Protein N -Glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease

Abstract

Key Points:

1. Multiomics performed on diabetic kidney disease biopsies revealed five N-glycan signatures of sclerotic glomeruli that significantly differed compared with healthy glomeruli.

2. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in diabetic kidney disease.

Background: Diabetes is expected to directly affect renal glycosylation; yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD).

Methods: We used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in formalin-fixed paraffin-embedded sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single-nucleus RNA sequencing (snRNAseq) data were used to inform on transcript levels of glycosylation machinery in different cell types and states.

Results: We detected 120 N-glycans, and among them, we identified 12 of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that were common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and N-acetyllactosamine synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay.

Conclusions: Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

Keywords: diabetic glomerulosclerosis; diabetic kidney disease.

Graphical abstract

Figure 1

Spatial N-glycomics and regional proteomics of HR kidney biopsies. (A) Genes involved in the specific biosynthetic steps during N-glycan maturation. (B) The regional proteomic table shows the relative ratio of enzymes involved in N-glycans' biosynthesis, between glomeruli and tubulointerstitial regions. (C) Brightfield microscopy (where white dashed lines outline glomeruli) and overlayed MALDI-MS ion images show the distribution of a sialylated tetra-antennary glycan (m/z 2852.9810) in glomeruli and a bisecting, nonsialylated N-glycan (m/z 2158.7766) in tubular regions outside of the glomeruli. Glom, glomeruli; HR, healthy reference; MALDI-MS, matrix-assisted laser desorption/ionization–mass spectrometry; TI, tubulointerstitial.

Figure 2

Discriminant and nondiscriminant N-glycans between healthy and sclerotic glomeruli. Composition (Comp), m/z values, and tentative structures of 12 glomeruli specific N-glycans that are (A) enriched is healthy glomeruli compared with sclerotic glomeruli, (B) enriched in sclerotic compared with healthy glomeruli, and (C) equally abundant in healthy glomeruli and sclerotic glomeruli. *Some sialic acid N-glycans are visualized at two m/z values that correspond to two different adducts ([M+Na] and [M–H+2Na]). HG, healthy glomeruli; SG, sclerotic glomeruli.

Figure 3

Example of high-resolution MALDI-MSI of N-glycans in DKD human biopsies. (A) Relative abundance of N-glycan at m/z 2539.9037 (Hex:7 HexNAc:6 dHex:1) over the entire kidney biopsy section (s-1908-000922). (B) Relative abundance of N-glycan (m/z 2830.9991; Hex:7 HexNAc:6 dHex:1 NeuAc:1) that is upregulated in healthy glomeruli. (C) Relative abundance of N-glycan (m/z 2539.9037, Hex:7 HexNAc:6 dHex:1), which is nondiscriminant between healthy and sclerotic glomeruli. (D) Relative abundance of N-glycan (m/z 1976.6587, Hex:5 HexNAc:4 NeuAc:1) that is upregulated in sclerotic glomeruli. (E) Blended ion images of upregulated (magenta) and downregulated (green) N-glycans in sclerotic glomeruli. (F) Normalized ion image of m/z 2539.9037 N-glycan signal with m/z 1976.6587 N-glycan signal, which clearly distinguishes healthy from sclerotic glomeruli. (G) Post–MALDI PAS-stained microscopy image with clear visualization of neighboring sclerotic and healthy glomeruli. (H) The ratio of MS signal intensities at m/z 1976.6587 and m/z 2539.9037 in every glomerulus within the biopsies (there are multiple glomerulus per biopsy). The ratio of signals was normalized with corresponding ratio in reference sample that was run parallel with each biopsy. Microscopy images of glomeruli with the specific normalized ratio are presented as inserts. Pixel size of MALDI-MSI images is 25×25 µm. DKD, diabetic kidney disease; MALDI-MSI, matrix-assisted laser desorption/ionization–mass spectrometry imaging; MS, mass spectrometry; PAS, periodic acid–Schiff.

Figure 4

Comparative multiomics analysis of healthy and diseased kidney biopsies. (A) Regional proteomics data include averaged values of ratios between abundance in DKD (DG) and healthy glomeruli, as well as DKD glomeruli and D TI. (B) An atlas of healthy and injured cell states and niches in the human kidney. SnRNAseq shows the relative expression of selected genes in POD and dPOD in all samples analyzed so far by snRNAseq as part of KPMP and in a subgroup of patient samples whose biopsies were also analyzed by MALDI-MSI–based spatial N-glycomics. dPOD, degenerative podocytes; D TI, DKD tubulointerstitium; KPMP, Kidney Precision Medicine Project; POD, healthy podocytes; SnRNAseq, single-nucleus RNA sequencing.