Dose-dependent titration of prorenin and blood pressure in Cyp1a1ren-2 transgenic rats: absence of prorenin-induced glomerulosclerosis.

OBJECTIVE: Prorenin has been associated with cardiovascular disease and the development of glomerulosclerosis via a renin/prorenin receptor. In cyp1a1ren-2 transgenic rats, prorenin levels and arterial pressure can be increased by oral administration of indole-3-carbinol (I3C). The transgenic strain has been used as a model of malignant hypertension. METHODS: The present study was designed to test the hypotheses that (i) low doses of I3C would result in dose-dependent sustained increases in arterial pressure without signs of malignancy, making cyp1a1ren-2 transgenic rats a useful model to study nonmalignant hypertension, and (ii) cyp1a1ren-2 transgenic rats would develop glomerulosclerosis when they were chronically exposed to 0.125% I3C in their diet. RESULTS: I3C treatment for 2 weeks resulted in increases of plasma prorenin concentrations and arterial pressure in a dose-dependent manner. Rats thrived well over a period of 12 weeks on dietary I3C concentrations (wt/wt) of 0.125%. Plasma prorenin concentration rose from 0.1 +/- 0.1 microg to 17.9 +/- 5.0 mug angiotensin I/ml per h (P < 0.01) and mean arterial pressure increased to a plateau of 170 +/- 5 mmHg (P < 0.001) between weeks 6 and 12. After 12 weeks of 0.125% I3C, rats exhibited moderate hypertensive renal vasculopathy, but no histological signs of glomerulosclerosis. CONCLUSIONS: The cyp1a1ren-2 transgenic rat model allows for chronic dose-dependent titration of arterial pressure by a simple and non-invasive intervention, making this strain a useful model to study malignant and nonmalignant arterial hypertension. High circulating prorenin levels per se do not cause glomerulosclerosis.

Time-based analysis of silver-stained proteins in acrylamide gels.

Silver staining of proteins after PAGE often remains the method of choice in many laboratories. Nevertheless, it is known that quantification of protein levels is keenly restricted to a small range of protein concentrations leading to an over- or underestimation of protein amounts. To overcome this, a time-based analysis method was developed to avoid the saturation effect of the silver-staining reaction, thus resulting in an improved dynamic range of the gel image produced and therefore better quantification of proteins. Instead of the well-known end-point image analysis, gray intensities of time series images of a developing gel are determined and times until a threshold gray value is reached are calculated. These times are used to calculate a new grayscale image which can be analyzed using commercial image processing software.

Differential haemoglobin gene expression in the crustacean Daphnia magna exposed to different oxygen partial pressures.

The quantity and quality of the haemoglobin (Hb) of Daphnia magna is related to oxygen partial pressure in the water. Both the dynamics of hypoxia-induced Hb gene transcription, as well as Hb properties in animals incubated long-term at hyperoxia, normoxia and hypoxia, were investigated. Examination of Hb gene (dhb1-dhb3) transcription showed the expression of dhb2 and especially dhb3 to increase markedly approximately one hour after the onset of hypoxia, whereas dhb1 was expressed more or less constitutively. At an incubation close to anoxia, an onset of dhb3 transcription was found already after two minutes. In long-term incubated animals, concentration and oxygen affinity of Hb were lower at higher oxygen partial pressures. With decreasing oxygen availability, the subunit composition of Hb macromolecules changed. The share of the dhb2-encoded subunit, DHbF, increased already during moderate hypoxia. The increase of dhb3 mRNA (encoding DHbC) may be related to a transient increase of DHbC in the first days of hypoxia and/or to an additional coding of dhb3 for DHbD. The rise of DHbD, and particularly DHbA, only at severe hypoxia coincided with the increase of Hb oxygen affinity. The dhb1-encoded subunits DHbB and DHbE showed either a relatively moderate increase or even a decrease in concentration at hypoxia. In small animals with restricted homeostasis capabilities such as Daphnia, adaptation of the protein equipment seems to be a more effective strategy than allosteric modulator control.

The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties.

The process of oxygen-dependent hemoglobin induction in Daphnia magna was studied over an 11-day period of hypoxia (ambient oxygen partial pressure: 3 kPa). Along with the increase of hemoglobin concentration in the hemolymph, hemoglobin became the dominant protein fraction in gel filtration experiments using extracts of whole animals. The size of the native aggregates was constant. However, subunit composition depended on the duration of hypoxia: the pattern of predominantly expressed subunits under hypoxia deviated from that of normoxic individuals. The varying degree of hypoxic induction for different hemoglobin subunits was confirmed by autoradiography. Along with changes in hemoglobin subunit composition, oxygen affinity of the respiratory protein increased. The dynamics of the hemoglobin induction process was analysed. Newly synthesized hemoglobin can be detected within 18 h after the onset of hypoxia. A marked increase in hemoglobin concentration is evident from the third day of hypoxia, and a steady state of hemoglobin concentration is reached within 11 days. The changes of hemoglobin subunit expression in response to hypoxia form the structural basis for the observed adjustments of hemoglobin function leading to enhanced oxygen transport at low ambient oxygen concentrations.