A renin transcript lacking exon 1 encodes for a non-secretory intracellular renin that increases aldosterone production in transgenic rats

Renin transcripts lacking exon 1 and thus the signal sequence for co-translational transport to the endoplasmatic reticulum encode for a protein (exon[2-9]renin), that is confined to the cytoplasm. The function of exon(2-9)renin is currently unknown. Mitochondrial renin increases under conditions which stimulate aldosterone production. We hypothesized that exon(2-9)renin (1) is translated into a functionally active protein in vivo, (2) is not secreted but remains within the cytoplasm and (3) stimulates aldosterone production. To test these hypotheses we generated transgenic rats overexpressing exon(2-9)renin. Four transgenic lines were obtained expressing the transcript in various tissues including the heart and the adrenal gland. Renin was enriched particularly in the cytoplasm of transgenic rats. Renin was not elevated in plasma, indicating that exon(2-9)renin is produced but not secreted. The ratio of aldosterone to renin concentrations in plasma (PAC/PRC) was elevated in all transgenic lines except line 307, which also did not exhibit elevated cytoplasmatic renin levels in the adrenal gland (PAC/PRC in controls: 2.8±2.3; line 307: 1.9±0.8; n. s.; line 284: 5.8±1.9; P<0.02; line 294: 5.0±2.3; P<0.001; line 276: 10.3±5.1; P<0.001). We conclude that the exon(1A-9) renin transcript (1) is translated into a functionally active intracellular protein; (2) is targeted to the cytoplasm rather than being sorted to the secretory pathways and (3) is functionally active, regulating aldosterone production. The CX-(exon2-9)renin transgenic rat appears to be a useful model to study the role and the mechanisms of action of cytoplasmatic renin derived from exon(1A-9) transcripts.

a second renin transcript, termed exon(1A-9)-renin, which codes for a non-secretory protein [8]. Similar transcripts have been found in other species as well, including humans [9,10]. These transcripts lack exon 1 and thus the signal for the co-translational transport to the ER. In humans one of the renin transcripts directly starts with exon 2 [10]. In rats, exon 2 of the exon(1A-9)renin transcript is preceded by a short sequence of about 80 base pairs derived from intron A [8]. This sequence is non-coding and therefore can only have regulatory functions.

Determination of prorenin, renin and aldosterone and electrolyte concentrations
Plasma prorenin, renin and aldosterone levels were determined as described previously from ethylenediaminetetraacetic acid (EDTA) blood obtained from the retro-orbital plexus after light ether anaesthesia [18], except that ANGI determination was based on a solid phase radioimmunoassay. Sub-cellular fractions were prepared by differential centrifugation as described previously [13]. Protein, renin and prorenin levels were determined as described previously [18]. The specificity of the enzyme reaction was controlled using the renin inhibitor CH732 [13]. Serum sodium and potassium concentrations were determined using the ISE Analysator AVL 988-4 (AVL Medical Instruments, Bad Homburg, Germany).

Cardiac histology
Hearts were preserved in 4% formalin, embedded in paraffin. Coronal slices (2-3 µm) were stained with haematoxylin eosin. Sections were investigated by light microscopy at magnifications of x 200 and x 400 (Axioplan, Zeiss) in a blinded manner by an observer who was unaware of the different study groups.

Blood pressure measurements
Systolic blood pressure was determined by tail plethysmography in conscious trained and pre-heated wild-type (WT) rats (10 female, 9 male) and TGR (8 female, 18 male). Rats were trained on five consecutive days. Measurements were performed on three consecutive days.

Statistical analyses
Prorenin, renin and aldosterone levels were compared between WT and TGR of the same line by t-test. Blood pressure levels in male and female rats were compared by two way analysis of variance. Data are reported as mean ± SEM. Statistical significance was accepted at P<0.05.

Generation of TGR and transgene expression in various tissues
Independent microinjection experiments resulted in four lines of TGR. The lines were termed TGR276, TGR284, TGR294 and TGR307. The latter two lines (TGR294 and TGR307) reproduced well, whereas reproduction was limited in TGR276 and TGR284. TGR294 and TGR307 could therefore be investigated in more detail than TGR276 and TGR284. Transgene expression was detected by means of RT-PCR in a number of tissues including kidney, adrenal gland, heart, brain, liver and spleen (

Intracellular renin activities in the heart
Cardiac renin levels were low, approaching the detection limit in three out of the four lines investigated, including both lines of WT controls and TGR294 (Figs. 1B and 2). In TGR307, cardiac renin activity was fivefold higher than in the appropriate WT control line (Fig. 1B). More detailed analyses revealed that cardiac renin activity in TGR307 was elevated predominantly in the cytosolic fraction, but also in the 200 x g fraction, representing the nuclei together with some mitochondria and cytosol (Fig. 2C). There were no differences between TGR307 and their WT controls with respect to renin activities in the low density fractions (3000 x g and 15,000 x g ) where secretory renin is expected. Cardiac prorenin (trypsin-activatable inactive renin) levels were increased in the 200 x g fraction of TGR294 (Fig. 2B) and in the cytosol of TGR307 (Fig. 2D) compared to their respective WT controls. Renin levels in the 200 x g fraction of TGR307 and prorenin levels in the 200 x g fractions of TGR307 and TGR294 were significantly higher than in the 1000 x g fraction. Thus, there was a specific enrichment in the 200 x g fraction and contamination from the fractions of lower density, 3000 x g and 15,000 x g (Fig. 2B-D) was excluded.

Intracellular renin activities in the adrenal gland
Adrenal renin activities were considerably higher than cardiac renin activities and considerably above the detection limit in all groups (Fig. 3B). TGR294 showed decreased and TGR307 normal adrenal renin activities when compared to their respective WT controls. To investigate the intracellular distribution of renin, we calculated the enrichment of renin in sub-cellular fractions normalized to renin activities in the total homogenates (Fig. 3C). There was significant enrichment of renin activities in the cytosol and the high-density fraction (200 x g) of TGR294 compared with their WT controls. Furthermore, there was less enrichment of renin activities in the low density fractions (3000 x g, 15,000 x g) of TGR294 than in the low-density fractions of WT controls (each P<0.05, Fig. 3C). Renin activities were similar in sub-cellular fractions of TGR307 and their WT controls. We failed to detect prorenin in adrenal tissue in any of the groups (not shown).

Exon(2-9)renin and the adrenal gland
In the rat adrenal gland both the exon(1-9)renin and the exon(1A-9) renin transcripts are expressed. It has been suggested that there is a functionally active adrenal RAS in the zona glomerulosa. This assumption was based on the fact that under some conditions, such as potassium load or bilateral nephrectomy, aldosterone levels correlate better with adrenal renin levels than with circulating renin levels [22][23][24]. The functional significance of intra-adrenal renin has been demonstrated in vitro and in vivo (for review see) [24,25]. Thus, in adrenal explant cultures or adrenal monolayers the potassium-stimulated aldosterone production is reduced by  [26][27][28][29]. In vivo, the AT1 receptor antagonist losartan inhibits aldosterone production after bilateral nephrectomy [30], a procedure which eliminates circulating renin, but increases renin in the adrenal cortex [22]. There is now considerable evidence that there is a local secretory renin system in the adrenal cortex, which regulates aldosterone production [24,25,31].
Taken together, the results obtained in four different lines of exon (2)(3)(4)(5)(6)(7)(8)(9)renin TGR collectively demonstrate that the protein encoded by the exon(1A-9)renin transcript (1) is translated into a functionally active intracellular protein; (2) is targeted to the cytoplasm and a high-density fraction rather than being sorted to the secretory pathways and (3) is functionally active, regulating aldosterone production. The CX-(exon2-9)renin transgenic rat appears to be a useful model to study the role and the mechanisms of action of cytoplasmatic renin derived from exon(1A-9) transcripts.