STAT3 drives the expression of ACSL4 in acute kidney injury

Summary Long-chain acyl-CoA synthetase family 4 (ACSL4) metabolizes long-chain polyunsaturated fatty acids (PUFAs), enriching cell membranes with phospholipids susceptible to peroxidation and drive ferroptosis. The role of ACSL4 and ferroptosis upon endoplasmic-reticulum (ER)-stress-induced acute kidney injury (AKI) is unknown. We used lipidomic, molecular, and cellular biology approaches along with a mouse model of AKI induced by ER stress to investigate the role of ACSL4 regulation in membrane lipidome remodeling in the injured tubular epithelium. Tubular epithelial cells (TECs) activate ACSL4 in response to STAT3 signaling. In this context, TEC membrane lipidome is remodeled toward PUFA-enriched triglycerides instead of PUFA-bearing phospholipids. TECs expressing ACSL4 in this setting are not vulnerable to ferroptosis. Thus, ACSL4 activity in TECs is driven by STAT3 signaling, but ACSL4 alone is not enough to sensitize ferroptosis, highlighting the significance of the biological context associated with the study model.


FIGURE S5. [Proximal tubule lipidome with tunicamycin] related to FIGURE 5
Relative contents in lipid classes with mol%<1 in isolated proximal tubules isolates from mouse kidney 48 hours after intraperitoneal injection of 1 mg/kg tunicamycin (Tun) or DMSO (Vh).(n=3 mice per group).p values were computed with the FDR approach with the method of Benjamini Krieger and Yekiuteli, with a FDR<1%.The y-axis indicates the relative expression of each lipid class (mol%), which is the percentage of total membrane lipids in the sample.

FIGURE S6. [Liperfluo staining of HK2 cells with RSL3] related to FIGURE 6
Fluorescence intensity of lipid hydroperoxides (LiperFluo staining) in HK-2 cells incubated with with RSL3 for 24 hours in the presence or absence of siRNA targeting ACSL4 (siACSL4) or a scrambled siRNA (siCtrl) (n=12 replicates per condition).p values were computed with the FDR approach with the method of Benjamini, Krieger and Yekiuteli, with a FDR<1%.

FIGURE S7. [HK2 cells lipidome with oncostatin] related to FIGURE 6
A. Relative contents in lipid classes with mol%>1 in HK-2 cells incubated with 20 ng/ml OSM for 48 hours compared to the vehicle condition (n=3 replicates per condition).p values were computed with the FDR approach with the method of Benjamini, Krieger and Yekiuteli, with a FDR<1%.The y-axis indicates the relative expression of each lipid species (mol%), which is the percentage of total membrane lipids in the sample.

B.
Relative contents in lipid classes with mol%<1 in HK-2 cells incubated with 20 ng/ml OSM for 48 hours compared to the vehicle condition (n=3 replicates per condition).p values were computed with the FDR approach with the method of Benjamini, Krieger and Yekiuteli, with a FDR<1%.The y-axis indicates the relative expression of each lipid species (mol%), which is the percentage of total membrane lipids in the sample.

FIGURE S8. [HK2 cells lipidome with IL-6] related to FIGURE 6
A. Relative contents in lipid classes with mol%<1 in HK-2 cells incubated with 100 ng/ml Il-6 for 48 hours compared to the vehicle condition (n=3 replicates per condition).p values were computed with the FDR approach with the method of Benjamini Krieger and Yekiuteli, with a FDR<1%.The y-axis indicates the relative expression of each lipid class (mol%), which is the percentage of total membrane lipids in the sample.B. Relative contents in PE (18:0 / 20:4) and PE (18:0 / 22:4) in HK-2 cells incubated with 100 ng/ml Il-6 for 48 hours compared to the vehicle condition (n=3 replicates per condition).p values were computed with the FDR approach with the method of Benjamini, Krieger and Yekiuteli, with a FDR<1%.PE: phosphatidylethanolamine, PC: phosphatidylcholine, PI: phosphatidylinositol.The y-axis indicates the relative expression of each lipid species (mol%), which is the percentage of total membrane lipids in the sample.

[Gpx4, Fsp1 and Slc7a11 transcripts levels] related to FIGURE 1 A, B and C.
Gpx4, Fsp1 and Slc7a11 transcripts levels measured by q-PCR in kidneys cortex of mice 48 and 96 hours after intraperitoneal injection of 1 mg/kg tunicamycin (Tun) or DMSO (Vh) (n=6 mice per group).p values were computed with one-way ANOVA followed by a Dunnett's multiple comparisons test.
D. Expression of Osm transcripts by q-PCR in kidneys cortex of mice 48 hours and 96 hours after intraperitoneal injection of 1 mg/kg Tunicamycin (Tun) or DMSO (Vh) (n=3 to 4 mice per group).pvalueswere computed with one-way ANOVA followed by a Dunnett's multiple comparisons test.FIGURE S4. [ACSL4 and IDO1 expression by IFNg] related to FIGURE 4A and B. ACSL4 and IDO1 transcripts levels measured by RT-qPCR in HK-2 cells incubated with 50 ng/ml IFNg for 24 hours (n=4-5 replicates per condition).p values were computed with a Student's T test.