Volume 88, Issue 6, Pages 1336-1344 (December 2015) Urinary mitochondrial DNA is a biomarker of mitochondrial disruption and renal dysfunction in acute kidney injury  Ryan M. Whitaker, L. Jay Stallons, Joshua E. Kneff, Joseph L. Alge, Jennifer L. Harmon, Jennifer J. Rahn, John M. Arthur, Craig C. Beeson, Sherine L. Chan, Rick G. Schnellmann  Kidney International  Volume 88, Issue 6, Pages 1336-1344 (December 2015) DOI: 10.1038/ki.2015.240 Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 1 Urinary mitochondrial DNA (mtDNA) copy number is associated with progression of renal dysfunction from collection in patients with cardiopulmonary bypass (CPB)-induced acute kidney injury (AKI).Urine was collected from patients following CPB. Urinary mtDNA levels were measured via real-time quantitative PCR for ND1 and corrected to the nuclear control gene β-actin. (a) Patients were stratified into three groups based on collection and maximum Acute Kidney Injury Network staging: no AKI (n=17), stable AKI (n=63), and progressive AKI (n=31). Data are expressed as median±IQR. Multiple comparisons were performed using Kruskal–Wallis test followed by Dunn’s test. *P<0.05 versus No AKI, #P<0.05 versus stable AKI. (b–d) Area under the receiver operator characteristic curve (AUC) analysis was performed comparing the three groups of patients. Kidney International 2015 88, 1336-1344DOI: (10.1038/ki.2015.240) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 2 Urinary mitochondrial DNA (mtDNA) copy number is not correlated with degree of cardiopulmonary bypass (CPB)-induced acute kidney injury in humans.Urinary mtDNA/nuclear DNA (nDNA) was not correlated with (a) maximum serum creatinine, (b) maximum percentage change in serum creatinine from baseline, (c) urinary angiotensinogen levels, (d) bypass time, (e) days to maximum creatinine, and (f) days to discharge (n=111 for all analyses). Linear regression was performed and Spearman’s rank-order coefficients were calculated. Kidney International 2015 88, 1336-1344DOI: (10.1038/ki.2015.240) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 3 Urinary mitochondrial DNA (mtDNA) levels are increased in mice following renal ischemia/reperfusion (I/R).Male C57BL/6 mice underwent sham surgery or ischemia (5, 10, or 15min) followed by reperfusion. Mice were placed in metabolic cages from 6 to 24h after reperfusion for urine collection. Blood was collected and mice were killed 24h after reperfusion. Renal function was evaluated by (a) blood urea nitrogen (BUN) and (b) 18-h urine output. Urinary mtDNA levels were measured by real-time PCR for ND1 and are expressed as (c) total urinary output in 18h and (d) urinary output corrected for urinary creatinine levels. Data are expressed are mean±s.e.m. #Indicates statistical significance at P<0.05 versus sham controls; sham (n=12), 5min I/R (n=4), 10min I/R (n=9), 15min I/R (n=11). Kidney International 2015 88, 1336-1344DOI: (10.1038/ki.2015.240) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 4 Total urinary output of mitochondrial DNA (mtDNA) is correlated with degree of ischemia but not blood urea nitrogen (BUN).Urinary mtDNA levels expressed as total urinary output per 18h were correlated with (a) BUN levels or (b) ischemia time (n=36). Linear regression was performed and Spearman’s rank-order coefficients were calculated. Kidney International 2015 88, 1336-1344DOI: (10.1038/ki.2015.240) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 5 Total 18h urinary output of mitochondrial DNA (mtDNA) is a biomarker of renal injury following ischemia/reperfusion (I/R) in mice.Receiver operator characteristic curves were constructed for total 18h urinary output of mtDNA comparing (a) sham surgical mice (n=12) to all mice that underwent I/R (n=24) or (b) only mice with BUN>2 s.d. above the historical sham average BUN (n=13). AKI, acute kidney injury. Kidney International 2015 88, 1336-1344DOI: (10.1038/ki.2015.240) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 6 Total 18h urinary output of mitochondrial DNA (mtDNA) correlates with reductions in renal mtDNA copy number and mitochondrial gene expression.Urinary mtDNA levels expressed as total urinary output per 18h were correlated with relative (a) renal mtDNA copy number, (b) PGC-1α, (c) COX1, and (d) NDUFB8 (n=36). Renal mtDNA and mRNA expression were corrected to the nuclear gene β-actin. Linear regression was performed and Pearson correlation coefficients were calculated. Kidney International 2015 88, 1336-1344DOI: (10.1038/ki.2015.240) Copyright © 2015 International Society of Nephrology Terms and Conditions