P53
URINARY ε- γ (GLUTAMYL) LYSINE: A NOVEL BIOMARKER OF CHRONIC
KIDNEYDISEASE
Da Silva-Lodge M¹, El Nahas A¹, Sultana S², Johnson T¹
¹Sheffield Kidney Institute, Academic Nephrology Unit, Medical School, University ofSheffield,²Pfizer
Pharmaceuticals, Sandwich, Kent, UK
BACKGROUND: Chronic Kidney Disease (CKD) is often progressive leading to End Stage Renal Disease
(ESRD). Early detection of CKD would almost certainly improve long term outcome. Current kidney
function tests have serious limitations as they only can detect dysfunction after significant damage has
occurred, provide limited help with the diagnosis or stage of the disease and are also unhelpful in assessing
early response to therapy. Therefore there is an urgent requirement for identification of early and reliable non
invasive biomarkers of kidney disease. Irrespective of the underlying kidney pathology, the final process of
CKD is characterised by excessive accumulation of Extracellular Matrix (ECM) that leads to fibrosis and
scar formation. The pathobiology of this is well described and as such mediators of the process that are
present in urine theoretically would make good biomarkers of CKD. Transglutaminase 2 (TG2) is well
described as a key mediator of kidney fibrosis, being instrumental in scar tissue formation by catalysing the
post-translational modification of ECM proteins by the formation of ε (γ-glutamyl) lysine bonds. Previous
studies have demonstrated that tissue levels of TG2 and ε (γ-glutamyl) lysine correlate well with histological
renal scarring. We therefore hypothesised that ε (γ-glutamyl) lysine can be measured in the urine and that it
may act as a specific, sensitive and early biomarker of CKD.
AIMS: To determine if ε (γ-glutamyl) lysine crosslink can be measured in urine and if so, correlate levels to
the development of kidney scarring to ascertain its potential as a urine biomarker of kidney fibrosis.
EXPERIMENTAL METHODS: Urines obtained from the rat 5/6th subnephrectomy (SNx) model of CKD
at 7, 28 and 84 days were screened for ε (γ-glutamyl) lysine di peptide. Controls consisted of sham-operated
rats. 10 mg of protein from rat urine precipitate (TCA precipitation followed by ether extraction ) was
resuspended in NH4CO3 , submitted to exhaustive proteolytic digestion (Subtilisin, Pronase E, Leucine
Aminopeptidase, Prolidase and Carboxypeptidase Y), freeze dried and fractionated onto a Biochrom 30
Amino acid Analyser (AAA) using a standard lithium and pH gradient. The concentration of ε (γ glutamyl)
lysine was obtained from a 570 nm profile by reference to a 5 nmols / 20ul ε (γ-glutamyl) lysine standard. ε
(γ-glutamyl) lysine had at a retention time of 77 minutes with sample spiking to confirm peak selection.
Urinary albumin excretion was measured by ELISA and creatinine clearance (CrCl) by autoanalyser.
Scarring Index was determined by multiphase image analysis of Masson`s Trichrome sections. MannWhitney U test was applied to estimate differences between groups.
RESULTS: ε (γ-glutamyl) lysine excretion was increased by 132% in the SNx group at 84 days when
expressed per mg of urine protein; however no change was noted prior to this. Total excretion of ε (γglutamyl) lysine in 24 hours was 3 fold higher in the SNx compared to the controls as early as 7 days, 2.2
fold by 28 days and 8 fold higher at 84 days (n = 7 SNx & n = 12 Controls) (p<0.05 at all time points). Total
urinary ε (γ-glutamyl)-lysine excretion showed a positive linear correlation with 24h proteinuria (r = 0.8791,
95% CI = 0.7593 to 0.9413, p < 0.0001, kidney scarring (Scarring Index) (r = 0.8551, 95% CI = 0.5750 to
0.9558, P = 0.002) and a negative correlation with Creatinine Clearance (r = -0.7228, 95% CI = -0.8652 to 0.4725, p < 0.001).
CONCLUSIONS: When expressed per mg of urine protein ε (γ-glutamyl) lysine was only elevated in
advanced kidney fibrosis indicating potential as a spot measurement for advanced disease only. 24 hours
secretion of ε (γ-glutamyl) lysine had large increases at all 3 time points suggesting that it may have potential
as a biomarker of progressive kidney scarring. 24 hours urine ε (γ-glutamyl) lysine may have advantages
over albuminuria and Scarring Index as an early biomarker as it was significantly increased at 7 and 28 days
whereas both conventional markers failed to have statistically significant changes in this model. Future work
will test this observation in human urines to ascertain whether it would have clinical value.