Ccl2 deficiency protects against chronic renal injury in murine renovascular hypertension

Inflammation plays an important role in the pathogenesis of renal and cardiovascular disease in renovascular hypertension (RVH). Ccl2 is an important mediator of inflammation, and is induced within 24 hours following surgery to establish RVH in the murine 2 kidney 1 clip model, a time prior to onset of interstitial inflammation, fibrosis, or tubular atrophy. We tested the hypothesis that Ccl2 deficiency protects the stenotic kidney (STK) from development of chronic renal damage in mice with renovascular hypertension due to renal artery stenosis (RAS). RAS surgery was performed on wild type (WT) and Ccl2 knock out (KO) mice; animals were studied for four weeks. Renal blood flow was reduced to similar extent in both WT and Ccl2 KO mice with RVH. Perfusion of the stenotic kidney was significantly reduced in Ccl2 KO mice as assessed by magnetic resonance imaging (MRI). Stenotic kidney volume in WT, but not in Ccl2 KO mice, was significantly reduced following surgery. Cortical hypoxia was observed in the stenotic kidney of Ccl2 KO mice, as assessed by blood oxygen level-dependent MRI (BOLD-MRI). Ccl2 KO mice showed less cortical atrophy than WT RAS mice. Ccl2 deficiency reduced the number of infiltrating mononuclear cells and expression of Ccl5, Ccl7, Ccl8, Ccr2 and Cd206. We conclude that Ccl2 is a critical mediator of chronic renal injury in RVH.

that pharmacological inhibition might have "off-label" effects that may not be due to specific CCR2 inhibition. Based on these considerations, we tested the hypothesis that Ccl2 deficiency alone can protect the stenotic kidney from the development of severe interstitial fibrosis, tubular atrophy, and interstitial inflammation in mice with homozygous deletion of the Ccl2 gene (Ccl2 KO mice) subjected to unilateral renal artery stenosis.

Materials and Methods
Animals. Male Ccl2 knockout (B6. 129S4-Ccl2 tm1Rol /J) (KO) and wild type (C57BL6/J) (WT) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). WT mice (N = 27 RAS, N = 9 sham) and Ccl2 KO (N = 24 RAS, N = 9 sham) mice underwent RAS or Sham surgery at 8-10 weeks of age. RAS surgery was done by placing a polytetrafluoroethylene cuff around the right renal artery using two sutures to secure the cuff in place as described previously 32 ; sham surgeries were performed by isolating and manipulating the right renal artery without cuff placement. All of the animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) prior to conducting any experiment. These animal procedures were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Imaging protocol and Analysis. MRI studies were performed on a subset of animals (N = 13 WT RAS, N = 4 WT Sham, N = 12 Ccl2 KO RAS, N = 4 Ccl2 KO Sham) employing a vertical 16.4T scanner equipped with a 38 mm inner diameter birdcage coil (Bruker Biospin, Billerica, MA). The images for the kidney volume determination were collected axially using respiratory gated 3D Fast Imaging with Steady Precession (3D-FISP) and reconstructed into 3D volumetric images. The kidney volume for cortex and medulla was then assessed from 3D-FISP using Analyze software (Biomedical Imaging Resource, Mayo Clinic, MN) and reported as sum of both cortex and medulla. Perfusion was assessed by arterial spin labelling (ASL) method by using a Flow-sensitive Alternating Inversion Recovery (FAIR) sequence with Rapid Acquisition with Relaxation Enhancement (RARE) as described earlier 33 . Perfusion of cortex and medulla in the stenotic kidney was calculated using the ASL tool module of Bruker-Paravision software and reported as the sum of both compartments. Blood flow for cortex and medulla was calculated by multiplying the perfusion times the volume of corresponding compartment and reported as their sum.
Renal oxygenation was assessed by BOLD-MRI imaging using a respiration-gated 3D multi-echo gradient echo sequence 33 . The imaging parameters used were: TR 200 ms; TE 3.5-24.5 ms; echo number 8; flip-angle 25°; slab thickness 1 mm; FOV 2.56 × 2.56 cm 2 ; matrix size 128 × 128 × 8; number of averages 2. " 8 images were reconstructed after zero-filling the k-space data to 256 × 256 for BOLD MRI. T 2 * was quantified by pixel-wise Animal Blood Pressure Measurement. Non-invasive method using the tail cuff and volume pressure recording (VPR) sensor technology was used on conscious mice to record the blood pressure measurements at baseline and following surgery (CODA 6, Kent Scientific Corporation, Torrington, Connecticut, USA). Plasma Renin Activity. Plasma was available from 21 WT RAS, 7 WT Sham, 21 Ccl2 KO RAS, and 8 Ccl2 KO Sham mice. Blood was collected via the inferior vena cava four weeks after surgery; plasma was separated from it by centrifugation and stored at −80 °C until the time of assay. Quantitative determination of plasma renin content was done by radioimmunoassay as described earlier 20 .
Renal Function. Renal function was assessed by the measurement of serum creatinine and blood urea nitrogen (BUN) levels at 24 h after ischemia using a Creatinine Analyzer 2 and a BUN Analyzer 2 (Beckman Instruments, Fullerton, CA) as previously described 34 . Histology and immunohistochemical analysis. Histologic and immunohistochemical analysis was performed on the subset of animals for which imaging studies were performed (see above). The kidney tissues were fixed in 10% neutral buffered formalin and processed using standard techniques. 5 µm histological sections were prepared and stained with hematoxylin-eosin (H&E). Immunohistochemical staining was done for

Results
Blood pressure was elevated to a similar extent in Ccl2 KO RAS and WT RAS mice. There were no differences in the total body weight of WT RAS, WT sham, Ccl2 KO RAS and Ccl2 sham mice. The mean systolic blood pressure in both WT and KO mice following surgery to establish unilateral renal artery stenosis was significantly higher compared to sham mice. However, there was no significant difference in blood pressure measured in WT RAS and Ccl2 KO RAS mice (Fig. 1a). As expected, the weight of the cuffed kidney was significantly reduced, compared to sham (Fig. 1b). In WT mice, the contralateral kidney became hypertrophic, as evidenced by a significant increase in weight (Fig. 1c). The heart weight was also increased in the WT RAS mice (Fig. 1d). In contrast, the contralateral kidney weights of the Ccl2 KO RAS mice was similar to that of Ccl2 sham mice (Fig. 1c) and the heart weights of Ccl2 KO RAS and Ccl2 KO sham mice were similar (Fig. 1d). There were no significant differences in plasma BUN, creatinine or plasma renin activity between any of the groups.

Renal Blood flow was reduced to a similar extent in WT and Ccl2 KO RAS mice as assessed by MRI.
The renal blood flow in both WT and Ccl2 KO RAS mice was reduced to similar extent following surgery to establish RVH (Fig. 2). The volume of the STK in WT RAS was significantly reduced compared to the pre-surgery baseline but no differences were observed between Ccl2 KO RAS and WT RAS mice at 3 weeks following surgery (1 week prior to the end of the experiment) (Fig. 2). Perfusion of the stenotic kidney in Ccl2 KO mice was significantly reduced, compared to its pre-surgery baseline (Fig. 3). There were no significant differences in perfusion of the stenotic kidneys of WT RAS or Ccl2 KO RAS mice. KO RAS mice showed significant elevation in cortical R 2 * , which is a measure of hypoxia following RAS at 3 weeks compared to its baseline. Although the stenotic kidneys of WT RAS mice showed higher R 2 * values compared to their baseline, this difference did not reach significance (Fig. 4). The cuffed kidneys from Ccl2 KO RAS mice were protected from development of chronic renal damage. The stenotic kidney of KO mice showed protection against renal damage as assessed by "% atrophy cortical area" compared to WT mice (Fig. 5a,b) despite a similar elevation in blood pressure (Fig. 1a).

The extent of interstitial fibrosis in Ccl2 KO RAS mice was less than that of WT RAS mice.
Quantitative assessment of interstitial fibrosis was performed on Sirius red stained sections of renal cortex. The extent of interstitial fibrosis was significantly greater in WT RAS mice than in Ccl2 KO RAS mice (Fig. 6).

Macrophage influx is decreased in Ccl2 KO mice compared to WT mice following RAS surgery.
Macrophage influx was lower in the stenotic kidney of Ccl2 KO mice than WT mice with RAS, as assessed by the percentage of cortical surface areas staining positively for the macrophage marker F4/80 (Fig. 7). We did not find any significant differences in CD3 + T cell influx between Ccl2 KO and WT mice with RAS (Fig. 8).
Expression of pro-inflammatory chemokines is lower in Ccl2 KO than WT mice following RAS surgery. Ccl2 deficiency was confirmed using real time PCR in KO mice (Fig. 9a). Ccl2 expression was significantly higher in WT RAS mice, compared to Ccl2 KO RAS and WT sham mice. Similar results were observed for the expression of Ccl5, Ccl7 and Ccl8 (Fig. 9b,c,d respectively), although we did not observe any difference in Ccl8 expression between WT and Ccl2 KO RAS.
Ccr2 expression was significantly higher in WT RAS mice, compared to Ccl2 KO RAS and WT sham mice (Fig. 10a). There were no significant differences in expression of iNos in any group (Fig. 10b). Cd206 expression was higher in WT RAS mice, compared to WT sham and KO RAS mice (Fig. 10c).
Il6 expression was significantly higher in WT RAS mice compared to WT sham (Fig. 11a). However, we did not find any differences in Il12, Gata3, Rorc expression in any of the groups (Fig. 11b,c,d respectively). Although, TNFα expression was significantly higher in WT RAS compared to sham (Fig. 12a), there were no differences in TNFα expression between WT and Ccl2 KO RAS. No difference in Tgfβ expression was observed in any group (Fig. 12b).
Acta2 and Col3A1 expression was significantly higher in WT RAS mice compared to its sham ( Fig. 13a  and b), whereas, Col3A1 expression was significantly elevated in the KO RAS mice compared to the KO sham group (Fig. 13b). Acta2 expression was significantly higher in WT RAS mice than Ccl2 KO RAS mice (Fig. 13a).

Discussion
Our studies provide further evidence that Ccl2 plays a key role in mediating renal inflammation in RVH and its deficiency alone can protect against the chronic renal injury. Despite a similar flow reduction in the stenotic kidneys of WT and Ccl2 KO mice, the stenotic kidney of Ccl2 KO mice showed significant protection against renal injury, as evidenced by less cortical atrophy, compared to WT RAS. The R2* value, a measure of renal hypoxia, was significantly higher in the STK of KO mice which could be due to the fact that perfusion was significantly reduced in KO RAS mice, whereas perfusion was maintained in WT RAS mice, albeit at a reduced cortical volume. Plasma BUN and creatinine were within normal limits in all groups, indicating that the contralateral kidney was able to compensate for the chronic damage which developed in the stenotic kidney. These findings are consistent with observations that BUN and creatinine are relatively normal in patients who undergo unilateral nephrectomy.
Although, blood pressure was increased to a similar extent in KO and WT RAS mice, the heart weight was significantly higher than sham in WT RAS but not Ccl2 KO RAS mice. Similarly, the contralateral (non-cuffed) kidney showed a higher weight in WT RAS but not Ccl2 KO RAS mice. Reduction of chronic renal injury in the STK of KO RAS mice was associated with less macrophage, but not T cell infiltration.
In our previous study, we found that pharmacologic inhibition of CCR2 reduced Ccl5 and TNFα expression, but not Ccl2, Il-6, Il-10, Il-12, or Tgfβ expression 20 . In the current study, we demonstrate that Ccl2 deficiency significantly reduces expression of other pro-inflammatory molecules, including Ccl5, Ccl7, Ccl8, Ccr2, and the M2 macrophage marker Cd206. Unlike the CCR2 inhibitor, TNFα expression was not significantly reduced in Ccl2 KO RAS mice, compared to WT RAS animals, raising the possibility that TNF expression may not directly involve Ccl2-CCR2 signaling.
The lack of cardiac hypertrophy in KO RAS mice contrasts with our studies of other signaling pathways culminating in renal atrophy in RAS. The STK of RAS mice treated with the p38 inhibitor SB203580 was protected from development of renal inflammation, tubular atrophy, and interstitial fibrosis. However, SB203580 had no effect on blood pressure, plasma renin activity, or heart weights compared to vehicle treated RAS mice 35 . Similarly, the STK of mice with deficiency of Smad3, a critical intermediate of TGF-β signaling, was protected from development of renal atrophy, interstitial fibrosis, and tubular atrophy. Although, blood pressure was increased to a similar extent in Smad3 KO and WT RAS mice at 3 days after surgery, blood pressure after that time point was significantly lower in Smad3 KO RAS mice than WT mice 32,36 . Despite this, the heart weight to body weight ratio was significantly higher in the Smad3 KO RAS mice than WT RAS mice. Furthermore, the Smad3 KO RAS mice had a severe cardiac phenotype characterized by myocardial inflammation with extensive myocyte necrosis, which was associated with sudden death in Smad3 KO RAS mice 36 . Based on these considerations, we propose that Ccl2 may provide a better therapeutic target to reduce chronic renal damage and to protect the heart than p38 or Smad3 inhibition.  Although, our studies support a central role for Ccl2 in reducing inflammatory responses to renal injury, there are several limitations. We demonstrated that mice with global Ccl2 deficiency are protected from development of renal atrophy in RVH; we have not established whether Ccl2 signaling in parenchymal cells or in bone marrow derived infiltrating inflammatory cells is responsible for this protective effect. Studies to address this issue will be important to determine whether more specific therapy should target immune/inflammatory cells or parenchymal cells to prevent development of chronic renal and cardiovascular disease in RVH. Not all studies support a protective role for Ccl2 deficiency in renal disease. In a renal ischemia-reperfusion model, Ccl2 deficiency exacerbated renal damage 37 . With that caveat in mind, human studies in patients with chronic renal disease show a strong correlation between urinary Ccl2 excretion, albuminuria, and renal infiltration of macrophages 23 . We conclude that therapy directed towards Ccl2 signaling may provide a novel therapeutic target to prevent progression renal and possibly cardiovascular disease in RVH.