Characterization of renal biomarkers for use in clinical trials: biomarker evaluation in healthy volunteers

Figures & data

Table 1 Current status and rationale of biomarkers included in the current study

Parameter	Preclinical	Clinical
A1M		Increased in AKI patients post cardiopulmonary bypass^Citation35
B2M	Increased with gentamicin sulfate or cisplatin treatment in rats^Citation36	Increased with fumaric acid treatment for psoriasis^Citation37
Calbindin	Increased with gentamicin sulfate treatment in rats^Citation36 Increased with a triple reuptake inhibitor in monkeys^Citation38	Increased with cisplatin treatment^Citation34
Clusterin	Increased with gentamicin sulfate, 2-bromoethylamine, or cisplatin treatment in rats^Citation36 Increased with a triple reuptake inhibitor in monkeys^Citation38 Increased with n-phenylanthranilic acid^Citation39 Had a higher diagnostic value than BUN and serum creatinine^Citation40
CTGF	Increased in diabetic rats^Citation41	Increased in diabetics with nephropathy and a small portion of diabetics without nephropathy^Citation41
Cystatin C	Increased with gentamicin sulfate, 2-bromoethylamine, or cisplatin treatment in rats^Citation36 Had a higher diagnostic value than BUN and serum creatinine^Citation40 Increased with paraquat treatment^Citation42	Increased in patients with AKI compared with healthy volunteers^Citation33 Predicted AKI in sepsis critically ill patients^Citation43
GSTα	Increased with gentamicin sulfate, 2-bromoethylamine, or cisplatin treatment in rats^Citation36 Increased with cisplatin treatment^Citation44	Increased in subjects treated with amphotericin B desoxycholate^Citation45
KIM-1	Increased with gentamicin sulfate, 2-bromoethylamine, or cisplatin treatment in rats^Citation36 Increased with paraquat treatment^Citation42 Increased with cisplatin treatment^Citation44	Increased in patients with AKI compared with healthy volunteers^Citation33
Microalbumin	Increased with cisplatin treatment^Citation44	Increased in AKI patients post cardiopulmonary bypass^Citation35
NAG	Increased with cisplatin treatment^Citation44	Increased in patients with AKI compared to healthy volunteers^Citation33 Increased in AKI patients post cardiopulmonary bypass^Citation35
NGAL	Increased with gentamicin sulfate, 2-bromoethylamine, or cisplatin treatment in rats^Citation36	Increased in patients with AKI compared with healthy volunteers^Citation33 Increased in AKI patients post cardiopulmonary bypass^Citation35
Osteopontin	Increased with gentamicin sulfate, 2-bromoethylamine, or cisplatin treatment in rats^Citation36 Increased with cisplatin treatment^Citation44	Subjects with AKI had higher values than subjects without AKI^Citation46
THP		Neonates with AKI had lower levels of THP (uromodulin)^Citation47
TIMP-1		Is higher in patient with renal disease than controls^Citation48
TFF3	Decreased in rats treated with cisplatin^Citation49	Higher levels may indicate ongoing repair in the kidney^Citation50
VEGF		Increased in patients with AKI compared with healthy volunteers^Citation33

Abbreviations: A1M, alpha-1-microglobulin; AKI, acute kidney injury; B2M, β2-microglobulin; BUN, blood urea nitrogen; CTGF, connective tissue growth factor; GSTα, glutathione S-transferase alpha; KIM-1, kidney injury marker-1; NAG, N-acetyl-β-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; THP, Tamm-Horsfall urinary glycoprotein; TIMP-1, tissue inhibitor of metalloproteinase 1; TFF3, trefoil factor 3; VEGF, vascular endothelial growth factor.

Table 2 Study demographics

Baseline characteristic	Biomarker population (n=39)
Age (years)
Mean, (SD) median	43.9 (12.37) 43
Body mass index (kg/m^2)
Mean (SD)	24.4 (2.65)
Sex, n (%)
Female	19 (49)
Male	20 (51)
Race
Non-Hispanic Caucasian	38 (97.4)
Other	1 (2.6)

Abbreviation: SD, standard deviation.

Table 3 Summary statistics of plasma and urine samples analyzed with routine kidney biomarkers in the current study

Parameter	Plasma		Urine
	Confidence interval	%CVinter/%CVintra	Confidence interval	%CVinter/%CVintra
Creatinine	73.5–112.5 μmol/L	11/6	1.09–12.46 mmol/L	73/38
Osmolality	287–307 mOs/kg	1/2	79.28–793.94 mOs/kg	60/37
Potassium	3.49–4.71 mmol/L	4/7	5.77–63.26 mmol/L	68/46
Sodium	140.6–145.4 mmol/L	1/2	10.97–99.41 mmol/L	78/42
Total protein	66.4–80.8 g/L	3/5	3.32–58.74 mg/L	147/56

Abbreviations: %CV_inter, intersubject coefficient of variation; %CV_intra, intrasubject coefficient of variation.

Table 4 Summary statistics of urine samples analyzed with renal biomarkers under characterization within the current study

Parameter	Concentration		Concentration normalized to creatinine
	Confidence interval	%CVinter%CVintra	Confidence interval	%CVinter/%CVintra
Single analyte assays (central laboratory)
Creatinine	1.09–12.46 mmol/L	73/38	NC	NC
NAG	0.05–2.24 U/L	117/62	NC	NC
Single analyte assays (Pacific Biomarkers)
B2M	20–83 μg/L	50/29	0.04–0.15 μg/mg	43/29
Creatinine	0.06–1.29 mg/mL	74/44	NC	NC
Cystatin C	0.003–0.042 mg/L	68/40	21.3–77.2 ng/mg	33/10
KIM-1	15–249 pg/mL	65/43	50.3–539.7 pg/mg	50/17
NAG	0.34–2.4 U/L	62/37	0.001–0.003 U/mg	32/17
NGAL	0.3–150.3 ng/mL	299/50	1.9–580.3 ng/mg	325/38
Multiplex assays (Rules Based Medicine)
A1M	0.12–3.00 μg/mL	109/43	0.45–4.58 μg/mg	63/23
B2M	0.01–0.09 μg/mL	51/43	0.03–0.18 μg/mg	40/21
Calbindin	0.31–114.0 ng/mL	158/82	1.6–133.3 ng/mg	107/60
Clusterin	0.00–0.07 μg/mL	74/54	0.01–0.15 μg/mg	75/24
CTGF	0.20–1.00 ng/mL	41/33	0.19–2.08 ng/mg	58/32
Creatinine	9.9–144.0 mg/dL	58/40	NC	NC
Cystatin-C	3.2–75.0 ng/mL	65/50	17.5–70.4 ng/mg	27/19
GSTα	1.1–64.0 ng/mL	78/61	1.8–169.0 ng/mg	94/56
KIM-1	0.05–0.48 ng/mL	49/39	0.11–0.90 ng/mg	42/15
Microalbumin	0.4–62.0 μg/mL	260/47	1.9–258.3 μg/mg	331/33
NGAL	3–254 ng/mL	233/48	6.4–271.2 ng/mg	264/45
Osteopontin	66–1,230 ng/mL	66/50	236–1,459 ng/mg	36/18
THP	0.23–1.90 μg/mL	38/40	0.75–4.17 μg/mg	38/20
TIMP-1	0.3–10.0 ng/mL	108/72	0.34–13.09 ng/mg	114/67
TFF3	0.03–3.10 μg/mL	205/43	0.09–9.06 μg/mg	230/32
VEGF	105–879 pg/mL	52/35	456–1,286 pg/mg	24/15

Abbreviations: NC, not calculated; A1M, alpha-1-microglobulin; B2M, β-2-microglobulin; CTGF, connective tissue growth factor; GSTα, glutathione S-transferase alpha; KIM-1, kidney injury marker-1; NAG, N-acetyl-β-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; THP, Tamm-Horsfall urinary glycoprotein; TIMP-1, tissue inhibitor of metalloproteinase 1; TFF3, trefoil factor 3; VEGF, vascular endothelial growth factor; %CV_inter, intersubject coefficient of variation; %CV_intra, intrasubject coefficient of variation.

Table 5 Effect of sex (20 males and 19 females) on the renal biomarkers under characterization within this study

Concentration		Concentration normalized to creatinine
Parameter	Sex with higher value	Parameter	Sex with higher value
A1M	Male
B2M	Male	B2M	Female
Calbindin	Male	Calbindin	Male
Clusterin	Male	Clusterin	Female
CTGF	Male	CTGF	Female
Creatinine	Male
Cystatin C	Male	Cystatin-C	Female
GSTα	Female	GSTα	Female
KIM-1	Male	KIM-1	Female
NGAL	Female	NGAL	Female
Osteopontin	Male	Osteopontin	Female
TFF3	Female	TFF3	Female
THP	Male	THP	Female
VEGF	Male	VEGF	Female

Abbreviations: A1M, alpha-1-microglobulin; B2M, β-2-microglobulin; CTGF, connective tissue growth factor; GSTα, glutathione S-transferase alpha; KIM-1, kidney injury marker-1; NAG, N-acetyl-β-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; THP, Tamm-Horsfall urinary glycoprotein; TFF3, trefoil factor 3; VEGF, vascular endothelial growth factor.

Figure 1 Plots of vendor assay correlations. (A) Coefficient of determination (r ²) for each biomarker with assays from two vendors. (B–I) are biomarkers with r ²≥0.700 (B) Creatinine concentration, (C) NAG concentration, (D) creatinine concentration, (E) KIM-1 concentration, (F) NGAL concentration, (G) cystatin C concentration, (H) KIM-1 concentration normalized to creatinine, and (I) NGAL concentration normalized to creatinine.

Notes:¹Comparison between Central and Pacific Biomarker assays, both single analyte assays; ²comparison between Pacific Biomarker (single analyte) and Rules Based Medicine (multiplex) assays.
Abbreviations: KIM-1, kidney injury marker-1; NAG, N-acetyl-β-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin.

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