Shear stress increases
nitric oxide (NO) production by endothelial cells, inner medullary collecting duct cells, and thick ascending limb. We postulated that the osmotic diuresis accompanying
type 1 diabetes is associated with increased
NO synthase (NOS) activity and/or expression in the renal medulla. Diabetes was induced by injection of
streptozotocin, with
insulin provided to maintain moderate
hyperglycemia (Hyp) or euglycemia (Eug) for 3 wk.
Sham rats received vehicle treatments. A separate group of rats (Phz) received
phlorizin to produce a
glucose-dependent osmotic diuresis. Renal medullary NOS1 and NOS2 activities did not differ between groups, whereas NOS3 activity was significantly increased in Hyp. Neither NOS1 nor NOS3
protein levels differed significantly between groups. Reduced phosphorylation of NOS3 at Thr(495) and Ser(633) was evident in medullary homogenates from Hyp rats, with no difference apparent at Ser(1177). Immunohistochemical analysis indicated prominent expression of pThr(495)NOS3 in the thick ascending limb and collecting duct of
Sham and Phz rats. Hyp rats displayed staining in the collecting duct but minimal thick ascending limb staining. Immunostaining with anti-pSer(1177)NOS3 was evident only in the thick ascending limb, with no apparent differences between groups. In summary,
glucose-dependent osmotic diuresis alone did not alter NOS activity or expression in the renal medulla. Diabetic
hyperglycemia increased medullary NOS3 activity without a concomitant increase in NOS3
protein levels; however, NOS3 phosphorylation was reduced at Thr(495) and Ser(633). Thus changes in the phosphorylation of NOS at known regulatory sites might represent the primary mechanism underlying increased renal medullary NOS activity in diabetic
hyperglycemia.