Renal albumin absorption in physiology and pathology

review

http://www.kidney-international.org & 2006 International Society of Nephrology

Renal albumin absorption in physiology and pathology H Birn1 and EI Christensen1 1

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

Albumin is the most abundant plasmaprotein serving multiple functions as a carrier of metabolites, hormones, vitamins, and drugs, as an acid/base buffer, as antioxidant and by supporting the oncotic pressure and volume of the blood. The presence of albumin in urine is considered to be the result of the balance between glomerular filtration and tubular reabsorption. Albuminuria has been accepted as an independent risk factor and a marker for renal as well as cardiovascular disease, and during the past decade, evidence has suggested that albumin itself may cause progression of renal disease. Thus, the reduction of proteinuria and, in particular, albuminuria has become a target in itself to prevent deterioration of renal function. Studies have shown albumin and its ligands to induce expression of inflammatory and fibrogenic mediators, and it has been hypothesized that increased filtration of albumin causes excessive tubular reabsorption, resulting in inflammation and fibrosis, resulting in the loss of renal function. In addition, it is known that tubular dysfunction in itself may cause albuminuria owing to decreased reabsorption of filtered albumin, and, recently, it has been suggested that significant amounts of albumin fragments are excreted in the urine as a result of tubular degradation. Thus, although both tubular and glomerular dysfunction influences renal handling of albumin, it appears that tubular reabsorption plays a central role in mediating the effects of albumin on renal function. The present paper will review the mechanisms for tubular albumin uptake and the possible implications for the development of renal disease. Kidney International (2006) 69, 440–449. doi:10.1038/sj.ki.5000141; published online 6 January 2006 KEYWORDS: kidney; proximal tubule; endocytosis; fibrosis; progression of chronic renal; failure

Correspondence: EI Christensen, Department of Cell Biology, Institute of Anatomy, University of Aarhus, University Park, Building 234, DK-8000 Aarhus C, Denmark. E-mail: [email protected] Received 27 October 2005; revised 27 October 2005; accepted 11 November 2005; published online 6 January 2006 440

Albumin is synthesized in the liver and is the most abundant plasma protein. It is an anionic, flexible, heart-shaped molecule with a molecular weight of B65 kDa.1 Although it is not essential to life, a number of important and very diverse functions have been ascribed to this protein, including the maintenance of the oncotic pressure and blood volume, acid/ base buffer functions, antioxidant functions and the transport of a number of different substances like fatty acids, bilirubin, ions such as Ca2 þ and Mg2 þ , drugs, hormones, and lipophilic as well as hydrophilic vitamins, for example, vitamin A, riboflavin, vitamin B6, ascorbic acid, and folate. Both serum and urinary albumin levels are important prognostic indicators in renal disease. Hypoalbuminemia in renal disease is associated with increased mortality and has been ascribed both to malnutrition and to inflammation, whereas the level of albumin in the urine is directly related to the progression of renal disease, with no known lower threshold to the relationship. These findings have prompted a strong interest in the mechanisms for renal handling of albumin and in the effects of albumin on renal function. Albumin filtered in the glomeruli is considered to be the major source of urinary albumin. Filtration of albumin is followed by tubular reabsorption, and thus the resulting albuminuria reflects the combined contribution of these two processes. Dysfunction of both these processes may result in increased excretion of albumin, and both glomerular injury and tubular impairment have been implicated in the initial events leading to proteinuria. Multiple theories have emerged to explain the clinical observation that once renal damage has reached a certain level, renal function will decline independent of the primary events initiating the process. Within the last decade there has been considerable focus on the potential pathogenic role of proteinuria, 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. Although new pathways of tubular albumin reabsorption have been hypothesized recently, receptor-mediated endocytosis has been extensively studied and characterized as an essential mechanism for proximal tubule uptake of albumin. The present review will include recent advances in the underKidney International (2006) 69, 440–449

H Birn and EI Christensen: Albumin absorption in physiology and pathology

standing of this process and the potential implications for albumin-induced changes in renal tubular function. GLOMERULAR FILTRATION OF ALBUMIN

The amount of albumin normally filtered in the glomeruli has been estimated using various techniques, including micropuncture of rats and dogs, estimating the concentration of albumin in the ultrafiltrate between 1 and 50 mg/ml.2 This corresponds to a filtered load of albumin between 170 mg and 9 g/24 h in normal humans. Inhibition of tubular albumin uptake in humans by lysine suggested filtration of at least B281 mg/min, corresponding to B400 mg/24 h.3 Similar studies in lysine-treated rats resulted in the excretion of 2.5–25 mg/24 h4,5 corresponding to 0.7–7 g/24 h in humans. These studies were based on immunoassays for detecting urinary albumin. It was recently suggested, based on the excretion of radiolabelled albumin and high-performance liquid chromatography, that significant amounts of albumin fragments are excreted in rat and human urine, possibly resulting from tubular degradation of filtered albumin, followed by luminal secretion of albumin fragments.6–9 Such fragments cannot be detected by conventional analyses, including radioimmunoassay. These studies, implicating a much greater amount of filtered albumin in the normal kidney, are still controversial. Renal catabolism and excretion of as much as 6.5 mg/100 g body weight/h in normal rats were estimated,8 apparently exceeding normal rat liver albumin synthesis rate.10 The findings are also in contrast to reports on urine protein excretion in Fanconi patients using proteomics.11 Thus, although the albumin filtration rate still remains controversial, it is clear that a significant amount of albumin is filtered and subsequently reabsorbed by the renal tubules. TUBULAR REABSORPTION OF ALBUMIN

Proximal tubular reabsorption of albumin by endocytosis was demonstrated almost 40 years ago.12,13 Endocytic uptake of proteins has been intensively studied and includes both nonspecific fluid-phase endocytosis as well as receptormediated endocytosis. In the kidney, proximal tubule fluidphase endocytosis of solutes is negligible.2,14 Receptormediated endocytosis, as originally described,15 involves the specific binding of a ligand to receptors in the apical plasma membrane. The receptor–ligand complex is internalized by invagination of the plasma membrane caused by adaptor molecule-mediated formation of a cytoplasmic coat.16–18 In clathrin-coated endocytosis, binding of adaptor molecules to the cytoplasmic tail of transmembrane receptors involves a characteristic NPXY motif17 identified in a number of endocytic receptors. Internalization is followed by dissociation of the invaginations from the plasma membrane forming vesicles. While the cytoplasmic coat dissociates, vesicles may fuse with other newly formed vesicles, or with an existing pool of larger vesicles, followed by acidification of the intravesicular lumen and the dissociation of the ligand from the receptor. The ligand may be further transported into Kidney International (2006) 69, 440–449

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lysosomes for storage or degradation, or into the cytosol for further processing/transport. The receptor is recycled back to the luminal membranes through a recycling compartment known as dense apical tubules,19 but may also be transported to lysosomes for degradation. Albumin binding sites on isolated proximal tubule segments and cells in vitro have been identified,14,20–22 and several receptors for tubular uptake of albumin have been identified. These include the multiligand receptors megalin and cubilin, responsible for the constitutive uptake of a vast variety of filtered plasma proteins. MEGALIN

Megalin is a multifunctional, endocytic receptor belonging to the low-density lipoprotein receptor family. It binds a number of structurally very different proteins. Megalin was originally identified as the antigen in Heymann nephritis, a rat model of membranous glomerulonephritis.23 It contains a large amino-terminal, extracellular domain, a single transmembrane domain and a short carboxy-terminal cytoplasmic tail (Figure 1). The latter harbors two NPXY sequences mediating the binding to adaptor proteins and the clustering into clathrin-coated pits,24–27 in addition to several Src homology 3 and one Src homology 2 recognition sites.24,25 The extracellular domain is composed of four cysteine-rich clusters of low-density lipoprotein receptor type A repeats constituting the ligand binding regions separated and followed by a total of 17 epidermal growth factor (EGF)type repeats and eight spacer regions containing YWTD repeats. Generally, binding to megalin is Ca2 þ dependent, and megalin itself binds calcium very strongly.28 Site-directed mutations of basic amino-acid residues in aprotinin, a ligand for megalin, decrease the affinity for the receptor, suggesting that binding is favored by cationic sites on the ligands.29 Many ligands are anionic proteins, indicating that the distribution of charge rather than the overall isoelectric point is important for binding. Renal expression

Megalin is heavily expressed in the kidney proximal tubule brush-border and luminal endocytic apparatus,23,28,30–32 as well as in lysosomal structures revealing smaller amounts of immmunoreactive megalin. Megalin has also been identified in the glomerular podocytes of Lewis rats,33 but not in other rat strains or other species. Extrarenal expression of megalin includes a number of other absorptive epithelia, including the ileum34,35 and the rodent yolk sac36 (reviewed in Christensen and Birn37). Much of our knowledge on the functions of megalin is based on data recovered from the study of megalin-deficient mice.38,39 Megalin-deficient, proximal tubule cells are characterized by a loss of endocytic invaginations, vesicles, and the membrane recycling compartment, dense apical tubules.40 So far, no significant changes in transport of water, electrolytes, glucose, or amino acids have been described in the megalin-deficient mice; however, an increased amount of several low-molecular weight serum proteins has been 441

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patients with low-molecular weight and tubular proteinuria, including Dent’s disease, have shown several analogies.51,54

Megalin NH2 Cubilin

CUBILIN

Amnionless NH2

Luminal plasmamembrane

NH2 COOH

NPXY VENQNY

COOH

CUB domain NPXY Complement-type repeat EGF-type repeat Spacer region containing YWTD Transmembrane domain NPXY motifs and VENQNY motif

Figure 1 | Schematic representation showing the structure of megalin, cubilin, and AMN. Megalin is a 4600-amino-acid transmembrane protein with a non-glycosylated molecular weight of 517 kDa. The extracellular domain (B4400 amino acids) contains four cysteine-rich clusters of low-density lipoprotein receptor type A repeats constituting the ligand binding regions separated and followed by a total of 17 EGF-type repeats and eight spacer regions containing YWTD repeats. A single transmembrane domain (22 amino acids) is followed by the cytoplasmic tail (213 amino acids), which contains two NPXY sequences and one VENQNY sequence in addition to several Src homology 3 and one Src homology 2 recognition sites. Cubilin is a 3600-amino-acid protein with no transmembrane domain and a non-glycosylated molecular weight of 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 acids followed by eight EGF-type repeats. The amino-terminal region contains a potential palmitoylation site and an amphipathic helix structure with some similarity to the lipid binding regions of apolipoproteins. AMN is a 434-amino-acid, single-transmembrane protein with a non-glycosylated molecular weight of B46 kDa. It has no known, closely related proteins, but a cysteine-rich stretch of 70 amino acids in the extracellular domain has similarity to modules present in a small group of proteins serving as bone morphogenic protein inhibitors. Both AMN and megalin bind cubilin and studies indicate that both are involved in the endocytosis of this receptor.

identified in the urine. The normal expression of megalin is dependent on receptor-associated protein (RAP)41 serving as a chaperone-protecting newly synthesized receptor from the early binding of ligands and possibly involved in folding of the receptor.42–46 RAP binds megalin with high affinity within the endoplasmic reticulum and functions as an intracellular ligand inhibiting the binding of most other ligands to megalin. A number of diseases characterized by proteinuria reveal decreased renal megalin expression.47–49 These include Dent’s disease, which is caused by mutations in the renal chloride channel ClC-5.50 Disruption of ClC-5 impairs proximal tubule endocytosis, and causes a reduction in megalin expression.49,51–53 Analyses on megalin knockout mice and 442

Cubilin is a multiligand, endocytic receptor also known as the intestinal intrinsic factor (IF)-B12 receptor.55 Cubilin was originally identified as the target of teratogenic antibodies in rats.56 It is a 460 kDa protein with little structural homology to known, endocytic receptors (Figure 1).57–59 Cubilin is 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) domains.61 No transmembrane domain has been identified in cubilin consistent with the observation that it is released from renal cortical membranes by nonenzymatic and nonsolubilizing procedures.58 The many CUB domains in cubilin support the ability of the receptor to bind a variety of ligands. Binding sites for selected ligands have been partially localized. Albumin, along with IF-B12, binding sites have been identified in a 113-residue amino-terminus, which includes the EGF-type repeats. Although IF-B12 inhibits binding of albumin to cubilin, the binding sites for the two proteins do not appear to be identical within this domain,62 and an additional binding site for IF-B12 has been identified in CUB domains 5–8.60 RAP binds to CUB domains 13–14,35,60 although its role for the processing of this receptor is unknown. Megalin also binds to cubilin, possibly within the amino-terminal region including CUB domains 1 and 2,34 supporting an interaction between the two receptors as discussed below. Renal expression

Cubilin is highly expressed in the renal proximal tubule brush-border and endocytic apparatus (Figure 2).36,55,63 As with megalin, immunoreactive cubilin can also be identified in lysosomes.55 Normal expression of cubilin is dependent on amnionless (AMN), a B45 kDa, transmembrane protein (Figure 1) identified as an important factor for the normal development of the middle portion of the primitive streak in mice.64 AMN co-localize with cubilin in kidney proximal tubule,65 interacts with the EGF-type repeats of cubilin and is essential for normal translocation of the cubilin–AMN complex from the ER to the plasmamembrane and for the subsequent endocytosis.65,66 Dogs with a mutation in the AMN gene as well as AMN-deficient mouse epithelial cells reveal defective apical insertion of cubilin.67–71 Mutations in either the cubilin or the AMN gene have been identified in Imerslund–Gra¨sbeck disease,72,73 a rare, inherited vitamin B12 deficiency syndrome characterized by defective intestinal absorption of IF-B12.74–77 Two affected Finnish families revealed a one-amino-acid substitution in CUB domain 8 affecting the binding of IF-B12 to cubilin or, alternatively, a point mutation expected to activate a cryptic intronic splice site causing an in-frame insertion and introducing stop codons predicting a truncation of the receptor in CUB Kidney International (2006) 69, 440–449

H Birn and EI Christensen: Albumin absorption in physiology and pathology

a

b

Figure 2 | Immunocytochemical localization of cubilin and uptake of endogenous albumin in cryosections of dog kidney cortex. In normal dog kidney, (a) intense labeling for cubilin (green fluorescence) is observed at the luminal brush border and in endocytic vesicles co-localizing with reabsorbed albumin (red fluorescence) reflecting endocytosis of filtered protein. Apical vesicles reveal colocalization of cubilin and albumin as indicated by the yellow label (arrows in a). In dogs with a mutation in the AMN, gene-causing defective insertion of cubilin into the apical membranes, (b) cubilin is retained in intracellular vesicles and no evidence of endocytic uptake of albumin is observed. Only a sparse labeling for albumin can be identified in the tubular lumen and along the apical membrane, the latter possibly reflecting binding to megalin (arrows in b). One micrometer cryosections from perfusion fixed dog kidneys were incubated with rabbit anti-dog cubilin (1:200) and sheep anti-rat albumin (1:200) primary antibodies followed by incubation with Alexa Flour 488 donkey anti-rabbit and Alexa Fluor 546 donkey anti-sheep fluorescence labelled secondary antibodies. Bars ¼ 10 mm.

domain 6.78 While the patient with the latter mutation has overt albuminuria, patients with amino-acid substitution reveal varying degrees of proteinuria ranging from little or no, to clearcut.77 Affected members of three Norwegian families, shown to have a nucleotide deletion causing the introduction of an early stop codon in the AMN gene,73 were all previously characterized, and had proteinuria at the time of diagnosis.74 The proteinuria observed in these patients support a role for cubilin in renal tubular protein reabsorption, and it may be hypothesized that the difference in the degree of albuminuria depends on the type of mutation and reflects the extent of inactivation of cubilin function, in particular whether the mutation affects the multiligand properties or only the IF-B12 binding site. OTHER RECEPTORS

Additional renal albumin receptors have been suggested including 55 and 31 kDa proteins identified by albuminaffinity chromatography and localized by immunocytochemistry. The sizes of these proteins are comparable to albumin binding proteins identified in endothelial cells and fibroblasts.79–81 Antibody cross-reactivity suggested that the lowmolecular weight receptors identified in rat proximal tubule are, in fact, fragments of cubilin,70 although the presence of multiple, structurally different albumin binding proteins cannot be excluded. The exact localization and functional importance of these, however, remains to be established. MEGALIN- AND CUBILIN-MEDIATED UPTAKE OF ALBUMIN

Cubilin plays an important role in normal proximal tubule endocytic reabsorption of filtered albumin (Figure 3). Kidney International (2006) 69, 440–449

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Albumin binds to cubilin with an estimated Kd B0.6 mM.70 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¨sbeck disease may also have albuminuria, supporting a role of cubilin. Albumin uptake in opossum kidney (OK) cells was inhibited by IF-B12, and anti-cubilin antibodies.82 The inhibitory constant for IF-B12 was 1.7 mM in line with the finding that IF-B12 and albumin both may bind to the same 113-residue amino-terminus of cubilin.62 Megalin-deficient mice also reveal an increase in urinary albumin excretion,70 suggesting that megalin is involved in tubular albumin reabsorption. Direct binding of labelled albumin to megalin has previously been identified using Sepharose columns;83 however, an additional, high-affinity binding between purified cubilin and megalin has been described in vitro.58 Based on this, it was suggested that megalin facilitates the endocytosis and intracellular trafficking of cubilin.58 This is further supported by studies showing that the in vitro uptake 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 antisense oligonucleotides.87 Thus, megalin may be involved in albumin reabsorption directly as a receptor for albumin, and/or indirectly by affecting the expression and/or endocytic function of cubilin.70 Albumin uptake in OK cells is inhibited by RAP, and by anti-megalin antibodies supporting this notion. The inhibitory constant for RAP is 17 nM;82 however, whether this reflects inhibition of albumin binding to cubilin, to megalin, or cubilin–megalin interaction is unknown. So far, RAP and albumin binding sites within the same region of cubilin have not been identified. It has been argued that the capacity of the megalin/ cubilin-mediated mechanism for tubular uptake of albumin is low,9 and that the urinary albumin excretion in megalindefective mice and cubilin-defective dogs is only slightly increased, representing merely a small increase in the excretion of non-degraded albumin.9 It must be considered, however, that megalin and cubilin are low-affinity albumin receptors serving in a high-capacity, reabsorptive mechanism. Assuming an albumin concentration of 10 mg/ml in the ultrafiltrate, this is about one-fourth of the estimated Kd for albumin binding to cubilin, and thus the tubular flow and the length of the proximal tubule becomes important for the amount of albumin reabsorbed. In mice and rats, with relatively short proximal tubules, the fraction of reabsorbed albumin compared to the filtered load is smaller than in larger species with longer proximal tubules. Megalin dysfunction caused a B1.5-fold increase in albumin excretion in mice compared to a sevenfold increase in cubilindefective dogs,70 and up to 425-fold increase in Imerslund–Gra¨sbeck patients.77 The low affinity of albumin binding to cubilin/megalin also implies that the amount of albumin 443

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Endocytic invaginations

Glomerulus 5 1

4

Dense apical tubules Amino acids Lysosome

Molecules carried by albumin

Megalin

Albumin

Proximal tubule

Cubilin

AMN

2a

2c 3

2b Lysosome

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, this is 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 highcapacity 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 concentration of albumin in the ultrafiltrate may be much higher as the reabsorbed amount is not limited by capacity, but rather by the flow and concentration of albumin in the ultrafiltrate. An important factor adding to the capacity of this system is the constant and fast regeneration of receptors by membrane recycling.88 Thus, the finding of only a small increase in albumin excretion in cubilin- or megalin-deficient animals is not inconsistent with the reabsorption and renal catabolism of significant amounts of protein by the same receptors during heavy proteinuria with filtration of large amounts of protein.

protein kinase C induces ectodomain shedding of megalin, also generated by the binding of the ligand vitamin Dbinding protein. It was suggested that this is accompanied by the release of an intracellular domain, possibly involved in intracellular signalling.92 The cytoplasmic tail of megalin has been shown to interact with a number of cytoplasmic proteins possibly modulating expression and/or function of the receptor including the adaptor protein autosomal recessive hypercholesterolemia27 and disabled protein 2 (Dab2).93–95 However, to what extent these potential, 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 of proteins is considered a constitutive process regulated only by changes in receptor expression levels identified in conditions such as Dent’s disease, polycystic kidney disease and possibly diabetes. Based on in vitro studies using OK cells, it has been suggested that albumin uptake may be regulated by G proteins,89 known to be involved in the regulation of vesicular transport, by phosphatidylinositol 3kinase,90 and by protein kinases.91 Recently, it was shown that 444

OTHER PATHWAYS

Alternative mechanisms of albumin reabsorption have been suggested (Figure 3), including a fast, high-capacity retrieval pathway for non-degraded albumin located distal to the glomerular basement membrane.9,96 This hypothesis is based on the analysis of the radioactive profile in renal venous blood following pulse injection of 3H-albumin in the isolated, perfused kidney.96 This revealed a second peak interpreted as the rapid return of filtered, labelled albumin Kidney International (2006) 69, 440–449

H Birn and EI Christensen: Albumin absorption in physiology and pathology

with an estimated capacity of 400–500 g/24 h.9 While it was suggested that micropuncture of early proximal tubule failed to identify this process because of its location in the glomerulus, or very early in the tubular system,96 the nature of this proposed mechanism has not been established. It is generally believed that tight junctions prevent paracellular transport of most proteins,97–101 and so far, no morphologic support for an alternative, transcellular albumin transport mechanism has been demonstrated. Nevertheless, defects in such a highly speculative, high-capacity albumin transport pathway may result in heavy albuminuria without any initial changes in glomerular function, and thus implicate a radical change in our understanding of albuminuric renal disease. ALBUMIN REABSORPTION AND INFLAMMATION

The concept of albumin-induced renal inflammation and scarring has been intensively explored using both clinical and experimental approaches. Albuminuria has been shown to be an independent risk factor for the progression of renal disease and the renoprotective effect of antiproteinuric treatment, whether by dietary or pharmacological intervention, has been shown to be correlated to the reduction in proteinuria.102,103 The hypothesis tested is that excess albumin in the tubular lumen leads to the induction of inflammation, possible tubular epithelial–mesenchymal transformation and interstitial fibrosis. Overload albuminuria in rats and mice causes interstitial inflammation and fibrosis.104,105 Studies on proximal tubule cells have shown that albumin exposure induces the expression of a number of inflammatory and fibrogenic mediators, including cytokines such as RANTES (regulated upon activation, normal T cell expressed and secreted),106 monocyte chemotactic protein-1,107 interleukin-8,108 fractalkine109 and tumor necrosis factor-a,110 as well as endothelin,111,112 transforming growth factor-b (TGF-b),112 collagen;113 and may also induce changes in tubular cell expression of surface integrins114 or, eventually, apoptosis.115,116 These data suggest that albumin itself and/or bound ligands such as fatty acids117–120 initiate a series of events that eventually leads to fibrosis. The exact cascade has not been established; however, evidence suggests that the process involves the initial endocytic uptake of albumin. Renal cell lines with low endocytic activity do not respond to albumin by the activation of intracellular pathways and collagen synthesis,113 and megalin-deficient cells appear to be less sensitive to hemoglobin-induced proximal tubule necrosis.121 Interestingly, TGF-b, which may be induced by albumin exposure,112 may also act in a feedback mechanism increasing albumin filtration122 and at the same time inhibiting megalin- and cubilin-mediated albumin endocytosis,123 both leading to increased albuminuria. Recent evidence indicates that albumin may interact with signalling receptors including apically expressed EGF receptors.124 In addition, it has been shown that members of the low-density lipoprotein receptor family may be involved directly in transmembrane signal transduction.125 The lowdensity lipoprotein and very low-density lipoprotein recepKidney International (2006) 69, 440–449

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tors, low-density lipoprotein receptor-related protein and the apolipoprotein ER2, have been shown to bind other cytoplasmic adaptor proteins, notably Dab1, at NPXY motifs on the cytoplasmic tail of these receptors.126,127 Dab1 is part of a signalling pathway involving the extracellular protein Reelin important for brain development.128 So far, no specific signalling events have been documented for megalin; however, as previously mentioned, the cytoplasmic tail of megalin has been shown to interact with Dab2,93,94 an intracellular protein that may be involved in signalling or regulating of cellular growth and differentiation.95 Recent evidence also indicates signalling by ligand-induced, enzymatic release of a carboxyl-terminal fragment of megalin.92 Thus, it may be that excessive binding of filtered proteins, including albumin, to megalin may initiate intracellular signalling events. Proposed intracellular signalling cascades include the activation of nuclear factor kb110,129–133 as well as STAT (signal transducers and activator of transcription)134 transcription factors, possibly through the formation of reactive oxygen species,131,134 a process that may also be dependent on protein kinase C.131 Interestingly, delipidated albumin has been suggested to protect against reactive oxygen species,135 indicating that the potentially protective versus damaging effect is determined at least, in part, by the substances carried by albumin.136 In addition to albumin, several other filtered proteins have been suggested to induce proximal tubule phenotypic changes, including iron carriers, proteins of the complement system, immunoglobulins, and growth factors. It is not clear to what extent each of these proteins or their combination contributes to the inflammation and fibrosis; however, some studies indicates differential and protein-specific effects,137,138 and studies in analbuminemic rats even suggested that albumin was not important to the progression of proteinuric, renal disease when evaluated for up to 20 weeks.139,140 Most of the studies exploring the mechanism of proteinuria-induced renal changes are based on proximal tubule cell lines. Cells are exposed to albumin or serum fractions and changes are studied by protein and gene analysis of the media and/or the cells. It is obvious that extrapolation from such data to the human in vivo situation may be difficult also considering the somewhat conflicting data observed with different proteins in different cell systems,116,137,138 as well as the reported changes in the expression of several genes of still unknown function.141 The obvious challenge to the overall hypothesis is the ability to specifically interfere with the cascade of events leading to irreversible renal damage. An established approach has been to prevent or reduce proteinuria notably by blocking the renin–angiotensin system.142 Another approach would be to inhibit tubular uptake of proteins either by blocking binding to receptors or by inhibiting endocytosis. As both megalin and cubilin are multiligand receptors serving other important functions, for example, for the uptake and metabolism of vitamins,37 including the activation of vitamin D,143,144 this approach may prove inappropriate. Recently, it has been 445

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shown that transfection with a monocyte chemotactic protein-1 antagonist145 or a truncated form of IkBa, thereby inhibiting nuclear factor-kb,133 inhibits albumin-overload induced tubulointerstitial injury supporting the possibility of therapeutic intervention at the tubular level. Additional evidence suggests that inhibition of the renin–angiotensin system in addition to a reduction of proteinuria may also attenuate albumin-induced signal activation in tubular cells.146 Further studies should evaluate the clinical significance and feasibility of the various approaches. An alternative hypothesis to explain the progression of renal disease has been proposed based on histological studies of the development and progression of animal and human renal disease.147 The analyses revealed differential changes in glomerular structure, including adhesions of the glomerular tuft to Bowman’s capsule causing the formation of crescents, either by misdirected filtration or by epithelial cell proliferation. It was suggested that progression of injury into the tubulointerstitium is caused by direct encroachment of the glomerular disease either by misdirected filtration into the space between the tubular epithelium and basement membrane (Figure 3)148,149 or by incorporation of the initial proximal tubule into the growing cellular crescent.149 In both cases, this results in the loss of nephrons and subsequent fibrosis, which is considered a reparative process important for the maintenance of renal structure rather than a determinant of further injury.147 While this hypothesis does not exclude a direct effect of proteins, including albumin, filtered into the tubular lumen, it is clear that the approach to prevent progression of renal disease according to this hypothesis should be aimed at the glomerular changes rather than the tubular reactive processes. CONCLUSIONS

Ample evidence suggests an association between the progression of renal disease and albuminuria reflecting an imbalance between glomerular filtration and tubular reabsorption of this protein. Modulation of the renin–angiotensin system has proven to reduce proteinuria and to attenuate the progression of renal disease. In vitro studies show that albumin induces changes in tubular cells stimulating the expression of inflammatory and fibrogenic mediators. Although inflammation appears to be an important pathogenic factor in a number of renal diseases characterized by albuminuria, the possible pathogenic role of albumin itself, however, is not fully elucidated. An attractive hypothesis consistent with the in vitro observations suggests that an initial glomerular dysfunction causing filtration of increased amounts of albumin and other potential proinflammatory proteins is followed by proximal tubule reabsorption and the induction of inflammation and, eventually, fibrosis mediated by the tubular cells. Whether this mechanism is important to the pathophysiology of human renal disease remains to be established, urging the need for means to specifically intervene at specific steps in this pathway. 446

ACKNOWLEDGMENTS

The work was supported by the Danish Medical Research Council, the University of Aarhus, the NOVO-Nordisk Foundation, Fonden til Lægevidenskabens Fremme, the Biomembrane Research Center, the Beckett Foundation, the Leo Nielsen Foundation, the Ruth Ko¨nig-Petersen Foundation, the Birn-Foundation and the European Commission (EU Framework 6, EureGene, Contract Number 05085).

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