Accumulating evidence suggests that autophagy is involved in the pathophysiological processes of kidney diseases. However, the role of autophagy in the formation of calcium oxalate (CaOx) nephrolithiasis remains unclear. In this study, we investigated the effects of autophagy on renal tubular epithelial cell injury induced by CaOx crystals in vivo and in vitro. We first observed that the expression levels of LC3-II and BECN1 and number of autophagic vacuoles were markedly increased in the renal tissue of CaOx stone patients. We subsequently found that exposure of HK-2 cells to CaOx crystals could increase LC3-II and BECN1 expression as well as the number of GFP-LC3 dots and autophagic vacuoles in a dose- and time-dependent manner. In addition, our results suggest that CaOx crystals induced autophagy, at least in part, via activation of the reactive oxygen species (ROS) pathway in HK-2 cells. Furthermore, inhibition of autophagy using 3-methyladenine or siRNA knockdown of BECN1 attenuated CaOx crystal-induced HK-2 cells injury. However, enhancing autophagic activity with rapamycin exerted an opposite effect. Taken together, our results demonstrate that autophagy is essential for CaOx crystal-induced renal tubular epithelial cell injury and that inhibition of autophagy could be a novel therapeutic strategy for CaOx nephrolithiasis.

Ferroptosis is an iron-dependent lipid peroxidation process. Although the involvement of ferroptosis in kidney diseases has recently been reported, the association between ferroptosis and urolithiasis remains unclear. The present study examined the effects of ferroptosis on calcium oxalate (CaOx) crystal-induced renal tubular epithelial cell injury in vivo and in vitro . First, renal tubular epithelial cells were exposed to various concentrations of CaOx. By measuring cell viability, Fe 2+ levels, lipid peroxidation levels and the levels of ferroptosis-related proteins, it was identified that the relative expression of the ferroptosis agonist proteins, p53, long-chain acyl-CoA synthetases (ACSL4), transferrin (TF) and trans-ferrin receptor (TRC), increased, while the relative expression of the ferroptosis inhibitory proteins, solute carrier family 7 member 11 (SLC7A11, XCT) and glutathione peroxidase 4 (GPX4), decreased significantly. Furthermore, the levels of Fe 2+ and lipid peroxidation gradually increased, while cell viability significantly decreased. From these results, it was noted that the extent of CaOx-induced ferroptosis activation and cell injury was dependent on the CaOx concentration. To further investigate the association between ferroptosis and renal tubular epithelial cell injury, the ferroptosis agonist, erastin, and the ferroptosis inhibitor, ferrostatin-1, were used to regulate the degree of ferroptosis at the same CaOx concen-tration in in vivo and in vitro experiments. CaOx-induced ferroptosis and damage to renal tubular epithelial cells and renal tissue were investigated. Finally, it was identified that through the regulation of ferroptosis levels, renal tubular epithelial cell injury increased significantly when the ferrop-tosis level increased, and vice versa. On the whole, the present results indicated that ferroptosis is essential for renal tubular epithelial cell injury induced by CaOx crystals. This finding is highly significant and promotes the further investigation of the association between ferroptosis and urolithiasis.

Renal tubular epithelial cell damage is the basis for the formation of kidney stones. Oxalate can induce human proximal tubular (HK-2) cells to undergo autophagy and ferroptosis. The present study was aimed at investigating whether the ferroptosis of HK-2 cells induced by oxalate is caused by the excessive activation of autophagy. We treated HK-2 cells with 2 mmol/L of oxalate to establish a kidney stone model. First, we tested the degree of oxidative damage and the level of autophagy and ferroptosis in the control group and the oxalate intervention group. We then knocked down and overexpressed the BECN1 gene and knocked down the NCOA4 gene in HK-2 cells, followed by redetection of the above indicators. We confirmed that oxalate could induce autophagy and ferroptosis in HK-2 cells. Moreover, after oxalate treatment, overexpression of the BENC1 gene increased cell oxidative damage and ferroptosis. In addition, knockdown of NCOA4 reversed the effect of oxalate-induced ferroptosis in HK-2 cells. Our results show that the effects of oxalate on the ferroptosis of HK-2 cells are caused by the activation of autophagy, and knockdown of the NCOA4 could ameliorate this effect.

Calcium oxalate nephrolithiasis is a common and highly recurrent disease in urology; however, its precise pathogenesis is still unknown. Recent research has shown that renal inflammatory injury as a result of the cell-crystal reaction plays a crucial role in the development of calcium oxalate kidney stones. An increasing amount of research have confirmed that inflammation mediated by the cell-crystal reaction can lead to inflammatory injury of renal cells, promote the intracellular expression of NADPH oxidase, induce extensive production of reactive oxygen species, activate NLRP3 inflammasome, discharge a great number of inflammatory factors, trigger inflammatory cascading reactions, promote the aggregation, nucleation and growth process of calcium salt crystals, and ultimately lead to the development of intrarenal crystals and even stones. The renal tubular epithelial cells (RTECs)-crystal reaction, macrophage-crystal reaction, calcifying nanoparticles, endoplasmic reticulum stress, autophagy activation, and other regulatory factors and mechanisms are involved in this process.