CHAC1 Mediates Endoplasmic Reticulum Stress-Dependent Ferroptosis in Calcium Oxalate Kidney Stone Formation

Abstract

The initiation of calcium oxalate (CaOx) kidney stone formation is highly likely to stem from injury to the renal tubular epithelial cells (RTECs) induced by stimulation from an aberrant urinary environment. CHAC1 plays a critical role in stress response mechanisms by regulating glutathione metabolism. Endoplasmic reticulum (ER) stress and ferroptosis are demonstrated to be involved in stone formation. This study attempted to elucidate the mechanism of ER stress-dependent ferroptosis and the role of CHAC1 in CaOx kidney stones. Here, regulating ER stress and CHAC1 expression are performed in in vivo and in vitro stone models. These findings indicated that 4-Phenylbutyric acid (4-PBA)treatment and CHAC1 deficiency alleviated the ferroptotic status, including restoring GSH content, suppressing Fe²⁺ and lipid peroxidation accumulation, as well as regulating ferroptosis-related proteins. Notably, 4-PBA treatment and CHAC1 deficiency both attenuated oxidative damage, improved renal function, importantly, decreased crystal deposition. Additionally, ChIP-seq and ChIP-qPCR analyses demonstrated that CHAC1 is the vital downstream target gene of ATF4. The results indicated that ATF4 depletion inhibited the upregulation of CHAC1 and pro-ferroptotic response induced by Ox stimulation. Overall, ATF4/CHAC1 axis mediating ER stress-dependent ferroptosis may be a promising research direction for identifying potential strategy to prevent and treat CaOx kidney stones.

Keywords: ATF4; CHAC1; endoplasmic reticulum stress; ferroptosis, kidney stone.

Figure 1

ER stress participated in mediating oxalate‐induced oxidative injury and ferroptosis in RTECs. a) Bubble plot for KEGG pathway analysis of DEGs. b) Western blotting results showed the protein expression levels of GRP78, IRE‐1, P‐IRE‐1, PERK, P‐PERK, ATF4, and CHOP for stone model group and control group. c) The violin plots show the protein relative expression according to the western blotting results. The content of T‐AOC d), SOD e), and LDH f) were detected in four groups, respectively. g) Images show the levels of cellular ROS and brighter red indicates higher levels of cellular ROS (magnification, ×200). The cellular GSH content h) and MDA content i) were measured. j) The iron assay kit was used to quantitatively detect the cellular Fe²⁺ content. k) The Liperfluo assay was used to detect the cellar degree of lipid peroxidation in four groups with flow cytometric analysis. l) Western blotting results show the protein expression levels of GPX4 and XCT for four groups and m) bar graphs show the protein relative expression. n) The ability of cell adhesion was detected. o) Images show the status of cell‐crystal adhesion between Ox crystals and HK‐2 cells (magnification, ×400). One set of representative images of three independent experiments is shown. Based on three independent experiments, data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 2

The positive role of regulating ER stress in inhibiting CaOx stone formation. a) A flowchart of the animal experiments. The bar graphs show the content of BUN b) and the content of CREA c) in blood for four groups, respectively. The Kim‐1 d) and NGAL content e) were measured using Elisa kits. f) Images show the 4‐HNE expression abundance in renal tissue for four groups (magnification, ×400). g) Images show the positive area of highly α‐SMA expression (magnification, ×400). h) Images show the degree of renal fibrosis using the Masson staining (magnification, ×400). i) Bar graph shows the mean red fluorescence intensity of 4‐HNE expression. j) Bar graph shows the positive area fraction of highly α‐SMA expression. k) Bar graph shows the positive area fraction of Masson staining. Immunohistochemical staining was performed to measure the expression abundance of CD44 l) (magnification, ×400) and ANXA2 m) (magnification, ×400). n) Bar graph shows the positive area fraction of CD44 protein expression. o) Bar graph shows the positive area fraction of ANXA2 protein expression. p) Bar graph shows the positive area fraction of crystal deposition. q) Von Kossa staining was used to display the status of crystal deposition in renal tissue (magnification, ×10 and ×400). Animal experiments were based on six independent experiments. One set of representative images of six independent experiments is shown. All data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 3

ER stress‐associated ATF4/CHAC1 axis was activated in CaOx kidney stone model. a) The heatmap plot shows differential expression changes of all transcription factors in the human transcription factors PCR Array plate. b) The bubble plot shows differential expression changes of all transcription factors related to ER stress. The size of bubbles represents the amount of P value. The color of bubbles represents the amount of fold change. c) Bubble plot of KEGG in ChIP‐seq. d) Western blotting results show the protein expression levels of CHAC1 for stone model group and control group and e) bar graph shows the protein relative expression. f) ChIP‐seq verified the distribution of ATF4 reads over the CHAC1 gene. g) ChIP‐qPCR verified the transcription of CHAC1 by ATF4 targeting. h) qPCR results show the transcriptional levels of ATF4 and CHAC1 followed by Ox concentration gradient incubating for 24 h. i) qPCR results show the transcriptional levels of ATF4 and CHAC1 followed by time gradient with 1.5 mM Ox incubation. j) Western blotting results show the protein expression levels of ATF4 and CHAC1 followed by Ox concentration gradient incubating for 24 h. k) Western blotting results show the protein expression levels of ATF4 and CHAC1 followed by time gradient with 1.5 mm Ox incubation. l) Western blotting results show the protein expression levels of ATF4 and CHAC1 for four groups. m) Immunohistochemical staining was performed to measure the expression abundance of ATF4 protein (magnification, ×400). n) Immunohistochemical staining was performed to measure the expression abundance of CHAC1 protein (magnification, ×400). One set of representative images is shown. In vitro experiments were based on three independent experiments and in vivo experiments were based on six independent experiments, data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 4

The ATF4/CHAC1/GPX4 pathway was involved in ER stress‐dependent ferroptosis in CaOx kidney stone model. a) Volcano plot of DEGs in 4D‐LFQ quantitative proteomic analysis. b) The linearity graph shows the association between CHAC1 and GPX4 in nephrolithiasis patients based on the nephrolithiasis gene expression datasets (GSE73680). c) Immunofluorescence staining was performed to simultaneously detect the CHAC1 and GPX4 protein expression abundance in renal tissue (magnification, ×400) and d) bar graph shows the protein relative expression. e) The validation of ATF4 knockdown by western blot. f) The CHAC1 mRNA expression in HK‐2 cells were determined using qRT‐PCR analysis for five groups. g) Western blotting results show the protein expression levels of CHAC1 and GPX4 for four groups and h,i) bar graphs show the protein relative expression. The GSH j) content and MDA k) content were detected in three groups. l) The FerroOrange assay was used to qualitatively detect the cellular Fe²⁺ content. Bar graph shows the mean red fluorescence intensity and m) images were taken under a dark field (magnification, ×400). Brighter red indicates higher cellular Fe²⁺ content. n) The BDP 581/591 C11 assay was used to qualitatively measure the degree of lipid peroxidation and bar graph shows the ratio of red to green fluorescence (magnification, ×400). The lower the ratio, the worse the degree of lipid peroxidation. o) Immunofluorescence staining was performed to simultaneously detect the cellular CHAC1 and GPX4 protein expression abundance (magnification, ×400) and bar graph shows the ratio of CHAC1 expression to GPX4 expression. One set of representative images of three independent experiments is shown. Based on three independent experiments, data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 5

CHAC1 mediated oxalate‐induced oxidative injury and apoptosis in RTECs. a) LV vector for CHAC1 knockdown and the plasmid for CHAC1 overexpression were used to construct sh‐CHAC1 and oe‐CHAC1 HK‐2 cell lines. b) Scatter plot for DEPs in RNA‐seq experiment. c) The cell viability for six groups was detected using CCK‐8 assay. The T‐AOC content d), SOD content e), and LDH content f) was measured in each group. g) Western blotting results show the protein expression levels of NQO‐1, NRF2, IL‐18, and IL‐1β for six groups. h) The JC‐1 assay was performed to determine the mitochondrial membrane potential of HK‐2 cells (magnification, ×400). The lower the ratio of red to green fluorescence, the worse the mitochondrial membrane potential. i) The flow cytometry was used detect the apoptotic condition for six groups. j) The heatmap shows differential expression changes of all DEPs related to apoptotic pathways. k) The Fluo‐4, AM and Rhod‐2, AM were used to detect intracellular Ca²⁺ content and mitochondrial Ca²⁺ content, respectively (magnification, ×1000). l) The cellular caspase 3 activity for six groups was detected. m) Western blotting results show the protein expression levels of BAX, BCL2 and MCU for six groups. n) Bar graphs show the MCU protein relative expression for six groups. One set of representative images of three independent experiments is shown. Based on three independent experiments, data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 6

CHAC1 deficiency ameliorated the renal impairment and improved renal function in the mouse kidney stone model. a) AAV‐sh‐CHAC1 was injected 30 days in advance to inhibit CHAC1 expression in the kidneys and then glyoxylic acid was used to establish mouse kidney stone model. b) Images show the mouse kidneys from four groups, respectively. c) The bar graph shows the mouse body weight for four groups. The bar graphs show the urine volume d) and urine pH e) for four groups, respectively. The bar graphs show the content of BUN f) and the content of CREA g) in blood for four groups, respectively. h) Images show the expression abundant of Kim‐1 for four groups. i) Immunohistochemical staining was used to measure NGAL expression in renal tissue (magnification, ×400). j) Bar graph shows the mean red fluorescence intensity of Kim‐1 expression. k) Bar graph shows the positive area fraction of highly NGAL expression. l) Images show the degree of renal fibrosis using the Masson staining (magnification, ×400). m) Images show the positive area of highly α‐SMA expression (magnification, ×400). n) Bar graph shows the positive area fraction of Masson staining. o) Bar graph shows the positive area fraction of highly α‐SMA expression. p) The TUNEL staining was performed to detect the apoptotic level in renal tissue (magnification, ×400) and q) bar graph show the mean green fluorescence intensity. In vivo experiments were based on six independent experiments. One set of representative images of six independent experiments is shown. All data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 7

CHAC1 modulated ER stress‐dependent ferroptosis in Ox‐induced RTECs. a) Bubble plot of KEGG pathway in RNA‐seq experiment. b) The content of GSH was detected for six groups. c) The content of MDA was detected for six groups. d) The BDP 581/591 C11 assay was used to qualitatively measure the degree of lipid peroxidation (magnification, ×400). e) The FerroOrange assay was used to qualitatively detect the cellular Fe²⁺ content. Images were taken under a dark field (magnification, ×400). f) Bar graph shows the ratio of red to green fluorescence. The lower the ratio, the worse the degree of lipid peroxidation. g) Bar graph shows the mean red fluorescence intensity. Brighter red indicates higher cellular Fe²⁺ content. h) Western blotting results show the protein expression levels of GPX4, XCT, CD71, and ACSL4 for six groups and i) bar graphs show the protein relative expression. j) Western blotting results show the protein expression levels of GPX4 and XCT for five groups and k) bar graphs show the protein relative expression. l) Images show the mitochondrial morphology alterations associated with ferroptosis under transmission electron microscopy. (scale bar = 2 µm, 0.5 µm). Red asterisks indicate mitochondria with reduced mitochondrial cristae and ruptured mitochondrial membrane, and red arrows indicate shrunk mitochondria. m) Western blotting results show the protein expression levels of ANXA2 and CD44 for six groups. n) Images show the status of cell‐crystal adhesion between Ox crystals and HK‐2 cells (magnification, ×400). One set of representative images of three independent experiments is shown. Based on three independent experiments, data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 8

CHAC1 deficiency suppressed ferroptosis and alleviated crystal deposition in the mouse kidney stone model. The content of GSH a) and the content of Fe²⁺ b) in renal tissue were detected for four groups. c) Images show the 4‐HNE expression abundance in renal tissue for four groups (magnification, ×400). d) Western blotting results show the protein expression levels of GPX4, XCT, CD71, and ACSL4 for four groups and e) bar graphs show the protein relative expression. f) Immunohistochemical staining was used to measure GPX4 protein expression in renal tissue (magnification, ×400). g) Immunohistochemical staining was used to measure ACSL4 protein expression in renal tissue (magnification, ×400). h) Bar graph shows the positive area fraction of highly GPX4 expression. i) Bar graph shows the positive area fraction of highly ACSL4 expression. j) Images show the mitochondrial morphology alterations associated with ferroptosis under transmission electron microscopy (scale bar = 2 µm, 1 µm). Red asterisks indicate mitochondria with reduced mitochondrial cristae and ruptured mitochondrial membrane, and red arrows indicate shrunk mitochondria. k) Von Kossa staining was used to display the status of crystal deposition in renal tissue (magnification, ×8 and ×400). l) Western blotting results show the protein expression levels of CD44 and ANXA2 for four groups and m) bar graphs show the protein relative expression. Animal experiments were based on six independent experiments. One set of representative images of six independent experiments is shown. All data are presented as means ± SEM. P value is directly displayed and P value < 0.01 is considered significant.

Figure 9

A schematic diagram illustrates the mechanism that CHAC1 mediates ER stress‐dependent ferroptosis in CaOx kidney stone formation.