Biomedical subjects

Heddwen L Brooks

Publications and source records attributed to Heddwen L Brooks.

13 recordsLinked to original sources

PubMed2006

Axial heterogeneity of vasopressin-receptor subtypes along the human and mouse collecting duct.

Vasopressin and vasopressin antagonists are finding expanded use in mouse models of disease and in clinical medicine. To provide further insight into the physiological role of V1a and V2 vasopressin receptors in the human and mouse kidney, intrarenal localization of the receptors mRNA was determined by in situ hybridization. V2-receptor mRNA was predominantly expressed in the medulla, whereas mRNA for V1a receptors predominated in the cortex. The segmental localization of vasopressin-receptor mRNAs was determined using simultaneous in situ hybridization and immunohistochemistry for segment-specific markers, including aquaporin-2, Dolichos biflorus agglutinin, epithelial Na channels, Tamm Horsfall glycoprotein, and thiazide-sensitive Na(+)-Cl(-) cotransporter. Notably, V1a receptor expression was exclusively expressed in V-ATPase/anion exchanger-1-labeled alpha-intercalated cells of the medullary collecting duct in both mouse and human kidney. In cortical collecting ducts, V1a mRNA was more widespread and detected in both principal and intercalated cells. V2-receptor mRNA is diffusely expressed along the collecting ducts in both mouse and human kidney, with higher expression levels in the medulla. These results demonstrate heterogenous axial expression of both V1a and V2 vasopressin receptors along the human and mouse collecting duct. The restricted expression of V1a-receptor mRNA in intercalated cells suggests a role for this receptor in acid-base balance. These findings further suggest distinct regulation of renal transport function by AVP through V1a and V2 receptors in the cortex vs. the medulla.

Animals↗

PubMed2006

CARMA: A platform for analyzing microarray datasets that incorporate replicate measures.

BACKGROUND: The incorporation of statistical models that account for experimental variability provides a necessary framework for the interpretation of microarray data. A robust experimental design coupled with an analysis of variance (ANOVA) incorporating a model that accounts for known sources of experimental variability can significantly improve the determination of differences in gene expression and estimations of their significance. RESULTS: To realize the full benefits of performing analysis of variance on microarray data we have developed CARMA, a microarray analysis platform that reads data files generated by most microarray image processing software packages, performs ANOVA using a user-defined linear model, and produces easily interpretable graphical and numeric results. No pre-processing of the data is required and user-specified parameters control most aspects of the analysis including statistical significance criterion. The software also performs location and intensity dependent lowess normalization, automatic outlier detection and removal, and accommodates missing data. CONCLUSION: CARMA provides a clear quantitative and statistical characterization of each measured gene that can be used to assess marginally acceptable measures and improve confidence in the interpretation of microarray results. Overall, applying CARMA to microarray datasets incorporating repeated measures effectively reduces the number of gene incorrectly identified as differentially expressed and results in a more robust and reliable analysis.

Analysis of Variance↗

PubMed2006

Effects of water restriction on gene expression in mouse renal medulla: identification of 3betaHSD4 as a collecting duct protein.

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Comparative effects of dehydroepiandrosterone sulfate on ventricular diastolic function with young and aged female mice.

The adrenal steroid hormone dehydroepiandrosterone (DHEA) and its sulfated derivative [DHEA(S)] have been extensively studied for their potential anti-aging effects. Associated with aging, DHEA levels decline in humans, whereas other adrenal hormones remain unchanged, suggesting that DHEA may be important in the aging process. However, the effect of DHEA(S) supplementation on cardiac function in the aged has not been investigated. Therefore, we administered to young and old female mice a 60-day treatment with exogenous DHEA(S) at a dose of 0.1 mg/ml in the drinking water and compared the effects on left ventricular diastolic function and the myocardial extracellular matrix composition. The left ventricular stiffness (beta) was 0.30 +/- 0.06 mmHg/mul in the older control mice compared with 0.17 +/- 0.02 mmHg/mul in young control mice. Treatment with DHEA(S) decreased left ventricular stiffness to 0.12 +/- 0.03 mmHg/mul in the older mice and increased left ventricular stiffness to 0.27 +/- 0.04 mmHg/mul in young mice. The mechanism for the DHEA(S)-induced changes in diastolic function appeared to be associated with altered matrix metalloproteinase activity and the percentage of collagen cross-linking. We conclude that exogenous DHEA(S) supplementation is capable of reversing the left ventricular stiffness and fibrosis that accompanies aging, with a paradoxical increased ventricular stiffness in young mice.

Aging↗

PubMed2005

Altered expression of selected genes in kidney of rats with lithium-induced NDI.

Lithium treatment is associated with development of nephrogenic diabetes insipidus, caused in part by downregulation of collecting duct aquaporin-2 (AQP2) and AQP3 expression. In the present study, we carried out cDNA microarray screening of gene expression in the inner medulla (IM) of lithium-treated and control rats, and selected genes were then investigated at the protein level by immunoblotting and/or immunohistochemistry. The following genes exhibited significantly altered transcription and mRNA expression levels, and these were compatible with the changes in protein expression. 11beta-Hydroxysteroid dehydrogenase type 2 protein expression in the IM was markedly increased (198 +/- 25% of controls, n = 6), and immunocytochemistry demonstrated an increased labeling of IM collecting duct (IMCD) principal cells. This indicated altered renal mineralocorticoid/glucocorticoid responses in lithium-treated rats. The inhibitor of cyclin-dependent kinases p27 (KIP) protein expression was significantly decreased or undetectable in the IMCD cells, pointing to increased cellular proliferation and remodeling. Heat shock protein 27 protein expression was decreased in the IM (64 +/- 6% of controls, n = 6), likely to be associated with the decreased medullary osmolality in lithium-treated rats. Consistent with this, lens aldose reductase protein expression was markedly decreased in the IM (16 +/- 2% of controls, n = 6), and immunocytochemistry revealed decreased expression in the thin limb cells in the middle and terminal parts of the IM. Ezrin protein expression was upregulated in the IM (158 +/- 16% of controls, n = 6), where it was predominantly expressed in the apical and cytoplasmic domain of the IMCD cells. Increased ezrin expression indicated remodeling of the actin cytoskeleton and/or altered regulation of IMCD transporters. In conclusion, the present study demonstrates changes in gene expression not only in the collecting duct but also in the thin limb of the loop of Henle in the IM, and several of these genes are linked to altered sodium and water reabsorption, cell cycling, and changes in interstitial osmolality.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2004

Pax2 expression occurs in renal medullary epithelial cells in vivo and in cell culture, is osmoregulated, and promotes osmotic tolerance.

Pax2 is a transcription factor that is crucial for kidney development, and it is also expressed in the normal adult kidney, where its physiological function is unknown. In the present study, we find by cDNA microarray analysis that Pax2 expression in second-passage mouse inner-medullary epithelial cells is increased by a high NaCl concentration, which is significant because NaCl levels are normally high in the inner medulla in vivo, and varies with urinary concentration. Furthermore, a high NaCl concentration increases Pax2 mRNA and protein expression in mouse inner medullary collecting duct (mIMCD3) cells, and its transcriptional activity. Pax2 mRNA and protein expression is high in normal adult mouse renal inner medulla but much lower in renal cortex. Pax2 protein is present in collecting duct cells in both renal medulla and cortex and in thin descending limbs of Henle's loop in inner medulla. Treating Brattleboro rats with desamino-Cys-1,d-Arg-8 vasopressin, which increases inner-medullary NaCl concentration, causes a 4-fold increase in inner-medullary Pax2 protein. Treatment with furosemide, which decreases inner-medullary NaCl, reduces inner-medullary Pax2 mRNA and protein. Pax2-specific short interfering RNA increases high NaCl concentration-induced activation of caspase-3 and apoptotic bodies in mIMCD3 cells. We thus conclude that (i) Pax2 is expressed in normal renal medulla, (ii) its expression is regulated there by the normally high and variable NaCl concentration, and (iii) it protects renal medullary cells from high NaCl concentration-induced apoptosis.

Animals↗

PubMed2004

Developmental expression of glucokinase in rat hypothalamus.

Neurons in the hypothalamus sense changes in glucose concentration. Glucokinase (GK), a key enzyme for pancreatic (beta)-cell glucose sensing, was found in both the embryonic and adult hypothalamus. GK activity accounted for approximately 20% of total hexokinase (HK) activity in both embryonic and adult hypothalamus with no activity measured in cortical samples, indicating that glucose sensing in the hypothalamus initiates early in development and precedes the maturation of glucose signaling in liver.

Age Factors↗

PubMed2004

Renal medullary gene expression in aquaporin-1 null mice.

Mice that lack the aquaporin-1 gene (AQP1) lack a functional countercurrent multiplier mechanism, fail to concentrate the inner medullary (IM) interstitium, and present with a urinary concentrating defect. In this study, we use DNA microarrays to identify the gene expression profile of the IM of AQP1 null mice and corresponding changes in gene expression resulting from a loss of a hypertonic medullary interstitium. An ANOVA analysis model, CARMA, was used to isolate the knockout effect while taking into account experimental variability associated with microarray studies. In this study 5,701 genes of the possible approximately 12,000 genes on the array were included in the ANOVA; 531 genes were identified as demonstrating a >1.5-fold up- or downregulation between the wild-type and knockout groups. We randomly selected 35 genes for confirmation by real-time PCR, and 29 of the 35 genes were confirmed using this method. The overall pattern of gene expression in the AQP1 null mice was one of downregulation compared with gene expression in the renal medullas of the wild-type mice. Heat shock proteins 105 and 94, aldose reductase, adenylate kinase 2, aldolase B, aldehyde reductase 6, and p8 were decreased in the AQP1 null mice. Carboxylesterase 3, matrilin 2, lipocalin 2, and transforming growth factor-alpha were increased in IM of AQP1 null mice. In addition, we observed a loss of vasopressin type 2 receptor mRNA expression in renal medullas of the AQP1 null mice. Thus the loss of the hyperosmotic renal interstitium, due to a loss of the concentrating mechanism, drastically altered not only the phenotype of these animals but also their renal medullary gene expression profile.

Animals↗

PubMed2003

Expression of osmotic stress-related genes in tissues of normal and hyposmotic rats.

TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved in organic osmolyte accumulation. Surprisingly, it is expressed in virtually all tissues, including many never normally exposed to hypertonicity. We measured TonEBP mRNA (real-time PCR) and protein (Western blot analysis) in tissues of control (plasma osmolality 294 +/- 1 mosmol/kgH2O) and hyposmotic (dDAVP infusion plus water loading for 3 days, 241 +/- 2 mosmol/kgH2O) rats to test whether the ubiquitous expression of TonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not in brain, and is not detected in heart and skeletal muscle. TonEBP mRNA decreases in brain and liver but is unchanged in other tissues. There are no general changes in mRNA of TonEBP-mediated genes: aldose reductase (AR) does not change in any tissue, betaine transporter (BGT1) decreases only in liver, taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70-1 and Hsp70-2 mRNA increase greatly in most tissues, which cannot be attributed to decreased TonEBP activity. The conclusions are as follows: 1) TonEBP protein or mRNA expression is reduced by hyposmolality in thymus, liver, and brain. 2) TonEBP protein and mRNA expression are differentially regulated in some tissues. 3) Although AR, SMIT, BGT1, and TauT are regulated by TonEBP in renal medullary cells, other sources of regulation may predominate in other tissues. 4) TonEBP abundance and activity are regulated by factors other than tonicity in some tissues.

Animals↗

PubMed2003

Long-term regulation of ENaC expression in kidney by angiotensin II.

We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the alpha subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased alphaENaC protein abundance in rat kidney cortex. The decrease in alphaENaC protein abundance in response to AT1 receptor blockade was associated with a fall in alphaENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT1 receptor blockade on alphaENaC expression was not blocked by spironolactone, suggesting a direct role of the AT1 receptor in regulation of alphaENaC gene expression. Candesartan administration was also found to increase the abundances of the beta and gamma subunits. The increase in beta and gammaENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in beta and gammaENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function.

Angiotensin II↗

PubMed2002

cDNA array identification of genes regulated in rat renal medulla in response to vasopressin infusion.

With the aim of identifying possible gene targets for direct or indirect regulation by vasopressin in the renal medulla, we have carried out cDNA array experiments in inner medullas of Brattleboro rats infused with the V(2) receptor-selective vasopressin analog desamino-Cys1,d-Arg8 vasopressin (dDAVP) for 72 h. Of the 1,176 genes on the array, 137 transcripts were increased by 2-fold or more, and 10 transcripts were decreased to 0.5-fold or less. Quantitative, real-time RT-PCR measurements confirmed increases seen for six selected transcripts (Wilms' tumor protein, beta-arrestin 2, neurofibromin, casein kinase IIbeta, aquaporin-3, and aquaporin-4). To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting for 28 of the proteins whose cDNAs were on the array. For several targets including aquaporin-2, transcript abundance and protein abundance changes did not correlate. However, for most genes examined, changes in mRNA abundances were associated with concomitant protein abundance changes. Targets with demonstrated increases in both protein and mRNA abundances included neurofibromin, casein kinase IIbeta, the beta-subunit of the epithelial Na channel (beta-ENaC), 11beta-hydroxysteroid dehydrogenase type 2, and c-Fos. Additional cDNA arrays revealed that several transcripts that were increased in abundance after 72 h of dDAVP were also increased after 4 h, including casein kinase IIbeta, beta-ENaC, aquaporin-3, UT-A, and syntaxin 2. These studies have identified several transcripts whose abundances are regulated in the inner medulla in response to infusion of dDAVP and that could play roles in the regulation of salt and water excretion.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2002

Targeted proteomic profiling of renal Na(+) transporter and channel abundances in angiotensin II type 1a receptor knockout mice.

The renal tubule transporters responsible for Na(+) and water transport along the nephron have been identified and cloned, permitting comprehensive analysis of transporter protein abundance changes in complex physiological models by using a "targeted proteomics" approach. Here, we apply this approach to screen renal homogenates from mice in which the gene for the angiotensin II type 1a (AT(1a)) receptor has been deleted (versus wild-type mice) to determine which sodium transporters and channels are regulated by the AT(1a) receptor at the protein abundance level. In mice maintained on a low NaCl diet (<0.02% NaCl), (1) the abundances of 2 aldosterone-regulated transporters were markedly decreased in knockout versus wild-type mice, namely, the thiazide-sensitive cotransporter and the alpha-subunit of the amiloride-sensitive Na(+) channel (alpha-ENaC); (2) the abundances of beta-ENaC and gamma-ENaC were markedly increased; and (3) there were no significant changes in the abundances of the proximal tubule Na+-H(+) exchanger or the Na(+)-K(+)-2Cl(-) cotransporter of the thick ascending limb. When the experiment was repeated on higher NaCl diets (0.4% or 6% NaCl), the decrease in alpha-ENaC abundance persisted, whereas the other changes were abolished. Analysis of serum aldosterone concentration in AT(1a) knockout mice and wild-type mice on the low NaCl diet revealed the absence of a decrease with AT(1a) gene deletion (11.8 +/- 2.3 nmol/L for knockout mice and 5.7 +/- 0.8 nmol/L for wild-type mice [significantly increased]). These results reveal that the AT(1a) receptor plays an important role in regulation of Na(+) transporter and channel proteins in the "post-macula densa" region of the renal tubule via a mechanism that is not dependent on altered circulating aldosterone concentrations.

Angiotensin II↗

PubMed2002

Posttranscriptional compensation for heterozygous disruption of the kidney-specific NaK2Cl cotransporter gene.

Mice homozygous for a loss of function mutation of the kidney-specific NaK2Cl cotransporter, BSC1/NKCC2, do not survive. Here the effects of loss of one copy of the gene are studied. NKCC2 mRNA of NKCC2 +/- kidney was 55 +/- 6% of +/+, yet no differences were found between NKCC2 +/+ and +/- mice in BP, blood gas, electrolytes, creatinine, plasma renin concentration, urine volume and osmolality, ability to concentrate and dilute urine, and response to furosemide. When mice were challenged with 180 mM NH(4)Cl, plasma ammonia and urinary ammonia excretion were increased twofold and fivefold, respectively, but there was still no difference between the two genotypes. NKCC2 +/- mice had a near-normal level of NKCC2 protein and no clear change in the distribution of NKCC2 in the thick ascending limb (TAL) cells. In vitro microperfusion of isolated TAL showed no significant difference between the two genotypes in the basal and vasopressin-stimulated capacity to reabsorb NaCl. There was no difference in the mRNA expressions of thiazide-sensitive NaCl cotransporter, epithelial Na channel (ENaC), aquaporin-2, ROMK, and NaKATPase. Halving the mRNA expression of NKCC2 does not affect BP or fluid balance because of compensatory factors that restore the protein level to near normal. One possible factor is a regulated increase in the movement of cytoplasmic protein to the luminal membrane leading to a restoration of functional transporter to an essentially wild type level.

Adaptation, Physiological↗