Name: a modified two kidney one clip mouse model of renin regulation in renal artery stenosis
Uploaded: 2020-10-26T13:00:00.000+00:00
Duration: 8 min 21 s
Description: Watch this Scientific Journal Video about A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis at JoVE.com

Research was supported by NHLBI Research Scientist Development Grant (1K01HL135461-01) to JAG.&#160;Thank you to David Carmona-Berrio, and Isabel Adarve-Rengifo for their technical assistance.

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.
1. Animal preparation and dissection
<ol>
	<li>Turn on the germinator and water pump of&#160;the heating pad about 30 min before starting surgery.</li>
	<li>Cut 0.5 mm length polyurethane tubing with a sharp scalpel. Remove 0.2 mm of the circumference by making a cut lengthwise to make a cuff. 
	NOTE: This is a critical part of the renal artery stenosis procedure that requires extreme precision, attention to details and patience. Try to cut several pieces of polyurethane tubing at a time. Perform all of this procedure under microscope.</li>
	<li>Before proceeding any further, put on surgical sterile gloves and a surgical mask.</li>
	<li>Use C57BL/6 wild-type (WT) mice of 6-8 weeks. Use an equal number of male and female mice to avoid any sex bias.</li>
	<li>Record the weight of the mice before performing surgery. The ideal weight to perform renal artery stenosis with polyurethane tube is 18-22 g. Handle mice gently and do not agitate while injecting the anesthesia. Administer pre-operative analgesia (Ketofgen, 5 mg/kg body wieght). 
	NOTE: While transferring the mice from their housing room to the surgery room, carry them with great gentleness and care to avoid agitation. Carrying mice cages in hands instead of a trolley is highly recommended.</li>
	<li>Anesthetize the mice with a mixture of ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW) via intraperitoneal (I.P.) injections.</li>
	<li>Place the mouse back inside the cage until fully anesthetized. It takes about 4-5 minutes before the mouse is fully unconscious. Pinch the tail or toe with forceps to check if the mouse is fully unconscious and ready for the surgery.</li>
	<li>Lay the mouse on its back on a paper towel. Remove the hair of the lateral abdomen using an electric hair clipper following the opposite direction of hair growth. After shaving the surgical site, clean the area with a surgical scrub containing iodine (or chlorhexidine) followed by a rinse with 70% ethanol (or sterile saline).&#160; Repeat 3 times. 
	NOTE: The hair removal procedure must be performed at some distance or preferably at a different bench than the surgery procedure bench to avoid any hair interference and hair contamination during the surgery procedure.</li>
	<li>Cover the heating pad with a sterile sheet. Bring the mouse to the surgical bench and place on to the sterile sheet, facing the mouse&#8217;s right lateral side towards the microscope. Maintain a constant pad temperature of 37 &#176;C with circulating water.</li>
	<li>Open the sterilized bag containing all the surgical equipment. Using a dissecting microscope and sterile sharps scissors, make a small flank incision (close to 13th thoracic rib, the last rib in mouse) and about 0.5 cm away from vertebrae. Proceed along the lumbar vertebrae and make a 1 inch incision.</li>
	<li>Pull back the skin and muscle to expose the kidney.</li>
	<li>Clean and remove the surrounding fat using sterile cotton swabs to isolate the renal artery. Isolate the renal nerve from the renal artery using curved forceps.</li>
	<li>When performing the sham surgery, apply sutures to close the skin&#160;and apply antibiotic to the closed wound, then&#160;proceed to Post-operative care. If not, proceed with the following section to stenose the artery. 
	NOTE: Every experiment should have sham animals as controls of the surgical procedure. Sham animals consist of mice that have gone through the surgical procedure of exposing the renal artery without placing a cuff on it.</li>
</ol>
2. Right renal artery stenosis
<ol>
	<li>Place two nylon sutures under the right renal artery, make loose knots, and then place the cuff around the main renal artery approximately equidistant between the kidney and aorta bifurcation</li>
	<li>Close the cuff using the nylon sutures. Make four&#160;knots for each suture to avoid the probability of losing the sutures after the surgery.</li>
	<li>Close the incision in the muscle by applying a simple continuous suture.</li>
	<li>Make simple interrupted sutures to close the skin.</li>
	<li>Administer IACUC approved analgesics. 
	NOTE: Autoclave surgical tools before every use. If more than one surgery is being performed at once, wipe all used tools with a sterile alcohol gauge and place them in hot germinator for 15-30 s after every surgery. Change sterile gloves also for each mouse.</li>
</ol>
3. Post-operative care
<ol>
	<li>Return mice to their cage and leave the cage half on, half off, a circulating water heating pad for 2 - 3 hours. Add gel diet recovery food inside the cage.</li>
	<li>Administer painkiller (ketoprofen) intraperitoneally (dose: 5 mg/kg BW)&#160;the next day.</li>
	<li>Weigh the mice for the next two days; if some mice lose more than 20% of their weight consult with the veterinarian and decide if the animal needs to be euthanized following the appropriate IACUC authorized procedure.</li>
	<li>Monitor the mice daily to assess for redness, swelling, pain or infection.</li>
	<li>Seek veterinary consultation for postoperative complications in accordance with the institutional IACUC policies..</li>
</ol>
4. Tissue harvest
<ol>
	<li>Harvest tissue 3 weeks after surgical procedure. Record the weight of each mouse.</li>
	<li>Euthanize the mouse with an IACUC approved procedure.</li>
	<li>Place the mouse on a sterile platform in supine position to dissect.</li>
	<li>Secure and extend the limbs to limit movement.</li>
	<li>Thoroughly spray the mice with 70% ethanol.</li>
	<li>Make a midline incision to open the abdomen and chest area using sharp scissors.</li>
	<li>Pull back the skin and peritoneal wall.</li>
	<li>Carefully expose the heart and puncture the right ventricle and exsanguinate the mouse.</li>
	<li>Remove both kidneys using forceps. Kidneys are located on the back of the mice. 
	NOTE: Do not mix both kidneys. Be aware of stenosed, sham, and contralateral kidneys.</li>
	<li>Remove the kidney capsule, clean them from any fat and record the weight of each kidney separately.</li>
	<li>Cut a longitudinal section of both kidneys and fixed in 4% PFA overnight at 4 &#176;C to be later processed for paraffin embedding, to perform in situ hybridization (ISH) and immunohistochemistry (IHC). Follow ISH and IHC protocols as reported23,24.</li>
	<li>Isolate the cortex of the remaining kidney and flash freeze in liquid nitrogen to perform western blot. Store samples at -80 &#176;C until analysis.</li>
	<li>Quantify renin, and N-GAL expressions with Western blot as described in literature23,24.</li>
</ol>
5. Statistics
<ol>
	<li>Use one-way or two-way ANOVA for experiments with three or more conditions followed by Bonferroni post-hoc tests for comparisons between individual groups. Consider a p-value equal or less than 0.05 significant. Use software (e.g., GraphPad Prism 8.2) to perform all statistical analysis.</li>
</ol>

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No conflicts of interest, financial or otherwise, are declared by the authors.

Renal artery stenosis is an important cause of secondary or resistant hypertension, and kidney injury1,29. The two kidney one clip (2K1C) Goldblatt model has been employed to study RAStenosis induced renovascular hypertension1,17,18,19. 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 injury18,30,31,32,33,34,35. Moreover, this model is used to study immune cell infiltration, fibrosis, inflammation, and acute and chronic kidney injury markers29.
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 stenosis36,37,38. 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.

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 disease1,2. 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 hypertension3,4. Known therapies targeting RAAS, such as ACE inhibitors or angiotensin receptor blockers, alleviate hypertension, but need close examining for kidney failure and hyperkalemia5,6,7. 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 damage8. 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 models2,9,10,11,12,13,14,15,16. 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 hypertension17,18,19,20. This model is generated by the constriction of the renal artery using a clip17,20,21, therefore producing renal artery occlusion that results in an increase in renin expression and hypertension17,19,20,21. 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&#160;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 diameter22. 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 model20,21.
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).

<table><tbody><tr><td>Diet Gel</td><td>Clear H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Diet-Gel 76A</td><td>Surgery recovery diet</td></tr><tr><td>EMC Heated Hard pad</td><td>Hallowell</td><td>000A2788B</td><td>Heating pads were used to keep mice warm</td></tr><tr><td>Ethilon Nylon Suture</td><td>Ethicon</td><td>662G</td><td>4-0 (1.5 metric), This suture was used to close the peritoneum, and skin</td></tr><tr><td>Ethilon Nylon Suture</td><td>Ethicon</td><td>2815 G</td><td>8-0 (0.4 metric), This suture was used to close cuff to tie and constrict the artery</td></tr><tr><td>Germinator 500</td><td>Braintree Scientific Inc.</td><td>GER 5287</td><td>Sterilize surgical tools between surgeries</td></tr><tr><td>Ketoprofen</td><td>Zoetis</td><td>Ketofen</td><td>Painkiller</td></tr><tr><td>Polyurethane</td><td>Braintree Scientific Inc.</td><td>MRE-025</td><td>This tube was used to initiate stenosis</td></tr><tr><td>Povidone-iodine antiseptic swabsticks</td><td>Medline</td><td>MDS093901</td><td>It was applied after hair removal and surgery on the skin</td></tr><tr><td>Reflex 7 Clip Applier</td><td>Roboz Surgical Instrument Co</td><td>204-1000</td><td>This clip applier was used to apply clip in case one or more sutures went off</td></tr><tr><td>Sterile towel drapes</td><td>Dynarex</td><td>4410</td><td>It was used as a bedsheet for mice during surgery</td></tr><tr><td>Triple antibiotic ointment</td><td>Medi-First</td><td>22312</td><td></td></tr><tr><td>Water pump</td><td>Stryker</td><td>T/pump Professionals</td><td>Used to warm and circulate water in the heating hard pad to keep mice warm during and post-surgery</td></tr></tbody></table>

a modified two kidney one clip mouse model of renin regulation in renal artery stenosis

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 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.&#160;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&#160;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 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 compared&#160;to the contralateral and sham kidneys (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61058/61058fig01.jpg" /> 
Figure 1: Renin expression.&#160;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 &#177; SD. P value calculated with two-way ANOVA followed by Tukey post-hoc test. *P&#60;0.05, **P&#60; 0.01, ***P&#60; 0.001, N=3-6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61058/61058fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61058/61058fig02.jpg" /> 
Figure 2: Immunohistochemistry analysis for visualization and localization of renin expression after renal artery stenosis.&#160;Kidneys were isolated after euthanizing mice from 3-day renal artery stenosis. One&#160;piece 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 &#181;m. 15x magnification. White dotted circles denote the location of glomeruli. G: Glomeruli, AfAr: Afferent arteriole, N=4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61058/61058fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61058/61058fig03.jpg" /> 
Figure 3: In situ hybridization analysis of renin mRNA expression after renal artery stenosis.&#160;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&#8217;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 &#181;m. White dotted circles denote glomeruli expressing renin. G: Glomeruli, AfAr: Afferent arteriole, N=4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61058/61058fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61058/61058fig04.jpg" /> 
Figure 4: Neutrophil gelatinase-associated lipocalin (N-GAL) expression after renal artery stenosis.&#160;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 &#946;-actin. Data are presented as the mean &#177; SD. P value calculated with two-way ANOVA followed by Tukey post-hoc test. **P&#60;0.01, ***P&#60; 0.001, N=3-5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61058/61058fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis at JoVE.com

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

A modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed using polyurethane tubing to initiate renal artery stenosis, inducing an increase in renin expression and kidney injury. Here, we describe a detailed procedure of preparing and placing the cuff onto the renal artery to generate a reproducible and consistent 2K1C mouse model.

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

4.6K Views.  Vanderbilt University Medical Center. Een gemodificeerd 2 nier 1 clip (2K1C) Goldblatt muismodel werd ontwikkeld met behulp van polyurethaan tubing om nierslagaderstenose te initiëren, waardoor een toename van renine-expressie en nierbeschadiging werd geïnduceerd. Hier beschrijven we een gedetailleerde procedure voor het voorbereiden en plaatsen van de manchet op de nierslagader om een reproduceerbaar en consistent 2K1C-muismodel te genereren.

Video: A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

A Murine Model of Hemodialysis Access-Related Hand Dysfunction

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.

This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology.

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Biology

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called "uremic cardiomyopathy". We describe here an experimental CKD mouse model, named 5/6th partial nephrectomy (PNx) with pole ligation, which developed uremic cardiomyopathy at four weeks post-surgery. This PNx model was performed by a two-step surgery. In step-one surgery, both poles of the left kidney were ligated. In step-two surgery, which was performed 7 days after the step-one surgery, the right kidney was removed. For the sham surgery, the same surgery procedures were performed but without pole ligation of the left kidney or removal of the right kidney. The surgical procedures are easier and less time-consuming, compared to other methods. However, the remnant functional renal mass is not as easily controlled as the renal artery ligation. Four weeks after surgery, in comparison with the sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

This manuscript provides a detailed two-step surgical procedure to perform mouse 5/6th partial nephrectomy (PNx) with pole ligation. Four weeks after surgery, in comparison with sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype ...

Standardized 5/6 Nephrectomy in Rats for Renal Insufficiency and Malperfusion

5/6 Nephrectomy Using Sharp Bipolectomy Via Midline Laparotomy in Rats

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the treatment of CKD have had a significant impact on patient outcomes. While in the past, CKD was often considered a death sentence, with many patients succumbing to the complications of end-stage renal disease, it is now increasingly being managed as a chronic condition with the availability of dialysis and kidney transplantation, as well as new pharmaceutical developments such as SGLT2 inhibitors or nonsteroidal mineralocorticoid receptor antagonists. 
Yet, there continues to be a growing demand for further exploration into the pathophysiological processes and potential therapeutic interventions. Reliable biological models play a crucial role in facilitating this research. Given the multifaceted nature of renal disease, which encompasses not only cell biology but also vascular microanatomy and endocrine signaling, an appropriate model must possess a level of biological complexity that only an animal model can offer, rendering rodents an obvious choice.
This manuscript, therefore, provides an intricate, systematic protocol for surgically reducing renal parenchyma through midline laparotomy and combined total and partial nephrectomy in rats for survival as well as non-survival applications. It emphasizes the critical role of precise surgical techniques in ensuring consistent and reliable outcomes. Prime examples of potential applications for this model include biomolecular and pharmaceutical studies as well as the development of innovative intraoperative imaging modalities, such as hyperspectral imaging, to objectively visualize and differentiate renal malperfusion.

This manuscript introduces a standardized protocol for a 5/6 nephrectomy by sharp bipolectomy via midline laparotomy in a rat model, aiming to induce a state of renal insufficiency via renal parenchyma reduction with a great degree of methodical precision and low risk of technical error.

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the ...

Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model

Source: Wang, M., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63719">Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion.</a> J. Vis. Exp. (2022)
This video demonstrates a procedure for renal artery sympathetic denervation in an angiotensin II-induced hypertensive mouse model, using phenol to disrupt sympathetic nerve function and reduce blood pressure.

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

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