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Renal Filtration, Transport, and Metabolism of Albumin and Albuminuria
Albuminuria is a sensitive marker of kidney dysfunction and associated with increased mortality and risk of cardiovascular disease. Urinary excretion of albumin is regulated by balance between glomerular filtration and tubular reabsorption. Our knowledge of the molecular mechanisms regulating the glomerular albumin permeability and proximal tubule, endocytic reabsorption of albumin has greatly expanded and animal models as well as genetic analyses in human diseases have established the significance of these mechanisms in normal renal handling of albumin. Furthermore, studies, primarily in vitro , have suggested that albumin may affect the function and phenotype of tubular cells indicating that albuminuria contributes to the progression of albuminuric, kidney disease. Despite this information, our knowledge of the exact molecular dysfunction leading to albuminuria in most cases of acquired renal diseases is still limited. Furthermore, the independent effect of albuminuria in disease progression and thus the importance of interventions targeted at tubular dysfunction remains to be established in human disease
Keywords
albuminuria, glomerular filtration, megalin/cubilin, proximal tubular endocytosis, interstitial fibrosis
Introduction
Albuminuria is one of the oldest yet remains one of the most sensitive and widely used markers of kidney dysfunction. Albumin is the most abundant plasma protein and its urinary excretion is determined by the combined effects of glomerular filtration and renal tubular processing ( Fig. 73.1 ). Dysfunction of both these processes may result in increased excretion of albumin, and glomerular injuries as well as tubular damage have been implicated in the initial events leading to albuminuria. Albuminuria not only indicates acute or chronic renal damage but is also a well established and independent marker of progression in chronic kidney disease (CKD). Interventions aimed at reducing albuminuria have proved effective in ameliorating the continuous loss of renal function in various form of CKD suggesting that albuminuria not only is a marker of kidney disease but in fact involved in the pathophysiology of progression. Experimental evidence points to direct and deleterious effects of albumin on renal tubular cells and identifies a number of downstream mediators initiating inflammation and eventually renal fibrosis.
Gene analyses in human diseases and animal knockout models have identified a number of key molecules regulating glomerular filtration and tubular reabsorption of albumin. In most cases of human disease, however, both the precipitating events and the accelerating mechanisms associated with albuminuria are unknown and may include several, different pathways. The relative importance of the various molecular mechanisms regulating glomerular filtration and tubular handling of albumin remain controversial both in normal physiology and in disease and the evidence for an independent pathogenic role of albumin in the development and progression of renal disease is debated.
This chapter will review the structures controlling glomerular filtration of albumin and discuss the molecular and pathophysiological mechanisms causing changes in glomerular permselectivity. Furthermore, the receptors regulating tubular reuptake of filtered albumin are presented and the possible pathways by which filtered albumin may cause tubular and interstitial damage are discussed in relation to acute and chronic kidney disease.
Albumin
Albumin is an anionic, flexible, heart-shaped, 585-amino acid, single polypeptide chain with a MW ~67 kDa present in plasma at a normal concentration of 35–50 mg/ml. While 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 including fatty acids, bilirubin, ions such as Ca 2+ and Mg 2+ , drugs, hormones, and lipophilic as well as hydrophilic vitamins, e.g., vitamin A, riboflavin, vitamin B6, ascorbic acid, and folate. Albumin undergoes posttranslational modification including glycation, acetylation, methylation, carbamylation and phosphorylation. Albumin is almost exclusively synthesized in the liver at a rate of 10–15 g per day in a healthy person and its normal half life is estimated to 19 days representing the balance between synthesis, transcapillary escape, and catabolism predominantly within muscle, liver and kidney. In kidney diseases such as nephrotic syndrome and end stage renal disease, including well managed patients on peritoneal dialysis, albumin synthesis appears to be increased compensating for increased losses. Normally the albumin gene is silent in the kidney, however, it has recently been shown that the gene is activated in cases of acute kidney failure leading to the renal synthesis of albumin. The local production of albumin is associated with albuminuria, however, the extent to which local synthesis of albumin contributes to the urinary excretion of albumin is not known.
Glomerular Filtration
Glomerular Filtration Barrier
The glomerular filtration barrier is structurally composed of three layers, the capillary endothelial cells, the glomerular basement membrane (GBM) and the podocyte filtration slit membrane ( Fig. 73.2 ). The barrier is freely permeable to water, solutes and small molecules however, increasing size of macromolecules causes increasing restriction to filtration as do negative charge.
The fenestrated endothelium ( Fig. 73.2 ) is unusual since the fenestrae generally are not closed by diaphragms, except as demonstrated in rat where the capillaries which are direct tributaries to the efferent arteriole do indeed have diaphragms closing the fenestrae. The pores in the endothelium appear not to be fully open holes. By special fixation procedures it has been demonstrated that the pores are filled with glycoproteins forming “sieve plugs” probably contributing to the endothelial part of the filtration barrier. The endothelial cells have a thick glycocalyx and an even thicker endothelial cell surface coat, which are believed by many authors to contribute significantly to the charge selectivity of the barrier (for a recent review see Haraldsson et al. ).
The basement membrane, which in man is about 300 nm thick, consists of three layers, a lamina densa, located between a lamina rara interna facing the endothelial cell and a lamina rara externa, facing the podocyte ( Fig. 73.2 ). In the 1970s the GBM was considered the major contributor to the charge selectivity of the filtration barrier (see also Kanwar et al. for references). In vitro studies, however, on isolated GBM showed no charge selectivity and removal of charged components of the GBM in mouse knock out studies in general did not change charge selectivity, for discussions see. A large variety of both genetic and acquired, albuminuric diseases affect the GBM, e.g., Alport syndrome and diabetes mellitus (see also later).
The third component of the barrier, the podocyte filtration slit membrane ( Fig. 73.2 ), has attracted great interest as a key part of the filtration barrier, especially since the findings that, for example, gene defects of nephrin ( NPHS1 ) induce the congenital nephrotic syndrome of the Finnish type and gene defects of podocin (NPHS2) induce nephrotic syndrome of the non-Finnish type. The porous structure of the filtration slit membrane was first described by Rodewald and Karnowsky measuring the dimensions of the pores to be 4×14 nm. In a recent publication the mean radius of the observed irregular circular or elipsoid pores were measured to be 12.1 nm. In proteinuric rats additional large pores were observed and suggested to contribute to the increased filtration of protein/albumin in pathologic conditions, a finding which awaits confirmation.
An elegant model for the charge restriction has recently been put forward, based on micropuncture experiments in glomeruli of Necturus maculosus . The authors identified a filtration dependant negative electrical charge in the Bowman’s space compared to the capillary lumen, a charge that would allow negatively charged proteins like albumin to be electrophoresed back to the blood and the opposite for positively charged proteins.
There is no doubt that all three structural components of the glomerular filter are necessary for maintaining the barrier, illustrated by the observations that damage to any part eventually leads to albuminuria, that the GBM is synthesized from both the epithelial- and endothelial cells and that vascular endothelial growth factor (VEGF)-A produced by the podocytes influences development and maintenance of the endothelial cells which possesses receptors for VEGF-A, VEGFR-1 and 2.
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 µg/ml. This corresponds to a filtered load of albumin between 170 mg and 9 g per 24 h in normal humans. Inhibition of tubular albumin uptake in humans by lysine suggested filtration of at least 281 µg/min, corresponding to ~400 mg/24 h. Similar studies in lysine treated rats resulted in the excretion of 2.5 mg to 25 mg/24 h corresponding to 0.7–7 g/24 h in humans. In rat the filtration fraction of albumin was estimated to 0.0006 by micropuncture studies in good agreement with the results mentioned above. This figure was, however, challenged by Comper and colleagues who estimated the filtration fraction by two-photon microscopy to be 0.034. As calculated by Gekle this implies a filtration of 225 g/24 h of albumin in humans and the results were immediately questioned by several groups. Subsequently, three studies have seriously questioned the technical approach applied by Russo et al. Thus, the notion of normal glomerular filtration of such large amounts of albumin remains highly controversial. For an excellent review comparing glomerular permselectivity of ficoll, dextran and globular proteins, see Venturoli and Rippe.
Albumin Uptake in Glomerular Cells
Albumin uptake has been demonstrated in vivo in podocytes from human, rat and mouse and in vitro in mouse and human podocytes. Accumulation of endocytosed protein in podocytes is also indicated by podocyte vacuolization in proteinuric patients and experimentally, endocytic uptake of tracer proteins in podocytes have been demonstrated in vivo . The albumin binding receptor megalin (see below) has been identified in rat podocytes and very recently also on human podocytes providing a mechanism for the endocytic uptake of albumin and other proteins. It has been proposed that unless removed, filtered proteins would clog the glomerular filter due to the podocyte slit membrane. Such a theoretical clogging of the slit diaphragm may be attenuated by megalin mediated, podocyte endocytosis of trapped protein, including albumin. It should be emphasized that the endocytic uptake of albumin in the podocytes is minimal compared to the subsequent uptake in the proximal tubule (see below).
Tubular Albumin Uptake
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