MITOCHONDRIAL BIOMARKERS IN ACUTE KIDNEY INJURY Rick G. Schnellmann, PhD Department of Drug Discovery and Biomedical Sciences SC Center for Therapeutic Discovery and Development

ACUTE KIDNEY INJURY Clinical Features Causes –Pre-renal: decreased perfusion –Intra-renal: ischemia, nephrotoxic event –Post-renal: obstruction Targets –Tubular epithelial cells –Inflammatory cells –Endothelial cells Recovery – 50% Diagnosis Treatments Supportive – dialysis

Bonventre et al., 2010 ACUTE KIDNEY INJURY BIOMARKERS

Relative changes in AKI biomarker concentration in humans over time after surgery. Alge and Arthur, JM, 2015 ACUTE KIDNEY INJURY BIOMARKERS

ACUTE KIDNEY INJURY I.Mitochondria as a Target in AKI II.Urinary Biomarkers of Renal Mitochondrial Dysfunction

MITOCHONDRIA AS A TARGET Mitochondrial dysfunction is recognized as a mediator of acute and chronic diseases: Acute: acute organ failure (stroke, myocardial infarction, acute liver and kidney injury) Chronic: neurodegenerative diseases, chronic kidney disease, cancer, epilepsy Mitochondrial Diseases: muscular dystrophies

MITOCHONDRIAL DYSFUNCTION IN ACUTE KIDNEY INJURY I.Animals: Ischemia/reperfusion Antibiotics (e.g. aminoglycosides) Nucleoside Reverse Transcriptase inhibitors (NRTIs) Chemotherapeutics (e.g. cisplatin) Environmental/Industrial (e.g. HgCl 2 ) II.Humans: Minimal data

MITOCHONDRIAL DYSFUNCTION IN ACUTE KIDNEY INJURY I.Rat model using glycerol-induced rhabdomyolysis II.Mouse model using renal-induced ischemia/reperfusion III.Mouse model using folic acid

MITOCHONDRIAL DYSFUNCTION IN A RAT MODEL OF RHABDOMYOLYSIS-INDUCED AKI

SUMMARY There is a sustained decrease in proximal tubular mitochondrial proteins in AKI. The sustained decrease in mitochondrial proteins is throughout renal cortex, outside the necrotic areas. Mitochondrial homeostasis is disrupted within hours of an injury and remains disrupted for over a week.

ACUTE KIDNEY INJURY I.Mitochondria as a Target in AKI II.Urinary Biomarkers of Renal Mitochondrial Dysfunction a) mtDNA b) ATP synthase 

BIOMARKERS OF MITOCHONDRIAL FUNCTION At this time, few tools are available to measure mitochondrial dysfunction in vivo. Current biomarkers of organ dysfunction do not focus on mitochondrial dysfunction. Historical/current biomarkers of mitochondrial dysfunction are limited to invasive muscle biopsies, organ ATP measurements, or functional measurements in isolated mitochondria intravital microscopy and urinary metabolomics. Consequently, new non-invasive assays are needed that focus on mitochondrial dysfunction.

mtDNA AS A BIOMARKER OF RENAL MITOCHONDRIAL DYSFUCTION

URINARY mtDNA INCREASED IN MICE WITH AKI Whitaker et al., 2015

URINARY mtDNA INCREASED IN MICE WITH AKI

Sham vs. I/R AUC = 0.88 p= URINARY mtDNA INCREASED IN MICE WITH AKI

URINARY mtDNA INCREASED IN HUMANS WITH AKI

URINARY ATP SYNTHASE  (ATPS  ) IN HUMANS WITH ACUTE KIDNEY INJURY John Arthur

NGAL Naive IR 15 AB DFE FULL LENGTH ATP SYNTHASE β (adjusted to total protein) CLEAVED ATP SYNTHASE β (adjusted to total protein) NGAL (adjusted to total protein) 24 hours after sham or I/R SERUM CREATININE (mg/dL) BUN (mg/dL) 24 hours after sham or I/R Naive Sham IR 5 IR 10 IR MW C Sham IR 5 min IR 10 min IR 15 min Sham IR 5 min IR 10 min IR 15 min IR 10 min IR 5 min Sham Naive Sham IR 5 IR 10 IR 15 min IR 10 min IR 5 min Sham Naive IR 15 min IR 10 min IR 5 min Sham Naive URINARY ATPS  INCREASED IN MICE WITH AKI Whitaker et al., 2015

B Sham IR 5 min IR 10 min ATP Synthase β GAPDH ATP Synthase β GAPDH Sham IR 15 min A 24 hours after sham or I/R RENAL ATP SYNTHASE β (Normalized to GAPDH) Sham IR 5 min IR 10 min IR 15 min RENAL ATPS  IS DECREASED IN MICE WITH AKI

RENAL MITOCHONDRIAL FUNCTION IS DECREASED IN MICE WITH AKI

A 50 kDa 25 kDa NGAL ATPSβ SERUM ALT (U/L) SERUM CREATININE (mg/dL) B C 5 months after initial treatment D Control diet NASH diet IR 15 min NASH diet Control diet NASH diet URINARY FULL LENGTH ATPS  IS NOT INCREASED IN MICE WITH HEPATIC INJURY Whitaker et al., 2015

1.5d AFTER CARDIAC SURGERY AB DC AKI 50 kDa No AKI 25 kDa FULL LENGTH ATP SYNTHASE β (adjusted to total protein) SERUM CREATININE (mg/dL) CLEAVED ATP SYNTHASE β (adjusted to total protein) No AKI AKI No AKI AKI No AKI AKI URINARY FULL LENGTH ATPS  IS INCREASED IN HUMANS WITH AKI Whitaker et al., 2015

CONCLUSION These proof of principle studies provide evidence that ATPS  and mtDNA may be novel and sensitive urinary biomarkers of renal mitochondrial dysfunction.

ACKNOWLEDGEMENTS Ryan Whitaker Jay Stallons, PhD Craig Beeson, PhD John Arthur, MD, PhD Sherine Chan, PhD Jason Funk, PhD Midhun Korrapati, PhD Jenny Rahn, PhD Funding NIH T32 HL NIH T32 ES NIH GM NIH ES VA Merit Award