Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane

Abstract

The mechanism of renal glucose transport involves the reabsorption of filtered glucose from the proximal tubule lumen across the brush border membrane (BBM) via a sodium-dependent transporter, SGLT, and exit across the basolateral membrane via facilitative, GLUT-mediated, transport. The aim of the present study was to determine the effect of streptozotocin-induced diabetes on BBM glucose transport. We found that diabetes increased facilitative glucose transport at the BBM by 67.5 % (P < 0.05)--an effect that was abolished by overnight fasting. Western blotting and immunohistochemistry demonstrated GLUT2 expression at the BBM during diabetes, but the protein was undetectable at the BBM of control animals or diabetic animals that had been fasted overnight. Our findings indicate that streptozotocin-induced diabetes causes the insertion of GLUT2 into the BBM and this may provide a low affinity/high capacity route of entry into proximal tubule cells during hyperglycaemia.

Figure 1. Time dependency of glucose uptake by BBM vesicles prepared from renal cortex of normal rats

Uptake was measured at room temperature using buffer containing 200 mm NaSCN and 100 µmd-[³H]glucose with (▪, dashed line) or without (•, continuous line) 1 mm phlorizin (PZ) to block SGLT-mediated transport.

Figure 2. Detection and quantification of renal BBM facilitative glucose transporters

A, detection of GLUT1, GLUT2 and GLUT5 by Western blotting using BBM vesicles prepared from renal cortex of control (C), 2–4 week diabetic (D) and 2–4 week diabetic rats subjected to an overnight fast (FD). B, quantification of levels of GLUT1, GLUT2 and GLUT5 from bands produced by Western blotting of cortical BBM vesicles prepared from normal (open bars), 2–4 week diabetic (black bars) and overnight fasted, 2–4 week diabetic rats (grey bars). Results are expressed as mean ± s.e.m. Values were obtained from Western blots for each GLUT isoform carried out on six vesicle preparations. * P < 0.05, ** P < 0.001 compared to control; + P < 0.001 compared to 2–4 week diabetic.

Figure 3. Localization of GLUT2 protein in kidneys from control (A and E), 2–4 week diabetic (B and D) and overnight fasted, 2–4 week-diabetic rats (C)

GLUT2 is localized at the BLM in control and overnight fasted kidneys (arrows), but can be detected at both the BLM (arrows) and BBM (arrowheads) of diabetic kidneys. Antibody specificity was confirmed using sections probed with antibody pre-absorbed with an excess of antigenic peptide (D). E, low power image demonstrating that GLUT2 protein is expressed in the outer cortex of the kidney. Scale bars: A-D = 50 µm, E = 500 µm.

Figure 4. Confocal imaging of GLUT2 protein in kidneys from control, 2-4 week diabetic and overnight fasted, 2-4 week diabetic rats

The images obtained were produced as an overlay of one to three images selected from the Z-stack as the most representative of the entire section. GLUT2 was localized at the BLM (arrows) in control kidneys, but was detected at both the BLM and BBM (arrowheads) of diabetic kidneys. Overnight fasting of diabetic rats shows intracellular and BLM staining of GLUT2. Scale bars = 10 µm.

Figure 5. Localization of GLUT1 (A and B) and GLUT5 (C and D) protein in kidneys from control (A and C) and diabetic rats (B and D)

In both control and diabetic kidneys, GLUT1 displays intracellular as well as membrane staining, whilst, GLUT5 can be detected exclusively at the BBM. Scale bars = 50 µm.