Figure 4

CpG methylation analyzed by bisulfite sequencing: Results at the KLK1 promoter, as well as a global control (LINE-1 repetitive elements), in genomic DNA from urine or blood (mononuclear cells). Results (shown as mean ± one SEM), from established AKI cases or healthy controls, were analyzed by ANOVA, factoring for age, sex and race. LINE-1 reagents (Pyromark, Biotage) were used to analyze the 3 CpG sites in LINE-1 repetitive elements, while the KLK1 gene was analyzed by a separate PCR covering 4 promoter CpGs. Promoter KLK1 specific methylation was substantially higher in blood than urine DNA (blood 66.38 ± 1.00 vs. urine 33.43 ± 4.67%; ANOVA p < 0.0001). Promoter KLK1 methylation in blood DNA was higher in AKI than controls (AKI, 70.32 ± 2.27 vs. controls, 65.36 ± 1.05%; ANOVA p = 0.011). Promoter KLK1 methylation in urine DNA did not differ in AKI versus controls (AKI, 40.95 ± 7.06 vs. controls, 30.35 ± 5.88%; ANOVA p = 0.22). Global LINE-1 methylation was greater in both blood than urine DNA (blood 73.11 ± 0.38 vs. urine 69.37 ± 0.86%, p = 0.0002). LINE-1 methylation in blood DNA did not differ in AKI and controls (AKI, 71.71 ± 0.44 vs. controls, 73.67 ± 0.41%; ANOVA p = 0.08). LINE-1 methylation in urine DNA did not differ in cases/controls (AKI, 69.53 ± 1.54 vs. controls, 69.29 ± 1.05%; ANOVA p = 0.79).