The sulfilimine cross-link of collagen IV contributes to kidney tubular basement membrane stiffness

Abstract

Basement membranes (BMs), a specialized form of extracellular matrix, underlie nearly all cell layers and provide structural support for tissues and interact with cell surface receptors to determine cell behavior. Both macromolecular composition and stiffness of the BM influence cell-BM interactions. Collagen IV is a major constituent of the BM that forms an extensively cross-linked oligomeric network. Its deficiency leads to BM mechanical instability, as observed with glomerular BM in Alport syndrome. These findings have led to the hypothesis that collagen IV and its cross-links determine BM stiffness. A sulfilimine bond (S = N) between a methionine sulfur and a lysine nitrogen cross-links collagen IV and is formed by the matrix enzyme peroxidasin. In peroxidasin knockout mice with reduced collagen IV sulfilimine cross-links, we find a reduction in renal tubular BM stiffness. Thus this work provides the first direct experimental evidence that collagen IV sulfilimine cross-links contribute to BM mechanical properties and provides a foundation for future work on the relationship of BM mechanics to cell function in renal disease.

Keywords: basement membrane; collagen IV; elastic modulus; peroxidasin; sulfilimine cross-link.

Fig. 1.

Cantilever calibration procedure. A: spring constant of reference cantilevers (k_r) was determined by hanging lengths of platinum wire from the end of the reference cantilever (F = force applied to the cantilever) and measuring cantilever deflection (d_r). Image is shown with an overlay of the original cantilever position. B: spring constant of the measurement cantilever was determined by displacing the reference cantilever relative to the measurement cantilever and measuring the displacement of the reference and measurement cantilevers (x_r and x_m, respectively). Measurement cantilever stiffness is calculated from the slope of the relative deflection plot and the known reference cantilever stiffness.

Fig. 2.

Microcantilever method for measuring the stress-strain behavior of renal tubules. A–E: isolated tubule, measurement cantilever, and holding pipette at 0, 10, 21, 31, and 40% strain, respectively. F: stress-strain curve.

Fig. 3.

Elastic modulus in cellularized and decellularized renal tubules. No significant difference in the elastic modulus was found between cellularized and decellularized tubules (n = 3). Elastic modulus was normalized to the cellularized elastic modulus at the corresponding strain range.

Fig. 4.

Sulfilimine cross-link content in wild-type (WT) and peroxidasin knockout (KO) mice. A: Coomassie blue-stained gels after SDS-PAGE under nonreducing conditions of purified NC1 hexamers from WT and peroxidasin KO mice. Under denaturing conditions, hexamers dissociate into singly cross-linked dimeric (D1), doubly cross-linked dimeric (D2), and un-cross-linked monomeric (M) subunits. B: number of sulfilimine cross-links per hexamer was quantified using densitometry of D1, D2, and M subunits for WT and KO mice. Individual data points (n = 4) are displayed with mean and SE. *P < 0.05 (by unpaired t-test).

Fig. 5.

Renal tubular basement membrane structure in WT and peroxidasin KO mice. A: representative hematoxylin-eosin-stained images of WT and peroxidasin KO mouse kidneys. B: transmission electron microscopy images from WT and peroxidasin KO mouse kidney. Insets: magnified images demonstrating tubular basement membrane (TBM) width measurements. C: for measured TBM width, each data point represents mean of 40–100 replicate measurements of sections from 1 WT and 1 peroxidasin KO mouse kidney. Individual animal data points (n = 3 with 40–100 replicates) are displayed with mean and SE.

Fig. 6.

Mechanical properties of TBM from WT and peroxidasin KO mice. A: stress-strain curves for WT and KO TBM. Stress data were pooled in 2.5% increments of strain, and mean stress and strain were calculated from the pooled data. Values are means ± SE. Error bars for strain are obscured by individual data points. B: elastic modulus of TBMs as measured from the slope of the individual tubule stress-strain curves at 10% increments of strain. Values are means ± SE for 16 tubules from 4 different animals. *P < 0.05.