Animals
Eight-week-old C57BL/6 mice (20-25 grams, half male) were purchased from the Experimental Animal Center in Zhejiang Medical Academy of Sciences and housed in a temperature-controlled room with 12-hour day/night cycles. The mice had free access to standard food and water throughout the study. All animal studies were done in compliance with the regulations and guidelines of Zhejiang University institutional animal care and conducted according to the AAALAC and the IACUC guidelines (Permit Number: 2016-205, date of approval: 26 Feb 2016). Animals were euthanized with overdose anesthesia after experimentation.
Retrograde Ureteral Lentivirus Delivery and Unilateral Ureteral Obstruction (UUO)
Mice were anesthetized using 50 mg/kg sodium pentobarbital by intraperitoneal injection. Mice were then infused with lentivirus 7 days prior to UUO as previously described. Briefly, mice were anesthetized, and a midline abdominal incision was performed on the left kidney and the terminal ureters were obstructed. Then, 5 × 107 IU/100 μl filter-purified scrambled-shRNA (Scr-sh group, n=6) or NR1-shRNA (NR1-sh Group, n=6) lentivirus cocktail (forward: 5’-CACCGGTACCCATGTCATCCCAAATCGAAATTTGGGATGACATGGGTACC-3’ and reverse: 5’-AAAAGGTACCCATGTCATCCCAAATTTCGATTTGGGATGACATGGGTACC-3’) purchased from NovoBio Biotechnology Co., Shanghai, China [22] was infused through the ureters for 5 min via an intrathecal catheter attached to a micro-syringe (Hamilton, Franklin, Massachusetts) pump (WPI). After 7 days, mice were re-anesthetized and a midline abdominal incision was performed on the left kidney. The left ureters were double ligated with 4-0 silk surgical sutures [30].
Human proximal tubule (HK-2) cell culture and drug treatment
HK-2 cells (American Type Culture Collection, Manassas, Virginia) were grown in keratinocyte medium (Thermofisher, Waltham, Massachusetts) with 10% FBS in a 5% CO2 humidified incubator at 37°C. Cells were then treated with recombinant human TGF-β (100 ng/ml, R&D System, Minneapolis, Minnesota) for 48 h with a combination of NMDA (50 μM, Tocris, Minneapolis, Minnesota), MK-801 (10 μM, Tocris), or KN-93 (10 μM, Tocris).
Ischemia/reperfusion (IR) mouse model and DXM Administration
Mice were anesthetized and then a midline abdominal incision was performed on the right kidney. The right renal artery was isolated from the renal vein carefully and then clapped for 60 min. Mice were then divided randomly into the IR group (n=6), low-dose DXM group (LD group, 1 mg/ml in drinking water, n=6), moderate-dose DXM group (MD group, 2 mg/ml in drinking water, n=6), and high-dose DXM group (HD group, 3 mg/ml in drinking water, n=6). Mice were then euthanized 28 days post-IR.
Masson's Trichrome Staining, Immunohistochemistry and Immunofluorescent assays
Seven days post-UUO, mice were anesthetized, and the obstructed kidneys were harvested. Paraffin sections were stained with Masson's trichrome or labeled with antibodies against TGF-β1 (Cell Signaling Technology, Danvers, Massachusetts), alpha-smooth muscle actin (Abcam, Cambridge, Massachusetts), COL1A1 (Abcam), S100A4 (Abcam) or Fibronectin (Abcam). Frozen sections and fixed cells were labeled with antibodies against NR1 (ThermoFisher), p- or total CaMKII (Abcam), p- or total extracellular signal-regulated kinase (ERK, Abcam), Snail (Abcam) or E-cadherin (Abcam) and then incubated with the relevant secondary antibodies for immunohistochemistry or immunofluorescence (Abcam). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, ThermoFisher) for immunofluorescent assays.
Western blotting Analysis
Kidneys and cells were homogenized in RIPA lysis buffer with protease and phosphatase inhibitor cocktail (Cell Signaling Technology). Total protein was then separated by SDS-PAGE and blotted with antibodies against α-SMA, COL1A1, S100A4, Fibronectin, NR1, p- or total CaMKII, p- or total ERK. The membranes were scanned and analyzed using the Gel Doc XR imaging system (Bio-Rad Laboratories, Hercules, California).
Statistical analysis
Data values were presented as mean ± SD. Area of fibrosis and positive staining in tissue sections was measured with Image J software and was shown as percentages. Standard ANOVA with Bonferroni test was performed using GraphPad Prism 6.0. Two-tailed Student’s t test was used for other data types. Differences were considered statistically significant at P <0.05.