- Email: [email protected]
Mechanisms of albumin uptake by proximal tubular cells
Mechanisms of Albumin Uptake by Proximal Tubular Cells Nigel J. Brunskill, PhD, FRCP ● The likely role of albumin in the induction tubulo-interstitial injury in proteinuria has stimulated considerable interest in the entry of albumin into the proximal tubule and its subsequent uptake by proximal tubular cells. Currently, there is considerable controversy over the degree of glomerular permeability to albumin. After filtration, however, albumin binds to megalin and cubulin, two giant receptors in the apical membrane of proximal tubular cells. Albumin is subsequently re-absorbed by proximal tubular cells by receptor-mediated endocytosis, a process subject to complex regulation. The interaction of albumin with proximal tubule cells also leads to the generation of intracellular signals. The understanding of these pathways may provide important insights into the pathogenesis of renal scarring in proteinuria. © 2001 by the National Kidney Foundation, Inc. INDEX WORDS: Albumin; proximal tubule; endocytosis; megalin; cubulin.
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ROTEINURIA REPRESENTS an adverse clinical finding in patients with renal disease. Patients with heavy proteinuria are more likely to develop tubulointerstitial inflammation, scarring, and fibrosis and progress to end-stage renal failure. For many years, it was believed that excess albuminuria was simply a marker of more severe renal disease, which was more likely to progress as a result of this severity rather than as a result of the albuminuria itself. However, this view has been challenged recently with the accumulation of a body of evidence suggesting that albuminuria may injure proximal tubular epithelial cells (PTCs), thus precipitating a proinflammatory and profibrotic environment in the renal tubulointerstitium. The potential pathophysiological importance of albuminuria has stimulated considerable interest in the entry of albumin into the proximal tubule and the mechanisms of its subsequent interaction with the proximal tubular epithelium. GLOMERULAR FILTRATION OF ALBUMIN
The glomerular, as opposed to tubular, origin of urinary protein was established after a number of classical experiments performed in the early 20th century.1 The conventional paradigm of glomerular function indicates that permeability to albumin is low, with filtration of albumin abrogated by both size and charge selectivity of the glomerular basement membrane. Nonetheless, appreciable amounts of albumin enter the proximal tubule normally, and in rodents, albumin has been measured in proximal tubular fluid by means of micropuncture techniques. Although results have been variable, it is generally accepted that healthy proximal tubular fluid albu-
min concentrations are in the range of 10 to 30 g/mL.2 In recent years, this traditional view of glomerular filtration has been disputed. Osicka et al3 and Eppel et al4 suggested that albumin is very freely filtered by the glomerulus, with the consequence that proximal tubular albumin concentrations are very high, in the 2- to 3-mg/mL range. These same investigators indicated that the vast majority of this albumin is reabsorbed intact into the circulation early in the proximal tubule through a transtubular cell retrieval pathway. However, this pathway remains speculative and, to date, has not been shown biochemically or morphologically. It is not discussed further in this review. ALBUMIN BINDING TO PTCS
Binding studies in vitro have identified two albumin-binding sites in PTCs; one with high affinity but low capacity, and the other with low affinity but high capacity.5,6 The equilibrium dissociation constant (KD) of the higher affinity site suggests that such a receptor would be well placed to mediate binding and uptake of proximal tubular fluid albumin in health, whereas the site with lower affinity but higher From the Departments of Cell Physiology and Pharmacology and Nephrology, University of Leicester, Faculty of Medicine and Biological Sciences, Leicester, UK. Address reprint requests to Nigel J. Brunskill, PhD, FRCP, Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, Medical Sciences Bldg, University Rd, Leicester LE1 9HN, UK. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3701-0204$3.00/0 doi:10.1053/ajkd.2001.20733
American Journal of Kidney Diseases, Vol 37, No 1, Suppl 2 (January), 2001: pp S17-S20
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NIGEL J. BRUNSKILL
capacity binding may be responsible for increased albumin binding and uptake in nephrosis. Identification of albumin receptors has been the focus for researchers investigating the endothelium and kidney proximal tubule. In endothelia, a 60-kd glycoprotein receptor for albumin has been partially characterized and named “albondin.”7 This protein exists in endothelia together with a number of lower molecular-weight proteins in the range of 14 to 30 kd. The identities of the lower molecular-weight proteins are unclear. Early studies of PTCs identified similar low-molecular-weight proteins with albumin-binding activity, but albondin has not been found in PTCs.6 More recently, it has been convincingly shown in PTCs that albumin binds to two giant protein receptors, megalin and an associated protein, cubulin.8,9 At least some of the lower molecularweight albumin-binding proteins detected in earlier studies represent breakdown products of cubulin, but the possibility remains that some represent discrete albumin receptors in their own right. More work is required to clarify this issue. UPTAKE OF ALBUMIN BY PTCS
Binding of albumin to the receptors on the apical surface of PTCs is a prelude to its uptake by receptor-mediated endocytosis (RME). A number of elegant morphological studies have shown this pathway both in vivo and in vitro by using cultured PTCs, in which albumin can be followed from its binding in apical invaginations through an endosomal compartment culminating in the lysosome, where it is broken down to constituent amino acids.10 Park and Maack11 used perfused rabbit proximal tubular segments to show both highaffinity low-capacity and low-affinity highcapacity uptake systems for albumin. This model is obviously compatible with the results of the albumin-binding experiments previously described. Opossum kidney (OK) cells have provided a particularly useful in vitro model for the detailed study of the regulatory mechanisms of albumin reabsorption in the proximal tubule. Albumin RME in OK cells is dependent on both cyclic adenosine monophosphate and extracellular calcium ions, with in-
creasing intracellular cyclic adenosine monophosphate and removal of extracellular calcium ions, both leading to a reduction in albumin uptake.12 Data suggest the involvement of several protein kinases, notably protein kinase A, protein kinase C, and phosphatidylinositol 3-kinase (PI 3-kinase) in the regulation of albumin RME in the proximal tubule.13,14 Furthermore, the heterotrimeric guanosine triphosphate– binding protein ␣-subunit, G␣i-3, is also involved in regulating the endocytic uptake of albumin in OK cells.15 Not surprisingly, disruption of the actin cytoskeleton and inhibition of microtubule polymerization also block albumin RME.16 Alkalinization of endosomes interferes with intracellular ligand handling and trafficking and recycling of receptors. Inhibition of the vacuolar adenosinetriphosphatase with bafilomycin leads to an increase in endosomal pH, and a similar effect can be achieved by incubation of cells with NH4Cl. Endosomal alkalinization precipitated by either of these agents is accompanied by a marked reduction in both the affinity constant (Km) and maximal transport activity (Jmax) for albumin RME in OK cells.13 However, fluid-phase endocytosis is unaffected by these maneuvers. The Na⫹-H⫹ exchanger isoform 3 (NHE3) is an important regulator of endosomal pH homeostasis. In particular, NHE3 activity is crucial for the maintenance of early endosomal acidification. It therefore is noteworthy that albumin RME may be blocked by inhibitors of NHE3.17 A wide variety of physiological and pathophysiological stimuli have been described to regulate NHE3 activity, therefore suggesting that these same stimuli may act through NHE3 to regulate albumin endocytosis in PTCs. These data show the complexity of the regulation of albumin RME. Exactly how the activity of the various enzymes and transporters is coordinated at the cellular level requires further study. Unraveling the mechanistic aspects of this endocytic pathway is likely to provide important insights into the pathophysiological nature of proteinuria. OTHER PTC RESPONSES TO ALBUMIN EXPOSURE
As indicated, a significant portion of albumin binding to PTCs is believed to be mediated by
ALBUMIN UPTAKE BY PROXIMAL TUBULAR CELLS
megalin. This is an interesting molecule, and the structure of megalin merits some discussion. Megalin, a single polypeptide of 4,660 amino acids, is member of the low-density lipoprotein– receptor family.18 The generally accepted function of these receptors is the internalization of a variety of ligands before their lysosomal degradation. The extracellular and ligand-binding domains of megalin are very similar to those of other members of this receptor family, and megalin clearly is a very attractive candidate for the role of a PTC-albumin receptor. However, the cytoplasmic portion of megalin shows little homology to other low-density lipoprotein–receptor family members other than short internalization sequences. This portion of the molecule contains Src-homology domains, PDZ domains, and protein kinase phosphorylation sites.18 Such sequences strongly indicate a potential role for megalin in signal transduction. The potential for megalin to signal into the cell interior may explain a number of interesting effects of albumin on PTC cell function. Incubation of PTCs with albumin in vitro stimulates cell proliferation.19 This effect is dependent on PI 3-kinase activity, and not only is RME of albumin regulated by PI 3-kinase, but albumin is also able to stimulate the activity of this enzyme when incubated with cultured PTCs.14,19 Albumin also stimulates p70 ribosomal protein S6 kinase (pp70s6) in a PI 3-kinase–dependent manner, and inhibition of this enzyme also inhibits albumin-induced PTC proliferation.19 Appreciable stimulation of PI 3-kinase occurs at low concentrations of albumin, suggesting a potential role for this signaling pathway in PTC homeostasis in health and disease. The mechanism of the stimulation of this kinase cascade by albumin is not yet clear, but binding of albumin to megalin and subsequent signal transduction through megalin may be the explanation. SUMMARY
Albumin filtered by glomeruli binds to receptors in PTCs. Binding of albumin to PTCs not only precedes endocytosis, but also appears to elicit intracellular signals that may be important in the pathophysiological state of renal disease. Fuller understanding of the complexities of binding and endocytic regulation may facilitate the
S19
manipulation of these processes and the amelioration of proteinuria-induced PTC injury. REFERENCES 1. Waldherr R, Ritz E: Edmund Randerath (1899-1961): Experimental proof for the glomerular origin of proteinuria. Kidney Int 56:1591-1596, 1999 2. Oken DE, Flamenbaum W: Micropuncture studies of the proximal tubule albumin concentrations in normal and nephrotic rats. J Clin Invest 50:1498-1505, 1971 3. Osicka TM, Pratt LM, Comper WD: Glomerular capillary wall permeability to albumin and horseradish peroxidase. Nephrology 2:199-212, 1996 4. Eppel GA, Osicka TM, Pratt LM, Jablonski P, Howden B, Glasgow EF Comper WD: The return of glomerularfiltered albumin to the rat renal vein. Kidney Int 55:18611870, 1999 5. Gekle M, Mildenberger S, Freudinger R, Silbernagl S: Functional characterization of albumin binding to the apical membrane of OK cells. Am J Physiol 271:F286-F291, 1996 6. Brunskill NJ, Nahorski S, Walls J: Characteristics of albumin binding to opossum kidney cells and identification of potential receptors. Pflugers Arch 433:497-504, 1997 7. Schnitzer JE, Oh P: Albondin mediated capillary permeability to albumin. Differential role of receptors in endothelial transcytosis and endocytosis of native and modified albumins. J Biol Chem 269:6072-6082, 1994 8. Cui S, Verroust PJ, Moestrup SK, Christensen EI: Megalin/gp330 mediates uptake of albumin in renal proximal tubule. Am J Physiol 271:F900-F907, 1996 9. Birn H, Fyfe JC, Jacobsen C, Mounier F, Verroust PJ, Orskov H, Willnow TE, Moestrup SK, Christensen EI: Cubulin is an albumin binding protein important for renal tubular albumin reabsorption. J Clin Invest 105:1353-1361, 2000 10. Christensen EI, Neilson S: Structural and functional features of protein handling in the kidney proximal tubule. Semin Nephrol 11:414-439, 1991 11. Park CH, Maack T: Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit. J Clin Invest 73:767-777, 1984 12. Gekle M, Mildenberger S, Freudinger R, Silbernagl S: Kinetics of receptor-mediated endocytosis of albumin in cells derived from the proximal tubule of the kidney (opossum kidney cells): Influence of Ca2⫹ and cAMP. Pflugers Arch 430:374-380, 1995 13. Gekle M, Mildenberger S, Freudinger R, Silbernagl S: Endosomal alkalinization reduces Jmax and Km of albumin receptor-mediated endocytosis in OK cells. Am J Physiol 268:F899-F906, 1995 14. Brunskill NJ, Stuart J, Tobin AB, Walls J, Nahorski S: Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase. J Clin Invest 101:2140-2150, 1998 15. Brunskill NJ, Cockcroft N, Nahorski S, Walls J: Albumin endocytosis is regulated by heterotrimeric GTPbinding protein G␣i-3 in opossum kidney cells. Am J Physiol 271:F356-F364, 1996 16. Gekle M, Mildenberger S, Freudinger R, Schwerdt G, Silbernagl S: Albumin endocytosis in OK cells: Dependence
S20
on actin and microtubules and regulation by protein kinases. Am J Physiol 272:F668-F677, 1997 17. Gekle M, Drumm K, Mildenberger S, Freudinger R, Gassner B, Silbernagl S: Inhibition of Na⫹-H⫹ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum. J Physiol 520:709-721, 1999
NIGEL J. BRUNSKILL
18. Hussain MM, Strickland DK, Bakillah A: The mammalian low-density lipoprotein receptor family. Annu Rev Nutr 19:141-172, 1999 19. Dixon R, Brunskill NJ: Activation of mitogenic pathways by albumin in kidney proximal tubule epithelial cells: Implications for the pathophysiology of proteinuric states. J Am Soc Nephrol 10:1487-1497, 1999
P
ROTEINURIA REPRESENTS an adverse clinical finding in patients with renal disease. Patients with heavy proteinuria are more likely to develop tubulointerstitial inflammation, scarring, and fibrosis and progress to end-stage renal failure. For many years, it was believed that excess albuminuria was simply a marker of more severe renal disease, which was more likely to progress as a result of this severity rather than as a result of the albuminuria itself. However, this view has been challenged recently with the accumulation of a body of evidence suggesting that albuminuria may injure proximal tubular epithelial cells (PTCs), thus precipitating a proinflammatory and profibrotic environment in the renal tubulointerstitium. The potential pathophysiological importance of albuminuria has stimulated considerable interest in the entry of albumin into the proximal tubule and the mechanisms of its subsequent interaction with the proximal tubular epithelium. GLOMERULAR FILTRATION OF ALBUMIN
The glomerular, as opposed to tubular, origin of urinary protein was established after a number of classical experiments performed in the early 20th century.1 The conventional paradigm of glomerular function indicates that permeability to albumin is low, with filtration of albumin abrogated by both size and charge selectivity of the glomerular basement membrane. Nonetheless, appreciable amounts of albumin enter the proximal tubule normally, and in rodents, albumin has been measured in proximal tubular fluid by means of micropuncture techniques. Although results have been variable, it is generally accepted that healthy proximal tubular fluid albu-
min concentrations are in the range of 10 to 30 g/mL.2 In recent years, this traditional view of glomerular filtration has been disputed. Osicka et al3 and Eppel et al4 suggested that albumin is very freely filtered by the glomerulus, with the consequence that proximal tubular albumin concentrations are very high, in the 2- to 3-mg/mL range. These same investigators indicated that the vast majority of this albumin is reabsorbed intact into the circulation early in the proximal tubule through a transtubular cell retrieval pathway. However, this pathway remains speculative and, to date, has not been shown biochemically or morphologically. It is not discussed further in this review. ALBUMIN BINDING TO PTCS
Binding studies in vitro have identified two albumin-binding sites in PTCs; one with high affinity but low capacity, and the other with low affinity but high capacity.5,6 The equilibrium dissociation constant (KD) of the higher affinity site suggests that such a receptor would be well placed to mediate binding and uptake of proximal tubular fluid albumin in health, whereas the site with lower affinity but higher From the Departments of Cell Physiology and Pharmacology and Nephrology, University of Leicester, Faculty of Medicine and Biological Sciences, Leicester, UK. Address reprint requests to Nigel J. Brunskill, PhD, FRCP, Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, Medical Sciences Bldg, University Rd, Leicester LE1 9HN, UK. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3701-0204$3.00/0 doi:10.1053/ajkd.2001.20733
American Journal of Kidney Diseases, Vol 37, No 1, Suppl 2 (January), 2001: pp S17-S20
S17
S18
NIGEL J. BRUNSKILL
capacity binding may be responsible for increased albumin binding and uptake in nephrosis. Identification of albumin receptors has been the focus for researchers investigating the endothelium and kidney proximal tubule. In endothelia, a 60-kd glycoprotein receptor for albumin has been partially characterized and named “albondin.”7 This protein exists in endothelia together with a number of lower molecular-weight proteins in the range of 14 to 30 kd. The identities of the lower molecular-weight proteins are unclear. Early studies of PTCs identified similar low-molecular-weight proteins with albumin-binding activity, but albondin has not been found in PTCs.6 More recently, it has been convincingly shown in PTCs that albumin binds to two giant protein receptors, megalin and an associated protein, cubulin.8,9 At least some of the lower molecularweight albumin-binding proteins detected in earlier studies represent breakdown products of cubulin, but the possibility remains that some represent discrete albumin receptors in their own right. More work is required to clarify this issue. UPTAKE OF ALBUMIN BY PTCS
Binding of albumin to the receptors on the apical surface of PTCs is a prelude to its uptake by receptor-mediated endocytosis (RME). A number of elegant morphological studies have shown this pathway both in vivo and in vitro by using cultured PTCs, in which albumin can be followed from its binding in apical invaginations through an endosomal compartment culminating in the lysosome, where it is broken down to constituent amino acids.10 Park and Maack11 used perfused rabbit proximal tubular segments to show both highaffinity low-capacity and low-affinity highcapacity uptake systems for albumin. This model is obviously compatible with the results of the albumin-binding experiments previously described. Opossum kidney (OK) cells have provided a particularly useful in vitro model for the detailed study of the regulatory mechanisms of albumin reabsorption in the proximal tubule. Albumin RME in OK cells is dependent on both cyclic adenosine monophosphate and extracellular calcium ions, with in-
creasing intracellular cyclic adenosine monophosphate and removal of extracellular calcium ions, both leading to a reduction in albumin uptake.12 Data suggest the involvement of several protein kinases, notably protein kinase A, protein kinase C, and phosphatidylinositol 3-kinase (PI 3-kinase) in the regulation of albumin RME in the proximal tubule.13,14 Furthermore, the heterotrimeric guanosine triphosphate– binding protein ␣-subunit, G␣i-3, is also involved in regulating the endocytic uptake of albumin in OK cells.15 Not surprisingly, disruption of the actin cytoskeleton and inhibition of microtubule polymerization also block albumin RME.16 Alkalinization of endosomes interferes with intracellular ligand handling and trafficking and recycling of receptors. Inhibition of the vacuolar adenosinetriphosphatase with bafilomycin leads to an increase in endosomal pH, and a similar effect can be achieved by incubation of cells with NH4Cl. Endosomal alkalinization precipitated by either of these agents is accompanied by a marked reduction in both the affinity constant (Km) and maximal transport activity (Jmax) for albumin RME in OK cells.13 However, fluid-phase endocytosis is unaffected by these maneuvers. The Na⫹-H⫹ exchanger isoform 3 (NHE3) is an important regulator of endosomal pH homeostasis. In particular, NHE3 activity is crucial for the maintenance of early endosomal acidification. It therefore is noteworthy that albumin RME may be blocked by inhibitors of NHE3.17 A wide variety of physiological and pathophysiological stimuli have been described to regulate NHE3 activity, therefore suggesting that these same stimuli may act through NHE3 to regulate albumin endocytosis in PTCs. These data show the complexity of the regulation of albumin RME. Exactly how the activity of the various enzymes and transporters is coordinated at the cellular level requires further study. Unraveling the mechanistic aspects of this endocytic pathway is likely to provide important insights into the pathophysiological nature of proteinuria. OTHER PTC RESPONSES TO ALBUMIN EXPOSURE
As indicated, a significant portion of albumin binding to PTCs is believed to be mediated by
ALBUMIN UPTAKE BY PROXIMAL TUBULAR CELLS
megalin. This is an interesting molecule, and the structure of megalin merits some discussion. Megalin, a single polypeptide of 4,660 amino acids, is member of the low-density lipoprotein– receptor family.18 The generally accepted function of these receptors is the internalization of a variety of ligands before their lysosomal degradation. The extracellular and ligand-binding domains of megalin are very similar to those of other members of this receptor family, and megalin clearly is a very attractive candidate for the role of a PTC-albumin receptor. However, the cytoplasmic portion of megalin shows little homology to other low-density lipoprotein–receptor family members other than short internalization sequences. This portion of the molecule contains Src-homology domains, PDZ domains, and protein kinase phosphorylation sites.18 Such sequences strongly indicate a potential role for megalin in signal transduction. The potential for megalin to signal into the cell interior may explain a number of interesting effects of albumin on PTC cell function. Incubation of PTCs with albumin in vitro stimulates cell proliferation.19 This effect is dependent on PI 3-kinase activity, and not only is RME of albumin regulated by PI 3-kinase, but albumin is also able to stimulate the activity of this enzyme when incubated with cultured PTCs.14,19 Albumin also stimulates p70 ribosomal protein S6 kinase (pp70s6) in a PI 3-kinase–dependent manner, and inhibition of this enzyme also inhibits albumin-induced PTC proliferation.19 Appreciable stimulation of PI 3-kinase occurs at low concentrations of albumin, suggesting a potential role for this signaling pathway in PTC homeostasis in health and disease. The mechanism of the stimulation of this kinase cascade by albumin is not yet clear, but binding of albumin to megalin and subsequent signal transduction through megalin may be the explanation. SUMMARY
Albumin filtered by glomeruli binds to receptors in PTCs. Binding of albumin to PTCs not only precedes endocytosis, but also appears to elicit intracellular signals that may be important in the pathophysiological state of renal disease. Fuller understanding of the complexities of binding and endocytic regulation may facilitate the
S19
manipulation of these processes and the amelioration of proteinuria-induced PTC injury. REFERENCES 1. Waldherr R, Ritz E: Edmund Randerath (1899-1961): Experimental proof for the glomerular origin of proteinuria. Kidney Int 56:1591-1596, 1999 2. Oken DE, Flamenbaum W: Micropuncture studies of the proximal tubule albumin concentrations in normal and nephrotic rats. J Clin Invest 50:1498-1505, 1971 3. Osicka TM, Pratt LM, Comper WD: Glomerular capillary wall permeability to albumin and horseradish peroxidase. Nephrology 2:199-212, 1996 4. Eppel GA, Osicka TM, Pratt LM, Jablonski P, Howden B, Glasgow EF Comper WD: The return of glomerularfiltered albumin to the rat renal vein. Kidney Int 55:18611870, 1999 5. Gekle M, Mildenberger S, Freudinger R, Silbernagl S: Functional characterization of albumin binding to the apical membrane of OK cells. Am J Physiol 271:F286-F291, 1996 6. Brunskill NJ, Nahorski S, Walls J: Characteristics of albumin binding to opossum kidney cells and identification of potential receptors. Pflugers Arch 433:497-504, 1997 7. Schnitzer JE, Oh P: Albondin mediated capillary permeability to albumin. Differential role of receptors in endothelial transcytosis and endocytosis of native and modified albumins. J Biol Chem 269:6072-6082, 1994 8. Cui S, Verroust PJ, Moestrup SK, Christensen EI: Megalin/gp330 mediates uptake of albumin in renal proximal tubule. Am J Physiol 271:F900-F907, 1996 9. Birn H, Fyfe JC, Jacobsen C, Mounier F, Verroust PJ, Orskov H, Willnow TE, Moestrup SK, Christensen EI: Cubulin is an albumin binding protein important for renal tubular albumin reabsorption. J Clin Invest 105:1353-1361, 2000 10. Christensen EI, Neilson S: Structural and functional features of protein handling in the kidney proximal tubule. Semin Nephrol 11:414-439, 1991 11. Park CH, Maack T: Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit. J Clin Invest 73:767-777, 1984 12. Gekle M, Mildenberger S, Freudinger R, Silbernagl S: Kinetics of receptor-mediated endocytosis of albumin in cells derived from the proximal tubule of the kidney (opossum kidney cells): Influence of Ca2⫹ and cAMP. Pflugers Arch 430:374-380, 1995 13. Gekle M, Mildenberger S, Freudinger R, Silbernagl S: Endosomal alkalinization reduces Jmax and Km of albumin receptor-mediated endocytosis in OK cells. Am J Physiol 268:F899-F906, 1995 14. Brunskill NJ, Stuart J, Tobin AB, Walls J, Nahorski S: Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase. J Clin Invest 101:2140-2150, 1998 15. Brunskill NJ, Cockcroft N, Nahorski S, Walls J: Albumin endocytosis is regulated by heterotrimeric GTPbinding protein G␣i-3 in opossum kidney cells. Am J Physiol 271:F356-F364, 1996 16. Gekle M, Mildenberger S, Freudinger R, Schwerdt G, Silbernagl S: Albumin endocytosis in OK cells: Dependence
S20
on actin and microtubules and regulation by protein kinases. Am J Physiol 272:F668-F677, 1997 17. Gekle M, Drumm K, Mildenberger S, Freudinger R, Gassner B, Silbernagl S: Inhibition of Na⫹-H⫹ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum. J Physiol 520:709-721, 1999
NIGEL J. BRUNSKILL
18. Hussain MM, Strickland DK, Bakillah A: The mammalian low-density lipoprotein receptor family. Annu Rev Nutr 19:141-172, 1999 19. Dixon R, Brunskill NJ: Activation of mitogenic pathways by albumin in kidney proximal tubule epithelial cells: Implications for the pathophysiology of proteinuric states. J Am Soc Nephrol 10:1487-1497, 1999