2.1 Animals

Seven-week-old male littermates were procured and acclimated to the new environment for 3 weeks. Subsequently, the mice were randomly divided into two groups. The omeprazole (Ome) group received intraperitoneal injections of omeprazole (10.4 mg/kg × day, dissolved in saline) for 28 consecutive days, whereas the control group received equivalent doses of saline. The omeprazole was delivered through parenteral injection instead of oral gavage, consistent with the administration protocol described earlier [4]. The omeprazole dose was determined on the basis of the body surface area, according to previous studies [15, 16]. Typically, the maximum dose of omeprazole administered to humans is 20 mg/day orally or 80 mg/day intravenously. As this study utilised intraperitoneal injection, a standard dose of 80 mg/day was selected. The resulting equation is as follows: Mouse dose = Human dose (mg/kg × day) × Mean body weight (70 kg) × Equivalence factor (0.0026)/Mean body weight of mice (0.02 kg) = (80 mg/day) × (0.0026/0.02 kg) = 10.4 mg/kg × day. Biological samples were collected there after.

2.2 Cell Culture

HK2 (CRL-2190, Procell, China), NRK (CRL-0173, Procell, China), 293 T (CL0571&CL0005, Fenghui, China) and HPC (XY-h1247, XYBiotechnology, China) are commercially available. The HK2 cell proliferation culture conditions consisted of 95% F12 + 5% fetal bovine serum (FBS) + penicillin/streptomycin and were maintained in an atmosphere of 95% air and 5% CO₂. The remaining cells were cultured in 90% Dulbecco's modified Eagle medium (DMEM) + 10% FBS + penicillin/streptomycin. The cells were passaged every 3 days. The culture conditions for the cell assays and omeprazole treatment (O118724, Aladdin, China) were described separately for each assay.

2.3 Cell Counting Kit-8 (CCK-8)

Cell viability was evaluated using a CCK-8 assay (BS350A, Biosharp, China) according to the manufacturer's protocol [17]. Briefly, cells were cultured with omeprazole in 96-well plates at 37°C. Following a 7-day incubation period, the culture medium was replaced with fresh medium and the CCK-8 reagent was added. After a 2-h incubation, absorbance was measured at 450 nm using a microplate reader.

2.4 Hoechst 33342/Propidium Iodide (PI)

Cell death was assessed using Hoechst 33342/PI staining (BL116A, Biosharp, China) following the manufacturer' protocol. Briefly, cells were cultured in Petri dishes at 37°C (100% F12 + omeprazole). After 7 days, the cells were rinsed with PBS and stained with Hoechst 33342/PI for 20 min. PI which emits red fluorescence, can permeate cells with compromised plasma membranes, thereby staining necrotic cells. Hoechst 33342, which emits blue fluorescence, penetrates living cells and binds specifically to DNA. Images were acquired using a Leica microscope.

2.5 Fluorescence-Activated Cell Sorting (FACS) Analysis

FACS was employed to evaluate the effects of various omeprazole concentrations on cells following a 48-h incubation. The cells were stained with Annexin V-FITC/PI (G1511, Servicebio, China) and analysed for apoptosis using a CytoFLEX LX flow cytometer (Beckman Coulter, USA). Data from 10 000 cells per sample were processed using FlowJo software (version 10), with each experiment repeated at least three times.

2.6 RNA-Seq

The cells were cultured in Petri dishes at 37°C (100% F12 + omeprazole). After 7 days, the samples were collected, preserved in TRIzol and shipped on dry ice to a third-party company for RNA-seq. The entire process, including RNA isolation, integrity verification, library preparation and RNA-seq, was carried out by Guangzhou Gene Denovo Biotechnology Co. Ltd. RNA-seq was conducted utilising the T7 sequencing platform at Beijing Genomics Institute. The company provided the test report. The result of the RNA-seq is in the Supporting Information document.

The RNA extraction protocol using TRIzol reagent (Thermo Fisher Scientific) was performed as follows. Cells were lysed in TRIzol, followed by thorough homogenisation. Chloroform was added (0.2 mL per 1 mL TRIzol) and the mixture was vortexed for 30 s. After centrifugation at 12 000g for 15 min at 4°C, the colourless upper aqueous phase was carefully transferred to a fresh tube. RNA was precipitated by adding 0.5 mL isopropanol per 1 mL initial TRIzol volume (incubate 10 min), followed by centrifugation at 12 000g for 10 min at 4°C. The supernatant was discarded, and the RNA pellet was washed twice with 75% ethanol (prepared with DEPC-treated water). After air-drying for 5–10 min, the RNA was resuspended in RNase-free water (DEPC-treated). RNA quality was assessed by measuring A260/A280 ratios (expected range: 1.8–2.1) using a NanoDrop spectrophotometer, and integrity was verified by agarose gel electrophoresis (28S:18S ribosomal RNA ratio ≈ 2:1).

2.7 qRT-PCR

RNA was extracted using the TRIzol method and subsequently reverse-transcribed into complementary DNA (cDNA) using a synthesis kit (K1622, Thermo Fisher Scientific, USA). Quantitative real-time PCR (qRT-PCR) was performed using a commercial kit (B21203, Bimake, China) in accordance with the manufacturer's instructions. The qRT-PCR analysis was conducted on an ABI-Q3 instrument (ABI, USA), with Gapdh used as the reference gene. The sequence of the primers is in the Supporting Information document. Additional methodological details can be found in previously published studies [17].

The cDNA synthesis procedure involves first preparing high-quality, DNase-treated total RNA (1–5 μg) in nuclease-free water. The RNA is then mixed with either random hexamers or oligo(dT) primers, incubated at 65°C for 5 min to denature secondary structures and immediately chilled on ice. For the reverse transcription reaction, a master mix is prepared containing 4 μL of 5× RT buffer, 1 μL of 10 mM dNTP mix, 1 μL of Ribolock RNase inhibitor and 1 μL of RevertAid Reverse Transcriptase, brought to a final volume of 20 μL with nuclease-free water. The reaction proceeds through three temperature phases: 25°C for 5 min (primer annealing), 42°C for 60 min (cDNA synthesis) and 70°C for 5 min (enzyme inactivation). The resulting cDNA can be diluted 1:5–1:10 for downstream applications and stored at −20°C for short-term preservation.

The qRT-PCR amplification was performed in 20 μL reactions containing: 10 μL of 2× SYBR Green Master Mix, 2 μL of diluted cDNA template, 0.8 μL of 10 μM primer mix (0.4 μL each of forward and reverse primers) and 7.2 μL of nuclease-free water. The thermal cycling protocol comprised: initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s (with fluorescence acquisition) and a final melt curve analysis (60°C–95°C). Data were analysed by determining the threshold cycle (C_t) values for both target and reference genes, with relative expression levels calculated using the $$$ {2}^{-\Delta \Delta {C}_{\mathrm{t}}} $$$ method.

2.8 Biochemical Indicators

Blood samples were collected after anticoagulation with sodium heparin. Plasma was isolated by centrifugation at 4°C and 3000 rpm (RPM) for 5 min. The levels of creatinine, urea nitrogen (BUN) and uric acid were quantified using fully automated analysers. Urine analysis was conducted using 24-h random urine samples collected prior to the conclusion of the experiment. Following centrifugation, the samples were transferred to the Department of Laboratory Medicine for further analysis.

2.9 Histology

Fresh samples were fixed in 4% paraformaldehyde (PFA) for 72 h and subsequently processed through dehydration and paraffin embedding. Tissue sections with a thickness of 3 μm were prepared for subsequent staining procedures. Haematoxylin and eosin (H&E, G1076, Servicebio, China) and immunohistochemistry (IHC, ZLI-9017 and PV-6000, ZSGB-Bio, China) staining were performed using commercially available kits [18]. Antibodies targeting proliferating cell nuclear antigen (PCNA, A12427, ABclonal, China), F4/80 (28463-1-AP, Proteintech, USA) and kidney injury molecule 1 (KIM1, BA3537, Boster, China) were obtained from third-party suppliers. Images were acquired using a microscope imaging system.

2.10 Statistics

The data are presented as the mean ± standard deviation (SD). The sample size for each experimental group is detailed in the corresponding figure legends. Differences between group means were analysed using an unpaired Student's two-tailed t test (GraphPad Prism 6, USA). The normality of the data was assessed using the Shapiro–Wilk test, Q-Q plots and histograms. For data that deviated from a normal distribution, the Mann–Whitney U test was employed to re-evaluate the results, ensuring the accuracy of the statistical analysis. To ensure statistical rigour, Dunnett's test was implemented to evaluate significant differences between the control group and multiple treatment groups. Comprehensive methodological details of the statistical analyses are available in the Supporting Information. A p-value < 0.05 was considered statistically significant.

3.1 Omeprazole Dose-Dependently Disrupts Cell Morphology and Proliferation

To investigate the effects of omeprazole on cell proliferation and morphology, HK2, HPC, NRK and HEK293T cells were exposed to varying concentrations of the drug (Figure 2A,B). The results demonstrated a concentration-dependent reduction in cell density within the 5–50 μM range. Notably, NRK cells exhibited minimal sensitivity to omeprazole. At concentrations above 50 μM, omeprazole induced cell death, resulting in the detachment and floating of cells. Furthermore, the characteristic paving-stone morphology of normal HK2 cells was disrupted in a concentration-dependent manner, leading to irregular or rounded cell shapes. Similar morphological changes were observed in HPC, NRK and HEK293T cells. Cell viability was subsequently evaluated using the CCK-8 assay (Figure 2C), which revealed a dose-dependent decline in viability within the 5–50 μM range. At concentrations exceeding 50 μM, cell viability was almost completely abolished in HK2. Interestingly, NRK cells maintained significant drug tolerance. This discrepancy may be attributed to the fact that HK2 cells are derived from human kidney tubular epithelial cells, whereas NRK cells originate from rat kidney tubular epithelial cells. Species differences likely account for the significant variations in the observed results. To further validate the intergroup differences and the reliability of the t-test results, we performed additional verification using Dunnett's test (Statistics S1–S7). The findings from this analysis aligned with the t test outcomes, reinforcing the conclusion that omeprazole induces dose-dependent impairments in cell morphology, proliferation and viability. Moreover, due to the extensive death and detachment of HEK293T cells, they could not be reliably detected by the CCK-8 assay and were therefore excluded from subsequent analyses.

3.2 Omeprazole Dose-Dependently Induces Cell Death

Next, the effect of omeprazole on cell death was investigated. In HK2 cells (Figure 3A,B), Hoechst 33342/PI staining revealed minimal cell death at a 5 μM concentration of omeprazole, as no significant red fluorescence was observed. However, the intensity of red fluorescence progressively increased with higher drug concentrations, with 15 and 50 μM concentrations being sufficient to induce cell death. At concentrations exceeding 50 μM, a substantial number of cells died and detached, making staining impractical. A similar trend was observed in HPC cells (Figures S1 and 3B), although the mortality rate of HPC was significantly lower than that of HK2. Additionally, NRK cells appeared to be relatively insensitive to the drug concentration (Figures S1 and 3B). Notably, NRK cells exhibited baseline mortality even in the absence of the drug, but this mortality rate did not change significantly at concentrations below 50 μM. To further validate the intergroup differences and the reliability of the t test results, we performed additional verification using Dunnett's test (Statistics S8–S10). The findings from this analysis aligned with the t test outcomes. These findings were further validated using Annexin V staining and the results were consistent with those of Hoechst 33342/PI staining (Figure 3C,D). Collectively, these experiments demonstrated that different cell types exhibit varying sensitivities to omeprazole, with HK2 cells appearing to be the most sensitive in this study. In contrast, NRK cells were the least sensitive to omeprazole.

3.3 Omeprazole Disrupts Gene Expression in HK2

To elucidate the mechanisms underlying the suppression of cell proliferation and viability by omeprazole, the effects of 5 μM omeprazole on HK2 cells were evaluated using RNA-seq. The analysis identified significant differential expression in 82 genes, with 51 genes being markedly downregulated and 31 genes upregulated in the omeprazole-treated group compared to the control group (Figure 4A,B). Gene Ontology (GO) enrichment analysis of these genes demonstrated that omeprazole primarily impacts the cell cycle, metabolism and cytokine receptor activity (Figure 4C). Notably, one of the most striking changes was observed in vitamin D-24 hydroxylase activity. In other words, omeprazole treatment downregulated both cytochrome P450 and vitamin D 24-hydroxylase expression (Supporting Information RNA-seq data), potentially elevating circulating calcium and vitamin D levels through this mechanism. While the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis also revealed differences in enriched pathways between the two groups, these results did not reach statistical significance (Figure 4D). Furthermore, gene set enrichment analysis (GSEA) supported these findings, highlighting significant alterations in pathways associated with the cell cycle and metabolism (Figure 4E,F). Representative images are provided here, with additional results available in the Supporting Information. The accuracy of the RNA-seq data was further validated by qRT-PCR, which yielded nearly identical results (Figure 4G,H), confirming significant differences in the expression of genes related to the cell cycle, metabolism and cytokine receptors. Collectively, these findings suggest that omeprazole disrupts gene expression.

3.4 Omeprazole Increases the Environmental Susceptibility of HK2

Changes in gene expression can significantly influence the environmental susceptibility of cells. To investigate the effects of omeprazole, HK2 cells were treated with 5 μM omeprazole for a specified duration and subsequently exposed to H₂O₂ (Figure 5A). The CCK-8 assay results revealed that preculturing with omeprazole significantly enhanced the cytotoxic effects of H₂O₂ on the cells (Figure 5B). This finding was further supported by visual observations, which showed that H₂O₂ induced cell death and detachment and these effects were markedly exacerbated by prior omeprazole treatment (Figure 5C). These findings suggest that chronic exposure to omeprazole increases the susceptibility of cells to environmental stressors.

3.5 Omeprazole has no Effect on Body Weight or Kidney Function

The direct effects of omeprazole (10.4 mg/kg × day) on body weight and organ function were examined in mice. Omeprazole administration did not significantly alter body weight, kidney weight or heart weight (Figure 6A–C). Furthermore, omeprazole had no discernible impact on kidney function, as evidenced by unchanged levels of BUN, plasma creatinine, plasma uric acid, urinary total protein/creatine and urinary microalbumin/creatine (Figure 6D–H). Similarly, no significant differences were observed in liver function markers, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (Figure 6I,J). Additionally, no omeprazole-associated changes in calcium/vitamin D were detected (Figure 6K,L).

3.6 Omeprazole has No Effect on the Kidneys Other Than Minor Inflammation

Next, we investigated the effects of omeprazole on the kidneys of mice. Kidney morphology, injury, proliferation and inflammation were assessed using H&E, KIM-1, PCNA and F4/80 staining, respectively. The results demonstrated that omeprazole had no significant impact on kidney pathology scores and no detectable expression of KIM-1 or PCNA was observed in either group (Figure S1). Additionally, although omeprazole increased the expression of F4/80, the overall levels of F4/80 remained exceptionally low (Figure 7A,B).