##Improved renal function recovery after DN via the inhibition of ferroptosis
To explore whether SRS 16-86 could improve renal function recovery after DN, we examined the expression of 24-hour urine volume and CRE2U and UTP content in urine and uric acid (UA) as well as the UREA and creatinine (CRE) content in blood (Figure 7A,7B,7C; Figure 8A,8B,8C). As was shown in Figure 2, 24-hour urine volume and the content of CRE2U and UTP in the DN-SRS group were remarkably lower than those in the DN group. We observed the same pattern of change in assessments of UA, UREA and CRE. These results showed SRS 16-86 significantly promoted renal function recovery after DN.
#Discussion
Ferroptosis is a newly discovered cell death pathway, which has been confirmed in stroke, Parkinson disease, and spinal cord injury(14-15). However, ferroptosis in DN has not been reported on. We found that the key regulatory factors of ferroptosis, including GPX4, GSH, and xCT were reduced in DN. Meanwhile, tests for ROS and 4HNE indicated that lipid peroxidation level was added. By analyzing tissue structure and renal function after SRS 16-86 inhibited ferroptosis, we found that inhibiting ferroptosis could increase the survival of normal tissue structure and improve the recovery of renal function. Inflammatory cytokines also decreased after SRS 16-86 treatment. Experimental evidence supporting the beneficial effect of ferroptosis interference opens new avenues of treatment for reducing cell death and promoting DN repair.
Excess iron in tissue cells induces cell death by producing ROS through the Fenton reaction. Additionally, GPX4 inactivation due to GSH depletion can also lead to ROS accumulation through lipid peroxidation(16-17). ROS can react with polyunsaturated fatty acids (PUFAs) in lipid membranes and induce lipid peroxidation. A study has shown that ferroptosis is closely regulated by the combination of several signaling pathways, including the regulation of iron homeostasis, the RAS/rapidly accelerated fibrosarcoma (RAF) signaling pathway and the glutamine-cystine transport signaling pathway(18-19). Factors such as GSH and GPX4 play key roles in the ferroptoptic process. GSH removes excessive ROS from the body through GPX4, thus protecting the body from damage. Once the dynamic GSH-GPX4-ROS balance is destroyed, the excessive ROS generated by the body cannot be removed in time, which causes certain damage to the body. GPX4 deficiency has been found to lead to significantly elevated iron death in the epithelial cells of the renal tubules, resulting in acute renal failure(20). It has been found that the increase of glutamate can promote the activation of glutamate-glutathione transporter, allowing glutamate to enter the cell to produce excessive ROS and induce ferroptosis(21). SRS 16-86 is a newly more stable and effective synthesized iron-droop inhibitor(22-23). It has a strong protective effect on renal ischemia-reperfusion injury. In our DN model, SRS 16-86 increased the concentration of GSH in renal tissue and decreased the lipid ROS marker 4HNE. GPX4 and xCT are markers of ferroptosis. The expression of GPX4 and xCT was downregulated after injury and increased after inhibition treatment. These results indicate that SRS 16-86 inhibiting the ferroptosis process. HE staining showed that more tissues were retained after inhibitor treatment.
Inflammation is an immune response that is produced by the body according to changes in the internal and external environment of cells. Moderate immune reaction can protect the body, while excessive immune reaction can cause harm to the body. The process of ferroptosis is often accompanied by inflammation(24-26). In a mouse model of folic acid induced acute renal injury, necrosis and inflammation accompanied by ferroptosis led to the death of a large number of renal tubular cells, causing acute renal failure and early death(27). In our study, SRS 16-86 treatment reduced the expression of proinflammatory cytokine IL-1 β, TNF-α, and ICAM-1 which suggesting that inhibition of ferroptosis may also lead to the blocking of the inflammatory cascade in DN. Lipid peroxidation in ferroptosis can produce inflammatory signal molecules. This was consistent with the effect of Fer-1 on reducing proinflammatory cytokines in the acute renal injury model. However, whether ferroptosis is related to the inflammatory microenvironment of DN is a question that remains to be further studied.
Indications that the ferroptosis pathway is related to the secondary injury of DN has opened up exploration of this problem. (I) GSH exhaustion and lipid peroxidation have been observed in DN, but GPX4 and other essential factors for iron removal in DN is still unclear. The exploring of these factors could provide new ways into the pathophysiology of DN. (II) Moreover, the sensitivity of the different types of cells in DN to ferroptosis is unclear. In our study, we found that in the DN group, the glomerular morphology was abnormal, the gap between renal tubules was enlarged, and renal fibrosis was enhanced. However, the extent to which cells in each tissue are affected remains unknown. It’s important to determine whether other known drugs could promote DN by inhibiting ferroptosis. Studying ferroptosis may elucidate the mechanism of traditional medicine and provide a new direction for treatment.