The conventional model of renal glucose reabsorption involves uptake from the proximal tubule lumen across the brush-border membrane (BBM) by a sodium-dependent transporter, SGLT, followed by exit across the basolateral membrane (BLM) via a facilitative, GLUT-mediated transporter. Our previous studies have shown that streptozotocin (STZ)-induced diabetes has no effect on SGLT-mediated transport but increases GLUT-mediated transport across the renal BBM (Debnam et al. 1997), which is associated with increased levels of GLUT2, demonstrated by Western blotting. However, immunohistochemistry performed on unfixed tissue sections failed to detect this protein at the BBM. The present study investigated whether kidney fixation in vivo yielded the same pattern of GLUT2 expression as that obtained in unfixed tissue.
Diabetes was induced in male Sprague-Dawley rats using a single tail vein injection of STZ (45 mg kg-1). Three-week diabetic, overnight fasted diabetic and weight-matched control animals (n = 4 per experimental group) were terminally anaesthetised with pentobarbitone sodium (90 mg kg-1 I.P.) and the left kidney perfused with periodate-lysine-paraformaldehyde (PLP) (Mclean & Nakane, 1974). Fixed kidneys were removed, embedded in OCT and snap frozen in liquid N2. Cryostat sections (7 µm) were mounted onto polysine-coated slides and a standard peroxidase-DAB immunohistochemical protocol used for the localisation of GLUT2 protein. Experiments were performed in accordance with the Animals (Scientific Procedures) Act, 1986.
Immunohistochemistry performed on kidneys fixed in vivo with PLP showed GLUT2 labelling in the S1 segment of the proximal convoluted tubule (PCT). In control kidneys, GLUT2 was localised exclusively to the BLM, whereas in diabetic kidneys it was detected at both the BLM and BBM. Overnight fasting of the diabetic animals reduced blood glucose levels and returned the distribution of GLUT2 immunoreactivity to that of controls.
In vivo fixation seems crucial in the detection of GLUT2 at the BBM of PCT cells in diabetes. GLUT2 protein appears to be rapidly shuttled in or out of the BBM in response to the glycaemic status of the animal. It is noteworthy that in intestinal enterocytes, where the transport process of glucose is very similar to that in the PCT, BBM levels of GLUT2 also increase in response to high luminal glucose concentrations (Kellet et al. 2000).
This work was supported by The Wellcome Trust and the St Peter’s Trust.