Heterogeneous distribution of ion transporters in enteroids enriched in different intestinal epithelial cell types

Abstract

The small intestine which is characterized by different cell types lining specific sites along the crypt‐villus axis plays a critical role in water, electrolyte and mineral balance. Normally, net fluid absorption by the small intestine occurs mainly because ion‐dependent fluid absorption in the villus column is greater than ion‐driven fluid secretion in the crypt. To generate these site‐specific ion gradients, appropriate transporters must be strategically located in cells lining the crypt or villus regions. The multivalent ions calcium and iron also function as essential minerals, and deficiency in these minerals is highly prevalent worldwide. The crypt‐villus site of many electrolyte and mineral transporters has been localized by immunocytochemistry, but their expression in rare cell types like stem cells (ISC) is unknown. To test the hypothesis that representative ion and mineral transporters are distributed heterogeneously among the various intestinal cell types, we made enteroids enriched in ISC, enterocytes (ENT), goblet (GOB), and Paneth (PAN) cells. We then confirmed by cell‐type specific biomarkers that ISC, ENT, GOB and PAN enteroids were enriched in stem cells (~90% of all cells per organoid), enterocytes (90%), goblet (80%) and Paneth (65%) cells, respectively. ENT and GOB enteroids had the highest mRNA levels of the chloride‐anion exchanger DRA and sodium‐hydrogen exchanger NHE3, while the sodium‐bicarbonate cotransporter NBCe1 was expressed primarily in ENT enteroids. Conversely, the chloride channel CFTR and sodium‐potassium‐chloride cotransporter NKCC1 mRNA were mainly expressed in ISC and PAN enteroids. Findings confirm previous immunocytochemical localization of NBCe1 and DRA in the villus region lined by enterocytes, with CFTR and NKCC1 mainly in intestinal crypts containing Paneth and stem cells. Basolateral NKCC1 staining was observed in ISC and PAN, but not in ENT and GOB, enteroids. The membrane location and expression of NKCC1 in crypt‐dwelling cells reflect its prominent role in intestinal fluid secretion. Members of the intestinal epithelial transport system for iron (DMT1, FTL, IREG1 and HEPH) showed homogeneous distribution in all cell types. The calcium transporters TRPV6 and PMCA1 were expressed least in ENT and GOB, respectively. Our findings depict a mostly heterogeneous distribution of ion transporters in different cell types, reflecting their likely distribution along the crypt villus axis in vivo, suggesting that appropriate expression in different cell types may be essential for ion and mineral homeostasis.Support or Funding InformationSupported by NSF 1456673, 1754783 (RF) and NIH R01AT010243 (RF, NG)