Stephen A. Kempson

Molecular regulation of renal phosphate transport.

M. Levi , S.A. Kempson, M. Lö tscher, J. Biber, H. Murer 3 Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, and Department of Veterans Affairs Medical Center, Dallas, Texas Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana Institutes of Physiology and Anatomy, University of Zu ̈rich-Irchel, Zürich, Switzerland

Renal Adaptation to a Low Phosphate Diet in Rats: BLOCKADE BY ACTINOMYCIN D

The major renal adaptive changes in response to selective dietary phosphate restriction are a marked reduction in urinary excretion of phosphate and an increased urinary excretion of calcium; at the cellular level, there is selective increase in renal cortical brush border membrane phosphate uptake and increase in specific activity of alkaline phosphatase. In the present study we examined whether these functional and biochemical adaptive changes could be blocked by drugs known to inhibit protein synthesis. Administration of actinomycin D or cycloheximide to rats switched from a diet with normal phosphate content (0.7%) to a diet with low (0.07%) phosphate content either completely (actinomycin D) or partially (cycloheximide) prevented the expected decrease in urinary excretion of phosphate and increase in the urinary excretion of calcium. The specific activity of alkaline phosphatase measured in crude membrane fraction (washed 100,000 g pellet) from renal cortical homogenate in animals fed a low phosphate diet and treated with actinomycin D or with cycloheximide was significantly lower than in control animals also on a low phosphate diet receiving placebo; but there were no differences between treated and untreated animals in the activities of two other brush border enzymes, gamma-glutamyltransferase and leucine aminopeptidase. Actinomycin D administered to rats maintained on a normal phosphate diet throughout the course of the experiment caused an increase in the urinary excretion of phosphate on the last (6th) day of the experiment but did not change urinary excretion of calcium. In acute clearance experiments, infusion of actinomycin D to rats adapted to a low phosphate diet did not increase fractional excretion of phosphate. In separate experiments, using the same dietary protocol as above, brush border membrane fraction (vesicles) was prepared from renal cortex of rats sacrificed at the end of the experiment. In this preparation Na(+)-dependent (32)Pi and d-[(3)H]glucose uptake and activities of brush border enzymes membrane were determined. Brush border membrane vesicles prepared from rats fed a low phosphate diet showed significantly higher Na(+)-dependent (32)Pi uptake compared with rats fed a normal phosphate diet. This increase in (32)Pi uptake was completely prevented when rats on a low phosphate diet were simultaneously treated with actinomycin D. These differences were specific for (32)Pi transport as no differences were observed in d-[(3)H]glucose uptake among the three groups. There was a positive correlation (r = 0.82, P < 0.01) between (32)Pi uptake and specific activity of alkaline phosphatase measured in aliquots of the same brush border membranes, whereas no such correlation was observed with two other brush border membrane enzymes gamma-glutamyltransferase and leucine aminopeptidase. These observations show that actinomycin D prevents both the functional and cellular renal adaptive changes induced by a low phosphate diet. Taken together, these observations suggest that renal adaptation to a low phosphate diet could be prevented by inhibition of de novo protein synthesis.

The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface

The Na(+)- and Cl(-)-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodium-myo-inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver.

Osmotic regulation of renal betaine transport: transcription and beyond.

Cells in the kidney inner medulla are routinely exposed to high extracellular osmolarity during normal operation of the urinary concentrating mechanism. One adaptation critical for survival in this environment is the intracellular accumulation of organic osmolytes to balance the osmotic stress. Betaine is an important osmolyte that is accumulated via the betaine/γ-aminobutyric acid transporter (BGT1) in the basolateral plasma membrane of medullary epithelial cells. In response to hypertonic stress, there is transcriptional activation of the BGT1 gene, followed by trafficking and membrane insertion of BGT1 protein. Transcriptional activation, triggered by changes in ionic strength and water content, is an early response that is a key regulatory step and has been studied in detail. Recent studies suggest there are additional post-transcriptional regulatory steps in the pathway leading to upregulation of BGT1 transport, and that additional proteins are required for membrane insertion. Reversal of this adaptive process, upon removal of hypertonic stress, involves a rapid efflux of betaine through specific release pathways, a reduction in betaine influx, and a slower downregulation of BGT1 protein abundance. There is much more to be learned about many of these steps in BGT1 regulation.

DIFFERENTIAL ACTIVATION OF SYSTEM A AND BETAINE/GABA TRANSPORT IN MDCK CELL MEMBRANES BY HYPERTONIC STRESS

Accumulation of osmolytes by renal cells is due in part to increased uptake via specific transporters. These include amino acid transport system A and the betaine/GABA transporter (BGT1). Transport changes have been characterized using intact cells which makes the intracellular mechanisms difficult to determine. In this study the hypertonic upregulation of system A and BGT1 was studied directly at the membrane level in Madin-Darby canine kidney (MDCK) cells. Both system A and BGT1 transport systems were detected in an isolated membrane fraction containing plasma membranes. System A transport was increased in membranes prepared from cells after 6 h hypertonic stress (449 mosmol/kg) but BGT1 activity was minimal and not different from isotonic controls. The increase in system A was blocked by inhibitors of RNA and protein synthesis. BGT1 transport was induced in membranes prepared after 24 h hypertonicity. At this time system A activity in the membrane fraction remained increased, unlike the downregulation observed in intact MDCK cells. We conclude that differential upregulation of system A and BGT1 by hypertonic stress is due to intrinsic changes in these transporters at the membrane level. In contrast, the downregulation of system A in intact cells when hypertonicity is prolonged for 24 h is likely due to the action of an intracellular repressor that is not present in the isolated membranes.

Betaine chemistry, roles, and potential use in liver disease

BACKGROUND Betaine is the trimethyl derivative of glycine and is normally present in human plasma due to dietary intake and endogenous synthesis in liver and kidney. Betaine is utilized in the kidney primarily as an osmoprotectant, whereas in the liver its primary role is in metabolism as a methyl group donor. In both organs, a specific betaine transporter mediates cellular uptake of betaine from plasma. The abundance of both betaine and the betaine transporter in liver greatly exceeds that of other organs. SCOPE OF REVIEW The remarkable contributions of betaine to normal human and animal health are summarized together with a discussion of the mechanisms and potential beneficial effects of dietary betaine supplements on liver disease. MAJOR CONCLUSIONS A significant amount of data from animal models of liver disease indicates that administration of betaine can halt and even reverse progression of the disruption of liver function. Betaine is well-tolerated, inexpensive, effective over a wide range of doses, and is already used in livestock feeding practices. GENERAL SIGNIFICANCE The accumulated data indicate that carefully controlled additional investigations in humans are merited. The focus should be on the long-term use of betaine in large patient populations with liver diseases characterized by development of fatty liver, especially non-alcoholic fatty liver disease and alcoholic liver disease.

The betaine/GABA transporter and betaine: roles in brain, kidney, and liver

The physiological roles of the betaine/GABA transporter (BGT1; slc6a12) are still being debated. BGT1 is a member of the solute carrier family 6 (the neurotransmitter, sodium symporter transporter family) and mediates cellular uptake of betaine and GABA in a sodium- and chloride-dependent process. Most of the studies of BGT1 concern its function and regulation in the kidney medulla where its role is best understood. The conditions here are hostile due to hyperosmolarity and significant concentrations of NH4Cl and urea. To withstand the hyperosmolarity, cells trigger osmotic adaptation, involving concentration of a transcriptional factor TonEBP/NFAT5 in the nucleus, and accumulate betaine and other osmolytes. Data from renal cells in culture, primarily MDCK, revealed that transcriptional regulation of BGT1 by TonEBP/NFAT5 is relatively slow. To allow more acute control of the abundance of BGT1 protein in the plasma membrane, there is also post-translation regulation of BGT1 protein trafficking which is dependent on intracellular calcium and ATP. Further, betaine may be important in liver metabolism as a methyl donor. In fact, in the mouse the liver is the organ with the highest content of BGT1. Hepatocytes express high levels of both BGT1 and the only enzyme that can metabolize betaine, namely betaine:homocysteine –S-methyltransferase (BHMT1). The BHMT1 enzyme removes a methyl group from betaine and transfers it to homocysteine, a potential risk factor for cardiovascular disease. Finally, BGT1 has been proposed to play a role in controlling brain excitability and thereby represents a target for anticonvulsive drug development. The latter hypothesis is controversial due to very low expression levels of BGT1 relative to other GABA transporters in brain, and also the primary location of BGT1 at the surface of the brain in the leptomeninges. These issues are discussed in detail.

Osmoregulation of Neutral Amino Acid Transport

Abstract Neutral amino acids are important organic osmolytes found in most osmotically stressed cells. During prolonged hypertonic stress, accumulation of neutral amino acids contributes to the cell volume recovery and may protect cellular proteins against salt denaturation. Hypertonicity activates system A transport, a major neutral amino acid transporter in mammalian cells, by a microtubule-dependent mechanism. Similar to the regulation of amino acid efflux induced by hypotonic-swelling, influx via system A may be under the control of intracellular ionic strength. An osmotically sensitive repressor may negatively regulate the gene expression of a regulatory protein which activates the preexisting system A transporter protein. Hypertonically activated protein kinases may be involved in this osmoregulation of amino acid transport.

Current concepts of regulation of phosphate transport in renal proximal tubules.

The wealth of new information on BBM transport of Pi which has accumulated in recent years gives an indication of the importance and intellectual challenge that the mechanism of this process poses to investigators. In this brief reflection on the field, we have tried to draw attention to some general principles and features which may be helpful as working hypotheses in the development of the field. To date, a disproportionate amount of effort may have been spent on deciphering putative intracellular regulatory mechanisms, without knowing some essential fundamental properties of the Na+-Pi-COT. We suggest that a major effort should be exerted towards elucidating biogenesis of the Na+-Pi-COT, the possible existence of a membrane cycling mechanism, and a refined analysis of the Na+-Pi-COT in specific subsegments of proximal tubules. Advances in these areas together with studies of both the rapid and long-term adaptive regulation of Pi transport are needed, given the central role of the kidney in total body Pi homeostasis both in health and disease.

Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells

Extracellular ATP interacts with purinergic P2 receptors to regulate a range of physiological responses, including downregulation of transport activity in the nephron. ATP is released from cells by mechanical stimuli such as cell volume changes, and autocrine signaling by extracellular ATP could occur in renal medullary cells during diuresis. This was tested in Madin-Darby canine kidney (MDCK) cells, a model used frequently to study P1 and P2 receptor activity. ATP was released within 1 min after transfer from 500 to 300 mosmol/kgH2O medium. A 30-min incubation with ATP produced dose-dependent inhibition (0.01-0.10 mM) of the renal betaine/GABA transporter (BGT1) with little effect on other osmolyte transporters. Inhibition was reproduced by specific agonists for P2X (alpha,beta-methylene-ATP) and P2Y (UTP) receptors. Adenosine, the final product of ATP hydrolysis, also inhibited BGT1 but not taurine transport. Inhibition by ATP and adenosine was blocked by pertussis toxin and A73122, suggesting involvement of inhibitory G protein and PLC in postreceptor signaling. Both ATP and adenosine (0.1 mM) produced rapid increases in intracellular Ca2+, due to the mobilization of intracellular Ca2+ stores and Ca2+ influx. Blocking these Ca2+ increases with BAPTA-AM also blocked the action of ATP and adenosine on BGT1 transport. Finally, immunohistochemical studies indicated that inhibition of BGT1 transport may be due to endocytic accumulation of BGT1 proteins from the plasma membrane. We conclude that ATP and adenosine, through stimulation of PLC and intracellular Ca2+, may be rapidly acting regulators of BGT1 transport especially in response to a fall in extracellular osmolarity.