Human primary renal tubuloids as tools for calcineurin inhibitor nephrotoxicity assessment
Bingxuan Zheng1, Puxun Tian1, Meng Dou1, Ge Deng1, Chenguang Ding1, Yuting Shi1, Yang Gao1, Xingzhe Zhang1.
1Department of Kidney Transplantation, Hospital of Nephropathy, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, People's Republic of China
Background: Although calcineurin inhibitor(CNIs) is the most widely used immunosuppressive agent in kidney transplantation, its nephrotoxicity affects the long-term survival of kidney transplant recipients. To assess the individualized nephrotoxicity of CNIs, we performed the assessment using a method of generating renal tubuloids from the renal tissue of patients.
Methods: Primary human kidney tissue derived from biopsy specimens of living donor kidneys. Mince the tissue into pieces of~1 mm3 size using scalpels and digest pieces of tissue into cell pellets using collagenase. The cell pellets were then transferred into media containing matrigel, B27 supplement, R-spondin,epidermal growth factor, fibroblast growth factor-10, N-acetylcysteine, A83-01and Y-27632. Tubuloids were cultured for several days, re-seeded in 96-well plates, and treated with CNIs(tacrolimus) at doses of 0, 20, 40,or 60μM for 24 hours. Morphological changes, Cell viability and KIM-1 expression were evaluated.
Results: Generation of human tubuloid cultures is identified by immunohistochemical staining for markers expressed in the tubular epithelium, such as paired box 8 protein (PAX8). The 3D structure of the kidney tubuloids and cell viability decreased in dose-dependent manners after treatment with CNIs(tacrolimus). Treatment with CNIs(tacrolimus) increased KIM-1 expression in a dose-dependent manner.
Conclusions: We generated renal tubuloids from patient kidney tissue. This method can evaluate the nephrotoxicity of CNIs in renal transplant patients and provide a possible new way for the personalized medicine of immunosuppressive agent.
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