564 - Preclinical Imaging of Lysine Metabolism in Kidney Transplant Injury: Implications for Pediatric Allograft Health

Publication Number: 4552.564

Justin Adler, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; Alexander I. Zavriyev, University of Pennsylvania, Philadelphia, PA, United States; Molly M. Sheehan, University of Pennyslvania, Philadelphia, PA, United States; Erum A. Hartung, Children’s Hospital of Philadelphia, Philadelphia, PA, United States; Stephen Kadlecek, University of Pennsylvania, Philadelphia, PA, United States; Terence Gade, University of Pennsylvania, Philadelphia, PA, United States

Background: Acute cellular rejection and ischemia-reperfusion injury (IRI) are major causes of renal allograft failure, yet current diagnostic tools are invasive and nonspecific. Hyperpolarized (HP) MRI offers a real time non-invasive method for imaging of metabolic pathways that can help detect altered cellular metabolism following injury. Preliminary studies suggest that IRI leads to lysine accumulation, while acute cellular rejection increases tryptophan metabolites. However, in vivo metabolic signatures in the post-transplant kidney remain poorly characterized.

Objective: This study aims to characterize baseline lysine metabolism in healthy murine kidneys using hyperpolarized MRI, establishing foundational metabolic data to support future investigations of transplant rejection and ischemia-reperfusion injury.

Design/Methods: Experiments utilized hyperpolarized MRI to assess lysine metabolism in n=6 healthy C57BL/6 mice. Spectral data from NMR in vitro studies of lysine and its downstream metabolites, including crotonyl-, formyl-, methyl-, and butyryl-lysine, were analyzed for key chemical shifts. In vitro NMR spectroscopy of lysine was performed at pH's of 7, 8.5, and 10 to evaluate pH-dependence. Metabolite identification was validated using pyruvate reference standards and comparison with known chemical shifts in the literature.

Results: A rapid conversion of lysine to an unidentified downstream metabolite was observed on hyperpolarized imaging of healthy mice (Figure 1). NMR spectra showed no significant differences between lysine with different N-terminal lysine modifications, indicating that the spectral changes observed in vivo are not due to addition of a functional group to lysine (Figure 2). A single lysine peak (~183 ppm) demonstrated notable pH sensitivity between pH 8.5-10 in vitro, though this was not reproducible in vivo (Figure 3). The findings indicate complex lysine metabolism throughout the kidney that cannot be explained solely by pH or lysine functional group modification.

Conclusion(s): Hyperpolarized MRI can visualize lysine metabolism in vivo, revealing metabolic alterations of lysine that may hold the potential to serve as a clinical diagnostic biomarker of IRI after kidney transplantation. Future studies will examine lysine metabolism under varied pH states in mice that underwent left renal warm ischemia to compare metabolic differences in the injured kidney to the healthy contralateral kidney.

In vivo hyperpolarized MRI of [¹³C]-lysine metabolism in a healthy murine model
Overlaid dynamic spectra across the kidney reveal regional metabolic heterogeneity within the cortex, medulla, and perirenal tissue. This suggests altered lysine conversion pathways associated with microenvironmental differences.

Thermal equilibrium MR spectra of lysine derivatives under baseline conditions
Overlaid spectra of butyryl-, crotonyl-, formyl-, and methyl-lysine show closely aligned chemical shifts ( < 0.2 ppm difference) across the 170-205 ppm range, indicating minimal variation in carbonyl resonance despite N-terminal modifications. These findings suggest that microenvironmental chemistry at the N terminus alone cannot explain the spectral imaging differences that were observed in vivo.

NMR spectra of lysine acquired under controlled pH conditions (7.0, 8.5, and 10.0) at thermal equilibrium
Small but notable chemical-shift variations are observed in select carbonyl resonances, indicating modest pH-dependent alterations in the lysine microenvironment. These in vitro findings suggest that local physicochemical conditions can influence spectral profiles, though the magnitude of change does not fully account for the in vivo spectral results.