PMC

Paraffin sections were prepared from 2-month-old mice kidneys, and immunofluorescence staining was performed to detect the expression of opiate receptors. Nephrin was used as a marker of podocytes.

Human podocytes were starved in serum free medium for 12 h, and then 10⁻⁶ M morphine was added. Cell lysates were collected at different time points for Western blot.

Two-month old mice were administered with either normal saline or morphine by subcutaneous implantation of a 75-mg slow-release morphine pellet. After 72 h urine was collected, and the amount of albumin (A) and albumin/creatinine ratio (B) were determined.

Total RNAs were prepared from human podocytes (labeled as P1, P2), and were used for RT-PCR to detect the expression of delta opiate receptor (DOR), kappa opiate receptor (KOR), and mu opiate receptor (MOR). RNAs from human fetal brain (B) were used as positive control. GAPDH was used as internal control.

A. Human podocytes were treated with agonists specifically for DOR (labeled as D), KOR (K), or MOR (M) for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin (B), synaptopodin (C), CD2AP (D), and podocin (E) in A, and the results (mean ± SD) represent three independent samples. * p<0.05 compared with control (CN).

A. Differentiated human podocytes were treated with 10⁻⁸ or 10⁻⁶ M morphine for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin (B), synaptopodin (C), CD2AP (D), and podocin (E) in A, and the results (mean ± SD) represent three independent samples. * p<0.05 compared with control (0 M).

A. Human podocytes were treated with 1, 10, or 50 µM H₂O₂ for 24 h, and the cell lysates were collected for Western blots. B–E. Quantification of the expression of nephrin (B), synaptopodin (C), CD2AP (D), and podocin (E) in A, and the results (mean ± SD) represent three independent samples. * p<0.05, ** p<0.01 compared with control (0 µM).

A. Human podocytes were pretreated with SOD or catalase for 1 h before 10⁻⁶ M morphine was added. After another 24 h incubation at 37°C, the cell lysates were collected for Western blot. B–E. Quantification of the expression of nephrin (B), synaptopodin (C), CD2AP (D), and podocin (E) in A, and the results (mean ± SD) represent three independent samples. * p<0.05 compared with blank control, while # p<0.05 compared with morphine treatment alone.

A. Human podocytes were pretreated with of LY294002 (LY), SB203580 (SB), or SP600125 (SP) for 1 h before 10⁻⁶ M morphine was added. After another 24 h incubation at 37°C, the cell lysate were collected for Western blot. B–E. Quantification of the expression of nephrin (B), synaptopodin (C), CD2AP (D), and podocin (E) in A, and the results (mean ± SD) represent three independent samples. * p<0.05 compared with blank control, while # p<0.05 compared with morphine treatment alone.

A. Human podocytes were treated with 10⁻¹⁰ to 10⁻⁶ M morphine for 12 h, and were labeled with DCFH for 30 min. After washing with PBS, the cells were incubated at room temperature for 2 h, and the ROS generation was determined. * p<0.001 compared with control (0 M). B. Kinetics of ROS generation for 10⁻⁶ M morphine treatment. C. Paraffin sections were prepared from control or morphine-receiving mice, and immunofluorescent staining was performed to detect the nuclear expression of 8-OHdG, a molecular marker of oxidative damage to DNA. WT1 was used as a marker of podocytes.

A. Two-month-old mice were administrated either normal saline or morphine for 3 days. Frozen sections were prepared for immuno-fluorescence staining. B. kidneys were homogenized, and the tissue lysates were subjected to Western blot to detect the changes of nephrin and synaptopodin. C–D. Quantification of the expression of nephrin (C) and synaptopodin (D), and the results (mean ± SD) represent three independent samples. E. A representative microphotograph from transmission electron microscopy studies. Foot processes are indicated by arrows. Foot processes are well delineated and are not fused in the control, while they are fused (effaced) in morphine-receiving mice. The scale bar is 500 nm. Mag. 270,000X.