The LIPUS device consists of an arbitrary waveform function generator (SDG1022X, Siglent, Shenzhen, China), an oscilloscope (TDS2022B, Tektronix, Shanghai, China) and ultrasound transducers (Φ = 3 cm). Among them, the ultrasound transducers were specially manufactured and tested by the Academy of Electronics and Engineering (the University of Electronic Science and Technology of China, Chengdu, China) for rat in vitro experiments. Before LIPUS stimulation, hair on the back of each rat was removed, the positions of bilateral kidneys were determined and marked by ultrasound imaging. Then, the ultrasound transducers were placed over the back marker with an ultrasonic gel pad (Mibo, Mp-A, Shenzhen, China) in between. Rats in the CKD + LIPUS group were stimulated by LIPUS for 20 min every 24 h, treatment duration was four weeks. During this period, isoflurane (R510-22-10, RWD, Shenzhen, China) was produced by the gas anaesthesia machine (MSSVAP01, MSS, Keighley, England) to keep rats under anaesthesia. According to previous studies [10 (link),11 (link)] and our preliminary experimental results, the parameters of LIPUS are set as follows: intensity of 60 mW/cm2 with a 50% duty cycle, and pulse frequency of 1.0 MHz with 1 kHz repetition rate. As for the Control group and the CKD group, which received the same treatments except LIPUS stimulation.