The Effect of Cortex/Medulla Proportions on Molecular Diagnoses in Kidney Transplant Biopsies: Rejection and Injury Can Be Assessed in Medulla

Abstract

Histologic assessment of kidney transplant biopsies relies on cortex rather than medulla, but for microarray studies, the proportion cortex in a biopsy is typically unknown and could affect the molecular readings. The present study aimed to develop a molecular estimate of proportion cortex in biopsies and examine its effect on molecular diagnoses. Microarrays from 26 kidney transplant biopsies divided into cortex and medulla components and processed separately showed that many of the most significant differences were in glomerular genes (e.g. NPHS2, NPHS1, CLIC5, PTPRO, PLA2R1, PLCE1, PODXL, and REN). Using NPHS2 (podocin) to estimate proportion cortex, we examined whether proportion cortex influenced molecular assessment in the molecular microscope diagnostic system. In 1190 unselected kidney transplant indication biopsies (Clinicaltrials.govNCT01299168), only 11% had <50% cortex. Molecular scores for antibody-mediated rejection, T cell-mediated rejection, and injury were independent of proportion cortex. Rejection was diagnosed in many biopsies that were mostly or all medulla. Agreement in molecular diagnoses in paired cortex/medulla samples (23/26) was similar to biological replicates (32/37). We conclude that NPHS2 expression can estimate proportion cortex; that proportion cortex has little influence on molecular diagnosis of rejection; and that, although histology cannot assess medulla, rejection does occur in medulla as well as cortex.

Keywords: biopsy; clinical research/practice; kidney biology; kidney transplantation/nephrology; microarray/gene array; molecular biology; molecular biology: mRNA/mRNA expression; rejection.

© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1

Diagram showing the sampling strategy for the technical replicate pairs, biological replicate pairs, and cortex–medulla pairs.

Figure 2. Volcano plot of fold change between cortex and medulla versus negative log of adjusted p-value with false discovery rate

NPHS2 had the highest association and fold change between cortex and medulla of 55 000 probe sets. A selection of highly significant probe sets distinguishing cortex from medulla is labeled.

Figure 3

Predicted proportion of cortex in a sample histologically called cortex or medulla using either the principal component 1 score based on 29 of the top 30 probe sets (excluding NPHS2) (y-axis) or using NPHS2 expression alone (x-axis).

Figure 4

Boxplot showing log of NPHS2 expression in medulla and cortex samples as established by histology (A) and the predicted proportion cortex distribution across all samples (B). Box shows the interquartile range, horizontal bar—median and whiskers—1.5 × standard deviation.

Figure 5. Density plot of NPHS2 expression in 1190 nonbisected biopsy cores

Black symbols show the distribution of NPHS2 expression values in cortex and medulla divided pair samples, respectively.

Figure 6. Scatter plots with predicted proportion cortex (y-axis) versus molecular ABMR, TCMR, and rejection scores in the biological replicate set, the cortex and medulla set, and the reference set (x-axis)

Vertical dotted line indicates the positive/negative cutoffs for the molecular scores; horizontal dotted line indicates the 0.2/0.8 split for the proportion of cortex. A) ABMR - biological replicates, B) ABMR - cortex/medulla, C) ABMR - reference set, D) TCMR - biological replicates, E) TCMR - cortex/medulla, F) TCMR - reference set, G) Rejection - biological replicates, H) Rejection - cortex/medulla, I) Rejection - reference set. Refer to Table 3 for further details. ABMR, antibody-mediated rejection; TCMR, T cell–mediated rejection.

Figure 7. Investigating the relationship between injury (IRRAT score) and proportion of cortex

Vertical dotted line indicates the arbitrary positive/negative cutoff for the molecular IRRAT scores; horizontal dotted line indicates an arbitrary 0.2/0.8 split for the proportion of cortex. A) IRRATs - cortex/medulla, B) IRRATs - biological replicates, C) IRRATs - reference set. Refer to Table 3 for further details. IRRAT, acute kidney injury transcripts.

Figure 8. Reproducibility plots of the molecular scores of rejection (TCMR, ABMR, all rejection) and acute kidney injury (IRRAT) in the technical and biological replicates and in the cortex–medulla divided pairs

Dotted horizontal line indicates the positive/negative cutoffs for the molecular scores. The y-axes are the scores for the two samples compared for the TCMR, ABMR, and Rejection classifiers (a number between 0 and 1.0) or the geometric mean of the expression of the AKI transcripts (IRRATs). The x-axis is the mean of classifier or IRRAT scores. ABMR, antibody-mediated rejection; AKI, acute kidney injury; IRRAT, acute kidney injury transcripts; TCMR, T cell–mediated rejection.