Authors: Mohammad Saleem (1Department of Medicine/Clinical Pharmacology Division, Vanderbilt University Medical Center), Pierina Barturen-Larrea (1Department of Medicine/Clinical Pharmacology Division, Vanderbilt University Medical Center), Luz Saavedra (1Department of Medicine/Clinical Pharmacology Division, Vanderbilt University Medical Center), Jose A. Gomez (1Department of Medicine/Clinical Pharmacology Division, Vanderbilt University Medical Center)
Categories: Article
Source: Journal of visualized experiments : JoVE
Doi: 10.3791/61058
Authors: Mohammad Saleem, Pierina Barturen-Larrea, Luz Saavedra, Jose A. Gomez
Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) is overactivated. In this context, there is a narrowing of the renal arteries that stimulate an increase in the expression and release of renin, the rate-limiting protease in RAAS. The resulting rise in renin expression is a known driver of renovascular hypertension, frequently associated with kidney injury and end organ damage. Thus, there is a great interest in developing novel treatments for this condition. The molecular and cellular mechanism of renin control in renal artery stenosis is not fully understood and warrants further investigation. To induce renal artery stenosis in mice, a modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed. The right kidney was stenosed in wild type mice and sham operated mice were used as control. After renal artery stenosis, we determined renin expression and kidney injury. Kidneys were harvested, and fresh cortices were used to determine protein and mRNA expression of renin. This animal model is reproducible and can be used to study pathophysiological responses, molecular and cellular pathways involved in renovascular hypertension and kidney injury.
Renal artery stenosis (RAStenosis) is an intractable problem affecting about 6% of people over 65 and in up to 40% of people with coronary or peripheral vascular disease^1,2^. Current treatments for the disease are limited; therefore, there is a critical need to develop new therapies to treat renovascular hypertension or resistant hypertension induced by RAStenosis. Renin angiotensin aldosterone system (RAAS) is the key pathway involved in the pathogenesis of RAStenosis induced hypertension or renovascular hypertension^3,4^. Known therapies targeting RAAS, such as ACE inhibitors or angiotensin receptor blockers, alleviate hypertension, but need close examining for kidney failure and hyperkalemia^5,6,7^. Renin catalyzes the rate-limiting step in RAAS; it converts angiotensinogen to angiotensin I. In atherosclerosis, plaque formation causes the narrowing of renal artery that drives renin secretion, resulting in renovascular hypertension and kidney damage^8^. A number of studies have reported increased levels of oxidative stress during renovascular hypertension in humans, which were corroborated with the two kidney one clip (2K1C) mice model as well as other hypertensive animal models^2,9,10,11,12,13,14,15,16^. The molecular mechanism of renin expression control during RAStenosis induced renovascular hypertension is not well understood and warrants further investigation.
Experimental animal models that reliably and reproducibly recapitulate RAStenosis are important in elucidating the cellular and molecular mechanisms of renin expression control for the development of novel therapies. The 2K1C mouse model is a well-established experimental model to study the pathogenesis of renovascular hypertension^17,18,19,20^. This model is generated by the constriction of the renal artery using a clip^17,20,21^, therefore producing renal artery occlusion that results in an increase in renin expression and hypertension^17,19,20,21^. However, there are no technical reports available, which describe a step by step procedure to generate renal artery stenosis in animal models.
Conventional U-shaped silver clips, polyurethane tubes and other clips have been used to constrict the renal artery to induce renal artery stenosis. Some studies have shown that the design and material of the clip are critical to obtaining reliable and reproducible data with the 2K1C animal model. According to Lorenz et al., the use of conventional U-designed silver clips induces a low success rate of hypertension (40–60%)^21^. Due to the clip design, the renal artery is press laterally, triggering a few constrictions and greater probability to be dislodged from the renal artery. Silver malleability and ductility may allow changes in clip widths; therefore, causing different hypertension levels among mice. Silver dioxides on the clip can cause perivascular inflammation, intimal proliferation, and tissue granulation, altering the renal artery diameter^22^. Due to the variability in the levels of hypertension obtained with the conventional U-design silver clip, Warner et al. and Lorenz et al. have successfully used a rounder-design polyurethane tubing to initiate renal artery stenosis in mice, generating a more reliable and consistent induction of the two kidney one clip animal model^20,21^.
In this report, we describe a surgical protocol to generate experimental RAStenosis in mice, using the polyurethane tubing to constrict the renal artery. The polyurethane round-design cuff is a more reproducible, reliable and low-cost clip to generate stenosis in mouse. The goal of this experimental model is to study and define the molecular and cellular mechanism of renin expression control during renal artery stenosis. We confirmed the success of RAStenosis mice model by measuring renin expression and kidney injury marker neutrophil gelatinase-associated lipocalin (N-GAL).
Mice were housed and cared at the Vanderbilt University Medical Center (VUMC) Division of Animal Care following the National Institutes of Health (NIH) guidelines and the Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services. All animal procedures were approved by the VUMC Institutional Animal Care and Use Committee prior to starting the experiments.
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 compared to 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 hypertension^2,25,26^. Neutrophil gelatinase-associated lipocalin (NGAL) is a well characterized acute injury marker and is overexpressed during kidney injury^27,28^. Therefore, acute kidney injury marker NGAL was measured using immunoblotting. Immunoblotting data showed that N-GAL was highly upregulated in the stenosed kidney when compared to the contralateral and sham kidneys (Figure 4).
Renal artery stenosis is an important cause of secondary or resistant hypertension, and kidney injury^1,29^. The two kidney one clip (2K1C) Goldblatt model has been employed to study RAStenosis induced renovascular hypertension^1,17,18,19^. A number of previous studies using various animals models have shown that stenosis in the renal artery is a strong stimulator of renin overexpression and release, and kidney injury^18,30,31,32,33,34,35^. Moreover, this model is used to study immune cell infiltration, fibrosis, inflammation, and acute and chronic kidney injury markers^29^.
Here, we described a detailed and step by step procedure to generate reproducible, reliable and consistent renal artery stenosis model in mice. Earlier, metal clips have been employed to initiate renal artery stenosis^36,37,38^. As an alternative, we used polyurethane round tubing (MRE 025; internal diameter (ID) = 0.30 mm; outside diameter (OD) = 0.63 mm; wall thickness, (WT) = 0.16 mm). We used tubing, since placement of a polyurethane cuff would result constriction in two dimensions (constriction) rather than one (flattening), as with a metal clip. Also, using polyurethane round tubing provides an advantage of uniform constriction in the renal artery. The critical step and challenges are to cut the right size of polyurethane tubing, which requires extreme attention to details that must be performed using a microscope. Another critical criterion is to keep mice between 18–22 g to fit the tubing onto the renal artery. Mice within this weight range normally have a renal artery outer diameter (OD) that is consistently within the range of the tubing cuff diameter. A limitation of the method is that heavy (above 25 g) or small (below 16 g) mice are difficult to perform surgery on because of the size of the tube and cuff made in it. However, when required, changes in the polyurethane tubing can be made to accommodate younger or older mice.
We have conducted 3-day and 15-day studies to initiate renal artery stenosis in mice with about 95% success rate. In our experience, induction of the renal artery stenosis using this method produced reliable, reproducible, and consistent results among the mice regardless of the sex. To confirm the constriction of renal artery, we measured renin expression and kidney injury. Our data suggest that renin expression significantly increased in the stenosed kidney.