O objetivo foi determinar se aldosterona e corticosterona agem sobre a acidificação do túbulo proximal e se esses efeitos são genômicos e/ou não-genômicos. A reabsorção de HCO3- foi avaliada por microperfusão estacionária. Aldosterona e corticosterona perfundidas na luz tubular causaram aumento significante do JHCO3-. Na presença de etanol, actinomicina D, cicloheximida ou espironolactona, o JHCO3- foi estatisticamente igual ao valor controle (2,84 ± 0,079 nmol.cm-2.s-1). RU486 sozinho inibiu o efeito estimulador da aldosterona e corticosterona. Losartan não alterou o JHCO3-. Concanomicina ou S3226 diminuiram o efeito estimulador da corticosterona. A aldosterona perfundida nos capilares peritubulares aumentou o JHCO3-. Assim, a aldosterona e corticosterona tem um efeito rápido, não-genômico, estimulante do JHCO3-, provavelmente com a participação do GR e pela ativação do NH3 e da H+-ATPase luminais. Além disto, a aldosterona e corticosterona endógenas estimulam o JHCO3- no túbulo proximal. / The purpose was to determine if aldosterone and corticosterone act on the acidification of proximal tubule and if these hormonal effects are genomic and/or nongenomic. Bicarbonate reabsorption was evaluated by microperfusion. Aldosterone and corticosterone caused a significant increase in JHCO3-. In the presence of ethanol, actinomycin D, cycloheximide or espironolactone, the JHCO3- was not different from the control value (2.84 ± 0.079 nmol.cm-2.s-1). However, in the presence of RU486 a decrease on JHCO3- was observed. Losartan inhibited the JHCO3-. Concanamicyn or S3226 decreased the stimulatory effect of corticosterone. Aldosterone perfused into peritubular capillaries also increased JHCO3-. Our results indicate that: aldosterone and corticosterone has a rapid, nongenomic, stimulatory effect on JHCO3-; probably, GR participates in this process and; this effect, probably, occurs by activation of luminal NH3 and H+-ATPase. Besides, endogenous aldosterone and corticosterone stimulate JHCO3-.

Os experimentos indicam, como já demonstrado em estudos prévios do laboratório, que angiotensina II (Ang II) apresenta efeito estimulatório sobre a cinética de recuperação de pHi em células OKP. Tal estímulo não é acentuado pela super-expressão de AT1aR recombinante, ao contrário do que imaginávamos inicialmente. Acreditamos que, por conta da capacidade de amplificação de sinal característica dos receptores acoplados a proteína G, um aumento de expressão do receptor AT1aR em relação ao nível endógeno seja redundante para o fenômeno biológico estudado. Por outro lado, os resultados para o grupo com super-expressão de ATRAP corroboram nossa hipótese inicial, ao indicar uma atenuação do efeito de Ang II sobre a recuperação de pHi, em comparação aos demais grupos experimentais tratados com Ang II. Considerando que a recuperação de pHi em células OKP reflete essencialmente a atividade de troca Na+/H+ mediada pelo contra-transportador NHE3, podemos concluir que a regulação positiva de NHE3 via AT1aR/AngII é prejudicada pelo aumento de expressão da proteína ATRAP. / The experimental data suggests that, as shown in previous works from our laboratory, angiotensin II (Ang II) raises the pHi recovery rate in OKP cells. This upregulation is not enhanced by recombinant AT1aR overexpression, contrary to our initial hypothesis. We believe that, due to signal amplification mediated by G-protein coupled receptors, any increase in AT1aR would be redundant considering the biological phenomenon of interest. On the other hand, results from the ATRAP overexpression group supports our initial hypothesis, pointing an attenuated effect of Ang II over pHi recovery in relation to the remaining groups treated with Ang II. Considering that pHi recovery in OKP cells primarily reflects the Na+/H+ exchange activity mediated by NHE3 antiporter, we can conclude that NHE3 upregulation mediated by AT1aR/AngII is impaired by an increase in ATRAP protein expression.

O objetivo do presente trabalho foi analisar o papel do ANP na ação não genômica da Aldosterona sobre o trocador Na+/H+ no segmento S3 do túbulo proximal de rato, isolado, in vitro. Os resultados indicam que o pHi basal do segmento S3 proximal de ratos é 7.20 + ou - 0.009 (n = 47/209). O valor médio da velocidade de extrusão celular de H+ na condição controle é de 0.195 + ou - 0.012 pHi/min (n = 16/96). Os dados confirmam que a aldosterona apresenta um efeito bifásico sobre o NHE1: em baixas doses (10-12 M) o estimula, enquanto que em altas doses (10-6M), o inibe. O ANP (10-6 M) não possui efeito sobre o NHE1; contudo, o ANP previne ambos os efeitos da aldosterona sobre esse trocador. O valor médio da concentração do cálcio no citosol ([Ca2+]i) na condição controle é 100 ± 1 (n = 5) hM Adicionalmente, nossos estudos mostram que o ANP diminui a [Ca2+]i e inibe o efeito estimulatório de ambas as doses de aldosterona sobre esse parâmetro. / The effects of aldosterone and ANP(2 min preincubation) on the intracellular pH recovery rate (pHirr) after the acid load induced by NH4Cl and on the [Ca2+]i were investigated in isolated rat S3 segment. The basal pHi was 7.20 + ou - 0.009(n=47/209) and the basal pHirr via the Na+/H+ exchanger was 0.195 + ou - 0.012 pHi/min(n=16/96). Aldosterone(10-12M) caused an increase in the pHirr, but aldosterone(10-6M) decreased it. ANP(10-6M) alone or plus aldosterone(10-12 or 10-6 M) had no effect on pHirr. The basal [Ca2+]i was 100 + ou - 1(n=5)hM. After 1 min of Aldosterone pi there was a transient and dose-dependent increase of the [Ca2+]i and after 6 min pi there was a new increase of [Ca2+]i. ANP alone decreased the [Ca2+]i and prevented the stimulatory effects of aldosterone(10-12 or 10-6M) on this parameter. The data indicate a nongenomic action of aldosterone and ANP on the Na+/H+ exchanger and on [Ca2+]i and are compatible with stimulation of the this exchanger by increases in [Ca2+]i in the lower range (at10-12M aldosterone) and inhibition by increases at high levels (at10-6M aldosterone).

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Le récepteur du facteur de libération de l’hormone de croissance (GHRHR) est un récepteur de la famille des récepteurs couplés aux protéines G. Il est fortement exprimé dans les cellules somatotropes de l’hypophyse antérieure de plusieurs mammifères. Ce récepteur exerce un rôle primordial dans la stimulation de la synthèse et de la sécrétion de l’hormone de croissance ainsi que dans la prolifération des somatotropes. Au niveau extrahypophysaire, les niveaux les plus élevés d’ARNm du GHRHR se retrouvent dans le rein. Toutefois, aucune analyse immunohistochimique n’existe encore sur la localisation précise et la quantification sur les niveaux de GHRHR dans les différents segments du rein de rat et sa dynamique d’expression en situation normale et pathologique telle que l’ischémie. De plus, dans le rein humain normal, aucune information n’est présentement disponible. Le premier article de ce mémoire a pour objectif d’identifier, par immunofluorescence directe, la localisation du GHRHR à travers le système tubulaire rénal, chez le rat jeune en bonne santé. Nos résultats mettent en évidence que dans le rein de rat sain, le GHRHR est exprimé dans les cellules du tubule proximal contourné et droit, de l’anse de Henlé ascendante épaisse corticale et médullaire et de l’anse de Henlé ascendante mince. Le cortex et la bande externe de la médulla externe seraient les deux régions où l’expression est la plus élevée. À la suite d’une insulte rénale comme l’ischémie-reperfusion (IR) chaude, nos résultats démontrent que l’expression du GHRHR est régulée à la baisse dans ces mêmes régions. De plus, une augmentation de certains marqueurs de détérioration cellulaire est présente comme l’enzyme initiatrice, la caspase-9 clivée et effectrice (caspase-3 clivée), des fragments d’ADN et la surexpression d’indication d’injure tissulaire comme la protéine Kidney Injury Molecule 1 (KIM-1). L’ensemble de ces résultats ouvre plusieurs pistes d’études concernant l’importance du GHRHR en rénoprotection. Le deuxième article de ce mémoire a pour objectif d’identifier, par immunofluorescence directe, la localisation du GHRHR à travers le système tubulaire rénal humain. Nos résultats suggèrent que dans le rein humain sain, le GHRHR est davantage exprimé dans le cortex, plus précisément, au niveau du tubule proximal droit et contourné et l’anse de Henlé corticale ascendante épaisse. L’expression du GHRHR est aussi notable au niveau de la région médullaire, pour être plus spécifique, au niveau de l’anse de Henlé médullaire ascendante épaisse et de la médulla. Ainsi, comme chez le rat, l’expression du GHRHR rénal est régio-spécifique. Finalement, le troisième article de ce mémoire est une revue de la littérature ayant pour but d’établir un lien entre les mécanismes connus du stress oxydant dans un contexte d’IR rénale et son impact spécifique dans la médulla. Cet article met en évidence que les différents segments du rein réagissent différemment à une agression oxydante et que la médulla est la région la plus vulnérable. De plus, cette revue de la littérature souligne que les différents types de mécanismes connus du stress oxydant dans un contexte d’IR rénale, tel que la production de dérivés réactifs de l’oxygène, ciblent principalement deux structures du néphron : le tubule proximal et l’anse de Henlé ascendante épaisse médullaire. Les principales répercussions de ces mécanismes observables sont l’inflammation, l’apoptose cellulaire et la diminution des fonctions rénales. Ces mécanismes peuvent aussi être utilisés comme un outil de diagnostic ou de détermination de la santé de l’organe. / The growth hormone-releasing factor receptor (GHRHR) is a receptor of the family of G- protein-coupled receptors. It is highly expressed in the somatotropic cells of the anterior pituitary of several mammals. This receptor plays an essential role in the stimulation of the synthesis and secretion of growth hormone as well as in the proliferation of somatotrophs. At the extra- pituitary level, the highest levels of GHRHR mRNA are found in the kidney. However, no immunohistochemical analysis yet exists on precise localization and quantification of GHRHR levels in the different segments of the rat kidney and its expression dynamics in normal and pathological situations such as ischemia. Additionally, in the normal human kidney, no information is currently available. The first article of this thesis aims to identify, by direct immunofluorescence, the localization of the GHRHR through the renal tubular system, in young healthy rats. Our results show that in the healthy rat kidney, GHRHR is expressed in the cells of the convoluted and right proximal tubule, of the cortical and medullary thick ascending loop of Henle and of the thin ascending loop of Henle. The cortex and the outer band of the outer medulla would be the two regions where the expression is the highest. Following a renal insult such as warm ischemia-reperfusion (RI), our results demonstrate that GHRHR expression is down-regulated in these same regions. In addition, an increase in certain markers of cellular damage is present including initiating enzymes, cleaved and effector caspase-9 (cleaved caspase-3), DNA fragments and overexpression indicative of tissue injury such as protein Kidney Injury Molecule 1 (KIM-1). All these results open up several avenues of study concerning the importance of GHRHR in renoprotection. The second article of this thesis aims to identify, by direct immunofluorescence, the localization of the GHRHR through the human renal tubular system. Our results suggest that in the healthy human kidney, the GHRHR is more expressed in the cortex, more precisely at the level of the right, convoluted proximal tubule and the thick ascending cortical loop of Henle. The expression of GHRHR is also appreciable at the level of the medullary region, more precisely at the level of the thick ascending medullary loop of Henle, and the medulla. Thus, as in rats, the expression of renal GHRHR is region specific. Finally, the third article of this thesis is a review of the literature aimed at establishing a link between the known mechanisms of oxidative stress in the context of renal IR and its specific impact in the medulla. This article highlights that the different segments of the kidney react differently to an oxidative attack and that the medulla is the most vulnerable region. In addition, this review of the literature underlines that the different types of known mechanisms of oxidative stress in a context of renal IR, such as the production of reactive oxygen species, mainly target two structures of the nephron: the proximal tubule and the thick ascending loop of Henle. The main repercussions of these observable mechanisms are inflammation, cellular apoptosis and reduced renal function. These mechanisms can also be used as a diagnostic tool or to determine the health of the organ.

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