Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury

Abstract

Noninvasive biomarkers are needed to assess immune risk and ultimately guide therapeutic decision-making following kidney transplantation. A requisite step toward these goals is validation of markers that diagnose and/or predict relevant transplant endpoints. The Clinical Trials in Organ Transplantation-01 protocol is a multicenter observational study of biomarkers in 280 adult and pediatric first kidney transplant recipients. We compared and validated urinary mRNAs and proteins as biomarkers to diagnose biopsy-proven acute rejection (AR) and stratify patients into groups based on risk for developing AR or progressive renal dysfunction. Among markers tested for diagnosing AR, urinary CXCL9 mRNA (odds ratio [OR] 2.77, positive predictive value [PPV] 61.5%, negative predictive value [NPV] 83%) and CXCL9 protein (OR 3.40, PPV 67.6%, NPV 92%) were the most robust. Low urinary CXCL9 protein in 6-month posttransplant urines obtained from stable allograft recipients classified individuals least likely to develop future AR or a decrement in estimated glomerular filtration rate between 6 and 24 months (92.5-99.3% NPV). Our results support using urinary CXCL9 for clinical decision-making following kidney transplantation. In the context of acute dysfunction, low values can rule out infectious/immunological causes of injury. Absent urinary CXCL9 at 6 months posttransplant defines a subgroup at low risk for incipient immune injury.

Keywords: Acute rejection; biomarker; chemokines; kidney allograft; kidney graft function.

Figure 1

Consort diagram illustrating the outcome of patients throughout the course of the study including numbers and results of biopsies performed and numbers of patients who reached the 24-month endpoint.

Figure 2. Urinary mRNAs as biomarkers for biopsy-proven acute rejection (AR) within the first 6 months posttransplant

(A) Box and whisker plots depicting the median (dash), 25 and 75 percentiles (top and bottom of box)with whiskers extending to 1 and 100 percentile for each gene in patients with infection (Inf), Banff grade suspicious rejection (Susp) Banff grade ≥1A AR or other/no rejection/infection (Other). Results are absolute copy number normalized to the 18S RNA. p-Values are from an ANOVA with Tukey–Kramer multiple comparison corrections. (B) Receiver operator characteristics curves for granzyme B mRNA, CXCL9 mRNA and combined granzyme B + CXCL9 mRNA as diagnostic for Banff grade ≥1A AR using optimal thresholds of granzyme B mRNA = 1.017 × 10⁻⁶ and CXCL9 mRNA= 1.925 × 10⁻⁶ normalized units (copy number gene/copy number of 18S mRNA). Also see Tables 2, S4 and S5.

Figure 3. Urinary CXCL9 and CXCL10 protein as biomarkers for biopsy-proven acute rejection (AR) and an incipient decrement in GFR

(A) Box and whisker plots depicting the median (dash), 25 and 75 percentiles (top and bottom of box) with whiskers extending to 0 and 100 percentile for urinary CXCL9 and CXCL10 protein in patients infection (Inf), Banff grade suspicious rejection (Susp), Banff grade ≥1AAR or other/no rejection/infection (Other). p-Values are from an ANOVA with Tukey–Kramer multiple comparison corrections. (B) Receiver operator characteristics curves CXCL9 protein ± granzyme B RNA or CXCL9 mRNA as diagnostic for Banff grade ≥1AAR (optimal threshold for CXCL9 protein is 35pg/mL). See Tables 2, S4 and S5 for additional details. (C) Dot plot depicting individual urinary CXCL9 protein values obtained within 30 days prior to a for-cause biopsy, at the time of the biopsy or within 30 days after the for-cause biopsy in patients biopsied for suspected rejection, stratified by presence (red) or absence (blue) of Banff grade ≥1A AR on the biopsy. Dashed line depicts the mean for each set. *p < 0.0001 versus values for AR (red) at that time point. (D) Dot plot correlating 6-month urinary CXCL9 obtained during clinical quiescence with subsequent diagnosis of AR between 6 and 24 months. (E) Dot plot correlating individual values of 6-month urinary CXCL9 with individual “i” and “t” scores from simultaneous surveillance biopsies. p-Value of <0.0001 refers to an ANOVA comparing the means across Banff scores for each panel.