Development of a Multiplexed LC-MS/MS Assay for the Quantitation of Podocyte Injury Biomarkers Nephrin, Podocalyxin, and Podocin in Human Urine

CKD is frequently diagnosed only after a significant
progression.
GFR is the most common indicator of kidney function but is limited
in detecting early CKD cases and distinguishing glomerular, tubular,
and global CKD. Aiming to provide a glomeruli specific biomarker assay,
we developed a peptide immunoaffinity targeted mass spectrometry method
for the quantitation of three podocyte specific proteins in human
urine: nephrin, podocalyxin, and podocin. Proteins in urine were precipitated,
stable isotope labeled peptide standards incorporated, and digested
with trypsin. Target peptides were enriched using an online antibody
column prior to LC-MS/MS. The performance metrics for nephrin, podocalyxin,
and podocin were evaluated: The lower limits of quantitation were
3.8, 22.0, and 5.4 pM, respectively. The intraplate relative error
(RE) was within ±10.6%, ± 10.4%, and ±16.1%, and coefficient
of variation (CV) was ≤27.2%, ≤ 14.1%, and ≤20.7%
accordingly. The interplate RE was within ±7.0%, ± 3.8%,
and ±3.0%, and CV was ≤17.2%, ≤ 12.1%, and ≤20.0%
for the three analytes. The urinary nephrin, podocalyxin, and podocin
concentrations in 60 healthy volunteers and 20 disease samples was
measured, thereby establishing the basal levels of these protein and
enabling future evaluation of their roles as noninvasive biomarkers
of glomerular injury in the clinic.