Molecular Physiology Lab
The mission of the laboratory is to study the role of dopamine, and adrenergic receptor subtypes, dopamine regulatory genes (e.g., G protein-coupled receptor kinase 4, GRK4) and cholecystokinin receptors on sodium transport in specific nephron segments and their roles in the pathogenesis of genetic hypertension and metabolic syndrome in humans and in animal models of these diseases. The experiments are designed to study (a) the integrative physiology of these receptors in vivo, as well as (b) the molecular and cellular biology of these receptors in (1) specific nephron segments, (2) stably transfected cell lines (COS, CHO, HEK, LTK-, and OK), (3) primary and immortalized cultures of human renal proximal tubule cells from hypertensive and normotensive subjects and rat juxtaglomerular cell, (4) immortalized renal proximal tubule cells from normotensive and spontaneously hypertensive rats, (5) immortalized renal proximal tubule and distal convoluted tubule cells from mice, and in vivo studies include: (i) “knock-out and knock-in” mice and (ii) gene transfer/rescue in rodents using adeno-asssociated virus (AAV) vectors. There is active collaboration among researchers doing genetic and functional studies in humans.
The signal transduction mechanisms involved in these receptor-mediated transport mechanisms are studied by measuring: (1) activities of adenylyl cyclase, phospholipase C, phospholipase D, protein kinase C, protein kinase A, protein phosphatase and G protein-coupled receptor kinases, (2) expression of adenylyl cyclase, phospholipase C, phospholipase D, protein kinase C, protein kinase A and G protein-coupled receptor kinase isoform RNA expression by in-situ hybridization and RT/PCR, and protein expression by immunohistochemistry, immunoblotting and ELISA, (3) phosphorylation of dopamine receptor subtypes and sodium channels, exchangers, pump, and transporters, (4) activities and expression of proteins related to oxidative stress and inflammation as related to hypertension, and (5) interaction among G protein-coupled receptors, G proteins, G protein-coupled receptor kinases, sorting nexins and other effector and adaptor proteins. The transport of sodium is studied by measuring NHE3 activity in brush border membrane vesicles, and Na+/K+ ATPase activity by measuring enzyme activity, ouabain-inhibitable 86Rb+ uptake in microdissected specific nephron segments, and in apical and basolateral membranes in polarized cells grown on semipermeable support or alginate beads in 3D. There are also projects studying the mechanisms by which renal dopamine receptors regulate oxidative stress and inflammation in health and hypertension. Moreover, the detailed biophysical characterization of sodium-hydrogen exchanger is performed by a combination of electrophysiology (patch-clamp) and self-referencing ion-selective electrodes. Cloning of novel genes that may regulate blood pressure and regulation of these genes at the 5' and 3' untranslated regions are also being studied. Protein-protein interactions are studied by biochemical and morphological methods, including confocal microscopy and biophysical methods, such as Förster resonance energy transfer, and fluorescence life time imaging. Reagents include selective agonists and antagonists, antibodies, antisense oligonucleotides, siRNA/shRNA, targeted gene expression using AAV vectors, and dominant negative mutants, and cells/mice over-expressing (transgenics), under-expressing (siRNA/shRNA) or non-expressing (knockouts) the dopamine and adrenergic receptors and regulators. The laboratory has also developed a novel method of organ-selective reversible gene silencing (e.g., kidney, stomach and duodenum).
Trainees working in the Jose laboratory will learn techniques of integrative renal physiology, cell biology, and applied molecular biology. Emphasis is placed on studying genes that regulate blood pressure and contribute to the pathogenesis of spontaneous hypertension in animals and in humans and pharmacogenomics. The trainees will also be taught the generation of hypothesis-driven research, grantsmanship, and pathways to become independent investigators.
The studies are supported by grants from the National Institutes of Health (NHLBI [7R37HL023081, 1R01HL092196, 5P01HL068686, 5P01HL074940], and NIDDK [DK039308]), and from the pharmaceutical industry (BIAL, Portugal).