Abstract:
Introduction
Renal biomarkers in current practice, urine albumin and serum creatinine, are inaccurate for early
detection of Chronic Kidney Disease of uncertain etiology (CKDu) and discrimination of CKDu from other
chronic renal diseases. Aim of the pilot study is to evaluate “fit for purpose” biomarkers or biomarkers that
manifest in CKDu. Novel candidate renal biomarkers in clinical validation phase can be used for this
purpose. Luminex xMAP, a robust analytical platform can assay multiple analytes in a small sample
volume (< 50 µL) at picogram level that comply with this study.
Methods
80 biopsy proven stage 1-3 CKDu patients (Nanayakkara, 2011) were selected from renal clinics using a
systematic sampling method. Urine and serum samples were collected and stored in -80°C refrigerator
according to biological sample collection for clinical proteomics SOPs. Total of 27 renal candidate
biomarkers were included in customized magnetic bead based multiplexed assay kits (four serum and
two urine Millipore kits). Samples were prepared with duplicates of blank (1), standards (6), quality
controls (2), and patients (39) on a 96 well microtiter plate, according to the manufacturer's instructions.
Samples were analyzed on Luminex MAGPIX analyzer. The biomarker concentrations in the samples
were determined from the 5-parameter logistic fit standard curves created in Milliplex analyst software.
The manifestation of a biomarker in CKDu was deduced from number of samples within detectable range.
The percentages for each biomarker was tested at 5% of significant level (H P ≤ 0.8 Vs H : P > 0.8) to
extract the biomarkers which exceeds significantly > 80% of cases within detectable range (One sample
proportion test).
Results
Results of candidate renal biomarkers in 80 CKDu patients
Analyte Detectable Concentration Range% detected within range*
min-DC max-DC
Serum
TGF-β 5.01 ρ 3789 ρ 100
RBP-4 0.13 ρ 18978 ρ 0.00 (all > max-DC)
β2MG 0.035 ρ 29453 ρ 100
Cystatin C 0.062 ρ 5283 ρ 100
NGAL 0.05 ρ 4442 ρ 57 (43 > max-DC)
TIMP-1 5.14 ρ 655.91 ρ 99
Collagen-IV0.52 ρ 988.42 ρ 100
IL-10 13.10 ρ 738.13 ρ 21 (79 < min-DC)
IL-6 2.45 ρ 221.41 ρ 29 (69 < min-DC)
TNF R11 3.58 ρ 6555 ρ 96
OPG 5.27 ρ 793.05 ρ 96
KIM-1 24.89 ρ 865.44 ρ 24 (76 < min-DC)
Pentaxin-3 9.66 ρ 4904 ρ 96
Renin 86.04 ρ 25632 ρ 88
PTH 1.99 ρ 251.98 ρ 96
FGF-23 23.68 ρ 1044 ρ 34 (66 < min-DC)
Urine
NGAL 0.0088 η 559.32 η 100
RBP-4 0.043 η 7511 η 24 (76 > max-DC)
β2MG 0.56 η 19252 η 48 (52 > max-DC)
Cystatin C 0.02 η 1981 η 99
OPN 28.74 η 2198 η 99
α1MG 125.55 η 42130 η 83
TIMP-1 0.25 η 58.76 η 93
KIM-1 0.05 η 1.34 η 92
FABP-1 8.49 η 53.34 η 49 (51 < min-DC)
Collagen-IV018 η 340.92 η 100
TFF-3 0.25 η 1170 η 99
* number of cases within detectable concentration (DC) as a percentage of the total number of cases, ρ =
pg/ml, η = ng/ml.
Conclusions
Serum (n = 10) TGFβ-1, β2MG, Cystatin C, TIMP-1, Collagen-IV, TNF RII, OPG, Pentaxin-3, Renin, PTH
and urine (n=8) NGAL, Cystatin C, OPN, α1MG, TIMP-1, KIM-1, Collagen-IV, TFF-3 are fit for validation
of CKDu. Serum IL-10, IL-6, KIM-1, FGF-23 and urine FABP-1 are not manifested in majority whereas
serum NGAL, urine β2MG and RBP-4 in both matrices are detected above the max-DC in all cases, and
we may further dilute the samples to get within the range.