Megalin is essential for renal proximal tubule reabsorption and accumulation of transcobalamin-B12

Megalin has previously been shown to bind and mediate endocytosis of transcobalamin (TC)-B12. However, the physiological significance of this has not been established, and other TC-B12binding proteins have been suggested to mediate renal uptake of this vitamin complex. The present study demonstrates by the use of megalin-deficient mice that megalin is, in fact, essential for the normal renal reabsorption of TC-vitamin B12and for renal accumulation of this highly conserved vitamin. Megalin-deficient mice excrete increased amounts of TC and B12in the urine, revealing a defective renal tubular uptake of TC-B12. The urinary B12excretion is increased ∼4-fold, resulting in an ∼28-fold higher renal B12clearance. This is associated with an ∼4-fold decrease in B12content in megalin-deficient kidney cortex. Thus megalin is important to prevent urinary loss of vitamin B12. In addition, light- and electron-microscopic immunocytochemistry demonstrate lysosomal accumulation of B12in rat and mouse proximal tubules. In rats this accumulation is correlated with vitamin intake. Thus renal lysosomal B12accumulation is dependent on vitamin status, indicating a possible reserve function of this organelle in the rat kidney.

Active transport of photoactivated tubulin molecules in growing axons revealed by a new electron microscopic analysis.

To determine whether tubulin molecules transported in axons are polymers or oligomers, we carried out electron microscopic analysis of the movement of the tubulin molecules after photoactivation. Although previous optical microscopic analyses after photobleaching or photoactivation had suggested that most of the axonal microtubules were stationary, they were not sufficiently sensitive to allow detection of actively transported tubulin molecules which were expected to be only a small fraction of total tubulin molecules in axons. In addition, some recent studies using indirect approaches suggested active polymer transport as a mechanism for tubulin transport (Baas, P.W., F.J. Ahmad. 1993. J. Cell Biol. 120:1427-1437; Yu, W., V.E. Centonze, F.J. Ahmad, and P.W. Bass, 1993, J. Cell Biol. 122:349-359; Ahmad, F.J., and P.W. Bass. 1995. J. Cell Sci. 108:2761-2769). So, whether transported tubulin molecules are polymers or not remain to be determined. To clear up this issue, we made fluorescent marks on the tubulin molecules in the axons using a photoactivation technique and performed electron microscopic immunocytochemistry using anti-fluorescein antibody. Using this new method we achieved high resolution and high sensitivity for detecting the transported tubulin molecules. In cells fixed after permeabilization, we found no translocated microtubules. In those fixed without permeabilization, in which oligomers and heterodimers in addition to polymers were preserved, we found much more label in the regions distal to the photoactivated regions than in the proximal regions. These data indicated that tubulin molecules are transported not as polymers but as heterodimers or oligomers by an active mechanism rather than by diffusion.