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. 2023 Mar 30;24(7):6500.
doi: 10.3390/ijms24076500.

Effects of Mineralocorticoid Receptor Blockade and Statins on Kidney Injury Marker 1 (KIM-1) in Female Rats Receiving L-NAME and Angiotensin II

Jiayan Huang  1 , Ezgi Caliskan Guzelce  1 , Shadi K Gholami  1 , Kara L Gawelek  2 , Richard N Mitchell  2 , Luminita H Pojoga  1 , Jose R Romero  1 , Gordon H Williams  1 , Gail K Adler  1
Affiliations

Effects of Mineralocorticoid Receptor Blockade and Statins on Kidney Injury Marker 1 (KIM-1) in Female Rats Receiving L-NAME and Angiotensin II

Jiayan Huang et al. Int J Mol Sci. .

Abstract

Kidney injury molecule-1 (KIM-1) is a biomarker of renal injury and a predictor of cardiovascular disease. Aldosterone, via activation of the mineralocorticoid receptor, is linked to cardiac and renal injury. However, the impact of mineralocorticoid receptor activation and blockade on KIM-1 is uncertain. We investigated whether renal KIM-1 is increased in a cardiorenal injury model induced by L-NAME/ANG II, and whether mineralocorticoid receptor blockade prevents the increase in KIM-1. Since statin use is associated with lower aldosterone, we also investigated whether administering eiSther a lipophilic statin (simvastatin) or a hydrophilic statin (pravastatin) prevents the increase in renal KIM-1. Female Wistar rats (8-10 week old), consuming a high salt diet (1.6% Na+), were randomized to the following conditions for 14 days: control; L-NAME (0.2 mg/mL in drinking water)/ANG II (225 ug/kg/day on days 12-14); L-NAME/ANG II + eplerenone (100 mg/kg/day p.o.); L-NAME/ANG II + pravastatin (20 mg/kg/day p.o.); L-NAME/ANG II + simvastatin (20 mg/kg/day p.o.). Groups treated with L-NAME/ANG II had significantly higher blood pressure, plasma and urine aldosterone, cardiac injury/stroke composite score, and renal KIM-1 than the control group. Both eplerenone and simvastatin reduced 24-h urinary KIM-1 (p = 0.0046, p = 0.031, respectively) and renal KIM-1 immunostaining (p = 0.004, p = 0.037, respectively). Eplerenone also reduced renal KIM-1 mRNA expression (p = 0.012) and cardiac injury/stroke composite score (p = 0.04). Pravastatin did not affect these damage markers. The 24-h urinary KIM-1, renal KIM-1 immunostaining, and renal KIM-1 mRNA expression correlated with cardiac injury/stroke composite score (p < 0.0001, Spearman ranked correlation = 0.69, 0.66, 0.59, respectively). In conclusion, L-NAME/ANG II increases renal KIM-1 and both eplerenone and simvastatin blunt this increase in renal KIM-1.

Keywords: CVD (cardiovascular disease); N-omega-nitro-L-arginine methyl ester (L-NAME); angiotensin II (ANG II); kidney injury molecule (KIM-1); mineralocorticoid receptor (MR); pravastatin; simvastatin; statin.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Experimental Study Timeline. All the rats received food containing 1.6% Na+ from day (−2) through day 14. The rats received Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) in drinking water from day 1 through day 14 and ANG II infusion from day 12 through day 14 via osmotic minipumps. Blood pressures were measured noninvasively via tail cuffs three times at baseline (B1, B2 and B3) and on days 6, 11, and 14 during the study. The 24-h urine collections were performed from day 11 through day 14. Blood collection and organ harvest (hearts, kidneys, adrenals, and aortas) were performed on day 15.
Figure 2
Figure 2
Each circle represents systolic blood pressure (SBP) for an animal. Data are presented as mean ± SEM. Data were analyzed by one-way ANOVA with subsequent pair wise comparison (Tukey–Kramer HSD) between treatment groups. Each of the following time points were analyzed separately; baseline blood pressure, average of day 6 and 11 blood pressure, and day 14 blood pressure. p values for comparison with the control group (Tukey–Kramer HSD) are as follows: ✭✭ p < 0.01; ✭✭✭ p < 0.001; # p = 0.06, ## p = 0.07.
Figure 3
Figure 3
Day 14, 24-h urinary KIM-1/creatinine ratios (ng/mg). Data are shown as box plots (minimum value, first quartile, median, third quartile, and maximum value). The lines are superimposed in the control group. There were significant differences between the five treatment groups (p < 0.0001) by Wilcoxon/Kruskal Wallis (Rank sums) test. Nonparametric pairwise comparisons were performed using Wilcoxon method. There was a significant increase in day 14 urinary KIM-1/creatinine ratio (ng/mg) in all L-NAME/ANG II groups as compared with the control group (✭✭ p < 0.01, ✭✭✭ p < 0.001). Day 14 urinary KIM-1/creatinine ratio (ng/mg) was significantly lower in L-NAME/ANG II groups receiving eplerenone (p = 0.0046) and simvastatin (p = 0.031) as compared with the L-NAME/ANG II group.
Figure 4
Figure 4
Immunostaining of KIM-1 in kidneys from a control treated animal (A.1.,A.2.) and L-NAME/ANG II treated animal (B.1.,B.2.). (A.1.) shows minimal to no KIM-1 immunostaining. B.1 shows KIM-1 immunostaining of proximal renal tubules. Boxes indicated the area in (A.1.) magnified in (A.2.) and the area in (B.1.) magnified in (B.2.). (A.1.,B.1.) 4× magnification, (A.2.,B.2.) 20× magnification. C indicates the renal cortex and M indicates the renal medulla.
Figure 5
Figure 5
Semi-quantitative evaluation of kidneys with KIM-1 immunostaining. Data are shown as box plots. There were significant differences between the five treatment groups (p < 0.0001) by Wilcoxon/Kruskal Wallis (Rank sums) test. Nonparametric pairwise comparisons were then performed using Wilcoxon method. Higher KIM-1 immunostaining scores were observed in all L-NAME/ANG II groups as compared to the control group, ✭✭✭ p < 0.001. KIM-1 immunostaining scores were significantly lower in the L-NAME/ANG II/eplerenone (p = 0.004) and L-NAME/ANG II/simvastatin (p = 0.037) groups as compared with the L-NAME/ANG II group.
Figure 6
Figure 6
KIM-1 expression relative to expression in control group’s kidney. Data are shown as box plots (minimum value, first quartile, median, third quartile, and maximum value). The lines are superimposed in the control group. There were significant differences between the five treatment groups (p < 0.0006) by Wilcoxon/Kruskal Wallis (Rank sums) test. Nonparametric pairwise comparisons were then performed using Wilcoxon method. All L-NAME/ANG II groups had higher KIM-1 mRNA expression in renal cortex as compared with the control group, ✭✭ p < 0.01. KIM-1 mRNA expression was significantly lower in the L-NAME/ANG II/eplerenone (p = 0.012) group as compared with the L-NAME/ANG II group.
Figure 7
Figure 7
Cardiac Ventricle Histopathology. Representative section of cardiac ventricle stained with H&E from: (A) control group showing normal cardiac histology and (B) L-NAME/ANG II group showing prototypical cardiac damage characterized by myocyte eosinophilia, nuclear drop-out, myocyte necrosis, and inflammatory infiltrates; 4× magnification.
Figure 8
Figure 8
Cardiac Injury/Stroke Composite Score Comparison. Data are shown as box plots (minimum value, first quartile, median, third quartile, and maximum value). The lines are superimposed in the control group. There were significant differences between the five treatment groups (p < 0.001) by Wilcoxon/Kruskal Wallis (Rank sums) test. Nonparametric pairwise comparisons were performed using Wilcoxon method. All L-NAME/ANG II groups had significantly higher cardiac injury/stroke composite scores as compared to the control group, ✭✭✭ p < 0.001. The L-NAME/ANG II/eplerenone group (p = 0.044) had a significantly lower cardiac injury/stroke composite score compared with the L-NAME/ANG II group.
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