Interplay between transglutaminases and heparan sulphate in progressive renal scarring

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.


Suppl. Fig. 2. Versican and TG2 dual immunostaining
Versican and TG2 immunostaining of unfixed cryostat sections with rabbit monoclonal antiversican antibody and mouse monoclonal anti-TG2 antibody IA12 followed by donkey anti-

PCR primers, QPCR amplification and analysis
Primers (Sigma Genosys) were designed manually or using the Primer3 software (available at SDSC Biology workbench 3.2; http://workbench.sdsc.edu/), and their level of secondary structure evaluated by the DNA calculator software (available at http://www.sigmagenosys.com/calc/DNACalc.asp). All primer-set were planned to amplify genes across intervening sequences whenever possible (Suppl. For all transcripts studied, a ramp temperature from 72C to 95C was used to generate the melt curves, which were used to check the homogeneity of the amplified transcripts. Each sample was run in triplicate and a no-template control was included to rule out contamination. Absolute quantifications were performed using standard curves of transcript-specific RT-PCR products, purified from low melting gel (0.7% W/V, Bioline) using SpinX columns (Corning), according to the manufacturer's instructions. The gel-purified cDNA standards were quantified spectrophotometrically at 260nm using NanoDrop 8000 (Thermo Scientific) and the copy number calculated from the concentration using the Avogadro number (6.02 × 10 23 mol -1 ).
Serial dilutions of the transcript-specific cDNA standards with known amounts of input copy (10 10 -10 2 molecules) were amplified by real time PCR in triplicate and the corresponding threshold cycle values plotted against the log copy number to generate standard curves. The reaction efficiency (E= [10 (-1/M) ] -1), slope (M) and r squared (r 2 ) were determined by the Corbett Rotor-Gene 6000 series software (optimal values for reaction efficiencies, slope and r squared are 1, -3.322 and 1 respectively). In each experimental set, each target kidney sample was analysed by real time RT-PCR for Ppia and Gapdh expression. To normalize for RNA input and possible inefficiencies in cDNA synthesis copy numbers obtained for the target genes (TG and Syndecan family) were divided by the corresponding Ppia value. Absolute quantifications were performed using standard curves of purified RT-PCR products which showed high test linearity (r, 0.991-0.999) and efficiency (0.63-1.1) over the wide quantification range (10 2 -10 8 molecules).

Western blotting of TG family members
The following primary antibodies were used to detect the TG members at the stated dilutions

Analysis of active TGF-β1 in NRK52 cells with and without HS antagonism
Active and total TGF-β were determined using the MLEC luciferase TGF-β quantitative bioassay, provided by Prof Martin Griffin (Aston University) 48 . Cells were seeded at 1.5 × 10 5 grown in an 8-well chamber slide and treated with 12 µM Surfen before addition of pre activated Rh-TG2 as described above. After fixation in (3% PFA) and permeabilisation (0.1% (w/v) Triton X100), active Smad3 was detected by rabbit anti-Smad3(pSer425) polyclonal antibody (Sigma) followed by donkey anti rabbit Alexa 488. Total Smad3 was detected by rabbit anti-Smad3 polyclonal antibody (Cell Signalling) followed by donkey anti rabbit Alexa 488. Active Smad 3 was quantified by ImageJ intensity analysis.