Kidney disease is a global health concern, affecting approximately 500 million individuals, or one in every ten adults worldwide in 2010 (7). Over 1.5 million of these individuals rely on life-sustaining treatments such as hemodialysis or kidney transplantation due to end-stage renal failure (4). Kidney transplantation remains the optimal therapeutic strategy, significantly enhancing survival rates and the overall quality of life in patients with end-stage kidney disease (3). Over the past few decades, survival rates have been bolstered by advances in surgical techniques, refinement of immunosuppressive regimens, and the implementation of comprehensive cellular and humoral immune response monitoring protocols, leading to an impressive one-year survival rate exceeding 95% for renal allografts (10). However, chronic allograft injury, fueled by various pathological mechanisms, persistently hampers long-term graft survival, despite the marked improvements in immunosuppression (5).

The development of a mouse model for renal transplantation has been pivotal to the understanding of the pathophysiological processes underlying human renal transplantation (14). This model has proven to be an indispensable research tool (2). This is primarily due to the thoroughly characterized mouse genome and the availability of a plethora of experimental methods and techniques that can be employed in this model. The murine model of renal transplantation has been instrumental in studying various facets of the transplantation process, including acute rejection, cellular and humoral rejection, ischemia-reperfusion injury, and the assessment of innovative therapeutic interventions (13). However, the utility of this model is contingent upon the acquisition of advanced microsurgical skills, especially for performing mouse kidney transplantation. Although there is a wealth of literature and video resources available on the topic (9,13), mastering the intricacies of the surgical procedure, particularly venous anastomosis, remains a significant challenge. Notably, studies have highlighted that venous anastomosis poses a greater difficulty than arterial anastomosis, given the weaker and more pliable structure of veins in microvascular anastomosis (6,11). Moreover, the challenges related to the limited visibility of venous anastomosis may not be restricted to the mouse renal vein alone. In the realm of hand and digit replantation, comparable difficulties arise in venous anastomosis (12). Venous anastomosis is not a task for novices. The primary rationale for this assertion lies in the inherently thinner nature of vein walls which predisposes the vein edges to inward rolling, thereby introducing the potential for errors in stitch placement (8). The author has also encountered instances where limited visibility of the mouse renal vein such as concurrent suture between anterior and posterior walls, anastomosis with the renal vein rotated 180 degrees, or hard to first suture into renal vein due to poor visualization of renal vein orifice resulted in surgical failure. To mitigate this challenge, the author proposes a two stay suture technique, designed to enhance the visualization of the renal vein’s anterior and posterior walls, thereby potentially improving the completion rate and reducing the time required for renal vein anastomosis. This study, therefore, aims to compare the effectiveness of the two stay suture technique with the conventional anchoring suture technique in facilitating renal vein anastomosis, not cover the graft function for the two suture techniques in mouse kidney transplantation.

Overview of study design

This study used 44 male Balb/c mice (8 weeks old, 25 g each) for kidney transplantation, divided equally into donor and recipient groups (22 mice each). Two surgical techniques were used for anastomosis: 11 mice received the conventional suture technique, and the other 11 mice had the two stay suture technique. This study was conducted under the approval and guidance of the IACUC of Seoul National University Hospital (AAALAC-accredited facility; IACUC number: 21-0055).

Donor kidney procurement and transplantation procedure

Anesthesia

The induction was initiated with 4% isoflurane. The absence of the withdrawal reflex was confirmed by pinching the toe, and isoflurane (1-2%) was used during the surgery (1). Throughout the procedure, the respiratory rate was monitored to prevent deep anesthesia.

Surgical approach

With the exception of the two stay suture technique for vein anastomosis, all surgical procedures below have been briefly described based on the methodology employed by the group led by Tse et al. (13).

A 2 cm midline incision was made in the abdominal cavity. The left kidney was carefully isolated by blunt dissection of surrounding tissues. The left renal vein was prepared by ligating and dividing adjacent veins, and the ureter was also ligated and divided. The renal vein was separated from the renal artery, creating an opening for anastomosis to the recipient’s inferior vena cava (IVC). Heparin (5 units) was administered to the IVC. The left kidney was perfused with a heparin saline solution. The renal vein was resected, and the adjacent aorta was divided to widen the anastomosis. The left kidney, associated vessels, and ureter were then removed from the donor.

Bench procedure

The extracted kidney was immediately placed in a 4°C saline solution (Fig. 1A, D). For the conventional anchoring suture technique, 10-0 nylon sutures were inserted from outside to inside the renal vein lumen superiorly and then separately inferiorly (Fig. 1B, E). An alternative two stay suture technique involved additional sutures on the anterior and posterior walls of the renal vein (Fig. 1C, F), enhancing the visualization and separation of the renal vein during anastomosis.

Figure 1. Bench surgery images of resected mouse donor kidney for transplantation. (A, D) depict harvested mouse donor kidneys. (B, E) illustrate the conventional anchoring suture technique for end-to-side anastomosis of the renal vein to the recipient’s IVC. (C, F) showcase the two stay suture technique, which enhances visualization and separation of the anterior and posterior walls of the renal vein during anastomosis. (D-F) represent magnified images of (A-C), respectively. Symbols: arrow, abdominal aorta; arrow head, renal vein; s, superior wall; i, inferior wall; a, anterior; p, posterior wall of the renal vein.

Preparation of recipient and its anastomosis site

The recipient’s preparation mirrored the donor surgery, including anesthesia, laparotomy, and abdominal wall retraction. The right nephrectomy was performed by ligating and removing the right renal artery, vein, and ureter. The abdominal aorta and inferior vena cava (IVC) were isolated below the renal vessels. If lumbar vessels were present at the expected anastomosis site, they were identified and ligated using 10-0 nylon suture. Finally, microvascular clamps were applied above and below the anastomosis site on both the aorta and IVC.

Vein anastomosis

An end-to-side anastomosis of the donor renal vein to the recipient IVC was performed. The IVC was punctured with a 30 G needle and the incision was further extended using fine micro scissors. The donor renal vein was affixed to the inferior corner of the IVC incision with 10-0 nylon, followed by the establishment of a running suture line between the renal vein and the IVC. Depending on the visualization objective, the stay sutures were removed either during or after the suture line was completed. This anastomosis was achieved using either a conventional anchoring stay suture technique or a two stay suture technique.

Evaluation criteria for completion or failure of vein anastomosis

The completion is determined when there are no apparent issues with the anterior and posterior aspects between the renal vein and the inferior vena cava (IVC) after anastomosis, and blood flowing smoothly into the renal vein upon reperfusion without leakage. In cases where these criteria are met, it is classified as a completion. Otherwise, if issues persist, it is classified as a failure.

Artery anastomosis

The donor aorta, including the renal artery, was connected to the recipient aorta by making an aortotomy with a 30 G needle. A 10-0 nylon running suture line was made along the lateral and medial wall of the aorta, secured against the stay stitch.

Ureteric anastomosis and abdominal closure

Two holes were made in the bladder using a 20 G needle. Curved forceps were used to pull the donor ureter through these holes, and the proximal end of the ureter was anchored to the bladder wall with two 10-0 nylon sutures.

Post transplantation managements and euthanasia

Cefazolin (300 mg/kg twice daily) and meloxicam (5 mg/kg once daily) were administered subcutaneously for up to 3 days. Upon completion of the experimental period, the mice were euthanized under deep anesthesia using 5% isoflurane. Euthanasia was performed by exsanguination through the inferior vena cava, collecting 1 cc of blood, inducing euthanasia through hypovolemic shock.

Statistical analysis

The duration of end-to-side anastomosis of the renal vein to recipient IVC was expressed as the mean ± standard deviation. Statistical differences were assessed using non-parametric Mann-Whitney U test, with significance set at p < 0.05.

The two stay suture technique for end-to-side anastomosis between the renal vein and recipient’s IVC demonstrated both expedited procedural times and a higher completion rate when compared to the conventional anchoring suture technique.

The venous anastomosis time observed for the conventional anchoring suture technique averaged at 29 ± 7 minutes. This significantly contrasted with the two stay suture technique which reported an average of 14 ± 1 minutes (Table 1). Furthermore, based on the assessment of reperfusion after anastomosis such as blood flow into the renal vein and the occurrence of blood leakage, completion or failure of vein anastomosis were evaluated. The completion rate for the venous anastomosis using the conventional anchoring suture technique was found to be 64%. The primary cause for failure in venous anastomosis was attributed to suboptimal visualization of both the anterior and posterior walls of the renal vein. This limitation prolonged the duration of venous anastomosis and led to inadvertent simultaneous suturing of the anterior and posterior walls of the renal vein during anastomosis.

Table 1 . Comparison of conventional anchoring suture technique and two stay suture technique for end-to-side anastomosis of the renal vein to the recipient’s inferior vena cava (IVC).

	Time for end-to-side anastomosis of renal vein to recipient IVC (minutes, 11 cases per each group, mean ± SD)	Completion rate of renal vein to recipient IVC anastomosis (%)	Reasons for failure of venous anastomosis
Conventional anchoring suture technique	29 ± 7	64	Four instances of anastomosis failure were observed, attributable to the inability to accurately distinguish the anterior and posterior walls of the renal vein. Consequently, both walls were concurrently sutured during anastomosis. Even among the 7 successful venous anastomoses, differentiating the anterior and posterior walls of the renal vein proved challenging, leading to extended anastomosis times (23-25 minutes) relative to the two stay suture technique.
Two stay suture technique	14 ± 1*	100

*p < 0.05..

The current study underscores the efficacy of a two stay suture technique in enhancing renal vein visibility, thereby improving the speed and completion rate of renal vein anastomosis. The characteristic thin and pliable walls of the mouse renal vein pose a challenge to its visualization post-resection. Furthermore, the traditional approach of suturing the superior and inferior walls of the renal vein during bench surgery fails to guarantee a successful anastomosis to the recipient’s inferior vena cava. This limitation arises due to tension-induced adhesion of the anterior and posterior walls of the renal vein, complicating their differentiation during anastomosis. The two stay suture technique mitigates this challenge by facilitating a clear distinction between the anterior and posterior walls via the employment of stay sutures. This innovation simplifies the placement of running sutures, thereby expediting and enhancing the likelihood of successful anastomosis.

Mouse kidney transplantation surgery, classified as a microsurgery, demands a steep learning curve. Prior research suggests that a novice requires approximately 40 transplantation practices to consistently achieve vascular anastomosis times, with an average venous anastomosis time approximating 14 minutes due to vascular anastomosis time about 28 ± 0.47 minutes (13). Such a level of proficiency is attainable under the tutelage of an experienced surgeon within a well-equipped laboratory. The author of the present study also performed over 40 non-survival surgeries on mice to acquire proficiency in the conventional suture technique. This study found that the conventional anchoring suture technique required an average of 29 minutes for venous anastomosis. In contrast, the two stay suture technique required an average of 14 minutes, comparable to the time taken by a well-resourced research team.

A limitation of this study is the reliance on visual assessment alone to determine the completion or failure of vascular anastomosis in the transplanted kidney, without the application of ultrasound examination. Regarding the evaluation of blood flow in the transplanted kidney, the author used Doppler to evaluate blood flow of graft in rat kidney transplantation. Doppler is used during the procedure to confirm blood flow with an 8 MHz probe in rat kidney transplantation. However, in mice, blood flow cannot be confirmed at 8 MHz, and a 40 MHz ultrasound device is required. Given the limitations of the current research conditions, it is practically challenging to conduct examinations using a 40 MHz ultrasound probe. Instead, the author visually assesses reperfusion by confirming arterial pulsation and observing the kidney’s color change, as well as checking for venous distension and leakage after reperfusion. It is important to note that this method does not conclusively establish blood flow in the kidney, and utilizing a 40 MHz ultrasound probe to assess renal blood flow in mouse kidney transplantation is considered the gold standard.

The two stay suture technique can be deemed a beneficial tool for murine kidney transplantation model, enabling them to visualize venous anastomosis and carry out successful venous anastomosis in a more efficient, expedient, and successful manner.

The author would like to thank Dr. S.H. Yang in SNUH for motivation for murine kidney transplantation to me.

J.M. Kim contributed to data acquisition, analysis, and interpretation, study conception and design as well as writing of the manuscript. J.M. Kim is responsible for the final approval of the manuscript.

The author has no conflicting interests.