5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Summary

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

Abstract

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

Introduction

Due to its progressive nature, ensuing end stage kidney disease, and associated cardiovascular morbidity and mortality, CKD is a growing public health problem¹. Slowing CKD progression is therefore a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. The kidney consists of a broad range of different cell types that interact with each other. This complexity cannot be mimicked in vitro.

To study novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well-known experimental model of progressive renal disease, resembling several aspects of human CKD^2,3. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is associated with progressive intrarenal capillary loss⁴, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function⁵. We used a rat strain (Lewis) that is relatively resistant to development of CKD and therefore we combined removal of 5/6th of renal mass with nitric oxide (NO) depletion^{6, 7, 8} and a high salt diet⁹. After arrival and acclimatization, animals receive a NO synthase inhibitor (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy (UNX) with continuation of L-NNA after two days. One week later, subtotal nephrectomy (SNX) i.e. removal of 2/3rds of renal mass is performed on the left side. After SNX, animals are allowed to recover for 2 days followed again by 20 mg/L LNNA in drinking water for a period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. The reason to perform the UNX on the right side and the SNX on the left side is that the renal vessels are longer on the left side which makes it easier to access the kidney without stretching the vessels too much when the kidney is exposed outside the body. In literature, models are described in which the poles of the left kidney are removed first, followed by UNX of the right kidney one week later^10,11,12. In our hands this model showed a much more rapid development of renal failure, but also a much larger variation in loss of renal function. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed, characterized by marked reduction in glomerular filtration rate (69%) and effective renal plasma flow (62%)¹³ hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD, providing a window of opportunity for evaluation of therapeutic interventions.

Protocol

All experiments are executed in accordance to the animal experimental ethical guide lines of the Utrecht experimental animal committee. The protocol is performed under the guidance and approval of the author's institution's animal care and use committee.

CKD is induced in male inbred Lewis rats (Charles River, Sulzfeld, Germany) at the age of 8 weeks. Rats are housed under standard conditions in a light-, temperature- and humidity-controlled environment.

1. Surgery Preparation

1. Sterilize surgical instruments:
   • 1 student tissue forceps 1-2 teeth 12 cm
   • 1 student standard pattern forceps
   • 2 Semken forceps
   • 1 Mayo scissors
   • 1 student iris scissors
   • 1 Olsen-Hegar needle holder with scissors
   • Blanket
2. Check inventory list:
   • Operating table with warming pad and lamp
   • O₂, isoflurane
   • Tissues
   • Shaver
   • Scales
   • Sterile operating set
   • Sterile gauze 5x5 and 10x10
   • 70% alcohol
   • 0.9% NaCl solution
   • 1 ml syringe
   • Needles (25G)
   • Gel foam pads: spongostan
   • Vicryl 4.0 and 5.0
   • Buprenorphine 0.03 mg/kg (1:10 diluted in physiological salt)
   • Clean cage for rats after surgery

2. Right Side Uninephrectomy

1. Disinfect the table with 70% alcohol.
2. Place rat in induction box and induce anesthesia with 4% isoflurane and 1 liter flow of O₂. Note: depending on the rat strain used, pre- and perioperative analgesia are prescribed, this needs to be discussed with the local veterinarian.
3. Transfer rat to table and place nose into cone. The rat and nose cone are placed on a heating pad to maintain the rat's body temperature. The rat is ventilated with a mixture of 2% isoflurane and 1 liter O₂.
4. Shave the flank of the rat and disinfect with alcohol. Wash and disinfect hands and wear sterile surgical gloves. Wear a head cap and a mask. Make a 1- 1.5 cm incision parallel to the ribs using anatomical forceps and blunt scissors. Expose the kidney by blunt dissection of the back muscles.
5. The lower pole of the kidney is visible, carefully use the small blunt forceps to grip the perirenal fat tissue. Externalize the kidney by gently pulling on the perirenal fat with forceps. Take care to not disturb the adrenal gland during this procedure. The adrenal gland can be easily removed by placing a forceps at the medial site in the fat tissue and gently moved upwards between the adrenal gland and the kidney as depicted in Figure 2.
6. Place kidney gently on gauze and clear of surrounding fat and connective tissue. Identify the renal artery and vein and place a ligature (5.0 vicryl) with a single knot around the vessels, but do not tie off the knot.
7. Move the loose knot carefully along the vessels towards the aorta by approximately 0.5 cm, to create space between the kidney and the knot. This is done to prevent the ligature from slipping off after cutting.
8. Tie knot with two double knots. If perfusion is halted, the color of the kidney will immediately change to brown. Do not cut the ends of the ligature.
9. Cut the renal vessels close to the kidney and remove the kidney. Gently pull on the long ends of the ligature to check for bleeding of the renal vessels. The ligature should stay in place on the vessel. Cut the long ends of the ligature. Note, the remnant renal vessels will retract into the abdominal cavity immediately.
10. Blot excised kidney dry and weigh it.
11. Close the skeletal muscle incision with running sutures with Vicryl 5.0. Inject 0.10 ml (0.03 mg/kg) buprenorphine i.m in the hind limb. Close the skin with Vicryl 4.0 with intracutaneous sutures. This prevents the rats from opening the wound. Note: due to respiratory depression in Lewis rats, we only give post-operative analgesia just before switching off the isoflurane.
12. House the rat in a clean solitary cage with easy access to food and water. Place the cage half on warming pad O/N and check the rat carefully the next day. When the wound is closed and the rat is active, which is normally between 6 and 12 hr after surgery, the rat can be placed back into group housing.

3. Left Side Subtotal Nephrectomy

1. Seven days later, the same preparations are made on the left side as for the right side as described from 2.1 to 2.5. Before surgery, the amount of renal tissue that needs to be removed corresponding to approximately 2/3rds of the weight of the right kidney is calculated. Prepare small pieces of gel foam (Spongostan, Johnson & Johnson, New Jersey) before surgery.
2. Place sharp scissors around the upper pole of the left kidney and resect the upper pole in one stroke (for scissor location, see Figure 3). Cover immediately with a piece of gel foam and exert mild pressure with sterile gauze.
3. Repeat with the lower pole of the kidney as in 3.3.
4. Lift kidney to prevent clotting on skin. Maintain mild pressure on both gel foam pads with sterile gauze until the bleeding stops. When bleeding persists, add another foam pad.
5. Place remnant kidney with adherent gel foam pads inside the abdomen. Close the muscle and skin and follow 2.9-2.12.

4. Sham Surgery

1. Sham operated controls undergo the same procedure in order to expose the kidneys. Instead of extirpating the kidney or cutting the poles one week later, both the kidneys are decapsulated at a one week interval, taking care not to disturb the adrenal glands. Wound closure and post-operative care are identical to 2.11 and 2.12.

Representative Results

After subtotal nephrectomy, approximately 1/6th of total renal mass is left. Figure 4 shows the weight of the removed part of the right kidney with mean and standard deviation in two previous experiments. One should keep in mind that in the week after UNX, hypertrophy of the left kidney occurs; indicating that the weight that needs to be removed calculated based on the weight of the right kidney always results in less than 5/6th removal. However, since it is not possible to determine the weight of the left kidney during surgery; this is the most accurate way to remove approximately 5/6th of the original renal mass.

Over time, rats with CKD develop hypertension, uremia, anemia, proteinuria and a significant decrease in GFR and ERPF. After 6 weeks, established CKD has developed, a suitable time-point to test rescue interventions. Over time, strong progression of hypertension and proteinuria is observed while hematocrit and renal function (GFR and ERPF) show mild deterioration (Figure 5). Other symptoms that we do not focus on in our experiments include derangements in calcium-phosphate and lipid metabolism, and many others. Depending on the strain of rats, development of CKD can vary markedly^{14, 15, 2}. We added the high salt diet and a NO blocker to induce hypertension and endothelial dysfunction.

Progression of renal failure is tracked by collection of urine (for measurement of the amount of protein and creatinine), blood (plasma urea and creatinine) and systolic blood pressure. We realize that development of hypertension can be followed more precisely when using telemetry instead of tail cuff plethysmography. Creatinine clearance can be calculated but tends to underestimate the decline in GFR due to extensive tubular creatinine secretion in rats¹⁶, underlining the importance of the gold standard method to determine renal function by inulin and PAH clearance^{17, 18, 19}. Koeners et al. described the complete procedure²⁰. Inulin and PAH clearance are calculated from their concentration in the urine sample (U), urine flow rate (V), and their plasma concentration (P). We previously showed a marked reduction of GFR and ERPF in this model using classic clearance technology, while this was less apparent from changes in plasma creatinine, plasma urea, or creatinine clearance¹³.

Figure 1. Time line of L-NNA, high salt diet, UNX and SNX. Longitudinal measurements to determine renal function and termination time-point are indicated. When longer follow-up is needed after SNX, 6% NaCl diet can be continued over time.

Figure 2. Adrenal gland dissection. To remove the adrenal gland without disturbing it, place forceps in the fat between the kidney and the adrenal gland and move from the vessel pole upwards to the top as indicate by the red line. Figure adapted from the 20th U.S. edition of Gray's Anatomy of the Human Body, 1918.

Figure 3. Subtotal nephrectomy. Red lines indicate cutting edges. Figure adapted from the 20th U.S. edition of Gray's Anatomy of the Human Body, 1918.

Figure 4. Percentage of removed renal tissue after subtotal nephrectomy in two different experiments. Exp 1; n=16, exp 2; n=23.

Figure 5. Development of renal failure in CKD rats terminated 6 weeks after SNX (sdashed lines n=5) vs. healthy controls (pdashed lines n=5) and CKD rats terminated 12 weeks after SNX (£n= 8) vs. healthy controls (•n=8). CKD rats received L-NNA until week 4 and high salt diet from week 1 until termination (not shown in graphs). SNX induces hypertension (measured by tailcuff plethysmography) (A), uremia (B), anemia (C) and proteinuria (D) and marked reduction of GFR (E) and ERPF (F)¹³. * Indicated p<0.05 vs. healthy controls for each time point tested by two-way ANOVA with Bonferroni post-test for graphs A, B and D. Graphs C, E and F are tested with a two-tailed t-test for 6 and 12 weeks time point. Click here to view larger figure.

Discussion

Surgical removal of 5/6th of renal mass in the Lewis rat, combined with a high-salt diet and temporary NOS inhibition leads to a model of CKD that resembles human CKD and allows study of causal mechanisms and efficacy of therapeutic interventions in CKD.

The 5/6th nephrectomy model is a well-known and extensively described model for CKD. However, simply removing 5/6th of renal mass does not lead to immediate renal failure in all rat strains. We use Lewis rats to study the effects of cell-based therapies in CKD as the availability of GFP+ Lewis rats²¹ allows cell-tracking of administered donor cells in the recipient (nonGFP⁺) rat. The Lewis rat is relatively resistant to development of kidney injury and development of CKD is slow compared to other strains^{22, 15}. Therefore we combined removal of 5/6th of renal mass with NO depletion and a high salt diet since this resembles several aspects of human CKD like high salt intake and endothelial dysfunction. Readers should keep in mind that the need for combining 5/6th nephrectomy with high salt diet and/or NO depletion depends on the strain of rats used for the experiments.

We performed 5/6th nephrectomy in a two-step procedure instead of a one-step procedure as this is regarded as less burdensome for the animal, is associated with less surgery-related mortality and is preferred by our animal experimental committee. We preferred flank incision rather than laparatomy to reach the kidneys as laparotomy is associated with a higher risk of wound infection, loosening of stitches, subcutaneous herniation and adhesions to the intestines compared to flank incision. Furthermore, if the experimental design involves an intervention with laparotomy - as is the case in our experimental studies for the administration of bone marrow cells into the renal artery of the remnant kidney- performing laparotomies for SNX is not preferable as repeated laparotomies should be avoided.

Following surgical removal of 5/6th of renal mass, several critical problems can occur. During UNX there can be difficulties stabilizing the kidney since the surrounding fat will detach easily. Two methods can be used to get the kidney exposed. 1) Use smaller forceps to get grip on the renal vessels by gently moving down from the lower pole of the kidney towards the vasculature and carefully pull until you can stabilize the kidney. 2) Use blunt forceps to carefully pull the kidney out of the abdomen. When the kidney is exposed, gauze can be used to stop the bleeding that may occur during this procedure. To prevent bleeding, start pulling the fat at the lower pole of the kidney, where the fat is strongly attached to the kidney.

It is important to create space between the knot around renal vessel and kidney to prevent bleeding after removal of the kidney as the knot can slip off the renal vessels due to the incoming blood flow, quickly filling the abdomen with blood. A gauze can be used to remove the blood and pressure should be applied to stop bleeding. Use forceps to grip the renal and place a new ligature. When renal vessels cannot be traced back, maintain pressure until bleeding is stopped. Add 1 ml of physiological salt to prevent dehydration due to bleeding in the abdominal cavity and wait approximately 5 min before closing the muscle and skin layer. Monitor rat extra carefully for the following days. When the ligature is still in place after removal of the kidney but renal vessels are bleeding, the long ends of the ligature can be used to tie off the vessels.

When after dissection of the poles, despite holding pressure on gel foam pads if bleeding persists, this is likely due to injury to a large renal artery or the renal pelvis. This bleeding can be stopped by placing new gel foam pads on the wound, taking care not to move the foam pad during lifting the remnant since the larger vessels are close to the hilum, and waiting for a longer period.

Blood can be traced in the urine until 2 days post-surgery. When the renal pelvis is damaged or a large renal vessel persists to bleed, there will be a persistent trace of hematuria. Rats with persistent hematuria after surgery need to be euthanized since it is not possible to stop the bleeding. If hematuria is observed at a later time-point after surgery, which can occur up to 2 weeks after surgery, the rat should also be euthanized since blood has clotted inside the renal pelvis and will eventually clog the ureter and bladder, leading to obstruction.

When more rapid development of renal failure is required, the protocol can be reversed, i.e. first remove the poles followed by uninephrectomy a week later. This reversed model has been used in other rat strains, see for example Liu et al.²³ who describes a three fold increase in serum urea and an almost four fold decrease in serum creatinine at two weeks post-surgery. Alternatively, more renal mass can be removed. Keep in mind that removal of more renal mass will increase the risk of hemorrhages and death. Cortex can safely be removed from the top of the kidney curvature. Do not remove more medullary tissue to avoid bleeding of the large renal arteries. Variants on the SNX model are renal mass reduction by one-sided nephrectomy and either ligation of two of the three renal artery branches (infarction model) or resection or ligation of the poles of both kidneys. The differences between these models have been extensively investigated by Griffin and Bidani²⁴. The infarction model is accompanied by higher renin release, a more acute and pronounced rise in blood pressure and more initial glomerular injury³. In both models, a state of chronic stable kidney disease develops over the course of 4 to 8 weeks. Coagulation of the renal cortex can also be used to reduce renal mass ²⁵.

Materials

Name	Company	Catalog Number	Comments
			Reagent
L-NNA	Sigma-aldrich	N5501
Spongostan dental: gel foam pads 1x1x1 cm	Johnson&Johnson	Ms0005
Ethicon Vicryl FS-2S naald 4/0 V392H p/36	Ethicon	V303H
Ethicon Vicryl RB-1+ naald 5/0 V303H p/36	Ethicon	V392H
Buprenorphine (0.3 mg/ml)	Via local pharmacist ordered by Reckitt Benckiser pharmaceuticals	unknown
			Equipment
Student Tissue Forceps - 1x2 Teeth 12 cm	Fine Science Tools (FST)	91121-12
Student Standard Pattern Forceps	FST	91100-12
Mayo Scissors	FST	14010-15
2X Semken Forceps	FST	11008-13
Student Iris Scissors	FST	91460-11
Olsen-Hegar Needle Holder	FST	12002-14