animals from each group as described above (section 2.4.).

2.5.5 Histological examination

For histological examination, randomly selected animals were perfused with fixative as described above (section 2.4.3). The left kidney was removed and after a further 24 h

fixation period, dehydrated by passing through an increasing concentration of EtOH and subsequently embedded into paraffin, by an experienced renal pathologist, Stuart Fleming of the Centre for Genome Research, Edinburgh, Scotland. 3 pm sections were cut and stained with haematoxylin and eosin (H & E) (Stevens, 1990). Sections (three per rat) were examined by an experienced renal pathologist (SF) who was blinded to the treatment status of each animal. Tissues were examined from randomly selected untreated survivors (controls, n=8), ramipril treated survivors (n=9) and rats which were culled after exhibiting

features of established MH (n=7). Three elements, namely arterial intimai proliferation, arteriolar necrosis and nephron injury were scored on a scale of 1-5 as follows:

1: 0-5 % scored 1 for trend analysis

2: 6-25 % scored 2 for trend analysis

3: 26-50 % scored 3 for X^ trend analysis

4: >75 % scored 4 for X^ trend analysis

5: >76 % affected scored 5 for X^ trend analysis

In addition, vascular medial thickening was graded 1 to 5 based on the degree of hypertrophy observed in medium-sized arteries on each section.

2.6 TISSUE TRANSGENE EXPRESSION IN THE TGR(mRen2)27 rat

The TGR(mRen2)27 rat may be a useful model in which to study cardiac and vascular responses to hypertension and the role of the RAS in such responses. However, such studies have been hampered by debate concerning the level of Ren-2 transgene and native

renin expression in both the heart and vascular tissue (Ekker et al, 1989, Hilgers et al,

ill-defined numbers and animals of mixed sex and age might account for much of the confusion. Therefore, expression of the transgene in the aorta, carotid and in specific cardiac chambers of adult male TGR(mRen2)27 heterozygote rats of defined number, age, weight and sex was examined in this study.

The RNase protection assay is a sensitive method of identifying transgene expression in animal models and was therefore used in this study. Confirmation of low-level expression was sought using the much more sensitive technique of reverse transcription-polymerase

chain reaction (RT-PCR) amplification of transgene mRNA. In order to prevent

contamination and subsequent false positive results disposable equipment was used throughout with all non disposable equipment being sterilized (using HCl where appropriate) between handling each tissue and each animal type.

2.6.1 Extraction using the RNAzol B method

Tissues

Fifteen male heterozygous TGR(mRen2)27 rats and 15 SD controls (all 250-300g) were anaesthetised with an intraperitoneal injection as described above (section 2.4.3). The abdomen was opened, the abdominal aorta tied with 3/0 braided silk, the animals perfused with iced saline for 1 min (180 mmHg for transgenics, 120 mmHg for controls) through a proximally placed 18-gauge cannula. The left lung, both carotids and proximal 1-2 cm of aorta were rapidly removed, cleaned of surrounding tissue on ice, washed in iced saline, blotted dry, weighed and snap frozen in liquid nitrogen. The heart was removed, cleaned and rinsed, the RV dissected free from the LV and septum and the tissues similarly treated.

RNA extraction

RNA extraction was performed by Dr Hugh Montgomery at the Centre for Genome

Research, Edinburgh, UK in a laboratory previously unexposed to Ren-2 mRNA or

complementary DNA (cDNA). RNAzol contains phenol and guanidium thiocyanate

(Chomczynski & Sacchi, 1987), promotes the formation of RNA-guanidium-water complexes, abolishes the hydrophilic interactions of DNA and proteins and thus selectively leaves RNA in aqueous solution. Yields are usually approximately 5-6 pg RNA per 1 mg tissue, but can be as low as 1 pg per 1 mg of tissue. The weights of each tissue sample varied, therefore the number of animals used as a source for RNA varied with the tissue being studied (12 lungs, 6 RV, 3 LV, 15 carotids and 15 aortic samples). Like tissues

were pooled and processed together, although the lung samples were processed in two batches of 6 (lung^ and lung^). The samples were homogenized in 50 ml Coming tubes

(Ultra-Turrax Drive T25 350 Watt homogenizer (8000-24000 revolutions/min; DCA Labortechnik, Germany), with the addition of approximately 2 ml RNAzol per 100 mg tissue (15 aortas in 15 ml, 15 carotids in 15 ml, 12 lungs in 50 ml, 6 RV in 25 ml and 3 LV

in 25 ml) for 0.5-1.0 min. Each homogenizer was stripped and cleaned in 2 M HCl (5 min soak) before use and between samples, before being rinsed in dH2 0 (RNase free). 0.1

volumes of chloroform were added and the samples covered, shaken for 15 s, left on ice (4 °C) for 5 min and centrifuged at 2 1 0 0 g- for 15 min at 4 °C. The aqueous phase (excluding

interface) was transferred to a 30 ml glass Corex tube, an equal volume of isopropanol added, the samples left on ice for 15 min and then centrifuged at 16500 g for 15 min at 4 °C. A yellow-white pellet of RNA was identified at the bottom of the tube. After removal of the supernatant, the RNA pellet was washed with 1 ml 70 % v/v EtOH added to the RNA (>0.8 ml EtOH per 50-100 pg RNA) and the sample vortexed and re-centrifuged at 16500 g, for 8 min at 4 °C. This process was then repeated. The RNA pellet was

incompletely air-dried (10-15 min) and re-dissolved by vortexing in pure formamide (Chomczynski, 1992). The use of pure chemicals and the strong denaturing effect of RNAzol meant that diethylpyrocarbonate (DEPC)-treated solutions were not required. Large grey pellets were derived from the ventricular samples, suggesting glycogen

contamination. The process of RNAzol extraction was therefore repeated on these

samples.

RNA quantity and qualitv

Final RNA concentrations are usually in the range of 4 mg/ml. The RNA samples were diluted 1:200 for quantification by ultraviolet (UV) absorption spectrophotometry at 260 nm. Absorption at 280 nm was also quantified, as higher absorption at this wavelength suggests protein contamination. The concentration of RNA was calculated from the following equation: Absorbance at 260 nm x dilution x 40 = pg RNA/ml. RNA was shown to be intact by agarose gel electrophoresis.

2.6.2 RNAse protection assay for Ren-2 mRNA

Making the riboprobe (Figure 2.3)

cDNA for the Ren-2 gene has been cloned into the Bluescript II KS plasmid vector. The

full-length cDNA was cloned into the Sal I and Hind III sites of the vector (Dr Matthew Sharp, personal communication). This plasmid was linearized using Nco I, which cuts once in the Ren-2 cDNA and transcribed using bacteriophage T7 DNA-dependent RNA

Figure 2.3: RNase protection assay for mouse Ren-2 gene mRNA.

Ren-2 cDNA T7 Promotor

Hind Site Sa! I Site Ren-2 DNA Cleavage Site (Ncol)

17 RNA polymerase produces

32P-labelled cRNA strand

2

Ren2 mRNA