Characterization of glomerular extracellular matrix by proteomic analysis of laser-captured microdissected glomeruli

Abstract

Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with that available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin fixed paraffin embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.

Mammalian hemicentin 1 is assembled into tracks in the extracellular matrix of multiple tissues

“…HMCN1 was identified as a normal matrix component in one proteomic analysis of human glomeruli, but not in a subsequent analysis . Here we generated CRISPR/Cas9‐mediated Hmcn1 and Hmcn2 knockout mice to test whether HMCNs function to join the two separate epithelial and endothelial BMs into the thick GBM during glomerulogenesis, and to confirm a previous report that the lack of HMCN1 causes preimplantation lethality, a phenotype that has not been observed in other mammalian extracellular matrix gene mutants.…”

PLA2R and THSD7A: Disparate Paths to the Same Disease?

“…Mass spectrometry has proved a powerful technique by which to identify proteins enriched in glomeruli, using laser capture techniques. 86,87 Although candidate autoantigens may be intrinsic glomerular proteins, they may be enriched within immune deposits of MN, compared with control sections, and thus may be detectable using such approaches. Another potential methodology uses human serum to identify reactive spots on a protein microarray, 88 and could potentially identify novel candidates in samples from patients with dual negative primary MN.…”

Protein Mass Spectrometry Made Simple

“…7 Laser capture has been applied to proteomic analysis of kidney tissue as outlined in Figure 1. 8 The value of laser capture microscopy is that allows us to enrich for structures in the kidney that are only affected by specific processes. Alternatively, proteins can be enriched by simple resolution on acrylamide gels.…”