Colloidal phenomena in porous media, natural or engineered, are important in a breadth of science and technology applications, but fundamental understanding is hampered by the difficulty in measuring colloid deposit morphology in situ. To partially address this need, this paper describes a static light scattering apparatus using a flow cell filled with refractive index matched (RIM) porous media, allowing real-time measurement of colloidal phenomena as a function of depth within the flow cell. A laser interacts with the colloids in the pore space and their structures, but not with the RIM media. The intensity of scattered light is measured as a function of scattering angle, which allows characterization of colloid deposit morphology as a fractal dimension and a radius of gyration. In parallel, fluid discharge rate and pressure drop are recorded to determine permeability, a key parameter for any application involving flow through porous media. This apparatus should prove useful in any application requiring characterization of colloidal phenomena within porous media. Additionally, this paper describes how to use granular Nafion as RIM porous media.

Although in vivo and cell culture studies have provided useful information about the regulation of the sodium phosphate (NaPi) cotransporters, such studies are unable to provide information at the molecular level about interactions between proteins. The NaPi proteins are found within both intestinal and renal brush border microvilli, and previous work has shown that these microvilli contain scaffolding proteins (PDZ proteins) and myosin motors. The recent development of several advanced imaging techniques has allowed detailed analysis of how NaPi proteins interact with scaffolding proteins and myosin motors. Using techniques such as apical total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, raster image correlation spectroscopy, and fluorescence lifetime imaging-Förster resonance energy transfer, we have found that a myosin motor is involved in trafficking of the NaPi cotransporters and also that Npt2a and Npt2c seem to have different affinities for the PDZ protein Na+/H+ exchanger regulatory factor 1. Further application of these techniques will provide additional insights into NaPi trafficking and regulation.

In renal proximal tubule (PT) cells, sodium-phosphate cotransporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ∼70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low-Pi conditions, NaPiIIa is retained within the apical membrane. Under high-Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney cells, a PT cell model, knockdown of Shank2 in cells maintained in low-Pi media resulted in a marked decrease in NaPiIIa abundance. After being transferred into high-Pi media, live-cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the subapical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the subapical domain in temporal synchrony. Raster image cross-correlation spectroscopy demonstrated these two proteins course through the subapical domain in temporal-spatial synchrony. In the microvilli of cells under low-Pi conditions and in the subapical domain of cells under high-Pi conditions, fluorescence lifetime imaging microscopy-Forster resonance energy transfer analysis of Cer-NaPiIIa and EYFP-Shank2E found these fluors reside within 10 nm of each other. Demonstrating a complexity of functions, in cells maintained under low-Pi conditions, Shank2 plays an essential role in the apical retention of NaPiIIa while under high-Pi conditions Shank2 remains associated with NaPiIIa and escorts NaPiIIa through the cell interior.

The sodium-dependent phosphate (Na/P(i)) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P(i). The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P(i) reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P(i) transporters. Pdzk1(-/-) mice adapted to chronic low P(i) diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P(i) transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P(i) transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P(i) concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P(i) transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c.

The kidney is a key regulator of phosphate homeostasis. There are two predominant renal sodium phosphate cotransporters, NaPi2a and NaPi2c. Both are regulated by parathyroid hormone (PTH), which decreases the abundance of the NaPi cotransporters in the apical membrane of renal proximal tubule cells. The time course of PTH-induced removal of the two cotransporters from the apical membrane, however, is markedly different for NaPi2a compared with NaPi2c. In animals and in cell culture, PTH treatment results in almost complete removal of NaPi2a from the brush border (BB) within 1 h whereas for NaPi2c this process in not complete until 4 to 8 h after PTH treatment. The reason for this is poorly understood. We have previously shown that the unconventional myosin motor myosin VI is required for PTH-induced removal of NaPi2a from the proximal tubule BB. Here we demonstrate that myosin VI is also necessary for PTH-induced removal of NaPi2c from the apical membrane. In addition, we show that, while at baseline the two cotransporters have similar diffusion coefficients within the membrane, after PTH addition the diffusion coefficient for NaPi2a initially exceeds that for NaPi2c. Thus NaPi2c appears to remain "tethered" in the apical membrane for longer periods of time after PTH treatment, accounting, at least in part, for the difference in response times to PTH of NaPi2a versus NaPi2c.