Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1

ABSTRACT The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl 2 inhibited AQP1 water permeability by .95%. We found that none of the tested compounds at 50 mM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority

Hypertension in children with chronic kidney disease: pathophysiology and management

Arterial hypertension is very common in children with all stages of chronic kidney disease (CKD). While fluid overload and activation of the renin–angiotensin system have long been recognized as crucial pathophysiological pathways, sympathetic hyperactivation, endothelial dysfunction and chronic hyperparathyroidism have more recently been identified as important factors contributing to CKD-associated hypertension. Moreover, several drugs commonly administered in CKD, such as erythropoietin, glucocorticoids and cyclosporine A, independently raise blood pressure in a dose-dependent fashion. Because of the deleterious consequences of hypertension on the progression of renal disease and cardiovascular outcomes, an active screening approach should be adapted in patients with all stages of CKD. Before one starts antihypertensive treatment, non-pharmacological options should be explored. In hemodialysis patients a low salt diet, low dialysate sodium and stricter dialysis towards dry weight can often achieve adequate blood pressure control. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers are first-line therapy for patients with proteinuria, due to their additional anti-proteinuric properties. Diuretics are a useful alternative for non-proteinuric patients or as an add-on to renin–angiotensin system blockade. Multiple drug therapy is often needed to maintain blood pressure below the 90th percentile target, but adequate blood pressure control is essential for better renal and cardiovascular long-term outcomes

Aquaporins: important but elusive drug targets.

The aquaporins (AQPs) are a family of small, integral membrane proteins that facilitate water transport across the plasma membranes of cells in response to osmotic gradients. Data from knockout mice support the involvement of AQPs in epithelial fluid secretion, cell migration, brain oedema and adipocyte metabolism, which suggests that modulation of AQP function or expression could have therapeutic potential in oedema, cancer, obesity, brain injury, glaucoma and several other conditions. Moreover, loss-of-function mutations in human AQPs cause congenital cataracts (AQP0) and nephrogenic diabetes insipidus (AQP2), and autoantibodies against AQP4 cause the autoimmune demyelinating disease neuromyelitis optica. Although some potential AQP modulators have been identified, challenges associated with the development of better modulators include the druggability of the target and the suitability of the assay methods used to identify modulators

Estudi de transportadors de sodi i aquaporines renals en trastorns del balanç hidrosalí

En aquesta tesi s'han estudiat l'excreció de transportadors de sodi i canals d'aigua o aquaporines renal en diverses patologies del balanç hidrosalí, així com l'expressió d'aquestes proteïnes en ronyó de rates en un model experimental d'hipertensió induïda per ciclosporina.En el primer treball, en un model experimental de rata d'hipertensió induïda per ciclosporina vam detectar que les rates que desenvolupaven hipertensió tenien augmentada l'expressió renal del transportador de sodi NKCC2. Aquest increment d'NKCC2 podria afavorir la reabsorció de sodi a nivell de la nansa de Henle amb la conseqüent expansió del volum plasmàtic, i per tant, un augment de la pressió arterial. L'augment detectat de l'aquaporina-1 detectat a les rates tractades podria estar afavorint una reabsorció més efectiva d'aigua al túbul proximal, mentre que la reducció de l'expressió d'aquaporina-2 a les rates tractades conduiria a una disminució de la permeabilitat del túbul col·lector i podria representar un mecanisme compensatori a l'increment de la reabsorció de sodi i aigua.Més endavant vam estudiar l'excreció de les aquaporines-1 i -2 a l'orina de pacients amb cirrosi en les diferents fases de la retenció de sodi i aigua (cirrosi sense ascites, cirrosi amb ascites i síndrome hepatorenal). En aquesta malaltia a mesura que evoluciona la hepatopatia apareix una retenció hidrosalina. Aquesta es manifesta inicialment per edemes i ascites, passant posteriorment al desenvolupament d'hiponatrèmia com a conseqüència del predomini de la retenció d'aigua respecte a la sal. Vam observar que l'excreció urinària d'aquaporina-2 disminuïa de forma progressiva a mesura que progressava la retenció hidrosalina. Aquestes dades apunten a que hi hauria menor quantitat d'aquaporina-2 al túbul col·lector i una menor permeabilitat osmòtica d'aquest epiteli que podria representar una resposta adaptativa del ronyó a la retenció hidrosalina i a l'expansió del volum del fluid extracel·lular. D'altra banda, no es van trobar diferències en l'excreció urinària d'aquaporina-1 entre individus controls i pacients cirròtics amb o sense ascites, tot i que aquest canal d'aigua s'ha trobat disminuït en pacients amb síndrome hepatorenal.Per a estudiar amb major profunditat l'excreció de les proteïnes renals de la membrana apical en exosomes a l'orina vam estudiar en primer lloc la localització de les proteïnes NKCC2 i les aquaporines-1 i -2, així com les formes fosforilades pNKCC2 i pS256-Aquaporina-2, a nivell ultraestructural en ronyó humà. Així per primera vegada vam detectar les proteïnes NKCC2 i les aquaporines-1 i -2, així com les formes fosforilades, associades a unes estructures vesiculars que podrien correspondre's als cossos multivesiculars. D'altra banda vam confirmar totes les proteïnes anteriorment esmentades associades a exosomes urinaris en orina humana ultracentrifugada. Finalment en l'últim treball amb pacients amb hipertensió essencial vam estudiar com la ingesta de sal modificava la pressió arterial i l'excreció de diferents transportadors de sodi (NKCC2 i NCC) en exosomes urinaris. Tot i que en aquest estudi es van detectar per primera vegada els transportadors NKCC2 i NCC en exosomes urinaris pacients hipertensos, l'excreció en exosomes urinaris d'aquests transportadors no permetia preveure quina regió tubular podria estar implicada en la patogènia de la sal sensibilitat.En definitiva, els resultats d'aquess estudis ens han permès aprofundir en els mecanismes fisiopatològics que intervenen en la susceptibilitat d'aquestes patologies i més important encara, ens han donat les bases per posa a punt nous projectes en patologies humanes amb una orientació fisiopatològica i sobretot terapèutica.In this thesis we have studied the urinary excretion of renal sodium transporters and water channels in several pathologies of the water and salt balance, likewise the expression of these proteins in the kidney of rats in an experimental model of hypertension induced by cyclosporine.At the first work, in an experimental model of rat with hypertension induced by cyclosporine we detect that the rats with hypertension had an increased expression of the renal sodium cotransporter NKCC2. This increase could facilitate the sodium reabsorption in the thick ascending limb, so there would be an increase of the plasmatic volume and the consequent rise of the blood pressure. The observed augment of aquaporin-1 in the rats treated with cyclosporine could be increasing a more effective water reabsorption in the proximal tubule, while the reduction of the expression of the aquaporin-2 in the treated rats would lead to a diminished permeability of the collecting duct and it could represent a compensatory mechanism to the increased reabsorption of sodium and water.Furthermore we studied the urinary excretion of aquaporin-1 and -2 of cirrhotic patients in the different phases of the sodium and water retention (cirrhosis without ascites, cirrhosis with ascites and hepatorenal syndrome). This disease progresses to sodium retention which is often associated with an impaired ability to eliminate solute-free water, which may lead to dilutional hyponatremia from a disproportionate increase in total body water relative to total sodium content. We detected a progressive decrease in urinary aquaporin-2 excretion as the severity of cirrhosis increased, from compensated cirrhosis to cirrhosis with ascites and hepatorenal syndrome. The reduction of aquaporin-2 would mean a decreased abundance of renal aquaporin-2 and therefore lower water permeability in the collecting tubules. This may represent an adaptive renal response to sodium retention, with expansion of extracellular fluid volume and dilutional hyponatremia observed in those who have cirrhosis with ascites. On the other hand, there were no differences between healthy subjects and patients with cirrhosis with or without ascites in urinary excretion of aquaporin-1, but urinary aquaporin-1 excretion of those with hepatorenal syndrome was extremely low. To study in depth the excretion of apical renal proteins via exosomes to the urine, first of all, we investigated the localization of the proteins NKCC2 and aquaporins-1 and -2, as well as the phosphorylated proteins pNKCC2 and pS256-aquaporin-2 at ultrastructural level in human kidney. So, for first time we could detect all these proteins, but aquaporin-1, associated to vesicles that could be the multivesiculars bodies. On the other side, we also confirmed the presence of all those proteins, including aquaporin-1, associated to urinary exosomes from ultracentrifuged human urine. Eventually, at the last work with patients with essential hypertension we studied how the salt intake could modify the arterial blood pressure and the excretion of two renal sodium transporters (NKCC2 and NCC) in urinary exosomes. Even though it was the first time NKCC2 and NCC were detected in urinary exosomes from hypertensive patients, the excretion of these transporters through exosomes in the urine could not predict which part of the renal tubule might be implicated in the pathogenesis of salt sensitive hypertension