1 Section of Nephrology, Department of Medicine, Robert Wood Johnson School of Medicine, New Brunswick, New Jersey, 2 Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, Illinois, USA
Keywords: albumin; apoptosis; chronic renal disease; oxidant injury; proteinuria
Introduction
Over the last two decades a shift in paradigm has taken place regarding the role of proteinuria in the progression of renal disease. Across a wide spectrum of glomerular and non-glomerular diseases (especially those characterized by tubulointerstitial fibrosis), the degree of proteinuria has been shown to correlate with the risk of progression to end-stage renal disease [1]. Initially, proteinuria was thought simply to be a marker of the severity of glomerular injury. Recent evidence, however, suggests that proteinuria, and particularly albuminuria, may itself promote tubular injury, with interstitial inflammation and eventual fibrosis [2,3].
While several mechanisms have been put forward to explain how albuminuria can produce renal injury, a direct causal relationship, even in animal models, has yet to be established. To complicate matters further, recent in vitro data indicate that albumin can inhibit apoptosis and promote the survival of primary cultures of mouse proximal tubular epithelial (MPT) cells and macrophages [46]. These results present a seeming paradox: how can albumin act as a potent survival factor for tubular epithelial cells in culture and yet at the same time mediate their injury in vivo? We will attempt, in this brief review, to reconcile these seemingly contradictory findings.
Inhibition of apoptosis by albumin: carriage of non-cytokine survival factors
Albumin is a circulating plasma protein of 69 kDa with a variety of homeostatic functions. These include maintenance of oncotic pressure, buffering of acid-base changes, and transport to and from tissues of multiple bioactive substances [7]. Among the many molecules carried by albumin are free fatty acids, phospholipids such as lysophosphatidic acid (LPA), prostaglandins, heavy metals, and steroid-based hormones and vitamins [7].
Recently we showed that albumin-bound LPA is a major serum survival factor for primary cultures of MPT cells and peritoneal macrophages [4,5]. LPA is the smallest and structurally simplest of all glycerophospholipids and is found in serum at concentrations of 220 µmol/l. Nearly all serum LPA exists tightly bound to albumin in a biologically active form. LPA is an extremely potent inhibitor of apoptosis. For both MPT cells and macrophages, protection from apoptosis by LPA is equivalent to that seen with 10% serum or conventional survival factors such as epidermal growth factor (EGF) or macrophage colony stimulating factor (M-CSF). A significant effect on survival is seen with LPA concentrations as low as 6 µmol/l in MPT cells and as low as 50 nmol/l in macrophages. The survival activity of LPA is dependent upon activation of the lipid kinase phosphatidylinositol 3-kinase (PI3K) [4,5]. Downstream mediators of PI3K include Akt and pp70s6k. The latter kinase plays an important role in proliferation and is the highly specific target of the immunosuppressant rapamycin. As nearly all survival factors described to date are proteins or steroid-based lipids, LPA therefore represents a new class of cell survival factors.
Two additional points merit emphasis. First, LPA is also a potent proliferative factor for MPT cells [4]. Stimulation of proliferation occurs at concentrations as low as 3 µmol/l and, as expected, is largely mediated through pp70s6k. Second, LPA may not be the only non-cytokine survival factor carried by albumin. Other phospholipids, such as phosphatidic acid, are also capable of inhibiting apoptosis [5]. In addition, we have recently found that albumin-bound unsaturated (but not saturated) fatty acids, including linoleic and oleic acids, are potent survival factors for cells in culture [8].
Inhibition of apoptosis by albumin: scavenging of reactive oxygen species
During the course of our studies on LPA, we observed that 99.95% delipidated albumin alone, which is devoid of LPA or other lipids, is itself able to inhibit apoptosis of MPT cells and peritoneal macrophages [6]. This lipid-independent survival activity of albumin appears to depend on albumin's ability to act as an anti-oxidant [6].
Many plasma proteins can act as anti-oxidants by serving as sacrificial sinks for attack by reactive oxygen species (ROS), either directly through oxidation of amino-acid side-chains or indirectly through reaction with lipid species and radicals arising from the peroxidation of cell-membrane lipids [9]. The amino acids cysteine, histidine, methionine, tyrosine, and tryptophan are particularly susceptible to direct oxidative attack, whereas lysine is most susceptible to attack by malonyldialdehyde, one of the principal products of lipid peroxidation [9]. Albumin and other plasma proteins such as ceruloplasmin and transferrin also protect against oxidative injury by binding the transition metals Fe+2 and Cu+2, thereby preventing generation of the hydroxyl radical OH· via the Fenton reaction [10].
Among plasma proteins, albumin is unique in possessing a free sulph-hydryl group (at Cys-34), making it a particularly effective scavenger of ROS [6,7]. Addition of delipidated albumin (dBSA) alone, in the absence of any other survival factors, inhibits apoptosis and promotes the survival of MPT cells and macrophages [6]. A significant effect on survival by dBSA is seen at concentrations as low as 0.5 mg/ml (7.25 µmol/l), or 1% of albumin's concentration in serum. Importantly, dBSA does not activate PI3K [6], implying that its survival activity occurs via a mechanism distinct from that for most cytokines. Based on the following evidence [6], we proposed that dBSA inhibits apoptosis via scavenging of ROS. First, apoptosis of MPT cells and macrophages following withdrawal of survival factors is accompanied by accumulation of ROS, an effect that is inhibitable by dBSA. Second, during protection from apoptosis, there is a progressive decrease in the free sulph-hydryl content of dBSA, consistent with scavenging of ROS. Third, peroxidation of dBSA with H2O2 or chemical blockade of free sulph-hydryl groups by carboxyamidation almost completely eliminates the survival activity of dBSA. Moreover, dBSA completely protects both MPT cells and macrophages from cell death during exposure to xanthine/xanthine oxidase, a well-established model of oxidative injury. Peroxidation of dBSA abrogated its ability to inhibit the death of cells exposed to xanthine/xanthine oxidase.
Structural modifications of albumin: oxidative and non-oxidative
Over the past two decades a growth of knowledge has occurred implicating oxidative and non-oxidative structural modifications of proteins in the molecular pathogenesis of ageing and disease states such as diabetes, atherosclerosis, and acute and chronic inflammation [11,12]. Albumin and other circulating plasma proteins can undergo a variety of structural alterations through interaction of their amino-acid side-groups with various reactive species. For example, as previously discussed, a variety of ROS and oxidative free radicals can react directly with the amino-acid side-chains of albumin to generate an assortment of oxidatively modified forms of albumin [6,7,9]. In addition, albumin may be indirectly modified by oxidation through reaction with various carbonyl intermediates to produce so-called carbonyl stress end-products [13]. Highly reactive carbonyl intermediates arise via the non-enzymatic auto-oxidation of carbohydrates, lipids, and amino acids. Once formed, they can then react with, and significantly alter the structure of, proteins. Advanced glycation end-products (AGE) and advanced lipoxidation end-products (ALE) are examples of carbonyl stress end products, produced via the interaction of albumin with carbonyl compounds derived from carbohydrates and lipids respectively [13].
In the case of carbohydrates, carbonyl compounds such as glyoxal, methylglyoxal, and glycoaldehyde react with free amine groups on albumin or other proteins to form reversible adducts called Schiff bases [13]. Over time, these reversible adducts undergo a series of chemical rearrangements to form less reversible Amadori-type early glycation products [13]. Although Amadori products are still chemically reversible, their reaction kinetics are extremely slow, requiring weeks to achieve equilibrium, and so for nearly all purposes Amadori products may be regarded as irreversible [13]. Haemoglobin A1c, used to monitor average blood glucose level, is an example of an Amadori product. Amadori products eventually undergo a further series of chemical rearrangements to yield irreversible products termed AGE [13]. Examples of AGE are pentosidine, pyrraline, and N-(carboxymethyl)lysine (CML). The formation of AGE is enhanced by oxidation, a process that has been termed glycoxidation [13].
Fatty acids bound to albumin or other proteins can also undergo auto-oxidation to yield reactive carbonyl compounds [13,14]. Susceptibility to auto-oxidation is proportional to the number of carbon double bonds, so that polyunsaturated fatty acids comprise the most important source of lipid carbonyl compounds [14]. Once formed, lipid hydroperoxides can break down to yield highly reactive aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE), which may then react with exposed lysine residues on albumin and other proteins to yield MDA- and HNE-lipid adducts termed ALE [13].
Amino acids are an additional source of carbonyl intermediates. Auto-oxidation of serine, threonine, and the hydroxyamino acids hydroxyproline and hydroxylysine yields the carbonyl intermediates glycoaldehyde and acrolein [13]. Reaction of glycoaldehyde with lysine residues on albumin and other proteins results in the formation of the carbonyl stress end-product CML [13]. As CML is also generated through the reaction of carbohydrate-derived carbonyl intermediates, CML is categorized as an AGE. It is of note that serum CML is over 90% bound to albumin [13].
Structurally modified albumin as inflammatory signalling intermediates
Oxidative and carbonyl stress end products have both been shown to initiate inflammatory cascades via their interaction with scavenger receptors on macrophages [13,15]. For example, engagement of RAGE (receptors for advanced glycation end products) on macrophages by AGE-modified ß2-microglobulin results in all of the following: macrophage activation; release of cytokines such as tumour necrosis factor- (TNF-
), platelet-derived growth factor (PDGF), and transforming growth factor-ß (TGF-ß); release of chemokines, leading to the recruitment of macrophages and other inflammatory cells; production and release of proteases, including matrix metalloproteinases; and generation of ROS [13,1517]. RAGE-mediated activation of macrophages not only promotes, but is also in part dependent on, oxidant stress [13]. In the case of AGE-modified ß2-microglobulin, an important consequence of RAGE-mediated macrophage activation is the inflammatory degradation of ß2-microglobulin into intermediates involved in dialysis-related amyloidosis [15].
Expression of RAGE has been demonstrated on renal tubular epithelial and mesangial cells in patients with diabetic nephropathy and a variety of other renal diseases [18]. Engagement of mesangial cell RAGE leads to up-regulation of a number of stress response genes including nuclear factor kappa B (NF-B), peroxisome proliferator-activated receptor-
(PPAR-
), and haemoxygenase [19,20]. In addition, AGE have been shown to stimulate mesangial cell synthesis of fibronectin and type IV collagen [17]. In light of these observations, it is feasible that structurally modified albumin, generated by passage through an inflamed glomerulus and/or during prolonged travel throughout the systemic circulation, could bind to mesangial and tubular cell RAGE, thereby inducing release of inflammatory mediators, renal tubular cell damage, and fibrogenesis.
Hypothesis
In the majority of proteinuric states, albumin comprises the major protein fraction found in the urine. Epithelial cells lining the proximal tubule reabsorb filtered albumin through receptor-mediated endocytosis (e.g. via megalin) [21]. Albuminuria results when the filtered load of albumin exceeds the tubular reabsorptive capacity. Thus, urinary albumin represents an underestimate of the actual quantity of albumin to which tubular epithelial cells are exposed. Even in normal states, in which the glomerular filtration coefficient for albumin is quite low, the daily filtered load of albumin reaches an average of 12 g, virtually none of which appears in the urine [22]. Thus, the tubular epithelial cells of even healthy individuals reabsorb a significant quantity of albumin. This raises the possibility that albumin may play a role in normal tubular homeostasis.
We return now to the question posed at the beginning of this review. How can albumin act as a potent survival factor to prolong the viability of renal tubular epithelial cells in culture and yet at the same time promote tubulointerstitial injury in vivo? We hypothesize that the answer lies in the balance of beneficial vs potentially injurious factors carried by albumin, as well as in the multiple structural modifications to which albumin is susceptible. Under normal physiological conditions, albumin may contribute to tubular cell homeostasis through its anti-oxidant activity and through delivery of LPA and other putative non-cytokine growth factors. Conversely, in pathological states such as glomerulonephritis, in which, for a variety of reasons, albumin may be subject to increased oxidative stress, albumin could function as a sort of Trojan horse, carrying oxidized lipids as well as reactive carbonyl groups in the form of AGE and ALE. According to this model, the opening of the gates of Troy would be mediated by receptors such as megalin or RAGE and other scavenger receptors, which bind unmodified and modified forms of albumin respectively. Damage to renal tubular epithelial cells would then ensue through a variety of mechanisms: oxidative damage through reaction with oxidized phospholipids and fatty acids bound to albumin; structural alteration of cell membrane proteins and lipids through reaction with carbonyl groups of modified albumins such as AGE and ALE; and RAGE-mediated activation of macrophages and other cells, with release of pro-inflammatory chemokines and cytokines. Oxidized lipoproteins and other oxidized lipids that also cross into the tubular space may further oxidize urinary albumin, enhancing its potential toxicity.
In conclusion, while current evidence suggests a causal role for urinary albumin in tubulointerstitial inflammation, fibrosis, and progression of renal failure, we believe that a more plausible culprit is a structurally modified albumin that, along with its bound moieties, has been chemically altered by oxidative and carbonyl stress.
Acknowledgments
The authors would like to acknowledge import- ant contributions from Wilfred Lieberthal, Jason Koh, and Vivian Elizabeth Abernethy. This work was supported by NIH Grant AR/AI42732 (JSL), a Clinical Investigator Award from the National Kidney Foundation (JSL), and a Career Enhancement Award from the American Society of Nephrology (JSL).
Notes
Correspondence and offprint requests to: Jerrold S. Levine, Nephrology Section, The University of Chicago, 5481 South Maryland Avenue, MC5100, S-506, Chicago, IL 60637, USA.
References