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

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Role of Cholesterol in the Regulation of Renal Phosphate Transport

4. Cellular Mechanisms as How Cholesterol Modulates Renal Phosphate Transport

5. Summary

6. Acknowledgments

7. References

The kidney plays a critical role in the regulation of inorganic phosphate (Pi) homeostasis through changes in the proximal tubular apical membrane Na-dependent Pi (Na/Pi) transport activity. In response to alterations in dietary Pi intake and during the aging process, changes in renal Na/Pi transport activity are inversely correlated with apical membrane cholesterol content. Cholesterol regulates Na/Pi transport activity by fluidity-dependent and fluidity-independent mechanisms, including regulation of Na/Pi protein transcription, synthesis, and trafficking to and from the plasma membrane.

2. INTRODUCTION

Lipids play an essential structural role as a barrier between intra and extracellular medium and as a matrix providing a suitable hydrophobic environment for membrane proteins. The functional role of lipids as a selective barrier to fully or moderately water soluble compounds, and as modulators of membrane transport proteins, enzymes, and channels activity have been well recognized (1-4).

In this report we will discuss the evidence that has been obtained during the past decade which indicates an important role for alterations in renal proximal tubular cell membrane cholesterol content in the regulation of renal phosphate transport activity.

The kidney plays a critical role in the regulation of inorganic phosphate (Pi) homeostasis. Pi is freely filtered across the glomerules, and most of the Pi is reabsorbed along the proximal tubule via a sodium gradient-dependent process (Na/Pi co-transport). The Na/Pi cotransporter is located on the apical brush border membrane (BBM) of the proximal tubule. The evidence, to date, indicates that regulation of the overall renal tubular Pi transport by dietary, hormonal, or metabolic factors occurs at the level of the proximal tubular BBM Na/Pi cotransport system (5-8).

3. Role of Cholesterol in the Regulation of Renal Phosphate Transport

The first evidence that alterations in BBM cholesterol content may modulate Na/Pi cotransport activity was provided by Molitoris et al. (9) who showed that in rats chronically fed a low Pi diet the adaptive increase in the renal tubular reabsorption of phosphate was associated with a decrease in BBM cholesterol content. In fact, the authors demonstrated an inverse linear relationship between the tubular reabsorption of Pi and BBM cholesterol content. Since a decrease in BBM cholesterol also resulted in an increase in BBM fluidity, this study suggested that either a decrease in BBM cholesterol content or an increase in BBM fluidity could play a role in the regulation of Na/Pi transport. Subsequent studies with isolated BBM and/or renal tubular cells grown in culture demonstrated that a direct increase in membrane fluidity indeed results in an increase in Na/Pi cotransport activity (10-12). Further studies in our laboratory confirmed the findings of Molitoris et al., and also showed that in aged rats the decrease in the renal tubular reabsorption of Pi was associated with an increase in BBM cholesterol content (Figure 1) and a decrease in BBM fluidity (13).

Figure 1: There is an inverse relationship between BBM Cholesterol content and BBM Na/Pi cotransport activity in young versus aged rats and in rats fed a low Pi versus a high Pi diet. Data adapted from References 13 and 14.

In studies designed to determine if changes in BBM cholesterol content per se do play a role in the modulation of BBM Na/Pi cotransport (14), we first showed that in BBM isolated from young rats chronically fed a low Pi diet in vitro enrichment with cholesterol, to levels present in young rats chronically fed a high Pi diet, completely reversed the adaptive increase in Na/Pi cotransport activity (Figure 2).

Figure 2: In rats which have been fed a low Pi diet, in vitro enrichment of the isolated BBM with cholesterol, to levels found in rats fed a normal Pi diet, reverses the adaptive increases in BBM Na/Pi cotranport activity (right panel) and BBM fluidity (inversely related to the fluorescence polarization of diphenylhexatriene, r_DPH, left panel). Adapted from Reference 14.

In subsequent studies, we showed that in BBM isolated from young rats in vitro enrichment with cholesterol, to levels present in aged rats, reproduced the age-related decrease in Na/Pi cotransport activity (Figure 3). Of interest, this effect of cholesterol was specific and selective for Na/Pi cotransport, as cholesterol enrichment had no effect on BBM Na/glucose, Na/proline, and Na/sulfate cotransport, or Na/H exchange activities (14).

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).

5. SUMMARY

In summary, in addition to the chemical and physical effects on the Na/Pi cotransport proteins at the level of the plasma membrane, in intact renal tubular cells cholesterol could also regulate Na/Pi cotransport activity by modulating Na/Pi mRNA level and/or BBM Na/Pi protein abundance (Table 1).

Further studies in renal tubular cells grown in culture should elucidate the cellular and molecular mechanisms how perturbations in cholesterol metabolism and cholesterol content modulate Na/Pi cotransport activity.

6. ACKNOWLEDGMENTS

This work was supported by grants from Department of Veterans Affairs Merit Review and the National Kidney Foundation of Texas. The authors thank Sandra Nickerson for Secretarial Assistance and the Biomedical Media Service at the Dallas VAMC for the illustrations.

7. REFERENCES

1. Le Grimellec, C., Friedlander, G., Hossain, E., Yandouzi, E., Zlatkine, P., & M. Giocondi: Membrane fluidity and transport properties in epithelia. Kidney Int 42, 825-36, (1992)

2. Aloia, R., Tian, H., & F. Jansen: Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membrane. Proc Nat Acad Sci USA 90, 5181-85, (1993)

3. C. Grant: Lateral phase separations and the cell membrane. In Membrane Fluidity in Biology, Vol 2, R. C. Aloia editor, Acad Press NY 131-50, (1983)

4. Maresca, B., & A. Cossins: Cell physiology - fatty feedback and fluidity. Nature (London) 365, 606-7, (1993)

5. T. Dousa: Modulation of renal Na/Pi cotransport by hormones acting via genomic mechanism and by metabolic factors. Kidney Int 49, 997-1004, (1996)

6. S. Kempson: Peptide hormone action on renal phosphate handling. Kidney Int 49, 1005-9, (1996)

7. Lotscher, M., Wilson, P., Nguyen, S., Kaissling, B., Biber, J., Murer, H., & M. Levi: New aspects of adaptation of rat renal Na/Pi cotransporter to alterations in dietary phosphate. Kidney Int 49, 1012-18, (1996)

8. Levi, M., Kempson, S., Lotscher, M., Biber, J., H. Murer: Molecular Regulation of Renal Phosphate Transport. J Membrane Biol 154, 1-9, (1996)

9. Molitoris, B., Alfrey, A., Harris, R., & F. Simon: Renal apical membrane cholesterol and fluidity in regulation of phosphate transport. Am J Physiol 249, F12-19, (1985)

10. Friedlander, G., Le Grimellec, C., Giocondi, M., & C. Amiel: Benzyl alcohol increases membrane fluidity and modulates cyclic AMP synthesis by renal proximal tubular cells in primary culture. Biochim Biophys Acta 1022, 1-7, (1990)

11. Friedlander, G., Shahedi, M., Le Grimellec, C., & C. Amiel: Increase in membrane fluidity and opening of tight junctions have similar effects on sodium- coupled uptakes in renal epithelial cells. J Biol Chem 263, 11183-88, (1988)

12. Yusufi, A., Szczepanska-Konkel, M., Hoppe, A., & T. Dousa: Different mechanisms of adaptive increase in Na⁺-Pi cotransport across renal brush-border membrane. Am J Physiol 256, F852-61, (1989)

13. Levi, M., Jameson, D., & W. Van der Meer: Role of BBM lipid composition and fluidity in impaired renal Pi transport in aged rat. Am J Physiol 256, F85-94, (1989)

14. Levi, M., Baird, B., & P. Wilson: Cholesterol Modulates Rat Renal Brush Border Membrane Phosphate Transport. J Clin Invest 85, 231-37, (1990)

15. Levi, M., Wilson, P. V., Cooper, O. J., & E. Gratton: Lipid Phases in Renal Brush Border Membranes Revealed by Laurdan Fluorescence. Photochemistry and Photobiology 57, 420-25, (1993)

16. Blitterswijk, W., Van der Meer, B., & H. Hilkman: Quantitative contributions of cholesterol, and the individual classes of phospholipids and their degree of fatty acyl (un) saturation to membrane fluidity measured by fluorescence polarization. Biochemistry 26, 1746-56, (1987)

17. Blitterswijk, V.,Van Hoeven,R., & B. Van der Meer: Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence depolarization measurements. Biochim Biophys Acta 644, 323-32, (1981)

18. Parasassi, T., De Stasio, G., d'Ubaldo, A., & E. Gratton: Phase fluctuation in phospholipid membranes revealed by LAURDAN fluorescence. Biophys J 57, 1179-86, (1990)

19. Parasassi, T., Giusti, A., Raimondi, M., & E. Gratton: Abrupt modification of phospholipid bilayer properties at critical cholesterol concentrations. Biophys J 68, 1895-1902, (1995)

20. Levi, M., Shayman, J., Abe, A., Gross, S., McCluer, R., Biber, J., Murer, H., Lotscher, M., & R. Cronin: Dexamethasone Modulates Rat Renal Brush Border Membrane Phosphate Transporter mRNA and Protein Abundance and Glycosphingolipid Composition. J Clin Invest 96, 207-16, (1995)

21. So, P., French, T., Yu, W., Berland, K., Dong, C., & E. Gratton: Time-resolved microscopy using two-photon excitation. Bioimaging 3, 49-63, (1995)

22. Yu, W., So, P., French, T., E. Gratton: Fluorescence Generalized Polarization of Cell Membranes: A Two-Photon Scanning Microscopy Approach. Biophys J 70, 626-36, (1996)

23. Parasassi, T., Gratton, E., Yu, W., Wilson, P., M. Levi: Two-photon fluorescence microscopy of LAURDAN GP-domains in model and natural membranes. Biophys J, (In press).

24. Levi, M., Lotscher, M., Sorribas, V., Custer, M., Arar, M., Kaissling, B., Murer, H., & J. Biber: Cellular Mechanisms of Acute and Chronic Adaptation of Rat Renal Phosphate Transporter to Alterations in Dietary Phosphate. Am J Physiol 267, F900-8, (1994)

25. Sorribas, V., Lotscher, M., Loffing, J., Biber, J., Kaissling, B., Murer, H., M. Levi M: Cellular mechanisms of the age-related decrease in renal phosphate reabsorption. Kidney Int 50, 855-863, (1996)

26. G. Gasic: Basic-Helix-Loop-Helix Transcription Factor and Sterol Sensor in a Single Membrane-Bound Molecule. Cell 77, 17-19, (1994)

27. Wang, X., Sato, R., Brown, M., Hua , X., J. Goldstein: SREBP-1, a Membrane- Bound Transcription Factor Released by Sterol-Regulated Proteolysis. Cell 77, 53-62, (1994)

28. Boulanger, C., Tanner, F., Béa, M., Hahn, A., Werner, A., T. Lotscher: Oxidized Low Density Lipoproteins Induce mRNA Expression and Release of Endothelin From Human and Porcine Endothelium. Circulation Research 70, 1191-7, (1992)

29. Ding, G., Pesek-Diamond, I., J. Diamond: Cholesterol, macrophages, and gene expression of TGF-ß1 and fibronectin during nephrosis. Am J Physiol 264, F577- 584, (1993)

30. Chang, W., Rothberg, K., Kamen, B., Anderson: Lowering the Cholesterol Content of MA104 Cells Inhibits Receptor-mediates Transport of Folate. J Cell Biol 118, 63-9, (1992)

31. Cerneus, D., Ueffing, E., Posthuma, G, Strous, G., A. van der Ende: Detergent insolubility of Alkaline Phosphatase during biosynthetic Transport and Endocytosis. Role of Cholesterol. J Biol Chem 268, 3150-55, (1993)