Representative Results
Renal artery constriction increases renin expression in the stenosed kidney while repressing expression in the contralateral kidney. The two kidney one clip (2K1C) or Goldblatt model of stenosis induces increased renin expression and kidney injury.This is recognized as the best representative model of unilateral renal artery stenosis in humans.
Expression of renin and prorenin (precursor of renin) were measured using immunoblotting. The data show that renin and prorenin expression increased in the stenosed kidney comparing to contralateral and sham kidneys, suggesting that the cuff was constricting the renal artery causing changes in renal perfusion (Figure 1). To visualize the localization of renin expression, IHC was performed. IHC corroborated immunoblotting data showing increased expression of renin in the clipped kidney (Figure 2). Moreover, juxtaglomerular (JG) cells recruitment along the afferent arteriole was seen in the stenosed kidney (Figure 2). To investigate the effect on renin mRNA expression levels, ISH was performed. The ISH data suggest increased renin mRNA and JG cells recruitment in the stenosed kidney when comparedto contralateral and sham kidneys (Figure 3).
Another characteristic of renal artery stenosis is the upregulation of kidney injury markers due to changes in kidney perfusion, superoxide production and hypertension2,25,26. Neutrophil gelatinase-associated lipocalin (NGAL) is a well characterized acute injury marker and is overexpressed during kidney injury27,28. Therefore, acute kidney injury marker NGAL was measured using immunoblotting. Immunoblotting data showed that N-GAL was highly upregulated in the stenosed kidney when comparedto the contralateral and sham kidneys (Figure 4).
Figure 1: Renin expression.After 15- and 3-days of renal artery stenosis, mice were euthanized. Kidneys were harvested, and renin expression was determined by western blot. (A) is showing representative western blots images from 15-days stenosed (left panel) and sham mice (right panel). (B) is showing the densitometric analysis of prorenin (left panel) and renin (right panel) protein bands. Beta-actin was used as loading control. (C) is showing the representative western blots images from 3-day stenosed (left panel) and sham mice (right panel). (D) is showing densitometric analysis of prorenin (left panel) and renin (right panel) protein bands. Beta-actin was used as loading control. Data are presented as the mean ± SD. P value calculated with two-way ANOVA followed by Tukey post-hoc test. *P<0.05, **P< 0.01, ***P< 0.001, N=3-6. Please click here to view a larger version of this figure.
Figure 2: Immunohistochemistry analysis for visualization and localization of renin expression after renal artery stenosis.Kidneys were isolated after euthanizing mice from 3-day renal artery stenosis. Onepiece from longitudinal cut of the whole kidney was fixed with 4% neutral buffered formalin solution, after that dehydrated in a graduated ethanol series, and embedded in paraffin. Green staining represents renin protein expression; blue, nuclei. (A) Representative microscopy images of stenosed kidney (left side), contralateral kidney (right side) from stenosed mice. (B) Representative microscopy images of sham kidney (left side), and contralateral kidney (right side) from sham mice. Scale bar 30 microns. 90X magnification. (C) Representative microscopy images of stenosed kidney (left side), contralateral kidney (right side) from stenosed mice. (D) Representative microscopy images of sham kidney (left side), and contralateral kidney (right side) from sham mice. These images were mainly taken from cortex region. Scale bar 50 µm. 15x magnification. White dotted circles denote the location of glomeruli. G: Glomeruli, AfAr: Afferent arteriole, N=4. Please click here to view a larger version of this figure.
Figure 3: In situ hybridization analysis of renin mRNA expression after renal artery stenosis.After 3-days of renal artery stenosis, mice were euthanized and kidneys were isolated and perfusion-fixed with 4% neutral buffered formalin solution, dehydrated in a graduated ethanol series, and embedded in paraffin. In situ hybridization was performed following the manufacturer’s instructions. Dark red staining represents mRNA renin expression; blue, nuclei. (A). Representative microscopy image of stenosed kidneys (left side), and contralateral kidney (right side) from stenosed mice. (B). Representative microscopy image of sham kidney (left side), and contralateral kidney (right side) from sham mice. Scale bar 50 µm. White dotted circles denote glomeruli expressing renin. G: Glomeruli, AfAr: Afferent arteriole, N=4. Please click here to view a larger version of this figure.
Figure 4: Neutrophil gelatinase-associated lipocalin (N-GAL) expression after renal artery stenosis.After 3-day renal artery stenosis, mice were euthanized and kidneys were harvested, and N-GAL expression measure by Western blot. (A) Representative Western blots images from 3-day stenosed (left panel) and sham mice (right panel). Beta-actin was used as loading control. (B) Densitometric analysis of N-GAL bands. Protein density values of N-GAL were normalized to β-actin. Data are presented as the mean ± SD. P value calculated with two-way ANOVA followed by Tukey post-hoc test. **P<0.01, ***P< 0.001, N=3-5. Please click here to view a larger version of this figure.
