A Novel Multi-Biomarker Assay for Non-Invasive Quantitative Monitoring of Kidney Injury

Abstract

The current standard of care measures for kidney function, proteinuria, and serum creatinine (SCr) are poor predictors of early-stage kidney disease. Measures that can detect chronic kidney disease in its earlier stages are needed to enable therapeutic intervention and reduce adverse outcomes of chronic kidney disease. We have developed the Kidney Injury Test (KIT) and a novel KIT Score based on the composite measurement and validation of multiple biomarkers across a unique set of 397 urine samples. The test is performed on urine samples that require no processing at the site of collection and without target sequencing or amplification. We sought to verify that the pre-defined KIT test, KIT Score, and clinical thresholds correlate with established chronic kidney disease (CKD) and may provide predictive information on early kidney injury status above and beyond proteinuria and renal function measurements alone. Statistical analyses across six DNA, protein, and metabolite markers were performed on a subset of residual spot urine samples with CKD that met assay performance quality controls from patients attending the clinical labs at the University of California, San Francisco (UCSF) as part of an ongoing IRB-approved prospective study. Inclusion criteria included selection of patients with confirmed CKD and normal healthy controls; exclusion criteria included incomplete or missing information for sample classification, logistical delays in transport/processing of urine samples or low sample volume, and acute kidney injury. Multivariate logistic regression of kidney injury status and likelihood ratio statistics were used to assess the contribution of the KIT Score for prediction of kidney injury status and stage of CKD as well as assess the potential contribution of the KIT Score for detection of early-stage CKD above and beyond traditional measures of renal function. Urine samples were processed by a proprietary immunoprobe for measuring cell-free DNA (cfDNA), methylated cfDNA, clusterin, CXCL10, total protein, and creatinine. The KIT Score and stratified KIT Score Risk Group (high versus low) had a sensitivity and specificity for detection of kidney injury status (healthy or CKD) of 97.3% (95% CI: 94.6–99.3%) and 94.1% (95% CI: 82.3–100%). In addition, in patients with normal renal function (estimated glomerular filtration rate (eGFR) ≥ 90), the KIT Score clearly identifies those with predisposing risk factors for CKD, which could not be detected by eGFR or proteinuria (p < 0.001). The KIT Score uncovers a burden of kidney injury that may yet be incompletely recognized, opening the door for earlier detection, intervention and preservation of renal function.

Non‐radiological assessment of kidney stones using the kidney injury test (KIT), a spot urine assay

“…A,B), with mean (±SEM) values of 10.93 ± 0.80 vs 18.25 ± 1.64 vs 29.89 ± 1.39, respectively. At the pre‐established threshold of 18.5 , KIT stone score discriminated between healthy controls and those with radiologically confirmed stones with an area under the receiver‐operating characteristic curve of 0.9522 (Fig. A).…”

“…( C ) The score is highest in patients with radiologically confirmed kidney stones (KSC, teal), intermediate in those with a history of kidney stones (KSP, turquoise), and lowest in the HC group (grey; P < 0.0001 by Kruskal–Wallis test, pairwise comparisons corrected by Dunn’s post hoc test). With a threshold detection for stone risk set at a KIT stone score of >18.5 based on our prior publication , the sensitivity and specificity were found to be 81.94% and 92.59% respectively.…”

“…The KIT assay, which measures the six biomarkers of cell‐free DNA (cfDNA), methylated cfDNA, clusterin, creatinine, total protein and CXCL10, was performed as previously described . For measurement of cfDNA, we have previously developed a proprietary 5′ biotinylated oligonucleotide complementary chemiluminescent immunoprobe for the measurement of specific target cfDNA fragments.…”

“…Development, threshold determination, and calculation of the KIT stone score were as previously described . Comparisons involving two groups were performed using the non‐parametric Mann–Whitney U ‐test.…”

“…The kidney injury test (KIT) assay, consisting of a panel of urinary biomarkers measured on a random urine sample, was recently validated for early detection of kidney injury, inclusive of early detection of kidney stone disease . In the present study, we evaluate the utility of the KIT assay and KIT stone score as a non‐invasive method for the detection of urinary stone disease, without the need for 24‐h urine collection.…”