Tripeptides isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP) act as ACE inhibitors in vitro. Double transgenic rats (dTGR) harbouring human renin and human angiotensinogen genes develop malignant hypertension due to increased angiotensin II formation. The present study was aimed to evaluate possible antihypertensive effect of IPP and VPP in this severe model. Four-week-old dTGR were randomized in three groups to receive: (1) water (control), (2) fermented milk containing IPP and VPP, and (3) IPP and VPP dissolved in water for three weeks. Fermented milk, but not peptides in water, attenuated the development of hypertension in dTGR by 19 mmHg versus the control group (
Fermented milk products containing biologically active tripeptides, isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), have been shown to attenuate the development of hypertension in spontaneously hypertensive rats (SHRs) [
Despite these findings, the exact antihypertensive mechanisms behind IPP and VPP have not been confirmed. As described previously, double transgenic rats (dTGR) harbouring human renin and human angiotensinogen genes develop malignant hypertension, cardiac hypertrophy, renal damage, and endothelial dysfunction due to increased angiotensin II (Ang II) formation by the transfected human genes [
The aim of the present study was to evaluate the possible antihypertensive effect of fermented milk preparation containing IPP and VPP, and the pure peptides in drinking water, for the first time in this severe model of hypertension, dTGR, which overexpresses RAS. The role of ACE inhibitory activity of IPP and VPP, and their effects on hypertension related biochemical markers, were also studied.
The study protocol was approved by the Animal Experimentation Committee of the Institute of Biomedicine, Helsinki, Finland. Four-week-old body-weight-matched (
The concentration of the peptides in the drinking fluid was based on our previous experience on the fluid consumption of spontaneously hypertensive rats in similar experiments
Systolic blood pressure was measured by using a tail-cuff blood pressure analyser (IITC Life Science, Woodland Hills, CA, USA). Before measurement, the rats were kept at
Fluid consumption was monitored daily and feed consumption weekly. The energy nutrient and mineral contents in the fermented milk, as well as the IPP and VPP contents to control their preservation, were analysed by the peptide research group in Valio Ltd (Helsinki, Finland). The tripeptide content of
The mesenteric artery was cleaned of adherent connective tissue. Three-mm-long arterial rings were placed between stainless steel hooks and suspended in an organ bath chamber in Krebs-Ringer buffer (pH 7.4) of the following compositions (mM):
During weeks 1 and 3 of the treatment period, the rats were housed individually in metabolic cages for 24 hours. The consumption of drinking fluids and feed was measured and the intake of energy nutrients, minerals, and IPP and VPP was calculated during the 24-hour period (Table
Body weight, urine volume, and the intake of feed, fluids and minerals during the 24-hour period in metabolic cages at the end of the three-week treatment period (mean ± SE,
Variable | Control | Fermented milk | Peptide | Difference between the groups, |
---|---|---|---|---|
Body weight, g | 235 ± 13 | 221 ± 11 | 239 ± 7 | .53 |
Urine volume, mL | 33 ± 3 | 33 ± 4 | 44 ± 4 | .085 |
Feed intake, g | 26 ± 1 | 16 ± 1 | 26 ± 0.5 | <.001 |
Fluid intake, mL | 37 ± 4 | 24 ± 8 | 47 ± 4 | .032 |
Energy, kJ | 352 ± 15 | 285 ± 27 | 353 ± 7 | .014 |
Sodium, mg | 59 ±2 | 44 ± 3 | 60 ± 1 | <.001 |
Potassium, mg | 175 ± 7 | 188 ± 28 | 175 ± 3 | .76 |
Calcium, mg | 260 ± 11 | 213 ± 21 | 261 ± 5 | .025 |
Magnesium, mg | 51 ± 2 | 36 ± 2 | 52 ± 1 | <.001 |
At the end of the study, the rats were rendered unconscious with
Urinary albumin excretion was measured by ELISA, using rat albumin as a standard (Celltrend GmbH, Luckenwalde, Germany). Plasma renin activity (PRA) was measured using a radioimmunoassay of angiotensin I (Medix Angiotensin I test, Medix Biochemica, Kauniainen, Finland); aldosterone was measured by radioimmunoassay (Diagnostic Products Co, Los Angeles, CA, USA); and plasma B-type natriuretic peptide (BNP) was measured by radioimmunoassay as described in detail earlier [
For morphological studies, the renal samples were fixed in 4% buffered paraformaldehyde at room temperature, dehydrated in graded alcohol, and embedded in paraffin. Two-to-three-
The results are expressed as means with standard errors (SE). Statistically significant differences between the groups were tested by permutation type ANOVA with Hommel’s adjustment if appropriate. Permutation type test was used because there are a small number of observations in each group and assumptions underlying to corresponding parametric test cannot be relied on.
Body weight gain development was similar between the groups during the 3-week treatment (Table
The mean (
During the treatment period, six rats from the controls, two rats from the peptide group, and four rats from the fermented milk group died during nights and therefore could not be autopsied.
In daily measurements of the fluid intake, there were no differences between the groups and also the energy intake was similar between the groups (data not shown). This explains the similar body weight at the end of the experiment. The mean estimated daily peptide intake (IPP+VPP) during the whole study period was 5.4 mg/kg in the fermented milk group and 10.9 mg/kg in the peptide group. However, the 24-hour monitoring in metabolic cages, showed differences in the feed and fluid intake being lowest in the fermented milk group (Table
To test the ACE inhibitory activity of IPP and VPP in the in vitro bioassay, mesenteric artery rings of normotensive SD rats were used. IPP and VPP (0.33 mM) administered together inhibited 84% of the angiotensin I (1
The dTGRs showed marked cardiac and renal hypertrophy compared to the normotensive SD controls. Neither fermented milk nor peptides affected the heart or kidney weight to body weight ratios (Table
Biochemical variables and cardiac and renal hypertrophies at the end of the three-week treatment period (mean
Variable | Control | Fermented milk | Peptide | Difference between the groups, |
---|---|---|---|---|
Urinary albumin, mg/d | 22 | 35 | 30 | .20 |
PRA, ng Ang I/mL/h | 90 | 99 | 95 | .98 |
BNP, pmol/L | 3.1 | 2.3 | 3.5 | .24 |
Aldosterone, pg/mL | 1228 | 2551 | 1494 | .18 |
Cardiac hypertrophy index (mg/g) | 5.3 | 5.1 | 5.1 | .59 |
Renal hypertrophy index (mg/g) | 4.6 | 4.8 | 4.8 | .61 |
The arteriales of untreated dTGRs showed perivascular inflammation and concentric hypertrophy with medial and intimal proliferations. The glomeruli had notable mesangial expansion. The tubules were only minimally affected.
The damage score was 3.3
During the treatment period, all the dTGR groups developed severe albuminuria, compared to the healthy SD controls (
Aldosterone values tended to be highest in the fermented milk group. However, the difference did not reach statistical significance obviously due to large variations. There was no statistical difference between the groups in PRA or BNP (Table
The present study with double transgenic rats harbouring human renin and angiotensinogen genes aimed to evaluate the blood-pressure-lowering effect and its mechanisms of the two casein-derived tripeptides IPP and VPP in a fermented milk product. According to previous studies by us [
The difference from the control group was of the same magnitude (about 20 mmHg) as in our previous studies on SHRs [
One explanation for the fairly weak antihypertensive and lack of biochemical effects in the fermented milk group and the lack of effect in the peptide group may be the short treatment period, low dose of peptides, and the fast development of severe hypertension with death of the animals. In our earlier experimental [
The intake of tripeptides in the fermented milk group and the peptide group remained lower than in our earlier studies [
Biochemical determinations did not show clear differences between the groups in any of the markers. With this rat model, therefore, ACE inhibition does not seem to explain the blood pressure lowering effect in the fermented milk group. According to a previous study [
There are two major concerns in the study. Firstly, due to fast development of severe hypertension, part of the animals, mainly from the untreated (control) group, died during the intervention thus weakening the power of the study. Furthermore, from the same reason the intervention period remained shorter than that in our previous studies where the antihypertensive effect has been more evident [
The present study shows that the weak ACE inhibitory activity of IPP and VPP in drinking water is not able to prevent the severe hypertension in this rapidly developing hypertension model, although the antihypertensive effect is evident in SHRs. However, the peptides in fermented milk clearly antagonized the increase in blood pressure suggesting improved kinetics of the peptides in this product or other active components present.
This work is supported by the National Technology Agency (TEKES) Finland and The Academy of Finland. The authors wish to thank Elina Lausvaara for preparing the study products, Anneli von Behr, and Anne Hakala for expert technical assistance.