Authors thank E. Ferrantelli and G. Liappas for their support setting the 5/6 nephrectomy protocol, R. S&#225;nchez-D&#237;az and P. Mart&#237;n for the assistance with ureic nitrogen assessments, and E. Hevia and F. N&#250;&#241;ez for the assistance with mice care. This work was supported by grants SAF2016-80648R from the &#34;Ministerio de Econom&#237;a y Competitividad&#34;/Fondo Europeo de Desarrollo Regional (MINECO/FEDER) to Manuel L&#243;pez-Cabrera and PI 15/00598 from&#160;Fondo de Investigaciones Sanitarias (FIS)-FEDER funds, to Abelardo Aguilera.

All methods described here have been approved by the Institutional Animal Care and Use Committee of the Molecular Biology Center Severo Ochoa (Madrid, Spain).
Note: C57BL/6J female mice aged 12 to 14 weeks and weighing approximately 20 g at the start of the study were used. All the animals were housed under standard conditions and were given food and water ad libitum. Health conditions were checked daily.&#160;The material required, such as gloves, drape, catheter, suture and needles, should be sterile.
1. Placing the Catheter
Note: If the kidneys are not removed, they remain completely functional, so it does not consider the effect of uremia, thus permitting the study of PDF exposure in isolation. The surgery consists on introducing only the distal extreme of the catheter into the peritoneal cavity and placing the access port on the back of the animal, providing access to it. The procedure to place the catheter is as follows:
<ol>
	<li>Place the mouse in an induction chamber and provide anesthesia using 4% isoflurane and oxygen with a flow rate of 0.4 L/min until loss of righting reflex.
	<ol>
		<li>Maintain the animal with 2% isoflurane in 100% oxygen with a flow of 0.3 L/min by means of a nosecone tube connected to the anesthesia apparatus. Confirm proper anesthetization by assessing muscular tone and response to stimulation.</li>
		<li>Check the rate and depth of respiration during all the process. Use vet ointment on eyes to prevent dryness while under anesthesia. It is preferable if the procedures are done in a flow cabinet to ensure the maintenance of sterile conditions during surgeries.</li>
	</ol>
	</li>
	<li>Shave the right flank and the back of the animals in order to perform the surgery and to later inject the fluid at the access port in a clean area. Place the animal in a lateral position resting on its left flank in the surgical table, with a thermal system to ensure that its temperature will not fall.</li>
	<li>Disinfect the area with 1% chlorhexidine gluconate solution. Make a small cut (0.5 cm) with blunt scissors in the skin on the right flank of the body, and separate it carefully with the help of the scissors from the adjacent muscle layer, so that the whole area of the back of the animal is well separated to later be able to introduce the port of access with ease. Please reference Figure 1 to see the materials required to follow the procedure.</li>
	<li>Make a small incision of about 1 mm in diameter through the muscle layer and insert the tip of the catheter and the first plastic ring. The peritoneal damage caused is minimal.</li>
	<li>Suture the peritoneal wall tightly around the catheter, with a 5.0 or 6.0 non-absorbable suture. One plastic ring is then located inside the peritoneal cavity, and the other between the muscle and the skin. The catheter is thereby fixed to prevent fluid from leaking into the subcutaneous space.</li>
	<li>Insert the access port into the subcutaneous space towards the tail of the mouse, without securing it to a fix position to the skin, as it could itch and the animals may scratch and bit their skin.</li>
	<li>Close the wound of the skin with a 5.0 or 6.0 non-absorbable suture. Remove the inhalation anesthesia and allow the animal to recover consciousness. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. When fully recovered, mouse can be returned to the company of other animals. 
	Note: The experiments can begin after 4 to 7 days of postoperative recovery.</li>
	<li>Provide analgesia by dissolving 3 mL of ibuprofen (20 mg/mL) in 250 mL of drinking water for the day of the surgery.</li>
	<li>During the post-surgical period, check the health status of the animal daily, checking that there are no reddened areas on the skin, bristly hair or wounds.</li>
	<li>Inject the fluid by holding the animal (without anesthetizing it) by the tail and grabbing the access port with one hand, and the needle with the other. Disinfect the area with 1% chlorhexidine gluconate solution before injection. It is interesting to use special needles (Huber needles), that are beveled in order to part instead of pierce the silicone septum of the access port (Figure 1A). Two injections per day during 40 days are enough to observe peritoneal alterations (Figure 3).</li>
	<li>When finishing the experiment, euthanize the mouse by carbon dioxide asphyxiation or cervical dislocation.</li>
</ol>
2. Performing a 5/6 nephrectomy and placing the catheter
Note: To better resemble the situation in PD patients it is possible to perform a 5/6 nephrectomy, allowing only a residual renal function. In this case, serum samples should be taken to analyze urea levels by extracting 250&#8201;&#956;L of blood via facial vein puncture, at least one day before starting the surgeries, at the middle of the treatment and when sacrificing the animals.It is preferable if the procedures are done in a flow cabinet to ensure the maintenance of sterile conditions during surgeries.
<ol>
	<li>Anesthetize the mice by using isoflurane as in Step 1.1.</li>
	<li>Provide analgesia with 0.1 mg/kg of Buprenorphine, injected subcutaneously at the neck of the animal&#160;and by dissolving 3 ml of ibuprofen (20mg/ml) in 250 ml of drinking water for the day before and the day of the surgery.</li>
	<li>Shave the laterals and the back of the animals in order to perform the surgeries and to later inject the fluid at the access port in a clean area.</li>
	<li>Perform an incision of approximately 0.5 cm in the skin, at the left side, close to the ribs, to have direct access to the left kidney.</li>
	<li>Open a small incision in the muscle to take the left kidney out of the peritoneum, removing the capsule and the adrenal gland. To remove the capsule is necessary to better hold the kidney outside the peritoneal cavity.</li>
	<li>Burn and cut the extremes of the kidney with a cauterizer (see Figure 1 for materials needed).</li>
	<li>Reintroduce the kidney into the peritoneal cavity and suture the wounds at the muscle and the skin with insoluble 5.0 or 6.0 suture.</li>
	<li>The day after, completely remove the right kidney and insert the catheter using the same incision as to remove the kidney. Again, anesthetize the mouse with isoflurane and subcutaneously inject 0.1 mg/kg of Buprenorphine just before the surgical procedure.&#160;Also dissolve 3 ml of ibuprofen (20mg/ml) in 250 ml of drinking water the day before and the day of the surgery.</li>
	<li>Make an incision in the skin of about 0.5 cm and, with the help of the scissors, separate the skin at the back of the animal from the muscle to open the space where the access port will be located.</li>
	<li>Perform a cut in the muscle (about 0.3-0.4 cm) to take the right kidney out of the peritoneal cavity.</li>
	<li>Remove the capsule and the adrenal gland to have better access to the kidney. Ligate&#160;kidney vein, artery, and ureter with non-absorbable 5.0 or 6.0 suture and remove the kidney completely.</li>
	<li>Suture the wound at the peritoneal muscle, introducing the end of the catheter so that the muscle must remain between the two plastic rings, as explained before (step 1.5).</li>
	<li>Introduce the access port into the subcutaneous space and suture the skin as explained in steps 1.6 and 1.7. 
	Note: Mice should rest for at least 10 days from these surgeries to ensure that the wounds at the peritoneal muscle are completely healed and there will be no leakage into the subcutaneous space while injecting the fluid. Inject the fluid as in step 1.10.</li>
	<li>When finishing the experiment, euthanize the animals by carbon dioxide asphyxiation or cervical dislocation.</li>
</ol>

The authors have nothing to disclose.

The first published data analyzing PD alterations by using a &#34;close system&#34; technique was performed in 200929 . This close system means that the entire device is located inside the body and fluid is injected with a needle through an access port. The most important technical problem in long-term animal models of fluid infusion through a catheter is the occurrence of obstruction. Possible options are to perform omentectomy or add heparin to the PDFs to reduce peritoneal adhesions. Neverthele...

Kidney Function and Renal Disease
Kidneys are essential organs involved in homeostasis, blood filtration and hormone production. There are various conditions that lead to kidney failure and to the subsequent onset of uremia, which has been defined as the group of systemic symptoms due to the accumulation of waste products in the blood retained due to kidney function disorders1. Moreover, since homeostatic capability is also affected when there is a renal failure, hypertension due to volume overload may occur, which is also dangerous as it can lead to heart failure1. When the functional capability of kidneys is less than 10%-15%, the patient must undergo one of the following therapeutic options: hemodialysis, peritoneal dialysis (PD) or renal transplantation.
PD is an interesting option that allows patients to continue treatment from the comfort of their home or practically anywhere, thus avoiding the need for frequent hospital visits and stays. The PD technique eliminates small toxic molecules and excess water generated by the body2 through the instillation of an osmotic fluid (peritoneal dialysis fluid, PDF) into the peritoneal cavity. This instillation generates the osmotic gradient necessary for the exchange of solutes and water between the peritoneal capillary and PDF, a process known as ultrafiltration (UF).
Peritoneal Injury Induced by Peritoneal Dialysis
The peritoneal cavity is covered by a membrane (PM) composed of a monolayer of mesothelial cells resting on a matrix, which also houses few blood vessels, fibroblasts, macrophages and other cell populations. Unfortunately, the peritoneal membrane always suffers some alterations during PD treatment, such as apoptosis and loss of mesothelial cells, mesenchymal transition of mesothelial (MMT) and endothelial (end-MT) cells, recruitment of inflammatory cells and fibrocytes, vascular alterations, angiogenesis, lymphangiogenesis and/or fibrosis3,4,5,6,7,8,9. These alterations are responsible for the development of an UF capacity failure10, which precludes the continuation of the therapy, requiring that the patient must receive an alternative treatment to survive (hemodialysis or renal transplantation). Therefore, for these patients, it is essential to delay or control the development of these peritoneal alterations.
It has been speculated that uremia alone may cause inflammation11, but the most important local factor is PDF bioincompatibility. Most PDFs use glucose as the osmotic agent, which causes inflammation. Due to PDF storage times and sterilization, glucose suffers a process of degradation, and new products from this reaction appear, generating more inflammation, MMT and apoptosis12,13. Moreover, there is also the possibility of mechanical damage due to the instillation method. All these factors, acting continuously, may generate a persistent and recurrent inflammatory state, leading to chronic inflammation, which drives to membrane deterioration and, conclusively, UF failure. How this damage could be reduced or avoided is still a matter of study.
Analyzing the Development of Lesions: From Human Samples to Animal Models
Working with human biopsies is a limiting factor due to the difficulty in obtaining tissue samples. These samples can only be obtained from surgeries performed due to catheter malfunction or transplantation, usually after years of PD treatment. This approach is useful for the analysis of pathological changes suffered by a peritoneal membrane exposed to PDF, but is not sufficient to study the development of the process. Another possibility is to analyze cells drained from dialysis effluent, but this still fails to provide a complete scenario. Merging both techniques is only possible with animal models. The peritoneal structure is similar among mammals, and therefore there are models with different animal species. There are a few studies based on sheep (Rodela et al.14 and Barrell et al.15) and rabbit16,17 models; however, smaller animals are preferable as they are easier to house and maintain, and are also more economical. The use of rats18,19,20,21,22,23,24 offers a shorter treatment time needed to observe morpho-functional alterations. It has represented a very useful model to explore different issues such as the effect of anti-fibrotic drugs as for example BMP-7 (bone morphogenic protein-7)25 and RAS (renin-angiotensin system) targeting26,27,28.
However, the murine model has emerged as an ideal model with many benefits over others. The most interesting advantage is the possibility of using genetically modified mice to study the molecular and cellular basis of peritoneal damage. In fact, mice are often employed for the analysis of numerous diseases, as there are many different strains with various well-known genetic backgrounds. Other advantages include the reduced space required for housing, reduced cost of experiments (due to the animals&#39; smaller size), ease of handling, the availability of reagents and the increasing amount of available information on the different strains of mice since they have been most commonly used animals in research.
A mice-based model employing an implanted device has been the most recently established model for PD29,30, and has been shown to mimic peritoneal deterioration suffered by PD patients due to exposure to PDFs. This model has collaborated to understand the pathological processes implicated31,32,33. Moreover, it has been used to validate various potential treatments for ameliorating this deterioration using immune modulators and anti-inflammatory drugs and other anti-fibrotic and anti-angiogenic agents, such as COX-2 (cyclooxygenase-2) inhibitors34, PPAR-&#947; (peroxisome proliferator-activated receptor-&#947;) agonists35, Tamoxifen36, Paricalcitol (a vitamin D receptor activator that modulates the immune reaction)37, Rapamycin38 and Nebivolol39.
Developing the Mouse Model with an Implanted Catheter
The goal of this model is to resemble, as much as possible, the technique used in human PD patients, allowing to perform extended treatments of PD in small animals. So far, three techniques for instillation of dialysis fluid into the peritoneum have been tested in mice. The first one, blind puncture of the front abdominal wall, is controversial due to the multiple risks that it may incur, such as peritoneal damage, bleeding and, as is blindly performed, visceral puncture. The second technique is the so-called &#34;open permanent system&#34;, in which the device for injecting the fluid is placed outside the body. This procedure is most similar to that performed in humans. However, it does not allow the development of long-term experiments, as it may increase the chances of infection, and generally requires the use of anesthesia to instill PDF, which may interfere with the results. The third technique is the &#34;closed system&#34;. With this approach, the entire device used for fluid instillation is located inside the animal&#39;s body. Fluid is injected with a needle through an access port, which is placed subcutaneously. This procedure reduces the risk of peritoneal infection and bleeding as well as the need for anesthesia.
To study the effect of uremia in PD, a recent murine model has also been stablished40 based on the PDF infusion model with catheter. This model brings in a novel technique to perform a nephrectomy in mice, thus reducing renal function. In the present article, a modification of the protocol employed by Ferrantelli et al. in 201540 has been developed. This new protocol allows catheter implantation while nephrectomy, reduces the length of the wound inflicted during surgery and facilitates access to the kidneys.

<table><tbody><tr><td>Minute Mouse Port 4French with retention beads and cross holes</td><td>Access technologies</td><td>MMP-4S-061108A</td><td></td></tr><tr><td>Posi-Grip Huber point needles 25 ga. X 1/2&amp;acute;&amp;acute;&amp;nbsp;</td><td>Access technologies</td><td>PG25-500</td><td></td></tr><tr><td>High Temperature Cautery Kit</td><td>Bovie</td><td>18010-00</td><td></td></tr><tr><td>Forane</td><td>abbVie</td><td>880393.4 HO</td><td></td></tr><tr><td>non absorbable suture 6/0</td><td>Laboratorio Agar&amp;oacute;</td><td>6121</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14079-10</td><td></td></tr><tr><td>forceps</td><td>Fine Science Tools</td><td>11002-12</td><td></td></tr><tr><td>clamp</td><td>Fine Science Tools</td><td>13002-10</td><td></td></tr><tr><td>Buprenorphine 0,3 mg/ml</td><td></td><td></td><td>pharmaceutical product</td></tr><tr><td>cotton swabs</td><td></td><td></td><td>pharmaceutical product</td></tr><tr><td>Dalsy (Ibuprofen) 20mg/mL oral suspension</td><td>AbbVie&amp;nbsp;S.R.L.&amp;nbsp;</td><td></td><td>pharmaceutical product</td></tr></tbody></table>

surgical techniques for catheter placement and 5/6 nephrectomy in murine models of peritoneal dialysis

Peritoneal dialysis (PD) is a renal replacement therapy consistent on the administration and posterior recovery of a hyperosmotic fluid in the peritoneal cavity to drain water and toxic metabolites that functionally-insufficient kidneys are not able to eliminate. Unfortunately, this procedure deteriorates the peritoneum. Tissue damage triggers the onset of inflammation to heal the injury. If the injury persists and inflammation becomes chronic, it may lead to fibrosis, which is a common occurrence in many diseases. In PD, chronic inflammation and fibrosis, along with other specific processes related to these ones, lead to ultrafiltration capacity deterioration, which means the failure and subsequent cessation of the technique. Working with human samples provides information about this deterioration but presents technical and ethical limitations to obtain biopsies. Animal models are essential to study this deterioration since they overcome these shortcomings.
A chronic mouse infusion model was developed in 2008, which benefits from the wide range of genetically modified mice, opening up the possibility of studying the mechanisms involved. This model employs a customized device designed for mice, consisting of a catheter attached to an access port that is placed subcutaneously at the back of the animal. This procedure avoids continuous puncture of the peritoneum during long-term experiments, reducing infections and inflammation due to injections. Thanks to this model, peritoneal damage induced by chronic PD fluid exposure has been characterized and modulated. This technique allows the infusion of large volumes of fluids and could be used for the study of other diseases in which inoculation of drugs or other substances over extended periods of time is necessary.
This article shows the method for the surgical placement of the catheter in mice. Moreover, it explains the procedure for a 5/6 nephrectomy to mimic the state of renal insufficiency present in PD patients.

Figure 1 shows all the materials required to follow the procedures described in protocol section. For this example, mice submitted or not to nephrectomy (8 animals per group) (Figure 2) were exposed during 40 days (two injections per day, waiting at least 2 hours between both) to a mixture of two different PDFs, commonly used in the clinical practice: Extraneal (icodextrin-based PDF) and Dianeal (glucose-based PDF). A group with ...

Watch this Scientific Journal Video about Surgical Techniques for Catheter Placement and 5/6 Nephrectomy in Murine Models of Peritoneal Dialysis at JoVE.com

Surgical Techniques for Catheter Placement and 5/6 Nephrectomy in Murine Models of Peritoneal Dialysis

This article shows the method for the surgical placement in mice of an intraperitoneal catheter attached to an access port that is positioned at the back of the animal. Moreover, it explains the procedure for a 5/6 nephrectomy to resemble the uremic state of PD patients.

Peritoneal dialysis (PD) is a renal replacement therapy consistent on the administration and posterior recovery of a hyperosmotic fluid in the peritoneal ...

surgical-techniques-for-catheter-placement-56-nephrectomy-murine

Research

JoVE Journal

Engineering

15.2K Views.  Spanish National Research Council. This article shows the method for the surgical placement in mice of an intraperitoneal catheter attached to an access port that is positioned at the back of the animal. Moreover, it explains the procedure for a 5/6 nephrectomy to resemble the uremic state of PD patients.

Video: Surgical Techniques for Catheter Placement and 5/6 Nephrectomy in Murine Models of Peritoneal Dialysis

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Biochemistry

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Medicine

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Bioengineering

A Modified Surgical Technique for Kidney Transplantation in Mice

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

This protocol presents a new surgical technique of mouse kidney transplantation focusing on a modified arterial anastomosis strategy. A vascular suture technique including a simple and safer ureter-bladder anastomosis method is also presented. These modifications shorten the operation time and improve the success rate of the mouse kidney transplantation procedure.

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this ...

Immunology and Infection

A Retrograde Implantation Approach for Peritoneal Dialysis Catheter Placement in Mice

Murine models are employed to probe various aspects of peritoneal dialysis (PD), such as peritoneal inflammation and fibrosis. These events drive peritoneal membrane failure in humans, which remains an area of intense investigation due to its profound clinical implications in managing patients with end-stage kidney disease (ESKD). Despite the clinical importance of PD and its related complications, current experimental murine models suffer from key technical challenges that compromise the models&#39; performance. These include PD catheter migration and kinking and usually warrant earlier catheter removal. These limitations also drive the need for a greater number of animals to complete a study. Addressing these drawbacks, this study introduces technical improvements and surgical nuances to prevent commonly observed PD catheter complications in a murine model. Moreover, this modified model is validated by inducing peritoneal inflammation and fibrosis using lipopolysaccharide injections. In essence, this paper describes an improved method to create an experimental model of PD.

This article describes modifications of a procedure to implant a peritoneal dialysis catheter in a murine model to avoid major technical issues observed with the conventional techniques.

Murine models are employed to probe various aspects of peritoneal dialysis (PD), such as peritoneal inflammation and fibrosis. These events drive ...

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 ...

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 ...

Biology

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 ...

Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells

Source: Murphy, K. A. et al. <a href="https://www.jove.com/video/55080" target="_blank">A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies.</a> J. Vis. Exp. (2017)
In this video, we develop an orthotopic mouse model of renal cell carcinoma. This model results in reproducible primary tumors in the injected kidney and metastatic tumors in the lung, allowing the evaluation of disease progression and treatment.

Generowanie modelu raka nerkowokomórkowego: protokół rozwoju ortotopowego mysiego modelu RCC poprzez donerkową implantację komórek rakowych

Source: Murphy, K. A. et al. A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical ...

Surgical Techniques for Catheter Placement and 5/6 Nephrectomy in Murine Models of Peritoneal Dialysis

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Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

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Generowanie modelu raka nerkowokomórkowego: protokół rozwoju ortotopowego mysiego modelu RCC poprzez donerkową implantację komórek rakowych