Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance

Abstract

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin’s reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Albumin uptake and processing by the proximal tubule: physiological, pathological, and therapeutic implications

“…The publication by Sandoval et al ( 148 ) addresses the technical aspects of photon detection and bit depth. The same laboratory has used four different microscope systems over time, all with increasingly superior components, and all have produced the same GSC values for albumin ( 3 , 4 , 6 , 38 , 143 , 151 , 153 – 155 ).…”

“…The proximal tubule cell, with its highly organized and specialized apical domains, may restrict or enhance interactions based on location, concentration, and a multitude of regulatory processes not fully represented in some of the in vitro cell culture models ( 73 ). For instance, both glycated and carbamylated albumin have shown reduced to no binding to cubilin’s albumin binding domain, yet PT uptake of these modified albumins is unaltered when quantified by rat intravital imaging ( 6 , 155 ). Understanding how this is occurring in vivo is important.…”

“…Of critical importance for understanding albumin dynamics may be how modified albumins, i.e., glycated, carbamylated, oxidized, and albumin bound to molecules such as drugs or fluorophores, affect the albumin-FcRn pH-dependent binding interaction ( TABLE 4 ). Recent studies have documented that glycated and carbamylated albumin have reduced binding to FcRn ( 6 , 155 ). In addition, vascular clearance is increased as binding to FcRn is reduced ( FIGURE 11 A ), and evidence for interstitial/endothelial kidney accumulation of the modified albumins was observed ( FIGURE 11 B ).…”

“…Proximal tubule cells, especially the S1 segment, efficiently and effectively reabsorb and transcytose albumin ( FIGURE 1 ) ( 4 , 5 ). Alterations to albumin such as glycation, carbamylation, and bound ligands affect the renal handling of filtered albumin and likely play a role in PT toxicity and progressive chronic kidney disease ( 6 , 7 ). Finally, there is increasing use of albumin as a molecular chaperone for specific therapeutics.…”

Intravital Multiphoton Microscopy as a Tool for Studying Renal Physiology, Pathophysiology and Therapeutics

“…Transcytosis of albumin was confirmed using molecular techniques, but the amount of albumin undergoing transcytosis remains unknown ( Tenten et al, 2013 ). A potential role for PT sorting of glycated, carbamylated and other potentially toxic albumins, mediated by FcRn binding, for catabolism via lysosomal trafficking has been proposed as a mechanism to rid the body of altered albumins while preserving physiologic albumin for transcytosis ( Dickson et al, 2014 ; Wagner et al, 2016b ; Yadav et al, 2021 ). Proximal tubule transcytosis of folic acid and other vitamins is known to occur ( Sandoval et al, 2004 ).…”