Crosstalk Between SMPDL3b and NADPH Oxidases Mediates Radiation-Induced Damage of Renal Podocytes

Abstract

Patients undergoing radiotherapy (RT) for various tumors localized in the abdomen or pelvis often suffer from radiation nephrotoxicity as collateral damage. Renal podocytes are vulnerable targets for ionizing radiation and contribute to radiation-induced nephropathies. Our prior work previously highlighted the importance of the lipid-modifying enzyme sphingomyelinase acid phosphodiesterase like 3b (SMPDL3b) in modulating the radiation response in podocytes and glomerular endothelial cells. Hereby, we investigated the interplay between SMPDL3b and oxidative stress in mediating radiation injury in podocytes. We demonstrated that the overexpression of SMPDL3b in cultured podocytes (OE) reduced superoxide anion generation and NADPH oxidase activity compared to wild-type cells (WT) post-irradiation. Furthermore, OE podocytes showed downregulated levels of NOX1 and NOX4 after RT. On the other hand, treatment with the NOX inhibitor GKT improved WTs' survival post-RT and restored SMPDL3b to basal levels. in vivo, the administration of GKT restored glomerular morphology and decreased proteinuria in 26-weeks irradiated mice. Taken together, these results suggest a novel role for NOX-derived reactive oxygen species (ROS) upstream of SMPDL3b in modulating the response of renal podocytes to radiation.

Keywords: NADPH oxidases (NOX); ionizing radiation (IR); nephropathies; podocytes (MeSH: D050199); reactive oxygen species (ROS); smpdl3b; sphingolipids (SLs).

Figure 1

Overexpression of SMPDL3b protects podocytes from radiation-induced reactive oxygen species. (A) Quantification of DHE mean immunofluorescence (left panel) at baseline (control), 10 and 30 minutes, 1hr, 12hrs, and 24 hours post-irradiation at 8 Gy (WT vs OE: *p = 0.007 at 1hr, #p = 0.0156 at 12 hrs, $p = 0.0039 at 24hrs). Immunofluorescence staining with DHE and DAPI of podocytes (right panel) at baseline 0 Gy (control) and 8 Gy at 1hr post-irradiation. Objective 20x. (B) Superoxide anion generation measured via lucigenin mediated NADPH oxidase assay in both WT and OE human podocytes at baseline (control) and 10min, 1hr, 2hrs, 3hrs after 8 Gy irradiation (WT vs. OE: **p = 0.0036 at 1hr, #p = 0.0340 at 3hrs). (C) Conventional PCR analysing expression of NOX1 (730bp),−2,−3,−4 (253bp), and−5 (762bp), ß-actin (106bp) in WT human podocytes cell lysate. The results shown are the mean values of at least three independent experiments.

Figure 2

Differential expression of NOX1 and NOX4 in WT and OE podocytes upon radiation. Transcriptional RT-PCR analysis of NOX1 (A) (*p = 0.007 for WT vs. OE at 2 hrs, #p = 0.0142 at 24hrs) and NOX4 (B) mRNA expression in WT and OE human podocytes at baseline, 30 min, 2 hrs, 4 hrs, 12 hrs, and 24 hrs after 8 Gy irradiation. Translational immunoblotting analysis of NOX1 (C) (WT vs. OE: 1hrs **p < 0.0041; 3 hrs &p < 0.0303; 12 hrs ##p < 0.0052; 24hrs $p < 0.0321), and NOX4 (D) (WT vs. OE: 24 hrs *p < 0.0245) in WT and OE human podocytes at baseline, 1 hr, 3 hrs, 6 hrs, 12 hrs, 24 hrs following 8 Gy irradiation. The results shown are the mean values of at least three independent experiments.

Figure 3

SMPDL3b protein levels are restored upon administration of both NAC and GKT after 24 hours of radiation in podocytes. SMPDL3b protein (A) (24 hrs vs. 24 hrs+GKT: *p = 0.0382) and mRNA expression (B) at baseline and 24 hrs after 8 Gy irradiation in pre-treated WT podocytes with ROS scavenger N-acetylcysteine (100 μM) and/or dual NOX1/4 inhibitor GKT (10 μM). (C) Survival profile of WT podocytes at baseline and 24hrs post-irradiation pre-treated with NAC and/or GKT via MTT assay detected by spectrophotometry (Ctrl vs. 24 hrs **p = 0.0045; Ctrl vs. 24 hrs+NAC+GKT **p = 0.0083; 24 hrs vs. 24 hrs+NAC **p = 0.0047; 24 hrs vs. 24 hrs+GKT *p = 0.0401; 24 hrs+NAC vs. 24 hrs+NAC+GKT **p = 0.0088). The results shown are the mean values of at least three independent experiments.

Figure 4

Radiation causes acute upregulation of NADPH oxidases protein expression in kidneys of 24hrs post-RT C57BLC/6 mice. Increase of protein levels of NOX1 (A) (Ctrl vs. Rad *p = 0.0493), and NOX4 (B) (Ctrl vs. Rad *p = 0.0196) detected by immunoblotting in renal cortices at 24hrs post-irradiation. Post-irradiation cleavage of caspase 3 reduced upon administration of GKT (Ctrl+GKT vs. Rad+GKT *p = 0.0498, Rad vs. Rad+GKT **p = 0.0075) (C). The results shown are the mean values of at least three independent experiments.

Figure 5

Administration of GKT restores radiation injury markers in 26 weeks post-irradiation mice. (A) Proteinuria measured in C57BLC/6 mice at 22 weeks post-RT by urine collection (Ctrl vs. Ctrl+GKT *p = 0.0308; Ctrl vs. Rad **p = 0.0452; Ctrl vs. Rad+GKT *p = 0.0484; Ctrl+GKT vs. Rad ****p < 0.0001; Rad vs. Rad+GKT ***p = 0.0001). (B) Systolic pressure measured by non-invasive tail-cuff method (Ctrl vs. Rad+GKT **p = 0.0089; Ctrl+GKT vs .Rad *p = 0.0134; Rad vs. Rad+GKT**p = 0.0018). GKT treatment ameliorates partially morphological parameters of irradiated glomeruli in mice. Representative photomicrographs of glomeruli from Ctrl (1), Ctrl+GKT (2), Rad (3), Rad+GKT (4) kidneys harvested from C57BLC/6 mice at 24 weeks post-radiation; Quantification of all parameters of at least 20 glomeruli in all groups (C) Paraffin-embedded sections, 5 μm thick, were stained with H&E (20x), scale bar 5 μm (Ctrl vs. Rad **p = 0.0010; Ctrl+GKT vs. Rad **p = 0.0027; Rad vs. Rad+GKT **p = 0.0099). (D) Paraffin-embedded sections, 5 μm thick, were stained with Masson Trichrome (blue stain) for collagen deposits (20x), scale bar 5 μm (Ctrl vs. Rad ***p = 0.0008; Ctrl vs. Rad+GKT *p=0.0222; Ctrl+GKT vs. Rad **p=0.0055; Rad vs. Rad+GKT *p=0.0347). (E) Immunoblotting of SMPDL3b in renal cortices homogenates of 26 weeks irradiated and non-irradiated C57BL6 mice. SMPDL3b is restored upon GKT treatment (Ctrl vs. Rad *p = 0.0214; Rad vs. Rad+GKT ns p = 0.0724). The results shown are the mean values of at least three independent experiments.