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

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.