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Background/Aims: Total body phosphate homeostasis is regulated by expression of type IIa sodium phosphate cotransporter (NpT2a) in the apical membrane (BBM) of renal proximal tubule cells. NpT2a expression is regulated by dietary phosphate and PTH but the mechanisms for trafficking of the protein are unknown. Based on 2D gel electrophoresis and mass spectroscopy data that changes in dietary phosphate stimulated changes in BBM expression of vacuolar H+-ATPase, we hypothesized that vacuolar H+-ATPase plays a significant role in regulation of NpT2a in opossum kidney (OK) cells, a model for renal proximal tubule transport. Methods: Role of vacuolar H+-ATPase was studied in opossum kidney (OK) cells by examining the effect of inhibition of vacuolar H+-ATPase on Pi uptake and NpT2a expression. Results: Pretreatment of OK cells with bafilomycin A1 and concanamycin A, inhibitors of vacuolar H+-ATPases, blocked high phosphate- and PTH-induced degradation of NpT2a, but had no effect on high phosphate or PTH induced inhibition of sodium-dependent phosphate transport. Exposure of the cells to bafilomycin A1 significantly decreased phosphate transport and apical membrane expression of NpT2a. Treatment with brefeldin A, an inhibitor of Golgi transport, decreased phosphate transport and apical membrane expression of NpT2a while treatment of cells with both brefeldin A and bafilomycin A1 had no additive effect. Conclusion: We conclude that vacuolar H+-ATPase plays a significant role in exocytosis of NpT2a into the apical membrane and in degradation of NpT2a but has no role in endocytosis. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Acute renal failure (ARF) is associated with hyperphosphatemia and decreased urinary phosphate excretion. The present study was undertaken to characterize the effects of ARF due to ischemia and reperfusion on renal phosphate transport and on gene and pro- tein expression of type IIa NaPi cotransporter (Npt2) the physiologically most relevant renal sodium-dependent phosphate cotransporter. Methods: The following groups of rats with intact parathyroid glands were studied: (1) sham operated (sham); (2) after 1 h ischemia by bilateral renal artery clamping (I), and after 1 h ischemia and reperfusion of 1 h (I + R 1 h); (3) 24 h (I + R 24 h); (4) 48 h (I + R 48 h), and (5) 72 h (I + R 72 h) duration. The effect of ARF on Npt2 mRNA and protein expression was also examined after parathyroidectomy (PTX) of 2 and 4 days’ duration. Results: Ischemia and reperfusion were associated with increases in plasma creatinine, hyperphosphatemia, and with decreased tubular phosphate reabsorption. Npt2 mRNA was significantly downregulated in the cortex, maximal at 24 and 48 h of reperfusion. The degree of Npt2 mRNA downregulation was not affected by PTX of 2–4 days’ duration. The abundance of Npt2 protein in proximal tubular apical brush border membrane was markedly decreased after reperfusion. Npt2 protein, however, was more abundant in PTX animals than in those with intact parathyroids and a similar degree of renal insufficiency. The immunohistochemical analysis of proximal tubular apical brush border membrane showed a progressive decrease of Npt2 protein labeling after ischemia and reperfusion, with progressive regeneration after 72 h. Conclusion: These results suggest that downregulation of Npt2 protein may contribute to the decreased tubular reabsorption of phosphate in acute ischemic renal failure and hyperphosphatemia. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample allow kinetic and diffusion properties of fluorescently labeled molecules to be determined, as well as their concentration and aggregation state. Data from multiple points of the sample can be acquired using scanning-FCS, image correlation spectroscopy, and raster image correlation spectroscopy. These techniques cover phenomena with characteristic durations from sub-microsecond to second time scales. The power of FLIM lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. FLIM is a robust means to quantify Förster resonance energy transfer and thus determine protein-protein interactions or protein conformational changes. In addition, FLIM is very valuable for functional imaging of ion concentrations in cells and tissues as it can be applied in heterogeneously labeled samples. In summary, FCS and FLIM allow information to be gathered beyond localization, including diffusional mobility, protein clustering and interactions, and molecular environment. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]