Each day, people are exposed to a number of potentially toxic organic materials. Metabolism of certain foods and drugs creates toxins within the body, while potentially harmful substances such as herbicides, pesticides, and exhaust fumes are taken into the body from the environment. One of the factors that determines how much damage these toxic substances can cause is how effectively they are eliminated from the body. The longer toxins remain in the body, the greater the potential for their accumulation and damage to bodily tissues. The members of the Comparative Membrane Pharmacology Section of the Laboratory of Cellular and Molecular Pharmacology at the NIEHS are using techniques in cell biology, physiology, and molecular biology to study the mechanisms responsible for excretion of xenobiotics from the body.
Physiology
The end products of metabolism are generally more polar than are the starting materials. In many cases, organic ions are produced. Organic ions destined for renal excretion are carried to the kidneys via the blood. The toxic ions diffuse out of blood vessels and into the extracellular fluid bathing the cells of the kidney tubule. Research physiologist John Pritchard has focused much of his research on the mechanisms by which negatively-charged organic ions, called organic anions (OA), enter the kidney epithelial cells from the extracellular fluid. To understand this problem, he first had to determine the energetics of OA entry into the cell.
Organic Anion and Organic Cation Transport
Credit: David Miller
Water-soluble OA are too large to pass easily through cellular membranes, and the negative charges of these compounds make passage even more difficult. The inside of kidney cells, like most cells, is negatively charged relative to the surrounding extracellular fluid. Negatively-charged chemicals must enter the cell against an electrochemical gradient; thus, OA uptake into the cell must be coupled with an energetically-favorable process. For some time, scientists noticed that the inflow of positively-charged sodium ions--a process driven by the out-to-in sodium gradient and, thus, energetically downhill--correlated with the entrance of OA into kidney epithelial cells. However, Pritchard and others studying the problem discovered that OA transport was not directly coupled with sodium-ion influx. An intermediary step was required. In 1987 Pritchard and German scientist Gerhardt Burkhardt determined independently that a ubiquitous cellular metabolite called -ketoglutarate (-KG) played this intermediary role. Their work showed that sodium-ion entry drives -KG accumulation. In the next step, -KG exits the cell, releasing energy, and OA enters in exchange. In this way, the energy derived from admitting sodium is indirectly used to admit OA.
However, because the ions are still too large to pass through the membrane unaided, they must be transported by a protein embedded in the membrane. One of Pritchard's current interests is identifying the transporter protein, or proteins, that admit OA into kidney epithelial cells. Conversely, while the entrance of positively-charged ions, called organic cations (OC), is energetically favorable, special transporter proteins are also necessary for their entrance into the cell. Pritchard hopes to study these proteins as well.
While one side of the kidney epithelial cell is responsible for taking up toxins from the extracellular fluid, the other side mediates the secretion of these toxins into the tubular lumen, where urine is formed. The energetics of expelling organic ions is the mirror-image of admitting them. Eliminating OC requires the input of energy, while eliminating OA may not. In addition to the various transporter proteins required for these processes there may be other mechanisms at work as well. Pritchard and colleagues are also studying the proteins and auxiliary mechanisms of organic-ion expulsion.
Molecular Biology
Molecular biologist Doug Sweet is working to characterize the various transporters at the molecular level. He hopes to use molecular biology techniques to identify the genes for the various transporters. By determining the genetic sequence of the transporters, Sweet will be able to derive the amino acid sequences and, thus, the structures of the various transport proteins.
Sweet's research continues the work of his predecessors Natasha Wolff and Rosemary Walsh. Using the technique of expression cloning, Walsh searched for the transporter protein (or proteins) that mediates the excretion of OC by the kidney epithelium. To do this, she created a library of genes expressed in the kidney. These genes were then amplified on plasmids and inserted into bacteria. Sweet screens these bacteria to identify the group that contains the gene or genes of interest--in this case, the one encoding the OC transporters. The bacteria are then divided into groups and the plasmids containing the various kidney genes are purified. Using in vitro transcription, the DNA of each group of plasmids is made into messenger RNA, which is microinjected into the oocytes of Xenopus laevis (the African clawed toad). This mRNA directs production of the proteins encoded in each of the genes of the kidney library. Finally, the oocytes are assayed for their ability to support OC transport. With each subsequent round of screening, the number of bacteria in each group is narrowed, eventually leading to a single bacterial colony containing a single kidney gene--in this case the OC transporter gene.
Sweet is now in the final stages of that screening process and believes he is close to locating the gene. In the next phase of his work, Sweet plans to search the library for genes encoding the OA transporters. Identifying these genes will help the researchers to learn more about how ions are admitted into kidney cells.
Having a detailed understanding of the gene and protein sequences of the transporters may help the researchers learn about the precise nature of the interactions between these proteins and organic ions. It may also help decipher interactions between organic ions competing for the same binding sites on the transporters, which can contribute to the relative toxicity of a compound. Sweet also plans to study the genomic sequences for the transporter genes in order to understand how the genes respond transcriptionally to changes in the toxic load present in a cell's environment.
Translocation
Transporting toxins into a kidney cell is only the first step in their elimination. In fact, the presence of these toxic substances inside the kidney epithelial cells raises the question of how the kidney protects itself from these toxins. Studies performed by research physiologist David Miller may provide an answer. Miller has found that once organic ions have entered the kidney cell, some of the ions--both positive and negative--are sequestered into small membrane-bound vesicles. Miller has discovered that the vesicles serve not only to protect the cell, but may also ferry toxins from one side of the cell to the other. Miller has evidence that once the vesicles reach the exit site, they release their contents outside of the cell. This might be accomplished, he says, by a fusion between the vesicular and cellular membranes.
Miller can actually observe some of these processes as they occur by attaching a small TV camera to his microscope and projecting the images into his computer. By using organic ions that are linked to fluorescent dyes, Miller can watch the flow of ions through and out of kidney epithelial cells on his computer screen.
Killifish kidney tubules. Fluorescent organic anion (bright areas) is secreted into the tubular lumen and taken up by adjacent cells. (green bar=10 micrometers)
Credit: David Miller
For some of this research, Miller uses the killifish, a type of minnow that he predicts could become the "laboratory rat of the fish world" because it is so hardy and adaptable to a wide range of environmental conditions. Furthermore, the killifish provides a model in which the entire excretion process can be studied in the intact kidney tubule.
Choroid Plexus
Whereas the killifish is good for studying gross features of renal organic-ion transport, the choroid plexus allows Miller, in collaboration with physiologist Alice Villalobos, to look at the passage of individual ion-transport vesicles across and out of cells.
One function of the choroid plexus is analogous to what the kidney does for the entire body: mediating removal of organic ions from the brain into the bloodstream for subsequent renal or hepatic excretion from the body. The choroid plexus is located within the brain's ventricles and forms the interface between the cerebrospinal fluid and the body's blood supply. The cells of the choroid plexus share much of their basic physiology with kidney epithelium. Both cell types have transporter proteins regulating the influx and outflow of organic ions, and both cells shuttle the ions across the cells in vesicles.
Villalobos has succeeded in culturing cells from the rat choroid plexus. In culture, these cells form a layer only one cell thick, making them easily visible under the microscope. Villalobos has observed that vesicles filled with fluorescent dye clearly cross the choroid plexus cell until they appear to burst, releasing their fluorescent contents outside of the cell.
Miller and Villalobos have performed several experiments whose results suggest that the cytoskeleton is crucial to vesicular transport. Specifically, they have shown that disrupting the filaments called microtubules halts transport. This, notes Miller, suggests that the microtubules provide a sort of "railroad track for the vesicles, where the end of the line is fusion of the membranes and release of vesicular contaminants."
Characterizing transport mechanisms--the transporter proteins and the vesicular transport system--is crucial to understanding xenobiotic excretion. Understanding the factors that determine whether or not a compound will be taken up and excreted by the kidney or the choroid plexus helps toxicologists to predict the compound's toxicity. In addition, these studies may provide insight into the factors that determine the use of drugs and other beneficial organic ions within the body.
Michelle Hoffman
Last Update: June 10, 1997