Deletion of Alox15 improves kidney dysfunction and inhibits fibrosis by increased PGD2 in the kidney

Background Lipid-metabolizing enzymes and their metabolites affect inflammation and fibrosis, but their roles in chronic kidney disease (CKD) have not been completely understood. Methods To clarify their role in CKD, we measured the mRNA levels of major lipid-metabolizing enzymes in 5/6 nephrectomized (Nx) kidneys of C57BL/6 J mice. Mediator lipidomics was performed to reveal lipid profiles of CKD kidneys. Results In 5/6 Nx kidneys, both mRNA and protein levels of Alox15 were higher when compared with those in sham kidneys. With respect to in situ hybridization, the mRNA level of Alox15 was higher in renal tubules of 5/6 Nx kidneys. To examine the role of Alox15 in CKD pathogenesis, we performed 5/6 Nx on Alox15−/− mice. Alox15−/− CKD mice exhibited better renal functions than wild-type mice. Interstitial fibrosis was also inhibited in Alox15−/− CKD mice. Mediator lipidomics revealed that Alox15−/− CKD mouse kidneys had significantly higher levels of PGD2 than the control. To investigate the effects of PGD2 on renal fibrosis, we administered PGD2 to TGF-β1-stimulated NRK-52E cells and HK-2 cells, which lead to a dose-dependent suppression of type I collagen and αSMA in both cell lines. Conclusion Increased PGD2 in Alox15−/− CKD mouse kidneys could inhibit fibrosis, thereby resulting in CKD improvement. Thus, Alox15 inhibition and PGD2 administration may be novel therapeutic targets for CKD. Supplementary Information The online version contains supplementary material available at 10.1007/s10157-021-02021-y.


Cell culture
Rat kidney epithelial cells  were cultured in Dulbecco's modified eagle medium (4.5 g/L glucose) (Nacalai Tesque) supplemented with 5% fetal bovine serum, whereas human renal proximal tubule cells (HK-2 cells) were cultured in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum. Both NRK 52E cells and HK-2 cells were purchased from ATCC. We seeded these cells on 6-well culture plates to 80% confluence in a complete medium for 24 h and then transferred them to a serum-free medium. Then, they were preincubated with lipid metabolites for 30 min, followed by the treatment of recombinant human TGF-β1 (Pepro Tech) at 5 ng/mL and the lipid metabolites for different dosage as indicated for 24 h. We cultivated all cells at 37 °C under 5% CO2 condition in a humidified incubator.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis
Total RNA extracted from the mouse kidneys was reverse-transcribed using ReverTra Ace (TOYOBO), and the qRT-PCR analysis was performed in the Thermal Cycler Dice Real Time System (Takara Bio). The primers and templates were mixed with SYBR Premix Ex Taq II (Takara Bio). Thereafter, the mRNA contents were normalized to GAPDH and then calculated using the comparative CT method. Sequence-specific primers for mice, NRK-52E cells and HK-2 cells are listed below.
Sequence-specific primers for mice

Histological analysis
We fixed the mouse kidneys in 10% formalin neutral buffer solution (Wako) and histologically analyzed them by using the Masson's trichrome method as described previously [1].

In situ hybridization
The kidneys were fixed by perfusion with periodate lysine (0.2 M) and paraformaldehyde (2%) in phosphate-buffered solution.
The fixed samples were embedded in an optimum cutting temperature compound (Tissue Tek) and then cryosectioned (5 µm thickness). RNA in situ hybridization was performed using the RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, #322300) according to the manufacturer's instructions. We used the Target Probe Mm-Alox15 (Advanced Cell Diagnostics, #539781) for Alox15.

Statistical analysis
Statistical significance was evaluated using an unpaired t test. For multiplex comparisons, the one-way analysis of variance test with Tukey's test was used. P < 0.05 was considered statistically significant. Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software).