Ageing alters the global composition of glomerular and tubulointerstitial extracellular matrix

Introduction: Chronic kidney disease (CKD) affects at least 1 in 10 individuals in the UK and this increases to 1 in 4 over the age of 65. It is therefore critical to understand aging processes in the normal kidney. The extracellular matrix (ECM) is a major component of glomeruli and the tubulointerstitium, determining cell shape, structure and ultimately, function. Dysregulation of the ECM underpins pathology across the spectrum of kidney disease but the mechanisms remain poorly understood. Recent studies have used global analysis to reveal the complexity of the human glomerular ECM but detail of the tubulointersitial ECM is lacking. Here we aimed to use global mass spectrometry (MS)-based proteomics to compare the glomerular and tubulointerstitial ECM and investigate the temporal composition of ECM derived from normal human glomeruli and tubulointerstitium.

Methods: Kidneys technically unsuitable for transplantation were obtained from six male donors (15, 29, 37, 61, 67 and 69 years of age) with ethical approval. For each kidney the cortex was separated into tubulointerstitial and glomerular fractions by differential sieving and centrifugation. Tubular and glomerular fractions were subsequently fractionated using a 4-step buffer extraction to produce cellular and ECM-enriched samples1. Proteins were resolved by SDS-PAGE and visualised by InstantBlue staining. Gel lanes were sliced and subjected to in-gel trypsin digestion and liquid chromatography–tandem mass spectrometry (MS) using an Orbitrap Elite (ThermoScientific).

Results: By MS, 246 known matrisome proteins were detected in the glomerular and tubulointerstitium ECM. Analyses of these data revealed distinct glomerular and tubulointerstitial ECM signatures; 37 proteins were glomerular-specific and 25 were specific to the tubulointerstitium. In the glomerular ECM, known glomerular basement membrane (GBM) components type IV collagen (α4) and laminin (α5β2γ1) were abundant. Whereas the tubulointersitial ECM was enriched for type IV collagen (α1, α2), type I collagen (α1), type III collagen (α1) and type XVI collagen (α1). Furthermore, we found increased glomerular type VI collagen (α1α2α3), lumican, versican, mimecan, biglycan and aggrecan with increasing age. In contrast, GBM proteins laminin β2 and γ1, decreased with age. In the tubulointerstitial ECM, latent-transforming growth factor (TGF)β-binding protein, TGFβ-induced protein and fibronectin all increased with age. In addition, in both glomerular and tubulointerstitial ECMs, we identified common proteins that increased in abundance with age (tissue inhibitor of metalloproteinase (TIMP) 3 and fibulin-1) and decreased with age (angiotensinogen).

Conclusions: These studies reveal unique matrisome signatures in normal adult human glomeruli and tubulointersititium and highlight the complexity of the ECM in both compartments. Ageing leads to marked changes in the ECM, which are likely to impact on cell and organ function. Greater understanding of ECM regulation may lead to new therapeutic strategies for kidney disease.

1Lennon R, Byron A, Humphries JD, Randles MJ, Carisey A, Murphy SM, Knight D, Brenchley PA, Zent RA, Humphries MJ. J Am Soc Nephrol 25:939-951, 2014