FABP5 suppression impairs the adaptive response to acute kidney injury

Technical Summary: FABP5 Suppression Impairs the Adaptive Response to Acute Kidney Injury

Problem Statement
Acute kidney injury (AKI) is a critical clinical condition associated with high mortality and accelerated progression to chronic kidney disease (CKD). Current therapeutic strategies are largely supportive, reflecting an incomplete understanding of the molecular mechanisms governing tubular cell injury and recovery. While metabolic dysfunction, specifically the suppression of mitochondrial fatty acid oxidation (FAO) and a shift to ineffective glycolysis, is recognized as a driver of AKI severity, the role of intracellular fatty acid trafficking in limiting mitochondrial oxidation during acute stress remains poorly defined. Fatty acid binding proteins (FABPs) act as cytosolic chaperones for lipid transport, yet the specific contribution of FABP5 to renal resilience and metabolic adaptation during AKI had not been addressed.

Methodology
The study employed an integrated in vitro and in vivo approach to investigate the role of FABP5 in renal tubular metabolism and AKI pathophysiology.

• In Vivo Models: The researchers utilized a folic acid-induced AKI (FA-AKI) model in C57BL/6 wild-type (WT) mice and FABP5-deficient (FABP5-/-) mice. Additionally, WT mice were treated with the specific FABP5 inhibitor SBFI-26 prior to AKI induction to assess the effects of acute pharmacological suppression. A subset of mice was analyzed at 15 days post-injury to evaluate the transition to chronic kidney disease (CKD).
• In Vitro Models: Murine proximal tubular cells (MCT), human HK-2 cells, and primary proximal tubular epithelial cells (PTEC) were used. Cells were subjected to serum starvation to simulate nutrient stress and treated with SBFI-26.
• Analytical Techniques:
  • Metabolic Flux & Imaging: Fluorescent pulse-chase assays using Red C12 tracked fatty acid trafficking from lipid droplets (LDs) to mitochondria. Seahorse XF analysis measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR).
  • Molecular Profiling: Transcriptomic analysis (RNA-seq) was performed on kidney tissues to identify differentially expressed genes (DEGs) and pathway enrichment (GSEA, Metascape). RT-qPCR and Western blotting validated gene and protein expression.
  • Functional Assays: Cell viability (CCK8, Annexin V/7-AAD), proliferation (BrdU), oxidative stress (MitoSOX, TBARS), and mitochondrial membrane potential (TMRM) were assessed. Immunohistochemistry and immunofluorescence evaluated immune cell infiltration, vascular markers, and fibrosis.

Key Contributions and Results

1. FABP5 Downregulation in AKI: The study established that FABP5 expression is rapidly and significantly downregulated in the kidney during nephrotoxic AKI (FA-AKI) in both mice and human tissue contexts. FABP5 is expressed in renal tubules, endothelial cells, and pericytes.

2. Disruption of Lipid Trafficking and Energy Metabolism:

   • In cultured tubular cells, FABP5 inhibition (via SBFI-26) or genetic deficiency disrupted the delivery of fatty acids from lipid droplets to mitochondria.
   • This blockade led to the depletion of lipid droplets without a corresponding increase in mitochondrial fatty acid oxidation. Instead, fatty acids were extruded from cells, suggesting a compensatory mechanism to prevent cytosolic lipotoxicity.
   • Consequently, FABP5 suppression caused rapid ATP depletion, mitochondrial membrane depolarization, and a significant increase in mitochondrial and cytoplasmic reactive oxygen species (ROS).
   • While AMPK was activated and transcriptional programs for FAO (e.g., Ppara, Ppargc1a) were upregulated, these compensatory mechanisms failed to restore energy homeostasis or prevent cell death. Glycolytic enzymes did not show overt compensatory upregulation to rescue the energy deficit.

3. Exacerbation of AKI Severity:

   • Both FABP5 deficiency and pharmacological inhibition in mice resulted in significantly worse AKI outcomes compared to WT controls, evidenced by elevated plasma urea and creatinine, increased tubular injury markers (Ngal), and higher rates of tubular cell death (TUNEL positive).
   • Contrary to the expectation that reduced inflammation might be protective, FABP5 suppression led to a "maladaptive" phenotype characterized by severe tissue injury despite reduced immune cell infiltration (neutrophils, macrophages, lymphocytes) and lower chemokine expression. This suggests that FABP5 is required for the adaptive inflammatory response necessary for tissue repair.

4. Transcriptomic Reprogramming:

   • Transcriptomic analysis revealed that FABP5 deficiency in the context of AKI led to a distinct gene expression profile compared to WT AKI.
   • Pathways related to lipid metabolism, mitochondrial biogenesis, and tubular homeostasis were further downregulated in FABP5-deficient kidneys.
   • Conversely, stress responses, including the Unfolded Protein Response (UPR) and oxidative stress pathways, were more pronounced.
   • The data indicated a failure to mount protective metabolic and reparative programs, with a specific impairment in FAO-related genes and mitochondrial DNA copy number maintenance.

5. Vascular and Pericyte Involvement:

   • FABP5 expression was noted in pericytes and endothelial cells. FABP5 deficiency was associated with reduced Vegfa expression and prevented the upregulation of VCAM1, potentially limiting angiogenic and immune recruitment responses.
   • In pericyte-like cells, FABP5 inhibition promoted a transition to a myofibroblast phenotype (increased α-SMA), linking FABP5 loss to potential fibrotic activation via FAO suppression.

6. AKI-to-CKD Transition:

   • At 15 days post-AKI, while functional recovery (urea/creatinine) and histological fibrosis were similar between WT and FABP5-deficient mice, the latter group showed persistent metabolic adaptations (increased glycolytic gene expression) and sustained upregulation of certain fibrogenic mediators. This suggests FABP5 primarily modulates the acute phase of injury resilience rather than the long-term repair process itself, though it may influence CKD progression indirectly.

Significance and Claims
The paper identifies FABP5 as a critical "metabolic gatekeeper" that links fatty acid trafficking to mitochondrial energy production and tissue resilience during AKI. The authors claim that the loss of FABP5 is a maladaptive mechanism that exacerbates AKI severity by impairing lipid metabolization and mitochondrial function, leading to energy failure and oxidative stress.

The study concludes that preserving FABP5 activity may confer resilience to AKI. Conversely, the authors highlight a potential clinical risk: FABP5 inhibitors currently under development for malignancy or pain management could inadvertently decrease kidney resilience and increase susceptibility to AKI or the nephrotoxicity of chemotherapeutic agents. The findings suggest that future clinical trials of FABP5 inhibitors should consider monitoring renal function and resilience as a safety parameter. The paper does not propose new therapeutic interventions to increase FABP5 but emphasizes the need to account for its protective role in the context of existing drug development pipelines.