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Abstract: PO0509

Nephrotoxicity Assessment with Human Kidney Tubuloids Using Spherical Nucleic Acid-Based mRNA Nanoflares

Session Information

Category: Bioengineering

  • 300 Bioengineering

Authors

  • Wiraja, Christian, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
  • Mori, Yutaro, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
  • Ichimura, Takaharu, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
  • Hwang, Jangsun, Nanyang Technological University, Singapore, Singapore, Singapore
  • Xu, Chenjie, City University of Hong Kong, Kowloon, Hong Kong
  • Bonventre, Joseph V., Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
Background

Drug-induced nephrotoxicity represents an important cause of acute kidney injury with associated patient morbidity and mortality and is often responsible for termination of drug development, after extensive resource allocation. Current platforms for testing nephrotoxicity are limited and require disruptive end-point molecular assays. We have paired a 3D human kidney tubuloid system which phenocopies kidney proximal tubules with spherical nanoflare (NF) mRNA nanosensors to achieve facile, real-time assessment of drug nephrotoxicity.

Methods

Primary human tubuloids were generated from tubule cells isolated from patients’ kidney tissue and cultured in 3D matrigel settings using serum-free media. NF nanosensors targeting kidney injury molecule-1 (KIM-1) mRNA and GAPDH mRNA were assembled and used to label tubuloids through overnight incubation. KIM-1 NF signal on tubuloids were monitored over time following drug exposure through fluorescence microscopy imaging. Quantitated NF signals were compared with quantitative polymerase chain reaction (qPCR) to verify the accuracy of NF signals. To demonstrate utility, the proposed NF-tubuloid platform was applied to screen the nephrotoxicity of 10 representative anti-cancer drugs.

Results

Specificity of KIM-1 NF sensors were demonstrated by studying a cell line constitutively expressing KIM-1 mRNA and adenovirus-transfected tubuloids. NF ability to thoroughly label kidney tubuloids and report tubular injury were evaluated by applying several concentrations of aristolochic acid and cisplatin, two well-known nephrotoxicants. Compared against its respective mRNA expression from qPCR, quantitated NF fluorescence showed a positive linear correlation with R2=0.931, indicating its accurate representation of tubuloid status. Finally, the platform was used to facilitate nephrotoxicity screening of anti-cancer drugs, with significant tubuloid KIM-1 upregulation induced by 5-fluorouracil and paclitaxel.

Conclusion

We demonstrated the utilization of NF nanosensors to monitor injury on human kidney tubuloids. This platform enables facile and personalized nephrotoxicity assessment in vitro.

Funding

  • NIDDK Support