Huamin Wang, Hubert Zajicek, Vijay Kumar, Paul Wilson, and Moshe Levi

Department of Medicine, The University of Texas Southwestern Medical Center at Dallas and Department of Veterans Affairs Medical Center, Dallas, Texas 75216, USA

Received 1/13/97; Accepted 1/13/97; On-line 02/01/96

4. Cellular Mechanisms as How Cholesterol Modulates Renal Phosphate Transport

Since changes in BBM cholesterol content causes simultaneous changes in BBM fluidity (15,16), in the next series of studies we tried to determine if changes in BBM cholesterol content per se, or changes in BBM fluidity, or both modulate Na/Pi cotranport activity. In our studies, this was achieved by first modulating BBM cholesterol content in vitro, and then measuring BBM fluidity and BBM Na/Pi transport activity at different temperatures. Our measurements indicated that there was a direct correlation between an increase in BBM fluidity and an increase in BBM Na/Pi cotransport activity. However, at a given level of BBM fluidity, increasing BBM cholesterol content still caused a decrease in BBM Na/Pi cotransport activity (Figure 3). The data, therefore, indicates that perturbations in BBM cholesterol content and/or BBM fluidity each, independent of each other, modulate BBM Na/Pi cotransport activity.

In these studies BBM fluidity was measured by the steady-state fluorescence polarization of 1, 6-diphenyl 1,2,5,-hexatriene (DPH), which measures the time-averaged structural and dynamic properties which determine the relative order and motions of the lipid molecules in the membrane bilayer (17). In subsequent studies, when we have also performed time resolved measurements with DPH, and steady-state and time-released measurements with the phase sensitive probe 6-dodecanoyl-2-diethylamino-naphthalane (Laurdan) ( 15, 18, 19, 20), we have found evidence for the presence of lipid microdomains in the BBM. In fact, in recent studies using the two-photon fluorescence microscopy of Laurdan (21-23), we have provided evidence for the presence of lipid domains within the BBM lipid bilayer of differing fluidity values (Figure 4). We have further shown that cholesterol strongly modulates the distribution of these lipid domains. It is therefore possible that cholesterol modulates BBM Na/Pi cotransport, in part, by influencing the distribution of lipid microdomains surrounding the Na/Pi cotransporter proteins, which in turn could modulate their transport function by influencing their diffusion within the BBM lipid bilayer. Alternatively, cholesterol could also modulate Na/Pi cotransport activity by direct biochemical modification of the Na/Pi cotransporters.

Figure 4: Generalized Polarization (GP) histogram of BBM labeled with Laurdan. Excitation GP is calculated according to GP = (I₄₄₀ - I₄₉₀/I₄₄₀ + I₄₉₀), where I₄₄₀ and I₄₉₀ are the fluorescence emission intensities at 440 and 490 nm. Adapted from reference 23.

The major question is whether the decrease in renal cholesterol content during chronic dietary Pi deprivation and/or the age-related increase in renal cholesterol content can modulate Na/Pi cotranport activity through transcriptional and translational regulation. Recent studies indicate that alterations in cholesterol content control cellular functions by diverse mechanisms. In human HeLa cells and fibroblasts, a decrease in membrane cholesterol induces proteolysis of a membrane-bound transcription factor and activates transcription of the genes for the LDL receptor and HMG CoA synthase (26-27). Cholesterol also regulates endothelin gene expression in endothelial cells (28). In rats, a high cholesterol diet causes increases in the glomerular transforming growth factor ß (TGF-ß₁) and fibronectin mRNA levels (29). In MA 104 cells, a monkey kidney epithelial cell line, lowering the cell cholesterol content decreases the clustering of the GPI-anchored folate receptors and inhibits receptor-mediated transport of folate (30). Cholesterol also modulates the apical membrane domain formation and internalization of alkaline phosphatase, another GPI-anchored protein (31).