All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed SMG rennin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue- specific expression. In an effort to understand the molecular basis for this differential expression, a detailed analysis of the transcripts originating from these loci was undertaken. S1 analysis of SMG and kidney RNA populations indicated that the majority of transcripts initiate at one major site on Ren-1 and Ren-2. Interestingly a minor fraction of transcripts in the SMG initiate at two upstream sites. These transcripts encode an upstream ORF which is in frame with that of the renin precursor. The precise tissue-specificities of Ren-1 and Ren-2 were also examined: in the kidney, SMG, and also in several extrarenal tissues, since there is increasing evidence of renin expression in a number of extrarenal sites. To distinguish between the two highly homologous transcripts, an assay was developed exploiting established base sequence differences between Ren-1 and Ren-2 mRNA's by extension of a primer downstream of such a base difference in the presence of the appropriate ddNTP. Using this assay, Ren-1 and Ren-2 were found to be equally well expressed in the kidney, whereas in the SMG only Ren-2 is efficiently expressed. Interestingly in the other extrarenal tissues examined, testis, liver and heart, it is the Ren-1 allele that is preferentially expressed. The assay was also able to demonstrate the similarity in response of Ren-l/Ren-2 to certain physiological stimuli, such as sodium depletion. Thus, this study of the regulation of mouse renin gene expression has demonstrated further striking differences in the tissue-specific expression of Ren-l/Ren-2, and added to the increasingly compelling evidence of extrarenal renin gene expression.

The renin-angiotensin-aldosterone system (RAAS) plays an essential role in maintaining plasma volume and arterial blood pressure by regulating angiotensin II levels. Dysregulation of the RAAS can result from an underlying disorder that results in a severe and untreatable form of hypertension, known as secondary hypertension. Measurement of plasma renin activity is a commonly employed method of diagnosing secondary hypertension. Plasma renin activity is quantified by determining the amount of angiotensin I generated through the enzymatic cleavage of angiotensinogen by renin. Radioimmunoassay is routinely used to measure plasma renin activity, however there are limitations to the method. With the prevalence of hypertension on the rise, there is need for a more accurate and rapid method of assessing the RAAS for diagnostic purposes and therapeutic monitoring. Multiplexed measurement of angiotensin I and angiotensin II would provide comprehensive understanding of the RAAS by determining dysregulation in the production of either molecule. In this thesis, the relationship between endogenous angiotensin I concentrations and plasma renin activity are studied in order to examine the research hypothesis that measurement of angiotensin I concentration correlates with plasma renin activity and whether this may provide a more accurate and rapid method of screening for hypertension when multiplexed with angiotensin II. To overcome the current limitations of radioimmunoassay for measuring plasma renin activity, a mass spectrometric-based method was developed to measure angiotensin I and plasma renin activity. Evaluation of the assay against radioimmunoassay demonstrates that the assay is reproducible and provides a linear response over a diagnostically relevant concentration range. Comparison of endogenous levels of angiotensin I with normal plasma renin activity levels show a correlation in this study (R=0.74). Comparison of plasma renin activity values by radioimmunoassay and iMALDI also show correlation (R=0.98), indicating that the iMALDI assay may provide an improved method for diagnosing secondary hypertension.

O aporte de nutrientes ingerido pela mãe durante a gestação e o transporte dos nutrientes da placenta para o feto são essenciais para o crescimento fetal. A sobrecarga de sódio durante a gestação tem demonstrado ser um dos fatores responsáveis pela hipertensão na vida adulta da prole. Um estudo anterior em nosso laboratório demonstrou que além do aumento da pressão arterial, o sistema renina angiotensina (SRA) da prole de mães que receberam sobrecarga de sódio durante a gestação e lactação, não respondeu adequadamente em resposta a um teste com dieta hipersódica. O SRA é importante para a homeostase e a manutenção de eletrólitos no organismo. As enzimas ciclooxigenase-2 (COX-2) e óxido nítrico sintase neuronal (nNOS) interagem com o SRA. O objetivo deste estudo foi avaliar o efeito da sobrecarga de sódio durante a gestação no SRA renal e sistêmico, COX-2 e nNOS da prole ao nascimento e na idade adulta. O grupo materno também foi investigado. Para tanto, ratas Wistar foram alimentadas com dieta normossódica (NS) ou hipersódica (HS) durante a gestação. Foram divididos em dois grupos: no primeiro grupo, foram estudados a mãe e o recém-nascido e no segundo grupo, a dieta foi trocada para NS ao nascimento independente da dieta materna e a prole masculina avaliada com 12 semanas de vida. A pressão arterial caudal (tcBP) foi medida na prole adulta e no grupo materno. Atividade da renina renal e plasmática, aldosterona sérica, expressão gênica da renina renal, COX-2, nNOS foram analisadas no rim da mãe, recém-nascido e na prole adulta avaliada após a administração de dieta NS, hipossódica (LS) ou hipersódica (HS) por 7 dias. A dieta hipersódica durante a gestação induziu: 1-) no grupo materno: maior pressão arterial e expressão protéica da COX-2 na medula renal, e diminuição da atividade da renina plasmática e renal e a aldosterona sérica; 2-) no grupo neonato: menor expressão do mRNA da COX-2 mRNA e maior expressão protéica de nNOS nos rins, e nenhuma diferença foi observada na aldosterona sérica, na atividade plasmática, renal, e expressão gênica da renina renal 3-) na prole adulta: a menor expressão gênica do mRNA de renina, atividade da renina plasmática e aldosterona sérica, maior atividade da renina renal, maior expressão gênica e protéica da COX-2 na medula renal e menor expressão gênica da nNOS no córtex renal. Foi observada uma resposta exacerbada da atividade da renina plasmática e renal após a administração das dietas HO ou HR. Em conclusão, a sobrecarga de sódio materna induz a ativação do SRA e da COX-2 na medula renal na prole adulta, tornando-as mais sensíveis ao sal. Essas características foram herdadas de fenótipo materno / Maternal nutrient intake and its transportation from the placenta to the fetus are essential for fetal growth during pregnancy. The sodium overload during pregnancy has been shown to be one of the factors responsible for hypertension and disturbance of renin angiotensin system (RAS) in adulthood offspring. The RAS is important for homeostasis and maintenance of electrolytes in the body. The enzymes cyclooxygenase-2 (COX-2) and neuronal nitric oxide synthase (nNOS) interact with RAS, especially in renin release. The aim of this study was to evaluate effects of maternal sodium overload during pregnancy on renal and systemic RAS, COX-2 and nNOS of offspring at birth and in adult age. A mother group was also investigated. For this, female Wistar rats were fed normossódica (NS) or hipersódica (HS) diet during pregnancy. It was divided in two groups: as soon as offspring´s birth one group was euthanized and studied the mother and new born. In second group, the diet was changed to NS diet independent of maternal diet after mother delivered and male offspring was studied at twelve weeks old. The tail cuff blood pressure (tcBP) of offspring and maternal group were evaluated. Plasma and renal renin activity, plasma aldosterone, gene expression of renal renin, COX-2, nNOS of mother, newborn and adult offspring were evaluated. The adult offspring RAS was evaluated after fed NS (control), low sodium (LS) or high sodium (HS) for 7 days. The high salt diet during pregnancy induced in 1) mother group: increased blood pressure and protein expression of COX-2 in renal medulla, and decreased plasma and renal renin activity and serum aldosterone. 2) newborn group: decreased gene expression of COX-2 mRNA and increased protein expression of nNOS in the kidney, and any difference was observed in serum aldosterone and plasma or renal renin activity or gene expression 3) adult offspring group: reduced gene expression of renin mRNA, plasma renin activity and serum aldosterone and increased renal renin. Also, increased gene and protein expression of COX-2 in the renal medulla and decreased nNOS gene expression in renal cortex. An exacerbated response of plasma or renal renin activity was observed after RAS stimulation or inhibition. In conclusion the maternal sodium overload induces an exacerbated response of the RAS and COX-2 activation in renal medulla in adult offspring. These characteristics were inherited from maternal phenotype

O aporte de nutrientes ingerido pela mãe durante a gestação e o transporte dos nutrientes da placenta para o feto são essenciais para o crescimento fetal. A sobrecarga de sódio durante a gestação tem demonstrado ser um dos fatores responsáveis pela hipertensão na vida adulta da prole. Um estudo anterior em nosso laboratório demonstrou que além do aumento da pressão arterial, o sistema renina angiotensina (SRA) da prole de mães que receberam sobrecarga de sódio durante a gestação e lactação, não respondeu adequadamente em resposta a um teste com dieta hipersódica. O SRA é importante para a homeostase e a manutenção de eletrólitos no organismo. As enzimas ciclooxigenase-2 (COX-2) e óxido nítrico sintase neuronal (nNOS) interagem com o SRA. O objetivo deste estudo foi avaliar o efeito da sobrecarga de sódio durante a gestação no SRA renal e sistêmico, COX-2 e nNOS da prole ao nascimento e na idade adulta. O grupo materno também foi investigado. Para tanto, ratas Wistar foram alimentadas com dieta normossódica (NS) ou hipersódica (HS) durante a gestação. Foram divididos em dois grupos: no primeiro grupo, foram estudados a mãe e o recém-nascido e no segundo grupo, a dieta foi trocada para NS ao nascimento independente da dieta materna e a prole masculina avaliada com 12 semanas de vida. A pressão arterial caudal (tcBP) foi medida na prole adulta e no grupo materno. Atividade da renina renal e plasmática, aldosterona sérica, expressão gênica da renina renal, COX-2, nNOS foram analisadas no rim da mãe, recém-nascido e na prole adulta avaliada após a administração de dieta NS, hipossódica (LS) ou hipersódica (HS) por 7 dias. A dieta hipersódica durante a gestação induziu: 1-) no grupo materno: maior pressão arterial e expressão protéica da COX-2 na medula renal, e diminuição da atividade da renina plasmática e renal e a aldosterona sérica; 2-) no grupo neonato: menor expressão do mRNA da COX-2 mRNA e maior expressão protéica de nNOS nos rins, e nenhuma diferença foi observada na aldosterona sérica, na atividade plasmática, renal, e expressão gênica da renina renal 3-) na prole adulta: a menor expressão gênica do mRNA de renina, atividade da renina plasmática e aldosterona sérica, maior atividade da renina renal, maior expressão gênica e protéica da COX-2 na medula renal e menor expressão gênica da nNOS no córtex renal. Foi observada uma resposta exacerbada da atividade da renina plasmática e renal após a administração das dietas HO ou HR. Em conclusão, a sobrecarga de sódio materna induz a ativação do SRA e da COX-2 na medula renal na prole adulta, tornando-as mais sensíveis ao sal. Essas características foram herdadas de fenótipo materno / Maternal nutrient intake and its transportation from the placenta to the fetus are essential for fetal growth during pregnancy. The sodium overload during pregnancy has been shown to be one of the factors responsible for hypertension and disturbance of renin angiotensin system (RAS) in adulthood offspring. The RAS is important for homeostasis and maintenance of electrolytes in the body. The enzymes cyclooxygenase-2 (COX-2) and neuronal nitric oxide synthase (nNOS) interact with RAS, especially in renin release. The aim of this study was to evaluate effects of maternal sodium overload during pregnancy on renal and systemic RAS, COX-2 and nNOS of offspring at birth and in adult age. A mother group was also investigated. For this, female Wistar rats were fed normossódica (NS) or hipersódica (HS) diet during pregnancy. It was divided in two groups: as soon as offspring´s birth one group was euthanized and studied the mother and new born. In second group, the diet was changed to NS diet independent of maternal diet after mother delivered and male offspring was studied at twelve weeks old. The tail cuff blood pressure (tcBP) of offspring and maternal group were evaluated. Plasma and renal renin activity, plasma aldosterone, gene expression of renal renin, COX-2, nNOS of mother, newborn and adult offspring were evaluated. The adult offspring RAS was evaluated after fed NS (control), low sodium (LS) or high sodium (HS) for 7 days. The high salt diet during pregnancy induced in 1) mother group: increased blood pressure and protein expression of COX-2 in renal medulla, and decreased plasma and renal renin activity and serum aldosterone. 2) newborn group: decreased gene expression of COX-2 mRNA and increased protein expression of nNOS in the kidney, and any difference was observed in serum aldosterone and plasma or renal renin activity or gene expression 3) adult offspring group: reduced gene expression of renin mRNA, plasma renin activity and serum aldosterone and increased renal renin. Also, increased gene and protein expression of COX-2 in the renal medulla and decreased nNOS gene expression in renal cortex. An exacerbated response of plasma or renal renin activity was observed after RAS stimulation or inhibition. In conclusion the maternal sodium overload induces an exacerbated response of the RAS and COX-2 activation in renal medulla in adult offspring. These characteristics were inherited from maternal phenotype