Chronic kidney disease (CKD) is defined as a decrease in renal function or glomerular filtration rate (GFR), and proteinuria is often present. Proteinuria increases with age and can be caused by glomerular and/or proximal tubule (PT) alterations. PT cells have an apical brush border membrane (BBM), which is a highly dynamic, organized, and specialized membrane region containing multiple glycoproteins required for its functions including regulating uptake, secretion, and signaling dependent upon the physiologic state. PT disorders contribute to the dysfunction observed in CKD. Many glycoprotein functions have been attributed to their N- and O-glycans, which are highly regulated and complex. In this study, the O-glycans present in rat BBMs from animals with different levels of kidney disease and proteinuria were characterized and analyzed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A principal component analysis (PCA) documented that each group has distinct O-glycan distributions. Higher fucosylation levels were observed in the CKD and diabetic groups, which may contribute to PT dysfunction by altering physiologic glycoprotein interactions. Fucosylated O-glycans such as 1-1-1-0 exhibited higher abundance in the severe proteinuric groups. These glycomic results revealed that differential O-glycan expressions in CKD progressions has the potential to define the mechanism of proteinuria in kidney disease and to identify potential therapeutic interventions.

Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments.

A complex physiological culture medium (Plasmax) was introduced recently, composed of nutrients and metabolites at concentrations normally found in human plasma to mimic the in vivo environment for cell line cultivation. As glycosylation has been proved to be involved in cancer development, it is necessary to investigate the glycan expression changes in media with different nutrients. In this study, a breast cancer cell line, MDA-MB-231BR, and a brain cancer cell line, CRL-1620, were cultivated in Plasmax and commercial media to reveal cell line glycosylation discrepancies prompted by nutritional environments. Glycomics analyses of cell lines were performed using LC-MS/MS. The expressions of multiple fucosylated N-glycans, such as HexNAc₄Hex₃DeoxyHex₁ and HexNAc₅Hex₃DeoxyHex₁, derived from both cell lines exhibited a significant increase in Plasmax. Among the O-glycans, significant differences were also observed. Both cell lines cultivated in EMEM had the lowest amounts of O-glycans expressed. The original work described the development of Plasmax, which improves colony formation, and resulted in transcriptomic and metabolomic alterations of cancer cell lines, while our results indicate that Plasmax can significantly impact protein glycosylation. This study also provides information to guide the selection of media for in vitro cancer cell glycomics studies.

Aiming to reduce the gap between in vitro and in vivo environment, a complex culture medium, Plasmax, was introduced recently, which includes nutrients and metabolites with concentrations normally found in human plasma. Herein, to study the influence of this medium on cellular behaviors, we utilized Plasmax to cultivate two cancer cell lines, including one breast cancer cell line, MDA-MB-231BR, and one brain cancer cell line, CRL-1620. Cancer cells were harvested and prepared for transcriptomics and proteomics analyses to assess the discrepancies caused by the different nutritional environments of Plasmax and two commercial media: DMEM, and EMEM. Total RNAs of cells were extracted using mammalian total RNA extract kits and analyzed by next-generation RNA sequencing; proteomics analyses were performed using LC-MS/MS. Gene oncology and pathway analysis were employed to study the affected functions. The cellular invasion and cell death were inhibited in MDA-MB-231BR cell line when cultured in Plasmax compared to DMEM and EMEM, whereas the invasion, migration and protein synthesis of CRL-1620 cell line were activated in Plasmax in relative to both commercial media. The expression changes of some proteins were more significant compared to their corresponding transcripts, indicating that Plasmax has more influence upon regulatory processes of proteins after translation. This work provides complementary information to the original study of Plasmax, aiming to facilitate the selection of appropriate media for in vitro cancer cell studies.

Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin’s albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin–albumin interaction.