#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.