Rat albumin

NRK-52E cells, a rat renal tubular cell line, were obtained from ATCC and cultured in the presence of DMEM/Ham’s F12 (DMEM/F12) medium supplemented with 10% fetal calf serum (FCS), glutamine (2 mM), penicillin (100 IU/ml), and streptomycin (100 μg/ml). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in air. For EMT experiments, cells were treated with 20μg/ml rat albumin (Sigma) for 48 h; for NF-κB translocation experiments, cells were treated with 20μg/ml rat albumin for 3 h.
Mesenchymal stem cells (MSCs) were generous gifts from Texas A&M Health Science Center. Passenger 7 MSCs were cultured according to the instruction in Eagle’s alpha minimum essential medium (α-MEM; Sigma), supplemented with 20% FBS (FBS, Invitrogen), 4mM L-glutamine (Invitrogen-Gibco), 100U/ml penicillin and 100ug/ml streptomycin (Invitrogen-Gibco). After 72 h, the medium was collected and used as stem cell conditioned media (SCM). The control conditioned media (CCM) were obtained from culturing rat renal medullary interstitial cells for 72 h using the same medium. Rat renal medullary interstitial cells were prepared as we described previously [22 (link), 23 (link)]. In preliminary experiments, cells treated with CCM did not show significant difference in the EMT markers described below compared with naive cells.

In contrast to humans, wild-type rats do not have a receptor for diphtheria toxin. In this model of podocyte injury, homozygous F344/NHsd-Tg(NPHS2-HBEGF) transgenic rats of both sexes expressing the human diphtheria toxin (DTx) receptor under the podocin promoter (kindly provided by Prof. R. Wiggins, University of Michigan; Wharram et al., 2005; Sato et al., 2009) [19 (link),20 (link)] were administered 12.5 ng of DTx (Merck KGaA, Darmstadt, Germany)/kg of body weight that was intravenously diluted in PBS supplemented with 0.1 mg/mL of rat albumin (Sigma Aldrich Chemie GmbH) as a carrier under isoflurane anesthesia. Survival biopsy was performed 21 days after toxin administration after analgesia with 0.05 mg of buprenorphine/kg of body weight and isoflurane anesthesia by removing the upper pole of the left kidney with a scalpel and immediately closing the wound with a collagen sponge (KOLLAGENresorb, Resorba Medical GmbH, Nürnberg, Germany). Finally, the experiment was terminated either on day 21 or day 42 after model induction, and the kidneys were harvested for analysis.