Generation and characterization of sodium/myo-inositol cotransporter (SMIT) knockout mice

Abstract

Cells that experience prolonged hypertonic environment undergo various adaptations. One of these changes is the accumulation of small organic osmolytes which do not perturb the behavior of key metabolic enzymes. Myo-inositol is one of these osmolytes. Mammalian cells accumulate myo-inositol through an active transporter, the Sodium/Myo-inositol cotransporter (SMIT), which is also called SLC5A3 for solute carrier family 5, number 3 protein. Previous observations suggest that SMIT may be involved in osmoregulation, renal function, production of phosphatidylinositol and neurological disorders. Although inhibitors against SMIT can be used to understand its function, the in vivo efficacy and specificity of these drugs are not clear. Therefore, we generated SMIT knockout mice to delineate the role of SMIT under both normal and disease conditions. There is no apparent abnormality with the heterozygous SMIT knockout mice. However, breeding between the heterozygous mice (37 litters) generated pups with the following genotypes: 22 homozygous(-/-), 161 heterozygous (+/-) and 90 wildtype (+/+). This indicates that only 25% of (-/-) survived into adulthood. In order to determine whether the mice die in utero or after birth, we sacrificed 8 pregnant heterozygous females, which were mated with heterozygous males, at embryonic day 18.5 (E18.5). The average litter size was 9.4 and the genotypic ratio followed mandellian ratio (16 -/- : 41 +/- : 18 +/+). This indicates all homozygous SMIT knockout can survive up to E18.5 but lethality occurred after birth. Northern blot hybridization showed that the 12kb transcript of SMIT mRNA is absent in the survival adult SMIT knockout mice. HPLC analysis showed that there was approximately 70% reduction in tissue myo-inositol and changes in osmolytes profile was also observed in the brain and kidney of homozygous knockout mice. However, no significant difference was observed in the volume of 24 hr water intake and urination.