Part Ⅲ：Renal Handling Of Albumin—From Early Findings To Current Concepts

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Part Ⅲ：Renal Handling of Albumin—From Early Findings to Current Concepts

Jakub Gburek, Bogusława Konopska and Krzysztof Goł ˛ab

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Abstract: Albumin is the main protein of blood plasma, lymph, cerebrospinal and interstitial fluid. The protein participates in a variety of important biological functions, such as maintenance of proper colloidal osmotic pressure, transport of important metabolites, and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in the vascular endothelium of muscle, skin, and liver, as well as in the kidney tubular epithelium. Long-lasting investigation in this area has delineated the principal route of its catabolism involving glomerular filtration, tubular endocytic uptake via the multiligand scavenger receptor tandem—megalin and cubilin-amnion less complex, as well as lysosomal degradation to amino acids. However, the research of the last few decades indicates that also additional mechanisms may operate in this process to some extent. Direct uptake of albumin in glomerular podocytes via the receptor for the crystallizable region of immunoglobulins (neonatal FCreceptor)was demonstrated. Additionally, luminal recycling of short peptides into the bloodstream and/or back into tubular lumen or transcytosis of whole molecules was suggested. The article discusses the molecular aspects of these processes and presents the major findings and controversies arising in the light of the research concerning the last decade. Their better characterization is essential for further research into the pathophysiology of proteinuric renal failure and the development of effective therapeutic strategies.

Keywords: albumin; renal catabolism of proteins; renal proximal tubule; proteinuria; megalin;cubilin

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4. Intracellular Transport and Degradation of Albumin

4.1.Transport to Lysosomes and Degradation

The intracellular flow of albumin proceeds through vesicular transport. Thanks to the early experiments, it has been already discovered that the sorting of albumin, its receptors and the vesicles themselves is closely related to their environment acidification. Alkalization of vesicles with bafilomycin Al or NHACl solution led to a rapid reduction in albumin absorption, even at low concentrations. At the same time, both the breakdown of albumin in lysosomes and recycling of receptors to the cell membrane was slowed down [48,49,86].

Vesicles acidification is initially accompanied by clathrin coat dissociation and the formation of early endosomes. Further acidification results in the dissociation of albumin from complexes with megalin and cubilin, and the budding of dense apical tubules that turn the receptors back to the cell surface. Dissociation of complexes is already observed below pH=6.5 [26,75,81].

The transporters involved in the acidification of endocytic vesicles were identified in recent years. V-ATPase located in the membrane of this organelle is involved in the process of protons transfer inside the endosome [87]. On the other hand, it is necessary to introduce negative ions to compensate for the potential of the endosomal membrane. In proximal tubule cells, chloride CLC-5 channels play an important role in the transport of the opposite ion. CLC-5 was demonstrated to be associated with albumin-containing endosomes. It cannot be found in dextran-containing endosomes, although V-ATP-also is present in both types of endosomes. Lowering the CLC-5 expression inhibits albumin endocytosis in the proximal tubule but does not affect the first phase of dextran endocytosis. The study carried out on mice with the CLC-5 gene knockout confirmed its importance in megalin and cubilin recirculation. The participation of CLC-5 in the endocytosis process is also of interest for inherited renal diseases. Dent's disease, in which one of the first symptoms is proteinuria, is caused by a mutation of the CLC-5 coding gene [88,89].

The Na*/Ht antiporters (sodium/proton exchangers—NHEs), which remove H ions from the inside of the cell with simultaneous Nat ion uptake, are also involved in the acidification of the endosomal vesicle environment. They are transporting molecules found in the plasma membrane of many cells, and their task is to maintain the correct pH inside the cell. These proteins consist of two functional domains, namely a hydrophobic domain containing several transmembrane sequences and a hydrophilic domain responsible for the regulation of their transport activity [90]. The NHE-3 protein is expressed in intestinal epithelial cells and in proximal renal tubules, always on the apical side. It is able to circulate between the cell membrane and the early endosomal compartment, contributing to the acidification of the vesicle contents. Internalization of the NHE-3 transporter occurs via clathrin vesicles [91]. NHE-3 is probably involved in the absorption of Na ions in these cells. Endosomal NHE-3s play a major role in the process of albumin molecules endocytosis. Na+gradient across the endosomal environment is supposed to dissipate along the endosomal pathway, and thus NHE3 should be important for the early steps of endocytosis, possibly prior to the stage(s) where H+-ATPase exerts its function [91]. NHE-3 inhibitors cause disturbances in the acidification of early vesicles, which considerably delays the process of albumin endocytosis. These disorders are partially explained by the slowed return of receptors to the cell membrane due to disturbances in the process of their dissociation. Another reason may be the disruption of the formation and fusion of transporting vesicles through alkalization. In addition, NHE-3 was found to be responsible only for lowering the pH in the very early phase of endocytosis. Its pharmacological inhibition leads to a significant reduction in the efficiency of this process [92]. Additionally, obesity may downregulate the NHE-3 and Na*/K+ATPase expression due to lowered levels of p-Akt factor and consequently reduce albumin reabsorption and endocytosis [93]. Moreover, the significant pool of NHE-3 exists as a multimeric complex with megalin in the brush border of the proximal tubule [94]. There are several mechanisms of endocytosis regulation. However, in the case of albumin reabsorption, they are poorly understood.

Calcium ions do not directly affect this process. Change in the cytoplasmic concentration of Ca²ions only slightly affects albumin uptake. However， their complete removal from the extracellular space results in a marked reduction in the ability of protein uptake (Km increases from 0.1.10-6to 2.5.10-6M), without an effect on the maximum rate of absorption. The presence of calcium ions is likely to determine the effectiveness of the first stage of endocytosis, i.e., the ligands binding by receptors [95]. It is known that stimulation of protein kinase A(PKA)by cyclic adenosine monophosphate(cAMP), forskolin, or parathyroid hormone results in a reduction of the total albumin reuptake capability but does not change the protein's affinity for the receptor. This underlying mechanism related to the alkalization of early endosomes depends on the cAMP presence. The effect of phosphatidylinositol 3-kinase(PI-3K) on albumin endocytosis was also reported [92,96]. Inhibition of its activity through the use of wortmannin significantly weakens this process efficiency. An activity of phosphatidylinositol3-kinase is related to the phase of ligands internalization. Proteins G are also involved in the signaling cascade associated with albumin internalization. The uptake of albumin is increased in the epithelial opossum kidney cells (OK cells) transfected with the gene of the G. i3 subunit. This effect was suppressed by the pertussis toxin as the concentration and incubation time increased. Thus, the role of G proteins seems to be closely related to the regulation of vesicular transport processes [56,97,98].

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4.2. Lysosomal Degradation

Lysosomal hydrolysis of proteins takes place with the participation of a set of lysosomal proteases, collectively referred to as cathepsins. The most important role in proteins degradation is attributed to cathepsins B, H and L. These hydrolases are transported in the form of proenzymes to early endocytic vesicles involving the mannose-6-phosphate receptor and are activated as they maturate. Partial hydrolysis of proteins occurs already in endosomes, however, massive degradation takes place only in lysosomes. Immuno-cytochemistry studies show that at least several different populations of endosomes and lysosomes, in terms of cathepsin composition, can be distinguished in the epithelial cells of the proximal tubule [99]. Recent studies indicate that the fusion of albumin-containing endosomes with lysosomes depends on the membrane receptors of these organelles. Proteinuria occurs in mice with lysosomal SCARB2/Limp-2 knockout, belonging to the family of B-class scavenging receptors, despite the correct expression and internalization of albumin receptors. It was found in these mice that fluorescently labeled albumin was effectively absorbed after intravenous administration but did not collocate with lysosomal cathepsin B and was not degraded. A similar effect was also observed in wild-type mice with massive albuminuria induced by i.p. albumin administration, indicating that, at the level of the proximal tubule, the limited capacity of lysosomal degradation plays a more important role in the development of albuminuria than the ability of its reabsorption [100,101]. However, the study of Nielsen et al. [102] revealed that the lysosomal capacity is increased under proteinuria in a murine model of focal segmental glomerulosclerosis (FSGS), supporting previous findings that lysosomal digestion capacity can be increased under proteinuria [99].

Much of the experimental data suggest that proteins——including albumin—are completely degraded in lysosomes to free amino acids, which are then transported through the lysosomal and basolateral membrane to the bloodstream. In experiments using isolated, perfused proximal tubules of rabbit and 125I labeled proteins, almost all perfusate radioactivity is associated with their catabolite2I-mono tyrosine. Similar results were obtained when measuring the efflux of radioactivity from kidney sections perfused with radiolabeled protein preparations [26,47].

However, the results of studies in this respect are not consistent. Some authors suggest that lysosomal degradation of albumin is not complete, and its products are predominantly polypeptide chains of varying lengths, which are returned to the lumen of the tubule and excreted in the urine. Osicka et al.[103,104] demonstrated that significant amounts of peptides derived from degraded albumin are found in the urine after intravenous administration of 3H-albumin to rats. However, they did not specify the size of the observed fragments in their study.

Similar results were obtained by Gudehithlu et al. [105] by intravenous administration of 125I-labeled albumin to rats. Urine proteins were precipitated with trichloroacetic acid and subjected to gel filtration allowing the determination of both fragments and whole albumin molecules. Small amounts of native albumin (2%) and significant amounts of its fragments (98%)with a molecular weight of 5-14 kDa were found in urine. These results also confirmed in vitro experiments on human HK-2 proximal tubule cells and ex vivo using perfused rat kidneys. The amount of albumin excreted in the form of whole molecules and polypeptides was about 70-fold higher than the values determined so far: Low-molecular fragments of albumin in urine were also found in the studies in rats with diabetes caused by streptozotocin administration [35].

These discrepancies may be related to the use of radioimmunological methods in previous studies that do not detect polypeptide fragments lacking specific epitopes to the applied antibodies, which could happen in the case of albumin degradation products. It is worth noting that such amounts of albumin excreted in urine would correspond to the amounts of filtered albumin with the assumption of a relatively large GSC, as discussed above.

Recent studies suggest that the albumin fragments observed in urine are not renal catabolites, but are the effect of free glomerular filtration, escaping the tubular resorption in a way not recognized until now. Both in control mice as well as in mice with cubilin or megalin knockout, mainly low-molecular albumin metabolites were found in the urine after 125I albumin administration. The inhibition of tubular endocytosis did not affect the form of albumin present in urine. Generation of low-molecular albumin fragments was also observed in the plasma [106].

4.3. TranSCytosis

The role of transcytosis as an alternative way of internalized albumin targeting was proposed by Russo et al. in 2009[33]. In their study, using electron microscopy and colloidal gold-labeled albumin, the authors observed the presence of albumin not only in lysosomes but also in large vesicles with a diameter of around 500 nm. These structures occurred throughout the cell and were often fused to the invagination of the basolateral membrane, releasing its contents into the peri-tubular network of capillary vessels. The integrity of the molecules was checked by means of antibodies directed against various albumin fragments. The authors suggest the co-existence of two mechanisms of intracellular albumin sorting depending on the integrity of molecules. Intact protein molecules return to circulation through a fast and efficient transcytosis, while a small part of molecules exhibiting structural changes are transported to lysosomes, where, according to the classic model, they are broken down into amino acid residues and in this form, they return to the circulation [33,38]. The model aroused numerous controversies. Opponents of this theory, taking into account many years of microscopic examination of proximal tubule cells, undermine the existence of these types of vesicles and the possibility of their fusion with the basolateral membrane. They suggest that the structures observed are the result of artifacts related to tissue fixation by immersion. Methods based on perfusion, considered to be optimal, have been used so far [41,107l. On the other hand, recent studies on nephrotic mice with podocin, megalin, and cubilin knockout indicate that endocytosis, dependent on these receptors, is not significant for maintaining albumin homeostasis. Despite total inhibition of reabsorption depending on these receptors, plasma albumin levels remained the same [108].

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5. Conclusions

The kidney is responsible for about 10% of the albumin pool catabolized daily. This fraction is significant for homeostasis. Possible kidney function abnormalities, initially caused by ongoing damage to glomeruli, can eventually lead to severe failure of this organ and consequently to irreversible tubular damage. The secondary toxicity is attributed to dysregulation of epithelial cell functions due to protein overload under conditions of excessive endocytosis in proximal tubules [10]. Renal catabolism of proteins includes filtration in the glomerulus, adsorption and endocytosis in proximal tubules, and intracellular hydrolysis or transcytosis of absorbed molecules.

The commonly accepted model assumes a relatively low level of glomerular albumin filtration due to its relatively large size and low isoelectric point, efficient reabsorption by endocytosis, involving megalin and cubilin receptors, vesicular transport to lysosomes, and hydrolysis to single amino acid residues. In this model, only a negligible amount of albumin is excreted in the urine, and degradation products mostly return to circulation.

The studies that have been carried out over the last decade yielded many unexpected observations that can dramatically change views on the topic of renal albumin catabolism. Alternative mechanisms that affect both the quantitative and qualitative image of this process emerge from them. New studies indicate, inter alia, that albumin permeation can occur with a 50-fold higher yield than suggested by previous studies. The proposed value of the GSC would then be 0.04, not below 0.001 like in the currently accepted model [33]. Other controversies concern the degree of lysosomal degradation of albumin and the direction of metabolites excretion. Some observations support the view that lysosomal proteolysis of albumin mainly produces polypeptides with a molecular weight of 5-14 kDa, which are largely recycled to the lumen of the tubule and excreted in the urine. Reports about the phenomenon of albumin transcytosis are also surprising. This mechanism would explain the efficient absorption and transport of larger amounts of filtered albumin. At the same time, these reports suggest that proximal tubule dysfunctions mainly contribute to albuminuria in the course of various diseases, and not, as previously thought, glomerulopathies. The current concepts and new findings concerning renal albumin handling are summarized in Figure2.

New reports have sparked a heated discussion in the world of science. If confirmed in further studies, they may change the views on the mechanism of albumin homeostasis and the pathomechanisms of diseases associated with albuminuria, such as nephrotic syndrome, renal failure against hypertension, diabetic nephropathy, and cardiovascular diseases.

It is worth mentioning that a variety of data in the field of proximal tubular reabsorption, lysosomal metabolism, and intracellular processing have been collected during basic studies on the structure of kidney epithelial cells. The achievements have been summarized in several excellent reviews [109-112].

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6. Perspectives

Taking into account the available data, we believe that alternative models are not well documented so far and the development of new therapeutic strategies should be based on the currently accepted concept.

In order to verify the significance of currently accepted and alternative concepts, further investigations are needed. Development of new animal models and more detailed clinical studies, involving relevant molecules, such as glomerular proteins, endocytic receptors, vesicular transporters, and other endosomal/lysosomal proteins are warranted.

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