PROJECT SUMMARY Mitochondrial dysfunction is recognized as a key early driver for disease progression in diabetic nephropathy (DN), among other chronic diseases, yet there exist very few tools for non-invasive assessment of mitochondrial status. Given that DN is an important source of diabetes-related morbidity and mortality (through its impact on cardiovascular outcomes), the ability to better understand and potentially even predict the progression of patients with diabetes to DN could have a large impact on clinical management of diabetes. The purpose of this R21 proposal (“Catalytic Tool and Technology Development in Kidney, Urologic, and Hematologic Diseases”) is to develop a new imaging probe with mitochondria-specific reactivity for hyperpolarized (HP) 13C MRI, [1,3- 13C2]acetoacetate (AcAc), providing non-invasive readout of mitochondrial metabolic status. This mitochondria- targeted ketone body probe has several favorable features with respect to HP 13C technology including long T1 signal lifetime, low toxicity, and rapid cellular uptake, but also has major associated challenges which have hampered development. To address these, we propose several technical innovations including: 1) improved chemical synthesis with increased purity and stability, 2) first-ever hyperpolarization on the state-of-the-art human-scale GE SPINlab 5T instrument, 3) improved formulation for dynamic nuclear polarization (DNP) with potentially higher polarization levels and shorter buildup times, and 4) highly efficient data acquisition with a novel pulse sequence employing multi-band spectral-spatial radiofrequency (SSRF) pulse excitation on a new MR/PET preclinical imaging platform (MR Solutions). The cumulative impact of these innovations is expected to provide a sensitivity gain of more than an order of magnitude over recent limited “proof of concept” work, propelling HP [1,3-13C2]AcAc into a powerful new imaging-based tool for assessing mitochondrial health. The tool will be evaluated in a well-characterized rodent model of DN, to directly test the hypothesis that HP [1- 13C]AcAc can detect early DN prior to clinically detectable manifestations. Lastly, consistent with the FOA, we are committed to a clear plan for sharing the newly developed “Catalytic Tool” with potential users (>30 labs with the necessary equipment for HP 13C MRI), including sharing all protocols for chemical synthesis and formulation, as well as sharing all pulse sequence source code, novel SSRF pulse waveforms, and data from the study online.

Public Health Relevance Statement (Project Narrative) The goal of this project is to develop new medical imaging-based markers for directly assessing mitochondrial function, which is affected early in the course of numerous chronic diseases, especially diabetic nephropathy (DN). Because of the limitations of existing clinical markers of DN, the proposed developments have the potential to make a significant impact on the clinical management of patients with diabetes and ultimately other chronic diseases.