Biomedical subjects

Andreas Rohrwasser

Publications and source records attributed to Andreas Rohrwasser.

10 recordsLinked to original sources

PubMed2006

Hypervolemia of pregnancy is not maintained in mice chronically overexpressing angiotensinogen.

OBJECTIVE: Women who develop pre-eclampsia show significantly less hypervolemia of pregnancy, compared with controls. We have shown that chronically elevated angiotensinogen expression increases a woman's risk of developing pre-eclampsia. Our objective was to determine whether increased angiotensinogen expression is sufficient to cause failed hypervolemia. STUDY DESIGN: To isolate the effects of elevated angiotensinogen expression, we studied transgenic mice with either 2 or 3 copies of the murine angiotensinogen gene. Plasma volume was measured by Evans blue dye dilution, and kidney sections were immunostained for angiotensinogen and renin. RESULTS: Three-copy mice failed to maintain hypervolemia after midgestation (P < .01) and failed to up-regulate renin expression in the distal nephron, compared with 2-copy controls. Intrarenal angiotensinogen was up-regulated during pregnancy in both genotypes. CONCLUSION: Chronically elevated angiotensinogen expression is sufficient to cause failed hypervolemia of pregnancy. Whether this observation is related to failed up-regulation of distal tubule renin expression requires further study.

Angiotensinogen↗

PubMed2006

Automated method for the isolation of collecting ducts.

The structural and functional heterogeneity of the collecting duct present a tremendous experimental challenge requiring manual microdissection, which is time-consuming, labor intensive, and not amenable to high throughput. To overcome these limitations, we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large-particle-based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD), or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large-particle-based flow cytometry or a complex object parametric analyzer and sorter (COPAS). A LIVE/DEAD assay demonstrates that the tubules were >90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Similarly, mRNA and protein from sorted tubules were analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules were related to sort stringency. Four to six replicates of 100 collecting ducts (9.68+/-0.44-14.5+/-0.66 cm or 9.2+/-0.7 mg tubular protein) were routinely obtained from a single mouse in under 1 h. In conclusion, large-particle-based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.

Animals↗

PubMed2005

Transcriptional diversity and expression of NEDD4L gene in distal nephron.

The ubiquitin ligase NEDD4L participates in plasma volume and blood pressure regulation by controlling expression of the epithelial sodium channel (ENaC). Genetic impairment of EnaC-Nedd4L-Proteasome system caused a rare mendelian hereditary human hypertension, Liddle syndrome. This finding suggested that Nedd4L is playing an important role in pathogenesis for hypertensive disorders. This prompted us to test a possible involvement of NEDD4L for the development of sodium-sensitive hypertension in Dahl salt-sensitive (DS) rats and its normotensive littermate Dahl salt-resistant (DR) rats. First, we analyzed the transcriptional diversity of rat Nedd4L gene and observed several isoforms with and without calcium-dependent membrane binding (C2) domain at the N-terminal of the protein as we found in human and mouse before. Then, we analyzed the expression of rat NEDD4L in the kidney of both DS and DR under high and low sodium regimens. NEDD4L expression examined by quantitative PCR technique revealed lower expression of NEDD4L transcripts in DS rats under either diet compared to DR animals; additionally, NEDD4L expression was significantly increased with sodium loading. Using in situ hybridization experiments, rat NEDD4L was predominantly expressed in distal nephron in a manner dependent on both sodium regimen and genetic background. A similar histological distribution pattern was observed in human kidney. The expression of NEDD4L in distal nephron and its response to chronic sodium loading suggest that it participates in the functioning of this segment in sodium reabsorption. This response was impaired in genetically sodium-sensitive animals. These findings suggested that Nedd4L gene products were involved in the development of salt-sensitive hypertension.

Adrenal Glands↗

PubMed2005

Significance of urinary angiotensinogen in essential hypertension as a function of plasma renin and aldosterone status.

OBJECTIVE: This study was performed to test the significance of urinary angiotensinogen (UAGT) in essential hypertensive patients stratified as a function of plasma renin and aldosterone. METHODS AND RESULTS: A sample of 248 essential hypertensives, investigated under their usual sodium diet and either off-medication or under a standardized treatment, was separated into two groups on the basis of upright plasma active renin and aldosterone medians. Patients with plasma active renin and aldosterone below medians are referred to as the low renin-aldosterone essential hypertensive group (LRA-EH). Others subjects are defined as other essential hypertensives (O-EH). Blood pressure (BP) was recorded by 24-h ambulatory monitoring. UAGT was measured by a specific enzyme-linked immunosorbent assay for total angiotensinogen. Because UAGT was markedly increased in the presence of overt proteinuria (>/= 300 mg/24 h), proteinuric patients (n = 29) were excluded from subsequent analyses. UAGT was a significant predictor of systolic and diastolic BP in LRA-EH females (P < 0.01 and P = 0.05, respectively) but not in males. By contrast, urinary sodium excretion (P < 0.001) and maintenance of treatment (P = 0.002) were significant predictors of systolic BP in males. These correlations were not observed in O-EH, whether males or females. CONCLUSIONS: In the present study, UAGT stands as a strong predictor of BP in women with low plasma renin/aldosterone, suggesting an involvement of the tubular renin-angiotensin system in these subjects. Higher sodium intake or the need to maintain treatment may account in part for the lack of a similar relationship in males.

Aged↗

PubMed2004

Expression of angiotensinogen in proximal tubule as a function of glomerular filtration rate.

BACKGROUND: Proximal tubule (PT) angiotensinogen (AGT) is part of a tubular renin-angiotensin system (RAS) that participates in the regulation of sodium reabsorption along the entire nephron. Physiologic maneuvers affecting AGT expression in PT also affect systemic RAS. Here, we tested the hypothesis that PT AGT is regulated by increased glomerular filtration rate (GFR). METHODS: Complete unilateral nephrectomy (UNX) in mice was used to induce a sustained increase in GFR in the remaining kidney. AGT expression was monitored by quantitative reverse transcription-polymerase chain reaction (RT-PCR). AGT protein in PT was investigated by semiquantitative histology. We also measured AGT concentration in plasma and in 24-hour urine by a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: Seven weeks after nephrectomy, UNX animals exhibited a 2-fold increase in tubular AGT mRNA (P <.001) compared with sham-operated control animals. The proportion of PT sections exhibiting AGT immunostaining was significantly increased at day 3 (P <.05), and remained elevated at seven weeks (UNX = 0.63 +/- 0.09, sham = 0.38 +/- 0.02, P <.01), revealing recruitment of AGT-producing cells along the PT. AGT excretion in final urine corrected for creatinine and kidney weight was also elevated by UNX at seven weeks (UNX = 209 +/- 42 pmol/mg/g, sham = 147 +/- 29 pmol/mg/g, P <.05), with no difference in plasma AGT between UNX and control animals. CONCLUSION: These observations suggest that AGT expression in PT adapts in the long-term to changes in GFR. In the UNX model, urinary AGT excretion is also elevated as a consequence of increase in net tubular flow.

Adaptation, Physiological↗

PubMed2003

Renin and kallikrein in connecting tubule of mouse.

BACKGROUND: The observation of renin expression in connecting tubule, a segment that also expresses tissue kallikrein (KLK-1), raises two questions. Are the genes expressed in the same or in different cells of connecting tubule? Does this topography support the hypothesis that KLK-1 activates prorenin or is it more likely that it affords coordinated gene regulation? METHODS: Renin and KLK-1 were examined by immunostaining and in situ hybridization. Renin activation by KLK-1 was investigated in vitro. In vivo, excretion of prorenin and active renin was compared in mice homozygous for targeted inactivation of KLK-1 (TK(-/-)) and normal littermates (TK(+/+)). RESULTS: Using in situ immunostaining for renin and in situ hybridization for KLK-1 mRNA, we found that connecting tubule cells expressing renin also expressed KLK-1. We confirmed in vitro activation of prorenin by KLK-1, but found no difference in the ratio of active renin to prorenin in urine of TK(-/-) and TK(+/+) animals. Compared to TK(+/+) controls, TK(-/-) mice exhibited significantly lower 24-hour excretion of prorenin (5.05 +/- 1.16 mg Ang I/hour vs. 9.39 +/- 1.96 mg Ang I/hour, P < 0.05) and active renin (1.98 +/- 0.25 mg Ang I/hour vs. 3.58 +/- 0.39 mg Ang I/hour, P < 0.05), with no difference in either urine volumes or plasma renin concentrations. CONCLUSION: Direct interaction between renin and KLK-1, not ruled out in vitro, is not supported in vivo. By contrast, lower excretion of active renin and prorenin in TK(-/-) compared to TK(+/+) suggest coordinated regulation of the two proteins in their participation to collecting duct function.

Animals↗

PubMed2002

Contribution of Sp1 to initiation of transcription of angiotensinogen.

Several genetic polymorphisms have been identified in the proximal promoter of angiotensinogen ( AGT). Gene titration experiments in transgenic animals have demonstrated that small increases in the basal expression of AGT can lead to elevated blood pressure. The direct proof that promoter variants of AGT can lead to elevated blood pressure will ultimately require the development of specific animal models. Before such work can be contemplated, however, a formal understanding of the mechanisms controlling transcriptional activation of AGT needs to be developed. Analysis of DNA-protein interactions in vitro and transactivation experiments in cultured cells reveal the critical role of an Sp1 binding site immediately upstream of the TATA box of AGT in both mouse and human. Both sites are required for transcription initiation in the mouse. By contrast, a minimal human AGT promoter can initiate transcription in the absence of either this Sp1 site or the TATA box, albeit at a lower level. Further analysis and consideration of these interspecific differences will be essential for the development of meaningful animal models to probe the mechanism by which AGT may predispose to human essential hypertension.

Angiotensinogen↗

PubMed2002

Power and replication in case-control studies.

The case-control study design, a common staple of epidemiology, is increasingly used to test for genetic association. The simplicity of the design accounts for both its appeal and its limitations. Too often, however, apparent controversy arises for lack of appreciation of basic tenets underlying statistical testing. Power and replication are two concepts most commonly ignored in evaluating such studies. We review the basic principles of statistical testing, recall simple means to calculate power, and provide numerical examples pertaining to the association between angiotensinogen and essential hypertension.

Alleles↗

PubMed2002

Effects of dietary sodium and genetic background on angiotensinogen and Renin in mouse.

Elements of a renin-angiotensin system expressed along the entire nephron, including angiotensinogen secreted by proximal tubule and renin expressed in connecting tubule, may participate in the regulation of sodium reabsorption at multiple sites of the nephron. The response of this tubular renin-angiotensin system to stepwise changes in dietary sodium was investigated in 2 mouse strains, the sodium-sensitive inbred C57BL/6 and the sodium-resistant CD1 outbred. Plasma angiotensinogen was not affected by sodium regimen, whereas plasma renin increased 2-fold under low sodium. In both strains, the variation in urinary parameters did not parallel the changes observed in plasma. Angiotensinogen and renin excretion were significantly higher under high sodium than under low sodium. Water deprivation, by contrast, induced significant activation in the tubular expression of angiotensinogen and renin. C57BL/6 exhibited significantly higher urinary excretion of angiotensinogen than did CD1 animals under both conditions of sodium intake. The extent to which these urinary parameters reflect systemic or tubular responses to challenges of sodium homeostasis may depend on the relative contribution of sodium restriction and volume depletion.

Aldosterone↗

PubMed2001

Angiotensinogen in essential hypertension: from genetics to nephrology.

There is general consensus that genetic variation accounts in part for individual susceptibilities to essential hypertension. In marked contrast to classic mendelian disorders, in which genetic alterations produce a gain or loss of function, genetic determinants of essential hypertension, high blood pressure of unknown cause, are expected to be small, achieving significance through the cumulative effects of environmental exposure over the course of a lifetime. Whether and how genetic factors that contribute to common diseases can be identified remain unclear. Research on a link between angiotensinogen and essential hypertension illustrates a path that began in genetics and is now leading toward nephrology. Various challenges encountered along the way may prove to be characteristic features of genetic investigations of the pathogenesis of common diseases. The implication of a gene by statistical analysis is only the beginning of a protracted process of functional analysis at increasing levels of biologic integration. The ultimate goal is to develop an understanding of the manner in which genetic variation at a locus can affect a physiologic parameter and to extract from this inference new knowledge of significance for the prevention or treatment of disease.

Alleles↗