Tissue specimens, Real Time-PCR, and ELISA analysis

RT-PCR was used to analyze the expression levels of hub genes. Bacterial strain UPEC307 was isolated from a 56-year-old female patient with acute pyelonephritis. In LB medium, we cultured UPEC307 overnight placed on a shaker at 180 r/min and at 37°C. Then 50 ul of the bacterial solution was transferred to 5 ml of fresh LB medium and cultured for 3 hours to reach the mid-log phase. The mid-log phase culture was centrifuged at 8000 r/min for 3 min to collect the bacteria and remove the supernatant.

C57BL/6 mice were purchased from Charles River Laboratories China (Beijing, China). The animal care and use procedures have been approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Science (AMMS, Beijing, China).the ethics committee, and all applicable institutional and government regulations regarding the ethical use of animals have been complied with. Mice were anesthetized and inoculated via the urethra with 100 ul UPEC307 resuspended in PBS. They were sacrificed at 6 h after infection, and their kidneys were taken aseptically. Then the mouse kidneys were weighed, PBS was added and the kidneys were crushed and ground, centrifuged at 4°C, 10,000 × g, 10 min, and the supernatant was taken.

The RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to extract total RNA from cells, and then the RNA was transcribed into cDNA using the MightyScript First Strand cDNA Synthesis Master Mix (Sangon Biotech, Shanghai, China). We used SYBR@Green Master Mix (Applied Biosystems) for RT-PCR, and the housekeeping gene GAPDH served as an internal control. The PCR primers used in this study are shown in Table 1.The TNF -α, IL-1β, IL-6 ELISA Kit were obtained from Neobioscience Technology (Shenzhen, China).

Primers used in this study