The water-soluble vitamin riboflavin (RF) is involved in key metabolic reactions and is essential for normal cellular functions and growth. Deficiency of RF occurs in humans during periods of physiological and pathological stress and leads to a variety of clinical abnormalities, including growth retardation and degenerative changes in the nervous system. The kidney is the main organ that eliminates RF from the body and therefore plays a key role in regulating body RF homeostasis. Depending on its plasma concentration, RF has been found to be transported bi-directionally in the renal tubule, i.e., reabsorbed and secreted. The reabsorption and secretion processes of RF represent movement of the vitamin across the highly polarized renal epithelial cells. These cells have two membrane domains-the brush border membrane (BBM) and basolateral membrane (BLM)- which are different in their composition, function and permeability. Little is known about the mechanisms involved in RF trans-epithelial transport processes (i.e., reabsorption and secretion) and of their individual trans-membrane transport events (influx and efflux) across the BBM and BLM. Also, nothing is known about the molecular nature of the transport systems involved. We will address these issues in this proposal. Membrane transport processes of many nutrients in different biological systems are regulated by their level in athe diet (i.e., in the body). The effect of RF deficiency and over-supplementation on RF renal reabsorption and secretion transport processes are not known and will be examined in this study. Thyroid hormone has profound effects on may aspects of RF metabolism and physiology, including the urinary excretion of the vitamin. The role of thyroid hormone in regulating RF renal transport processes is not known and will be investigated. Phenothiazine derivatives like chlorpromazine and antidepressant agents like imipramine and amitriptyline share structural similarities with RF and have been found to affect many aspects of normal RF metabolism nd physiology including its urinary excretion. The effects of these widely used therapeutic agents on RF renal transport processes are not known and will be examined. Transport studies will be performed using rabbit isolated perfused proximal tubules in vitro, purified BBM vesicles and BLM vesicles and primary cultures of renal proximal tubular epithelial cells. Expression cloning studies will be performed using Xenopus laevis oocytes. The results of these studies will provide valuable information regarding the cellular and molecular mechanisms of renal transport of an essential nutrient and of the factors that regulate and interfere with the transport processes involved. This should also significantly expand our knowledge in the general area of water-soluble vitamin nutrition and physiology.
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