CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 99 type II cardio-renal syndrome patients were enrolled from the medical centre of Guang’anmen Hospital of the China Academy of Chinese Medical Sciences in September 2009 and October 2016. The healthy subjects were enrolled from the medical centre of the southern district of Guang’anmen Hospital of the China Academy of Chinese Medical Sciences in 2016. The research protocol was in line with the ethics guidelines of the Declaration of Helsinki (1975). The protocol was approved by the ethics committee of Guang’anmen Hospital and informed consent was obtained from each patient. Patients with the following characteristics were excluded from the study: primary kidney disease; type 1 or type 2 diabetes; maglignancies; autoimmune diseases; congenital heart disease; severe arrhythmia, including frequent ventricular premature, second-degree type II atrioventricular block and third-degree atrioventricular block; cardiogenic shock combined with severe hypertension (systolic blood pressure ≥ 160 mmHg and/or diastolic blood pressure ≥ 100 mmHg); and underlying diseases that clearly affect heart and kidney function, such as hyperthyroidism, hypothyroidism, Cushing syndrome, primary aldosteronism and other endocrine diseases. The course of this study is shown in Fig. 1. All participants were examined by estimated glomerular filtration rate (eGFR) to verify the presence of type II cardio-renal syndrome. We first selected 20 type II cardio-renal syndrome patients and five healthy controls. Total RNAs were isolated from venous samples for micro-array analysis. Selected circRNAs were validated in the second cohort, which contained 12 control subjects and 15 patients with type II cardio-renal syndrome. There was no consensus definition for cardio-renal syndrome and expects proposed that evaluating chronic cardio-renal syndrome should include patients with at least moderate chronic kidney disease, which is defined as an eGFR < 60 ml/min/1.73 m2.15 In this research, patients who suffered from chronic heart failure, which was diagnosed according to the 2009 American College of Cardiology and American Heart Association,16 accompanied by eGFR < 60 ml/min/1.73 m2, were diagnosed as cardio-renal syndrome.17 Those patients who were diagnosed with chronic heart failure leading to chronic kidney disease were diagnosed as type II cardio-renal syndrome.18 The diagnosis was made independently by two experienced interventional cardiologists through visual observation. The healthy subjects were without any cardiac, renal, neuronal or liver diseases and did not meet any of the exclusion criteria. Blood samples (4 ml) were taken from the median cubital vein with an ethylenediaminetetraacetic acid (EDTA) anticoagulated vacutainer and total RNA was extracted immediately using a fast total RNA extraction kit (Biotech, Beijing, China), according to the manufacturer’s instructions. The RNA was then dissolved in RNAse-free water. Complementary DNA (cDNA) was generated by reverse transcription using the PrimeScript RT reagent kit (Takara Bio, Nojihigashi, Kusatsu, Japan), according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was amplified by 40 cycles of denaturing at 95°C for 10 seconds and 60°C for 30 seconds. The relative expression levels of circRNAs were determined by qPCR. The Ct (threshold cycle) value was the fractional cycle number at which the fluorescence exceeded the given threshold. Glyceraldehyde-3-phosphate dehydrogenase was used to normalise the RNA preparation. The relative expression levels of circRNAs were determined using the 2−ΔΔCT method. The RNAs of the peripheral blood of five control subjects and 20 type II cardio-renal syndrome patients were extracted for micro-array analysis. The purified RNAs were hybridised to a micro-array (Agilent human circRNA array v2.0) containing 170 340 human circRNA probes. The micro-array data of the circRNAs were then analysed using GeneSpring software v13.0 (Agilent Technologies, Santa Clara, CA, USA). The thresholds were as follows: fold change ≥ 2 or ≤ –2; p < 0.05 according to the t-test. Based on the results of the chip data, the miRanda software was used to predict the miRNAs that circRNA can bind to, and establish the possible association of circRNA with linear RNA targets. Then gene ontology annotation and biological signal pathway analysis [Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis] were performed for differentially expressed mRNAs, to clarify which biological pathways the differentially expressed mRNAs were mainly enriched in, and calculate the p-value of the pathway, then select the most closely related biological signal pathways of type II cardio-renal syndrome. To obtain the important circRNAs that would play an important role in type II cardio-renal syndrome, the candidates that were to be verified genes were selected from the 1 989 upregulated circRNAs utilising stricter screening criteria: fold change > 6 and p < 0.0001, and from the 895 downregulated circRNAs utilising stricter screening criteria: fold change > 2.6 and p < 0.0003. Statistical analysis Variables are expressed as mean ± standard deviation and median (quartiles) or percentages. The Shapiro–Wilk test was performed Inclusion criteria: 50–80 years old; with type II cardio-renal syndrome Exclusion criteria: (1) primary kidney disease; (2) type 1 or tye 2 diabetes; (3) malignancies; (4) autoimmune diseases; (5) congenital heart disease; (6) severe arrhythmia, including frequent ventricular premature; second-degree type II atrioventricular block; thirddegree atrioventricular block; (7) cardiogenic shock; (8) combined with severe hypertensive patients (systolic blood pressure ≥ 160 mmHg and/or diastolic blood pressure ≥ 100 mmHg); (9) underlying diseases that clearly affect heart and kidney function, such as hyperthyroidism, hypothyroidism, Cushing syndrome, primary aldosteronism and other endocrine diseases. Control group n = 5 CRS group n = 20 Control group n = 12 CRS group n = 15 Micro-array profile Quantitative real-time PCR Fig. 1. The study flow chart. CRS, cardio-renal syndrome.
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