Renal Albumin Absorption in Physiology and Pathology H Birn1 and EI Christensen1

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Renal albumin absorption in physiology and pathology H Birn1 and EI Christensen1

1Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus C, Denmark

Albumin is the most abundant plasmaprotein serving Albumin is synthesized in the liver and is the most abundant multiple functions as a carrier of metabolites, hormones, plasma protein. It is an anionic, flexible, heart-shaped vitamins, and drugs, as an acid/base buffer, as antioxidant molecule with a molecular weight of B65 kDa.1 Although it and by supporting the oncotic pressure and volume of the is not essential to life, a number of important and very diverse blood. The presence of albumin in urine is considered to be functions have been ascribed to this protein, including the the result of the balance between glomerular filtration and maintenance of the oncotic pressure and blood volume, acid/ tubular reabsorption. Albuminuria has been accepted as an base buffer functions, antioxidant functions and the transport independent risk factor and a marker for renal as well as of a number of different substances like fatty acids, bilirubin, cardiovascular disease, and during the past decade, evidence ions such as Ca2 þ and Mg2 þ , drugs, hormones, and has suggested that albumin itself may cause progression of lipophilic as well as hydrophilic vitamins, for example, vitamin renal disease. Thus, the reduction of proteinuria and, in A, riboflavin, vitamin B6, ascorbic acid, and folate. Both serum particular, albuminuria has become a target in itself to and urinary albumin levels are important prognostic indica- prevent deterioration of renal function. Studies have shown tors in renal disease. Hypoalbuminemia in renal disease is albumin and its ligands to induce expression of inflammatory associated with increased mortality and has been ascribed both and fibrogenic mediators, and it has been hypothesized that to malnutrition and to inflammation, whereas the level of increased filtration of albumin causes excessive tubular albumin in the urine is directly related to the progression of reabsorption, resulting in inflammation and fibrosis, resulting renal disease, with no known lower threshold to the relation- in the loss of renal function. In addition, it is known that ship. These findings have prompted a strong interest in the tubular dysfunction in itself may cause albuminuria owing to mechanisms for renal handling of albumin and in the effects of decreased reabsorption of filtered albumin, and, recently, it albumin on renal function. Albumin filtered in the glomeruli has been suggested that significant amounts of albumin is considered to be the major source of urinary albumin. fragments are excreted in the urine as a result of tubular Filtration of albumin is followed by tubular reabsorption, and degradation. Thus, although both tubular and glomerular thus the resulting albuminuria reflects the combined con- dysfunction influences renal handling of albumin, it appears tribution of these two processes. Dysfunction of both these that tubular reabsorption plays a central role in mediating processes may result in increased excretion of albumin, and the effects of albumin on renal function. The present paper both glomerular injury and tubular impairment have been will review the mechanisms for tubular albumin uptake and implicated in the initial events leading to proteinuria. Multiple the possible implications for the development of renal theories have emerged to explain the clinical observation that disease. once renal damage has reached a certain level, renal function Kidney International (2006) 69, 440–449. doi:10.1038/sj.ki.5000141; will decline independent of the primary events initiating the published online 6 January 2006 process. Within the last decade there has been considerable KEYWORDS: kidney; proximal tubule; endocytosis; fibrosis; progression of focus on the potential pathogenic role of proteinuria, chronic renal; failure including albuminuria. The hypothesis is based on the observations that the rate of progression in various renal diseases correlates with the degree of proteinuria and on studies using tubular cells in vitro showing that high concentrations of proteins induce the production of inflammatory and fibrogenic mediators. This points to a central role of the mechanisms regulating tubular uptake of albumin. Correspondence: EI Christensen, Department of Cell Biology, Institute of Although new pathways of tubular albumin reabsorption have Anatomy, University of Aarhus, University Park, Building 234, DK-8000 been hypothesized recently, receptor-mediated endocytosis has Aarhus C, Denmark. E-mail: [email protected] been extensively studied and characterized as an essential Received 27 October 2005; revised 27 October 2005; accepted 11 mechanism for proximal tubule uptake of albumin. The November 2005; published online 6 January 2006 present review will include recent advances in the under-

440 Kidney International (2006) 69, 440–449 H Birn and EI Christensen: Albumin absorption in physiology and pathology review

standing of this process and the potential implications for lysosomes for storage or degradation, or into the cytosol for albumin-induced changes in renal tubular function. further processing/transport. The receptor is recycled back to the luminal membranes through a recycling compartment GLOMERULAR FILTRATION OF ALBUMIN known as dense apical tubules,19 but may also be transported The amount of albumin normally filtered in the glomeruli to lysosomes for degradation. Albumin binding sites on has been estimated using various techniques, including isolated proximal tubule segments and cells in vitro have been micropuncture of rats and dogs, estimating the concentration identified,14,20–22 and several receptors for tubular uptake of of albumin in the ultrafiltrate between 1 and 50 mg/ml.2 This albumin have been identified. These include the multiligand corresponds to a filtered load of albumin between 170 mg receptors megalin and cubilin, responsible for the constitu- and 9 g/24 h in normal humans. Inhibition of tubular tive uptake of a vast variety of filtered plasma proteins. albumin uptake in humans by lysine suggested filtration of at least B281 mg/min, corresponding to B400 mg/24 h.3 MEGALIN Similar studies in lysine-treated rats resulted in the excretion Megalin is a multifunctional, endocytic receptor belonging to of 2.5–25mg/24 h4,5 corresponding to 0.7–7g/24 h in humans. the low-density lipoprotein receptor family. It binds a These studies were based on immunoassays for detecting number of structurally very different proteins. Megalin was urinary albumin. It was recently suggested, based on the originally identified as the antigen in Heymann nephritis, a excretion of radiolabelled albumin and high-performance rat model of membranous glomerulonephritis.23 It contains a liquid chromatography, that significant amounts of albumin large amino-terminal, extracellular domain, a single trans- fragments are excreted in rat and human urine, possibly membrane domain and a short carboxy-terminal cytoplasmic resulting from tubular degradation of filtered albumin, tail (Figure 1). The latter harbors two NPXY sequences followed by luminal secretion of albumin fragments.6–9 Such mediating the binding to adaptor proteins and the clustering fragments cannot be detected by conventional analyses, into clathrin-coated pits,24–27 in addition to several Src including radioimmunoassay. These studies, implicating a homology 3 and one Src homology 2 recognition sites.24,25 much greater amount of filtered albumin in the normal The extracellular domain is composed of four cysteine-rich kidney, are still controversial. Renal catabolism and excretion clusters of low-density lipoprotein receptor type A repeats of as much as 6.5 mg/100 g body weight/h in normal rats constituting the ligand binding regions separated and were estimated,8 apparently exceeding normal rat liver followed by a total of 17 epidermal growth factor (EGF)- albumin synthesis rate.10 The findings are also in contrast type repeats and eight spacer regions containing YWTD to reports on urine protein excretion in Fanconi patients repeats. Generally, binding to megalin is Ca2 þ dependent, using proteomics.11 Thus, although the albumin filtration and megalin itself binds calcium very strongly.28 Site-directed rate still remains controversial, it is clear that a significant mutations of basic amino-acid residues in aprotinin, a ligand amount of albumin is filtered and subsequently reabsorbed for megalin, decrease the affinity for the receptor, suggesting by the renal tubules. that binding is favored by cationic sites on the ligands.29 Many ligands are anionic proteins, indicating that the TUBULAR REABSORPTION OF ALBUMIN distribution of charge rather than the overall isoelectric Proximal tubular reabsorption of albumin by endocytosis was point is important for binding. demonstrated almost 40 years ago.12,13 Endocytic uptake of proteins has been intensively studied and includes both Renal expression nonspecific fluid-phase endocytosis as well as receptor- Megalin is heavily expressed in the kidney proximal tubule mediated endocytosis. In the kidney, proximal tubule fluid- brush-border and luminal endocytic apparatus,23,28,30–32 as phase endocytosis of solutes is negligible.2,14 Receptor- well as in lysosomal structures revealing smaller amounts of mediated endocytosis, as originally described,15 involves the immmunoreactive megalin. Megalin has also been identified specific binding of a ligand to receptors in the apical plasma in the glomerular podocytes of Lewis rats,33 but not in other membrane. The receptor–ligand complex is internalized by rat strains or other species. Extrarenal expression of megalin invagination of the plasma membrane caused by adaptor includes a number of other absorptive epithelia, including molecule-mediated formation of a cytoplasmic coat.16–18 In the ileum34,35 and the rodent yolk sac36 (reviewed in clathrin-coated endocytosis, binding of adaptor molecules to Christensen and Birn37). the cytoplasmic tail of transmembrane receptors involves a Much of our knowledge on the functions of megalin is characteristic NPXY motif17 identified in a number of based on data recovered from the study of megalin-deficient endocytic receptors. Internalization is followed by dissocia- mice.38,39 Megalin-deficient, proximal tubule cells are char- tion of the invaginations from the plasma membrane forming acterized by a loss of endocytic invaginations, vesicles, and vesicles. While the cytoplasmic coat dissociates, vesicles may the membrane recycling compartment, dense apical tu- fuse with other newly formed vesicles, or with an existing bules.40 So far, no significant changes in transport of water, pool of larger vesicles, followed by acidification of the electrolytes, glucose, or amino acids have been described in intravesicular lumen and the dissociation of the ligand from the megalin-deficient mice; however, an increased amount of the receptor. The ligand may be further transported into several low-molecular weight serum proteins has been

Kidney International (2006) 69, 440–449 441 review H Birn and EI Christensen: Albumin absorption in physiology and pathology

Megalin patients with low-molecular weight and tubular proteinuria, NH 2 including Dent’s disease, have shown several analogies.51,54 Cubilin CUBILIN Cubilin is a multiligand, endocytic receptor also known as 55 the intestinal intrinsic factor (IF)-B12 receptor. Cubilin was originally identiﬁed as the target of teratogenic antibodies in rats.56 It is a 460 kDa protein with little structural homology 57–59 Amnionless to known, endocytic receptors (Figure 1). Cubilin is

NH2 composed of an initial 110-amino-acid region necessary for membrane anchoring of the receptor60, followed by eight EGF-like repeats and 27 complement subcomponents C1r/ C1s, Uegf, and bone morphogenic protein-1 (CUB) 61 Luminal domains. No transmembrane domain has been identified NH 2 plasmamembrane in cubilin consistent with the observation that it is released NPXY COOH COOH from renal cortical membranes by nonenzymatic and non- VENQNY 58 CUB domain NPXY solubilizing procedures. The many CUB domains in cubilin Complement-type repeat EGF-type repeat support the ability of the receptor to bind a variety of ligands. Spacer region containing YWTD Binding sites for selected ligands have been partially localized. Transmembrane domain NPXY motifs and VENQNY motif Albumin, along with IF-B12, binding sites have been identified in a 113-residue amino-terminus, which includes Figure 1 | Schematic representation showing the structure of megalin, cubilin, and AMN. Megalin is a 4600-amino-acid trans- the EGF-type repeats. Although IF-B12 inhibits binding of membrane protein with a non-glycosylated molecular weight of albumin to cubilin, the binding sites for the two proteins do 517 kDa. The extracellular domain (B4400 amino acids) contains four not appear to be identical within this domain,62 and an cysteine-rich clusters of low-density lipoprotein receptor type A additional binding site for IF-B12 has been identified in CUB repeats constituting the ligand binding regions separated and 60 35,60 followed by a total of 17 EGF-type repeats and eight spacer regions domains 5–8. RAP binds to CUB domains 13–14, containing YWTD repeats. A single transmembrane domain (22 although its role for the processing of this receptor is amino acids) is followed by the cytoplasmic tail (213 amino acids), unknown. Megalin also binds to cubilin, possibly within the which contains two NPXY sequences and one VENQNY sequence in 34 addition to several Src homology 3 and one Src homology 2 amino-terminal region including CUB domains 1 and 2, recognition sites. Cubilin is a 3600-amino-acid protein with no supporting an interaction between the two receptors as transmembrane domain and a non-glycosylated molecular weight of discussed below. B400 kDa. The extracellular domain contains 27 CUB domains constituting molecular basis for interaction with multiple other proteins. The CUB domains are preceded by a stretch of 110 amino Renal expression acids followed by eight EGF-type repeats. The amino-terminal region Cubilin is highly expressed in the renal proximal tubule contains a potential palmitoylation site and an amphipathic helix brush-border and endocytic apparatus (Figure 2).36,55,63 As structure with some similarity to the lipid binding regions of with megalin, immunoreactive cubilin can also be identified apolipoproteins. AMN is a 434-amino-acid, single-transmembrane 55 protein with a non-glycosylated molecular weight of B46 kDa. It has in lysosomes. Normal expression of cubilin is dependent on no known, closely related proteins, but a cysteine-rich stretch of 70 amnionless (AMN), a B45 kDa, transmembrane protein amino acids in the extracellular domain has similarity to modules (Figure 1) identified as an important factor for the normal present in a small group of proteins serving as bone morphogenic development of the middle portion of the primitive streak in protein inhibitors. Both AMN and megalin bind cubilin and studies 64 indicate that both are involved in the endocytosis of this receptor. mice. AMN co-localize with cubilin in kidney proximal tubule,65 interacts with the EGF-type repeats of cubilin and is identified in the urine. The normal expression of megalin is essential for normal translocation of the cubilin–AMN dependent on receptor-associated protein (RAP)41 serving as complex from the ER to the plasmamembrane and for the a chaperone-protecting newly synthesized receptor from the subsequent endocytosis.65,66 Dogs with a mutation in the early binding of ligands and possibly involved in folding of AMN gene as well as AMN-deficient mouse epithelial cells the receptor.42–46 RAP binds megalin with high affinity within reveal defective apical insertion of cubilin.67–71 Mutations in the endoplasmic reticulum and functions as an intracellular either the cubilin or the AMN gene have been identified in 72,73 ligand inhibiting the binding of most other ligands to Imerslund–Gra¨sbeck disease, a rare, inherited vitamin B12 megalin. deficiency syndrome characterized by defective intestinal 74–77 A number of diseases characterized by proteinuria reveal absorption of IF-B12. Two affected Finnish families decreased renal megalin expression.47–49 These include Dent’s revealed a one-amino-acid substitution in CUB domain 8 disease, which is caused by mutations in the renal chloride affecting the binding of IF-B12 to cubilin or, alternatively, a channel ClC-5.50 Disruption of ClC-5 impairs proximal point mutation expected to activate a cryptic intronic splice tubule endocytosis, and causes a reduction in megalin site causing an in-frame insertion and introducing stop expression.49,51–53 Analyses on megalin knockout mice and codons predicting a truncation of the receptor in CUB

442 Kidney International (2006) 69, 440–449 H Birn and EI Christensen: Albumin absorption in physiology and pathology review

70 Albumin binds to cubilin with an estimated Kd B0.6 mM. Furthermore, in dogs with a dysfunction of AMN and a resulting defective apical expression of cubilin, urinary albumin excretion is significantly increased and associated with the lack of tubular endocytosis of albumin, demonstrated by the absent labelling for albumin in endocytic vesicles (Figure 2).70 Similarly, patients with Imerlund–Gra¨s- ab beck disease may also have albuminuria, supporting a role of cubilin. Albumin uptake in opossum kidney (OK) cells was Figure 2 | Immunocytochemical localization of cubilin and uptake 82 inhibited by IF-B12, and anti-cubilin antibodies. The of endogenous albumin in cryosections of dog kidney cortex. In mM normal dog kidney, (a) intense labeling for cubilin (green fluores- inhibitory constant for IF-B12 was 1.7 in line with the cence) is observed at the luminal brush border and in endocytic finding that IF-B12 and albumin both may bind to the same vesicles co-localizing with reabsorbed albumin (red fluorescence) 113-residue amino-terminus of cubilin.62 Megalin-deficient reflecting endocytosis of filtered protein. Apical vesicles reveal co- mice also reveal an increase in urinary albumin excretion,70 localization of cubilin and albumin as indicated by the yellow label (arrows in a). In dogs with a mutation in the AMN, gene-causing suggesting that megalin is involved in tubular albumin defective insertion of cubilin into the apical membranes, (b) cubilin is reabsorption. Direct binding of labelled albumin to megalin retained in intracellular vesicles and no evidence of endocytic uptake has previously been identified using Sepharose columns;83 of albumin is observed. Only a sparse labeling for albumin can be however, an additional, high-affinity binding between identified in the tubular lumen and along the apical membrane, the 58 latter possibly reflecting binding to megalin (arrows in b). One purified cubilin and megalin has been described in vitro. micrometer cryosections from perfusion fixed dog kidneys were Based on this, it was suggested that megalin facilitates the incubated with rabbit anti-dog cubilin (1:200) and sheep anti-rat endocytosis and intracellular trafficking of cubilin.58 This is albumin (1:200) primary antibodies followed by incubation with Alexa further supported by studies showing that the in vitro uptake Flour 488 donkey anti-rabbit and Alexa Fluor 546 donkey anti-sheep fluorescence labelled secondary antibodies. Bars ¼ 10 mm. of the cubilin ligands transferrin, Clara cell secretory protein and apolipoprotein A-I/high-density lipoprotein is inhibited by anti-megalin antibodies,84–86 as well as by megalin domain 6.78 While the patient with the latter mutation has antisense oligonucleotides.87 Thus, megalin may be involved overt albuminuria, patients with amino-acid substitution in albumin reabsorption directly as a receptor for albumin, reveal varying degrees of proteinuria ranging from little or and/or indirectly by affecting the expression and/or endocytic no, to clearcut.77 Affected members of three Norwegian function of cubilin.70 Albumin uptake in OK cells is inhibited families, shown to have a nucleotide deletion causing the by RAP, and by anti-megalin antibodies supporting this 73 82 introduction of an early stop codon in the AMN gene, were notion. The inhibitory constant for RAP is 17 nM; however, all previously characterized, and had proteinuria at the time whether this reflects inhibition of albumin binding to cubilin, of diagnosis.74 The proteinuria observed in these patients to megalin, or cubilin–megalin interaction is unknown. So support a role for cubilin in renal tubular protein reabsorp- far, RAP and albumin binding sites within the same region of tion, and it may be hypothesized that the difference in the cubilin have not been identified. degree of albuminuria depends on the type of mutation and It has been argued that the capacity of the megalin/ reflects the extent of inactivation of cubilin function, in cubilin-mediated mechanism for tubular uptake of albumin particular whether the mutation affects the multiligand is low,9 and that the urinary albumin excretion in megalin- properties or only the IF-B12 binding site. defective mice and cubilin-defective dogs is only slightly increased, representing merely a small increase in the OTHER RECEPTORS excretion of non-degraded albumin.9 It must be considered, Additional renal albumin receptors have been suggested however, that megalin and cubilin are low-affinity albumin including 55 and 31 kDa proteins identified by albumin- receptors serving in a high-capacity, reabsorptive mechanism. affinity chromatography and localized by immunocytochem- Assuming an albumin concentration of 10 mg/ml in the istry. The sizes of these proteins are comparable to albumin ultrafiltrate, this is about one-fourth of the estimated Kd for binding proteins identified in endothelial cells and fibro- albumin binding to cubilin, and thus the tubular flow and the 79–81 blasts. Antibody cross-reactivity suggested that the low- length of the proximal tubule becomes important for the molecular weight receptors identified in rat proximal tubule amount of albumin reabsorbed. In mice and rats, with 70 are, in fact, fragments of cubilin, although the presence of relatively short proximal tubules, the fraction of reabsorbed multiple, structurally different albumin binding proteins albumin compared to the filtered load is smaller than in cannot be excluded. The exact localization and functional larger species with longer proximal tubules. Megalin importance of these, however, remains to be established. dysfunction caused a B1.5-fold increase in albumin excretion in mice compared to a sevenfold increase in cubilin- MEGALIN- AND CUBILIN-MEDIATED UPTAKE OF ALBUMIN defective dogs,70 and up to 425-fold increase in Imerslund–- Cubilin plays an important role in normal proximal tubule Gra¨sbeck patients.77 The low affinity of albumin binding to endocytic reabsorption of filtered albumin (Figure 3). cubilin/megalin also implies that the amount of albumin

Kidney International (2006) 69, 440–449 443 review H Birn and EI Christensen: Albumin absorption in physiology and pathology

Glomerulus Endocytic invaginations 5 1

4 Dense apical tubules Amino acids

Lysosome

Molecules carried by albumin Cubilin AMN Albumin Megalin

Proximal tubule 2a

2c 2b Lysosome 3

Figure 3 | Pathways of albumin handling in the kidney. Albumin is filtered in the glomeruli (1) and reabsorbed by the proximal tubule cells by receptor-mediated endocytosis (2a). Internalization by endocytosis is followed by transport into lysosomes for degradation. Classically, thisis considered to result in the formation of free amino acids released into the circulation (2b); however, it has been recently suggested that significant amounts of albumin fragments are excreted in the urine, possibly resulting from tubular degradation of filtered albumin (2c). Some intact albumin may escape tubular reabsorption (3), the amount being greater as the glomerular filtration fraction of albumin increases or tubular function is compromised. The upper right shows a schematic representation of the intracellular pathways following endocytic uptake of albumin and possible associated substances. Following binding to the receptors, cubilin or megalin, the receptor–albumin complex is directed into coated pits for endocytosis, a process that may also involve AMN. The complex dissociates following vesicular acidification most likely also leading to the release of any bound substances. Albumin is transferred to the lysosomal compartment for degradation. Some albumin may be degraded within a late endocytic compartment and recycled as fragments to be released at the luminal surface. Alternatively, albumin fragments may be recycled from the lysosomal compartment by a yet unknown route. Receptors recycle through dense apical tubules, whereas released substances carried by albumin may be released into the cytosol or transported across the tubular cell. An alternative high- capacity retrieval pathway for non-degraded albumin located immediately distal to glomerular basement membrane has been proposed (4); but yet not characterized. Misdirected filtration of albumin into the interstitium resulting from pathological, glomerular changes has been proposed as a pathway for progression of renal disease (5). reabsorbed during heavy proteinuria with a high concentra- protein kinase C induces ectodomain shedding of megalin, tion of albumin in the ultrafiltrate may be much higher as the also generated by the binding of the ligand vitamin D- reabsorbed amount is not limited by capacity, but rather by binding protein. It was suggested that this is accompanied by the flow and concentration of albumin in the ultrafiltrate. An the release of an intracellular domain, possibly involved in important factor adding to the capacity of this system is the intracellular signalling.92 The cytoplasmic tail of megalin has constant and fast regeneration of receptors by membrane been shown to interact with a number of cytoplasmic recycling.88 Thus, the finding of only a small increase in proteins possibly modulating expression and/or function of albumin excretion in cubilin- or megalin-deficient animals is the receptor including the adaptor protein autosomal not inconsistent with the reabsorption and renal catabolism of recessive hypercholesterolemia27 and disabled protein 2 significant amounts of protein by the same receptors during (Dab2).93–95 However, to what extent these potential, heavy proteinuria with filtration of large amounts of protein. regulatory mechanisms are part of an active pathway regulating endocytosis in vivo is not known. REGULATION OF TUBULAR ALBUMIN UPTAKE Generally renal cubilin and megalin mediated tubular uptake OTHER PATHWAYS of proteins is considered a constitutive process regulated only Alternative mechanisms of albumin reabsorption have been by changes in receptor expression levels identified in suggested (Figure 3), including a fast, high-capacity retrieval conditions such as Dent’s disease, polycystic kidney disease pathway for non-degraded albumin located distal to the and possibly diabetes. Based on in vitro studies using OK glomerular basement membrane.9,96 This hypothesis is based cells, it has been suggested that albumin uptake may be on the analysis of the radioactive profile in renal venous regulated by G proteins,89 known to be involved in the blood following pulse injection of 3H-albumin in the regulation of vesicular transport, by phosphatidylinositol 3- isolated, perfused kidney.96 This revealed a second peak kinase,90 and by protein kinases.91 Recently, it was shown that interpreted as the rapid return of filtered, labelled albumin

444 Kidney International (2006) 69, 440–449 H Birn and EI Christensen: Albumin absorption in physiology and pathology review

with an estimated capacity of 400–500g/24 h.9 While it was tors, low-density lipoprotein receptor-related protein and the suggested that micropuncture of early proximal tubule failed apolipoprotein ER2, have been shown to bind other to identify this process because of its location in the cytoplasmic adaptor proteins, notably Dab1, at NPXY motifs glomerulus, or very early in the tubular system,96 the nature on the cytoplasmic tail of these receptors.126,127 Dab1 is part of this proposed mechanism has not been established. It is of a signalling pathway involving the extracellular protein generally believed that tight junctions prevent paracellular Reelin important for brain development.128 So far, no specific transport of most proteins,97–101 and so far, no morphologic signalling events have been documented for megalin; support for an alternative, transcellular albumin transport however, as previously mentioned, the cytoplasmic tail of mechanism has been demonstrated. Nevertheless, defects in megalin has been shown to interact with Dab2,93,94 an such a highly speculative, high-capacity albumin transport intracellular protein that may be involved in signalling or pathway may result in heavy albuminuria without any initial regulating of cellular growth and differentiation.95 Recent changes in glomerular function, and thus implicate a radical evidence also indicates signalling by ligand-induced, enzy- change in our understanding of albuminuric renal disease. matic release of a carboxyl-terminal fragment of megalin.92 Thus, it may be that excessive binding of filtered proteins, ALBUMIN REABSORPTION AND INFLAMMATION including albumin, to megalin may initiate intracellular The concept of albumin-induced renal inflammation and signalling events. Proposed intracellular signalling cascades scarring has been intensively explored using both clinical and include the activation of nuclear factor kb110,129–133 as well as experimental approaches. Albuminuria has been shown to be STAT (signal transducers and activator of transcription)134 an independent risk factor for the progression of renal disease transcription factors, possibly through the formation of and the renoprotective effect of antiproteinuric treatment, reactive oxygen species,131,134 a process that may also be whether by dietary or pharmacological intervention, has been dependent on protein kinase C.131 Interestingly, delipidated shown to be correlated to the reduction in proteinuria.102,103 albumin has been suggested to protect against reactive The hypothesis tested is that excess albumin in the tubular oxygen species,135 indicating that the potentially protective lumen leads to the induction of inflammation, possible versus damaging effect is determined at least, in part, by the tubular epithelial–mesenchymal transformation and inter- substances carried by albumin.136 In addition to albumin, stitial fibrosis. Overload albuminuria in rats and mice causes several other filtered proteins have been suggested to induce interstitial inflammation and fibrosis.104,105 Studies on proximal tubule phenotypic changes, including iron carriers, proximal tubule cells have shown that albumin exposure proteins of the complement system, immunoglobulins, and induces the expression of a number of inflammatory and growth factors. It is not clear to what extent each of these fibrogenic mediators, including cytokines such as RANTES proteins or their combination contributes to the inflamma- (regulated upon activation, normal T cell expressed and tion and fibrosis; however, some studies indicates differential secreted),106 monocyte chemotactic protein-1,107 interleukin-8,108 and protein-specific effects,137,138 and studies in analbumi- fractalkine109 and tumor necrosis factor-a,110 as well as nemic rats even suggested that albumin was not important to endothelin,111,112 transforming growth factor-b (TGF-b),112 the progression of proteinuric, renal disease when evaluated collagen;113 and may also induce changes in tubular cell for up to 20 weeks.139,140 expression of surface integrins114 or, eventually, apopto- Most of the studies exploring the mechanism of sis.115,116 These data suggest that albumin itself and/or bound proteinuria-induced renal changes are based on proximal ligands such as fatty acids117–120 initiate a series of events that tubule cell lines. Cells are exposed to albumin or serum eventually leads to fibrosis. The exact cascade has not been fractions and changes are studied by protein and gene established; however, evidence suggests that the process analysis of the media and/or the cells. It is obvious that involves the initial endocytic uptake of albumin. Renal cell extrapolation from such data to the human in vivo situation lines with low endocytic activity do not respond to albumin may be difficult also considering the somewhat conflicting by the activation of intracellular pathways and collagen data observed with different proteins in different cell synthesis,113 and megalin-deficient cells appear to be less systems,116,137,138 as well as the reported changes in the sensitive to hemoglobin-induced proximal tubule necrosis.121 expression of several genes of still unknown function.141 The Interestingly, TGF-b, which may be induced by albumin obvious challenge to the overall hypothesis is the ability to exposure,112 may also act in a feedback mechanism increasing specifically interfere with the cascade of events leading to albumin filtration122 and at the same time inhibiting irreversible renal damage. An established approach has been megalin- and cubilin-mediated albumin endocytosis,123 both to prevent or reduce proteinuria notably by blocking the leading to increased albuminuria. renin–angiotensin system.142 Another approach would be to Recent evidence indicates that albumin may interact with inhibit tubular uptake of proteins either by blocking binding signalling receptors including apically expressed EGF recep- to receptors or by inhibiting endocytosis. As both megalin tors.124 In addition, it has been shown that members of the and cubilin are multiligand receptors serving other important low-density lipoprotein receptor family may be involved functions, for example, for the uptake and metabolism of directly in transmembrane signal transduction.125 The low- vitamins,37 including the activation of vitamin D,143,144 this density lipoprotein and very low-density lipoprotein recep- approach may prove inappropriate. Recently, it has been

Kidney International (2006) 69, 440–449 445 review H Birn and EI Christensen: Albumin absorption in physiology and pathology

shown that transfection with a monocyte chemotactic ACKNOWLEDGMENTS The work was supported by the Danish Medical Research Council, the protein-1 antagonist145 or a truncated form of IkBa, thereby 133 University of Aarhus, the NOVO-Nordisk Foundation, Fonden til inhibiting nuclear factor-kb, inhibits albumin-overload Lægevidenskabens Fremme, the Biomembrane Research Center, induced tubulointerstitial injury supporting the possibility of the Beckett Foundation, the Leo Nielsen Foundation, the Ruth therapeutic intervention at the tubular level. Additional Ko¨nig-Petersen Foundation, the Birn-Foundation and the European evidence suggests that inhibition of the renin–angiotensin Commission (EU Framework 6, EureGene, Contract Number 05085). system in addition to a reduction of proteinuria may also attenuate albumin-induced signal activation in tubular 146 cells. Further studies should evaluate the clinical signifi- REFERENCES cance and feasibility of the various approaches. 1. 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