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US-20260125652-A1 - METHOD FOR EVALUATING NEPHROTOXICITY

US20260125652A1US 20260125652 A1US20260125652 A1US 20260125652A1US-20260125652-A1

Abstract

The present disclosure provides, as an improved in vitro evaluation system for evaluating nephrotoxicity of a substance such as a compound, a method for evaluating nephrotoxicity of a test substance comprising a step of contacting a test substance with a kidney organoid, wherein the higher cytotoxicity of test substance as compared with a control substance indicates the test substance is likely to have nephrotoxicity.

Inventors

  • Makoto Ikeya
  • Daisuke Kamiya
  • Teppei AKAHOSHI

Assignees

  • KYOTO UNIVERSITY
  • TAKEDA PHARMACEUTICAL COMPANY LIMITED

Dates

Publication Date
20260507
Application Date
20230928
Priority Date
20220929

Claims (14)

  1. 1 . A method for evaluating nephrotoxicity of a test substance comprising: a step of contacting a test substance with a kidney organoid, wherein the higher cytotoxicity of test substance as compared with a control substance indicates the test substance is likely to have nephrotoxicity.
  2. 2 . A method for screening a test substance likely to have nephrotoxicity comprising the steps of: (1) contacting a test substance with a kidney organoid; and (2) selecting a test substance showing higher cytotoxicity as compared with a control substance.
  3. 3 . The method according to claim 1 , wherein the kidney organoid is derived from a human induced pluripotent stem cell.
  4. 4 . The method according to claim 1 , wherein the kidney organoid is enriched in glomerular epithelial cell-like cells.
  5. 5 . The method according to claim 4 , wherein 30 to 80% of cells contained in the kidney organoid are glomerular epithelial cell-like cells.
  6. 6 . The method according to claim 4 further comprising: a step (a) of culturing a pluripotent stem cell in the presence of a 9.0 to 10.5 μM GSK3β inhibitor to obtain the kidney organoid enriched in glomerular epithelial cell-like cells.
  7. 7 . The method according to claim 1 , wherein the kidney organoid is enriched in renal proximal tubule epithelial cell-like cells.
  8. 8 . The method according to claim 7 , wherein 5 to 30% of cells contained in the kidney organoid are renal proximal tubule epithelial cell-like cells.
  9. 9 . The method according to claim 8 further comprising: a step (b) of culturing a pluripotent stem cell in the presence of a 5.5 to 8.5 μM GSK3β inhibitor to obtain the kidney organoid enriched in renal proximal tubule epithelial cell-like cells.
  10. 10 . A method for determining a site in the kidney to which a test substance shows toxicity comprising the steps of: (A) contacting a test substance with a kidney organoid enriched in glomerular epithelial cell-like cells; (B) contacting the test substance with a kidney organoid enriched in renal proximal tubule epithelial cell-like cells; and (C) comparing cytotoxicity obtained in the step (A) and cytotoxicity obtained in the step (B) to determine that a site showing toxicity is the glomerulus when the cytotoxicity obtained in the step (A) is higher, and that a site showing toxicity is the renal proximal tubule when the cytotoxicity obtained in the step (B) is higher.
  11. 11 . A method for screening nephrotoxicity of a drug candidate compound comprising the steps of: (1) contacting a test substance with a kidney organoid enriched in glomerular epithelial cell-like cells; and (2) selecting, as the drug candidate compound, a test substance showing no cytotoxicity or lower cytotoxicity as compared with a control substance.
  12. 12 . A kit for evaluating nephrotoxicity of a test substance comprising a kidney organoid.
  13. 13 . A method for producing a kidney organoid enriched in glomerular epithelial cell-like cells comprising: a step (a) of culturing a pluripotent stem cell in the presence of a 9.0 to 10.5 μM GSK3β inhibitor.
  14. 14 . A method for producing a kidney organoid enriched in renal proximal tubule epithelial cell-like cells comprising: a step (b) of culturing a pluripotent stem cell in the presence of a 5.5 to 8.5 μM GSK3β inhibitor.

Description

This application is based upon and claims the benefit of priority from Japanese patent application No. 2022-156017, filed on Sep. 29, 2022, the disclosure of which is incorporated herein in its entirety by reference. TECHNICAL FIELD The present disclosure relates to a method for evaluating nephrotoxicity of a test substance. More particularly, it relates to an in vitro nephrotoxicity evaluation method using a kidney organoid. BACKGROUND OF INVENTION An in vitro evaluation system using a renal proximal tubule epithelial cell line (RPTEC) has been proposed for evaluating nephrotoxicity of a substance such as a compound (see, for example, Non Patent Literature 1). In the in vitro evaluation system using RPTEC, it is difficult to evaluate toxicity to kidney constituent cells (such as a glomerular epithelial cells) except for those of the renal proximal tubule, and therefore, in some cases, a result different from that of a toxic reaction in the living kidney including other cells in addition to those of the renal proximal tubule is obtained. In relation to the present disclosure, Non Patent Literatures 2 to 4 disclose techniques for inducing a kidney organoid (KiO) from stem cells. CITATION LIST Non Patent Literature Non Patent Literature 1:“Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules”, Marianne K. Vormann, et. al., The AAPS Journal, 2018, 20, Art. No. 90Non Patent Literature 2:“Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis”, Minoru Takasato et. al., Nature, 2015, Volume 526, Page 564-568Non Patent Literature 3: “Generation of kidney organoids from human pluripotent stem cells”, Minoru Takasato et. al., Nature Protocols, 2016, Volume 11, Page 1681-1692Non Patent Literature 4:“Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk”, Sara E. Howden et. al., Cell Stem Cell, 2021, Volume 28, Issue 4, 1, Pages 671-684 SUMMARY OF INVENTION Technical Problem A main object of the present disclosure is to provide an improved in vitro evaluation system for evaluating nephrotoxicity of a substance such as a compound. Solution to Problem In order to solve the problem, the present disclosure provides the following [1] to [32]: [1] A method for evaluating nephrotoxicity of a test substance comprising a step of contacting a test substance with a kidney organoid, wherein the higher cytotoxicity of test substance as compared with a control substance indicates the test substance is likely to have nephrotoxicity.[2] A method for screening a test substance likely to have nephrotoxicity comprising the steps of: (1) contacting a test substance with a kidney organoid; and (2) selecting a test substance showing higher cytotoxicity as compared with a control substance.[3] A method for screening a test substance likely to have no nephrotoxicity or likely to have low nephrotoxicity comprising the steps of (1) contacting a test substance with a kidney organoid; and (2) selecting a test substance showing no cytotoxicity or lower cytotoxicity as compared with a control substance.[4] The method according to any one of [1] to [3], wherein the kidney organoid is derived from a human induced pluripotent stem cell.[5] The method according to any one of [1] to [4], wherein the kidney organoid is enriched in glomerular epithelial cell-like cells.[6] The method according to [5], wherein 30 to 80% of cells contained in the kidney organoid are glomerular epithelial cell-like cells.[7] The method according to [5] or [6] further comprising a step (a) of culturing a pluripotent stem cell in the presence of a 9.0 to 10.5 μM GSK3β inhibitor to obtain the kidney organoid enriched in glomerular epithelial cell-like cells.[7a] The method according to [7] comprising the step (a) before the step (1).[8] The method according to [7] or [7a], wherein the GSK3β inhibitor is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl) -5-(4-methyl-1H-imidazol-2-yl)2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile).[9] The method according to any one of [1] to [4], wherein the kidney organoid is enriched in renal proximal tubule epithelial cell-like cells.[10] The method according to [9], wherein 5 to 30% of cells contained in the kidney organoid are renal proximal tubule epithelial cell-like cells.[11] The method according to [9] or [10] further comprising a step (b) of culturing a pluripotent stem cell in the presence of a 5.5 to 8.5 μM GSK3β inhibitor to obtain the kidney organoid enriched in renal proximal tubule epithelial cell-like cells.[12] The method according to [11], wherein the GSK3β inhibitor is CHIR99021(6 -[[2-[[4-(2,4-dichlorophenyl) -5-(4-methyl-1H-imidazol-2-yl)2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile).[13 ] A method for predicting a site in the kidney to which a test substance shows toxicity comprising the steps of: (A) contacting a test substance with a kidney organoid enriched in glomerular epithelial cell-like cells;