Biomedical subjects
Publications and source records attributed to Victor Sorribas.
The renal regulation of phosphate is a complex process. Clinical disorders of phosphate handling have led to the identification of several genes and proteins involved in the maintenance of phosphate homeostasis. Further work is required to elucidate the precise pathways that regulate renal phosphate transport.
We previously showed an inverse correlation between membrane cholesterol content and Na-P(i) cotransport activity during the aging process and adaptation to alterations in dietary P(i) in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-P(i) cotransport activity and apical membrane Na-P(i) protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with beta-methyl cyclodextrin (beta-MCD) resulted in a significant increase in Na-P(i) cotransport activity accompanied by a moderate increase in apical membrane Na-P(i) protein abundance and no alteration of total cellular Na-P(i) protein abundance. Conversely, acute cholesterol enrichment achieved with beta-MCD/cholesterol resulted in a significant decrease in Na-P(i) cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-P(i) protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-P(i) cotransport activity by diverse mechanisms that also include significant interactions of Na-P(i) protein with lipid microdomains.
In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.
An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the NH2-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the NH2 terminus of NaPi-IIa within the PDZK1/NaPi-IIa/MAP17 complex, and 3) acts as an apical anchoring site for PDZK1.
Renal reabsorption is the main mechanism that controls mannose homeostasis. This takes place through a specific Na-coupled uphill transport system, the molecular identity of which is unknown. We prepared and screened a size-selected rat kidney cortex cDNA library through the expression of mannose transport in Xenopus laevis oocytes. We have identified a membrane protein that induces high-affinity and specific Na-dependent transport of d-mannose and d-glucose in X. laevis oocytes, most likely through stimulation of the capacity of an endogenous transport system of the oocyte. Sequencing has revealed that the cDNA encodes the counterpart of the human membrane-associated protein MAP17, previously known by its overexpression in renal, colon, lung, and breast carcinomas. We show that MAP17 is a 12.2-kDa nonglycosylated membrane protein that locates to the brush-border plasma membrane and the Golgi apparatus of transfected cells and that it is expressed in the proximal tubules of the kidney cortex and in the spermatids of the seminiferous tubules. It spans twice the cell membrane, with both termini inside the cell, and seems to form homodimers through intracellular Cys55, a residue also involved in transport expression. MAP17 is responsible for mannose transport expression in oocytes by rat kidney cortex mRNA. The induced transport has the functional characteristics of a Na-glucose cotransporter (SGLT), because d-glucose and alpha-methyl-d-glucopyranoside are also accepted substrates that are inhibited by phloridzin. The corresponding transporter from the proximal tubule remains to be identified, but it is different from the known mammalian SGLT-1, -2, and -3.