Identification of intestinal ion transport defects in microvillus inclusion disease

Abstract

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl Ϫ ) and sodium (Na ϩ ) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na ϩ ), CFTR (Cl Ϫ ), and SLC26A3 (DRA) (Cl Ϫ /HCO3 Ϫ ) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl Ϫ and Na ϩ stool loss in MVID diarrhea.CFTR; brush border; MVI; Myo5b; NHE3; MVID MICROVILLUS INCLUSION DISEASE (MVID) is a rare but life-threatening disease that affects newborns and children and leads to rapid death from severe secretory diarrhea. MVID clusters in the Middle East and Navajo Indian populations in the US and is associated with consanguinity (41,42,47,51,61). Stool volumes are greater than 125 ml·kg Ϫ1 ·day Ϫ1 with elevated levels of chloride (Cl Ϫ ) and sodium (Na

Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations

“…The same phenomenon of selectively affected cargo transport/traffic has been observed in certain cases of MYO5B/STX3-linked MVID (26). This aspect needs further detailed investigation, since other MVID patients showed somehow different localization patterns of apical markers (41,42).…”

125Iodide as a surrogate tracer for epithelial chloride transport by the mouse large intestine in vitro

“…This price gap has greatly expanded, and 125 I − is now >1/500th the cost of 36 Cl − . More recently, 125 I − has been used to measure Cl − –HCO 3 − exchange activity and Cl − absorption by cultured Caco‐2 cells (Gujral et al., ; Kravtsov et al., ; Kumar et al., ) and everted gut sacs of mouse large intestine (Saksena et al., ), respectively. However, neither reported a direct comparison of I − with Cl − and, although the latter study was the first instance in which 125 I − was used to examine Cl − transport by epithelia, it presented only a single unidirectional flux.…”

AP2 α modulates cystic fibrosis transmembrane conductance regulator function in the human intestine

“…T84 cells grown on snapwell filters possessing a TEER above 1000 ohms/cm 2 were mounted on the Ussing chamber and Cl − current was measured as described before [22]. …”