Animals
Male C57BL/6 mice aged 8 to 10 weeks were acquired from Charles River Laboratories (Beijing). Animals were maintained individually in vivo with 12-h light/dark cycles and access to food and water ad libitum. The animals were allowed to acclimate for 7 to 10 days before the experimental procedures were conducted. Approval was obtained from the institutional animal care use committee before study commencement. Only healthy mice were used for the experiments; the glomerular filtration rate was measured for each mouse before the start of the experiment, and mice with abnormal renal function were excluded.
Acute kidney injury model
Acute kidney injury models were established as follows:
1. Acute kidney injury in sepsis
1.1. Lipopolysaccharide (LPS): 10 mg/kg, intraperitoneal (IP) injection.
1.2. LPS: 2 mg/kg, ip.
2. Ischemia‒reperfusion acute kidney injury
2.1. Ischemia‒reperfusion: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg, ip), and the lateral abdominal hair of the mice was removed with hair clippers to expose the surgical area. Bilateral lateral abdominal incisions were identified to expose the kidneys, and the bilateral renal pedicles were clamped for 30 minutes using a noninjury-free microvascular clip. The fixture was then removed, and renal ischemia and reperfusion were monitored visually. Finally, surgical sutures were used to close the bilateral abdominal musculature. The body temperature of the mice was maintained at 37°C throughout the operation, and the body temperature was monitored [6]. After surgery, the mice had free access to food and water.
3. Platinum chemotherapy-associated kidney injury
3.1. Cisplatin: 30 mg/kg, ip.
4. Antibiotic-associated kidney injury
4.1. Vancomycin: 150 mg/kg, iv (Intravenous injections), qd (Once daily).
4.2. Piperacillin: 1400 mg/kg, ip, qd.
4.3. Vancomycin combined with piperacillin: Vancomycin: 150 mg/kg, iv, qd; piperacillin: 1400 mg/kg, ip, qd.
GFR measurement in conscious animals
Transcutaneous measurements were performed as previously described [5]. One day before the experiment, the area on the flank was shaved, and a depilation cream was applied to remove the remaining fur to facilitate transdermal readout. We briefly anesthetized the mice with isoflurane before the real-time GFR was measured. An optical device (MediBeacon GmbH, Mannheim, Germany) was attached to the shaved area and held in place with an elastic gauze bandage. Approximately 3-5 minutes after the mice had regained consciousness, the background signal was measured, and FITC-sinistrin was injected through the tail vein. The optical device and data were downloaded and analyzed using MB Studio v.22 (MediBeacon GmbH) after removal of the device under isoflurane anesthesia following continuous monitoring for approximately 1 hour. In addition, for the sepsis model, FITC-sinistrin was continuously injected into the tail vein every 2 hours to monitor the glomerular filtration rate at 2, 4, 6 and 8 hours after the model was established. The real-time GFR was calculated from the FITC-sinistrin clearance using a three-compartment model with linear baseline correction. To minimize the influence of other factors on the real-time GFR, the animals were measured at the same time of day under conditions such as the same ambient temperature and light.
Serum creatinine and blood urea nitrogen
Serum creatinine levels were assessed using reagent kits (Scr; cat. no. c011‑2‑1) according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute). Blood urea nitrogen was measured by an automatic biochemical analyzer (IDEEX, Catalyst One*Analyzer, USA).
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT–PCR)
Total RNA was isolated from renal tissues using TRIzol reagent (Invitrogen, San Diego, CA, USA). cDNA was obtained from 1 µg of RNA according to the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Relative levels of the target genes were determined via qPCR using Ultra SYBR Mixture (2x SYBR Green PCR Master Mix, Bimake, no. B21203). The PCR sequences of primers used are as follows: GAPDH (forward: 5’-ACTCCACTCACGGCAAATTC-3’, reverse: 5’-TCTCCATGGTGGTGAAGACA-3’); Lcn2 (forward: 5’-GGACCAGGGCTGTCGCTACT’, reverse: 5’-GGTGGCCACTTGCACATTGT-3’); and KIM-1 (forward: 5’-GTCTGTATTGTTGTCGAGTGGAG-3’, reverse: 5’-CGTGTGGGAATCTCTGGTTTAAC’). Transcript expression was calculated using the comparative Ct method and was normalized to that of GAPDH (2−ΔΔCt).
Statistical analysis
The data are presented as the mean ± SD. One-way analysis of variance (ANOVA), two-way ANOVA, and three-way ANOVA were used to compare the differences among multiple groups when needed plus Tukey’s post hoc test. All the statistical analyses were performed using GraphPad Prism 9.4.1. P values < 0.05 were considered to indicate statistical significance.