The elusive field of systems biology.
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Animals↗
Biomedical subjects
Allen W Cowley
Publications and source records attributed to Allen W Cowley.
Influence of diet and genetics on hypertension and renal disease in Dahl salt-sensitive rats.
Experiments examined the influence of diet and genetics on hypertension and renal disease in inbred Dahl salt-sensitive (SS/Mcw) rats and consomic rats in which chromosomes 16 (SS.BN16) or 18 (SS.BN18) of the normotensive Brown Norway rat were inserted into the genetic background of the SS/Mcw. Dahl SS/Mcw breeders and offspring were randomly placed on a purified AIN-76A diet or a grain-based diet, and male offspring were screened for cardiovascular and renal phenotypes following 3 wk on a 4.0% NaCl diet. High-salt arterial blood pressure (162 +/- 5 mmHg, n = 10), urinary protein excretion (147 +/- 16 mg/day, n = 14), and albumin excretion (72 +/- 9 mg/day, n = 14) were significantly elevated in the Dahl SS/Mcw maintained on the purified diet compared with rats fed the grain-based diet. Rats fed the purified diet also exhibited significantly more renal glomerular and tubular damage than rats fed the grain diet. Moreover, feeding the purified diet to the parents led to a significant increase in blood pressure in the offspring, regardless of offspring diet. Similar dietary effects were observed in SS.BN16 and SS.BN18 rats. In rats fed the purified diet, substitution of chromosomes 16 or 18 led to a significant decrease in arterial blood pressure, albumin excretion, and protein excretion compared with the SS/Mcw. Chromosomal substitution did not, however, affect albumin or protein excretion in the consomic rats compared with the SS/Mcw when the rats were maintained on the grain diet. These data demonstrate a significant influence of diet composition on salt-induced hypertension and renal disease in the Dahl SS/Mcw rat.
Animals↗
Renal oxidative stress in medullary thick ascending limbs produced by elevated NaCl and glucose.
The effects of NaCl, glucose, and thyroid hormone on the production of superoxide (O2*-) within the renal medulla of Sprague-Dawley rats were examined. Responses of intracellular superoxide [O2*-]i in isolated medullary thick ascending limbs (mTALs) were studied using real-time fluorescent microscopy with measurement of the dehydroethidium (DHE) to ethidium (Eth) conversion ratio (Eth/DHE ratio unit). The results demonstrated that elevations of extracellular NaCl (from 152 to 252 mmol/L), D-glucose (from 5 to 25 mmol/L), and triiodo-thyronine (T3; 10 micromol/L) significantly increased [O2*-]i levels. Preincubation with superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (1 mmol/L) significantly inhibited these responses. Stimulation with equamolar amounts of choline chloride or L-glucose failed to increase [O2*]i, indicating that these O2*- responses were not determined by changes in osmolality. The responses to NaCl, D-glucose, and T3 were abolished by pretreatment with the Na+/K+-ATPase pump inhibitor ouabain (4 mmol/L) and with Na+/H+ -exchanger inhibitor dimethylamiloride (100 micromol/L). We conclude that elevations of extracellular NaCl, D-glucose, or T3 levels can activate both the Na+/K+-ATPase pump and Na+/H+ exchanger in mTAL, which, in turn, is associated with increased intracellular concentrations of superoxide.
Animals↗
High throughput gene expression profiling: a molecular approach to integrative physiology.
Integrative physiology emphasizes the importance of understanding multiple pathways with overlapping, complementary, or opposing effects and their interactions in the context of intact organisms. The DNA microarray technology, the most commonly used method for high-throughput gene expression profiling, has been touted as an integrative tool that provides insights into regulatory pathways. However, the physiology community has been slow in acceptance of these techniques because of early failure in generating useful data and the lack of a cohesive theoretical framework in which experiments can be analysed. With recent advances in both technology and analysis, we propose a concept of multidimensional integration of physiology that incorporates data generated by DNA microarray and other functional, genomic, and proteomic approaches to achieve a truly integrative understanding of physiology. Analysis of several studies performed in simpler organisms or in mammalian model animals supports the feasibility of such multidimensional integration and demonstrates the power of DNA microarray as an indispensable molecular tool for such integration. Evaluation of DNA microarray techniques indicates that these techniques, despite limitations, have advanced to a point where the question-driven profiling research has become a feasible complement to the conventional, hypothesis-driven research. With a keen sense of homeostasis, global regulation, and quantitative analysis, integrative physiologists are uniquely positioned to apply these techniques to enhance the understanding of complex physiological functions.
Animals↗
Application of chromosomal substitution techniques in gene-function discovery.
A consomic rat strain is one in which an entire chromosome is introgressed into the isogenic background of another inbred strain using marker assisted selection. The development and initial physiologic screening of two inbred consomic rat panels on two genetic backgrounds (44 strains) is well underway. The primary uses of consomic strains are: (1) to assign traits and quantitative trait loci (QTL) to chromosomes by surveying the panel of strains with substituted chromosomes; (2) to rapidly develop congenic strains over a narrow region using several approaches described in this review and perform F2 linkage studies to positionally locate QTL in a fixed genetic background. In addition, consomic strains overcome many of the problems encountered with segregating crosses where, even if linkage is found, each individual in the cross is genetically unique and the combination of genes cannot be reproduced or studied in detail. Consomic strains provide greater statistical power to detect linkage than traditional F2 crosses because of their fixed genetic backgrounds, and can produce sufficient numbers of genetically identical rats to validate the relationship between a trait and a particular chromosome. These strains allow studies to be performed in a replicative or longitudinal manner to elucidate in greater detail the sequential changes responsible for the observed phenotypes of these animals, and they enable one to assess the impact of a causal gene region in a genome by allowing comparisons of the effect of replacement of a specific chromosome upon a disease susceptible or resistant genomic background. Consomics can be used to quickly develop multiple chromosome substitution models to investigate gene-gene interactions of complex traits or diseases. Finally, they often provide the best available inbred control strain for particular physiological comparisons with the inbred parental strains. Consomic rat strains are proving to be a unique scientific resource that greatly extends our understanding of genes and complex normal and pathological function.
Animals↗
Physiological genomics: tools and concepts.
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Genomics↗
Genomic map of cardiovascular phenotypes of hypertension in female Dahl S rats.
Genetic linkage analyses in human populations have traditionally combined male and female progeny for determination of quantitative trait loci (QTL). In contrast, most rodent studies have focused primarily on males. This study represents an extensive female-specific linkage analysis in which 236 neuroendocrine, renal, and cardiovascular traits related to arterial pressure (BP) were determined in 99 female F2 rats derived from a cross of Dahl salt-sensitive SS/JrHsdMcwi (SS) and Brown Norway normotensive BN/SsNHsdMcwi (BN) rats. We identified 126 QTL for 96 traits on 19 of the 20 autosomal chromosomes of the female progeny. Four chromosomes (3, 6, 7, and 11) were identified as especially important in regulation of arterial pressure and renal function, since aggregates of 8-11 QTL mapped together on these chromosomes. BP QTL in this female population differed considerably from those previously found in male, other female, or mixed sex population linkage analysis studies using SS rats. Kidney weight divided by body weight was identified as an intermediate phenotype that mapped to the same region of the genome as resting diastolic blood pressure and was correlated with that same BP phenotype. Seven other phenotypes were considered as "potential intermediate phenotypes, " which mapped to the same region of the genome as a BP QTL but were not correlated with BP. These included renal vascular responses to ANG II and ACh and indices of baroreceptor responsiveness. Secondary traits were also identified that were likely to be consequences of hypertension (correlated with BP but not mapped to a BP QTL). Seven such traits were found, notably heart rate, plasma cholesterol, and renal glomerular injury. The development of a female rat systems biology map of cardiovascular function represents the first attempt to prioritize those regions of the genome important for development of hypertension and end organ damage in female rats.
Animals↗
Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla.
The source of superoxide (O2*-) production and cell-to-cell interactions of O2*- and nitric oxide (NO) in response to angiotensin II (AngII) were studied by fluorescence microscopic techniques to image rat renal outer medullary microtissue strips. Changes in intracellular O2*- were determined by dihydroethidium-ethidium ratios, and NO was determined with 4,5-diaminofluorescein diacetate. AngII (1 micromol/L) significantly increased O2*- in the isolated, medullary thick ascending limb (mTAL). These responses were inhibited by the superoxide dismutase mimetic 4-hydroxytetramethylpiperidine-1-oxyl (TEMPOL) and by the NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin. AngII did not increase O2*- in either pericytes of isolated, intact vasa recta (VR) or pericytes of VR with a disrupted endothelium, even when surrounded by mTAL. However, AngII did increase O2*- when the tissue strips were preincubated with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), indicating that cross-talk of O2*- from mTAL to the VR occurred but was normally inhibited by NO. Also, tissue O2*- reduction by TEMPOL increased the diffusion of NO from mTAL to the pericytes, indicating that cross-talk of NO from the mTAL to the VR is also inhibited by O2*-. We conclude that AngII stimulates O2*- production in mTAL via the NAD(P)H oxidase pathway and that interactions of O2*- and NO ultimately determine the effectiveness of in situ free-radical cross-talk between the mTAL and the VR.
Angiotensin II↗
Physiological Genomics: the next three years.
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Forecasting↗
Quantitative assessment of the importance of dye switching and biological replication in cDNA microarray studies.
Dye switching and biological replication substantially increase the cost and the complexity of cDNA microarray studies. The objective of the present analysis was to quantitatively assess the importance of these procedures to provide a quantitative basis for decision-making in the design of microarray experiments. Taking advantage of the unique characteristics of a published data set, the impact of these procedures on the reliability of microarray results was calculated. Adding a second microarray with dye switching substantially increased the correlation coefficient between observed and predicted ln(ratio) values from 0.38 +/- 0.06 to 0.62 +/- 0.04 (n = 12) and the outlier concordance from 21 +/- 3% to 43 +/- 4%. It also increased the correlation with the entire set of microarrays from 0.60 +/- 0.04 to 0.79 +/- 0.04 and the outlier concordance from 31 +/- 6% to 58 +/- 5% and tended to improve the correlation with Northern blot results. Adding a second microarray to include biological replication also improved the performance of these indices but often to a lesser degree. Inclusion of both procedures in the second microarray substantially improved the consistency with the entire set of microarrays but had minimal effect on the consistency with predicted results. Analysis of another data set generated using a different cDNA labeling method also supported a significant impact of dye switching. In conclusion, both dye switching and biological replication substantially increased the reliability of microarray results, with dye switching likely having even greater benefits. Recommendations regarding the use of these procedures were proposed.
Algorithms↗
Increased H(2)O(2) counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla.
A membrane-permeable SOD mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (tempol), has been used as an antioxidant to prevent hypertension. We recently found that this SOD mimetic could not prevent development of hypertension induced by inhibition of renal medullary SOD with diethyldithiocarbamic acid. The present study tested a hypothesis that increased H2O2 counteracts the effects of tempol on renal medullary blood flow (MBF) and Na+ excretion (UNaV), thereby restraining the antihypertensive effect of this SOD mimetic. By in vivo microdialysis and Amplex red H2O2 microassay, it was found that interstitial H2O2 levels in the renal cortex and medulla in anesthetized rats averaged 55.91 +/- 3.66 and 102.18 +/- 5.16 nM, respectively. Renal medullary interstitial infusion of tempol (30 micromol x min-1x kg-1) significantly increased medullary H2O2 levels by 46%, and coinfusion of catalase (10 mg x min-1x kg-1) completely abolished this increase. Functionally, removal of H2O2 by catalase enhanced the tempol-induced increase in MBF, urine flow, and UNaV by 28, 41, and 30%, respectively. Direct delivery of H2O2 by renal medullary interstitial infusion (7.5-30 nmol x min-1x kg-1) significantly decreased renal MBF, urine flow, and UNaV, and catalase reversed the effects of H2O2. We conclude that tempol produces a renal medullary vasodilator effect and results in diuresis and natriuresis. However, this SOD mimetic increases the formation of H2O2, which constricts medullary vessels and, thereby, counteracts its vasodilator actions. This counteracting effect of H2O2 may limit the use of tempol as an antihypertensive agent under exaggerated oxidative stress in the kidney.
Animals↗
Increased renal medullary H2O2 leads to hypertension.
We have recently reported that exaggerated oxidative stress in the renal medulla due to superoxide dismutase inhibition resulted in a reduction of renal medullary blood flow and sustained hypertension. The present study tested the hypothesis that selective scavenging of O2*- in the renal medulla would prevent hypertension associated with this exaggerated oxidative stress. An indwelling, aortic catheter was implanted in nonnephrectomized Sprague-Dawley rats for daily measurement of arterial blood pressure, and a renal medullary interstitial catheter was implanted for continuous delivery of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETC, 7.5 mg x kg(-1) x d(-1)) and a chemical superoxide dismutase mimetic, 4-hydroxytetramethyl piperidine-1-oxyl (TEMPOL, 10 mg. kg-1. d-1). Renal medullary interstitial infusion of TEMPOL completely blocked DETC-induced accumulation of O2*- in the renal medulla, as measured by the conversion rate of dihydroethidium to ethidium in the dialysate and by urinary excretion of 8-isoprostanes. However, TEMPOL infusion failed to prevent DETC-induced hypertension, unless catalase (5 mg x kg(-1) d(-1)) was coinfused. Direct infusion of H2O2 into the renal medulla resulted in increases of mean arterial pressure from 115+/-2.5 to 131+/-2.1 mm Hg, which was similar to that observed in rats receiving the medullary infusion of both TEMPOL and DETC. The results indicate that sufficient catalase activity in the renal medulla is a prerequisite for the antihypertensive action of TEMPOL and that accumulated H2O2 in the renal medulla associated with exaggerated oxidative stress might have a hypertensive consequence.
Animals↗
Insights into Dahl salt-sensitive hypertension revealed by temporal patterns of renal medullary gene expression.
Dahl salt-sensitive SS and consomic, salt-resistant SS-13(BN)/Mcw rats possess a highly similar genetic background but exhibit substantial differences in blood pressure salt sensitivity. We used cDNA microarrays to examine sequential changes of mRNA expression of approximately 2,000 currently known rat genes in the renal medulla (a tissue critical for long-term blood pressure regulation) in SS and SS-13(BN)/Mcw rats in response to a high-salt diet (16 h, 3 days, or 2 wk). Differentially expressed genes in each between-group comparison were identified based on a threshold determined experimentally using a reference distribution that was constructed by comparing rats within the same group. A difference analysis of 54 microarrays identified 50 genes that exhibited the most distinct temporal patterns of expression between SS and SS-13(BN)/Mcw rats over the entire time course. Thirty of these genes could be linked to the regulation of arterial blood pressure or renal injury based on their known involvement in functional pathways such as renal tubular transport, metabolism of vasoactive substances, extracellular matrix formation, and apoptosis. Importantly, the majority of the 30 genes exhibited temporal expression patterns that would be expected to lower arterial pressure and reduce renal injury in SS-13(BN)/Mcw compared with SS rats. The phenotypic impact of the other 20 genes was less clear. These 50 genes are widely distributed on chromosome 13 and several other chromosomes. This suggested that primary genetic defects, although important, are unlikely to be solely responsible for the full manifestation of this type of hypertension and associated injury phenotypes. In summary, the results of this study identified a number of pathways potentially important for the amelioration of hypertension and renal injury in SS-13(BN)/Mcw rats, and these results generated a series of testable hypotheses related to the role of the renal medulla in the complex mechanism of salt-sensitive hypertension.
Animals↗
Gene expression reveals vulnerability to oxidative stress and interstitial fibrosis of renal outer medulla to nonhypertensive elevations of ANG II.
The present study was designed to determine whether nonhypertensive elevations of plasma ANG II would modify the expression of genes involved in renal injury that could influence oxidative stress and extracellular matrix formation in the renal medulla using microarray, Northern, and Western blot techniques. Sprague-Dawley rats were infused intravenously with either ANG II (5 ng. kg(-1). min(-1)) or vehicle for 7 days (n = 6/group). Mean arterial pressure averaged 110 +/- 0.6 mmHg during the control period and 113 +/- 0.4 mmHg after ANG II. The mRNA of 1,751 genes ( approximately 80% of all currently known rat genes) that was differentially expressed (ANG II vs. saline) in renal outer and inner medulla was determined. The results of 12 hybridizations indicated that in response to ANG II, 11 genes were upregulated and 25 were downregulated in the outer medulla, while 11 were upregulated and 13 were downregulated in the inner medulla. These differentially expressed genes, most of which were not known previously to be affected by ANG II in the renal medulla, were found to group into eight physiological pathways known to influence renal injury and kidney function. Particularly, expression of several genes would be expected to increase oxidative stress and interstitial fibrosis in the outer medulla. Western blot analyses confirmed increased expression of transforming growth factor-beta1 and collagen type IV proteins in the outer medulla. Results demonstrate that nonhypertensive elevations of plasma ANG II can significantly alter the expression of a variety of genes in the renal outer medulla and suggested the vulnerability of the renal outer medulla to the injurious effect of ANG II.
Angiotensin II↗
Genomics and homeostasis.
The Cannon lecture this year illustrates how knowledge of DNA sequences of complex living organisms is beginning to shape the landscape of physiology in the 21st century. Enormous challenges and opportunities now exist for physiologists to relate the galaxy of genes to normal and pathological functions. The first extensive genomic systems biology map for cardiovascular and renal function was completed last year as well as a new hypothesis-generating tool ("physiological profiling") that enables us to hypothesize relationships between specific genes responsible for the regulation of regulatory pathways. Techniques of chromosomal substitution (consomic and congenic rats) are beginning to confirm statistical results from linkage analysis studies, narrow the regions of genetic interest for positional cloning, and provide genetically well-defined control strains for physiological studies. Patterns of gene expression identified by microarray and mapping of expressed genes to chromosomal sites are adding to the understanding of systems physiology. The previously unimaginable goal of connecting approximately 36,000 genes to the complex functions of mammalian systems is indeed well underway.
Animals↗
Role of renal NO production in the regulation of medullary blood flow.
The unique role of nitric oxide (NO) in the regulation of renal medullary function is supported by the evidence summarized in this review. The impact of reduced production of NO within the renal medulla on the delivery of blood to the medulla and on the long-term regulation of sodium excretion and blood pressure is described. It is evident that medullary NO production serves as an important counterregulatory factor to buffer vasoconstrictor hormone-induced reduction of medullary blood flow and tissue oxygen levels. When NO synthase (NOS) activity is reduced within the renal medulla, either pharmacologically or genetically [Dahl salt-sensitive (S) rats], a super sensitivity to vasoconstrictors develops with ensuing hypertension. Reduced NO production may also result from reduced cellular uptake of l-arginine in the medullary tissue, resulting in hypertension. It is concluded that NO production in the renal medulla plays a very important role in sodium and water homeostasis and the long-term control of arterial pressure.
Animals↗
In memoriam. Arthur C. Guyton (1919-2003).
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History, 20th Century↗
Tubulovascular nitric oxide crosstalk: buffering of angiotensin II-induced medullary vasoconstriction.
Studies were designed to determine the source of NO responsible for buffering of the angiotensin II (Ang II)-mediated decrease of blood flow in the renal medulla. Intracellular Ca2+ concentration ([Ca2+]i) and NO production ([NO]i) of pericytes and endothelium of the vasa recta were independently measured with the use of fura 2-AM and 4,5-diaminofluorescein diacetate (DAF-2DA), respectively, in microtissue strips of the vascular bundles of the outer medullary vasa recta. Disruption of the endothelium of the vasa recta by perfusion with latex microspheres enabled imaging of the pericytes. Ang II (1 micromol/L) produced an increase of [NO]i of 19+/-6 U in pericytes of the vasa recta when the vessels were adjacent to medullary thick ascending limbs (mTALs). Pericytes of isolated vasa recta without surrounding mTALs showed a rapid peak increase in [Ca2+]i of 248+/-107 nmol/L, with a sustained elevation of 107+/-75 nmol/L, but did not show an increase in [NO]i to either Ang II (1 micromol/L) or the Ca2+ ionophore 4-bromo-A23187 (5 micromol/L). These observations indicated the lack of Ang II and Ca2+-sensitive NO production in pericytes of the vasa recta. In isolated vasa recta with intact endothelium, Ang II reduced [Ca2+]i from 128+/-28 to 62+/-13 nmol/L and failed to increase [NO]i. However, the Ca2+ ionophore did increase [NO]i in the endothelium (47+/-8 U), indicating the presence of Ca2+-sensitive NO production. Significant increases of [NO]i were observed in single isolated mTALs in response to both Ang II (33+/-6 U) and the Ca2+ ionophore (51+/-18 U). We conclude that Ang II increases [Ca2+]i in pericytes of the descending vasa recta as part of its constrictor action and that this vasoconstriction is buffered by the NO from the surrounding tubular elements, such as mTALs.
Angiotensin II↗