Zebrafish, Danio rerio, are able to survive exposure to extreme acidity (pH 4). Because previous studies demonstrated that disruption of ionic balance during exposure to acidic water is the major cause of mortality in acid-sensitive freshwater species, the focus of this thesis was to characterize the molecular mechanisms enabling zebrafish to maintain their Na+ homeostasis following exposure to acidic water. Initial findings (Chapter 2) demonstrated that branchial mRNA expression of selected isoforms of claudins, major components of tight junctions, are altered in an isoform-dependent manner, suggesting the potential regulation of epithelial permeability to minimize ion loss. Concurrently, a marked stimulation of Na+ uptake was observed in adults and larvae following acid-exposure. Because of the uniqueness of this response (increasing Na+ uptake in acidic water) among freshwater teleosts, the mechanisms related to Na+ uptake and its stimulation were investigated further (Chapters 3 - 7). Pharmacological treatments and gene knockdown approaches revealed that a functional metabolon consisting of an apically expressed Na+-H+-exchanger (NHE3b) in association with an apically expressed ammonia-conducting channel (Rhcg1), enables Na+ uptake in acidic water. During chronic (>1 day) exposure to acidic water, cortisol (via glucocorticoid receptors) and catecholamines (via β-adrenergic receptors) are involved in stimulating Na+ uptake. Although catecholamines may act on both NHE3b and Na+-Cl- co-transporter (NCC), the effects of cortisol on Na+ uptake are mediated primarily by activation of NHE3b. On the other hand, during acute (<3 h) exposure to acidic water, cortisol does not appear to affect Na+ uptake; rather, the stimulation of Na+ uptake appears to be mediated by angiotensin II and catecholamines. Cyclic AMP (cAMP), a signalling molecule synthesized following the activation of β-adrenergic receptors, is critically involved in stimulating Na+ uptake, likely via activation of NHE3b and NCC. In agreement with this idea, ionocytes that express NHE3b also express high levels of β-adrenergic receptor (propranolol binding sites) as well as trans-membrane adenylyl cyclase (forskolin binding sites). Taken together, the results of this thesis provide fresh insight into the mechanisms of osmoregulation in freshwater (FW) fish. In particular, the data reveal the presence of complex pathways regulating Na+ uptake in zebrafish exposed to acidic water. The relative importance of the various pathways depends in part on the duration of exposure; acute versus chronic.

Zebrafish, Danio rerio, are able to survive exposure to extreme acidity (pH 4). Because previous studies demonstrated that disruption of ionic balance during exposure to acidic water is the major cause of mortality in acid-sensitive freshwater species, the focus of this thesis was to characterize the molecular mechanisms enabling zebrafish to maintain their Na+ homeostasis following exposure to acidic water. Initial findings (Chapter 2) demonstrated that branchial mRNA expression of selected isoforms of claudins, major components of tight junctions, are altered in an isoform-dependent manner, suggesting the potential regulation of epithelial permeability to minimize ion loss. Concurrently, a marked stimulation of Na+ uptake was observed in adults and larvae following acid-exposure. Because of the uniqueness of this response (increasing Na+ uptake in acidic water) among freshwater teleosts, the mechanisms related to Na+ uptake and its stimulation were investigated further (Chapters 3 - 7). Pharmacological treatments and gene knockdown approaches revealed that a functional metabolon consisting of an apically expressed Na+-H+-exchanger (NHE3b) in association with an apically expressed ammonia-conducting channel (Rhcg1), enables Na+ uptake in acidic water. During chronic (>1 day) exposure to acidic water, cortisol (via glucocorticoid receptors) and catecholamines (via β-adrenergic receptors) are involved in stimulating Na+ uptake. Although catecholamines may act on both NHE3b and Na+-Cl- co-transporter (NCC), the effects of cortisol on Na+ uptake are mediated primarily by activation of NHE3b. On the other hand, during acute (<3 h) exposure to acidic water, cortisol does not appear to affect Na+ uptake; rather, the stimulation of Na+ uptake appears to be mediated by angiotensin II and catecholamines. Cyclic AMP (cAMP), a signalling molecule synthesized following the activation of β-adrenergic receptors, is critically involved in stimulating Na+ uptake, likely via activation of NHE3b and NCC. In agreement with this idea, ionocytes that express NHE3b also express high levels of β-adrenergic receptor (propranolol binding sites) as well as trans-membrane adenylyl cyclase (forskolin binding sites). Taken together, the results of this thesis provide fresh insight into the mechanisms of osmoregulation in freshwater (FW) fish. In particular, the data reveal the presence of complex pathways regulating Na+ uptake in zebrafish exposed to acidic water. The relative importance of the various pathways depends in part on the duration of exposure; acute versus chronic.

No description available.

No description available.

Caenorhabditis elegans has been studied as a model organism in various areas of biomedical research because it shares many conserved functions at molecular and genetic levels with humans. Specifically, it is an ideal organism to study heterogeneous metabolic syndromes such as Type 2 Diabetes Mellitus (T2DM) as C. elegans can be used to delineate molecular pathways that are at the core of its problems. A growing number of populations worldwide are faced with chronic T2DM, which also manifests several complications, such as blindness, neuropathy and cardiovascular diseases. Currently, metformin is the first-line drug of choice administered to treat T2DM. While the mechanism by which it alleviates the symptoms of diabetes is unknown, it has been found to reduce metabolic rate by partially inhibiting the mitochondrial complex I in mammals. Using C. elegans as a genetic model organism, we show that metformin reduces the mitochondrial activity through endosomal Na+/H+ exchanger, which a previous lab member has found to be a potential target of metformin. Furthermore, we show that high glucose diet−known to reduce the worm’s lifespan−alter the endosomal-lysosomal system and autophagy, providing insights to using C. elegans as a diabetic model. Based on these results, we propose that C. elegans can serve as a model organism to study T2DM as well as provide new ways to further investigate the pathophysiology of this disease.

O rim é tanto um órgão-alvo como a principal fonte de produção de ET-1, peptídio que regula a excreção de Na+ e água por este órgão que expressa os seus respectivos receptores, ETA e ETB, além dos trocadores NHE1 e NHE3 que são essenciais para o equilíbrio ácido base e hidroeletrolítico das células. Assim, o objetivo deste estudo foi investigar, em células IRPTC, o papel de ET-1 na atividade dos trocadores NHE1 e NHE3. Nossos resultados indicam que o tratamento agudo com ET-1 (10-9 M) aumenta a velocidade de recuperação do pHi (dpHi/dt) nos dois primeiros minutos após o pulso ácido, sugerindo aumento na atividade dos trocadores NHE1 e NHE3, que ocorre via ativação dos receptores ETA e ETB e parece ser secundária à atividade da p90RSk e p38MAPK. O tratamento crônico com ET-1 (10-9 M) reduz a dpHi/dt nos dois primeiros minutos após o pulso ácido, o que sugere redução na atividade dos trocadores NHE1 e NHE3, que pode ser secundária à inibição da Na+, K+-ATPase por ET-1. / The kidney is both a target organ and the main source of ET-1 production, a peptide that regulates the excretion of Na+ and water by this organ that expresses its respective receptors, ETA and ETB, in addition to the NHE1 and NHE3 exchanger which are Essential for the basic acid and electrolyte balance of cells. Thus, the objective of this study was to investigate, in IRPTC cells, the role of ET-1 in the activity of the NHE1 and NHE3 exchanger. Our results indicate that the acute treatment with ET-1 (10-9 M) increases the rate of recovery of pHi (dpHi/dt) in the first two minutes after the acid pulse, suggesting an increase in the activity of the NHE1 and NHE3 exchanger, which occurs via activation Of ETA and ETB receptors and appears to be secondary to the activity of p90RSk and p38MAPK. Chronic treatment with ET-1 (10-9 M) reduces dpHi/dt in the first two minutes after the acid pulse, suggesting a reduction in NHE1 and NHE3 exchanger activity, which may be secondary to inhibition of Na+, K+- ATPase by ET-1.

O presente estudo indica que o pHi basal do segmento S3 do túbulo proximal é 7.10 ? 0.007 (n = 444/2117), sendo a extrusão celular de H+ feita pelo trocador Na+/H+ (marjoritariamante) e pela H+-ATPase. Nossos resultados sugerem um papel do cálcio citosólico na regulação do processo de recuperação do pHi após carga ácida, mediada pelo trocador Na+/H+ e pela H+-ATPase. O trocador é estimulado por Aldosterona (10-12, 10-10 e 10-8 M) e inibido por Aldosterona (10-6 M) via ação genômica e não-genômica. Esses resultados são compatíveis com a estimulação do trocador por moderado aumento da [Ca2+]i citosólico (com Aldosterona 10-12 M) e sua inibição por pronunciado aumento da [Ca2+]i (com Aldosterona 10-6 M). A H+-ATPase é estimulada por Aldosterona em todas as doses utilizadas via ação genômica e não-genômica e esses resultados são coincidentes com um aumento da [Ca2+]i, dose dependente. Esses nossos achados são também compatíveis com a demonstração de uma ação hormonal não-genômica (após 1 ou 15 min) e genômica (após 1 hora) na [Ca2+]i, no trocador e na H+-ATPase. Adicionalmente, nossos resultados indicando que os efeitos hormonais genômicos ocorrem via receptor MR são coincidentes com nossos dados demonstrando a expressão desses receptores no segmento S3. Esses efeitos da Aldosterona que acabamos de descrever podem representar uma regulação fisiológica relevante, em condições de depleção e expansão de volume no animal intacto. / The present study indicate that the basal pHi of proximal S3 segment is 7.10 ? 0.007 (n = 444/2117), being made the extrusion by of Na+/H+ exchanger (mainly) and H+-ATPase. Our results suggest a role for cell calcium in regulating the process of pHi recovery after the acid load induced by NH4Cl, mostly mediated by a basolateral Na+/H+ exchanger, and stimulated by Aldosterone (10-12, 10-10 e 10-8 M) and impaired by Aldosterone (10-6 M) via a genomic and nongenomic action. They are compatible with stimulation of the Na+/H+ exchanger by increases in cell calcium in the lower range (at Aldosterone 10-12 M) and inhibition at high cell calcium levels (at Aldosterone 10-6 M). The H+-ATPase is stimulated in all the used doses via a genomic and nongenomic action, this is coincident with the dose-dependent increase in [Ca2+]i. This finding is also compatible with the demonstration of a hormonal nongenomic (after 1 min or 15 min) and genomic (after 1 h) action on [Ca2+]i, on the Na+/H+ exchanger and on H+-ATPase. Our results indicating that the genomics effects are via MR receptor are in accordanc with our finding showing expression of the receptors in the proximal S3 segment of rat. These Aldosterone effects may represent physiologically relevant regulation in conditions of volume depletion or expansion in the intact animal.

O presente estudo indica que o pHi basal do segmento S3 do túbulo proximal é 7.10 ? 0.007 (n = 444/2117), sendo a extrusão celular de H+ feita pelo trocador Na+/H+ (marjoritariamante) e pela H+-ATPase. Nossos resultados sugerem um papel do cálcio citosólico na regulação do processo de recuperação do pHi após carga ácida, mediada pelo trocador Na+/H+ e pela H+-ATPase. O trocador é estimulado por Aldosterona (10-12, 10-10 e 10-8 M) e inibido por Aldosterona (10-6 M) via ação genômica e não-genômica. Esses resultados são compatíveis com a estimulação do trocador por moderado aumento da [Ca2+]i citosólico (com Aldosterona 10-12 M) e sua inibição por pronunciado aumento da [Ca2+]i (com Aldosterona 10-6 M). A H+-ATPase é estimulada por Aldosterona em todas as doses utilizadas via ação genômica e não-genômica e esses resultados são coincidentes com um aumento da [Ca2+]i, dose dependente. Esses nossos achados são também compatíveis com a demonstração de uma ação hormonal não-genômica (após 1 ou 15 min) e genômica (após 1 hora) na [Ca2+]i, no trocador e na H+-ATPase. Adicionalmente, nossos resultados indicando que os efeitos hormonais genômicos ocorrem via receptor MR são coincidentes com nossos dados demonstrando a expressão desses receptores no segmento S3. Esses efeitos da Aldosterona que acabamos de descrever podem representar uma regulação fisiológica relevante, em condições de depleção e expansão de volume no animal intacto. / The present study indicate that the basal pHi of proximal S3 segment is 7.10 ? 0.007 (n = 444/2117), being made the extrusion by of Na+/H+ exchanger (mainly) and H+-ATPase. Our results suggest a role for cell calcium in regulating the process of pHi recovery after the acid load induced by NH4Cl, mostly mediated by a basolateral Na+/H+ exchanger, and stimulated by Aldosterone (10-12, 10-10 e 10-8 M) and impaired by Aldosterone (10-6 M) via a genomic and nongenomic action. They are compatible with stimulation of the Na+/H+ exchanger by increases in cell calcium in the lower range (at Aldosterone 10-12 M) and inhibition at high cell calcium levels (at Aldosterone 10-6 M). The H+-ATPase is stimulated in all the used doses via a genomic and nongenomic action, this is coincident with the dose-dependent increase in [Ca2+]i. This finding is also compatible with the demonstration of a hormonal nongenomic (after 1 min or 15 min) and genomic (after 1 h) action on [Ca2+]i, on the Na+/H+ exchanger and on H+-ATPase. Our results indicating that the genomics effects are via MR receptor are in accordanc with our finding showing expression of the receptors in the proximal S3 segment of rat. These Aldosterone effects may represent physiologically relevant regulation in conditions of volume depletion or expansion in the intact animal.

O objetivo do trabalho foi determinar o pHi, a capacidade tamponante do citosol na ausência de CO2/HCO3- (&#946;i) de neurônios dos gânglios das raízes dorsais e investigar a expressão de trocadores Na+/H+ e sua função na regulação do pHi. O pHi foi estimado com o indicador fluorescente BCECF. A operação do trocador era quantificada na recuperação da acidose intracelular induzida experimentalmente. Na ausência do tampão CO2/HCO3- a taxa de alcalinização (k) deve-se, por hipótese, ao transporte de H+ pelo trocador. A hipótese foi confirmada pela ação de agentes farmacológicos, e.g., amiloride. Em soluções tamponadas por CO2/HCO3- as células tem pHi de 7,24 e, em soluções tamponadas com HEPES, 7,04. A &#946;i foi de 8,17 mM/pH. As células se recuperam da acidose com k médio de 0,0138 s-1. O efeito inibitório do amiloride em concentração de 1 mM deve-se ao fato dos fenótipos celulares expressarem diferentes isoformas do trocador. Segundo RT-PCR, todas as 5 isoformas do trocador são expressas e a quantidade de RNAm, avaliada por qPCR, é maior para a NHE1, seguida de NHE5. / The objective here was to determine intracellular pH (pHi), cytosolic buffering power in CO2/HCO3- free medium (&#946;i) of neurons from dorsal root ganglia and to investigate the functional expression of the Na+/H+ exchangers in the regulation of pHi. pHi was estimated with fluorescence indicator BCECF. Exchanger operation was quantified during recovery from intracellular acidification induced experimentally. In CO2/HCO3- free medium the alkalinization rate (k) is due, hypothetically, H+ extrusion by the exchanger. This assumption was confirmed by action of pharmacologic agents, e.g., amiloride. In medium buffered with CO2/HCO3- cells have pHi of 7.24 and, in medium buffered with HEPES, 7.04. &#946;i calculated was 8.17 mM/pH. Cells recovery from acidosis with mean k of 0.0138 s-1. Inhibitory effect of amiloride in 1 mM concentration is due to cellular phenotypes expressing different Na+/H+ exchanger isoforms. According to RT-PCR, all the five exchanger isoforms are expressed and mRNA quantity, evaluated by qPCR, is greater to NHE1, followed by NHE5.

Em concentrações elevadas no plasma ou no tecido renal a Angiotensina II (Ang II) induz, alterações na hemodinâmica renal, injúria glomerular, aumento da síntese de componentes da matriz extracelular glomerular, estresse oxidativo e apoptose de células glomerulares, incluindo os podócitos. Os podócitos possuem um sistema reninaangiotensina (SRA) próprio e expressam os receptores AT1 e AT2 para o peptídeo, além do trocador Na+/H+ isoforma 1 (NHE1). O NHE1 está envolvido com a resistência e indução de apoptose, controle do volume celular e manutenção do fenótipo celular. Assim, o objetivo deste estudo foi investigar em podócitos, o papel da Ang II na indução de apoptose, e os eventos intracelulares associados à atividade do NHE1 nesta condição. Nossos resultados indicam que o tratamento com Ang II em alta concentração por 24 horas promove apoptose em podócitos. Nesta condição o NHE1, promove ativação da via de sinalização intracelular p38 MAPK e aumenta a atividade do NHE1 levando a alcalose, ativação da Bax e apoptose nos podócitos. / It has been observed that high plasma, or kidney tissue concentrations of angiotensin II (Ang II) leads to changes in renal hemodynamics, severe glomerular injury, increased synthesis of glomerular extracellular matrix components, oxidative stress and apoptosis in glomerular cells, including podocyte and mesangial cells. Podocytes a local renin-angiotensin system (RAS), expresses the AT1 and AT2 receptors for Ang II and the Na + / H + exchanger (NHE1). The NHE1 is involved with resistance and induction of apoptosis, cell volume control and maintenance of cell phenotype. Thus, the goal of this study was to investigate in podocytes the role of Ang II in the induction of apoptosis, and intracellular events linked to the NHE1 activity in this condition. Our results indicate that the treatment with Ang II, in a high dose, for 24 hours induces apoptosis in podocytes, and promotes oxidative stress. However, the activation of NADPH oxidase subunits Nox4 and p22 (phox) and pro- apoptotic pprotein Bax, came before the late apoptosis observed in 24 hours of treatment with Ang II. Under physiological conditions, the NHE1 activity contributes to cell survival by preventing cytosolic acidification. Moreover, Ang II via the AT1 receptor, activates intracellular signaling pathway p38 MAPK and increases the NHE1 activiy leading to alkalosis, Bax activation and apoptosis in podocytes.