Posttranslational regulation of NO synthase activity in the renal medulla of diabetic rats

Abstract

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.

Figure 1

Renal medullary NOS activity in kidneys from rats receiving vehicle treatments (SHAM), streptozotocin-treated rats receiving partial insulin replacement (HYP), streptozotocin-treated rats receiving insulin replacement to restore euglycemia (EUG), and normal rats receiving chronic phlorizin treatment (PHZ). A: Total NOS, NOS1, NOS2 and NOS3 activities in the cytosolic fraction of renal medulla. B: Total NOS, NOS1, NOS2 and NOS3 activities in the particulate fraction of renal medulla. Activities of each isoform were determined based on effects of isoform-specific inhibitors (see text for details). Values are means±SE; n=7−10. *P<0.05 vs SHAM.

Figure 2

NOS1 protein levels in cytosolic (C) and particulate (P) fractions of renal medulla from SHAM, EUG, HYP and PHZ rats. A: Representative Western blots. B: Summary of densitometric data, normalized to values observed in cytosolic fraction from SHAM rats. Values are means±SE. No significant between-group difference in NOS1 protein was apparent in either the cytosolic or the particulate fraction.

Figure 3

NOS3 protein levels in cytosolic (C) and particulate (P) fractions of renal medulla from SHAM, EUG, HYP and PHZ rats. A: Representative Western blots. B: Summary of densitometric data, normalized to values observed in cytosolic fraction from SHAM rats. Values are means±SE. No significant between-group difference in NOS3 protein was apparent in either the cytosolic or the particulate fraction.

Figure 4

Effect of T1D on NOS3 phosphorylation in renal medulla. A: Representative Western blots of medullary homogenates (without separation into cytosolic and membrane fractions). Phosphorylated and total NOS3 in each homogenate were probed on separate gels. As an equal loading control, each NOS3 and phospho-NOS3 blot was stripped and reprobed for β-actin (not shown). B: Densitometric values for phosphorylation site-specific NOS3 and NOS3 (each normalized to β-actin) were utilized to determine pNOS3/NOS3 for each phosphorylation site. Values are means±SE. n=8 rats in each group. *P<0.05 vs SHAM.

Figure 5

Representative immunohistochemical localization of pThr⁴⁹⁵NOS3 in the renal medulla from SHAM rats (A, B) and HYP rats (C - F). A, C & E: Outer medulla. B & D: Inner medulla. F: Intrarenal artery. Arrowheads, collecting duct; Arrows, thick ascending limb; Asterisks, vasa recta. Original magnification = 100×.

Figure 6

Immunohistochemical localization of pSer¹¹⁷⁷NOS3 in the renal medulla from SHAM rats (A & C) and HYP rats (B & D). A & B: Outer medulla. C & D: Inner Medulla. Arrowheads, collecting duct; Arrows, thick ascending limb. Original magnification = 100×.