but approximately 80% die of dehydration within a few days. The surviving Ren1c-/- mice have no renin mRNA expression in the kidney, hydronephrosis, thickening of renal arterial walls, and fibrosis in the kidney. Plasma renin and angiotensins I and II are undetectable. Urinary aldosterone is 6% wild-type. They have low tail-cuff BP (84 +/- 4 versus 116 +/- 5 mmHg in +/+) and excrete large amounts of urine (5.2 +/- 0.8 ml/d, 725 +/- 34 mOsm versus 1.1 +/- 0.1 ml/d, 2460 +/- 170 mOsm in +/+). After 5 d of drinking 5% dextrose, desmopressin does not increase the osmolality of the urine in -/- mice (624 +/- 19 to 656 +/- 25 mOsm), whereas in +/+, it increases severalfold (583 +/- 44 to 2630 +/- 174 mOsm). Minipump infusion of angiotensin II to Ren1c-/- mice restores BP to wild-type level, but preexisting damage to the medulla prevents complete restoration of the ability of the kidney to concentrate urine. Heterozygous Ren1c+/- mice, in contrast, are indistinguishable from +/+ in BP, urine volume, and osmolality. Kidney renin mRNA, the number of kidney cells producing renin, and plasma renin concentration in the Ren1c+/- mice are also indistinguishable from +/+. These results demonstrate that renin is the only enzyme capable of maintaining plasma angiotensins and that renin expression in the kidney is very tightly regulated at the mRNA level.

Angiotensin II↗

PubMed2004

Diabetic nephropathy is markedly enhanced in mice lacking the bradykinin B2 receptor.

Type I human diabetics and streptozotocin-induced diabetic mice with higher genetically determined levels of angiotensin-converting enzyme have an increased risk of developing nephropathy. However, previous experiments in mice and computer simulations indicate that modest increases in angiotensin-converting enzyme have minimal effects on blood pressure and angiotensin II levels, although bradykinin decreases significantly, inferring that bradykinin is critical for protecting the kidney in diabetics. Here, we confirm this inference by demonstrating that Akita diabetic mice lacking the bradykinin B2 receptor develop overt albuminuria, excreting the equivalent of >550 mg/day albumin in humans, which contrasts with the microalbuminuria (equivalent to <150 mg/day) seen in their simply diabetic littermates. The overt albuminuria is accompanied by a marked increase in glomerular mesangial sclerosis. The importance of bradykinin demonstrated here bears strongly on how current drugs reduce diabetic nephropathy and suggests that B2 receptor-specific agonists merit consideration in this context.

Animals↗

PubMed2004

A gammaGT-AT1A receptor transgene protects renal cortical structure in AT1 receptor-deficient mice.

To understand the physiological role of angiotensin type 1 (AT(1)) receptors in the proximal tubule of the kidney, we generated a transgenic mouse line in which the major murine AT(1) receptor isoform, AT(1A), was expressed under the control of the P1 portion of the gamma-glutamyl transpeptidase (gammaGT) promoter. In transgenic mice, this promoter has been shown to confer cell-specific expression in epithelial cells of the renal proximal tubule. To avoid random integration of multiple copies of the transgene, we used gene targeting to produce mice with a single-copy transgene insertion at the hypoxanthine phosphoribosyl transferase (Hprt) locus on the X chromosome. The physiological effects of the gammaGT-AT(1A) transgene were examined on a wild-type background and in mice with targeted disruption of one or both of the murine AT(1) receptor genes (Agtr1a and Agtr1b). On all three backgrounds, gammaGT-AT(1A) transgenic mice were healthy and viable. On the wild-type background, the presence of the transgene did not affect development, blood pressure, or kidney structure. Despite relatively low levels of expression in the proximal tubule, the transgene blunted the increase in renin expression typically seen in AT(1)-deficient mice and partially rescued the kidney phenotype associated with Agtr1a(-/-)Agtr1b(-/-) mice, significantly reducing cortical cyst formation by more than threefold. However, these low levels of cell-specific expression of AT(1) receptors in the renal proximal tubule did not increase the low blood pressures or abolish sodium sensitivity, which are characteristic of AT(1) receptor-deficient mice. Although our studies do not clearly identify a role for AT(1) receptors in the proximal tubules of the kidney in blood pressure homeostasis, they support a major role for these receptors in modulating renin expression and in maintaining structural integrity of the renal cortex.

Animals↗

PubMed2004

E-prostanoid-3 receptors mediate the proinflammatory actions of prostaglandin E2 in acute cutaneous inflammation.

PGs are derived from arachidonic acid by PG-endoperoxide synthase (PTGS)-1 and PTGS2. Although enhanced levels of PGs are present during acute and chronic inflammation, a functional role for prostanoids in inflammation has not been clearly defined. Using a series of genetically engineered mice, we find that PTGS1 has the capacity to induce acute inflammation, but PTGS2 has negligible effects on the initiation of this response. Furthermore, we show that the contribution of PTGS1 is mediated by PGE(2) acting through the E-prostanoid (EP)3 receptor. Moreover, in the absence of EP3 receptors, inflammation is markedly attenuated, and the addition of nonsteroidal anti-inflammatory agents does not further impair the response. These studies demonstrate that PGE(2) promotes acute inflammation by activating EP3 receptors and suggest that EP3 receptors may be useful targets for anti-inflammatory therapy.

Animals↗

PubMed2004

Cardiac hypertrophy and sudden death in mice with a genetically clamped renin transgene.

Several mouse models have already proved valuable for investigating hypertrophic responses to cardiac stress. Here, we characterize one caused by a well defined single copy transgene, RenTgMK, that genetically clamps plasma renin and thence angiotensin II at high levels. All of the transgenic males develop concentric cardiac hypertrophy with fibrosis but without dilatation. Over half die suddenly aged 6-8 months. Telemetry showed disturbances in diurnal rhythms a few days before death and, later, electrocardiographic disturbances comparable to those in humans with congestive heart failure. Expression of seven hypertrophy-related genes in this and two categorically different models (lack of atrial natriuretic peptide receptor A; overexpression of calsequestrin) were compared. Statistical analyses show that ventricular expressions of the genes coding for atrial natriuretic peptide, beta myosin heavy chain, medium chain acyl-CoA dehydrogenase, and adrenomedullin correlate equally well with the degree of hypertrophy, although their ranges of expression are, respectively, 50-, 30-, 10-, and 3-fold.

Animals↗

PubMed2004

Three-dimensional diffusion tensor microscopy of fixed mouse hearts.

The relative utility of 3D, microscopic resolution assessments of fixed mouse myocardial structure via diffusion tensor imaging is demonstrated in this study. Isotropic 100-microm resolution fiber orientation mapping within 5.5 degrees accuracy was achieved in 9.1 hr scan time. Preliminary characterization of the diffusion tensor primary eigenvector reveals a smooth and largely linear angular rotation across the left ventricular wall. Moreover, a higher level of structural hierarchy is evident from the organized secondary and tertiary eigenvector fields. These findings are consistent with the known myocardial fiber and laminar structures reported in the literature and suggest an essential role of diffusion tensor microscopy in developing quantitative atlases for studying the structure-function relationships of mouse hearts.

Animals↗

PubMed2004

Human genetics, animal models and computer simulations for studying hypertension.

Essential hypertension is probably caused by combinations of small quantitative changes in the expression of many genes together with environmental factors. In this article, strategies for studying hypertension using animal models are summarized with emphasis on the combined use of mouse models and computer simulations. We have chosen the rennin-angiotensin system as our main example. Future directions of hypertension research using gene targeting are also discussed.

Animals↗

PubMed2004

Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.

A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (approximately 36 kb) at the 5' end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.

Animals↗

PubMed2004

Altering the expression in mice of genes by modifying their 3' regions.

Polymorphic differences altering expression of genes without changing their products probably underlie human quantitative traits affecting risks of serious diseases, but methods for investigating such quantitative differences in animals are limited. Accordingly, we have developed a procedure for changing the expression in mice of chosen genes over a 100-fold range while retaining their chromosomal location and transcriptional controls. To develop the procedure, we first dissected the effects in embryonic stem (ES) cells of elements within and downstream of the 3' untranslated region (UTR) of a single copy transgene at the Hprt locus. As expected, protein expression varied with the steady-state level and half-life of the mRNA. The rank order of expression with various tested 3' regions is the same in ES cells, and in cardiomyocytes and trophoblastocytes derived from them. In mice having two functionally different native genes with modified 3'UTRs, the desired expression was obtained.

3' Untranslated Regions↗

PubMed2004

Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.

Renin-synthesizing cells are crucial in the regulation of blood pressure and fluid-electrolyte homeostasis. Adult mammals subjected to manipulations that threaten homeostasis increase circulating renin by increasing the number of renin-expressing/-releasing cells. We hypothesize that the ability of adult cells to synthesize renin does not occur randomly in any cell type, depending instead on the cell's lineage. To determine the fate of renin-expressing cells, we generated knockin mice expressing cre recombinase in renin-expressing cells and crossed them with reporter mice. Results show that renin-expressing cells are precursors for a variety of cells that differentiate into non-renin-expressing cells such as smooth-muscle, epithelial, mesangial, and extrarenal cells. In the kidney, these cells retain the capability to synthesize renin when additional hormone is required to reestablish homeostasis: specific subpopulations of apparently differentiated cells are "held in reserve" to respond (repeatedly) by de-differentiating and expressing renin in response to stress, and re-differentiating when the crisis passes.

Animals↗

PubMed2003

Physiological impact of increased expression of the AT1 angiotensin receptor.

To test the effect of increased AT1 receptor expression on blood pressure, we used gene targeting to generate mouse lines with a tandem duplication of the AT1A receptor gene locus (Agtr1a) along with >10 kb of 5' flanking DNA. By successive breeding, we generated mice with 3 and 4 copies of the Agtr1a gene locus on an inbred 129/Sv background. AT1A mRNA expression and AT1-specific binding of 125I-angiotensin II were increased in proportion to Agtr1a gene copy number. These animals survived in expected numbers, and their body, heart, and kidney weights were similar to wild-type, 2-copy control mice. Pressor responses to angiotensin II were blunted in the 4-copy mice compared with control mice. In male mice, there was no correlation between resting blood pressure and Agtr1a gene copy number or AT1A mRNA levels. However, in female mice, there was a highly significant positive correlation between blood pressure and AT1A receptor expression, paralleled by significant increases in aldosterone synthase expression with increase in gene copy number. Furthermore, in female but not male mice, there was a positive correlation between kallikrein and AT1A receptor mRNA levels and an inverse correlation between renin mRNA and Agtr1a copy number. Thus, in female but not male mice, genetic variants that increase expression of AT1 receptors affect blood pressure and gene expression programs. The impact of enhanced AT1 receptor expression on blood pressure may be blunted by systemic compensatory responses and altered signal-effector coupling in the vasculature.

Angiotensin II↗

PubMed2003

Natriuretic peptide receptor A mediates renal sodium excretory responses to blood volume expansion.

The deficiency of Npr1 [genetic determinant of natriuretic peptide receptor A (NPRA)] increases arterial pressures and causes hypertensive heart disease in mice similar to those seen in untreated human hypertensive patients. However, the quantitative role of NPRA in mediating the renal responses to blood volume expansion remains uncertain. To determine the specific contribution of NPRA in mediating the signaling mechanisms responsible for natriuretic and diuretic responses to nondilutional intravascular expansion, we administered whole blood to anesthetized Npr1 homozygous null mutant (0-copy), wild-type (2-copy), and gene-duplicated (4-copy) mice. In wild-type (2-copy) animals, urinary flow (microl x min-1 x g kidney wt-1) increased from 4.9 +/- 1.0 to 14.4 +/- 1.8 and sodium excretion (microeq x min-1 x g kidney wt-1) from 1.15 +/- 0.22 to 3.11 +/- 0.60, associated with a rise in glomerular filtration rate (GFR; ml x min-1 x g kidney wt-1) from 0.63 +/- 0.03 to 0.82 +/- 0.09 and renal plasma flow (RPF; ml x min-1. g kidney wt-1) from 2.96 +/- 0.17 to 4.36 +/- 0.41, whereas arterial pressure did not significantly increase. After volume expansion, 0-copy mice showed significantly lesser increases in urinary flow (P < 0.001) and sodium excretory (P < 0.001) responses even though the increases in arterial pressures were greater (P < 0.001) compared with 2-copy mice. The 4-copy mice showed augmented responses in urinary flow (P < 0.01) and sodium excretion (P < 0.001) along with rises in both GFR (P < 0.01) and RPF (P < 0.01) compared with 2-copy wild-type mice. These results establish that NPRA activation is the predominant mechanism mediating the natriuretic, diuretic, and renal hemodynamic responses to acute blood volume expansion.

Animals↗

PubMed2003

Why the kidney glomerulus does not clog: a gel permeation/diffusion hypothesis of renal function.

Current interpretations of kidney function in terms of a coarse filter followed by a fine filter have difficulty explaining why the glomerulus does not clog. I propose, as an alternative, a semiquantitative hypothesis that assumes that the size-selective property of the glomerulus is a consequence of the limited fraction of space in the glomerular basement membrane (a concentrated gel) into which macromolecules can permeate. The glomerular epithelial cell slits and slit diaphragms are assumed to impose substantial resistance to liquid flow across the glomerulus without acting as a molecular sieve. Calculations based on gel behavior show that proteins cross the glomerular basement membrane mainly by diffusion rather than by liquid flow, whereas water crosses entirely by flow. Thus, diffusion provides most of the protein, whereas flow provides the diluent. As a result, the single-nephron glomerular filtration rate (GFR) becomes a prime factor in (inversely) determining the concentration of proteins in early proximal tubular fluid. Because the reabsorption of proteins from the tubules is a saturable process, the gel permeationdiffusion hypothesis readily accounts for the albuminuria observed when single-nephron GFR is substantially reduced by severe pathological decreases in slit diaphragm length, such as occur in minimal-change nephrotic syndrome in humans, in animals treated with puromycin aminonucleoside, or in humans or animals with mutations in the gene coding for nephrin. My hypothesis predicts that albuminuria will ensue, even with a normal kidney, if the single-nephron GFR falls below approximately 50% of normal.

Animals↗

PubMed2003

Induction of hepatic differentiation in embryonic stem cells by co-culture with embryonic cardiac mesoderm.

BACKGROUND: Modifications in vitro have been used to direct embryonic stem (ES) cells toward endodermal phenotypes including hepatocytes; however, developmental correlates and evidence of biologic activity is lacking, and critical cell-cell interactions have not been investigated. In this study, we hypothesized that cardiac mesoderm (CM) signals ES cells in co-culture to undergo differentiation toward early hepatocyte lineage as determined by morphology and induction of genes essential for endodermal competence and hepatocyte development. METHODS: Green fluorescent protein ES derived from A129 mice were cultured with or without embryonic chick cardiac mesoderm. Cultures from day 1, 2, and 4 were analyzed for colony formation and ES morphology and 10(6) ES-derived cells were isolated for mRNA analysis. RESULTS: ES in co-culture with CM displayed colony formation, polymorphic appearance, and definitive interface with CM. In addition, ES + CM co-culture activated crucial transcription factors (sox 17alpha, HNF3beta, and GATA 4) required for hepatocyte development by day 1. mRNA for albumin and especially a-fetoprotein were also increased by culture days 2 and 4. CONCLUSIONS: ES cells co-cultured with CM display morphology and gene expression pattern required for hepatocyte differentiation and appear to recapitulate the molecular events of hepatogenesis.

Albumins↗

PubMed2003

Minireview: computer simulations of blood pressure regulation by the renin-angiotensin system.

Gene targeting experiments in mice have been used by us and others to test whether quantitative changes in gene expression in the renin-angiotensin system affect blood pressure. Surprisingly, these studies showed that blood pressure does not change with mild quantitative changes in the expression of the angiotensin converting enzyme (ACE). Yet, ACE inhibitors are widely used for the treatment of hypertension. This apparent paradox motivated us to develop a simple computer simulation, which qualitatively reconciled the paradox. We have now improved the simulation by including blood pressure as an explicit variable and by adding the kallikrein-kinin system and feedback control of plasma renin via plasma angiotensin II levels. The new simulation now matches quantitative aspects of the experimental data and suggests that a decrease in bradykinin plays an important role in the increased risk of diabetic nephropathy associated with genetically determined higher levels of ACE activity. This emphasizes that the value of these types of simulation lies in the thoughts that they provoke rather than in their ability to replicate experimental data.

Animals↗

PubMed2002

Targeted disruption of hsp70.1 sensitizes to osmotic stress.

The 70 kDa heat shock protein (Hsp70) plays a critical role in cell survival and thermotolerance in response to various stress stimuli. Two nearly identical genes, hsp70.1 and hsp70.3, in response to environmental stress, rapidly induce Hsp70. However, it remains unclear whether these two genes are differentially regulated by various stresses. To address the physiological role of the hsp70.1 and hsp70.3 genes in the stress response, we generated mice that specifically lack hsp70.1. In contrast to heat shock, which rapidly induced both hsp70.1 and hsp70.3 mRNA, osmotic stress selectively induced transcription of hsp70.1. In hsp70.1-deficient embryonic fibroblasts, osmotic stress markedly reduced cell viability. Furthermore, when osmotic stress was applied in vivo, hsp70.1-deficient mice exhibited increased apoptosis in the renal medulla. Taken together, our results demonstrate that differential expression of hsp70 genes contributes to the stress response and that the hsp70.1 gene plays a critical role in osmotolerance.

3' Untranslated Regions↗