[The strategy for research on tubular transport in the kidney].
The present publication summarises the experimental strategies that have been developed to identify, localize and characterize renal tubular transport mechanisms on the nephron, cellular and molecular level. 1) Identification and localization of transporters can be achieved on single ne (mRNA hybridization) techniques. 2) The functional properties of transporters and their regulation can be investigated on individual nephron segments in vivo or in vitro, on cell cultures, isolated cells, or on isolated membrane vesicles, applying different analysis techniques: net flux and tracer take-up studies, microscopic volume determinations, microelectrode measurements (conventional and ion-selective), patch-clamp measurements, microspectrofluorometry, X-ray microanalysis of frozen tissues, nuclear magnetic resonance, etc. 3) The primary structure (amino acid sequence) of transport proteins can be determined by protein purification with subsequent amino acid sequencing. More powerful, however, is expression cloning, e.g. screening of cDNA libraries for expression of a given transport protein in a model cell system (e. g. Xenopus laevis oocytes) with subsequent base sequence analysis. 4) The molecular function of transport properties can best be studied in heterologous expression experiments (overexpression, easy technical, e. g. in oocytes). This also allows monomeric and multimeric transporters to be distinguished and is a prerequisite for mutagenic studies in which the function of individual amino acid constituents of the protein can be determined. Such studies eventually allow detailed molecular models of transport function to be developed.
Frömter
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Zentrum Physiologie, Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D006801:Humans; D007684:Kidney Tubules / Q000502:physiology*; D008967:Molecular Biology / Q000379:methods; D012107:Research Design