Isotopic Nitrogen-15 Labeling of Mice Identified Long-lived Proteins of the Renal Basement Membranes

Abstract

The kidney is comprised of highly complex structures that rely on self-maintenance for their functions, and tissue repair and regeneration in renal diseases. We devised a proteomics assay to measure the turnover of individual proteins in mouse kidney. Mice were metabolically labeled with a specially formulated chow containing nitrogen-15 (15N) with the absence of normal 14N atoms. Newly synthesized proteins with 15N contents were distinguished from their 14N counterparts by mass spectrometry. In total, we identified over 4,000 proteins from the renal cortex with a majority of them contained only 15N. About 100 proteins had both 14N- and 15N-contents. Notably, the long-lived proteins that had large 14N/15N ratios were mostly matrix proteins. These included proteins such as type IV and type VI collagen, laminin, nidogen and perlecan/HSPG2 that constitute the axial core of the glomerular basement membrane (GBM). In contrast, the surface lamina rara proteins such as agrin and integrin had much shorter longevity, suggesting their faster regeneration cycle. The data illustrated matrix proteins that constitute the basement membranes in the renal cortex are constantly renewed in an ordered fashion. In perspective, the global profile of protein turnover is usefully in understanding the protein-basis of GBM maintenance and repair.

3D mapping reveals a complex and transient interstitial matrix during murine renal development

“…87,89 A common kidney matrisome was observed across multiple proteomic studies. The ECM proteins we identified had a large percentage of overlap with previous proteomic studies; [9][10][11][12][13][14][15][16][17][18]36,37 however, there were some differences (Table S3). Variations could be due to different tissue fractionation methods, protein abundance, and sample type.…”

“…35 The Venn diagram and figures were compiled using Adobe Illustrator. 35 The matrisome components identified in this study were compared with previously published studies analyzing the adult kidney, [9][10][11][12] fetal kidney, 12 adult glomerular tissue, [13][14][15][16][17] renal artery, 18 and kidney culture models, 36,37 when a list of matrisome proteins identified was available using a combined human and mouse matrisome list (Table S3). 32 Imaging.…”

“…5 Recently, fractionation techniques were developed to isolate ECM proteins, termed the matrisome, using increasingly stringent solutions to solubilize cellular proteins and characterize the matrix in diverse tissues, 8 including fetal and adult kidneys. [9][10][11][12][13][14][15][16][17][18][19][20] Despite these reports, there has yet to be a study that interrogates ECM dynamics across murine embryonic, fetal, and adult kidney development.…”

Aminoglycoside-induced premature termination codon readthrough of COL4A5 nonsense mutations that cause Alport syndrome

“…5). In addition, from the viewpoint specific to Alport syndrome, type IV collagen α3α4α5 incorporated into the GBM should be stable for a long period of time (56). This means that once enough type IV collagen α3α4α5 is induced by PTC-RT and incorporated into the GBM, continuous treatment should not be necessary, though intermittent treatments would likely be required.…”

Peroxidasin-mediated bromine enrichment of basement membranes

“…While this phenomenon could be due to increased expression of collagen IV and/or other basement membrane proteins, we are attracted to the idea that it is caused, at least in part, by reduced turnover of basement membrane proteins. Collagen IV as well as other basement membrane proteins (e.g., laminin, nidogen) are long-lived proteins (45), but a slower turnover rate would presumably increase tyrosine bromination by prolonging exposure to the HOBr produced by peroxidasin. It is possible, perhaps even likely, that the thickening of basement membranes in diabetes, along with peroxidasin-mediated bromination of basement membrane proteins, underlies higher bromotyrosine levels in the urine of patients with diabetes mellitus (46).…”