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

Summary

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.

Abstract

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.

Introduction

Chronic kidney disease (CKD) is a progressive condition that affects a significant portion of the global population. It is characterized by the gradual loss of endocrine and filtrative kidney function over time, leading to the accumulation of waste products and fluid in the body and an imbalance in the endocrine system. Recent data suggest that 9.1% to 13.4% of the worldwide population (between 700 million and one billion people) has CKD¹. The prevalence of CKD increases with age, affecting around 34% of people aged 65 years or older in the United States, compared to 12% in those aged 45-64 years and 6% in those aged 18-44 years².

Therefore, CKD is a significant contributor to the global burden of disease and mortality rates. Early detection and management of CKD are crucial in slowing its progression and reducing the risk of complications, such as cardiovascular disease, anemia, and ultimately, end-stage renal disease, which requires dialysis or kidney transplantation for survival³.

Therapeutic interventions for end-stage CKD have undergone a remarkable evolution over the past few decades. Historically, the management of end-stage CKD was limited to supportive care, with dialysis emerging as a life-sustaining modality in the 1960s. Since then, significant advancements have been made in dialysis techniques, including the development of more biocompatible membranes, improved vascular access, and the advent of peritoneal dialysis⁴. Additionally, kidney transplantation has emerged as the optimal treatment for end-stage CKD, offering improved survival and quality of life compared to dialysis⁵. However, the shortage of donor organs remains a significant challenge, driving research into novel strategies such as xenotransplantation and regenerative medicine approaches. Furthermore, the management of end-stage CKD-associated complications, such as secondary hyperparathyroidism, has been enhanced by the introduction of calcimimetic agents like etelcalcetide, which effectively modulate parathyroid hormone levels⁶.

Despite these advancements, the quest for more effective and targeted therapies continues, fueled by ongoing research into the molecular mechanisms underlying end-stage CKD progression and associated comorbidities. Therefore, CKD persists as a significant concern in patient care, prompting a continued need for extensive research into biomedical processes and therapeutic approaches. Robust biological models are essential to facilitate such investigations. Given the multifaceted nature of CKD, which encompasses aspects ranging from cellular biology to interorgan endocrine signaling, vascular functional anatomy, and rheology, an ideal model must possess a level of biological complexity that only a comprehensive model organism can provide. Thus, rodents emerge as the preferred model due to their capacity to encompass these various biological dimensions effectively.

The 5/6 nephrectomy remnant kidney model serves as a common tool in CKD research for rat and murine experiments due to its stable induction of renal insufficiency^{7,8,9,10,11,12,13,14}. This model entails the removal of one entire kidney and 2/3 of the other. The creation of the remnant kidney can be achieved through the surgical resection of renal poles, termed the polectomy model, or by ligating superior and inferior segmental renal arteries, resulting in pole infarction^{7,15,16,17,18,19,20}.

While this 5/6 nephrectomy model with polectomy is an established technique, it has only been introduced as a transparent and comprehensible protocol with a dorsolateral retroperitoneal access²¹. This access can be advantageous for a unilateral procedure with renal parenchyma reduction on just one side or for a two-stage procedure with a temporal distance of a few days in order to increase postoperative survival of the animal²². However, the utilization of a midline laparotomy approach offers distinct advantages over the conventional laterodorsal retroperitoneal access route.

By employing a single midline abdominal incision, the surgeon gains unimpeded access to the entire abdominal cavity, thereby facilitating a comprehensive exploration and manipulation of the intra-abdominal organs. This expanded surgical field not only streamlines the nephrectomy procedure, but also enables the concurrent execution of additional interventions that may be required for specific experimental protocols, for example, procedures on the ureters, such as ligation, resection, or reconstruction, which may be essential for studying the pathophysiology of obstructive uropathy. Furthermore, this approach permits the simultaneous resection or manipulation of other abdominal organs, such as the liver, spleen, or gastrointestinal tract, thereby expanding the scope of experimental investigations into multi-organ interactions or systemic disease models.

Moreover, the midline laparotomy approach facilitates the construction of an ileum conduit or neobladder, a surgical procedure that involves the creation of a urinary diversion using a segment of the ileum, which is particularly relevant in studies investigating bladder dysfunction or reconstructive urology techniques. This versatility in combining nephrectomy with other surgical interventions within the same operative field not only streamlines experimental protocols but also minimizes cumulative surgical trauma and associated risks to the animal subjects. Therefore, in the case of single-stage bilateral renal surgery or simultaneous additional intraabdominal procedures, the ventral access via midline laparotomy should be the preferred option.

Currently, there is no publication or protocol available describing this surgical strategy. Therefore, with this work, our objective is to present a detailed procedural guide for conducting renal resection and surgical induction of CKD via midline laparotomy in rats, applicable to both survival and non-survival studies. This experimental model creates a regulated environment conducive to investigating the complex dynamics of CKD, mimicking clinically significant scenarios. This protocol was specifically designed to illustrate the surgical technique. The intervention was therefore performed in a non-survival setting on a homogeneous group of 10 male rats. As there was no meaningful reason for the comparison to a baseline or alternative intervention, the inclusion of a control group was not necessary. 5/6 nephrectomy explicitly refers to the extent of surgical parenchyma resection. This certainly translates to a functional reduction in the sense of a reduction of glomerular filtration rate. However, the exact functional degree cannot be predicted but will have to be measured individually for each animal, for example, by using inulin or p-aminohippuric acid clearance^23,24 if required.

Protocol

All animal procedures outlined in this document were carried out within accredited facilities and have been granted approval by the institutional animal care and use committee (IACUC) of the Baden-Württemberg Regional Council in Karlsruhe, Germany (35-9185.81/G-62/23). Experimental animals were handled in accordance with institutional protocols and in compliance with German legislation governing animal welfare, as well as adhering to the guidelines set forth by the European Community Council (2010/63/EU) and the ARRIVE guidelines. Male Sprague Dawley rats with an initial weight of 400 g were utilized following a 1-week acclimatization period.

1. Anesthesia and analgesia

1. Narcotize the rat model with the pharmaceuticals of choice. To follow this protocol, perform volatile induction of sedation with isoflurane followed by an intraperitoneal injection of 100 mg/kg body weight ketamine for dissociative anesthesia and 4 mg/kg body weight xylazine. Achieve analgesia with subcutaneous injections of 5 mg/kg body weight carprofen.
   NOTE: A detailed protocol can be found in the cited literature²⁵.
2. Ensure adequate analgesic depth by looking for pain reflexes during the toe pinch test with surgical forceps and regularly reassess the depth of anesthesia during the procedure.
3. Apply ophthalmic lubricant to the eyes to prevent corneal desiccation.

2. Procedure preparation

1. Prepare the operating site with all the materials and instruments required, including polyfilament ligatures, silicone vessel loops, blunt overholt clamps, fine preparation scissors and forceps, as well as hemostatic patches cut into 0.8 x 0.6 cm pieces (Figure 1A-G). Prepare the rodent surgical exposure apparatus, a heating pad, and the surgical preparation hooks as specified in cited literature²⁵.
2. Shave the length of the desired access, disinfect the surgical site by three alternating scrubs with 70% ethanol and iodine-based or chlorhexidine-based scrubbing swabs in a circular motion, and achieve proper oxygenation via the inhalation of 100% oxygen using a neonatal face mask (Figure 1H-J). Cover the rest of the body outside the surgical situs with drapes to avoid contamination.
   NOTE: The representative animal that was used to obtain figure images was not draped in order to permit better anatomical landmark visualization.
3. Perform a median mini-laparotomy via an initial median cutaneous incision over the desired abdominal length of ~3 cm and a subsequent slightly smaller incision of the fascia along the linea alba (Figure 1K-M).
4. Achieve surgical exposure of the kidney using surgical compresses and surgical preparation hooks (Figure 1N-O). Only touch the renal parenchyma with atraumatic preparation instruments such as a moistened cotton swab or a moistened compress using forceps or blunt overholt clamps.

Figure 1: Experimental instruments, materials, and setup. (A) Surgical instruments required; (B) polyfilament ligature; (C) silicone vessel loop; (D) fine preparation scissors. (E-G) Hemostatic patch cut into 0.8 x 0.6 cm pieces. (H-J) Rat model shaven and oxygenated with face mask. The representative animal that was used to obtain figure images was not draped in order to permit better anatomical landmark visualization. (K,L) Median cutaneous incision over the desired abdominal length of ~3 cm. (M) Median mini-laparotomy; (N) exposure of the left kidney using a surgical compress, surgical preparation hooks, and a metal stand; (O) analogous exposure of the right kidney and resection of the Gerota's fascia. Please click here to view a larger version of this figure.

3. Partial nephrectomy

1. Expose the respective kidney, grab the perirenal fat that is attached to the thin Gerota's fascia, and apply some tension to locally lift the fascia off the renal parenchyma (Figure 2A,B.2).
2. Incise and undermine the fascia with the sharp end of one scissor's edge and continue with a longitudinal dissection of the Gerota's fascia (Figure 2B).
3. Perform a blunt degloving of the Gerota's fascia using closed scissors by gradually undermining the fascia all around the parenchyma and folding the fascial capsule medially-optionally partially resecting it (Figure 2C).
4. Sling the renal hilum using a silicone vessel loop for improved vascular control (Figure 2D).
5. Place the tip of the forceps into the retroperitoneal space to stabilize the kidney to avoid a dorsal escape of the kidney during the cutting process and perform the cranial sharp 1/3 renal polectomy in one precise aimed stroke using scissors (Figure 2E).
   NOTE: The dissection line should be chosen by clinical evaluation so that it separates the organ in 1/3 of organ height cranially and caudally.
6. Achieve hemostasis by applying a hemostatic patch, by manual compression, by compression using blunt instruments, or by hilar tension via the silicone vessel loop, effectively reducing hilar blood flow (Figure 2F-J).
   NOTE: Hemostasis was regularly achieved by simultaneous pulling of the vascular loop and the application of the hemostatic patch for 2.5-3 min.
7. Follow with a caudal sharp 1/3 renal polectomy in an analogous fashion (Figure 2K-N). For both scissor strokes, aim for a slightly angulated dissection plane that will leave more renal parenchyma at the hilar side and less tissue on the lateral side to avoid unintended hilar injury and reduce urinary leakage from the pelvicocaliceal system (Figure 2O). If a higher standardization of precise 5/6 parenchyma resection is desired, weigh the resectate of the total nephrectomy on the contralateral side described further below, weigh the resectates of the partial 2/3 nephrectomy, and repeat the dissection in a "salami-slicing" technique until exactly 2/3 of weight are obtained.

Figure 2: Partial nephrectomy. (A) Surgical exposure of one kidney. (B) Longitudinal incision of the Gerota's fascia using sharp scissors. (C) Blunt degloving of the Gerota's fascia using closed scissors. (D) Slinging the renal hilum using a silicone vessel loop. (E) Cranial sharp 1/3 polectomy using scissors and forceps as guidance. (F) Achieving hemostasis by applying an hemostatic patch; (G) achieving hemostasis by manual compression; (H-J) achieving hemostasis by compression using blunt instruments and hilar tension via the silicone vessel loop. (K-N) Caudal sharp 1/3 polectomy in analogy. (O) Schematic depiction of recommended dissection planes to avoid unintended hilar injury (black lines). Please click here to view a larger version of this figure.

4. Total nephrectomy

1. Mobilize the kidney in analogy to the steps described above and tunnel the renal hilum using blunt overholt clamps (Figure 3A,B).
2. Sling the renal hilum using a polyfilament ligature and place a secure sliding knot on the renal hilum rather closer to the abdominal vessels to occlude the renal blood flow and ureter (Figure 3C,D).
3. Sharply dissect the hilum using scissors and remove the kidney (Figure 3E,F).
4. Control for hemostasis and cut off the ligature ends (Figure 3G-J).

Figure 3: Total nephrectomy. (A) Surgical exposure of the contralateral kidney; (B) analogous removal of the Gerota's fascia and tunneling of the renal hilum using blunt overholt clamps; (C) slinging the hilum using a polyfilament ligature. (D) Placement of a sliding knot ligature on the renal hilum; (E,F) sharp dissection of the hilum using scissors and removal of the kidney; (G-I) control for hemostasis and cutting of the ligature ends. (J) Schematic depiction of recommended ligature height (dashed line) and dissection plane (black line). Please click here to view a larger version of this figure.

5. Abdominal wall closure

1. Place a corner suture on the abdominal fascia using a polyfilament suture (Figure 4A-D).
2. Continue suturing the abdominal fascia with a running suture with ~2 mm of tissue grabbed with each bite and about 4 mm between the bites (Figure 4E-I).
3. Suture the cutaneous layer using single stitches with ~3 mm of tissue grabbed with each bite and ~6 mm between the bites (Figure 4J-Q).

Figure 4: Abdominal wall closure. (A-D) Placement of a corner suture on the abdominal fascia using a polyfilament suture. (E-I) Running suture of the abdominal fascia; (J-Q) suturing of the cutaneous layer using single stitches. Please click here to view a larger version of this figure.

6. Further steps

1. Depending on the desired scenario, research purpose, and the degree of renal insufficiency desired, consider deviations from this protocol, including a variation in the amount of resected renal parenchyma, for example, unilateral total nephrectomy (3/6 nephrectomy), bilateral total nephrectomy (6/6 nephrectomy), unilateral monopolectomy (1/6 nephrectomy), bilateral monopolectomy (2/6 nephrectomy), unilateral bipolectomy (2/6 nephrectomy), and bilateral bipolectomy (4/6 nephrectomy).
2. In general, either euthanize the animals by sharp cardiectomy without prior abdominal wall closure for non-survival applications or perform stepwise abdominal closure as described above in case of planned survival experiments with follow-ups.
3. In the case of survival applications, maintain sterile conditions at all times during surgery. Before surgery, aseptically prepare skin at the surgical site using both a scrub (iodine-based or chlorhexidine-based) and alcohol. Use a fresh autoclaved pack of instruments for each animal.
4. Post surgery, monitor the animal until it is conscious enough to maintain sternal recumbency and isolate it until fully recovered.
5. As postsurgical treatment of the animal, include daily visits through medical staff as well as postsurgical pain treatment using subcutaneous injections of 5 mg/kg body weight carprofen 2x a day for 2 days.
6. Use score sheets with clearly defined termination criteria for the duration until full postoperative recovery. Use the Rat Grimace Scale²⁶ or the Body Condition Score²⁷ from cited literature. Terminate the experiment if Grimace Score ≥ 6 or a Body Condition score = 1 after application of carprofen. Additionally, terminate in case of a surgical complication such as a postoperative wound infection or abdominal wall closure insufficiency.

Results

This protocol was conducted in 10 male rats (mean weight 398 ± 35 g) in a non-survival setting and the procedure was performed by a third-year surgical resident. The success rate defined by survival over 20 min after abdominal wall closure was 100%. The mean duration of the preparation from skin incision until skin closure was 18 min 34 s ± 7 min 31 s.

Unfortunately, due to the non-survival nature of this manuscript, there are no data on the postoperative renal function. Future anima...

Discussion

CKD is defined by kidney damage or reduced kidney function for at least 3 months, regardless of the cause^28,29. Kidney damage encompasses pathologic anomalies in the native or transplanted kidney, identified via imaging, biopsy, or deduced from clinical markers like increased albuminuria (albumin-to-creatinine ratio > 30 mg/g or 3.4 mg/mmol) or urinary sediment alterations. Reduced kidney function implies a reduced glomerular filtration rate, which is usually...

Materials

Name	Company	Catalog Number	Comments
atraumatic preparation forceps	Aesculap	FB395R	DE BAKEY ATRAUMATA atraumatic forceps, straight
blunt overholt clamp	Aesculap	BJ012R	BABY-MIXTER preparation and ligature clamp, bent, 180 mm
cannula	BD (Beckton, Dickinson)	301300	BD Microlance 3 cannula 20 G
fixation rods	legefirm	‎500343896	tuning forks used as y-shaped metal fixation rods
heating pad	Royal Gardineer	IP67	Royal Gardineer Heating Pad Size S, 20 Watt
plastic perfusor tube	M. Schilling GmbH	S702NC150	connecting tube COEX 150 cm
polyfilament suture	Covidien	CL-769	Covidien Polysorb Braided Absorbable Suture 2-0 75 cm
preparation scissors	Aesculap	BC177R	JAMESON preparation scissors, bent, fine model, blunt/blunt, 150 mm (6")
sealing hemostat patch	Baxter	1506257	Hemopatch Sealing Hemopatch Baxter 45 x 90 mm
silicone vessel loop tie	SERAG WIESSNER	SL26	silicone vessel loop tie 2.5 mm red
Spraque Dawley rat	Janvier Labs	RN-SD-M	Spraque Dawley rat
steel plate	Maschinenbau Feld GmbH	C010206	Galvanized sheet plate, 40 x 50 cm, thickness 4.0 mm
Yasargil clip	Aesculap	FE795K	YASARGIL Aneurysm Clip System Phynox Temporary (Standard) Clip
Yasargil clip applicator	Aesculap	FE558K	YASARGIL Aneurysm Clip Applicator Phynox (Standard)