Identification and validation of urinary CXCL9 as a biomarker for diagnosis of acute interstitial nephritis

Abstract

BackgroundAcute tubulointerstitial nephritis (AIN) is one of the few causes of acute kidney injury with diagnosis-specific treatment options. However, due to the need to obtain a kidney biopsy for histological confirmation, AIN diagnosis can be delayed, missed, or incorrectly assumed. Here, we identify and validate urinary CXCL9, an IFN-γ-induced chemokine involved in lymphocyte chemotaxis, as a diagnostic biomarker for AIN.MethodsIn a prospectively enrolled cohort with pathologist-adjudicated histological diagnoses, termed the discovery cohort, we tested the association of 180 immune proteins measured by an aptamer-based assay with AIN and validated the top protein, CXCL9, using sandwich immunoassay. We externally validated these findings in 2 cohorts with biopsy-confirmed diagnoses, termed the validation cohorts, and examined mRNA expression differences in kidney tissue from patients with AIN and individuals in the control group.ResultsIn aptamer-based assay, urinary CXCL9 was 7.6-fold higher in patients with AIN than in individuals in the control group (P = 1.23 × 10-5). Urinary CXCL9 measured by sandwich immunoassay was associated with AIN in the discovery cohort (n = 204; 15% AIN) independently of currently available clinical tests for AIN (adjusted odds ratio for highest versus lowest quartile: 6.0 [1.8-20]). Similar findings were noted in external validation cohorts, where CXCL9 had an AUC of 0.94 (0.86-1.00) for AIN diagnosis. CXCL9 mRNA expression was 3.9-fold higher in kidney tissue from patients with AIN (n = 19) compared with individuals in the control group (n = 52; P = 5.8 × 10-6).ConclusionWe identified CXCL9 as a diagnostic biomarker for AIN using aptamer-based urine proteomics, confirmed this association using sandwich immunoassays in discovery and external validation cohorts, and observed higher expression of this protein in kidney biopsies from patients with AIN.FundingThis study was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) awards K23DK117065 (DGM), K08DK113281 (KM), R01DK128087 (DGM), R01DK126815 (DGM and LGC), R01DK126477 (KNC), UH3DK114866 (CRP, DGM, and FPW), R01DK130839 (MES), and P30DK079310 (the Yale O'Brien Center). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Keywords: Chemokines; Chronic kidney disease; Diagnostics; Nephrology.

Figure 1. Volcano plot demonstrating associations of proximity extension measurement of urine proteins with acute interstitial nephritis diagnosis.

Proteins with Q values of less than 0.05 using the Benjamini-Hochberg procedure are highlighted in red.

Figure 2. Urine CXCL9 levels are higher in acute interstitial nephritis compared with controls in the discovery cohort.

Box and whisker plots of CXCL9 by presence or absence of AIN (left panel) and by histological diagnosis in the discovery cohort (right panel). Boxes represent interquartile range and horizontal line within box represents median. Median and interquartile range values are presented in Table 2.

Figure 3. Urine CXCL9 improved the AUC for acute interstitial nephritis compared with existing information.

Comparison of AUC of CXCL9 for AIN diagnosis compared with clinicians’ prebiopsy diagnosis of acute interstitial nephritis obtained through chart review (left panel) and AIN statistical model as described in ref. (right panel)

Figure 4. CXCL9 levels were higher in AIN than in controls in the external validation cohorts.

Box plot of CXCL9 by presence or absence of AIN (A) and by histological diagnosis in the validation cohort (B). (C) AUC of CXCL9 for AIN diagnosis. AIN, acute interstitial nephritis; ANS, arterionephrosclerosis; ATN, acute tubular necrosis/injury; DKD, diabetic kidney disease; GN, glomerulonephritis

Figure 5. CXCL9 expression in kidney biopsies was higher in acute interstitial nephritis than in controls.

All native kidneys except ATI (transplant); Control, histologically normal biopsies; Nanostring analysis; Kruskal Wallis or Wilcoxon Rank-sum test