Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / La rétroaction tubuloglomérulaire (RTG) est un mécanisme important dans le contrôle de la filtration glomérulaire et de l'hémodynamique rénale. Ce phénomène est relié aux cellules de la macula densa (MD) qui ont la capacité de détecter des changements dans la composition du liquide tubulaire et de transmettre cette information aux artérioles afférentes (AA) qui, en se contractant, diminuent le débit de filtration glomérulaire et augmentent la résistance hydraulique du rein. La nature du signal qui est envoyé des cellules de la MD vers les AA reste encore inconnue. On sait cependant qu'une haute concentration de NaCl dans la lumière tubulaire déclenche la RTG, et que le cotransporteur Na+ +:2Cl^- est probablement le "sensor". Pour progresser dans l'étude de la RTG, une meilleure connaissance des cellules de la MD est absolument requise puisque c'est d'elles qu'origine le signal. La présente thèse a pour but de contribuer à une meilleure identification et à la caractérisation des propriétés membranaires des cellules de la MD. Dans un premier temps, à l'aide de la technique de patch clamp en "single channel" nous avons mis en évidence une grande densité de canaux K+ dans la membrane apicale. Nous montrons que ce type de canal est sensible au pH et au Ca2+ mais insensible à l'ATP. Dans un second temps, nous optons pour la technique de microperfusion de tubule isolé combinée à la microfluorimétrie. Nous démontrons ainsi la présence d'un échangeur Na+ + à la membrane apicale qui répond à toute variation de la concentration luminale de Na+ et de NaCl ([Na+]i et [NaCl]i). Par ailleurs, en utilisant les effets secondaires du cotransporteur Na+ +:2Cl^- sur le pH, nous démontrons que ce transporteur s'équilibre lorsque [NaCl]i atteint 18 mM. Ainsi, nous concluons que dans les conditions physiologiques, le cotransporteur produit une réabsorption de NaCl mais en opérant très près de son équilibre. Dans un troisième temps, nous utilisons une méthode dont nous avons établi la faisabilité pour les cellules de la MD, et qui est basée sur l'utilisation de NH4+ pour déterminer l'amplitude de certains flux. Nous démontrons que le NH4+ entre dans les cellules par le cotransporteur Na+ +:2Cl^- et par un transporteur sensible au Ba2+, et en sort, lors du retrait de NH4+ luminal, exclusivement sous forme de NH3 et de H+. Bien que la nature du transporteur sensible au Ba2+ n'ait pu être résolue de façon définitive, nous démontrons qu'elle ne correspond ni au canal K+ ni à l'échangeur K/(NH4)H. Ces études nous ont toutefois amenés à présenter pour la première fois un modèle qui permet d'évaluer quantitativement les mesures de taux d'acidification (d[pH]i/dt) et d'en tirer des mesures de flux. Grâce à ce modèle, nos données montrent que la membrane apicale est très perméable au NH3 et, contrairement à ce qu'il a été admis jusqu'à présent, le flux de NH4+ n'est pas égal au flux de protons mesuré mais lui est plutôt 2 à 3 fois supérieur. Enfin, dans un dernier temps, nous avons montré que le cotransporteur a une haute affinité pour le Na+ et pour le Cl-. Nous avons aussi commencé à étudier les facteurs régulateurs de ce cotransporteur en observant une inhibition de 40 % du cotransport suite à l'augmentation de la [Cl^-]i par inhibition de sa sortie basolatérale (10 μM NPPB). De plus, l'ajout de dbAMPc + forskoline stimule la conductance basolatérale au Cl^- mais inhibe faiblement le cotransporteur apical. En conclusion, nous considérons que ces études ont significativement contribué à l'amélioration de nos connaissances sur les cellules de la MD et à la progression dans la compréhension de la RTG, puisque nous sommes maintenant en position de spéculer avec un peu plus de précision sur les effets secondaires d'une augmentation de [NaCl]i.

<p>The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important.</p><p>In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect.</p><p>The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP >> 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. </p><p>In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells. </p>

The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important. In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect. The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP &gt;&gt; 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells.

<p>The kidney is the major organ regulating the extracellular fluid volume and thereby the arterial blood pressure. The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa cells. These cells are sensors for both renin release and the tubuloglomerular feedback mechanism (TGF), which is an important regulator of the glomerular filtration rate and afferent arteriole tone. The aim of this investigation was to elucidate the function of nNOS in the macula densa cells.</p><p>Acute nNOS inhibition in rats resulted in an increased TGF responsiveness and unchanged blood pressure while, after chronic inhibition, the TGF was normalised and the blood pressure was elevated. The plasma renin concentration was elevated in rats on long-term low salt diet, but was not significantly affected by chronic nNOS inhibition. On the other hand, nNOS inhibition for four days increased plasma renin concentration in rats treated with a low salt diet. The renal vasculature of rats exhibits a diminished renal blood flow and intracellular Ca2+ response to angiotensin II after one week blockade of nNOS while angiotensin II’s effect on the renal blood flow was abolished after four weeks treatment. Acute extracellular volume expansion diminish the TGF sensitivity thus assisting the elimination of excess fluid but after acute addition of nNOS inhibitor to volume expanded rats the TGF sensitivity restored.</p><p>In conclusion, the results from the present study suggest an important role for nNOS in the macula densa cells in the regulation of the arterial blood pressure and the modulation of the TGF response.</p>

The kidney is the major organ regulating the extracellular fluid volume and thereby the arterial blood pressure. The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa cells. These cells are sensors for both renin release and the tubuloglomerular feedback mechanism (TGF), which is an important regulator of the glomerular filtration rate and afferent arteriole tone. The aim of this investigation was to elucidate the function of nNOS in the macula densa cells. Acute nNOS inhibition in rats resulted in an increased TGF responsiveness and unchanged blood pressure while, after chronic inhibition, the TGF was normalised and the blood pressure was elevated. The plasma renin concentration was elevated in rats on long-term low salt diet, but was not significantly affected by chronic nNOS inhibition. On the other hand, nNOS inhibition for four days increased plasma renin concentration in rats treated with a low salt diet. The renal vasculature of rats exhibits a diminished renal blood flow and intracellular Ca2+ response to angiotensin II after one week blockade of nNOS while angiotensin II’s effect on the renal blood flow was abolished after four weeks treatment. Acute extracellular volume expansion diminish the TGF sensitivity thus assisting the elimination of excess fluid but after acute addition of nNOS inhibitor to volume expanded rats the TGF sensitivity restored. In conclusion, the results from the present study suggest an important role for nNOS in the macula densa cells in the regulation of the arterial blood pressure and the modulation of the TGF response.