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Abstract: TH-PO331

Differentiated Tubuloids to Model Human Distal Nephron (Patho) Physiology

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic


  • Pou Casellas, Carla, Universitair Medisch Centrum Utrecht, Utrecht, Utrecht, Netherlands
  • Yousef Yengej, Fjodor, Universitair Medisch Centrum Utrecht, Utrecht, Utrecht, Netherlands
  • Ammerlaan, Carola, Universitair Medisch Centrum Utrecht, Utrecht, Utrecht, Netherlands
  • Olde Hanhof, Charlotte, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Dilmen, Emre, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Hoenderop, Joost, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Rookmaaker, Maarten B., Universitair Medisch Centrum Utrecht, Utrecht, Utrecht, Netherlands
  • Clevers, Hans, Hubrecht Institute, Utrecht, Utrecht, Netherlands
  • Verhaar, Marianne C., Universitair Medisch Centrum Utrecht, Utrecht, Utrecht, Netherlands

The distal nephron segments are essential for electrolyte, acid-base and volume homeostasis. Tubuloid culture allows ample expansion (exp) of primary human renal epithelium from urine or tissue. Here we differentiate tubuloids into thick ascending limb of Henle (TAL) and principal cells (PC), characterize these at mRNA, protein and functional levels, and establish an in vitro human lithium (Li+) tubulopathy model.


Tubuloids were grown from kidney tissue of 4 donors in exp medium. After differentiation (diff) for 7 days, tubuloids were studied by qPCR, single cell RNA sequencing (scSeq) and immunofluorescence (IF)/-histochemistry (IHC). ScSeq data was compared with human kidney tissue. Electrolyte transport was tested using the FluxOR™ II assay. Diff tubuloids were exposed to 10 mM Li+ or Na+ (control) and analyzed by the same techniques.


Diff increased transcription of NKCC2 in the TAL (1269-fold, P<0.05), NCC (39-fold, P<0.05) in the distal convoluted tubule (DCT), and ENaCα (8-fold, P<0.05) and AQP2 (569-fold, P=0.06) in PC (n=4). Tubuloids in exp consisted of proliferative progenitors, whereas diff produced TAL and PC, with some DCT cells (n>3.000 cells/sample). Diff reduced progenitor markers and upregulated clinically relevant genes (e.g. NKCC2, ROMK, ENaC, AQP2, Na/K-ATPase, HNF1β, CAII) to levels reflecting tissue counterparts. IF/IHC confirmed polarized NKCC2, AQP2 and AQP3 protein expression. Luminal NKCC2 demonstrated furosemide-inhibitable Tl+ (replacing K+) uptake (n=3). Apical Li+ treatment suppressed AQP2 and upregulated the intercalated cell marker Pendrin, in contrast to Na+ or basolateral Li+. Li+ also upregulated proliferation, pro-inflammatory and tumor-associated genes. In addition, Li+ effects observed only in animal studies were seen, including downregulation of NKCC2, which was confirmed by IF and functional assays.


Tubuloid diff produces TAL and PC that resemble in vivo counterparts. TAL cells demonstrated electrolyte reabsorption, their main function. Treatment with Li+ caused changes in line with diabetes insipidus, tubulointerstitial nephritis and tumors seen in vivo. Taken together, differentiated tubuloids enable modeling of the human distal nephron in health and disease.


  • Government Support – Non-U.S.