#Abstract
Diabetic nephropathy (DN) is a common complication of diabetes mellitus
and cell death is a key issue in DN. Ferroptosis is a recently
discovered type of iron-dependent cell death and different from other
kinds of cell death including apoptosis and necrosis. However,
ferroptosis has not been described in the context of DN. This study was
to explore the role of ferroptosis in the DN pathophysiology and to
explore the efficacy of ferroptosis inhibitor SRS 16-86 on DN. The STZ
injection was used to establish the DM and DN animal models. We detected
the levels of iron, reactive oxygen species, and ferroptosis-specific
markers in a rat DN model to investigate whether there was ferroptosis
in the process of DN. The hematoxylin-eosin staining, blood
biochemistry, urine biochemistry and the of function kidney were used to
evaluate the efficacy of ferroptosis inhibitor-SRS 16-86 in repairing
DN. We found that SRS 16-86 could
improve the recovery of renal function after DN by improving the
antiferroptosis factors glutathione peroxidase 4, glutathione, and
system Xc-light chain and could lower the lipid peroxidation marker and
4-hydroxynonenal. SRS 16-86 treatment may improve the structure of renal
organization after DN. Inflammatory cytokines-interleukin 1β and tumor
necrosis factor α, and intercellular adhesion molecule 1 were decreased
significantly following SRS 16-86 treatment after DN. Results indicate
that there is a strong connection
between ferroptosis and the pathological mechanism of DN. The validity
of SRS 16-86, a ferroptosis inhibitor in DN repair, supports its
potential as a new therapeutic target for DN.
Keywords : Ferroptosis; diabetic nephropathy;
ferroptosis inhibitor
# Introduction
Diabetic nephropathy (DN) is a common complication of type 1 and type 2
diabetes mellitus (DM)(1). DN affects approximately 40% of people with
DM and is the leading cause of chronic kidney disease and end-stage
renal disease worldwide(2). Glomerular destruction leads to kidney
damage, proteinuria, and hypertension(3). Apoptosis, autophagy, and
necrosis are three forms of programmed cell death and are involved in
the pathogenesis of DN. Podocyte apoptosis leads to glomerular injury
and podocyte failure, which is related to proteinuria and glomerular
structural damage in DN(4). In the DN process, the epithelial cells of
the proximal convoluted tubules also undergo apoptosis, leading to
tubular atrophy, the reduction of tubular cells, and the formation of
glomeruli, which ultimately contributes to the loss of renal
function(5). On the other hand, the dysfunction of autophagy may also
cause the pathogenesis of DN. More specifically, the decrease of
podocyte autophagy activity in diabetes kidneys leads to changes in
podocyte function, which then destroys the glomerular filtration
barrier(6). In addition, the autophagic activity of renal proximal
tubular cells in diabetes is weakened, leading to the accumulation of
damaged molecules and organelles, which are usually decomposed by
autophagy, and this causes proteinuria(7). In addition, necrosis may
play a key role in podocyte injury and the subsequent worsening of
DN(8). Indeed, the pattern of cell death in DN has been intensely
researched. Apoptosis and necrosis are among of the causes of acute DN
cell injury, and autophagy seems to have beneficial effects on DN.
However, ferroptosis has not been
described in the context of DN.
In 2012, Dixon et al. studied the mechanism of erastin killing cancer
cells through renin-angiotensin system (RAS ) mutation and
formally named this cell death process ferroptosis (9). In the
process of ferroptosis, there is no morphological change in cell
membrane or chromatin, which primarily suggests that the volume and
cristae of mitochondria decreases while the density of the mitochondrial
membrane increases(10). Biochemically, the main manifestations of
ferroptosis are the decrease of glutathione peroxidase 4 (GPX4)
activity, the loss of intracellular glutathione, and the increase of
reactive oxygen species (ROS) level(11). The accumulation of iron and
the consumption of glutathione and lipid peroxidation are indispensable
and occur simultaneously in the process of ferroptosis(12). Inhibiting
lipid peroxidation can prevent the cell death stage of ferroptosis.
Ferroptosis is associated with
inflammatory processes in which cell-released substances are
significantly involved in the innate immune system and control cellular
inflammatory responses, signal transduction, and cell proliferation.
Ruptured ferroptosis associated cells release damage-related molecular
patterns. And ferroptosis leads to the infiltration of macrophages and
neutrophils and the release of inflammatory cytokines. When the
accumulation of lipid ROS exceeds a certain threshold, a large number of
pro-inflammatory cytokines are produced, such as interleukin 1β (IL-1β)
and tumor necrosis factor α (TNF-α).
Iron
overload and lipid ROS have been shown, in vitro, to be the two main
factors causing ferroptosis in tumor cells and brain slices, which have
been reported in DN. DN leads to ROS accumulation, and bleeding
increases the iron load in this process(13). Glutamic acid could also
induce the death of podocyte cells. After DN, the level of glutamic acid
increases, and glutamate excitotoxicity becomes apparent. GPX4
expression decreased and lipid peroxidation products increased in the
animal model of DN and the DN patients’ blood. Therefore, we speculated
that ferroptosis in DN contributes to its damage. And the inhibition of
ferroptosis could reduce functional damage and improve renal repair.
It is a question worthy of investigation that whether specific
inhibitors of ferroptosis could promote renal repair, and in vivo
research to identify effective and stable specific inhibitor of
ferroptosis is warranted. Ferrostatin 1 (Fer-1) is a first-generation
ferroptosis inhibitor, possessing demonstrated ability to inhibit
ferroptosis in vitro. However, due to the instability of plasma and
metabolism, its internal function is weak. SRS 16-86 is a
third-generation small-molecule which could restrain lipid ROS. It has
been reported to strongly inhibit ferroptosis in renal failure with
ischemia reperfusion injury.
We hypothesized that ferroptosis is an important damage mechanism after
DN. Specifically, we studied whether ferroptosis occurs in DN and SRS
16-86 to inhibit ferroptosis could improve the recovery of renal
function. The insights earned from our study could promote the
understanding of the process in DN and support new therapeutic method
for treating DN.