EET-Based Therapeutics Mitigate Sorafenib-Associated Glomerular Cell Damage

Abstract

Background: This study investigates how sorafenib induces toxicity in glomerular cells and examines the protective role of 8,9-epoxyeicosatrienoic acid (8,9-EET) analogs in reducing this kidney damage.

Methods: Human renal mesangial cells (HRMCs) and podocytes were treated with no treatment, sorafenib alone, or sorafenib combined with 8,9-EET analogs. Cell viability and apoptosis were measured in both cell types.

Results: Sorafenib (1-10 µM) lowered cell viability and increased caspase 3/7 activity in a dose-dependent way in HRMCs and podocytes. Five of twenty 8,9-EET analogs significantly enhanced cell survival and decreased apoptosis. RNA sequencing showed that sorafenib altered 1244 genes, including those involved in cell cycle and the Raf/MEK/ERK pathway. The 8,9-EET analog MDB-52a raised ANGPTL4 levels, linked to metabolism and vascular health, and reduced ACTA2, which could activate protective pathways. Nephroseq data correlated these gene changes with glomerulosclerosis.

Conclusions: MDB-52 appears to counteract gene disruptions and protect against sorafenib-induced kidney damage. Overall, 8,9-EET analogs targeting glomerular cells could be potential therapeutic agents to lessen sorafenib-related nephrotoxicity.

Keywords: epoxylipids; mesangial cells; nephrotoxicity; onconephrology; podocytes.

Figure 1

Sorafenib treatment reduces cell viability in a dose-dependent manner. (a) Cell viability measured in HRMCs treated with increasing concentrations of sorafenib (1, 3, 5 and 10 µM). The data demonstrate a dose-dependent reduction in cell viability, indicating the cytotoxic effects of sorafenib on this cell. (b) Cell viability measured in podocyte cells treated with serial dilutions of sorafenib. Similarly to HRMCs, podocytes show a dose-dependent decrease in viability, suggesting sensitivity to sorafenib-induced cytotoxicity across different glomerular cell types. These results highlight the dose-dependent impact of sorafenib on glomerular cell viability. White dots indicate individual samples; * p < 0.05, *** p < 0.01, and **** p < 0.001 indicates statistical significance.

Figure 2

Protective effects of an 8,9-EET analog on sorafenib treatment in preserving HRMC viability in a dose-dependent manner. (a) MDB-32 significantly increases cell viability, providing protection against sorafenib-induced cell death in HRMCs. (b) MDB-52a shows improved cell viability across all tested concentrations (1, 3, and 10 µM), with the 10 µM dose nearly restoring cell viability to the levels observed in control cells. (c–e) MDB-52b, MDB-77, and MDB-78 exhibit a dose-dependent increase in cell viability, with higher concentrations leading to greater protection against sorafenib-induced cytotoxicity. These results highlight the potential of these 8,9-EET analogs in enhancing cell survival under sorafenib treatment, with MDB-52a demonstrating the most significant effect at higher doses. White dots indicate individual samples; *** p < 0.01, and **** p < 0.001 indicates statistical significance.

Figure 3

8,9-EET Analog Mitigates Sorafenib-Induced HRMCs Death by Suppressing Caspase 3/7 Activity. Caspase 3/7 activity, a marker of sorafenib-induced apoptosis, is visualized as green, fluorescent spots. An increase in green fluorescence indicates elevated caspase 3/7 activity and greater cell death, while a decrease reflects reduced apoptotic activity. HRMCs were seeded into 96-well plates for compound screening. Each plate was used to test two to three compounds, alongside matched vehicle and sorafenib (10 µM) controls. Caspase 3/7 activation was assessed using a luminescent assay, with control datasets applied uniformly across all compounds tested on the same plate. MDB-32, MDB-52a, and MDB-52b were each tested on separate 96-well plates, with individual sets of vehicle and sorafenib controls specific to each compound. In contrast, MDB-77 and MDB-78 were tested concurrently on a single 96-well plate, sharing a common set of vehicle and sorafenib controls. This design enabled consistent intraplate comparisons while maintaining compound-specific control conditions. (a) Treatment with the 8,9-EET analog MDB-32 led to a 20–40% reduction in sorafenib-induced caspase 3/7 activity, suggesting a protective effect against apoptosis. (b) Cells treated with MDB-52a in combination with 10 µM sorafenib showed significantly lower caspase 3/7 activity compared to sorafenib alone. MDB-52a reduced activity in a dose-dependent manner by 60–90%, demonstrating strong anti-apoptotic efficacy in HRMC cells. (c) A similar reduction in caspase 3/7 activity was observed with MDB-52b, further supporting its protective role. (d,e) HRMC cells treated with MDB-77 and MDB-78 also exhibited decreased caspase 3/7 activity relative to sorafenib-treated controls, indicating potential anti-apoptotic effects of these compounds.

Figure 4

Protective effects of an 8,9-EET analog on sorafenib treatment in preserving podocyte cell viability in a dose-dependent manner. (a) MDB-52a significantly change in cell viability in podocyte cells, providing protection against sorafenib-induced cell death, with the 10 µM dose showing the most notable improvement. (b) MDB-52b enhances cell viability across all tested concentrations (1, 3, and 10 µM), with the 10 µM dose nearly restoring cell viability to 50%. (c–e) MDB-77, MDB-78, and RM-84 exhibit a dose-dependent increase in cell viability, with higher concentrations offering greater protection against sorafenib-induced cytotoxicity. These findings underscore the potential of 8,9-EET analogs in protecting cell survival during sorafenib treatment, with MDB-52a showing the most pronounced effect at 10 µM in podocyte cells. White dots indicate individual samples; **** p < 0.001 indicates statistical significance.

Figure 5

8,9 EET Analog Mitigates Sorafenib-Induced Cell Death of Podocyte by Suppressing Caspase 3/7 Activity. The protective potential of 8,9-EET analogs against sorafenib-induced apoptosis in podo-cytes was evaluated by measuring caspase 3/7 activity, a key marker of programmed cell death. Apoptotic activity was visualized as green, fluorescent spots, an increased number of spots indi-cates elevated caspase 3/7 activity, while fewer spots suggest reduced apoptosis. Human podo-cytes were seeded into 96-well plates to evaluate caspase 3/7 activation in response to various test compounds. Each plate was configured to include two to three compounds, along with matched vehicle and sorafenib (10 µM) controls. A single set of control data (vehicle and sorafenib) was used for all compounds tested on the same plate to ensure consistent intra-plate comparisons. MDB-52a, MDB-52b, and RM-84 were tested concurrently on one 96-well plate, sharing a common set of vehicle and sorafenib controls. Similarly, MDB-77 and MDB-78 were tested together on a separate plate, also using a single set of vehicle and sorafenib controls for both compounds. Treatment with 8,9-EET analogs significantly reduced caspase 3/7 activity induced by sorafenib, indicating their protective effects: (a) MDB-52a: Co-treatment with MDB-52a and 10 µM sorafenib resulted in a marked reduction in caspase 3/7 activity compared to sorafenib alone. The effect was dose-dependent, with a reduction of approximately 50–70%. (b) MDB-52b: Similar protective effects were observed, with caspase 3/7 activity reduced by 40–60% in a dose-dependent manner. (c,d) MDB-77 and MDB-78: Both compounds decreased caspase 3/7 activity when combined with sorafenib. MDB-77 was effective at 1 and 3 µM, while MDB-78 showed minimal effect at lower doses but was effective at 10 µM. (e) RM-84: This analog also demonstrated a dose-dependent reduction in caspase 3/7 activity, with the 10 µM dose significantly lowering apoptosis compared to sorafenib-only treatment.

Figure 6

Evaluation of the 8,9 EET analog mdb-52a for anticancer activity and tumor growth promotion. Human prostate cancer cells treated with MDB52a (1, 3 & 10 µM) showed no significant antitumor effects, indicating that MDB52a lacks inherent anticancer properties. Additionally, MDB52a did not promote tumor growth, suggesting it is a neutral compound in terms of cancer cell proliferation within this concentration range.

Figure 7

RNA-Seq analysis identifies key differentially expressed genes (DEGs) in HRMCs treated with 8,9-EET analog mdb-52a. This figure presents RNA sequencing (RNA-seq) analysis to identify key DEGs in HRMCs treated with 8,9-EET analogs. The volcano plot highlights the distribution of genes based on statistical significance. Red points represent genes that are statistically significant, meeting the threshold for both p-value (<0.05) and fold change (log2FC). The position along the x-axis indicates whether a gene is upregulated or downregulated. Red points on the right (positive log2 fold change) represent upregulated genes (i.e., genes expressed more in the experimental condition), while red points on the left (negative log2 fold change) represent downregulated genes (i.e., genes expressed less in the experimental condition). Green points represent non-significant genes: those on the left are non-significant downregulated genes, while those on the right are non-significant upregulated genes. While these genes meet the fold change threshold (either up or down), they fail to meet the significance threshold based on p-value (padj ≥ 0.05). This analysis provides an overview of how MDB-52a affect gene expression in HRMCs. (a) Heat map displaying the 50 most significantly differentially expressed genes across Groups A–E. (b) A DEG expression heatmap displaying the 50 most upregulated and downregulated genes between Groups A and B. These results highlight the extensive gene expression changes between the untreated group and the treatment group. (c) Heat map of Group D vs. Group B shows five significantly differentially expressed genes. These results indicate significant gene expression changes between the two groups, highlighting the impact of treatment with sorafenib alone compared to the combined treatment of sorafenib with MDB52a. (d) Heat map of Group E vs. Group A shows two significantly differentially expressed genes. These figures indicate significant gene expression changes in HRMCs treated with MDB52a compared to untreated cells. (e) The volcano plot comparing Group A and Group B shows that a total of 1244 genes were significantly differentially expressed. (f) Volcano plot displaying mRNA expression differences between Group D and Group B (g) Volcano plot displaying mRNA expression differences between Group E and Group A (h) Volcano plot displaying mRNA expression differences between Group C and Group B (i) Analysis of 1000 differentially expressed genes using a PCA plot revealed distinct clustering patterns. Groups B, C, and D exhibited similar gene expression levels, indicating related responses to their respective treatments. In contrast, Groups A (untreated) and E (treated with MDB52a) displayed similar gene reactivation patterns, suggesting that MDB52a may restore normal gene expression, mimicking the untreated condition. Group-A, without any treatment, Group-B, cells treated with 10 µM Sorafenib, Group-C (cells treated with 3 µM MDB-52a and 10 µM Sorafenib) Group-D (cells treated with 10 µM MDB-52a and Sorafenib) and Group-E (cells treated with 10 µM MDB-52a (8,9 EET analog).

Figure 8

MDB-52a effects on sorafenib-induced changes in podocyte-specific gene expression. Relative mRNA expression levels of key podocyte markers, including (a) NPHS1, (b) Desmin, (c) Synaptopodin, (d) NPHS2, (e) CD2AP, (f) ITGB, and (g) TJP1, were quantified using RT-PCR across different experimental groups. Group A represents untreated control cells with baseline expression levels. Group B (10 µM Sorafenib-treated cells) exhibited a significant reduction in protective markers (NPHS1, NPHS2, Synaptopodin, TJP1, CD2AP, ITGB) and a marked increase in the injury marker Desmin, indicating podocyte injury. Co-treatment with a low concentration of MDB-52a (Group C: 3 µM MDB-52a + 10 µM Sorafenib) showed minor improvements in some markers but was insufficient for substantial recovery. However, co-treatment with a higher concentration of MDB-52I (Group D: 10 µM MDB-52a + Sorafenib) significantly restored the expression of protective markers to near-normal levels and mitigated the upregulation of Desmin, highlighting the robust protective effects of MDB-52a at higher doses. Data are presented as mean ± SEM, and statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, NS: not significant).

Figure 9

Comparative expression analysis of selected genes in focal segmental glomerulosclerosis (FSGS) vs. healthy living donors. This figure presents a comparison of gene expression between patients with Focal Segmental Glomerulosclerosis (FSGS) (Group 2) and Healthy Living Donors (Group 1) using log2 median-centered intensity. The heatmap displays gene expression levels, with red indicating higher expression and blue indicating lower expression. The analysis highlights significant differences in gene expression between the two groups, helping to illustrate key molecular changes associated with FSGS. Data were obtained and visualized using Nephroseq (The Regents of The University of Michigan, Ann Arbor, MI, USA). For additional details, the Nephroseq resource can be accessed at: http://www.nephroseq.org/resource/main.html (accessed on 15 February 2024). Group 1 is Healthy Subjects and Group 2 is Focal Segmental Glomerulosclerosis (FSGS).