T between 6 and 20 weeks, in both cortex and medulla (Table 4 and Fig. 6, panels 1 vs. 4 and 7 vs. 5). 3.5 Correlation analysis The results obtained with Pearson and Spearman analyses were comparable. Spearman correlations with correlation coefficients |0.5| and p values 0.05 are presented in Supplementary Table 1. Degree of PAS staining in glomeruli (PTA) showed a positive correlation with GTA (Table S1, N 1). A positive correlation was observed between numbers of immune-positive tubules and degree of staining in cortex and medulla for eNOS (Table S1, N 2 and 3), nNOS (Table S1, N 4 and 5) and iNOS (Table S1, N 6). Also a positive correlation was seen between numbers of tubules immune-positive for iNOS and nNOS in cortex (Table S1, N 7). Mesangial matrix PAS staining and glomerular tuft expansion showed a number of Pan-RAS-IN-1 chemical information negative correlations with the immunostaining for NOS isoforms. For eNOS in medulla, a negativeActa Histochem. Author manuscript; available in PMC 2017 March 01.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptMG-132MedChemExpress MG-132 Slyvka et al.Pagecorrelation was observed with GTA (Table S1, N 8). A negative correlation was observed between nNOS in medulla and both PTA (Table S1, N 12) and PTA/GTA (Table S1, N 16 and 17) and for nNOS in cortex with GTA (Table S1, N 9), PTA (Table S1, N 11 and 13), and PTA/GTA (Table S1, N 15). A similar negative correlation was observed between iNOS in cortex and both GTA and PTA (Table S1, N 10 and 14). Please see Table S1 in the section on supplementary data given at the end of this article for details.Author Manuscript Author Manuscript4.2 eNOS4. Discussion4.1 Glomerular staining and morphology Mesangial matrix expansion, glomerular basement membrane thickening and glomerular hypertrophy are key histological findings in early DN (Abrass, 1995, Couser and Johnson, 1994, Kasiske et al., 1985, Schena and Gesualdo, 2005), followed later by glomerular contraction. In the present study, increases in both GTA and PTA were observed in both male and female obese diabetic Zucker rats with age and both GTA and PTA were less in rats on the AO diet indicating a correspondence between diet and matrix proliferation. As mesangial cells are important in regulation of glomerular filtration, this may be related to the improved GFR in 20 week females on the AO diet observed by Slyvka et al. (Slyvka, Inman, 2009). Treatment of STZ-induced diabetic rats (Wistar) with an NO donor has been shown to alleviate extracellular matrix proliferation (ECM) (Hsu et al., 2015), consistent with our finding that the AO-fortified diet also decreases the ECM in diabetic rats.Pathologic changes in the kidney are accompanied by changes in the expression and localization of constitutive and inducible NOS isoforms. In animal models of DN, endothelial damage caused by increased inflammation and excess production of ROS coupled with ineffective NO action leads to compensatory increases in constitutive eNOS and nNOS as well as iNOS (Prabhakar et al., 2007, Tan et al., 2007), eventually compounding and accelerating the damage. In the current experiment, levels of eNOS protein increased with duration of hyperglycemia in glomerular and vascular endothelium, in agreement with the findings of others (Veelken et al., 2000). It is known that eNOS levels are increased in glomerular endothelial cells of patients with DN (Hohenstein, Hugo, 2008). An increase in glomerular eNOS has also been seen in diabetic patients with microalbuminuria (.T between 6 and 20 weeks, in both cortex and medulla (Table 4 and Fig. 6, panels 1 vs. 4 and 7 vs. 5). 3.5 Correlation analysis The results obtained with Pearson and Spearman analyses were comparable. Spearman correlations with correlation coefficients |0.5| and p values 0.05 are presented in Supplementary Table 1. Degree of PAS staining in glomeruli (PTA) showed a positive correlation with GTA (Table S1, N 1). A positive correlation was observed between numbers of immune-positive tubules and degree of staining in cortex and medulla for eNOS (Table S1, N 2 and 3), nNOS (Table S1, N 4 and 5) and iNOS (Table S1, N 6). Also a positive correlation was seen between numbers of tubules immune-positive for iNOS and nNOS in cortex (Table S1, N 7). Mesangial matrix PAS staining and glomerular tuft expansion showed a number of negative correlations with the immunostaining for NOS isoforms. For eNOS in medulla, a negativeActa Histochem. Author manuscript; available in PMC 2017 March 01.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptSlyvka et al.Pagecorrelation was observed with GTA (Table S1, N 8). A negative correlation was observed between nNOS in medulla and both PTA (Table S1, N 12) and PTA/GTA (Table S1, N 16 and 17) and for nNOS in cortex with GTA (Table S1, N 9), PTA (Table S1, N 11 and 13), and PTA/GTA (Table S1, N 15). A similar negative correlation was observed between iNOS in cortex and both GTA and PTA (Table S1, N 10 and 14). Please see Table S1 in the section on supplementary data given at the end of this article for details.Author Manuscript Author Manuscript4.2 eNOS4. Discussion4.1 Glomerular staining and morphology Mesangial matrix expansion, glomerular basement membrane thickening and glomerular hypertrophy are key histological findings in early DN (Abrass, 1995, Couser and Johnson, 1994, Kasiske et al., 1985, Schena and Gesualdo, 2005), followed later by glomerular contraction. In the present study, increases in both GTA and PTA were observed in both male and female obese diabetic Zucker rats with age and both GTA and PTA were less in rats on the AO diet indicating a correspondence between diet and matrix proliferation. As mesangial cells are important in regulation of glomerular filtration, this may be related to the improved GFR in 20 week females on the AO diet observed by Slyvka et al. (Slyvka, Inman, 2009). Treatment of STZ-induced diabetic rats (Wistar) with an NO donor has been shown to alleviate extracellular matrix proliferation (ECM) (Hsu et al., 2015), consistent with our finding that the AO-fortified diet also decreases the ECM in diabetic rats.Pathologic changes in the kidney are accompanied by changes in the expression and localization of constitutive and inducible NOS isoforms. In animal models of DN, endothelial damage caused by increased inflammation and excess production of ROS coupled with ineffective NO action leads to compensatory increases in constitutive eNOS and nNOS as well as iNOS (Prabhakar et al., 2007, Tan et al., 2007), eventually compounding and accelerating the damage. In the current experiment, levels of eNOS protein increased with duration of hyperglycemia in glomerular and vascular endothelium, in agreement with the findings of others (Veelken et al., 2000). It is known that eNOS levels are increased in glomerular endothelial cells of patients with DN (Hohenstein, Hugo, 2008). An increase in glomerular eNOS has also been seen in diabetic patients with microalbuminuria (.