SRP14 Triggers Apoptosis in Renal Tubules to Exacerbate AKI Through an Interaction with RPS7

SRP14 Triggers Apoptosis in Renal Tubules to Exacerbate AKI Through an Interaction with RPS7 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SRP14 Triggers Apoptosis in Renal Tubules to Exacerbate AKI Through an Interaction with RPS7 Yi Li, Yun Tang, Liming Huang, Yanmei Wang, Qiao Tang, Zehui Liao, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7892365/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To explore potential targets for acute kidney injury (AKI), we exposed renal tubular epithelial cells (RTECs) to a hypoxia/reoxygenation environment and conducted labeling-free proteomics. This treatment significantly increased signal recognition particle 14 (SRP14) in apoptotic RTECs. SRP14 was elevated in the serum of patients with AKI. The SRP14 expression was increased in the renal tubules of patients with acute tubular necrosis, as well as in four AKI mouse models following the procedures of ischemia-reperfusion injury (IRI), cecal ligation and puncture, and treatment with lipopolysaccharide and cisplatin. SRP14 appears to play a crucial role in the apoptosis of RTECs, as evidenced by an IRI-induced AKI model in tubule-specific Srp14 knockout mice. Furthermore, SRP14 triggered apoptosis in renal tubules upon renal IRI via the ribosomal protein S7 (RPS7)-mediated tumor protein p53 (TP53)–MDM2 proto-oncogene (MDM2) pathway. We screened an apoptosis-specific library containing 356 US Food and Drug Administration–approved compounds to identify those that inhibit RPS7. We identified nafamostat mesilate as a potent RPS7 inhibitor that attenuated renal IRI by inhibiting RTEC apoptosis. These findings suggest that SRP14 triggers apoptosis in RTECs to exacerbating AKI through an interaction with RPS7, which may be a therapeutic target for nafamostat mesylate to alleviate AKI. Health sciences/Diseases/Kidney diseases/Acute kidney injury Health sciences/Medical research/Translational research SRP14 RPS7 renal tubular epithelial cells apoptosis AKI Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Points SRP14 induces apoptosis of renal tubular epithelial cells, thereby exacerbating AKI, by interacting with RPS7, which represents a potential target for nafamostat mesylate to intervene in AKI. Introduction Acute kidney injury (AKI) will ultimately progress to chronic kidney disease (CKD) with renal insufficiency, leading to heavy burdens for individuals, their families, and society (Kellum et al, 2021 ). Recently, the incidence of and mortality due to AKI have been increasing (Siew et al, 2015; Zhang et al, 2025 ). Due to its complex pathogenesis, there are no effective early interventions for AKI. Since previous studies have primarily focused on the post-development period of AKI, its initiation mechanism remains only partially understood. Therefore, there is an urgent need to thoroughly explore the mechanisms underlying AKI to advance the development of early prevention and therapy for it and delay its progression to CKD. One of the main pathological features of AKI is closely associated with renal tubule injury and the death of renal tubular epithelial cells (RTECs) (Maremonti et al, 2022 ), which are peculiarly prone to apoptosis, facilitating renal tubule injury to aggravate renal failure (Havasi et al, 2011; Fattori et al, 2017 ). In the early stages of AKI, the apoptosis of RTECs is induced by ischemia-reperfusion injury (IRI) and nephrotoxic substances, such as cisplatin (Wei et al, 2024 ; Pushpan et al, 2024 ), which is closely associated with damage to renal tubules, loss of renal function, and renal tissue injuries (Sanz et al, 2023 ;Xie et al, 2006). In patients with AKI, damaged kidneys secrete factors into the circulation to further provoke cell apoptosis and inflammation in the cardiovascular system, pulmonary tissues, liver, and brain, thereby leading to increased mortality (Havasi et al, 2011; Sanz et al, 2023 ; Doi et al, 2016; Grams et al, 2012). In contrast, inhibiting apoptosis in RTECs is believed to be pivotal for early prevention and treatment of AKI. Despite apoptosis-targeting therapies leading to decreased apoptosis in this model, clinically applicable treatments are far from realization. To explore potential therapeutic targets for AKI, we established an apoptosis model of RTEC using the hypoxia/reoxygenation (H/R) approach. Using label-free proteomics, differential protein expression profiles in RTECs after H/R revealed that signal recognition particle 14 (SRP14) was significantly elevated in these apoptotic RTEC populations. SRP14 is an important cytoplasmic ribosomal protein particle for the assembly of the Alu domain of the signal recognition particle (SRP) complex to block translation extension and guide the transport of eukaryotic secreted proteins to the rough endoplasmic reticulum membrane (Weichenrieder et al, 2000 ). However, the role of SRP14 in AKI remains unclear. Therefore, this study aimed to elucidate the role and underlying mechanism of SRP14 in AKI, which involves apoptosis in RTECs. Results IRI Elevates SRP14 and RPS7 in Renal Tubules Label-free quantitative proteomics performed on the human HK2 RTECs revealed the significant upregulation of SRP14 (fold change = 1.40, p = 0.0040) and RPS7 (fold change = 1.28, p = 0.0066) following exposure to H/R (Fig. 1A). TUNEL staining revealed evident apoptosis in the kidneys of mice with renal IRI (Figure S1A). The gene ontology (GO) enrichment analysis revealed that genes involved in apoptosis were significantly enriched in H/I-treated HK2 cells (Fig. 1B). Protein-protein interaction (PPI) analysis predicted an interaction between SRP14 and RPS7 (Figure S1B). Immunohistochemistry further confirmed the upregulation of both SRP14 and RPS7 in renal tissues from mouse models of AKI induced by IRI (Fig. 1C), LPS injection (Figure S1C), cisplatin injection (Figure S1D), and CLP (Figure S1E). Compared to the controls, immunofluorescence revealed increased colocalization of SRP14 and RPS7 with KIM-1 in the renal tubules of mice with renal IRI (Figs. 1D, S1F, and S1G). SRP14 is Associated with AKI and Renal Tubule Damage Of the 45 human subjects enrolled in the final analysis, 29 (64.4%) developed AKI. The detailed clinical characteristics of patients in both groups at the time of AKI diagnosis are shown in Table 1. Several variables differed significantly between groups, including serum creatinine, blood urea nitrogen (BUN), eGFR, uric acid, hemoglobin, total cholesterol, TIMP metallopeptidase inhibitor 2 (TIMP2), and SRP14. SRP14 levels were significantly higher in the AKI group than in the control group (Fig. 2A). In addition, SRP14 levels were lower among patients with AKI stage 3 than among patients with AKI stage 1 and 2 (Fig. 2B). The area under the receiver operating characteristic (ROC) curve (AUC) for serum SRP14 levels was 79.96% (95% confidence interval [CI]: 0.67–0.93, p = 0.001) with a cutoff of 15.29 ng/mL, a sensitivity of 0.621, and a specificity of 0.938 (Fig. 2C). Notably, SRP14 levels were significantly correlated with serum creatinine levels (Spearman’s rank correlation coefficient [ ρ ] = 0.31, p = 0.038) and eGFR ( ρ = −0.30, p = 0.043; Table S2, Figure S2A-G). Table 1. Comparison of clinical characteristics at diagnosis of AKI between AKI and control groups. AKI (n=29) Control (n=16) P value Male, n (%) 12 (41.4) 7 (43.8) 0.878 Age (y), mean±SD 67.6±18.3 48.3±11.5 0.078 Serum Cr (μmol/L), median (IQR) 170.6 (126.4, 248.0) 61.7 (54.2, 70.3) 3.78×10 -8 BUN (mmol/L), median (IQR) 13.7 (9.2, 19.3) 4.0 (3.2, 5.7) 1.82×10 -7 eGFR (mL/min/1.73 m 2 ), median (IQR) 32.6 (20.3, 42.3) 106.9 (99.9, 112.8) 4.94×10 -8 Uric acid (mmol/L), median (IQR) 466.0 (373.0, 627.5) 341.5 (244.5, 400.8) 0.003 Albumin (mmol/L), mean±SD 32.4±6.9 43.7±4.7 0.318 Hemoglobin (g/dL), mean±SD 108.2±24.2 137.9±14.4 0.044 TG (mmol/L), median (IQR) 1.3 (1.0, 2.0) 1.1 (0.8, 2.6) 0.297 TC (mmol/L), median (IQR) 4.1 (2.7, 4.7) 4.8 (4.2, 5.5) 0.006 Lymphocyte (%), median (IQR) 1.0 (0.5, 1.6) 1.9 (1.4, 2.4) 4.49×10 -3 Neutrophil (%), median (IQR) 8.3 (5.8, 11.7) 3.3 (2.7, 4.1) 9.19×10 -7 TIMP-2 (ng/ml), median (IQR) 3.3 (2.7, 4.4) 0.7 (0.6, 1.5) 1.07×10 -7 SRP14 (ng/ml), median (IQR) 17.5 (11.6, 20.6) 9.6 (7.4, 12.2) 9.80×10 -3 Abbreviations: Cr, creatinine; eGFR, estimated glomerular filtration rate; BUN, blood urea nitrogen; IQR, interquartile range; n, number; SD, standard deviation; TC, total cholesterol; TG, triglyceride; y, year. Table 2 The binding free energy between small molecules and proteins. Energy Component Average Std. Dev. Std. Err. of Mean VDWAALS -123.066 6.785 1.219 EEL 1139.232 79.066 14.201 EGB -1063.210 78.800 14.153 ESURF -20.151 0.795 0.143 DELTA G gas 1016.166 80.133 14.393 DELTA G solv -1083.360 78.733 14.141 DELTA TOTAL -67.194 8.838 1.588 Abbreviations: VDWAALS：van der Waals energy; EEL： Electrostatic energy; EGB： Polar solvation energy; ESURF： Non-polar solvation energy; Ggas： Total gas phase free energy; Gsolv： Total solvation free energy. Table 3. The potential binding domain of SRP14 and RPS7. Gene Method Start End Sequence RPS7 Mass spectrometry 59 70 AIIIFVPVPQLK RPS7 Mass spectrometry 184 194 DVNFEFPEFQL RPS7 Mass spectrometry 50 57 EIEVGGGR RPS7 Mass spectrometry 42 49 ELNITAAK RPS7 Mass spectrometry 91 98 HVVFIAQR RPS7 Mass spectrometry 8 37 IVKPNGEKPDEFESGISQALLELEMNSDLK RPS7 Mass spectrometry 121 142 TLTAVHDAILEDLVFPSEIVGK RPS7 Molecular docking 90 190 KHVVFIAQRRILPKPTRKSRTKNKQKRPRSRTLTAVHDAILEDLVFPSEIVGKRIRVKLDGSRLIKVHLDKAQQNNVEHKVETFSGVYKKLTGKDVNFEFP SRP14 Mass spectrometry 111 136 AAAAAAAAAPAAAATAPTTAATTAATAAQ SRP14 Molecular docking 70 136 VVSSKEVNKFQMAYSNLLRANMDGLKKRDKKNKTKKTKAAAAAAAAAPAAAATAPTTAATTAATAAQ Table 4. RPS7 binds to multiple hydrophilic residues of Nafamostat mesylate. Protein Small molecule Site RPS7 Nafamostat mesylate ASN165 RPS7 Nafamostat mesylate HIS168 RPS7 Nafamostat mesylate LYS169 RPS7 Nafamostat mesylate HIS91 RPS7 Nafamostat mesylate GLY89 RPS7 Nafamostat mesylate SER88 RPS7 Nafamostat mesylate GLU84 RPS7 Nafamostat mesylate VAL80 RPS7 Nafamostat mesylate GLN76 RPS7 Nafamostat mesylate VAL77 RPS7 Nafamostat mesylate ARG81 RPS7 Nafamostat mesylate PRO136 RPS7 Nafamostat mesylate PHE135 Table 5. The binding free energy between small molecules and proteins. Energy Component Average Std. Dev. VDWAALS -40.015 3.129 EEL -37.961 2.221 EGB 51.346 5.999 ESURF -5.330 0.821 DELTA G gas -77.976 8.979 DELTA G solv 46.016 3.435 DELTA TOTAL -31.959 3.221 Abbreviations: VDWAALS：van der Waals energy; EEL： Electrostatic energy; EGB： Polar solvation energy; ESURF： Non-polar solvation energy; Ggas： Total gas phase free energy; Gsolv： Total solvation free energy. To further elucidate the importance of SRP14 and RPS7 in renal tubular injury, renal tissue specimens were collected from patients with ATN ( n = 5), and normal ST renal tissue specimens were collected from patients with renal carcinoma ( n = 4; Table S2). Immunohistochemistry revealed increased SRP14 and RPS7 in the renal tubules of patients with ATN (Fig. 2D). Immunofluorescence staining for SRP14, RPS7, and KIM-1 showed increased colocalization of SRP14 and RPS7 with KIM-1 in the renal tubules of patients with ATN compared to normal ST renal tissues of patients with renal carcinoma (Figs. 2E, S2H, and S2I). SRP14 Deficiency in Renal Tubules Attenuates Renal IRI and Suppresses Apoptosis in RTECs To explore the role of SRP14 in renal tubules, tubule-specific Srp14 knockout ( Srp14 −/− , Ggt-Cre ) mice were established using a Cre- LoxP recombination system. Tail genotyping and immunohistochemical measurement of SRP14 confirmed the tubule-specific knockout of Srp14 in mice (Figure S3A, B). While the Srp14 −/− knockout mice did not differ significantly from the wild-type control mice in serum creatinine levels (Fig. 3A), BUN levels (Fig. 3B), and routine pathological characteristics (Fig. 3C), tubule-specific knockout of Srp14 moderately inhibited the expression of SRP14 and RPS7 (Fig. 3D) in the mouse kidney. Following IRI, tubule-specific knockout of Srp14 significantly reduced serum creatinine (Fig. 3A) and BUN (Fig. 3B) levels, attenuated tubular injury, and suppressed the expression of RPS7 in the mouse kidney (Fig. 3C). The number of TUNEL-positive cells in renal tissue was higher in mice with renal IRI than in the wild-type mice, while tubule-specific knockout of Srp14 decreased the number of TUNEL-positive cells following IRI (Fig. 3E). While IRI significantly facilitated the colocalization of SRP14 and RPS7 in murine renal tubules, tubule-specific knockout of Srp14 reduced the colocalization of SRP14 and RPS7 in murine renal tubules following IRI (Figs. 3F and S3C). Knockdown of Srp14 and Rps7 Inhibits Apoptosis of RTECs Following H/R The Cell Counting Kit-8 (CCK-8) assay was used to clarify the effects of SRP14 and RPS7 on the apoptosis of RTECs. In HK2 cells exposed to H/R, the silencing of SRP14 or RPS7 increased survival to 88.59% ± 9.25% and 89.10% ± 3.33%, compared to 68.26% ± 4.01% in control cells (Fig. 4A). Additionally, annexin V-FITC/PI staining revealed that knockdown of Srp14 or Rps7 inhibited the apoptosis of HK2 cells following H/R (Fig. 4B). The gating strategy for annexin V-FITC/PI staining is shown in Figure S4. In vitro , knockdown of Srp14 or Rps7 reduced the expression of p53, MDM2, cleaved CASP3, and BAX in HK2 cells following H/R (Fig. 4C, D). In addition, knockdown of Srp14 inhibited the expression of RPS7 in HK2 cells following H/R, whereas knockdown of Rps7 did not significantly affect the expression of SRP14 in HK2 cells following H/R (Fig. 4C, D). SRP14 Triggers Apoptosis in RTECs Upon IRI Involving RPS7 To elucidate the role of SRP14 and RPS7 in the renal tubule damage associated with IRI, recombinant adeno-associated virus (AAV) vectors harboring short hairpin RNA (shRNA)- Srp14 or a fusion gene of human RPS7 ( hRPS7 ) were delivered into the kidneys of C57BL/6 mice via intrarenal injection (Figure S5A). The optical in vivo imaging system confirmed the successful delivery of AAV-shRNA- Srp14 (Figure S5B). Immunohistochemical measurement further verified the reduction in SRP14 expression in murine kidneys infected with AAV-shRNA- Srp14 (Figure S5C). Some of the mice received intrarenal injection of the recombinant AAV harboring the fusion gene of hRPS7 . Notably, shRNA- Srp14 attenuated tubular injury and reduced the expression of SRP14 in murine kidneys with renal IRI. However, the presence of both recombinant hRPS7 and shRNA- Srp14 aggravated renal tubule injury and increased the expression of SRP14 in the kidneys of mice with renal IRI (Fig. 5A, B). In addition, shRNA- Srp14 inhibited the colocalization of SRP14 and RPS7 in the renal tubules of mice with renal IRI. Compared to wild-type mice with renal IRI, shRNA- Srp14 decreased the number of TUNEL-positive cells in renal tissues from mice with renal IRI; however, the presence of both recombinant hRPS7 and shRNA- Srp14 increased the apoptotic index (Fig. 5C) and moderately facilitated the colocalization of SRP14 and RPS7 in renal tubules of mice with renal IRI (Figs. 5D and S6). Moreover, shRNA- Srp14 decreased the expression of p53, MDM2, cleaved CASP3, and BAX in kidney tissues from mice with renal IRI. However, the presence of both recombinant hRPS7 and shRNA- Srp14 resulted in no significant decrease in the expression of p53, MDM2, cleaved CASP3, and BAX in kidney tissues of mice with renal IRI (Fig. 5E, F). SRP14 Interacts with RPS7 in RTEC With Hypoxia and Reoxygenation Confocal microscopy revealed significant colocalization of SRP14 and RPS7 in RTECs following H/R (Figs. 6A and S7A). Molecular docking showed that both SRP14 and RPS7 possess a core structural domain. The C-terminal region of RPS7 (residues 90–190) interacts with the C-terminal region of SRP14 (residues 70–136), which is vital for the interaction between SRP14 and RPS7 (Fig. 6B). In a molecular simulation of the SRP14–RPS7 complex lasting 20,000 ps, the RMSD indicated that the complex reached equilibrium at 8,000 ps. In addition, the dynamics simulation demonstrated a decreasing trend, suggesting strong interaction between SRP14 and RPS7. The trajectories from 8,000 to 20,000 ps were used to calculate the binding free energy. The total binding free energy change (ΔG) was − 67.19 kcal/mol, indicating tight binding between SRP14 and RPS7 (Figure S7B). The van der Waals potential energy was − 123.066 kcal/mol. Both the polar solvation (− 1063.21 kcal/mol) and non-polar solvation (− 20.1509 kcal/mol) were conducive to the binding of SRP14 and RPS7 (Table 2). Biolayer interferometry revealed a fast-binding and slow-dissociation trend between SRP14 and RPS7, with a dissociation constant ( K d ) of 2.43 × 10 − 8 M (Fig. 6C). Co-immunoprecipitation revealed an interaction between SRP14 and RPS7 in HK2 cells following H/R (Fig. 6D). To further establish the interaction between SRP14 and RPS7, immunoprecipitated samples were subjected to mass spectrometry analysis, revealing that the C terminal region of SRP14 (residues 111–136) and the C terminal region of RPS7 (residues 91–98 and 121–142) played a pivotal role in the interaction between SRP14 and RPS7 (Figs. 6E and S7C, Table 3). Screening of RPS7 Inhibiting Compounds from an Apoptosis-Specific Library Given the important role of RPS7 in SRP14-regulated apoptosis, recombinant RPS7 was used to screen an apoptosis-specific compound library comprised of 356 US Food and Drug Administration (FDA)-approved compounds via biolayer interferometry (Fig. 7A). After confirming the concentration gradient for the initially identified 15 small molecule compounds, the following eight small molecule compounds were identified as stably binding to RPS7 (Figs. 7B and S8): acitritin ( K d = 1.97 × 10 − 4 M), nafamostat mesilate ( K d = 6.45 × 10 − 4 M), tretinoin ( K d = 1.19 × 10 − 4 M), lapatinib ( K d = 2.11 × 10 − 2 M), benzbromarone ( K d = 9.77 × 10 − 5 M), embelin ( K d = 2.90 × 10 − 5 M), sanguinarine chloride ( K d = 1.52 × 10 − 4 M), and troglitazone ( K d = 9.14 × 10 − 5 M). CCK-8 assessments of the viability of HK2 cells determined half maximal inhibitory concentrations (IC 50 ) of 1.33 for acitritin (Fig. 7C), 22.58 for nafamostat mesilate (Fig. 7D), 86.66 for tretinoin (Fig. 7E), 33.13 for lapatinib (Fig. 7F), 64.22 for benzbromarone (Fig. 7G), 9.86 for embelin (Fig. 7H), 0.14 for sanguinarine chloride (Fig. 7I), and 5.35 for rroglitazone (Fig. 7J). In addition, CCK-8 assessment of the viability of HK2 cells following H/R determined half maximal effective concentrations (EC 50 ) of 6.67 for tretinoin (Fig. 7K), 1.79 for nafamostat mesilate (Fig. 7L), and 5.98 for benzbromarone (Fig. 7M). Moreover, 20 mg/kg/day of nafamostat mesilate or tretinoin attenuated renal tubule injury in mice with renal IRI (Fig. 7N). Nafamostat Mesilate Attenuates Renal IRI by Reducing RPS7-associated Apoptosis in RTECs To clarify the protective effects of nafamostat mesilate against renal IRI, mice were treated with 25 or 50 mg/kg/day of nafamostat mesilate following renal IRI. Nafamostat mesilate at 25 or 50 mg/kg/day decreased the levels of serum creatinine (Fig. 8A) and BUN (Fig. 8B) in mice with renal IRI. Routine histological assessments showed that at both 25 and 50 mg/kg/day, nafamostat mesilate attenuated tubular injury caused by renal IRI (Fig. 8C) and inhibited the expression of RPS7 in mouse kidney tissues (Fig. 8D). Compared to untreated control mice with renal IRI, the number of TUNEL-positive cells in renal tissues was lower in mice with renal IRI treated with 25 or 50 mg/kg/day nafamostat mesilate (Fig. 8E). At both 25 and 50 mg/kg/day, nafamostat mesilate significantly reduced the expression of RPS7, p53, MDM2, cleaved CASP3, and BAX in the renal tissues of mice with renal IRI (Fig. 8F). Molecular docking revealed an interaction between nafamostat mesilate and RPS7 involving residues Asn165, His168, Lys169, His91, Gly89, Ser88, Glu84, Val80, Gln76, Val77, Arg81, Pro136, and Phe135 (Fig. 8G, Table 4). The ΔG was − 31.9592 kcal/mol, indicating a stable interaction between nafamostat mesilate and RPS7 (Figure S9). The van der Waals potential energy was − 40.0145 kcal/mol. Collectively, these data show that both the polar solvation (51.3462 kcal/mol) and non-polar solvation (− 5.3299 kcal/mol) were conducive to the binding of nafamostat mesilate to RPS7 (Table 5). Discussion RTECs are sensitive to apoptosis, aggravating renal tubule injury and renal failure (Havasi et al, 2011; Maeda et al, 2024 ). Our study demonstrated that SRP14 was significantly elevated in RTECs following H/R. SRP14 is a cytoplasmic ribosomal protein involved in the assembly of the Alu domain of the SRP complex for signal recognition, thereby blocking translation elongation and guiding the transport of secreted proteins to the rough endoplasmic reticulum membrane (Weichenrieder et al, 2000 ; Brooks et al, 2009 ). The C-terminal region of SRP14 primarily inhibits translation elongation. Mutation of the C-terminal region of SRP14 prevents its blocking of translation elongation but, interestingly, does not affect its ribosome binding ability (Mason et al, 2000 ). These data suggest that SRP14 has additional, unknown functions in biological processes. We observed a marked increase in SRP14 in the serum of patients with AKI, with an AUC of 79.96%. Serum SRP14 levels correlated with serum creatinine levels, BUN levels, eGFR, uric acid levels, neutrophil counts, KIM-1 levels, and age. The expression of SRP14 was significantly elevated in the renal tubules of patients with ATN and four mouse models of AKI induced by IRI, LPS injection, CLP, and cisplatin injection. Thus, SRP14 is associated with AKI involving renal tubule damage. However, the effects of SRP14 on AKI remain unclear. To elucidate the role of SRP14 in AKI, tubule-specific Srp14 knockout mice were generated via a Cre- LoxP recombination strategy. Tubule-specific Srp14 knockout attenuated renal IRI, suppressed the apoptosis of RTECs, and reduced the expression of p53, MDM2, cleaved CASP3, and BAX in RTECs following H/R. Moreover, annexin V-FITC/PI staining revealed that Srp14 knockdown inhibited the apoptosis of HK2 cells following H/R. These results suggest that SRP14 may play a crucial role in the apoptosis of RTECs in IRI-induced AKI. As an interacting partner of MDM2, RPS7 contributes to the complex regulation of the p53-MDM2 feedback loop by stabilizing p53 protein and activating p53 function (Chen et al, 2007 ). As a substrate of MDM2, once bound to MDM2, RPS7 is ubiquitinated and degraded, sustaining the p53 response (Zhu et al, 2009 ). The p53-MDM2 feedback loop is crucial for apoptosis in renal IRI. Renal IRI significantly increased the expression of p53 in rat cortical tissue, and the p53 inhibitor Pifithrin-α inhibited apoptosis, protecting against renal IRI (Kelly et al, 2003 ). Zhang et al. found that p53 in proximal tubular cells promotes AKI, whereas p53 in other tubular cells does not contribute to AKI (Zhang et al, 2014 ). Our proteomic findings showed a significant increase in RPS7 in HK2 cells following H/R. Knockdown of Rps7 inhibited apoptosis involving the p53-MDM2 pathway in HK2 cells following H/R. Nonetheless, the role of RPS7 in renal IRI remains unknown. In addition, PPI analysis predicted an interaction between SRP14 and RPS7. Notably, SRP14 and RPS7 were observed to colocalize in RTECs following H/R. Molecular docking and simulation analyses suggested a direct interaction between SRP14 and RPS7. Biolayer interferometry and co-immunoprecipitation followed by mass spectrometry analysis revealed a direct interaction between SRP14 and RPS7 in RTECs following H/R. Knockdown of Srp14 inhibited the expression of RPS7 in HK2 cells following H/R, whereas knockdown of Rps7 did not significantly affect the expression of SRP14 in HK2 cells following H/R. Moreover, the presence of recombinant hRPS7 and shRNA- Srp14 resulted in no significant inhibition of apoptosis in the kidney tissues of mice with renal IRI. These results suggest that SRP14 triggers apoptosis in renal tubules upon renal IRI via a mechanism involving RPS7. While the MDM2-p53 pathway is well-established in global pharmaceutical research and development, the complex mechanism of the MDM2-p53 feedback loop presents a significant challenge for developing small-molecule inhibitors. While p53 can activate the expression of MDM2 , MDM2 can inhibit p53 through its degradation, blocking its transcription, and facilitating its nuclear export (Meng et al, 2014 ; Brummer et al, 2024). Ewa Langner et al has observed that significant accumulation of p53 protein and caspase-mediated apoptosis in small murine kidneys with tubular dilations upon centrosome loss (Langner et al, 2024 ). Therefore, it is necessary to explore novel therapeutic targets for the upstream mechanism of the MDM2-p53 feedback loop to prevent apoptosis. Given the important role of RPS7 in SRP14-regulated apoptosis involving the MDM2-p53 pathway, our study selected tretinoin, nafamostat mesilate, and benzbromarone from an apoptosis-specific compound library consisting of 356 FDA-approved compounds based on biolayer interferometry and CCK-8 assays. At 20 mg/kg/day, tretinoin and nafamostat mesilate attenuated renal tubule injury in mice with renal IRI. Routine histological measurements demonstrated a superior effect of nafamostat mesilate on murine renal tubules against renal IRI. As a synthetic broad-spectrum serine protease inhibitor, nafamostat mesylate is commonly used to treat intravascular coagulation during hemodialysis and pancreatitis (Iwashita et al, 2003 ; Davenport et al, 2011; Sundaram et al, 1996 ). Nafamostat mesylate can suppress the activation of the coagulation, contact, and complement systems during sepsis, thereby reducing injury to vital organs and improving prognosis (He et al, 2024 ). Treatment with nafamostat mesylate attenuated apoptosis involving the p38 pathway in the spinal cord tissues of rats with spinal cord injury (Duan et al, 2018 ). Xie et al. observed the potentially protective role of nafamostat mesylate against aristolochic acid-induced kidney injury in zebrafish involving protein glycosylation and amyloid aggregation (Xie et al, 2025 ). Guo et al. reported that intraperitoneal injection of nafamostat mesylate at 1 mg/kg to rats attenuated rhabdomyolysis-induced AKI (Guo et al, 2022 ). As they were unfortunately unaware of the pharmacokinetics and dosage-dependent effects of nafamostat mesylate, some biases exist in the conclusions of Guo et al., and the precise mechanism of nafamostat mesylate remains unknown. Our study showed that nafamostat mesylate attenuates renal IRI by targeting the RPS7-associated apoptosis of RTECs. It significantly reduced the expression of RPS7 in the kidney tissues of mice with renal IRI. Biolayer interferometry revealed a stable interaction between nafamostat mesylate and RPS7, characterized by fast binding and slow dissociation. Molecular docking suggested an interaction between nafamostat mesylate and RPS7 involving residues Asn165, His168, Lys169, His91, Gly89, Ser88, Glu84, Val80, Gln76, Val77, Arg81, Pro136, and Phe135. Therefore, RPS7 may be a potential target for nafamostat mesylate to slow the progression of AKI. However, our study had some limitations that should be addressed. Firstly, multicenter prospective cohort studies should be conducted to further elucidate the clinical features of SRP14 in AKI. Secondly, the tubule-specific Rps7 transgenic mouse model may be used to further explore the precise mechanism of the p53-MDM2 feedback loop and the cross-talk of multiple targets from nafamostat mesylate in RTEC apoptosis caused by AKI. Thirdly, the residues involved in the interaction of nafamostat mesylate with RPS7 could be functionally identified, and comparative analyses could be performed to validate the specificity and sensitivity of nafamostat mesylate, which was originally developed as a serine protease inhibitor, against RPS7 compared to other known serine proteases. In conclusion, our findings suggest that SRP14 triggers the apoptosis of RTECs, thereby exacerbating AKI, through an interaction with RPS7, which may be a potential target of nafamostat mesylate to slow the progression of AKI. Materials and Methods This section provides an overview of the materials and methods used in this study. For more detailed information and descriptions, please refer to the Supporting Information. Animals Male C57BL/6 mice (6–8 weeks old, 18–22 g) were obtained from Chengdu Dossy Experimental Animals Co., Ltd (Chengdu, China). Male tubule-specific Srp14 knockout ( Srp14 −/− , Ggt - Cre ) mice were obtained from Cyagen Biosciences (Suzhou, China). All mice were housed in the Animal Center of Sichuan Provincial People’s Hospital under specific pathogen-free conditions with a 12/12-hour light/dark cycle, humidity of 40%–70%, and ambient temperature of 18–22 ℃. The mice had free access to food and water. All animal studies were approved by the Ethics Committee of Sichuan Provincial People’s Hospital (approval numbers 2018 − 176 and 2020 − 215). To establish an IRI-induced mouse model of AKI, mice were anesthetized with 50 mg/kg pentobarbital sodium (Merck, Germany) administered via intraperitoneal injection, and their bilateral renal arteries were clamped with artery clamps (RS-5420; Roboz, USA) at 37℃ for 45 minutes. The mice in the sham group underwent the same procedure but without clamping. To establish the sepsis-induced mouse model of AKI, mice underwent cecal ligation and puncture (CLP), as described by Rittirsch et al (Rittirsch et al, 2009 ). The mice in the sham group underwent the same procedure but without CLP. To establish the drug-induced mouse models of AKI, mice were administered 10 mg/kg of lipopolysaccharide (LPS; L2630; Sigma-Aldrich, USA) or 15 mg/kg of cisplatin (479306; Sigma-Aldrich, USA) via intraperitoneal injection. The mice in the sham group were injected with equal volumes of saline. The mice with renal IRI were orally administered nafamostat mesylate (S1386; Selleck, USA) at doses of 20, 25, and 50 mg/kg/day and tretinoin (S1653; Selleck, USA) at a dose of 20 mg/kg/day. Subjects This study enrolled patients admitted to the Nephrology Department of Sichuan Provincial People’s Hospital from June to December 2018. Those with a confirmed diagnosis of end-stage kidney disease (defined as an estimated glomerular filtration rate [eGFR] of < 15 mL/min/1.73 m 2 ) at admission were excluded. Their demographic information was collected, including age and sex. Fast serum samples were collected 48 hours after admission, or earlier if necessary, and stored at − 20 ℃ until needed. Kidney biopsies were collected from patients with acute tubular necrosis (ATN); their clinical information is provided in Table S1 . Normal renal tissue surrounding the tumor (ST) was collected from patients with renal carcinoma as the normal control. Written informed consents were obtained from patients before conducting any study procedure. All study procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of Sichuan Provincial People’s Hospital (approval numbers 2018 − 176 and 2018 − 284). Cell Culturing and Treatments Human RTEC cell line HK2 was obtained from the National Collection of Authenticated Cell Cultures. The HK2 cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 containing 10% fetal bovine serum. The cells were exposed to hypoxic conditions (0% oxygen, 95% nitrogen, and 5% carbon dioxide) using a cell hypoxia/hyperoxia workstation (MiniStation Plus-MPS230418047; Gene Science, Chongqing, China). For regeneration, the cells were cultured under normoxic conditions (21% oxygen, 74% nitrogen, and 5% carbon dioxide). Routine Histology and Immunohistochemistry The kidney tissues obtained from human patients and mice were fixed in paraffin and then sliced into 2 µm-thick sections. The renal sections underwent routine histological examinations: hematoxylin and eosin (HE) staining (SC231202; Baso, China) and periodic acid-Schiff (PAS) staining (SC241001; Baso, China). They were also immunohistochemically stained with antibodies against SRP14 (1:100; NBP2-94184; Novus, USA) and RPS7 (1:50; SC-100834; Santa Cruz Biotechnology, USA) at 4℃ overnight. After washing, the sections were then incubated with an appropriate horseradish peroxidase (HRP)-labeled secondary antibody (K5007; Dako Products, Denmark) at 37 ℃ for 1 hour. Multiplex Immunofluorescence Staining and Multi-spectral Imaging The 2 µm renal tissue paraffin sections from human and mice underwent multiplex immunofluorescence staining with Opal 4-Color Manual IHC Staining Kits (FP1487001KT, FP1488001KT, and FP1495001KT; Akoya Bioscience, USA) using primary antibodies against SRP14 (1:100; NBP2-94184; Novus, USA), RPS7 (1:50; SC-100834; Santa Cruz Biotechnology, USA), and hepatitis A virus cellular receptor 1 (HAVCR1/KIM-1; 1:400; NBP-43761; Novus, USA) were used for multiplex immunofluorescence staining. The sections were observed under a confocal microscope (LSM900; ZEISS, Germany). TdT-mediated dUTP Nick-End Labeling (TUNEL) Murine renal tissues were fixed in paraffin and then sliced into 2 µm-thick sections. Then, the renal tissue slices underwent in situ apoptosis assessments using the DeadEnd Fluorometric TUNEL System (G3250; Promega, USA). The production of fluorescein-12-dUTP-labeled DNA was observed under a confocal microscope (LSM900, ZEISS, Germany). Flow cytometry Briefly, 2 × 10 5 HK2 cells were seeded into six-well plates and then transfected with SRP14 and RPS7 small interfering RNA. After exposure to H/R, the HK2 cells were collected and underwent flow cytometry using a flow cytometer (Becton Dickinson; for annexin A5 [ANXA5/annexin V]-fluorescein isothiocyanate [FITC]: excitation = 633 nm, emission = 660 nm; for propidium iodide [PI]: excitation = 488 nm, emission = 580 nm) by the Annexin V Alexa Fluor 488 & Propidium Iodide Cell Apoptosis Detection Kit (AD11; Dojindo Laboratories, Japan). Biolayer Interferometry We synthesized and expressed recombinant SRP14 and RPS7 proteins using the pET21b (+) prokaryotic expression plasmid. The recombinant RPS7 protein was biotinylated using the G-MM-IGT Biotinylation Kit (Genemore, China) according to the manufacturer’s protocol. The SA sensor (ForteBio/Pall Life Sciences, Menlo Park, CA, USA) was loaded with biotinylated RPS7 for 600 seconds. Next, after being washed with phosphate-buffered saline (PBS), the sensor was dipped into recombinant SRP14 protein at a concentration of 88.8–355 nM for 60 seconds for association, and then in PBS for 180 seconds for disassociation. The kinetics were recorded using the Octet K2 system (ForteBio/Pall Life Sciences, Menlo Park, CA, USA) at 1,000 rpm shaking and analyzed using the Octet Data Analysis HT 11.1 software. Immunoprecipitation and Immunoblotting Total proteins were extracted from murine renal tissues and cells using a lysis buffer (P0013B; Beyotime, China) containing a protease inhibitor (ST506-2; Beyotime, China). For immunoprecipitation, 20 µL of A/G agarose was added to each sample and incubated for 1 hour at room temperature. The total cell lysates (500 µg) were immunoprecipitated with antibodies against RPS7 (SC-100834; Santa Cruz Biotechnology, USA) or SRP14 (SC-377012; Santa Cruz Biotechnology, USA) overnight at 4℃. Then, the immunoprecipitants were separated using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to western blot analysis using specific antibodies against SRP14 and RPS7. For western blot analysis, the proteins were denatured by adding 5 × loading buffer and boiling at 100℃ for 5 minutes. Next, the proteins were separated by SDS-PAGE and electrotransferred to a polyvinylidene fluoride membrane (ISEQ00010; Merck Millipore, Germany). The membrane was then blocked with tris-buffered saline buffer containing 5% bovine serum albumin and 0.1% Tween-20 at room temperature for 1 hour. Next, the membrane was incubated with the primary antibodies at 4℃ overnight and then with the corresponding HRP-conjugated secondary antibodies at room temperature for 1 hour. Then, the protein bands were detected using actin beta (ACTB/β-actin; 1:1000; 6008; Proteintech, China) as the loading control. Primary antibodies against the following proteins were used: SRP14 (1:1000; NBP2-94184; Novus, USA), RPS7 (1:1000; PA5-77005; Invitrogen, USA), BCL2-associated X apoptosis regulator (BAX; 1:1000; ab182733; Abcam, UK), cleaved caspase 3 (CASP3; 1:1000; 9664s; Cell Signaling Technology), tumor protein p53 (TP53/p53; 1:1000; ab131442; Abcam, UK), and MDM2 proto-oncogene (MDM2; 1:1000; ab259265; Abcam, UK). The secondary antibodies included HRP-conjugated goat anti-rabbit (1:5000; 511203; ZenBio, China) and HRP-conjugated goat anti-mouse (1:5000; 511103; ZenBio, China). The signals were detected using the Immobilon Western Chemilum HRP Substrate (WBKLS-638173; Millipore/Merck, USA) and visualized and analyzed using a chemiluminescence image analysis system (5200; Tanon, Shanghai, China). Statistical Analysis Continuous variables are expressed as the mean ± standard deviation (SD) if normally distributed or the median (interquartile range [IQR]) if non-normally distributed. Variables were compared between the AKI and control groups using unpaired Student’s t -test, the Mann–Whitney U test, or the chi-square test. Correlation analyses were performed for variables that differed significantly between the AKI and control groups. SRP14 levels were compared among different AKI stages using one-way analysis of variance (ANOVA). A p -value of < 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS Statistics for Windows (version 29.0; IBM Corp., Armonk, NY, USA) and GraphPad Prism (version 8.0.1; GraphPad Software, La Jolla, CA, USA). Resource availability Lead contact Please request further information about resources and reagents associated with this study from the lead contact, Yi Li ( [email protected] ). Materials availability All reagents in this study are available from the lead contact with a completed materials transfer agreement. Data and code availability The label free proteomic data has been uploaded to iProX database (Project ID: IPX0011230000). All other data are presented in manuscript or the supplemental complete materials and methods. Please request material sharing associated with this study from the corresponding author Yi Li ( [email protected] ). Declarations Acknowledgments We would like sincerely thank Prof. Zhenglin Yang of Sichuan Provincial People’s Hospital for generously providing the research platform for this study. We thank Weishen Wu for his guidance with bioinformatics analysis. We also thank the generously technical supporting from Zhiying Wang, Jie Chen, and Lin Wang of Sichuan Provincial People’s Hospital. Author Contributions All authors have read and approved the final version of this manuscript. Yi Li designed the study, analyzed data, and prepared the manuscript. Min Wu, Li Wang and Guisen Li participated in consultation for study design and manuscript preparation. Yun Tang and Liming Huang conducted experiments, analyzed data, and prepared the manuscript. Yanmei Wang, Qiao Tang, Zehui Liao, Xueting Yang, Yangping Wu, Fang Wang, Yunlin Feng, Chanyu Geng, Sipei Chen, Qi Yao, Cihang Zhao, Jia Tang, Yilin Fu, Guoli Li, and Jun Gao conducted experiments and analyzed data. Declaration of interests All the authors declare no competing interests. Funding This work was supported by National Natural Science Foundation of China (82270729, U21A20349, 81700607, and 82470005), Projects from Department of Science and Technology of Sichuan Province (24NSFSC1735, 2023ZYD0170, and 2021YFS0370), Funds of Key Research and Development Grant of MOST (2023YFA095000), and Discipline construction foundation for Department of nephrology and institute from Sichuan Provincial People’s Hospital. References Kellum JA, Romagnani P, Ashuntantang G, Ronco C, Zarbock A, Anders HJ. (2021). Acute kidney injury. Nature reviews Disease primers. 7(1), 52. Siew ED, Davenport A. (2015). The growth of acute kidney injury: a rising tide or just closer attention to detail? Kidney international. 87(1), 46-61. Zhang Y, Xu D, Gao J, Wang R, Yan K, Liang H, Xu J, Zhao Y, Zheng X, Xu L, et al. (2025). Development and validation of a real-time prediction model for acute kidney injury in hospitalized patients. Nature communications. 16(1), 68. Maremonti F, Meyer C, Linkermann A. (2022). Mechanisms and Models of Kidney Tubular Necrosis and Nephron Loss. Journal of the American Society of Nephrology: JASN. 33(3), 472-486. Havasi A, Borkan SC. (2011). Apoptosis and acute kidney injury. Kidney international. 80(1), 29-40. Fattori V, Borghi SM, Guazelli CFS, Giroldo AC, Crespigio J, Bussmann AJC, Coelho-Silva L, Ludwig NG, Mazzuco TL, Casagrande R, et al. (2017). Vinpocetine reduces diclofenac-induced acute kidney injury through inhibition of oxidative stress, apoptosis, cytokine production, and NF-κB activation in mice. Pharmacol Res. 120, 10-22. 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Integration of transcriptomics and metabolomics reveals the molecular mechanisms underlying the effect of nafamostat mesylate on rhabdomyolysis-induced acute kidney injury. Frontiers in pharmacology. 13, 931670. Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA. (2009). Immunodesign of experimental sepsis by cecal ligation and puncture. Nature protocols. 4(1), 31-36. Additional Declarations There is NO Competing Interest. Supplementary Files ThewesternblotoriginaldataofFigure4DCompressed.pdf The western blot original data of Figure 4D ThewesternblotoriginaldataofFigure4CCompressed.pdf The western blot original data of Figure 4C ThewesternblotoriginaldataofFigure5ECompressed.pdf The western blot original data of Figure 5E ThewesternblotoriginaldataofFigure5FCompressed.pdf The western blot original data of Figure 5F ThewesternblotoriginaldataofFigure6DCompressed.pdf The immunoblot original data of Figure 6D ThewesternblotoriginaldataofFigure8FCompressed.pdf The western blot original data of Figure 8F SupplementaryTablesandFigureswithlegendsandsupplementalcompletematerialsandmethods.pdf Supplementary tables and figures with legends and supplemental complete materials and methods ReagentsToolsTableTEMPLATE.docx Reagents and tools table 9893RS.pdf Reporting Summary Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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The normal HK2 human proximal tubular epithelial cells were set as the normal control. The label-free quantitative proteomics performed on cells with hypoxia/reoxygenation (H/R) and normal control cells. Three biologically independent experiments were performed \u003cem\u003ein vitro\u003c/em\u003e. Aged male C57 BL/6 mice were clamped the bilateral renal arteries with artery clamps at 37 ℃ for 45 min and reperfusion for 24 hours to establish the murine AKI model induced by IRI. Mice in the sham group received the same procedure without clamp. Six biologically independent experiments were performed \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eA.\u003c/strong\u003e Heat map showed differentially expressed proteins with Fold change ³1.2, p ≤ 0.01 by label-free quantitative proteomics. The change with statistical significance is indicated either decrease (blue) or increase (red). \u003cstrong\u003eB.\u003c/strong\u003e Go enrichment analysis involving apoptosis with differentially expressed proteins by label-free quantitative proteomics. \u003cstrong\u003eC.\u003c/strong\u003e H\u0026amp;E, PAS staining, and immunohistochemistry measurement of SRP14 and RPS7 in murine renal tissues. Scale bar: black = 20 mm. \u003cstrong\u003eD. \u003c/strong\u003eImmunofluorescent staining of SRP14 and KIM-1, or RPS7 and KIM-1 in murine kidneys. Scale bar: white = 50 mm. “H/R” represents “hypoxia/reoxygenation”. “IRI” represents “ischemia reperfusion\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/73519f5630a6766486bd71fb.jpg"},{"id":95691671,"identity":"a3ab21e8-52c1-4549-bb3a-15c4024e50f4","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":478162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSRP14 was associated with AKI involving renal tubules damage.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e ELISA measuring serum SRP14 in AKI patients and normal controls. Serum SRP14 was tested in 45 candidates, among which 29 candidates were AKI patients. \u003cstrong\u003eB.\u003c/strong\u003e The level of serum SRP14 in different stages of AKI. AKI stages were defined by the KDIGO definition. \u003cstrong\u003eC.\u003c/strong\u003eThe ROC curve of serum SRP14. \u003cstrong\u003eD.\u003c/strong\u003e H\u0026amp;E, PAS staining, and immunohistochemistry measuring SRP14 and RPS7 in human renal tissues. Renal tissue specimens from patients with acute tubular necrosis (ATN) (n = 5) and Normal renal tissue surrounding the tumor (ST) (n = 4) from patients with renal carcinoma were collected. Scale bar: black = 20 mm. \u003cstrong\u003eE.\u003c/strong\u003eImmunofluorescent staining of SRP14 and KIM-1, or RPS7 and KIM-1 in human kidneys. Renal tissue specimens from patients with acute tubular necrosis (ATN) (n = 5) and Normal renal tissue surrounding the tumor (ST) (n = 4) from patients with renal carcinoma were collected. \u0026nbsp;Scale bar: white = 50 mm. “**” represents “p ≤ 0.01”.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/9e59a801e01ff626f347ada1.jpg"},{"id":95798811,"identity":"762fa260-66c7-46b5-b53b-477b8b404807","added_by":"auto","created_at":"2025-11-13 08:17:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":873799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSRP14\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deficiency of renal tubules suppressed apoptosis in renal tubular epithelial cells to attenuate renal IRI.\u003c/strong\u003e Male wild type C57 BL/6 mice and SRP14\u003csup\u003e-/-\u003c/sup\u003e, Ggt-Cre mice subjected to clamping bilateral renal arteries with artery clamps at 37 ℃ for 45 min and reperfusion for 24 hours to establish the murine AKI model induced by IRI. Wild type C57 BL/6 mice and SRP14\u003csup\u003e-/-\u003c/sup\u003e, Ggt-Cre mice in the sham group subjected to the same procedure without clamp.\u003cstrong\u003e A. \u003c/strong\u003eMeasuring serum creatinine in mice (n = 6). \u003cstrong\u003eB.\u003c/strong\u003e The detection of BUN in murine serum (n = 6). \u003cstrong\u003eC.\u003c/strong\u003e H\u0026amp;E and PAS staining in murine renal tissues (n = 6). Scale bar: black = 20 mm. \u003cstrong\u003eD.\u003c/strong\u003e Immunohistochemistry measuring SRP14 and RPS7 in murine kidneys (n = 6). Scale bar: black = 20 mm. \u003cstrong\u003eE.\u003c/strong\u003e TUNEL staining in murine kidneys (n = 6). Scale bar: white = 100 mm. \u003cstrong\u003eF.\u003c/strong\u003e Immunofluorescent staining SRP14, RPS7 and KIM-1 in murine kidneys (n = 6). Scale bar: white = 50 mm. “**” represents “p ≤ 0.01”. “WT” represents “wild type”. “IRI” represents “ischemia reperfusion injury”.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/ad74c33bd271e58881eefbe8.jpg"},{"id":95691681,"identity":"2679535e-ae43-4ff1-80b5-98e79663dc0d","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":373846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSRP14 and RPS7 silencing inhibited apoptosis in HK2 cells after H/R.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eCCK-8 determining cell viability for SRP14 and RPS7 silenced HK2 cells upon H/R. Six biologically independent experiments. \u003cstrong\u003eB.\u003c/strong\u003eFlowcytometry detecting Annexin V-FITC/PI of HK2 cells following H/R treatment. Three biologically independent experiments. \u003cstrong\u003eC.\u003c/strong\u003e Western blot measuring SRP14, RPS7, p53, MDM2, cleaved Caspase3, Bax, and b-actin in HK2 cells with H/R treatment and SRP14 silencing. Three biologically independent experiments. \u003cstrong\u003eD.\u003c/strong\u003e Western blot measuring SRP14, RPS7, p53, MDM2, cleaved Caspase3, Bax, and b-actin in HK2 cells with H/R treatment and RPS7 silencing. Three biologically independent experiments. “**” represents “p ≤ 0.01”. “H/R” represents “hypoxia/reoxygenation”.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/3c91da073f2d31a3e62cc1a8.jpg"},{"id":95691679,"identity":"a2c82fa1-bf83-4869-b8c0-e0d3c2198b3d","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":852768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSRP14 triggered apoptosis in renal tubular epithelial cells in mice upon renal IRI involving RPS7. \u003c/strong\u003eAAV-SRP14 shRNA was delivered into the kidney of C57 BL/6 male mice by intrarenal injection. Some of the mice subjected to intrarenal injection of the hRPS7 recombinant protein injection. The mice subjected to bilateral renal arteries with artery clamps at 37 ℃ for 45 min and reperfusion for 24 hours to establish the murine AKI model induced by IRI. Mice in the sham group received the same procedure without clamp. \u003cstrong\u003eA.\u003c/strong\u003e H\u0026amp;E and PAS staining murine renal tissues (n = 6). Scale bar: black = 20 mm. \u003cstrong\u003eB.\u003c/strong\u003e Immunohistochemistry measuring SRP14 and RPS7 in murine kidneys (n = 6). Scale bar: black = 20 mm. \u003cstrong\u003eC. \u003c/strong\u003eTUNEL staining in murine kidneys (n = 6). Scale bar: white = 100 mm. \u003cstrong\u003eD.\u003c/strong\u003eImmunofluorescent staining SRP14, RPS7 and KIM-1 in murine kidneys (n = 6). Scale bar: white = 50 mm. \u003cstrong\u003eE.\u003c/strong\u003eWestern blot measuring SRP14, RPS7, and b-actin in murine kidneys. Three biologically independent experiments. \u003cstrong\u003eF.\u003c/strong\u003eWestern blot measuring p53, MDM2, cleaved Caspase3, Bax, and b-actin in mice kidneys. Three biologically independent experiments. “shSRP14” represents “The AAV-SRP14 shRNA was delivered into the kidney of C57 BL/6 male mice by intrarenal injection”. “hRPS7” represents “The mice accept intrarenal injection of the RPS7 recombinant protein”. “IRI” represents “ischemia/reperfusion injury”.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/43d5674bdc8dc37d14241fdd.jpg"},{"id":95691682,"identity":"5e8c5d61-bef8-46d7-b256-a3c820354eb1","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":292016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSRP14 interacted to RPS7 in RTEC with H/R.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eConfocal microcopy showed significant colocalization of SRP14 and RPS7 in renal tubular epithelial cells after H/R treatment. \u003cstrong\u003eB.\u003c/strong\u003e Molecular docking about SRP14 and RPS7. \u003cstrong\u003eC.\u003c/strong\u003e Biolayer interferometry showed a direct interaction between SRP14 and RPS7 with fast binding and slow dissociation. \u003cstrong\u003eD.\u003c/strong\u003e Co-immunoprecipitation revealed an interaction between SRP14 and RPS7 in HK2 renal epithelial cells with hypoxia and reoxygenation. \u003cstrong\u003eE.\u003c/strong\u003e The mass spectrometry analysis on the immunoprecipitated samples. “H/R” represents “hypoxia/reoxygenation”.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/b72d8190e4b0a563a68a22fa.jpg"},{"id":95691687,"identity":"27a37d37-77d1-4569-97b3-cbeee0e16372","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":605775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening for RPS7 associated molecule from a compound library involving apoptosis.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eSchematic diagram shows the experimental procedure. \u003cstrong\u003eB. \u003c/strong\u003e\u0026nbsp;Biolayer interferometry assay selected 8 small molecule compounds that stably bind to RPS7. \u003cstrong\u003eC.\u003c/strong\u003e The IC50 of Acitretin in HK2 cells. \u003cstrong\u003eD.\u003c/strong\u003e The IC50 of Nafamostat mesylate in HK2 cells. \u003cstrong\u003eE.\u003c/strong\u003e The IC50 of Tretinoin in HK2 cells. \u003cstrong\u003eF.\u003c/strong\u003e The IC50 of Lapatinib in HK2 cells. \u003cstrong\u003eG.\u003c/strong\u003e The IC50 of Benzbromarone in HK2 cells. \u003cstrong\u003eH.\u003c/strong\u003eThe IC50 of Embelin in HK2 cells. \u003cstrong\u003eI.\u003c/strong\u003eThe IC50 of Sanguinarine chloride in HK2 cells. \u003cstrong\u003eJ.\u003c/strong\u003e The IC50 of Troglitazone in HK2 cells. \u003cstrong\u003eK.\u003c/strong\u003e The EC50 of Tretinoin in HK2 cells with H/R. \u003cstrong\u003eL.\u003c/strong\u003e The EC50 of Nafamostat mesylate in HK2 cells with H/R. \u003cstrong\u003eM.\u003c/strong\u003e The EC50 of Benzbromarone in HK2 cells with H/R. \u003cstrong\u003eN.\u003c/strong\u003eH\u0026amp;E and PAS staining in murine renal tissues (n = 6). Scale bar: black = 20 mm. “**” represents “p ≤ 0.01”. “H/R” represents “hypoxia/reoxygenation”. “IRI” represents “ischemia/reperfusion injury”.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/d4627e988cfbfbbb281c3532.jpg"},{"id":95691694,"identity":"a22a4991-1227-499f-bb9a-cacade6a856b","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":412371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNafamostat mesilate attenuated renal IRI against RPS7 associated apoptosis in RTEC cells.\u003c/strong\u003e We respectively treated mice with 25 mg/kg.d and 50 mg/kg.d Nafamostat mesilate upon renal IRI. The mice subjected to clamping bilateral renal arteries with artery clamps at 37 ℃for 45 min and reperfusion for 24 hours to establish the murine AKI model induced by IRI. Mice in the sham group subjected to the same procedure without clamp. \u003cstrong\u003eA.\u003c/strong\u003e The levels of serum creatinine in mice (n = 6). \u003cstrong\u003eB.\u003c/strong\u003e The levels of BUN in murine serum (n = 6).\u003cstrong\u003e C.\u003c/strong\u003e H\u0026amp;E and PAS staining in murine renal tissues (n = 6). Scale bar: black = 20 mm. \u003cstrong\u003eD.\u003c/strong\u003e Immunohistochemistry measuring RPS7 in murine kidneys (n = 6). Scale bar: black = 20 mm. \u003cstrong\u003eE. \u003c/strong\u003eTUNEL staining murine kidneys (n = 6). Scale bar: white = 100 mm. \u003cstrong\u003eF.\u003c/strong\u003eWestern blot measuring RPS7, p53, MDM2, Cleaved Caspase3, Bax, and b-actin in mice kidneys. Three biologically independent experiments. \u003cstrong\u003eG.\u003c/strong\u003e Molecular docking showed an interaction between Nafamostat mesilate and RPS7. “**” represents “p ≤ 0.01”. “NM” represents “Nafamostat mesilate”. “IRI” represents “ischemia/reperfusion injury”.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/d1bf5a73dbead72b724e38df.jpg"},{"id":96454332,"identity":"70351677-0522-4188-8f5a-516843989d8b","added_by":"auto","created_at":"2025-11-21 10:02:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6067930,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/d4bcf423-89c0-4253-bd82-17d801efc73f.pdf"},{"id":95691676,"identity":"b5df7835-3af7-4ddc-a80b-842b56095438","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":733659,"visible":true,"origin":"","legend":"The western blot original data of Figure 4D","description":"","filename":"ThewesternblotoriginaldataofFigure4DCompressed.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/dccecde89f2a0a5d341a7007.pdf"},{"id":95691675,"identity":"aa2f890d-6c34-4769-bf11-3accc00b5338","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":743786,"visible":true,"origin":"","legend":"The western blot original data of Figure 4C","description":"","filename":"ThewesternblotoriginaldataofFigure4CCompressed.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/7f4e837bef655c554ca16eae.pdf"},{"id":95798106,"identity":"f7f89989-f924-46b4-ab54-a97d46d6f693","added_by":"auto","created_at":"2025-11-13 08:15:29","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":284132,"visible":true,"origin":"","legend":"\u003cp\u003eThe western blot original data of Figure 5E\u003c/p\u003e","description":"","filename":"ThewesternblotoriginaldataofFigure5ECompressed.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/22be9e683fcaaed77a6d2909.pdf"},{"id":95691686,"identity":"7962384b-92e3-4bd6-bfa0-26cd2c7019f1","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":572571,"visible":true,"origin":"","legend":"The western blot original data of Figure 5F","description":"","filename":"ThewesternblotoriginaldataofFigure5FCompressed.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/ff2721f781151ab6d430ab4f.pdf"},{"id":95799861,"identity":"73695412-0a48-4cc0-953c-db2d447ae1db","added_by":"auto","created_at":"2025-11-13 08:21:00","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":157412,"visible":true,"origin":"","legend":"The immunoblot original data of Figure 6D","description":"","filename":"ThewesternblotoriginaldataofFigure6DCompressed.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/604f5c831aac1a38bc743735.pdf"},{"id":95691698,"identity":"5b8a0325-b096-401f-9191-fc2e21b99bfa","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":823586,"visible":true,"origin":"","legend":"The western blot original data of Figure 8F","description":"","filename":"ThewesternblotoriginaldataofFigure8FCompressed.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/a676659bb04262a15a1a6d88.pdf"},{"id":95691690,"identity":"21e31998-83d7-47a0-9dc7-40be2755c93b","added_by":"auto","created_at":"2025-11-12 02:20:58","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1463589,"visible":true,"origin":"","legend":"Supplementary tables and figures with legends and supplemental complete materials and methods","description":"","filename":"SupplementaryTablesandFigureswithlegendsandsupplementalcompletematerialsandmethods.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/97f59997d0d95b90d5e494b0.pdf"},{"id":95799790,"identity":"fcacb587-7846-4040-9dee-aa6e4c0fd7da","added_by":"auto","created_at":"2025-11-13 08:20:48","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":24816,"visible":true,"origin":"","legend":"Reagents and tools table","description":"","filename":"ReagentsToolsTableTEMPLATE.docx","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/d241f2fde9a20a6fe1ea2868.docx"},{"id":95691710,"identity":"eff62e52-09eb-4fb9-8e2f-66d53bb5eb6b","added_by":"auto","created_at":"2025-11-12 02:20:59","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":4682139,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"9893RS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7892365/v1/cf97336e6201327fddec2aab.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"SRP14 Triggers Apoptosis in Renal Tubules to Exacerbate AKI Through an Interaction with RPS7","fulltext":[{"header":"Key Points","content":"\u003cp\u003eSRP14 induces apoptosis of renal tubular epithelial cells, thereby exacerbating AKI, by interacting with RPS7, which represents a potential target for nafamostat mesylate to intervene in AKI.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAcute kidney injury (AKI) will ultimately progress to chronic kidney disease (CKD) with renal insufficiency, leading to heavy burdens for individuals, their families, and society (Kellum et al, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently, the incidence of and mortality due to AKI have been increasing (Siew et al, 2015; Zhang et al, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Due to its complex pathogenesis, there are no effective early interventions for AKI. Since previous studies have primarily focused on the post-development period of AKI, its initiation mechanism remains only partially understood. Therefore, there is an urgent need to thoroughly explore the mechanisms underlying AKI to advance the development of early prevention and therapy for it and delay its progression to CKD.\u003c/p\u003e\u003cp\u003eOne of the main pathological features of AKI is closely associated with renal tubule injury and the death of renal tubular epithelial cells (RTECs) (Maremonti et al, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which are peculiarly prone to apoptosis, facilitating renal tubule injury to aggravate renal failure (Havasi et al, 2011; Fattori et al, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the early stages of AKI, the apoptosis of RTECs is induced by ischemia-reperfusion injury (IRI) and nephrotoxic substances, such as cisplatin (Wei et al, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pushpan et al, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which is closely associated with damage to renal tubules, loss of renal function, and renal tissue injuries (Sanz et al, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Xie et al, 2006). In patients with AKI, damaged kidneys secrete factors into the circulation to further provoke cell apoptosis and inflammation in the cardiovascular system, pulmonary tissues, liver, and brain, thereby leading to increased mortality (Havasi et al, 2011; Sanz et al, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Doi et al, 2016; Grams et al, 2012). In contrast, inhibiting apoptosis in RTECs is believed to be pivotal for early prevention and treatment of AKI. Despite apoptosis-targeting therapies leading to decreased apoptosis in this model, clinically applicable treatments are far from realization.\u003c/p\u003e\u003cp\u003eTo explore potential therapeutic targets for AKI, we established an apoptosis model of RTEC using the hypoxia/reoxygenation (H/R) approach. Using label-free proteomics, differential protein expression profiles in RTECs after H/R revealed that signal recognition particle 14 (SRP14) was significantly elevated in these apoptotic RTEC populations. SRP14 is an important cytoplasmic ribosomal protein particle for the assembly of the Alu domain of the signal recognition particle (SRP) complex to block translation extension and guide the transport of eukaryotic secreted proteins to the rough endoplasmic reticulum membrane (Weichenrieder et al, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). However, the role of SRP14 in AKI remains unclear. Therefore, this study aimed to elucidate the role and underlying mechanism of SRP14 in AKI, which involves apoptosis in RTECs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eIRI Elevates SRP14 and RPS7 in Renal Tubules\u003c/h2\u003e\n \u003cp\u003eLabel-free quantitative proteomics performed on the human HK2 RTECs revealed the significant upregulation of SRP14 (fold change = 1.40, \u003cem\u003ep\u003c/em\u003e = 0.0040) and RPS7 (fold change = 1.28, \u003cem\u003ep\u003c/em\u003e = 0.0066) following exposure to H/R (Fig.\u0026nbsp;1A). TUNEL staining revealed evident apoptosis in the kidneys of mice with renal IRI (Figure S1A). The gene ontology (GO) enrichment analysis revealed that genes involved in apoptosis were significantly enriched in H/I-treated HK2 cells (Fig.\u0026nbsp;1B). Protein-protein interaction (PPI) analysis predicted an interaction between SRP14 and RPS7 (Figure S1B). Immunohistochemistry further confirmed the upregulation of both SRP14 and RPS7 in renal tissues from mouse models of AKI induced by IRI (Fig.\u0026nbsp;1C), LPS injection (Figure S1C), cisplatin injection (Figure S1D), and CLP (Figure S1E). Compared to the controls, immunofluorescence revealed increased colocalization of SRP14 and RPS7 with KIM-1 in the renal tubules of mice with renal IRI (Figs.\u0026nbsp;1D, S1F, and S1G).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSRP14 is Associated with AKI and Renal Tubule Damage\u003c/h3\u003e\n\u003cp\u003eOf the 45 human subjects enrolled in the final analysis, 29 (64.4%) developed AKI. The detailed clinical characteristics of patients in both groups at the time of AKI diagnosis are shown in Table\u0026nbsp;1. Several variables differed significantly between groups, including serum creatinine, blood urea nitrogen (BUN), eGFR, uric acid, hemoglobin, total cholesterol, TIMP metallopeptidase inhibitor 2 (TIMP2), and SRP14. SRP14 levels were significantly higher in the AKI group than in the control group (Fig.\u0026nbsp;2A). In addition, SRP14 levels were lower among patients with AKI stage 3 than among patients with AKI stage 1 and 2 (Fig.\u0026nbsp;2B). The area under the receiver operating characteristic (ROC) curve (AUC) for serum SRP14 levels was 79.96% (95% confidence interval [CI]: 0.67–0.93, \u003cem\u003ep\u003c/em\u003e = 0.001) with a cutoff of 15.29 ng/mL, a sensitivity of 0.621, and a specificity of 0.938 (Fig.\u0026nbsp;2C). Notably, SRP14 levels were significantly correlated with serum creatinine levels (Spearman’s rank correlation coefficient [\u003cem\u003eρ\u003c/em\u003e] = 0.31, \u003cem\u003ep\u003c/em\u003e = 0.038) and eGFR (\u003cem\u003eρ\u003c/em\u003e = −0.30, \u003cem\u003ep\u003c/em\u003e = 0.043; Table S2, Figure S2A-G).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Comparison of clinical characteristics at diagnosis of AKI between AKI and control groups.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAKI (n=29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eControl (n=16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMale, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12 (41.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7 (43.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.878\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAge (y), mean±SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67.6±18.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e48.3±11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSerum Cr (μmol/L), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e170.6 (126.4, 248.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e61.7 (54.2, 70.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.78×10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBUN (mmol/L), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.7 (9.2, 19.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.0 (3.2, 5.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.82×10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eeGFR (mL/min/1.73 m\u003csup\u003e2\u003c/sup\u003e), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32.6 (20.3, 42.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e106.9 (99.9, 112.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.94×10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUric acid (mmol/L), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e466.0 (373.0, 627.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e341.5 (244.5, 400.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAlbumin (mmol/L), mean±SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32.4±6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.7±4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.318\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHemoglobin (g/dL), mean±SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e108.2±24.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e137.9±14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTG (mmol/L), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.3 (1.0, 2.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1 (0.8, 2.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.297\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTC (mmol/L), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.1 (2.7, 4.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.8 (4.2, 5.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLymphocyte (%), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.0 (0.5, 1.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.9 (1.4, 2.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.49×10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNeutrophil (%), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.3 (5.8, 11.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.3 (2.7, 4.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.19×10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTIMP-2 (ng/ml), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.3 (2.7, 4.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.7 (0.6, 1.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.07×10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSRP14 (ng/ml), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17.5 (11.6, 20.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.6 (7.4, 12.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.80×10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: Cr, creatinine; eGFR, estimated glomerular filtration rate; BUN, blood urea nitrogen; IQR, interquartile range; n, number; SD, standard deviation; TC, total cholesterol; TG, triglyceride; y, year.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTable 2 The binding free energy between small molecules and proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEnergy Component\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAverage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStd. Dev.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStd. Err. of Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eVDWAALS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-123.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.785\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.219\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEEL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1139.232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e79.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.201\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEGB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-1063.210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e78.800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eESURF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.143\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDELTA G gas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1016.166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e80.133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.393\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDELTA G solv\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-1083.360\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e78.733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.141\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDELTA TOTAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-67.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.838\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.588\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: VDWAALS：van der Waals energy; EEL：\u0026nbsp;Electrostatic energy; EGB：\u0026nbsp;Polar solvation energy; ESURF：\u0026nbsp;Non-polar solvation energy; Ggas：\u0026nbsp;Total gas phase free energy; Gsolv：\u0026nbsp;Total solvation free energy.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTable 3. The potential binding domain of SRP14 and RPS7. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMethod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEnd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSequence\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAIIIFVPVPQLK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDVNFEFPEFQL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEIEVGGGR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eELNITAAK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHVVFIAQR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIVKPNGEKPDEFESGISQALLELEMNSDLK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTLTAVHDAILEDLVFPSEIVGK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMolecular docking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKHVVFIAQRRILPKPTRKSRTKNKQKRPRSRTLTAVHDAILEDLVFPSEIVGKRIRVKLDGSRLIKVHLDKAQQNNVEHKVETFSGVYKKLTGKDVNFEFP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSRP14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAAAAAAAAAPAAAATAPTTAATTAATAAQ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSRP14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMolecular docking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVVSSKEVNKFQMAYSNLLRANMDGLKKRDKKNKTKKTKAAAAAAAAAPAAAATAPTTAATTAATAAQ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTable 4. RPS7 binds to multiple hydrophilic residues of Nafamostat mesylate.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSmall molecule\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSite\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eASN165\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHIS168\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLYS169\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHIS91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGLY89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSER88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGLU84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVAL80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGLN76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVAL77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eARG81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePRO136\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRPS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNafamostat mesylate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePHE135\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eTable 5. The binding free energy between small molecules and proteins.\u003c/strong\u003e\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEnergy Component\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAverage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStd. Dev.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eVDWAALS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-40.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.129\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEEL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-37.961\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.221\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEGB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e51.346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eESURF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-5.330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.821\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDELTA G gas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-77.976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.979\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDELTA G solv\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.435\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDELTA TOTAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-31.959\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.221\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003eAbbreviations: VDWAALS：van der Waals energy; EEL： Electrostatic energy; EGB： Polar solvation energy; ESURF： Non-polar solvation energy; Ggas： Total gas phase free energy; Gsolv： Total solvation free energy.\n\u003c/div\u003e\n\u003cp\u003eTo further elucidate the importance of SRP14 and RPS7 in renal tubular injury, renal tissue specimens were collected from patients with ATN (\u003cem\u003en\u003c/em\u003e = 5), and normal ST renal tissue specimens were collected from patients with renal carcinoma (\u003cem\u003en\u003c/em\u003e = 4; Table S2). Immunohistochemistry revealed increased SRP14 and RPS7 in the renal tubules of patients with ATN (Fig.\u0026nbsp;2D). Immunofluorescence staining for SRP14, RPS7, and KIM-1 showed increased colocalization of SRP14 and RPS7 with KIM-1 in the renal tubules of patients with ATN compared to normal ST renal tissues of patients with renal carcinoma (Figs.\u0026nbsp;2E, S2H, and S2I).\u003c/p\u003e\n\u003ch3\u003eSRP14 Deficiency in Renal Tubules Attenuates Renal IRI and Suppresses Apoptosis in RTECs\u003c/h3\u003e\n\u003cp\u003eTo explore the role of SRP14 in renal tubules, tubule-specific \u003cem\u003eSrp14\u003c/em\u003e knockout (\u003cem\u003eSrp14\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eGgt-Cre\u003c/em\u003e) mice were established using a Cre-\u003cem\u003eLoxP\u003c/em\u003e recombination system. Tail genotyping and immunohistochemical measurement of SRP14 confirmed the tubule-specific knockout of \u003cem\u003eSrp14\u003c/em\u003e in mice (Figure S3A, B). While the \u003cem\u003eSrp14\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e knockout mice did not differ significantly from the wild-type control mice in serum creatinine levels (Fig.\u0026nbsp;3A), BUN levels (Fig.\u0026nbsp;3B), and routine pathological characteristics (Fig.\u0026nbsp;3C), tubule-specific knockout of \u003cem\u003eSrp14\u003c/em\u003e moderately inhibited the expression of SRP14 and RPS7 (Fig.\u0026nbsp;3D) in the mouse kidney.\u003c/p\u003e\n\u003cp\u003eFollowing IRI, tubule-specific knockout of \u003cem\u003eSrp14\u003c/em\u003e significantly reduced serum creatinine (Fig.\u0026nbsp;3A) and BUN (Fig.\u0026nbsp;3B) levels, attenuated tubular injury, and suppressed the expression of RPS7 in the mouse kidney (Fig.\u0026nbsp;3C). The number of TUNEL-positive cells in renal tissue was higher in mice with renal IRI than in the wild-type mice, while tubule-specific knockout of \u003cem\u003eSrp14\u003c/em\u003e decreased the number of TUNEL-positive cells following IRI (Fig.\u0026nbsp;3E). While IRI significantly facilitated the colocalization of SRP14 and RPS7 in murine renal tubules, tubule-specific knockout of \u003cem\u003eSrp14\u003c/em\u003e reduced the colocalization of SRP14 and RPS7 in murine renal tubules following IRI (Figs.\u0026nbsp;3F and S3C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of\u003c/strong\u003e \u003cstrong\u003eSrp14\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eRps7\u003c/strong\u003e \u003cstrong\u003eInhibits Apoptosis of RTECs Following H/R\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cell Counting Kit-8 (CCK-8) assay was used to clarify the effects of SRP14 and RPS7 on the apoptosis of RTECs. In HK2 cells exposed to H/R, the silencing of SRP14 or RPS7 increased survival to 88.59% ± 9.25% and 89.10% ± 3.33%, compared to 68.26% ± 4.01% in control cells (Fig. 4A). Additionally, annexin V-FITC/PI staining revealed that knockdown of \u003cem\u003eSrp14\u003c/em\u003e or \u003cem\u003eRps7\u003c/em\u003e inhibited the apoptosis of HK2 cells following H/R (Fig. 4B). The gating strategy for annexin V-FITC/PI staining is shown in Figure S4. \u003cem\u003eIn vitro\u003c/em\u003e, knockdown of \u003cem\u003eSrp14\u003c/em\u003e or \u003cem\u003eRps7\u003c/em\u003e reduced the expression of p53, MDM2, cleaved CASP3, and BAX in HK2 cells following H/R (Fig. 4C, D). In addition, knockdown of \u003cem\u003eSrp14\u003c/em\u003e inhibited the expression of RPS7 in HK2 cells following H/R, whereas knockdown of \u003cem\u003eRps7\u003c/em\u003e did not significantly affect the expression of SRP14 in HK2 cells following H/R (Fig. 4C, D).\u003c/p\u003e\n\u003ch3\u003eSRP14 Triggers Apoptosis in RTECs Upon IRI Involving RPS7\u003c/h3\u003e\n\u003cp\u003eTo elucidate the role of SRP14 and RPS7 in the renal tubule damage associated with IRI, recombinant adeno-associated virus (AAV) vectors harboring short hairpin RNA (shRNA)-\u003cem\u003eSrp14\u003c/em\u003e or a fusion gene of human \u003cem\u003eRPS7\u003c/em\u003e (\u003cem\u003ehRPS7\u003c/em\u003e) were delivered into the kidneys of C57BL/6 mice via intrarenal injection (Figure S5A). The optical \u003cem\u003ein vivo\u003c/em\u003e imaging system confirmed the successful delivery of AAV-shRNA-\u003cem\u003eSrp14\u003c/em\u003e (Figure S5B). Immunohistochemical measurement further verified the reduction in SRP14 expression in murine kidneys infected with AAV-shRNA-\u003cem\u003eSrp14\u003c/em\u003e (Figure S5C).\u003c/p\u003e\n\u003cp\u003eSome of the mice received intrarenal injection of the recombinant AAV harboring the fusion gene of \u003cem\u003ehRPS7\u003c/em\u003e. Notably, shRNA-\u003cem\u003eSrp14\u003c/em\u003e attenuated tubular injury and reduced the expression of SRP14 in murine kidneys with renal IRI. However, the presence of both recombinant hRPS7 and shRNA-\u003cem\u003eSrp14\u003c/em\u003e aggravated renal tubule injury and increased the expression of SRP14 in the kidneys of mice with renal IRI (Fig.\u0026nbsp;5A, B). In addition, shRNA-\u003cem\u003eSrp14\u003c/em\u003e inhibited the colocalization of SRP14 and RPS7 in the renal tubules of mice with renal IRI. Compared to wild-type mice with renal IRI, shRNA-\u003cem\u003eSrp14\u003c/em\u003e decreased the number of TUNEL-positive cells in renal tissues from mice with renal IRI; however, the presence of both recombinant hRPS7 and shRNA-\u003cem\u003eSrp14\u003c/em\u003e increased the apoptotic index (Fig.\u0026nbsp;5C) and moderately facilitated the colocalization of SRP14 and RPS7 in renal tubules of mice with renal IRI (Figs.\u0026nbsp;5D and S6). Moreover, shRNA-\u003cem\u003eSrp14\u003c/em\u003e decreased the expression of p53, MDM2, cleaved CASP3, and BAX in kidney tissues from mice with renal IRI. However, the presence of both recombinant hRPS7 and shRNA-\u003cem\u003eSrp14\u003c/em\u003e resulted in no significant decrease in the expression of p53, MDM2, cleaved CASP3, and BAX in kidney tissues of mice with renal IRI (Fig.\u0026nbsp;5E, F).\u003c/p\u003e\n\u003ch3\u003eSRP14 Interacts with RPS7 in RTEC With Hypoxia and Reoxygenation\u003c/h3\u003e\n\u003cp\u003eConfocal microscopy revealed significant colocalization of SRP14 and RPS7 in RTECs following H/R (Figs. 6A and S7A). Molecular docking showed that both SRP14 and RPS7 possess a core structural domain. The C-terminal region of RPS7 (residues 90–190) interacts with the C-terminal region of SRP14 (residues 70–136), which is vital for the interaction between SRP14 and RPS7 (Fig. 6B). In a molecular simulation of the SRP14–RPS7 complex lasting 20,000 ps, the RMSD indicated that the complex reached equilibrium at 8,000 ps. In addition, the dynamics simulation demonstrated a decreasing trend, suggesting strong interaction between SRP14 and RPS7. The trajectories from 8,000 to 20,000 ps were used to calculate the binding free energy. The total binding free energy change (ΔG) was − 67.19 kcal/mol, indicating tight binding between SRP14 and RPS7 (Figure S7B). The van der Waals potential energy was − 123.066 kcal/mol. Both the polar solvation (− 1063.21 kcal/mol) and non-polar solvation (− 20.1509 kcal/mol) were conducive to the binding of SRP14 and RPS7 (Table 2).\u003c/p\u003e\n\u003cp\u003eBiolayer interferometry revealed a fast-binding and slow-dissociation trend between SRP14 and RPS7, with a dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e) of 2.43 × 10\u003csup\u003e− 8\u003c/sup\u003e M (Fig. 6C). Co-immunoprecipitation revealed an interaction between SRP14 and RPS7 in HK2 cells following H/R (Fig. 6D). To further establish the interaction between SRP14 and RPS7, immunoprecipitated samples were subjected to mass spectrometry analysis, revealing that the C terminal region of SRP14 (residues 111–136) and the C terminal region of RPS7 (residues 91–98 and 121–142) played a pivotal role in the interaction between SRP14 and RPS7 (Figs. 6E and S7C, Table 3).\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eScreening of RPS7 Inhibiting Compounds from an Apoptosis-Specific Library\u003c/h2\u003e\n \u003cp\u003eGiven the important role of RPS7 in SRP14-regulated apoptosis, recombinant RPS7 was used to screen an apoptosis-specific compound library comprised of 356 US Food and Drug Administration (FDA)-approved compounds via biolayer interferometry (Fig.\u0026nbsp;7A). After confirming the concentration gradient for the initially identified 15 small molecule compounds, the following eight small molecule compounds were identified as stably binding to RPS7 (Figs.\u0026nbsp;7B and S8): acitritin (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 1.97 × 10\u003csup\u003e− 4\u003c/sup\u003e M), nafamostat mesilate (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 6.45 × 10\u003csup\u003e− 4\u003c/sup\u003e M), tretinoin (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 1.19 × 10\u003csup\u003e− 4\u003c/sup\u003e M), lapatinib (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 2.11 × 10\u003csup\u003e− 2\u003c/sup\u003e M), benzbromarone (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 9.77 × 10\u003csup\u003e− 5\u003c/sup\u003e M), embelin (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 2.90 × 10\u003csup\u003e− 5\u003c/sup\u003e M), sanguinarine chloride (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 1.52 × 10\u003csup\u003e− 4\u003c/sup\u003e M), and troglitazone (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 9.14 × 10\u003csup\u003e− 5\u003c/sup\u003e M).\u003c/p\u003e\n \u003cp\u003eCCK-8 assessments of the viability of HK2 cells determined half maximal inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) of 1.33 for acitritin (Fig.\u0026nbsp;7C), 22.58 for nafamostat mesilate (Fig.\u0026nbsp;7D), 86.66 for tretinoin (Fig.\u0026nbsp;7E), 33.13 for lapatinib (Fig.\u0026nbsp;7F), 64.22 for benzbromarone (Fig.\u0026nbsp;7G), 9.86 for embelin (Fig.\u0026nbsp;7H), 0.14 for sanguinarine chloride (Fig.\u0026nbsp;7I), and 5.35 for rroglitazone (Fig.\u0026nbsp;7J). In addition, CCK-8 assessment of the viability of HK2 cells following H/R determined half maximal effective concentrations (EC\u003csub\u003e50\u003c/sub\u003e) of 6.67 for tretinoin (Fig.\u0026nbsp;7K), 1.79 for nafamostat mesilate (Fig.\u0026nbsp;7L), and 5.98 for benzbromarone (Fig.\u0026nbsp;7M). Moreover, 20 mg/kg/day of nafamostat mesilate or tretinoin attenuated renal tubule injury in mice with renal IRI (Fig.\u0026nbsp;7N).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eNafamostat Mesilate Attenuates Renal IRI by Reducing RPS7-associated Apoptosis in RTECs\u003c/h3\u003e\n\u003cp\u003eTo clarify the protective effects of nafamostat mesilate against renal IRI, mice were treated with 25 or 50 mg/kg/day of nafamostat mesilate following renal IRI. Nafamostat mesilate at 25 or 50 mg/kg/day decreased the levels of serum creatinine (Fig.\u0026nbsp;8A) and BUN (Fig.\u0026nbsp;8B) in mice with renal IRI. Routine histological assessments showed that at both 25 and 50 mg/kg/day, nafamostat mesilate attenuated tubular injury caused by renal IRI (Fig.\u0026nbsp;8C) and inhibited the expression of RPS7 in mouse kidney tissues (Fig.\u0026nbsp;8D). Compared to untreated control mice with renal IRI, the number of TUNEL-positive cells in renal tissues was lower in mice with renal IRI treated with 25 or 50 mg/kg/day nafamostat mesilate (Fig.\u0026nbsp;8E). At both 25 and 50 mg/kg/day, nafamostat mesilate significantly reduced the expression of RPS7, p53, MDM2, cleaved CASP3, and BAX in the renal tissues of mice with renal IRI (Fig.\u0026nbsp;8F).\u003c/p\u003e\n\u003cp\u003eMolecular docking revealed an interaction between nafamostat mesilate and RPS7 involving residues Asn165, His168, Lys169, His91, Gly89, Ser88, Glu84, Val80, Gln76, Val77, Arg81, Pro136, and Phe135 (Fig. 8G, Table 4). The ΔG was − 31.9592 kcal/mol, indicating a stable interaction between nafamostat mesilate and RPS7 (Figure S9). The van der Waals potential energy was − 40.0145 kcal/mol. Collectively, these data show that both the polar solvation (51.3462 kcal/mol) and non-polar solvation (− 5.3299 kcal/mol) were conducive to the binding of nafamostat mesilate to RPS7 (Table 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRTECs are sensitive to apoptosis, aggravating renal tubule injury and renal failure (Havasi et al, 2011; Maeda et al, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our study demonstrated that SRP14 was significantly elevated in RTECs following H/R. SRP14 is a cytoplasmic ribosomal protein involved in the assembly of the Alu domain of the SRP complex for signal recognition, thereby blocking translation elongation and guiding the transport of secreted proteins to the rough endoplasmic reticulum membrane (Weichenrieder et al, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Brooks et al, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The C-terminal region of SRP14 primarily inhibits translation elongation. Mutation of the C-terminal region of SRP14 prevents its blocking of translation elongation but, interestingly, does not affect its ribosome binding ability (Mason et al, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). These data suggest that SRP14 has additional, unknown functions in biological processes.\u003c/p\u003e\u003cp\u003eWe observed a marked increase in SRP14 in the serum of patients with AKI, with an AUC of 79.96%. Serum SRP14 levels correlated with serum creatinine levels, BUN levels, eGFR, uric acid levels, neutrophil counts, KIM-1 levels, and age. The expression of SRP14 was significantly elevated in the renal tubules of patients with ATN and four mouse models of AKI induced by IRI, LPS injection, CLP, and cisplatin injection. Thus, SRP14 is associated with AKI involving renal tubule damage. However, the effects of SRP14 on AKI remain unclear.\u003c/p\u003e\u003cp\u003eTo elucidate the role of SRP14 in AKI, tubule-specific \u003cem\u003eSrp14\u003c/em\u003e knockout mice were generated via a Cre-\u003cem\u003eLoxP\u003c/em\u003e recombination strategy. Tubule-specific \u003cem\u003eSrp14\u003c/em\u003e knockout attenuated renal IRI, suppressed the apoptosis of RTECs, and reduced the expression of p53, MDM2, cleaved CASP3, and BAX in RTECs following H/R. Moreover, annexin V-FITC/PI staining revealed that \u003cem\u003eSrp14\u003c/em\u003e knockdown inhibited the apoptosis of HK2 cells following H/R. These results suggest that SRP14 may play a crucial role in the apoptosis of RTECs in IRI-induced AKI.\u003c/p\u003e\u003cp\u003eAs an interacting partner of MDM2, RPS7 contributes to the complex regulation of the p53-MDM2 feedback loop by stabilizing p53 protein and activating p53 function (Chen et al, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). As a substrate of MDM2, once bound to MDM2, RPS7 is ubiquitinated and degraded, sustaining the p53 response (Zhu et al, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The p53-MDM2 feedback loop is crucial for apoptosis in renal IRI. Renal IRI significantly increased the expression of p53 in rat cortical tissue, and the p53 inhibitor Pifithrin-α inhibited apoptosis, protecting against renal IRI (Kelly et al, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Zhang et al. found that p53 in proximal tubular cells promotes AKI, whereas p53 in other tubular cells does not contribute to AKI (Zhang et al, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our proteomic findings showed a significant increase in RPS7 in HK2 cells following H/R. Knockdown of \u003cem\u003eRps7\u003c/em\u003e inhibited apoptosis involving the p53-MDM2 pathway in HK2 cells following H/R. Nonetheless, the role of RPS7 in renal IRI remains unknown.\u003c/p\u003e\u003cp\u003eIn addition, PPI analysis predicted an interaction between SRP14 and RPS7. Notably, SRP14 and RPS7 were observed to colocalize in RTECs following H/R. Molecular docking and simulation analyses suggested a direct interaction between SRP14 and RPS7. Biolayer interferometry and co-immunoprecipitation followed by mass spectrometry analysis revealed a direct interaction between SRP14 and RPS7 in RTECs following H/R. Knockdown of \u003cem\u003eSrp14\u003c/em\u003e inhibited the expression of RPS7 in HK2 cells following H/R, whereas knockdown of \u003cem\u003eRps7\u003c/em\u003e did not significantly affect the expression of SRP14 in HK2 cells following H/R. Moreover, the presence of recombinant hRPS7 and shRNA-\u003cem\u003eSrp14\u003c/em\u003e resulted in no significant inhibition of apoptosis in the kidney tissues of mice with renal IRI. These results suggest that SRP14 triggers apoptosis in renal tubules upon renal IRI via a mechanism involving RPS7.\u003c/p\u003e\u003cp\u003eWhile the MDM2-p53 pathway is well-established in global pharmaceutical research and development, the complex mechanism of the MDM2-p53 feedback loop presents a significant challenge for developing small-molecule inhibitors. While p53 can activate the expression of \u003cem\u003eMDM2\u003c/em\u003e, MDM2 can inhibit p53 through its degradation, blocking its transcription, and facilitating its nuclear export (Meng et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Brummer et al, 2024). Ewa Langner \u003cem\u003eet al\u003c/em\u003e has observed that significant accumulation of p53 protein and caspase-mediated apoptosis in small murine kidneys with tubular dilations upon centrosome loss (Langner et al, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, it is necessary to explore novel therapeutic targets for the upstream mechanism of the MDM2-p53 feedback loop to prevent apoptosis. Given the important role of RPS7 in SRP14-regulated apoptosis involving the MDM2-p53 pathway, our study selected tretinoin, nafamostat mesilate, and benzbromarone from an apoptosis-specific compound library consisting of 356 FDA-approved compounds based on biolayer interferometry and CCK-8 assays. At 20 mg/kg/day, tretinoin and nafamostat mesilate attenuated renal tubule injury in mice with renal IRI. Routine histological measurements demonstrated a superior effect of nafamostat mesilate on murine renal tubules against renal IRI.\u003c/p\u003e\u003cp\u003eAs a synthetic broad-spectrum serine protease inhibitor, nafamostat mesylate is commonly used to treat intravascular coagulation during hemodialysis and pancreatitis (Iwashita et al, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Davenport et al, 2011; Sundaram et al, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Nafamostat mesylate can suppress the activation of the coagulation, contact, and complement systems during sepsis, thereby reducing injury to vital organs and improving prognosis (He et al, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Treatment with nafamostat mesylate attenuated apoptosis involving the p38 pathway in the spinal cord tissues of rats with spinal cord injury (Duan et al, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Xie et al. observed the potentially protective role of nafamostat mesylate against aristolochic acid-induced kidney injury in zebrafish involving protein glycosylation and amyloid aggregation (Xie et al, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Guo et al. reported that intraperitoneal injection of nafamostat mesylate at 1 mg/kg to rats attenuated rhabdomyolysis-induced AKI (Guo et al, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As they were unfortunately unaware of the pharmacokinetics and dosage-dependent effects of nafamostat mesylate, some biases exist in the conclusions of Guo et al., and the precise mechanism of nafamostat mesylate remains unknown.\u003c/p\u003e\u003cp\u003eOur study showed that nafamostat mesylate attenuates renal IRI by targeting the RPS7-associated apoptosis of RTECs. It significantly reduced the expression of RPS7 in the kidney tissues of mice with renal IRI. Biolayer interferometry revealed a stable interaction between nafamostat mesylate and RPS7, characterized by fast binding and slow dissociation. Molecular docking suggested an interaction between nafamostat mesylate and RPS7 involving residues Asn165, His168, Lys169, His91, Gly89, Ser88, Glu84, Val80, Gln76, Val77, Arg81, Pro136, and Phe135. Therefore, RPS7 may be a potential target for nafamostat mesylate to slow the progression of AKI.\u003c/p\u003e\u003cp\u003eHowever, our study had some limitations that should be addressed. Firstly, multicenter prospective cohort studies should be conducted to further elucidate the clinical features of SRP14 in AKI. Secondly, the tubule-specific \u003cem\u003eRps7\u003c/em\u003e transgenic mouse model may be used to further explore the precise mechanism of the p53-MDM2 feedback loop and the cross-talk of multiple targets from nafamostat mesylate in RTEC apoptosis caused by AKI. Thirdly, the residues involved in the interaction of nafamostat mesylate with RPS7 could be functionally identified, and comparative analyses could be performed to validate the specificity and sensitivity of nafamostat mesylate, which was originally developed as a serine protease inhibitor, against RPS7 compared to other known serine proteases.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings suggest that SRP14 triggers the apoptosis of RTECs, thereby exacerbating AKI, through an interaction with RPS7, which may be a potential target of nafamostat mesylate to slow the progression of AKI.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis section provides an overview of the materials and methods used in this study. For more detailed information and descriptions, please refer to the Supporting Information.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eMale C57BL/6 mice (6\u0026ndash;8 weeks old, 18\u0026ndash;22 g) were obtained from Chengdu Dossy Experimental Animals Co., Ltd (Chengdu, China). Male tubule-specific \u003cem\u003eSrp14\u003c/em\u003e knockout (\u003cem\u003eSrp14\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, \u003cem\u003eGgt\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e) mice were obtained from Cyagen Biosciences (Suzhou, China). All mice were housed in the Animal Center of Sichuan Provincial People\u0026rsquo;s Hospital under specific pathogen-free conditions with a 12/12-hour light/dark cycle, humidity of 40%\u0026ndash;70%, and ambient temperature of 18\u0026ndash;22 ℃. The mice had free access to food and water. All animal studies were approved by the Ethics Committee of Sichuan Provincial People\u0026rsquo;s Hospital (approval numbers 2018\u0026thinsp;\u0026minus;\u0026thinsp;176 and 2020\u0026thinsp;\u0026minus;\u0026thinsp;215).\u003c/p\u003e\u003cp\u003eTo establish an IRI-induced mouse model of AKI, mice were anesthetized with 50 mg/kg pentobarbital sodium (Merck, Germany) administered via intraperitoneal injection, and their bilateral renal arteries were clamped with artery clamps (RS-5420; Roboz, USA) at 37℃ for 45 minutes. The mice in the sham group underwent the same procedure but without clamping. To establish the sepsis-induced mouse model of AKI, mice underwent cecal ligation and puncture (CLP), as described by Rittirsch \u003cem\u003eet al\u003c/em\u003e (Rittirsch et al, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The mice in the sham group underwent the same procedure but without CLP. To establish the drug-induced mouse models of AKI, mice were administered 10 mg/kg of lipopolysaccharide (LPS; L2630; Sigma-Aldrich, USA) or 15 mg/kg of cisplatin (479306; Sigma-Aldrich, USA) via intraperitoneal injection. The mice in the sham group were injected with equal volumes of saline. The mice with renal IRI were orally administered nafamostat mesylate (S1386; Selleck, USA) at doses of 20, 25, and 50 mg/kg/day and tretinoin (S1653; Selleck, USA) at a dose of 20 mg/kg/day.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSubjects\u003c/h2\u003e\u003cp\u003eThis study enrolled patients admitted to the Nephrology Department of Sichuan Provincial People\u0026rsquo;s Hospital from June to December 2018. Those with a confirmed diagnosis of end-stage kidney disease (defined as an estimated glomerular filtration rate [eGFR] of \u0026lt;\u0026thinsp;15 mL/min/1.73 m\u003csup\u003e2\u003c/sup\u003e) at admission were excluded. Their demographic information was collected, including age and sex. Fast serum samples were collected 48 hours after admission, or earlier if necessary, and stored at \u0026minus;\u0026thinsp;20 ℃ until needed. Kidney biopsies were collected from patients with acute tubular necrosis (ATN); their clinical information is provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Normal renal tissue surrounding the tumor (ST) was collected from patients with renal carcinoma as the normal control. Written informed consents were obtained from patients before conducting any study procedure. All study procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of Sichuan Provincial People\u0026rsquo;s Hospital (approval numbers 2018\u0026thinsp;\u0026minus;\u0026thinsp;176 and 2018\u0026thinsp;\u0026minus;\u0026thinsp;284).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCell Culturing and Treatments\u003c/h2\u003e\u003cp\u003eHuman RTEC cell line HK2 was obtained from the National Collection of Authenticated Cell Cultures. The HK2 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/F-12 containing 10% fetal bovine serum. The cells were exposed to hypoxic conditions (0% oxygen, 95% nitrogen, and 5% carbon dioxide) using a cell hypoxia/hyperoxia workstation (MiniStation Plus-MPS230418047; Gene Science, Chongqing, China). For regeneration, the cells were cultured under normoxic conditions (21% oxygen, 74% nitrogen, and 5% carbon dioxide).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eRoutine Histology and Immunohistochemistry\u003c/h2\u003e\u003cp\u003eThe kidney tissues obtained from human patients and mice were fixed in paraffin and then sliced into 2 \u0026micro;m-thick sections. The renal sections underwent routine histological examinations: hematoxylin and eosin (HE) staining (SC231202; Baso, China) and periodic acid-Schiff (PAS) staining (SC241001; Baso, China). They were also immunohistochemically stained with antibodies against SRP14 (1:100; NBP2-94184; Novus, USA) and RPS7 (1:50; SC-100834; Santa Cruz Biotechnology, USA) at 4℃ overnight. After washing, the sections were then incubated with an appropriate horseradish peroxidase (HRP)-labeled secondary antibody (K5007; Dako Products, Denmark) at 37 ℃ for 1 hour.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMultiplex Immunofluorescence Staining and Multi-spectral Imaging\u003c/h2\u003e\u003cp\u003eThe 2 \u0026micro;m renal tissue paraffin sections from human and mice underwent multiplex immunofluorescence staining with Opal 4-Color Manual IHC Staining Kits (FP1487001KT, FP1488001KT, and FP1495001KT; Akoya Bioscience, USA) using primary antibodies against SRP14 (1:100; NBP2-94184; Novus, USA), RPS7 (1:50; SC-100834; Santa Cruz Biotechnology, USA), and hepatitis A virus cellular receptor 1 (HAVCR1/KIM-1; 1:400; NBP-43761; Novus, USA) were used for multiplex immunofluorescence staining. The sections were observed under a confocal microscope (LSM900; ZEISS, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eTdT-mediated dUTP Nick-End Labeling (TUNEL)\u003c/h2\u003e\u003cp\u003eMurine renal tissues were fixed in paraffin and then sliced into 2 \u0026micro;m-thick sections. Then, the renal tissue slices underwent in situ apoptosis assessments using the DeadEnd Fluorometric TUNEL System (G3250; Promega, USA). The production of fluorescein-12-dUTP-labeled DNA was observed under a confocal microscope (LSM900, ZEISS, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry\u003c/h2\u003e\u003cp\u003eBriefly, 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e HK2 cells were seeded into six-well plates and then transfected with SRP14 and RPS7 small interfering RNA. After exposure to H/R, the HK2 cells were collected and underwent flow cytometry using a flow cytometer (Becton Dickinson; for annexin A5 [ANXA5/annexin V]-fluorescein isothiocyanate [FITC]: excitation\u0026thinsp;=\u0026thinsp;633 nm, emission\u0026thinsp;=\u0026thinsp;660 nm; for propidium iodide [PI]: excitation\u0026thinsp;=\u0026thinsp;488 nm, emission\u0026thinsp;=\u0026thinsp;580 nm) by the Annexin V Alexa Fluor 488 \u0026amp; Propidium Iodide Cell Apoptosis Detection Kit (AD11; Dojindo Laboratories, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eBiolayer Interferometry\u003c/h2\u003e\u003cp\u003eWe synthesized and expressed recombinant SRP14 and RPS7 proteins using the pET21b (+) prokaryotic expression plasmid. The recombinant RPS7 protein was biotinylated using the G-MM-IGT Biotinylation Kit (Genemore, China) according to the manufacturer\u0026rsquo;s protocol. The SA sensor (ForteBio/Pall Life Sciences, Menlo Park, CA, USA) was loaded with biotinylated RPS7 for 600 seconds. Next, after being washed with phosphate-buffered saline (PBS), the sensor was dipped into recombinant SRP14 protein at a concentration of 88.8\u0026ndash;355 nM for 60 seconds for association, and then in PBS for 180 seconds for disassociation. The kinetics were recorded using the Octet K2 system (ForteBio/Pall Life Sciences, Menlo Park, CA, USA) at 1,000 rpm shaking and analyzed using the Octet Data Analysis HT 11.1 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eImmunoprecipitation and Immunoblotting\u003c/h2\u003e\u003cp\u003eTotal proteins were extracted from murine renal tissues and cells using a lysis buffer (P0013B; Beyotime, China) containing a protease inhibitor (ST506-2; Beyotime, China). For immunoprecipitation, 20 \u0026micro;L of A/G agarose was added to each sample and incubated for 1 hour at room temperature. The total cell lysates (500 \u0026micro;g) were immunoprecipitated with antibodies against RPS7 (SC-100834; Santa Cruz Biotechnology, USA) or SRP14 (SC-377012; Santa Cruz Biotechnology, USA) overnight at 4℃. Then, the immunoprecipitants were separated using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to western blot analysis using specific antibodies against SRP14 and RPS7.\u003c/p\u003e\u003cp\u003eFor western blot analysis, the proteins were denatured by adding 5 \u0026times; loading buffer and boiling at 100℃ for 5 minutes. Next, the proteins were separated by SDS-PAGE and electrotransferred to a polyvinylidene fluoride membrane (ISEQ00010; Merck Millipore, Germany). The membrane was then blocked with tris-buffered saline buffer containing 5% bovine serum albumin and 0.1% Tween-20 at room temperature for 1 hour. Next, the membrane was incubated with the primary antibodies at 4℃ overnight and then with the corresponding HRP-conjugated secondary antibodies at room temperature for 1 hour. Then, the protein bands were detected using actin beta (ACTB/β-actin; 1:1000; 6008; Proteintech, China) as the loading control. Primary antibodies against the following proteins were used: SRP14 (1:1000; NBP2-94184; Novus, USA), RPS7 (1:1000; PA5-77005; Invitrogen, USA), BCL2-associated X apoptosis regulator (BAX; 1:1000; ab182733; Abcam, UK), cleaved caspase 3 (CASP3; 1:1000; 9664s; Cell Signaling Technology), tumor protein p53 (TP53/p53; 1:1000; ab131442; Abcam, UK), and MDM2 proto-oncogene (MDM2; 1:1000; ab259265; Abcam, UK). The secondary antibodies included HRP-conjugated goat anti-rabbit (1:5000; 511203; ZenBio, China) and HRP-conjugated goat anti-mouse (1:5000; 511103; ZenBio, China). The signals were detected using the Immobilon Western Chemilum HRP Substrate (WBKLS-638173; Millipore/Merck, USA) and visualized and analyzed using a chemiluminescence image analysis system (5200; Tanon, Shanghai, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eContinuous variables are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) if normally distributed or the median (interquartile range [IQR]) if non-normally distributed. Variables were compared between the AKI and control groups using unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, the Mann\u0026ndash;Whitney U test, or the chi-square test. Correlation analyses were performed for variables that differed significantly between the AKI and control groups. SRP14 levels were compared among different AKI stages using one-way analysis of variance (ANOVA). A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were conducted using SPSS Statistics for Windows (version 29.0; IBM Corp., Armonk, NY, USA) and GraphPad Prism (version 8.0.1; GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eResource availability\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eLead contact\u003c/h2\u003e\u003cp\u003ePlease request further information about resources and reagents associated with this study from the lead contact, Yi Li ([email protected]).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eMaterials availability\u003c/h2\u003e\u003cp\u003eAll reagents in this study are available from the lead contact with a completed materials transfer agreement.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eData and code availability\u003c/h2\u003e\u003cp\u003eThe label free proteomic data has been uploaded to iProX database (Project ID: IPX0011230000). All other data are presented in manuscript or the supplemental complete materials and methods. Please request material sharing associated with this study from the corresponding author Yi Li ([email protected]).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like sincerely thank Prof. Zhenglin Yang of Sichuan Provincial People\u0026rsquo;s Hospital for generously providing the research platform for this study. We thank Weishen Wu for his guidance with bioinformatics analysis. We also thank the generously technical supporting from Zhiying Wang, Jie Chen, and Lin Wang of Sichuan Provincial People\u0026rsquo;s Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of this manuscript.\u0026nbsp;Yi Li designed the study, analyzed data, and prepared the manuscript. Min Wu, Li Wang and Guisen Li participated in consultation for study design and manuscript preparation. Yun Tang and Liming Huang conducted experiments, analyzed data, and prepared the manuscript. Yanmei Wang, Qiao Tang, Zehui Liao, Xueting Yang, Yangping Wu, Fang Wang, Yunlin Feng, Chanyu Geng, Sipei Chen, Qi Yao, Cihang Zhao, Jia Tang, Yilin Fu, Guoli Li, and Jun Gao conducted experiments and analyzed data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (82270729, U21A20349, 81700607, and 82470005), Projects from Department of Science and Technology of Sichuan Province (24NSFSC1735, 2023ZYD0170, and 2021YFS0370), Funds of Key Research and Development Grant of MOST (2023YFA095000), and Discipline construction foundation for Department of nephrology and institute from Sichuan Provincial People\u0026rsquo;s Hospital.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKellum JA, Romagnani P, Ashuntantang G, Ronco C, Zarbock A, Anders HJ. 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Structure and assembly of the Alu domain of the mammalian signal recognition particle. Nature. 408(6809), 167-173.\u003c/li\u003e\n\u003cli\u003eMaeda S, Sakai S, Takabatake Y, Yamamoto T, Minami S, Nakamura J, Namba-Hamano T, Takahashi A, Matsuda J, Yonishi H, et al. (2024). MondoA and AKI and AKI-to-CKD Transition. Journal of the American Society of Nephrology: JASN. 35(9), 1164-1182.\u003c/li\u003e\n\u003cli\u003eBrooks MA, Ravelli RB, McCarthy AA, Strub K, Cusack S. (2009). Structure of SRP14 from the Schizosaccharomyces pombe signal recognition particle. Acta crystallographica Section D, Biological crystallography. 65(Pt 5), 421-433.\u003c/li\u003e\n\u003cli\u003eMason N, Ciufo LF, Brown JD. (2000). Elongation arrest is a physiologically important function of signal recognition particle. The EMBO journal. 19(15), 4164-4174.\u003c/li\u003e\n\u003cli\u003eChen D, Zhang Z, Li M, Wang W, Li Y, Rayburn ER, Hill DL, Wang H, Zhang R. (2007). 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CNS neuroscience \u0026amp; therapeutics. 24(5), 429-438.\u003c/li\u003e\n\u003cli\u003eXie P, Liu H, Huo X, Chen J, Li Y, Huang Y, Yin Z. (2025). Nafamostat Mesylate Regulates Glycosylation to Alleviate Aristolochic Acid Induced Kidney Injury. Toxins (Basel). 17(3), 145.\u003c/li\u003e\n\u003cli\u003eGuo W, Wang Y, Wu Y, Liu J, Li Y, Wang J, Ou S, Wu W. (2022). Integration of transcriptomics and metabolomics reveals the molecular mechanisms underlying the effect of nafamostat mesylate on rhabdomyolysis-induced acute kidney injury. Frontiers in pharmacology. 13, 931670.\u003c/li\u003e\n\u003cli\u003eRittirsch D, Huber-Lang MS, Flierl MA, Ward PA. (2009). Immunodesign of experimental sepsis by cecal ligation and puncture. Nature protocols. 4(1), 31-36.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SRP14, RPS7, renal tubular epithelial cells, apoptosis, AKI","lastPublishedDoi":"10.21203/rs.3.rs-7892365/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7892365/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo explore potential targets for acute kidney injury (AKI), we exposed renal tubular epithelial cells (RTECs) to a hypoxia/reoxygenation environment and conducted labeling-free proteomics. This treatment significantly increased signal recognition particle 14 (SRP14) in apoptotic RTECs. SRP14 was elevated in the serum of patients with AKI. The SRP14 expression was increased in the renal tubules of patients with acute tubular necrosis, as well as in four AKI mouse models following the procedures of ischemia-reperfusion injury (IRI), cecal ligation and puncture, and treatment with lipopolysaccharide and cisplatin. SRP14 appears to play a crucial role in the apoptosis of RTECs, as evidenced by an IRI-induced AKI model in tubule-specific Srp14 knockout mice. Furthermore, SRP14 triggered apoptosis in renal tubules upon renal IRI via the ribosomal protein S7 (RPS7)-mediated tumor protein p53 (TP53)–MDM2 proto-oncogene (MDM2) pathway. We screened an apoptosis-specific library containing 356 US Food and Drug Administration–approved compounds to identify those that inhibit RPS7. We identified nafamostat mesilate as a potent RPS7 inhibitor that attenuated renal IRI by inhibiting RTEC apoptosis. These findings suggest that SRP14 triggers apoptosis in RTECs to exacerbating AKI through an interaction with RPS7, which may be a therapeutic target for nafamostat mesylate to alleviate AKI.\u003c/p\u003e","manuscriptTitle":"SRP14 Triggers Apoptosis in Renal Tubules to Exacerbate AKI Through an Interaction with RPS7","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 02:20:53","doi":"10.21203/rs.3.rs-7892365/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"541cca28-24e1-42c5-a22f-21ef7fe164d9","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57215564,"name":"Health sciences/Diseases/Kidney diseases/Acute kidney injury"},{"id":57215565,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2025-11-20T14:58:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-12 02:20:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7892365","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7892365","identity":"rs-7892365","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}