Optimization of Surgical Procedure and Efficacy Evaluation of Aortic Calcification Model in Rats with Chronic Kidney Disease

doi:10.12300/j.issn.1674-5817.2024.128

Abstract

Abstract:

Objective To establish a chronic kidney disease-associated aortic calcification model in SD rats using different nephrectomy surgical methods, and to compare and evaluate surgical duration and survival time to explore a more optimized modeling method. Methods According to different surgical methods, the SD rats were divided into four groups: Group A: intraperitoneal resection of 2/3 of the left kidney followed by right total nephrectomy in the second stage; Group B: intraperitoneal resection of 2/3 of the left kidney and simultaneous right total nephrectomy; Group C: dorsal approach right total nephrectomy followed by resection of 2/3 of the left kidney in the second stage; Group D: dorsal approach resection of 2/3 of the left kidney followed by right total nephrectomy in the second stage. After comparing survival curves of SD rats undergoing intraperitoneal versus dorsal approaches, and staged versus single-stage nephrectomy, the optimal nephrectomy surgical method was determined. Then, twenty-four 8-week-old SPF-grade male SD rats were selected for nephrectomy combined with calcitriol-induced calcification. Experimental group (12 rats): the dorsal approach left 2/3 nephrectomy followed by right total nephrectomy, with intraperitoneal injection of 1 μg/kg calcitriol administered one week later to induce aortic calcification. Control group (12 rats): the intraperitoneal injection of 250 μL/kg physiological saline containing 1% DMSO one week after sham surgery. After intraperitoneal injection of drugs for 3 months, the survival status of rats in each group was observed. Under anesthesia, blood samples were collected from each group to measure serum phosphorus and calcium ion concentrations, as well as serum urea nitrogen and creatinine levels. After euthanizing the rats, a post-mortem examination was performed to observe the residual kidney morphology, and HE staining was used to observe the pathological changes in the coronal section of the kidney. Additionally, the entire aorta of each group was taken, and the degree of aortic calcification was observed by staining with Alizarin red S and von Kossa. Real-time fluorescence quantitative PCR was used to detect the gene expression of smooth muscle actin-associated protein alpha (Sm22), Runt-related transcription factor 2 (Runx2), and osteopontin (OPN) in rat aortic tissue to evaluate the effectiveness of the model. Results The exploratory optimization experiment of different surgical procedures found that the survival rate of group D rats,which underwent 2/3 left kidney resection followed by right whole kidney resection via the dorsal approach, was the highest, indicating that this surgical procedure was the best method for establishing a chronic kidney disease model with renal dysfunction. The experimental group rats treated with this surgical procedure combined with high-dose calcitriol injection had significantly lower serum calcium ion concentration than those in the sham-operated control group (P<0.05), while serum phosphorus ion concentration, serum creatinine, and serum urea nitrogen levels were significantly higher than those of the control group (P<0.05). HE staining of the kidneys showed significant organic changes in the kidneys of the experimental group rats, with a significant decrease in glomerular count compared to that of the control group (P<0.05), indicating the successful establishment of a renal failure model. Alizarin red S staining showed significant pigment deposition in the aortic media of the experimental group rats, while von Kossa staining showed significant silver nitrate deposition in the aortic media of the experimental group rats, which was consistent with the manifestation of aortic calcification in renal failure. Real-time fluorescence quantitative PCR showed that the expression level of Sm22 in the aortic tissue of the experimental group rats decreased (P<0.05), while the expression levels of OPN and Runx2 increased (P<0.05), indicating a transition of aortic smooth muscle cells from smooth muscle phenotype to bone-like phenotype and successful induction of an aortic calcification model. Conclusion The method of establishing an aortic calcification model of chronic kidney disease in SD rats by first removing two-thirds of the left kidney via the dorsal approach followed by right total nephrectomy, combined with high-dose calcitriol administration, shortens the surgical time, improves the success rate of modeling, and increases the animal survival rate.

Key words: Vascular calcification, Chronic kidney disease, Nephrectomy, Animal models, SD rats

CLC Number:

Q95-33

PAN Yicong,JIANG Wenhong,HU Ming,et al. Optimization of Surgical Procedure and Efficacy Evaluation of Aortic Calcification Model in Rats with Chronic Kidney Disease[J]. Laboratory Animal and Comparative Medicine, 2025, 45(3): 279-289. DOI: 10.12300/j.issn.1674-5817.2024.128.

Figures/Tables 7

Figure1 Flowchart of the experimental process for modeling and evaluating aortic calcification in SD rats with chronic kidney diseaseNote：Eight-week-old SD rats were housed in SPF-grade animal barrier facilities and underwent left kidney two-thirds nephrectomy after three days of regular dietary adaptation. After surgery， they were routinely housed and underwent right kidney nephrectomy one week later. After one week， administer 1 μg/kg calcitriol injection every three days. After 12 weeks of injection， venous blood collection was performed， and samples were taken from the entire aorta and kidneys. Serum was used for blood biochemistry testing， and paraffin-embedded kidneys were sectioned in coronal side for HE staining. A portion of the aorta was sectioned in paraffin for Alizarin Red S staining and von Kossa staining， and a portion was frozen for real-time fluorescence quantitative PCR detection.

Table 1 Primers for real-time fluorescent quantitative PCR

目标基因名称 Target gene name	NCBI基因序列号 NCBI reference sequence	引物序列信息 Primer sequence information	扩增片段大小/bp Amplification fragment size/bp
Sm22 (Tagln)	NC-086026.1	F：5’-CAGATGGAACAGGTGGCTCAA-3’	161
		R：5’-GCCCAAAGCCATTACAGTCCTC-3’
OPN (Spp1)	NC-086032.1	F：5’-GCCGAGGTGATAGCTTGGCTTA-3’	145
		R：5’-TTGATAGCCTCATCGGACTCCTG-3’
Runx2	NC-086027.1	F：5’-GGATGCCTTAGTGCCCAAATG-3’	120
		R：5’-CACCCTGTGAGGTGGCTGAA-3’
β-actin	NC-086030.1	F：5’-CACCCGCGAGTACAACCTTC-3’	207
		R：5’-CCCATACCCACCATCACACC-3’

Figure 2 Kaplan-Meier survival curve analysis of rats in chronic renal insufficiency model groups constructed using different surgical methodsNote： The horizontal axis represents the time （in days） recorded from the second day after the removal of both kidneys. Upper of the figure shows that there is a significant difference in the cumulative survival rate of rats after 60 days of surgery using different surgical methods （log-rank test， P=0.007 7）， The survival rate of rats in group D was the highest， with statistically significant differences compared to groups A， B， and C （P=0.003 0， P=0.007 4， P=0.003 0， respectively）. Moreover， rats in groups A， B， and C experienced high mortality within 10 days after surgery.

Table 2 Comparison of biochemical indicators between the experimental group and the control group

血生化指标 Biochemistry index	实验组（n=9） Experimental group	对照组（n=12） Control group	t值 t value	P值 P value
血清钙 c/(mmol·L^-1) Serum calcium ions	1.876±0.036	1.929±0.042	3.431	0.004
血清磷c/(mmol·L^-1) Serum phosphate ions	2.059±0.333	1.580±0.271	3.046	0.014
血清肌酐c/(μmol·L^-1) Serum creatinine	119.960±35.640	21.164±8.076	8.984	<0.000 1
血清尿素氮c/(mmol·L^-1) Serum urea nitrogen	57.991±8.745	22.609±8.058	6.895	<0.000 1

Figure 3 HE staining observation of kidneys from rats in the experimental and control groupsNote： The experimental group consisted of rats undergoing nephrectomy and calcitriol calcification induction for 3 months， while the control group consisted of rats undergoing sham surgery and DMSO injection for 3 months. The white arrow in the picture represents the glomerulus. The number of glomeruli in the experimental group （CI group， n=9） was significantly less than that in the control group （sham group， n=12），***P<0.001.

Figure 4 Observation of von Kossa and Alizarin red S staining in the aorta of rats from the experimental and control groupsNote： IN represents the intima of the aorta； ME stands for the media of the aortic； AD represents the adventitia of the aorta. Figures A and B show von Kossa staining； Figures C and D show Alizarin red S staining. The proportion of stained area in the experimental group （CI， n=9） was significantly larger than that in the control group （sham， n=12）（***P<0.001）. The white arrow indicates arterial media calcification deposition under high magnification view.

Figure 5 Real-time fluorescence quantitative PCR detection of OPN， Runx2， and Sm22 gene expression in aortic tissue of rats from the experimental and control groupsNote： Compared with the control group （sham）， the expression level of Sm22， which reflects the vascular smooth muscle contraction phenotype， decreased in the experimental group （CI）（***P<0.001）， while the expression levels of OPN and Runx2， which reflects the osteogenic differentiation phenotype of vascular smooth muscle， increased significantly （ * P<0.05， ** P<0.01）.

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