## 4 Discussion  
To the best of the authors' knowledge, there is no previous study investigating the effects of NESP on animal models of renal injury; therefore, our study is the first report comparing the effects of orally administration of free SP and NESP in a rat model of renal I/R. Our results were quite promising in showing the beneficial effects of orally-administered NESP pre-treatment (20 mg/kg) for 2 days before ischemia induction compared to free SP, administered in the same period, on improvement of renal injury through normalizing oxidant/antioxidant imbalance, renal biomarkers and histological features in rats. Multiple factors including hypoxia, inflammatory responses and ROS production are involved in I/R-induced renal injury. Renal blood flow reduction during ischemia plays an important role in the pathophysiology of renal injury [40]. On the other hand, it has been in \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Markers** & **Control** & **I/R** & **SP** & **NESP** & **SP + I/R** & **NESP + I/R** \\ \hline Urea (mg/dL) & 5.97 \(\pm\) 0.99 & 49.11 \(\pm\) 3.11 *** & 6.21 \(\pm\) 0.42 *** & 7.31 \(\pm\) 0.97 *** & 32.34 \(\pm\) 3.08 *** & 12.14 \(\pm\) 1.00 *** \\ \hline Creatinine (mg/dL) & 0.38 \(\pm\) 0.04 & 1.98 \(\pm\) 0.23 *** & 0.19 \(\pm\) 0.02 *** & 0.29 \(\pm\) 0.01 *** & 1.12 \(\pm\) 0.09 *** & 0.41 \(\pm\) 0.01 *** \\ \hline TP (g/dL) & 6.84 \(\pm\) 0.21 & 2.98 \(\pm\) 0.09 *** & 5.78 \(\pm\) 0.43 *** & 7.58 \(\pm\) 0.51 *** & 4.54 \(\pm\) 0.09 *** & 6.11 \(\pm\) 0.07 *** \\ \hline Uric acid (mg/dL) & 1.35 \(\pm\) 0.30 & 5.98 \(\pm\) 0.39 *** & 0.97 \(\pm\) 0.02 *** & 1.31 \(\pm\) 0.11 *** & 4.20 \(\pm\) 0.07 *** & 1.98 \(\pm\) 0.25 *** \\ \hline \end{tabular}
\end{table} 
Table 1: Effects of administration of spironolactone (SP) and nano-encapsulated spironolactone (NESP) on the serum levels of urea, creatinine, total protein (TP) and uric acid in control, ischemic/reperfusion (I/R), spironolactone (SP), nano-encapsulated spironolactone (NESP), spironolactone (NESP), spironolactone-treated I/R (SP + I/R) and NESP-treated I/R (NESP + I/R) groups at the end of intervention (\(n=8\)). The data are represented as mean \(\pm\) SD. Asterisks indicate significant difference compared to control group (*** \(p<0.01\) and *** \(p<0.001\), analysis of variance (ANOVA), [PERSON]’s test for post-hoc comparisons). Hash marks indicate significant difference compared to I/R group (\({}^{\#}\)\(p<0.05\), *** \(p<0.01\) and *** \(p<0.001\), ANOVA, [PERSON]’s test for post-hoc comparisons). @ symbols show significant differences between SP + I/R and NESP + I/R groups (\({}^{\#}\)\(p<0.05\) and *** \(p<0.01\), ANOVA, [PERSON]’s test for post-hoc comparisons). dicated that mineralocorticoids have an important role in regulating renal vasculature tone and renal hemodynamic. Considering the role of aldosterone as a mineralocorticoid hormone in the pathophysiology of renal injury [45; 46], it seems that aldosterone antagonists such as SP have beneficial effects on renal injury induced by aldosterone [14; 47]. In the present study, the serum creatinine and urea levels in the I/R group increased significantly compared to the control group. The mechanism by which these two renal functional parameters increased is due to low renal blood flow and, consequently, decline in glomerular filtration rate. Treatment with SP and NESP led to a decrease in the level of urea and creatinine in the serum of ischemic rats. This supportive effect is due to the antagonistic action of these two substances against aldosterone. Previous studies showed similar findings regarding the effects of SP treatment in renal ischemia models on improving renal function [30]. In the present work, NESP showed an advantage over SP in renal biomarkers assays by correcting all the alterations in ischemic rats. In this regard, NESP decreased urea, creatinine and uric acid and increased TP compared to SP-treated I/R rats (Table 1).