Using the fly renal tubule system, we are currently studying SLC12 cation-chloride transporters, such as the potassium-chloride cotransporter and the sodium-potassium-2 chloride cotransporter, and their regulation by WNK and SPAK kinases, which have been implicated in hypertensive disorders in humans. We are also studying inwardly rectifying potassium channels, which have also been implicated in several human disorders. Our goal is to understand these transporters, channels and their regulation in greater mechanistic detail, identify new regulatory factors, and translate these insights into improved understanding of the mammalian kidney.
Pictorial representation of the fly renal tubules. As in humans, epithelial ion transport serves to regulate electrolyte concentration and water content in the fly. There are four tubules (only one is completely shown), two anterior and two posterior. Each tubule consists of multiple segments with different functional roles. From: Dow J.A.T. and Davies S.A. (2003) Physiol Rev 83: 687.
Cell model of the two cell types in the main segment, showing some of the transporters, pumps and channels. Cation flux occurs through the principal cell, whereas anion flux occurs through the stellate cell. The apically located vacuolar proton ATPase generates a lumen-positive transepithelial potential difference of about +40 mV under baseline conditions, which drives exchange of protons for potassium (or sodium) and allows potassium/sodium excretion. Of interest to our laboratory is the contribution of sodium-potassium-2 chloride cotransporters and inwardly rectifying potassium channels to basolateral membrane ion fluxes.
The Ramsay assay. This is one of the main assays used in the lab to study fly renal tubule function. Dissected fly renal tubules (also known as Malpighian tubules) are placed in a droplet of “bathing saline” and secrete fluid under mineral oil, preventing evaporation. The rate of fluid secretion can be determined by measuring the volume of secreted fluid/unit time. Ion-specific microelectrodes are used to measure ion concentrations (for example, potassium concentration). Rates of ion flux can then be calculated. From: O’Donnell M.J. (2009) J. Exp. Biol. 212: .363.
The GAL4/UAS system. Flies carrying the yeast transcriptional activator GAL4, expressed in a tissue-specific fashion (for example, in the fly renal tubule) depending on nearby enhancer elements, are crossed to flies of the opposite sex carrying a transgene of interest downstream of the GAL4 DNA-binding domain, UAS (Upstream Activating Sequence). Progeny flies will express the transgene in a tissue-specific fashion. If the transgene encodes a double-stranded RNA to a gene of interest, tissue-specific knockdown of the gene can be achieved. For genes in which global loss-of-function results in lethality, this allows study of loss of gene function in the renal tubules of adult flies. For example, we have used this technique to study the function of wnk and fray, the fly SPAK homolog, in the regulation of fluid secretion and potassium flux in the fly tubule.