Kidney-specific cell-free DNA as biomarker of kidney injury: Development and exploration of a multiplex digital droplet PCR assay.

INTRODUCTION Cell-free DNA (cfDNA) is a promising non-invasive biomarker of organ injury. In transplantation, cfDNA is typically quantified by distinguishing donor- from recipient-derived DNA. However, we aimed to develop a multiplex digital droplet PCR (ddPCR) assay to measure kidney-derived cfDNA independent of donor-recipient differences as a marker of kidney injury.

Methods

Five kidney-specific differentially methylated regions were identified in silico using The Cancer Genome Atlas and Gene Expression Omnibus data and combined into a multiplex ddPCR assay. The assay was validated in kidney implantation biopsies (n = 51), plasma from healthy controls (n = 10), and applied to plasma samples from 20 kidney transplant recipients without rejection, collected at six timepoints during the first posttransplant year (n = 120 samples).

Results

All five differentially methylated regions were detectable in kidney tissue, were minimally present in controls, and increased significantly within the first week post-transplant, reflecting ischemia-reperfusion induced kidney injury. No significant associations were found between cfDNA levels and immunosuppressive regimen, age, sex, eGFR, or delayed graft function.

Conclusions

We developed a robust multiplex ddPCR assay to detect kidney-specific cfDNA. The early post-transplant peak supports its potential as a marker of kidney injury. Unlike donor-recipient-based approaches, this method directly quantifies organ damage and may be broadly applicable beyond transplantation.