Doxorubicin Induces Subclinical and Therapeutically Reversible Mitochondrial Injury in Renal Tubular Cells Revealed by Ex Vivo Seahorse Assay and Structured Illumination Microscopy

Doxorubicin (DOX), a widely used anticancer agent, is known to cause acute heart failure through mitochondrial injury in cardiomyocytes. Recently, anthracycline exposure, including DOX, has also been reported to increase the risk of delayed-onset chronic kidney disease (CKD) in childhood cancer survivors, although the underlying mechanisms remain unclear. Conventional Seahorse assays for mitochondrial function have been limited to cultured cells, and mitochondrial morphology evaluation has required electron microscopy, which precludes wide-field quantitative analysis. In this study, we established a novel Seahorse assay method using freshly isolated renal tubules and applied a recently reported structured illumination microscopy (SIM) technique based on Masson’s trichrome-stained specimens to quantitatively evaluate mitochondrial morphology. We demonstrated that DOX induced subclinical and therapeutically reversible mitochondrial injury in renal tubular cells.

Eleven-week-old male C57BL/6J mice received intraperitoneal DOX (5 or 8 mg/kg) or vehicle once weekly for four weeks. One week after the final dose, the serum and kidneys were harvested. For the mitochondrial functional assay, the kidneys were minced and digested with collagenase IV. After one minute of sedimentation, supernatants were filtered, washed, and resuspended in Seahorse XF DMEM assay medium. Tubules were seeded onto Cell-Tak-precoated XF96 plates, equilibrated for 1 hour, and assayed with medium containing glucose, pyruvate, and glutamine. Oxygen consumption rates (OCR) were measured following sequential injections of oligomycin, FCCP, and a Rotenone/Antimycin A mixture, with values normalized to protein content. For mitochondrial morphological evaluation, the kidneys were fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with Masson’s trichrome. Images were acquired using SIM.

There was no significant difference in serum creatinine levels between the two groups (vehicle 0.55±0.06 vs. DOX 0.52±0.02 mg/dL, p > 0.05). Isolated tubules from the vehicle-treated mice demonstrated robust OCR increases by FCCP, whereas tubules from doxorubicin-treated mice exhibited reduced maximal OCR, indicating impaired mitochondrial respiratory capacity (Fig. 1a). SIM revealed that DOX induced mitochondrial fragmentation in renal tubular cells (Fig. 1b). This mitochondrial fragmentation was suppressed by the administration of nicotinamide mononucleotide (NMN), which has been previously shown to attenuate doxorubicin-induced cardiomyopathy.

Using newly established methods for functional and morphological evaluation of renal tubules, we demonstrated that doxorubicin induces mitochondrial injury in tubular cells. Furthermore, we showed that subclinical mitochondrial damage in renal tubules is reversible with treatment. Future studies will investigate its association with subsequent renal function decline and the potential of therapeutic interventions to prevent kidney dysfunction.

Figure 1(a). Ex vivo seahorse assay shows DOX-induced mitochondrial dysfunction. (b) SIM shows DOX-induced mitochondrial fragmentation.