Molecular and functional study of membrane transporters

Abstract

The sub-projects outlined in the present Thematic Project proposal all involve studying the transport of ions through cell membranes. With the exception of two that analyze 1) other aspects of glomerular cells and 2) the transport of the neutral molecule NH3 through cells involved with ammonia ammonia (NH3/NH4+) homeostasis or transport, such as the hepatocytes and collecting duct cells. These projects also focus on ion transport in the renal tubule or stem cells, with a particular interest in H+, Na+ and K+ coupled transporters. These projects include the study of the renin-angiotensin-aldosterone system (RAAS). In the glomerulus, the interactions of leptin and endothelin with the RAAS will be evaluated by using mesangial and podocyte cell lines. Another interest is to elucidate functional aspects of these interactions among angiotensin II (Ang II) receptor AT1R, ATRAP (AT1R Associated Protein), and HSC70 (Heat Shock Protein 70). The interaction between ATRAP and CAML (Ca2+ modulating cyclophilin ligand) is involved in activating the transcription factors NFATs via AngII. RdgB² (Retinal degenation type B²) - a phosphatidyl inositol (PI) transfer protein - will be also evaluated in proximal tubule and in Human Embrionic Kidney cells (HEK). The modulation of activity and expression of WNK1 and WNK4 - members of the WNK (With No lysine-K) kinase family - will be investigated in distal convoluted tubule and collecting duct cells. WNK 1 and 4 have been shown to be important regulators of Na+ and K+ transport in distal nephron, by AngII activating the AT1R/ATRAP/CAML/NFAT pathway. It is also important to evaluate the effect that calcineurin inhibitors have on immune-suppressors largely used in renal transplanted patients. The effects of Ang1-7 on the transport in proximal tubules, and the non-genomic effects of aldosterone will be investigated in isolated tubules and in the whole kidney. Finally, the effects of AngII and vasopressin on the transport of solutes and fluid in rat renal proximal tubules with water restriction will be investigated, with a focus on Na+/H+ exchanger NHE3.Another important aspect of the included sub-projects is to study the effect of simvastatin, sildenafil, and GLP-1 peptide - an incretin secreted by intestinal cells in postprandial period - on the activity and expression of NHE3 in proximal tubules. Another pharmacological approach is to determine the effect of phenylkenes on isoforms of a voltage sensitive potassium channel in stem cells. These projects have direct clinical interest, which is a new focus of our group. The methods proposed in these projects include measuring H+ and K+ activity using micropuncture techniques. Intracellular H+ and Ca2+ measurements will be determined using fluorescent probes in isolated tubules and in cultured cells using confocal or wide field fluorescence microscopes. The patch clamp technique will be used for the analysis of single channels. Analysis of tissue structure and protein immune-localization will be analyzed by optic microscopy and chemical or fluorescence techniques, respectively. We will also use molecular biology techniques to overexpress recombinant proteins, introduce mutations, induce gene silencing, study promoters with reporter genes, co-immune precipitation and western blot to study protein interactions, and RT-PCR for gene expression analysis. To analyze protein interactions, resonance energy transfer (RET) techniques will be employed, using yellow and cyan fluorescent proteins, for FRET, and Renilla luciferase and YFP, for BRET. The sub project involving the transport of ammonia is designed to understand how NH3 moves through channels, and, at a more integrated level, the physiological importance of gas channels. The oocyte NH3 permeability will be analyzed using microelectrodes to monitor changes in cell surface pH (pHS) caused by the influx of NH3 between extra and intracellular space of Rana oocytes after exposure of specific solutions. (AU)