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 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.
CKD, also known as chronic renal failure, is a progressive loss of kidney function over time that eventually develops into permanent kidney failure. CKD, from early stage renal disease states to end-stage renal disease (ESRD), is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called "uremic cardiomyopathy"1,2,3. The uremic cardiomyopathy in patients with CKD or ESRD is associated with cardiovascular abnormalities, mainly caused by overload of left ventricular (LV) pressure and/or volume, leading to LV hypertrophy (LVH), LV dilation, and LV systolic dysfunction4,5,6. Cardiac fibrosis is another common pathological process of uremic cardiomyopathy that reduces cardiac compliance resulting in LV diastolic dysfunction. Severe cardiac fibrosis can lead to sudden cardiac death even in those without cardiac symptoms7.
The 5/6th PNx is a commonly used CKD animal model for animal studies involving renal failure, uremic cardiomyopathy and hypertension. PNx is achieved by ablation of 5/6 renal parenchyma. The rat model was initially developed with the two most common protocols employed being surgical resection or infarction. The rat PNx model is an extremely useful model to study uremic cardiomyopathy with substantial elevations in blood pressure, cardiac hypertrophy and impaired diastolic function. Later, mouse PNx models, operated with the similar techniques as the rat model, were developed due to the wide availability and ease of making genetic manipulations in the mouse system.
It is well-documented that systemic oxidant stress is a constant feature of both clinical and experimental uremic cardiomyopathy8,9. Furthermore, oxidant stress contributes to the uremic syndrome10, and plays a critical role in the pathogenesis of the cardiac abnormalities seen in uremic cardiomyopathy11,12,13. To this point, we have demonstrated that the rodent 5/6th PNx model causes physiological, morphological, and biochemical features of uremic cardiomyopathy14,15,16,17. In the mouse PNx model described here, PNx-operated mice developed significant oxidative stress, at least partially mediated by Na/K-ATPase signaling function, which is critical in PNx-mediated experimental uremic cardiomyopathy. Attenuation of the Na/K-ATPase signaling not only reduces oxidative amplification, but also ameliorates the phenotypical changes in PNx-mediated experimental uremic cardiomyopathy18.
All animal care and experiments were approved by the Marshall University Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice (10-12 weeks old) were housed in a pathogen free animal facility in designated rooms equipped with cages that supply purified air under a 12 h light/dark cycle. Food and water were supplied ad libitum.
1. Surgery Preparation
NOTE: The surgical instruments and materials are obtained from different sources that are not specific to surgery operations. Instruments and materials from other sources can also be used for the same purpose. See the Table of Materials for a list of surgical instruments.
2. Step-one Surgery: Pole Ligation of Left Kidney
NOTE: Maintain sterile conditions during the surgery. Cover the autoclaved surgical instruments with a sterile pad. Keep more than one set of surgical instruments in hand for more than one surgery to prevent cross contamination during the surgery. If the instruments need to be used again, in the case of multiple surgeries, disinfect the instruments with betadine solution and 70% ethanol, and then sterilize in a germinator glass bead sterilizer for 5 min. Disinfect the operating area with 70% ethanol. Wear a gown, mask (to cover nose and mouth), cap (to cover head), and a pair of sterile gloves. Change gloves after each surgery.
3. Step-two Surgery: Removal of Right Kidney
4. Sham Surgery
5. Evaluation of Experimental Uremic Cardiomyopathy
The data indicated that this modified 5/6th PNx model by pole ligation is a simple and effective model to investigate uremic cardiomyopathy. At four weeks post-surgery, this PNx model presents impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function. The results are summarized below.
At four weeks post-surgery, the PNx mice developed impaired renal function in addition to cardiac morphological and biochemical changes that are consistent with the phenotype of human uremic cardiomyopathy. These PNx-mediated changes include the following observations as compared to the sham-operated mice. First, as expected, the PNx surgery caused significant impairment of renal function. As shown in Figure 2, the PNx surgery significantly increased plasma levels of Cystatin C (Figure 2a, n = 16), creatinine (Figure 2b, n = 16), BUN (Figure 2c, n = 16), when compared to the sham surgery. Second, there was PNx-stimulated anemia demonstrated by the significantly lower HCT in the PNx mice (Figure 2d, n=8). Third, the PNx-mediated cardiac hypertrophy and diastolic dysfunction were demonstrated by an increased heart weight/body weight ratio (Figure 3a), and transthoracic echocardiography (ECHO) analysis showing the significant increases in posterior wall thickness (PWT), anterior wall thickness (AWT), RWT (Figure 3b), MPI (Figure 3c), and LVMI (Figure 3d). There was no significant change of ejection fraction (EF, %). Fourth, the PNx-mediated cardiac fibrosis was demonstrated by PNx-induced increases in type I collagen expression in the LV homogenates (Figure 4a) and left remnant kidney homogenates (Figure 4b). Fifth, oxidant stress plays an important role in experimental uremic cardiomyopathy. In this PNx model, PNx significantly stimulated direct protein carbonylation modification of multiple proteins (Figure 4c) and c-Src activation (Figure 4d) in LV homogenates. PNx surgery did not cause significant loss of body weight at four weeks post-surgery. However, the PNx model by pole ligation method did not induce hypertension. There was no significant difference between sham and PNx mice in systolic BP, diastolic BP and mean BP (data not shown) measured at both one day before the step-one surgery and one day before sacrifice, which is consistent with the notion that the C57BL/6 mouse strain is not an established hypertensive model20,21. The data also suggest that this PNx model is unlikely a model of acute renal failure. Moreover, the PNx-mediated cardiac changes, but not impaired renal function, were attenuated by blockage of c-Src activation or induction of heme oxygenase-1 (HO-1)18.
Figure 1: Pole ligation of left kidney. The pictures showed left kidney before ligation (left) and after ligation (right). The doted lines in the left picture showed the estimated ligation lines. Please click here to view a larger version of this figure.
Figure 2: PNx with pole ligation caused impaired renal function and anemia. At four weeks post-surgery, the PNx surgery significantly increases plasma levels of (a) Cystatin C (n = 16), (b) creatinine (n = 16), (c) BUN (n = 16), compared to the sham surgery. PNx also significantly reduced (d) hematocrit (HCT) in PNx mice (n = 8). Data were expressed as Mean ± SEM. **, p <0.01, sham-operated mice vs. PNx-operated mice. Please click here to view a larger version of this figure.
Figure 3: PNx with pole ligation stimulated cardiac hypertrophy. At four weeks post-surgery, the PNx surgery significantly increases (a) heart weight/body weight ratio (a, n = 8), (b) relative wall thickness (RWT, n =1 9-21), (c) myocardial performance index (MPI, n = 19-21), and (d) left ventricle mass index (LVMI, n = 19-21). Data were expressed as Mean ± SEM. **, p <0.01, sham-operated mice vs. PNx-operated mice. Please click here to view a larger version of this figure.
Figure 4: PNx with pole ligation stimulated type I collagen expression, protein carbonylation and c-Src activation. At four weeks post-surgery, the PNx stimulated type I collagen expression in the left ventricle (LV) homogenates (a, n=11) and left remnant kidney homogenates (b, n = 7-10). Also in LV homogenates, the PNx stimulated direct protein carbonylation (c, n = 6) and c-Src activation (d, n = 6). Data were expressed as Mean ± SEM. **, p <0.01, sham-operated mice vs. PNx-operated mice. Please click here to view a larger version of this figure.
The rat 5/6th PNx model has been widely used to study CKD. Because of the much smaller kidney size in mouse, the classical artery ligation and pole resection are very challenging in mouse models with possible high mortality rates and unexpected bleeding/blood loss.
We adopted a mouse PNx model with pole ligation to overcome the bleeding/blood loss. This PNx model takes less time with improved survival rate and high reproducibility. This pole ligation model develops the phenotypic changes of human uremic cardiomyopathy at the time of four weeks post-surgery, and thus provides a model to study its mechanism(s) and therapeutic target(s).
In this PNx model, the 5/6th PNx is an approximate estimation of renal mass reduction since, and up until now, there is no method to exactly calculate the remnant renal mass of the two kidneys that have different sizes/masses. However, the ligation should be performed in a very similar way to reduce operation variation. We estimate that keeping about 40% of the left kidney (or about 20% of total renal mass) can significantly decrease the mortality rate (by about 20%) without sacrificing the development of the uremic cardiomyopathy phenotype. Another issue is the force of ligation which requires practice.
In comparison with the 5/6th PNx models by pole resection or renal artery ligation15,20, the pole ligation method is easy, quick, and less instrumentally demanding. The possible advantages of this PNx model include the following: 1) avoidance of possible blood bleeding as seen in the PNx model by pole resection or artery ligation, 2) impaired renal function with increase in oxidative stress caused by the inflammation response to the pole ligation, 3) lower mortality rate (about 20%), and 4) quick development of uremic cardiomyopathy phenotype. A successful pole ligation shows a progressive irreversible damage and degradation of the ligated poles. The ligated poles are visible at two weeks post-surgery (Figure 4), and mostly disappear at four weeks post-surgery (Figure 5). Comparison of the ligated poles at two weeks and four weeks post-surgery, indicates the inflammation reaction responding to the pole ligation.
This PNx model may be useful to study the mechanism(s) of drug action and therapeutic drug screening. We recently use this PNx model to investigate the role of the Na/K-ATPase signaling and oxidative stress in the development of uremic cardiomyopathy. Administration of pNaKtide, a specific antagonist of the Na/K-ATPase signaling, not only prevents the development of uremic cardiomyopathy but also reverses the onset of uremic cardiomyopathy18.
The critical steps within the protocol are as follows:1) A more aggressive ligation (remnant left kidney mass is less than 30%) will significantly increase the mortality rate (about 50%). 2) The force of ligation requires practice to reach the point that the ligation restricts the blood supply to the poles without breaking the capsule and kidney tissue. Breaking the kidney tissue leads to intra-renal bleeding that can cause death after removal of the right kidney. A successful ligated pole is discolored within 1 – 2 min after ligation, due to the limit of blood flow to the pole. 3) Special attention is required to avoid ligating the ureter of the left kidney or damaging the ureter when ligating the poles. A mouse with ligated or damaged ureter will not survive after removal of the right kidney. This may be the most noticeable disadvantage of the pole ligation method. 4) Maintenance of body temperature during surgery process and post-surgery care. 5) Control of possible infection using antibiotics.
The authors have nothing to disclose.
This work was supported by NIH R15 1R15DK106666-01A1 (to J. Liu) and NIH RO1 HL071556 (to J.I. Shapiro).
Iris Scissors, 11.5 cm, Straight | World Precision Instruments | 501758 | |
WPI Swiss Tweezers #5 11 cm, 0.1×0.06 mm Tips | World Precision Instruments | 504506 | |
Jewelers #5 Forceps, 11cm, Straight, Titanium | World Precision Instruments | 555227F | |
Iris Forceps, 10cm, Straight, Serrated | World Precision Instruments | 15914 | |
Tweezers #3, 11cm, 0.2×0.4mm Tips | World Precision Instruments | 501976 | |
Medesy Iris 4.5" Curved Scissors, Stainless Steel | Net23 | 3512 | www.Net32.com |
Dr. Slick Iris Scissors; 3.5" | Avid Max | 220-1-965-IrisScsrs-P | www.Avid Max.com |
Miltex Iris Scissors 4 1/8" Curved | 4mdmedical | V95-306 | www.4mdmedical.com |
5" Hemostat clap, curved jaw | PJTool | 4355 | www.pjtool.com |
sklar Knapp Iris scissors, straight and sharp/blunt 4" | Medical Device depot | 64-3430 | www.medicaldevicedepot.com |
Kelly Hemostatic Forceps straight 5.5" | Pilgtimmedical | FA710-50 | www.pilgtimmedical.com |
C57BL/6 mice | Hilltop Lab Animals Inc. | ||
Mouse Handling Table and rectal thermometer | Visualsonics | ||
MicroScan transducer | Vevo 1100 | MS400 | |
Vevo 1100 Imaging System | FUJIFILM VisualSonics Inc. | ||
Cystatin C ELISA kit and mouse creatinine kit | Crystal Chem. Inc. | ||
Mouse BUN ELISA kit | MyBioSource Inc |