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Renal Proximal Tubular Albumin Reabsorption: Daily Prevention of Albuminuria

Michael Gekle

M. Gekle is in the Physiologisches Institut, Universität Würzburg, Röntgenring 9, 97070 Würzburg, Germany.

	Abstract

 
Although the glomerular filtration coefficient of albumin is small, the daily filtered load can be as much as 8 g. To prevent such massive losses of albumin, quantitative reabsorption along the proximal tubules is accomplished by "receptor"-mediated endocytosis. Albumin reaches the lysosomes where it is degraded to amino acids.

	Introduction

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

 
What is albumin? The term albumin derives from albus, the Latin word for white (12), and dates back to the recognition that the portion of an egg that appears white after coagulation, consists mainly of proteins. This part of the egg is also called albumen. In the early days of plasma protein separation, the water-soluble fraction was called albumin. Proteins of the less water-soluble fraction were denominated globulins. Nowadays, albumin is generally regarded to mean serum or plasma albumin, a single protein species.

In quantitative terms, albumin is the most important plasma protein. At a concentration of 45 g/l (652 x 10^–6 mol/l), albumin represents ~60% of all plasma proteins. The molecular mass obtained from physical data (e.g., sedimentation equilibrium constant) is 69,000 Da (12). From physicochemical data, the following conformation of the albumin molecule in physiological environment can be derived. The amino acid chain is arranged in nine loops, stabilized by disulfide bonds, which form three spherical domains (denominated I-III, starting from the amino terminus) with different net charges. The net charges of the three domains are unevenly distributed, ranging from –9 and –8 to +2, leading to a net charge of –15. Thus albumin can be described as an ellipsoid molecule of 15 nm in length and 3.8 nm in diameter ("stubby cigar" shape) (12). One important function of albumin is the maintenance of an oncotic pressure difference between plasma and the interstitial space. Hence, albumin is involved in the regulation of fluid exchange across the capillary wall and prevents the formation of edema.

	Quantitative aspects of albumin filtration

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

 
Analysis of urine of healthy individuals shows that only very little albumin (30 mg/day) undergoes renal excretion (10). When one considers the plasma concentration of albumin and the high rate of renal blood flow, excretion of albumin with the final urine is very low. Nevertheless, this measurement shows that albumin reaches the tubular lumen. Furthermore, there are pathophysiological states in which the daily albumin excretion can be dramatically increased, resulting in the so-called albuminuria.

How can a molecule of the size and charge of albumin reach the tubular lumen? The composition of glomerular ultrafiltrate depends on the intrinsic permeability of the glomerular capillary wall (4). Molecules with effective radii >2 nm are retained with increasing efficiency as the radius rises. Furthermore, negatively charged macromolecules are restricted to a greater extent than neutral molecules of comparable size (4, 10). For albumin, the effective radius is in the range of 7.5 nm (4, 10), resulting in a low fractional filtration, which is even further impaired by the negative net charge. As a result, fractional filtration of albumin is in the range of 0.05–0.1% (10, 15). Multiplication of plasma albumin concentration (45 g/l), with the fractional filtration of 0.1%, results in an ultrafiltrate concentration for albumin of 45 mg/l. This value is in good agreement with albumin concentrations determined by micropuncture studies (10, 15). Assuming a daily glomerular filtration rate of 180 liters in a healthy adult, one can estimate the daily filtered load of albumin to be 8,100 mg. This corresponds to ~6.5% of total plasma albumin.

So far, the fate of albumin in the kidney can be summarized as follows. Per day, up to 8,100 mg of albumin reach the urinary space by glomerular filtration, but only ~30 mg are excreted with the final urine. Hence, more than 99% of filtered albumin (i.e., up to 8,070 mg/day) must be reabsorbed along the nephron. Extensive micropuncture studies show that reabsorption takes place almost exclusively along the proximal tubule (e.g., Refs. 2 and 15).

	Albumin is reabsorbed by endocytosis

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

 
Albumin seems to be almost evenly reabsorbed in early and late proximal convoluted tubules and straight tubules (15). Because of its size, albumin cannot leave the tubular lumen on the paracellular route across the tight junctions. Furthermore, albumin is not cleaved in the tubular lumen and therefore does not cross the apical membrane of the proximal tubular cell in the form of free amino acids (10, 15). Thus the only mechanism able to mediate albumin reabsorption is endocytosis. Hereby, small endocytic invaginations are formed at the microvillar base, at so-called clathrin-coated pits, which contain a certain volume of the extracellular (in this case tubular) fluid and thereby also molecules dissolved in the fluid. The endocytic invaginations are untied to form endocytic vesicles that transport their content to the endosomal compartment. From the endosomal compartment, part of the material taken up reaches the lysosomal compartment via late endosomes, whereas another part is brought back to the plasma membrane via recycling endosomes. One can roughly distinguish two forms of endocytosis: 1) fluid-phase endocytosis and 2) adsorptive endocytosis (10).

During fluid-phase endocytosis, the concentration of any given substrate in the endocytic invagination is the same as in the extracellular space, indicating that these substrates are not enriched at the plasma membrane, and thus uptake increases proportionally to the extracellular concentration of the substrate. Classical substrates for this kind of endocytic uptake are dextran or inulin, markers for determination of glomerular filtration rate. Obviously, uptake in the form of fluid-phase endocytosis is a very slow process, as can be easily derived from the fact that virtually all inulin filtered in the glomeruli reaches the final urine.

During adsorptive endocytosis, substances are bound to the cell membrane, and the concentration in the endocytic invagination exceeds the concentration in the extracellular space severalfold, e.g., in the case of albumin, up to 40-fold. The enrichment in the endocytic invaginations makes adsorptive endocytosis far more effective than fluid-phase endocytosis and renders quantitative uptake possible.

As pointed out above, albumin is reabsorbed quantitatively along the proximal tubule. Hence, reabsorption of albumin must be accomplished by adsorptive endocytosis. Comparison of the uptake rates of fluid-phase markers like dextran or inulin (Fig. 1A) with the uptake rate of albumin in cultivated proximal tubular cells (7, 14) or in isolated perfused proximal tubules (10) supports this notion. As shown in Fig. 1A, the rate of uptake of albumin 1) exceeds the rate of uptake of dextran by >20-fold and 2) is a saturable process, whereas the uptake of dextran increases linearly with the extracellular concentration. These data show that there is an enrichment of albumin with a limited capacity at the plasma membrane.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Functional characteristics of proximal tubular albumin reabsorption (according to Refs. 7, 9). A: endocytic uptake of albumin in proximal tubule-derived cells (OK cells) is a saturable process and exceeds rate of uptake of the fluid-phase marker dextran by severalfold. Inset: uptake of dextran with a different y scale. B: binding of albumin to apical membrane of OK cells shows an almost identical concentration dependence as albumin uptake. Binding in the physiological concentration range is >90% specific. C: macromolecules taken up by fluid-phase endocytosis should have a similar rate of endocytosis as dextran. Preferential uptake of albumin (A) over the fluid-phase marker dextran (D) cannot be explained by a charge-dependent accumulation because albumin has a negative net charge but can be explained by an albumin-binding protein in the apical membrane. Albumin interaction with a binding protein leads to accumulation in endocytic invaginations.

	What attracts albumin to the plasma membrane?

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

View larger version (16K): [in this window] [in a new window]   FIGURE 2. A: affinity constant (Km) of albumin binding is pH dependent. Km increases with decreasing pH. Sharp decrease of affinity is observed at pH values similar to those in endosomes and lysosomes. B: after detachment from apical membrane, endocytic vesicles deliver albumin-binding protein complex to the endosomal compartment (1). Albumin dissociates from the binding protein (2) due to low endosomal pH. Binding protein recycles to the apical plasma membrane (3). Albumin itself reaches the lysosomal compartment and is cleaved to single amino acids (4). Amino acids exit the lysosomes and leave the cell via the basolateral membrane (5). This mechanism prevents loss of valuable amino acids but does not deliver intact albumin to peritubular space.

	The importance of endosomal pH

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

 
Once albumin has been taken up by receptor-mediated endocytosis, it is directed to the endosomal compartment and its final destination, the lysosomes, where it will be cleaved to amino acids that exit the cell across the basolateral membrane (Fig. 2B). Hence, albumin must dissociate from the binding site before reaching the lysosomes, which could happen in the early endosomal compartment. The binding site returns to the apical membrane for further endocytic cycles, whereas albumin undergoes lysosomal degradation. A common mechanism triggering receptor-ligand dissociation is the drop in pH in the different endocytic compartments (11). Because binding affinity for albumin is pH dependent (Fig. 2A; Ref. 9), its dissociation from the binding site is most probably triggered by the acidic pH (pH <6.2). As long as pH ranges from 7.4 to 6.7, corresponding to the pH values at the beginning and at the end of proximal tubular lumens, binding affinity remains constant.

The functional importance of pH-dependent dissociation for effective albumin reabsorption has also been demonstrated experimentally (7). Endosomal alkalinization by either bafilomycin A₁ or NH₄Cl leads to a dramatic decrease of the apparent uptake affinity as well as of the maximal albumin uptake rate, whereas fluid-phase endocytosis is unaffected. At the same time, binding site recycling and albumin degradation are impaired. Reduction of the maximal uptake rate and uptake affinity is most probably due to reduced receptor recycling and enhanced reexocytosis (11). Enhanced renal albumin excretion in states of increased NH₄⁺ blood levels may be, at least in part, explained by endosomal alkalinization with the described changes of uptake affinity and rate (10).

As shown in Fig. 2B, the intracellular route of albumin can be summarized as follows. After detachment from the apical membrane, endocytic vesicles deliver the albumin-binding protein complex to the endosomal compartment. Albumin dissociates from the binding protein because of the low pH. The binding protein recycles to the apical plasma membrane, and albumin itself reaches the lysosomal compartment and is cleaved to single amino acids or small peptides. The amino acids exit the lysosomes and leave the cell via the basolateral membrane along an electrochemical gradient or in exchange for Na⁺. By this mechanism, >99% of filtered albumin is reabsorbed along the proximal tubules and <1% escapes into the final urine (Fig. 3A).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. A: summary of the fate of albumin. After filtration, albumin binds to brush border of proximal tubular cells and is taken up by endocytosis. In acidic endosomal compartment, albumin dissociates from binding protein and reaches lysosomal compartment where it is degraded to amino acids. Amino acids exit the cell via the basolateral membrane; <1% of filtered albumin escapes this mechanism and reaches final urine. B: basically, albuminuria can originate from enhanced glomerular filtration of albumin (glomerular albuminuria) or from reduced tubular reabsorption (tubular albuminuria). Loss of large amounts of albumin via urine can result in decreasing concentrations of serum albumin and thus in reduced intravascular oncotic pressure. As a consequence, capillary filtration of water into the interstitial space increases, leading to formation of edema.

	Regulation of albumin endocytosis

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

Overexpression of a G_i protein subunit in proximal tubule-derived OK cells results in an increase of albumin uptake to ~200% of control (1). This stimulation of endocytosis is inhibited by pertussis toxin, an inactivator of G_i and G_o proteins. Furthermore, endocytosis in control cells is reduced by pertussis toxin to ~50% of control, whereas binding of albumin is not affected by pertussis toxin. Activation of G_s proteins by cholera toxin has no effect on albumin endocytosis. The data presented in the study of Brunskill et al. (1) support a critical role for certain types of heterotrimeric G proteins in the regulation of albumin endocytosis, as was also shown for other endocytic processes. However, it is not known at present how and at which stage G proteins interact with albumin endocytosis. According to the widely accepted model of G protein action, an upstream receptor is linked to a downstream effector via G proteins. Further studies will have to be performed to show whether this is also true for albumin endocytosis regulation and, if so, which is the downstream regulator.

Stimulation of protein kinase A directly via adenosine 3',5'-cyclic monophosphate (cAMP) or by parathyroid hormone leads to a decrease of the maximum uptake rate in OK cells to ~75% of control without affecting the apparent affinity (8). Part of this effect may be attributed to a slight cAMP-induced endosomal alkalinization as discussed in "The importance of endosomal pH." Furthermore, phosphorylation of factors involved in the formation of endocytic invaginations and in budding possibly reduces the rate of albumin internalization. Because the formation of intracellular cAMP by the adenylate cyclase is under the negative control of G_i proteins, the downstream effector of G_i protein manipulation could well be protein kinase A (1). In this case, stimulation of the G_i protein would reduce the activity of adenylate cyclase and subsequently the stimulation of protein kinase A; thereby, one inhibitory effector for albumin endocytosis would be lowered, resulting in an increase of net endocytosis.

Ca²⁺ seems not to act as a regulator of albumin endocytosis but to be a prerequisite for physiological uptake rates (8). Increases in cytoplasmic Ca²⁺ concentration have only very minor effects on albumin endocytosis. In contrast, complete removal of extracellular Ca²⁺ leads to a dramatic decrease of the apparent affinity (K_m = 2.5 x 10^–6 mol/l) without affecting the maximal uptake rate, whereas a reduction of extracellular Ca²⁺ to ~10^–6 mol/l is without effect. Thus it seems that extracellular Ca²⁺ is an important prerequisite for albumin endocytosis. Additional removal of intracellular Ca²⁺ has no further effect, again indicating that cytoplasmic Ca²⁺ has no regulatory role. The underlying mechanism has not been elucidated so far. However, it is most probably different from the mechanism of action of cAMP, since removal of Ca²⁺ and addition of cAMP act additively (8).

These first studies on the regulation of albumin endocytosis show that this process is probably under the control of several positive (e.g., heterotrimeric G_i proteins) and negative (e.g., protein kinase A) regulators, which influence tubular albumin reabsorption, and thus renal albumin excretion, and possibly play some role in pathophysiological states with increased albumin loss.

	Pathophysiological considerations

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

 
In healthy subjects, filtration and reabsorption of albumin are in an equilibrium in which <1% of filtered albumin reaches the final urine (Fig. 3A). Pathophysiological states with increased renal albumin excretion, the so-called albuminurias, can result, in principle, from two malfunctions (Fig. 3B). The first malfunction is an increase of the filtered load, which results in glomerular albuminuria. Because the enrichment of albumin at the brush-border membrane has a limited capacity [e.g., maximum transport rate of high-affinity reabsorption of albumin in isolated proximal tubules is in the range of 0.05 ng min^–1 mm tubular length^–1 (10)], an increase in the filtered load leads to a growing disequilibrium between filtration and reabsorption. The second malfunction is renal excretion of albumin increasing without any change of the tubular load, as in the case of a reduced tubular reabsorption capacity. If the reabsorption capacity decreases at a constant filtered load, a growing disequilibrium again originates and causes a tubular albuminuria. Thus, for enhanced albumin excretion, one should always consider a malfunction of proximal tubular reabsorption.

As already described, impairment of endosomal acidification can affect endocytic albumin reabsorption. Simulation of affinity changes by a simple mathematical model (7, 10) shows the high susceptibility of the system toward small changes of its kinetic parameters. Increasing the K_m from 370 x 10^–9 to 1,300 x 10^–9 mol/l results in an increase in the percentage of albumin passing the proximal tubule, from values of ~1% to ~60% (corresponding to up to ~4,900 mg/day). However, changes in tubular uptake kinetics may also occur independently of vesicular pH homeostasis, as shown for the food contaminant ochratoxin A (6). Without affecting endosomal pH, ochratoxin A reduces the number of albumin-binding sites in the apical membrane by 30% and increases the rate of reexocytosis by 20%, thereby causing tubular proteinuria. The underlying molecular mechanism in this case has not yet been unveiled. Further points of attack for the generation of tubular albuminuria are the different mechanisms involved in the regulation of albumin endocytosis. Certainly, future studies will render more detailed insights into the regulatory processes and thereby broaden our pathophysiological knowledge.

	Acknowledgments

 
Work from the author's laboratory has been supported by the Deutsche Forschungsgemeinschaft.

I apologize to those whose work I was not able to cite due to space restrictions.

	References

Top Introduction Quantitative aspects of albumin... Albumin is reabsorbed by... What attracts albumin to... The importance of endosomal... Regulation of albumin... Pathophysiological... References

 

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