Characterization of Aldose Reductase from the Thick Ascending Limb of Henle’s Loop of Rabbit Kidney

Abstract

Background: The organic osmolyte sorbitol plays an important role in the osmoregulation of immortalized epithelial cells of the thick ascending limb of Henle’s loop (TALH) of rabbit. The intracellular sorbitol content seems to depend strongly on the extracellular osmolarity. To investigate the nature of the osmotic regulation we characterized the aldose reductase. Methods: We determined aldose reductase activity enzymatically and the content of organic osmolytes by HPLC. Results: The aldose reductase activity correlates with the extracellular tonicity. Elevating the osmolarity of the medium from 300 to 600 mosm/l by addition of NaCl or sucrose resulted in a significant increase of maximal velocity (V_max) of the adapted cells from 8 ± 1 µmol/g × min (300 mosm/l) to 322 ± 28 µmol/g × min (600 mosm/l, NaCl) or 54 ± 9 µmol/g × min (600 mosm/l, sucrose), respectively, while affinity (K_m) remained unchanged. But we found no rise of aldose reductase activity when extracellular urea concentration was elevated. Similar alterations in V_max were observed when the activity of the highly enriched enzyme was determined with glucose as substrate. Elevation of the extracellular osmolarity by NaCl and sucrose strongly induced the expression of aldose reductase protein with an apparent molecular weight of 39 kD. The affinity of glucose is characteristically low with a K_m above 300 mmol/l. Aldose reductase utilizes both NADPH and with lower affinity NADH as coenzymes. In vitro sulfate ions (0.4 mol/l) results in a two-fold activation of the aldose reductase activity whereas sodium (200–400 mmol/l) decreased the activity significantly (22–33%). Potassium and chloride up to 400 mmol/l did not alter the aldose reductase activity in vitro. Conclusions: These results indicate that the aldose reductase of TALH cells of the outer medulla is osmotically regulated and has many similarities with aldose reductase in renal inner medulla. Therefore, intracellular sorbitol synthesis seems to be of similar importance in the osmoregulation of TALH cells as in the inner medulla.