Hiragushi et al., 2001). We observed that treatment with an AO-fortified diet resulted in a decrease in eNOS staining in glomeruli in 3 groups of animals: females at 6 and 20 weeks of age and males at 20 weeks, possibly reflecting decreased inflammation or increased NO effectiveness. Few studies have been done comparing renal eNOS expression in the diabetic kidney of males and females. Male gender has been associated with more rapid progression of kidney disease (Baylis, 2009, Denton and Baylis, 2007, Neugarten and Silbiger, 1995) and overall, in the present study, levels of eNOS were PF-04418948 supplier higher in the kidneys of diabetic males than females. At 20 weeks females on the AO diet had lower glomerular scores for eNOS, butAuthor Manuscript Author ManuscriptActa Histochem. Author manuscript; available in PMC 2017 March 01.Slyvka et al.Pagehigher numbers of eNOS positive stained tubules and IHC scores in cortex. This group had better preservation of renal function as AZD4547MedChemExpress AZD4547 reported previously (Slyvka, Inman, 2009). Under physiological conditions renal NO is derived from constitutive eNOS and nNOS (Raij and Baylis, 1995). In healthy kidneys, eNOS is localized in the endothelium of glomeruli and afferent and efferent arterioles where it generates vasodilatory NO. It is not detected in other cell types or in the tubular epithelial cells (Bachmann et al., 1995, Furusu et al., 1998, Ishii et al., 2001, Jarry et al., 2003). We found eNOS expressed in distal and to a smaller degree in proximal tubular epithelial cells in the Zucker obese diabetic rat, and although others have found increased peroxynitrite in renal tubules with DM (Hsu, Lee, 2015), the functional significance of tubular expression of eNOS remains to be established. Clearly, additional studies are needed regarding the role of eNOS in specific renal cell types as it relates to glomerular and tubular nephropathy. 4.3 nNOS In the normal kidney, nNOS contributes to the constitutive generation of NO (Raij and Baylis, 1995). nNOS is found in the macula densa where it plays a role in tubule-glomerular feedback (Bachmann, Bosse, 1995, Ishii, Patel, 2001, Welch et al., 1999). It is also observed in efferent arterioles and in tubular epithelial cells all along the nephron with higher levels in the cortex and outer medulla than in the inner medulla (Bachmann, Bosse, 1995, Jarry, Renaudin, 2003). As mentioned above, renal damage in DN leads to compensatory increases in nNOS (Prabhakar, Starnes, 2007, Tan, Forbes, 2007). In the present study, nNOS was predominantly localized to the cortex (Table 3). It was also detected in epithelial cells of the thick ascending tubules, inner medullary collecting ducts and to a lesser extent the parietal epithelium of Bowman’s capsule. These findings are in agreement with those of others in the diabetic kidney (Fujihara, Mattar, 2002, Jarry, Renaudin, 2003, Komers et al., 2000, Yabuki, Tahara, 2006). The diminished expression of nNOS in epithelial cells of tubules observed here in the diabetic male rat on a REG diet with age may be related to pathological changes in renal hemodynamics and electrolyte and glucose transport regulation. Maintenance of nNOS in the 20 week males on the AO diet may indicate an effect of the diet to maintain compensatory NO production; unfortunately this did not correspond to augmentation of renal function in this group (Slyvka, Inman, 2009). Although higher levels of nNOS have been reported in medulla than in cortex in rodent models of T1DN (Shin,.Hiragushi et al., 2001). We observed that treatment with an AO-fortified diet resulted in a decrease in eNOS staining in glomeruli in 3 groups of animals: females at 6 and 20 weeks of age and males at 20 weeks, possibly reflecting decreased inflammation or increased NO effectiveness. Few studies have been done comparing renal eNOS expression in the diabetic kidney of males and females. Male gender has been associated with more rapid progression of kidney disease (Baylis, 2009, Denton and Baylis, 2007, Neugarten and Silbiger, 1995) and overall, in the present study, levels of eNOS were higher in the kidneys of diabetic males than females. At 20 weeks females on the AO diet had lower glomerular scores for eNOS, butAuthor Manuscript Author ManuscriptActa Histochem. Author manuscript; available in PMC 2017 March 01.Slyvka et al.Pagehigher numbers of eNOS positive stained tubules and IHC scores in cortex. This group had better preservation of renal function as reported previously (Slyvka, Inman, 2009). Under physiological conditions renal NO is derived from constitutive eNOS and nNOS (Raij and Baylis, 1995). In healthy kidneys, eNOS is localized in the endothelium of glomeruli and afferent and efferent arterioles where it generates vasodilatory NO. It is not detected in other cell types or in the tubular epithelial cells (Bachmann et al., 1995, Furusu et al., 1998, Ishii et al., 2001, Jarry et al., 2003). We found eNOS expressed in distal and to a smaller degree in proximal tubular epithelial cells in the Zucker obese diabetic rat, and although others have found increased peroxynitrite in renal tubules with DM (Hsu, Lee, 2015), the functional significance of tubular expression of eNOS remains to be established. Clearly, additional studies are needed regarding the role of eNOS in specific renal cell types as it relates to glomerular and tubular nephropathy. 4.3 nNOS In the normal kidney, nNOS contributes to the constitutive generation of NO (Raij and Baylis, 1995). nNOS is found in the macula densa where it plays a role in tubule-glomerular feedback (Bachmann, Bosse, 1995, Ishii, Patel, 2001, Welch et al., 1999). It is also observed in efferent arterioles and in tubular epithelial cells all along the nephron with higher levels in the cortex and outer medulla than in the inner medulla (Bachmann, Bosse, 1995, Jarry, Renaudin, 2003). As mentioned above, renal damage in DN leads to compensatory increases in nNOS (Prabhakar, Starnes, 2007, Tan, Forbes, 2007). In the present study, nNOS was predominantly localized to the cortex (Table 3). It was also detected in epithelial cells of the thick ascending tubules, inner medullary collecting ducts and to a lesser extent the parietal epithelium of Bowman’s capsule. These findings are in agreement with those of others in the diabetic kidney (Fujihara, Mattar, 2002, Jarry, Renaudin, 2003, Komers et al., 2000, Yabuki, Tahara, 2006). The diminished expression of nNOS in epithelial cells of tubules observed here in the diabetic male rat on a REG diet with age may be related to pathological changes in renal hemodynamics and electrolyte and glucose transport regulation. Maintenance of nNOS in the 20 week males on the AO diet may indicate an effect of the diet to maintain compensatory NO production; unfortunately this did not correspond to augmentation of renal function in this group (Slyvka, Inman, 2009). Although higher levels of nNOS have been reported in medulla than in cortex in rodent models of T1DN (Shin,.