##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.