The aim of this study was to determinate the gene expression levels of angiotensinogen, angiotensin converting enzyme, renin, (pro)renin receptor, and the final rennin-angiotensin system (RAS) products Angiotensin (Ang) II and Ang 1-7 inthe remnant kidney of 5/6 nephrectomized rats and its response to RAS pharmacological blockade. Male Wistar rats were divided into five groups: sham operated (SO), 5/6 nephrectomized (NFX), NFX + captopril (50 mg/ kg/day), NFX + losartan (10 mg/kg/day), and NFX + aliskiren (10 mg/kg/day). Animals were followed up for 60 days and protein urine excretion was measured. Systolic blood pressure, renal tissue RAS mRNA expression levels, plasma Ang II, and plasma Ang 1-7 were evaluated at day 60 after nephrectomy. Blood pressure and urine protein were increased after 5/6 nephrectomy. Ang II levels were increased 9.4 fold, whereas Ang 1-7 decreased 72.9% in NFX animals compared with SO rats. 5/6 nephrectomy increased renal angiotensinogen and (pro)renin receptor mRNA expression but down-regulated renin mRNA expression. RAS blockade restored the systolic blood pressure to normal values and slowed down urinary protein excretion, and also prevented changes in Ang II and Ang 1-7. RAS blockade reduced (pro)renin receptor, ACE, and AGT mRNA expression in the remnant kidney. However, renin mRNA expression increased compared with NFX rats. In conclusion these results suggest that inhibition of Ang II synthesis by RAS blockade is associated with renal regulation of RAS mRNA expression and this may be through a mechanism related with the Ang II/Ang 1-7 balance.
The Renin Angiotensin System (RAS) is one of the most phylogenetically conserved hormonal systems among vertebrates [
All animal procedures were conducted in accordance with Federal Regulations for animal experimentation and Care and were approved by the Animal Care Committee of CINVESTAV (CINVESTAV Zacatenco, D.F., México). Two months old male Wistar rats were housed in humidity and temperature controlled room with a 12-h light/ dark cycle. Rats were fed with a regular diet (LabDiet, USA) and had free access to tap water. The 5/6 nephrectomy was achieved by removal of the right kidney and infarction of two thirds of the left kidney as previously reported [
Rats were housed on metabolic cages 3 days prior urine collection. Consumed water and urine volume of 24 hours were measured. The urine was collected 14, 28, 42, and 56 days after surgery. Total protein was determined according to Bradford’s method [
At day 60 after surgery, blood pressure was measured by carotid artery cannulation as previously reported [
After SBP was obtained, blood was collected on tubes with EDTA (Vacutainer®) and centrifuged at 3000 g for 10 minutes at room temperature; plasma was stored at −70˚C. Kidneys were removed and immediately frozen in liquid nitrogen and stored at −70˚C for subsequent use.
Simultaneous detection and quantification of Ang II and Ang 1-7 were performed by capillary zone electrophoresis with UV detection by photodiode-array (CZEPDA) [
Total RNA was extracted from the kidneys using TRIZOL reagent (Invitrogen, USA), 5 μg of total RNA was reverse transcribed into cDNA in a total reaction mixture volume of 20 µL using the MMLV reverse transcriptase (Invitrogen, USA) according to the manufacturer’s instructions. To perform the real time quantitative PCR (Q-PCR) we used commercially available gene expression assays (Taqman®, Applied BiosystemsTM, USA) for AGT (Rn00593114_m1), REN (Rn00561847_m1), ACE (Rn00561094_m1), (pro)REN receptor (ATP6ap2, Rn01430718_m1), and 18S rRNA as endogenous control. Amplification was performed using the StepOneTM RealTime PCR system (Applied BiosystemTM). Q-PCR protocol was set up according to the manufacturer’s instructions. Each mRNA target was relative quantified by measuring the threshold cycle. Analysis of relative gene expression was performed using the ΔΔCT method [
Comparisons among the different treated groups were analyzed by ANOVA followed by Newman-Keuls test. Data are expressed as mean ± SEM, p < 0.05 was defined as statistically significant.
As we previously showed [
Plasma Ang II level was increased 9.7 fold, whereas Ang 1-7 was reduced ~72.9% in NFX animals compared with SO rats after 60 days of surgery (p = 0.001). RAS blockade clearly prevented the increase on Ang II
caused by 5/6 nephrectomy (
The RAS mRNA expression profiles were altered in the remnant kidney from NFX animals compared with SO rats, 60 days after surgery. AGT and (P)RR mRNA expression were increased 1.82 fold (p = 0.023) and 2.16 fold (p = 0.008) respectively, whereas REN mRNA ex-
pression was reduced 82.9% (p < 0.05). ACE mRNA expression was not affected by the 5/6 nephrectomy. RAS blockade also modified the renal RAS mRNA expression. Inhibition of REN (aliskiren), and ACE (captopril), or AT1 receptor blockade (losartan) in NFX rats reduced the mRNA expression of (P)RR, AGT, and ACE (p < 0.05) whereas REN mRNA expression was increased compared with NFX rats without treatment (p < 0.05) (
The 5/6 nephrectomy model is a classic animal representation of Ang II-dependent progressive renal impairment and hypertension. We demonstrated, in this study, that 5/6 nephrectomy modifies plasma Ang II and Ang 1-7 concentrations, as well as renal RAS mRNA expression. Moreover, RAS blockade was associated with restoration of plasma Ang ll and Ang 1-7 concentrations along with changes in renal RAS mRNA expression. Animals that underwent 5/6 nephrectomy developed high blood pressure and proteinuria after 60 days in comparison with the SO animals. As expected, RAS blockade with either AT1 receptor blocker (losartan), ACE (captopril) or renin (aliskiren) inhibitors was associated with normalization of SBP values after 60 days treatment post nephrectomy. Urine protein excretion, as kidney function indicator, showed that proteinuria in NFX rats was progressively elevated. Additionally, AT1 receptor blockade (losartan) and ACE inhibition (captopril) prevented the increment of urine protein excretion on NFX animals since day 28 after 5/6 nephrectomy. Our findings corroborate previous reports that showed the association of RAS blockade with normalization of blood pressure and kidney damage reduction [15,18]. Interestingly, decreased urine protein excretion was observed from day 14 after nephrectomy in animals treated with the REN inhibitor aliskiren, suggesting an early effect on kidney protection. REN inhibition in NFX animals showed a sustained reduction on proteinuria through this study. This efficient renoprotective effect by direct renin inhibition has been also reported by others [
Plasma Ang II was elevated in NFX animals whereas Ang 1-7 decreased 72.9% compared with SO rats. Previous reports that support our data have shown similar changes during renal damage [
RAS genes. Additionally, depletion of Ang II with ACE inhibitors, AT1 receptor blockers, or REN inhibitors modify renal RAS mRNA expression and further corroborate that Ang II levels are critical in the regulation of RAS mRNA intermediaries. The alterations in Ang 1-7 concentration in NFX rats suggest that Ang 1-7 may play a major role in the regulation of RAS mRNA expression during renal damage development. Other studies have shown the impact of Ang 1-7 on RAS genes expression in the kidney [25,26]. Our data might suggest that reduced Ang 1-7 levels could be associated with RAS over-activation via increased renal genes expression, whereas restoration of the Ang 1-7 by ACE inhibition, AT1 receptor blockade, and REN inhibition may participate in down-activation by regulation of RAS mRNA genes. Thus, it is possible that the two main RAS active peptides, Ang II and Ang 1-7 play an important role in the RAS gene mRNA autoregulation, Ang II stimulates and Ang 1-7 inhibits. Indeed, it has been reported that Ang 1-7 may antagonize the actions of Ang II especially in situations of an overactive RAS [5,25]. This heptapeptide interacts with the G protein-coupled recaptor Mas and exerts many actions that counteract those elicited by Ang II eg, vasodilation, improved renal blood flow, and enhanced pressure natriuresis. It also has antiproliferative actions on vascular smooth muscle and on cardiac myocytes stimulates nitric oxide and prostaglandin release [
All these data lead us to conclude that renal regulation of RAS mRNA expression is related with the Ang II/Ang 1-7 balance and that pharmacological blockade of the RAS pathway can be also associated with the regulation of renal RAS genes expression. These findings expand the view of RAS interactions within its own components and may contribute to elucidate new therapeutic responses.
This work was supported by the Mexican Council of Science and Technology (CONACYT), Research grant 81359 to Dr. Escalante and a CONACYT fellowship to Erika Cruz. We thank M.S. Azzael Hernández-Morán for technical assistance.