Glutamine

Mouse MCs were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and they were cultured in complete medium consisting of DMEM containing 2 mmol/l glutamine, 50 mmol/l β-mercaptoethanol, 20% fetal bovine serum (Sangon Biotech, Shanghai, China), penicillin/streptomycin antibiotics and 10 mmol/l HEPES, pH 7.4. Hyperglycaemia is a major stimulus for the development of nephropathy in diabetes patients.^{57 (link)} According to the previous results, MCs were stimulated with d-glucose at 5.5 mmol/l glucose plus 19.5 mmol/l mannitol (low-glucose group, L-MC) or at 25 mmol/l glucose (high-glucose group, H-MC) in HERAEUS instrument with 37 °C, 5% CO₂ and 95% humidity.^{3 (link)} HG stimulation had imitated the growth environment of MCs under the condition of DN, and LG stimulation had imitated normal growth environment.

Three-week-old ICR male mice (without receiving any solid food before the experiment) were divided randomly into two groups (n = 11 for control and 12 for experimental group): 1) mice that received a basal diet (18 (link), 24 (link)) and normal drinking water and 2) mice that received a basal diet and drinking water supplemented with glutamine (Sangon Biotech, Shangshai, China) at a dosage of 10 mg/ml. The dosage for glutamine supplementation was selected based on our previous study (25 (link)). The drinking fluid in both groups was changed daily. After 2 weeks of glutamine supplementation, the mice were sacrificed to collect the ileum after they were euthanized with CO₂ inhalation followed by cervical dislocation to ensure death. For collection of the ileum, the middle part of the ileum samples (about 2–3 cm) was collected after phosphate-buffered saline (PBS; pH = 7.2–7.4) washing. The ileum was fixed in fresh 4% paraformaldehyde for paraffin embedding or snap frozen in liquid nitrogen for mRNA analysis. The body weights of animals were regularly monitored during the treatment period.