Nephrectomy and high-salt diet inducing pulmonary hypertension and kidney damage by increasing Ang II concentration in rats

Abstract

Background: Chronic kidney disease (CKD) is a significant risk factor for pulmonary hypertension (PH), a complication that adversely affects patient prognosis. However, the mechanisms underlying this association remain poorly understood. A major obstacle to progress in this field is the lack of a reliable animal model replicating CKD-PH.

Methods: This study aimed to establish a stable rat model of CKD-PH. We employed a combined approach, inducing CKD through a 5/6 nephrectomy and concurrently exposing the rats to a high-salt diet. The model's hemodynamics were evaluated dynamically, alongside a comprehensive assessment of pathological changes in multiple organs. Lung tissues and serum samples were collected from the CKD-PH rats to analyze the expression of angiotensin-converting enzyme 2 (ACE2), evaluate the activity of key vascular components within the renin-angiotensin-aldosterone system (RAAS), and characterize alterations in the serum metabolic profile.

Results: At 14 weeks post-surgery, the CKD-PH rats displayed significant changes in hemodynamic parameters indicative of pulmonary arterial hypertension. Additionally, right ventricular hypertrophy was observed. Notably, no evidence of pulmonary vascular remodeling was found. Further analysis revealed RAAS dysregulation and downregulated ACE2 expression within the pulmonary vascular endothelium of CKD-PH rats. Moreover, the serum metabolic profile of these animals differed markedly from the sham surgery group.

Conclusions: Our findings suggest that the development of pulmonary arterial hypertension in CKD-PH rats is likely a consequence of a combined effect: RAAS dysregulation, decreased ACE2 expression in pulmonary vascular endothelial cells, and metabolic disturbances.

Keywords: Angiotensin converting enzyme 2; Chronic kidney disease; Metabolomics; Pulmonary hypertension; Renin–angiotensin–aldosterone system.

Fig. 1

Protocol for the establishment of CKD-PH rat model

Fig. 2

Progressive renal dysfunction and accelerated collagen deposition were confirmed in 5/6 Nx and high-salt diet-treated rats. A Bar graphs presenting the total urine protein, serum creatinine, and serum urea nitrogen. B bar graphs showing weight changes of kidney (normalized to body weight (BW)) during modeling. C-D Representative images of HE(C) and Masson’s(D) trichrome staining revealing renal structural modification and collagen deposition in the kidney sections from each group of rats. Data shown are means ± SEM; n = 7 in each group. *P < 0.05, **P < 0.01, significantly different as indicated

Fig. 3

Rats treated with 5/6 nephrectomy and high-salt diet exhibited increased hemodynamic measurements and right ventricular hypertrophy, but no pulmonary vascular remodeling was observed. Representative traces and bar graph (A) of the right ventricular systolic pressure (RVSP). Bar graph (B) showing the Fulton index [RV/(LV + S)] of sham and 5/6 Nx and high-salt diet-treated rats. Bar graphs of systemic blood pressures, including mABP, SBP, and DBP are presented in C. D Van Gieson Staining shows the histological and pathological changes in the pulmonary arteries in the lung sections of each group; analyzed data presents wall thickness (WT (%)) and vessel numbers per mm² of the pulmonary arteries in each group of rats. Typical immunofluorescence staining images (E) in lung sections from rats of each group. Staining of blue, green, and red is represented as Dapi, CD31, and α-SMA, respectively. The scale bar represents 20 μm as indicated. Representative images and summary data of pulmonary acceleration time/pulmonary ejection time (PAT/PET) and stroke volume (SV) are shown in F and I, respectively. Bar graphs of cardiac output (CO) and heartbeat (HR) from each group are presented in G and H. H&E staining of heart slides showing right and left myocardial cells shown in J and K. Summarized data showing cross-sectional area of right and left myocardial cells among the sham, 5/6 Nx-8-week group and 5/6 Nx-14-week group. Data shown are means ± SEM; n = 7 in each group. *P < 0.05, **P < 0.01, significantly different as indicated

Fig. 4

RASS dysregulation in CKD-PH rat model. Bar graphs of renin, AngII, aldosterone (ALD), and Ang (1–7) concentration from each group present in A (mean ± SEM; n = 8 in each group). Representative curves (B-D) presenting plasma/AngII-mediate contraction in isolated pulmonary artery (PA) rings precontracted with PE. E Summarized data (mean ± SEM; n = 3 in each group) showing the contraction of isolated PA rings in the presence of plasma or AngII treatment. Typical immunofluorescence staining images (F) and summary data (mean ± SEM; n = 6 in each group) of average immunofluorescent intensity for CD31, ACE2, and ACE2/CD31 (G) in lung sections from rats of each group. Staining of blue, green, and red is represented as Dapi, CD31, and ACE2, respectively. The scale bar represents 20 μm as indicated. Expression of ACE2 in lung tissue was measured by western blot of the sham, 5/6Nx-8-week group, and 5/6Nx-14-week group. n = 5–7. *P < 0.05, **P < 0.01, significantly different as indicated

Fig. 5

Potential differential metabolites were confirmed in rats treated with 5/6 Nx and a high-salt diet. Volcano Plots data obtained from R analysis using deSeq2, edgeR, and limma package showing differential metabolites among sham group and 5/6 Nx plus high-salt diet-induced groups (A-C). Heatmap (D) presenting differential metabolites between sham and operated groups. Venn chart indicating common increased differential metabolites, measured by different algorithm (E). The identified 17 up-regulated differential metabolites obtained from KEEG database were recorded, showing in heatmap (F)

Fig. 6

Further analysis of identified differential metabolites. Correlations of these identified differential metabolites were showing in A and B. Box plot (C) revealing the relative expression of identified differential metabolites among control and treatment groups. Violin plot (D) displaying the relative expression of top five differential metabolites between sham and treatment groups. Time-varied metabolites were divided into four clusters in accordance with time-series trend analysis (E). ROC (F) assessing the diagnostic reliability of top five differential metabolites. The Kruskal–Wallis analysis (G) illustrates a time-dependent increase in metabolite C00165