Experimental Renal Artery Stenosis Research Articles

Renal ischemia alters expression of mitochondria-related genes and impairs mitochondrial structure and function in swine scattered tubular-like cells.

Scattered tubular-like cells (STCs) are dedifferentiated surviving tubular epithelial cells that repair neighboring injured cells. Experimental renal artery stenosis (RAS) impairs STC reparative potency by inducing mitochondrial injury, but the exact mechanisms of mitochondrial damage remain unknown. We hypothesized that RAS alters expression of mitochondria-related genes, contributing to mitochondrial structural damage and dysfunction in swine STCs. CD24+/CD133+ STCs were isolated from pig kidneys after 10 wk of RAS or sham (n = 3 each). mRNA sequencing was performed, and nuclear DNA (nDNA)-encoded mitochondrial genes and mitochondrial DNA (mtDNA)-encoded genes were identified. Mitochondrial structure, ATP generation, biogenesis, and expression of mitochondria-associated microRNAs were also assessed. There were 96 nDNA-encoded mitochondrial genes upregulated and 12 mtDNA-encoded genes downregulated in RAS-STCs versus normal STCs. Functional analysis revealed that nDNA-encoded and mtDNA-encoded differentially expressed genes were primarily implicated in mitochondrial respiration and ATP synthesis. Mitochondria from RAS STCs were swollen and showed cristae remodeling and loss and decreased ATP production. Immunoreactivity of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and expression of the mitochondria-associated microRNAs miR-15a, miR-181a, miR-196a, and miR-296-3p, which target several mtDNA genes, were higher in RAS-STCs compared with normal STCs, suggesting a potential modulation of mitochondria-related gene expression. These results demonstrate that RAS induces an imbalance in mtDNA- and nDNA-mitochondrial gene expression, impairing mitochondrial structure and function in swine STCs. These observations support development of gene gain- and loss-of-function strategies to ameliorate mitochondrial damage and preserve the reparative potency of STCs in patients with renal ischemia.

Gene Expression Profiles Indicative of Reversible Injury in Experimental Renal Artery Stenosis

AimThe aim of this is to identify the gene expression profiles indicative of reversible versus irreversible injury in experimental (murine) renal artery stenosis (RAS).MethodTwo‐kidney‐one‐cuff hypertension was established by placement of a polytetrafluoroethylene cuff on the right renal artery (N=18). Sham surgery was performed by manipulating the right renal artery without cuff placement (N=6). To determine potential reversibility, the cuff was removed at 7 days (7D cuff‐off, N=6) and 14 days (14D cuff‐off, N=6) after initial RAS surgery. In a control RAS group, the cuff was left in place for 28 days (28D RAS, N=6). Kidneys were harvested at 28 days for assessment of atrophy and fibrosis. Renal blood perfusion and oxygenation were measured using 16.4T MRI. RNASeq analysis was performed on all the groups at 28 days following initial surgery.ResultsFollowing cuff placement, intra‐renal perfusion was reduced by 65% (p<0.0001 vs. baseline). In the 7D cuff‐off group intra‐renal perfusion was restored to baseline values after cuff removal, whereas in the 14D cuff‐off group perfusion was not significantly improved following cuff removal. In the 28D RAS group the reduction in perfusion was maintained throughout the study period.The 7D cuff‐off group had a significantly lower % atrophy (7 ± 2.2%, p=0.0042) and % area of interstitial fibrosis (0.57 ± 0.08 %, p<0.0001) compared to 28D RAS group (atrophy 66 ± 13%, area of fibrosis 7.79 ± 0.89 %). The 14D cuff‐off group had severe atrophy (56 ± 16.6%) and interstitial fibrosis (5.05 ± 1.22%) similar to that seen in 28D RAS group.RNASeq analysis data were normalized to appropriate sham controls in all the groups and was performed at 28 days following initial surgery. Induction of greater than Log2 ratio >2 and p values < 0.00001 were considered significant (following adjustment for multiple comparisons). Fibrosis associated genes were differentially regulated in the 7D cuff‐off compared to the 14D cuff‐off and 28D RAS group (table ).ConclusionWe conclude that cuff removal at 7 days is associated with relative preservation of renal structure, indicating that renal injury at this point is reversible. We have identified a number of fibrosis associated genes that are differentially regulated in the 7D cuff‐off group versus 14D cuff‐off group. Further analysis will determine the functional relevance of these differentially regulated genes.Support or Funding InformationThis project was funded by National Institutes of Health, NIAID R01 AI100911. Values of Log 2 Ratio Genes 7D Cuff‐off vs Sham 14D Cuff‐off vs Sham 28D RAS vs Sham COL1A1 0.96 2.62 2.23 COL1A2 0.79 2.07 1.91 COL3A1 1.26 2.60 2.35 MMP12 1.24 2.85 2.95 MMP14 1.34 2.70 2.55 MMP19 2.89 3.86 4.05 MMP2 1.55 3.49 3.22 MMP7 6.96 9.22 9.59 MTOR −0.46 −1.67 −2.06 SERPINE1 1.25 3.37 2.35 TGFB1i1 0.44 1.46 1.53 TGFB2 0.82 1.83 1.97 TGFB3 0.59 1.91 1.99 TGFBi 0.98 1.88 2.02 KLF15 −0.90 −1.76 −2.60 UBD 4.49 6.23 6.18 TIMP1 2.94 5.30 5.16 TIMP2 0.50 1.46 1.52 TIMP3 −0.35 −1.26 −1.36 HSPB1 0.16 2.00 2.13 HSPB8 1.51 2.26 2.30 CCL2 3.44 4.89 4.31 CCL12 2.88 3.71 4.12 SERPINE1 1.25 3.37 2.35 SERPINE2 1.17 2.39 2.35 SERPINF1 2.02 2.76 3.11 SERPINF2 −0.51 −2.03 −2.71 SERPING1 1.15 1.81 2.09 CLU 1.45 2.54 3.07 STC1 0.45 1.99 2.12 SPP1 1.75 3.06 3.37

CXCL16 regulates renal injury and fibrosis in experimental renal artery stenosis.

Recent studies have shown that inflammation plays a critical role in the initiation and progression of hypertensive kidney disease, including renal artery stenosis. However, the signaling mechanisms underlying the induction of inflammation are poorly understood. We found that CXCL16 was induced in the kidney in a murine model of renal artery stenosis. To determine whether CXCL16 is involved in renal injury and fibrosis, wild-type and CXCL16 knockout mice were subjected to renal artery stenosis induced by placing a cuff on the left renal artery. Wild-type and CXCL16 knockout mice had comparable blood pressure at baseline. Renal artery stenosis caused an increase in blood pressure that was similar between wild-type and CXCL16 knockout mice. CXCL16 knockout mice were protected from RAS-induced renal injury and fibrosis. CXCL16 deficiency suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the stenotic kidneys, which was associated with less expression of extracellular matrix proteins. Furthermore, CXCL16 deficiency inhibited infiltration of F4/80(+) macrophages and CD3(+) T cells in the stenotic kidneys compared with those of wild-type mice. Taken together, our results indicate that CXCL16 plays a pivotal role in the pathogenesis of renal artery stenosis-induced renal injury and fibrosis through regulation of bone marrow-derived fibroblast accumulation and macrophage and T-cell infiltration.

Blockade of CCR2 reduces macrophage influx and development of chronic renal damage in murine renovascular hypertension.

Renovascular hypertension (RVH) is a common cause of both cardiovascular and renal morbidity and mortality. In renal artery stenosis (RAS), atrophy in the stenotic kidney is associated with an influx of macrophages and other mononuclear cells. We tested the hypothesis that chemokine receptor 2 (CCR2) inhibition would reduce chronic renal injury by reducing macrophage influx in the stenotic kidney of mice with RAS. We employed a well-established murine model of RVH to define the relationship between macrophage infiltration and development of renal atrophy in the stenotic kidney. To determine the role of chemokine ligand 2 (CCL2)/CCR2 signaling in the development of renal atrophy, mice were treated with the CCR2 inhibitor RS-102895 at the time of RAS surgery and followed for 4 wk. Renal tubular epithelial cells expressed CCL2 by 3 days following surgery, a time at which no significant light microscopic alterations, including interstitial inflammation, were identified. Macrophage influx increased with time following surgery. At 4 wk, the development of severe renal atrophy was accompanied by an influx of inducible nitric oxide synthase (iNOS)+ and CD206+ macrophages that coexpressed F4/80, with a modest increase in macrophages coexpressing arginase 1 and F4/80. The CCR2 inhibitor RS-102895 attenuated renal atrophy and significantly reduced the number of dual-stained F4/80+ iNOS+ and F4/80+ CD206+ but not F4/80+ arginase 1+ macrophages. CCR2 inhibition reduces iNOS+ and CD206+ macrophage accumulation that coexpress F4/80 and renal atrophy in experimental renal artery stenosis. CCR2 blockade may provide a novel therapeutic approach to humans with RVH.

CCR2 deficiency fails to protect mice with renal artery stenosis from chronic renal injury (278.7)

Recent studies have implicated CCR2 ‐ CCL2 signaling in macrophage influx and development of chronic renal disease in human and experimental renal artery stenosis (RAS). We sought to test the hypothesis that development of chronic renal disease would be abrogated in CCR2 knockout (KO) mice. We established RAS in CCR2 KO and WT mice (n=10) through placement of a cuff on the right renal artery We have shown that the cuff reproducibly reduces blood flow by 65% to the kidney. Sham mice underwent manipulation of the right renal artery without cuff placement. Both CCR2 KO and WT mice developed hypertension, which persisted throughout the 4 week study. There was no difference in renal atrophy between CCR2 KO (53+//‐14.6% cortical surface area) and WT (61.2%+/‐14%) mice. Furthermore, there were no significant differences in F4/80+ macrophage infiltration (12.78+/‐4.9% surface area for CCR2 KO mice and 13.13+/‐ 3.1% for WT mice) or CD3+ T cell infiltration. Infiltrating macrophages were predominantly iNOS+ M1 phenotype; few arginase+ cells were observed in any group. Urine protein excretion was similar in CCR2 KO and WT mice. CCL was induced to a similar extent in CCR2 KO and WT mice. Furthermore, no differences in CCL5, CCL7, CCR5, CCR1, or iNOS expression was observed between CCR2 KO and WT mice. We conclude that macrophage infiltration and chronic renal damage occurs in mice with deletion of CCR2. Future studies will identify signaling pathway(s) responsible for macrophage influx and chronic renal damage in this model.Grant Funding Source: AI‐100911

Renal vein cytokine release as an index of renal parenchymal inflammation in chronic experimental renal artery stenosis

Renal parenchymal inflammation is a critical determinant of kidney injury in renal artery stenosis (RAS) but is difficult to assess in the single kidney without tissue samples. Whether renal vein (RV) levels of inflammatory markers reflect active parenchymal inflammation remains unknown. We evaluated the relationship between net RV cytokine release and tissue inflammation in the post-stenotic kidney. Pigs were studied after 10 weeks of RAS treated 4 weeks earlier with intra-renal vehicle or anti-inflammatory mesenchymal stem cells (MSCs) or normal control. Single-kidney renal blood flow was measured by fast computerized tomography. RV and inferior vena cava levels of tumor necrosis factor (TNF)-α, interferon (IF)-γ, monocyte chemoattractant protein (MCP-1) and interleukin (IL)-10 were measured by enzyme-linked immunosorbent assay, and their net release calculated. Renal expression of the same cytokines was correlated with their net release. Net release of TNF-α, IF-γ and MCP-1 was higher in RAS compared with normal and to the contralateral kidney (all P<0.05), decreased in MSC-treated pigs as was their tissue expression. Contrarily, the release of the anti-inflammatory IL-10 was lower in RAS and normalized in RAS+MSC. The net release of TNF-α, MCP-1 and IL-10 directly correlated with their tissue expression. The ratio of inflammatory-to-reparative macrophages directly correlated with the release of MCP-1, but inversely with the release of IL-10. In vitro cultured MSCs also induced a shift in the macrophage phenotype from inflammatory (M1) to reparative (M2). Our findings demonstrate that the release of inflammatory markers from the affected kidney provides an index of renal tissue inflammation in experimental RAS.

Improved Renal Tubular Function Detected by Magnetic Resonance Imaging after Revascularization of Experimental Renal Artery Stenosis

Renal artery stenosis (RAS) may lead to hypertension and renal injury. Reversibility of renal tubular dysfunction in RAS upon revascularization is unclear. We tested the hypothesis that percutaneous transluminal renal angioplasty (PTRA) would restore tubular function assessed as medullary oxygenation response to furosemide, a diuretic that inhibits medullary tubular transport. This was studied in 16 pigs after 6 wks of RAS, using blood oxygen level dependent magnetic resonance imaging (BOLD MRI) before and after furosemide. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined with multidetector computed tomography. Pigs were then randomized for PTRA or sham (n=8 each) and studied again 4 wks later; 8 normal pigs served as controls.ResultsStenotic kidney RBF, GFR, and BOLD MRI responses were reduced in RAS shams (Figure). PTRA decreased the degree of stenosis (11±7 vs. 71±5% in untreated RAS) and blood pressure (108±3 vs. 130±9 mmHg, p&lt;0.05). Renal and tubular function were improved in RAS+PTRA, although not to normal (Figure). Non‐stenotic kidneys showed no change in tubular function. Therefore, tubular solute transport‐related oxygen consumption can be partially restored and detected by BOLD MRI 4 wks after PTRA in post stenotic swine kidneys. This noninvasive method may be useful to assess the functional status of kidneys in RAS patients. Supported by NIH, HL085307.

The effect of captopril on glucoheptonate uptake in experimental renal artery stenosis

The effect of captopril on glucoheptonate uptake in experimental renal artery stenosis

The effect of captopril on glucoheptonate uptake in experimental renal artery stenosis

This study attempted to evaluate the role of glucoheptonate (GHA) in captopril renography in an in vivo laboratory investigation in which postcaptopril glucoheptonate uptake was analysed in awake 2KlC hypertensive rats. Clamped kidney uptake in a previous study was greater in the poststenotic kidney than in the normal kidney (P = 0.01) in rats with mild renal artery stenosis. A glucoheptonate renogram protocol was developed for use in rats anaesthetized with sodium pentobarbital. An 123I-hippuran scan was performed to determine the relative renal function, followed by a control 99Tcm-GHA scan. Five minutes after administering captopril, another 99Tcm-GHA scan was performed. Relative renal uptake was determined between 30 and 90 s postinjection. 99Tcm-GHA uptake in the clamped kidney was more than 50% of total uptake in 3/9 of the abnormal rats' control scans. No abnormal rats clamped kidney 99Tcm-GHA uptake was greater than 50% in the postcaptopril scans. Captopril reduced GHA uptake in all nine of the animals with baseline scans. These findings suggest that the laboratory observation of captopril induced paradoxically increased 99Tcm-GHA uptake in renal artery stenosis may not be observed scintirenographically. Moreover, the data support a potential value of glucoheptonate in captopril renography.

Influence of antihypertensive therapy on renal function.

Antihypertensive therapy influences kidney function by different mechanisms depending on the mode of action of the drug used. The GFR is improved by calcium entry blockers and ACE inhibitors, unaffected by vasodilators, alpha-blockers and centrally acting sympatholytics and impaired by beta-blockers. The same is true for renal blood flow and is due to changes of renal vascular resistance. Renal sodium excretion is impaired mostly by vasodilators, by alpha-blockers, sympatholytics and beta-blockers; in contrast, calcium entry blockers and ACE inhibitors acutely induce natriuresis. The RAAS is stimulated by vasodilators, unaffected by alpha-blockers and sympatholytics and suppressed by beta-blockers. Plasma catecholamines are stimulated by vasodilators and suppressed by centrally acting sympatholytics and unaffected by the others. Induction of acute renal functional impairment is reported for ACE inhibitors under conditions of compromised renal perfusion pressure such as in renal artery stenosis. These data from the literature reviewed are supported by our own experimental data on sodium balance under different drugs and micropuncture data in experimental renal artery stenosis. To achieve effective antihypertensive treatment with a low profile of side effects, careful monitoring of renal function seems to be mandatory.

A New Method for Induction of Fibrous Renal Artery Stenosis in the Pig Designed for the Study of Percutaneous Balloon Catheter Dilatation

An open surgical method for the creation of experimental renal artery stenosis by ligation with chromic catgut is described. Microscopic examinations at different intervals demonstrated the development of fibrosis throughout the arterial wall. These stenoses seem to provide a suitable model for the study of the effects of balloon catheter dilatation. Previously reported techniques for experimental artery stenosis induction are briefly reviewed.

Dilatation of Experimental Renal Artery Stenosis by Balloon Catheter

An experimental model has been developed in the pig in order to evaluate the effect of percutaneous transluminal angioplasty on fibrous renal artery stenosis. Stenoses were induced by bilateral subtotal ligation with chromic catgut. Six weeks following ligation unilateral transluminal dilatation was performed in 9 pigs using Grüntzig balloon catheters. In only one case a serious complication occurred with rupture of the renal artery. Seven pigs with dilated stenoses were re-examined by angiography 8 to 10 weeks after dilatation. In 6 cases the stenoses were practically completely eliminated while in one cases a re-stenosis developed. Microscopy of the dilated arteries revealed several small and large ruptures of th internal elastic membrane.

Use of sequential 197Hg chlormerodrin delayed scans to evaluate individual renal function : Experimental renal artery stenosis and ureteral obstruction

197Hg chlormerodrin delayed scans and conventional split renal clearances were performed on 6 dogs. Left renal artery stenosis was then created in 2 animals and left partial ureteral obstruction in the other 4. The studies were then repeated two and six weeks after surgery. There was a close correlation among the studies after the arterial stenosis, but the ureteral occlusion results were too variable to make the study meaningful or reliable.

The radioisotope renogram in dogs with experimental renal artery stenosis

The radioisotope renogram in dogs with experimental renal artery stenosis

OXYGEN SATURATION, OXYGEN TENSION, AND PH OF RENAL VEIN BLOOD IN EXPERIMENTAL UNILATERAL RENAL ARTERY STENOSIS.

The purpose of this investigation is to determine oxygen saturation, oxygen tension, and pH of renal vein blood in experimental renal artery stenosis. Unsatisfactory results are often obtained by the current procedures usually employed in patients with suspected renal artery stenosis. Thus, a more safe direct physiological method for identifying renal artery lesions is constantly being investigated. Acute and long term experimental renal artery stenosis has been performed by application of a modified Goldblatt clamp or silk ligature. Blood samples have been obtained from the femoral artery and both renal veins before and after stenosis.