Mitophagy regulates mitochondrial number following pharmacological induction of mitochondrial biogenesis in renal proximal tubule cells.

Background: Mitochondrial biogenesis (MB) induction through the activation of the 5-Hydroxytriptamine (5-HT) 1F receptor (HTR1F) is a promising mechanism for the treatment of diseases characterized by mitochondrial dysfunction, such as acute kidney injury (AKI). While several studies report pharmacological activation of MB in the proximal tubule, it is unclear how the proximal tubule regulates itself once the pharmacological activation is removed. Mitophagy is the process of selective mitochondria degradation. We hypothesize that mitophagy decreases mitochondrial number after pharmacological stimulation and restore mitochondrial homeostasis. Methods: Renal proximal tubules were treated at time 0hr with LY344864 or vehicle for 24 h and then removed. LY344864, a selective HTR1F agonist, induces MB in renal proximal tubules as previously reported (Gibbs et al., Am J Physiol Renal Physiol, 2018, 314(2), F260-F268). Vehicle and pharmacological reagents were added at the 24 h time point. Electron microscopy was used to assess mitochondrial morphology, number, and autolysosomes. Seahorse Bioscience XF-96 extracellular flux analyzer was used to measure maximal mitochondrial oxygen consumption rates (FCCP-OCR), a functional marker of MB. Results: LY344864 treatment increased FCCP-OCR, phosphorylation of protein kinase B (AKT), peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α), and mitochondrial number after 24 h. These endpoints decreased to baseline 24 h after LY344864 removal. Treatment with ROC-325, an autophagy inhibitor, increased Sequestosome-1 (SQSTM1/P62) and microtubule-associated protein-1 light chain 3 (LC3B) after 24 h of treatment. Also, ROC-325 treatment sustained the elevated mitochondrial number after LY344864 pre-treatment and removal. Conclusion: These data revealed that inhibition of autophagy extends elevated mitochondrial number and function by preventing the lysosomal degradation of mitochondria after the removal of LY344864.

Lasmiditan restores mitochondrial quality control mechanisms and accelerates renal recovery after ischemia-reperfusion injury.

BACKGROUND: Mitochondrial dysfunction is a well-established result of acute kidney injury (AKI). Previously, we identified that 5-hydroxytryptamine 1F (5-HT1F) receptor agonism with lasmiditan induces mitochondrial biogenesis (MB) and improves renal vasculature and function in an AKI mouse model. We hypothesize that lasmiditan also modulates mitochondrial dynamics and mitophagy in a mouse model of AKI. METHODS: Male mice were subjected to renal ischemia/reperfusion (I/R) and treated daily with lasmiditan (0.3 mg/kg) or vehicle beginning 24 h after injury for 3 or 6d. Serum creatinine was measured to estimate glomerular filtration. Electron microscopy was used to assess mitochondrial morphology and mitophagy. Mitochondrial-related protein were confirmed with immunoblotting. Mitochondrial function was assessed with ATP measurements. RESULTS: Lasmiditan treatment improved mitochondrial and kidney recovery as early as 3d post-AKI, as evidenced by increased ATP, and decreased serum creatinine, respectively. Electron micrographs of renal cortices revealed that lasmiditan also decreased mitochondrial damage and increased mitochondrial area and size by 6d after I/R injury. Additionally, lasmiditan treatment increased mitolysosomes by 3d, indicating induction of mitophagy. Phosphorylation of mitophagy-related proteins were also increased in the renal cortices of lasmiditan-treated AKI mice 3d after I/R injury, whereas fusion-related proteins were increased at 6d after I/R injury. CONCLUSION: These data reveal that lasmiditan accelerates renal recovery, restores normal mitochondrial membrane and cristae morphology, decreases excessive mitochondrial fission, and accelerates mitophagy post-AKI in a time-dependent manner, establishing mitochondrial function and recovery from AKI.

Lasmiditan promotes recovery from acute kidney injury through induction of mitochondrial biogenesis.

Acute kidney injury (AKI) involves rapid loss of renal function and occurs in 8-16% of hospitalized patients. AKI can be induced by drugs, sepsis, and ischemia-reperfusion (I/R). Hallmarks of AKI include mitochondrial and microvasculature dysfunction as well as renal tubular injury. There is currently no available therapeutic for AKI. Previously, our group identified that serotonin (5-HT)1F receptor agonism with lasmiditan accelerated endothelial cell recovery and induced mitochondrial biogenesis (MB) in vitro. We hypothesized that lasmiditan, a Federal Drug Administration-approved drug, would induce MB and improve microvascular and renal function in a mouse model of AKI. Male mice were subjected to renal I/R and treated with lasmiditan (0.3 mg/kg) or vehicle beginning 24 h after injury and then daily until euthanasia at 6 or 12 days. Serum creatinine was measured to estimate glomerular filtration rate. The renal cortex was assessed for mitochondrial density, vascular permeability and integrity, tubular damage, and interstitial fibrosis. Lasmiditan increased mitochondrial number (1.4-fold) in renal cortices. At 6 days, serum creatinine decreased 41% in the I/R group and 72% with lasmiditan. At 6 or 12 days, kidney injury molecule-1 increased in the I/R group and decreased 50% with lasmiditan. At 12 days, interstitial fibrosis decreased with lasmiditan by 50% and collagen type 1 by 38%. Evan's blue dye leakage increased 2.5-fold in the I/R group and was restored with lasmiditan. The tight junction proteins zonula occludens-1, claudin-2, and claudin-5 decreased in the I/R group and recovered with lasmiditan. At 6 or 12 days, peroxisome proliferator-activated receptor-γ coactivator-1α and electron transport chain complexes increased only with lasmiditan. In conclusion, lasmiditan treatment beginning AKI induces MB, attenuated vascular and tubular injury, decreased interstitial fibrosis, and lowered serum creatinine. Given that lasmiditan is a Federal Drug Administration-approved drug, these preclinical data support repurposing lasmiditan as a therapeutic for AKI.NEW & NOTEWORTHY AKI pathology involves a rapid decline in kidney function and occurs in 8-16% of hospitalized patients. There is currently no therapeutic for AKI. AKI results in mitochondria dysfunction, microvasculature injury, and loss of renal tubular function. In an I/R-induced AKI mouse model, treatment with the FDA-approved 5-HT1F receptor-selective agonist lasmiditan induced mitochondrial biogenesis, improved vascular integrity, reduced fibrosis, and reduced proximal tubule damage. These data support repurposing lasmiditan for the treatment of AKI.