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A SILAC-Based Approach Elicits the Proteomic Responses to Vancomycin-Associated Nephrotoxicity in Human Proximal Tubule Epithelial HK-2 Cells

Vancomycin, a widely used antibiotic, often induces nephrotoxicity, however, the molecular targets and underlying mechanisms of this side effect remain unclear. The present study aimed to examine molecular interactome and analyze the signaling pathways related to the vancomycin-induced nephrotoxicity in human proximal tubule epithelial HK-2 cells using the stable isotope labeling by amino acids in cell culture (SILAC) approach. The quantitative proteomic study revealed that there were at least 492 proteins interacting with vancomycin and there were 290 signaling pathways and cellular functions potentially regulated by vancomycin in HK-2 cells. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, EMT, and ROS generation. These findings suggest that vancomycin-induced proteomic responses in HK-2 cells involvefunctional proteins and pathways that regulate cell cycle, apoptosis, autophagy, and redox homeostasis. This is the first systemic study revealed the networks of signaling pathways and proteomic responses to vancomycin treatment in HK-2 cells, and the data may be used to discriminate the molecular and clinical subtypes and to identify new targets and biomarkers for vancomycin-induced nephrotoxic effect. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for drug-induced renal toxicity.


Introduction
Neonatal sepsis is common and is a major cause of morbidity and mortality [1]. Vancomycin is the preferred treatment for several neonatal staphylococcal infections. It remains the primary antibiotic treatment for multi-resistant Gram-positive infections, such as methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium [2]. Vancomycin pharmacokinetic estimates, which are different in neonates compared with adults, also exhibit extensive inter-neonatal variability [3]. In neonates, several vancomycin dosing schedules have been proposed, mainly based on age (i.e., postmenstrual and postnatal), body weight, or serum creatinine level. Although vancomycin has historically been linked to various toxicities, in particular nephrotoxicity, it was largely attributed to drug impurities in early formulations [4,5]. The incidence of such toxicities was drastically reduced after refinement of purification methods and the risk of nephrotoxicity was considered relatively low at less than 5% [5][6][7].
However, the molecular targets for vancomycin-associated nephrotoxicity are unclear. There is a lack of study which reveals the global targets of vancomycin with regard to its renal toxicity, although the characterization and identification of individual targets and related signaling pathways have provided important evidence for the mechanism of actions of vancomycin in vitro and in vivo. Stable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach to uncover the global proteomic responses to drug treatment and other interventions [8,9]. In particular, it can be used to systemically and quantitatively assess the target network of drugs, evaluate drug toxicity, and identify new biomarkers for the diagnosis and treatment of importance diseases such as cancer and Alzheimer's disease [8,10,11].
In this regard, in order to uncover the comprehensive and global understanding on the effect of vancomycin, we investigated the molecular targets of vancomycin in human proximal tubule epithelial HK-2 cells with a focus on cell cycle, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) pathways.

Overview of Proteomic Response to Vancomycin Treatment in HK-2 Cells
First, we performed a SILAC-based proteomic study to quantitatively determine the interactome of vancomycin in HK-2 cells treated with vancomycin at 50 µg/mL. There were 492 protein molecules identified as the potential targets of vancomycin in HK-2 cells (Table 1). These included a number of molecules involved in cell proliferation, cell metabolism, cell migration, cell invasion, cell survival, and cell death. Vancomycin increased the expression level of 178 protein molecules, but decreased the expression level of 314 protein molecules in HK-2 cells. Subsequently, these proteins were subject to IPA pathway analysis. A total of 486 molecular proteins were mapped using IPA (Table 2). Furthermore, as shown in and Table 3 and Figures 1 and 2 there were 290 signaling pathways and cellular functions that were potentially regulated by vancomycin in HK-2 cells. Additionally, there were 24 networks of molecular signaling pathways that were regulated in HK-2 cells when treated with vancomycin (Table 4 and Figures 3-26).

Vancomycin Induces Toxicity in HK-2 Cells
After we observed the effect of vancomycin on cellular functions and disease development, we also analysed the toxic effect of vancomycin in HK-2 cells. As shown in Table 6, there were 187 different toxic effects of vancomycin which were predicted in HK-2 cells. The proteomic results showed that treatment of vancomycin induced renal, heart, and liver toxicity, with cell necrosis and cell death. Moreover, toxic function analysis showed that there were 500 different diseases and functions which were regulated by vancomycin in HK-2 cells (Table 5). Notably, the analysis showed that vancomycin primarily induced renal toxicity, to a lesser extent, liver and heart toxicity. Vancomycin-induced renal toxicity included renal degradation, renal inflammation, renal nephritis, renal atrophy, renal hydronephrosis, kidney failure, renal hypoplasia, glomerular injury, renal fibrosis, renal necrosis/cell death, renal damage, and renal tubule injury. These are all observed in clinical studies.
Following the analysis of the effect of vancomycin on the cellular function and toxicity in HK-2 cells, we analyzed the effect of vancomycin on cellular signaling pathways, including the G 2 /M DNA damage check point signaling pathway, apoptosis signaling pathway, autophagy signaling pathway, endoplasmic reticulum (ER) stress signaling pathway, unfolded protein response (URP) signaling pathway, ERK-MAPK signaling pathway, and tight junction signaling pathway.

Vancomycin Regulates G 2 /M DNA Damage Check Point Signaling Pathway in HK-2 Cells
The proteomic results showed that treatment of HK-2 cells with 50 µg/mL vancomycin regulated G 2 /M DNA damage check point signaling pathway (Table 3)

Vancomycin Regulates EMT Pathways in HK-2 cells
EMT has a close association with cell migration and invasion and it plays an important role in fibrosis and cancer development [19]. Suppressing the progress of EMT may represent a useful approach for the treratment of fibrosis and cancer. We analyzed the effect of vancomycin on EMT-related proteins and signaling pathways using SILAC-based proteomic approach. The proteomic data showed that vancomycin regulated the epithelial adherent junction signaling pathway (Figure 32

Vancomycin Regulates ER Stress Pathways in HK-2 cells
The unfolded protein response (UPR) is a cellular stress response related to the ER [20]. It is a stress response that has been found to be conserved between all mammalian species, as well as yeast and worm organisms [21]. The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum [20,21]. The proteomic data showed that vancomycin regulated UPR signaling pathway ( Figure 35) and ER stress (Figure 36) in HK-2 cells. Incubation of HK-2 cells with vancomycin altered the expression level of several key proteins involved in UPR signaling pathway, including CALR, CANX, DNAJA2, HSP90B1, HSPA4, HSPA5, HSPA8, HSPA9, P4HB, UBXN4, and VCP. Furthermore, vancomycin altered the expression level of CALR, EIF2S1, HSP90B1, and HSPA5. Collectively, the regulatory effect of vancomycin on UPR and ER stress may contribute to vancomycin-associated nephropathy.

Vancomycin Regulates ERK-MAPK Signaling Pathway in HK-2 Cells
Additionally, treatment of HK-2 cells with vancomycin induced the ERK-MAPK signaling response ( Figure 37). There were a number of important proteins which were regulated by vancomycin in HK-2 cells. There was an increase in the expression level of H3F3A/H3F3B, PPP2CA, YWHAB, and YWHAQ, whereas there was a reduction in the expression level of HSPB1, ITGB1, MAPK1, PPP1CA, PPP2R1A, STAT1, TLN1, YWHAG, YWHAH, and YWHAZ. Taken together, the modulating effect of vancomycin on ERK-MAPK signaling pathway may contribute, at least in part, to vancomycin-induced nephrotoxicity.

Discussion
In the present study, we evaluated the global proteomic responses to vancomycin treatment with regard to cell cycle, programmed cell death, EMT and related molecular targets and signaling pathways in HK-2 cellsusing SILAC-based quantitative proteomic approach. The quantitative proteomic study showed that a large number of important proteins regulating cell proliferation, growth, cell death, and migration in HK-2 cells, with the involvement of a number of function proteins, such as CDK1, CDK2, E-cadherin, PI3K, Akt, mTOR, cytochrome c, caspase 9, caspase 3, Bcl-2, Bax, p53, PPAR, HSP, Erk1/2, Ras, and Rho.
This proteomic study also showed that vancomycin regulated mitochondrial function and cell death. Mitochondrial disruption and the subsequent release of cytochrome c initiate the process of apoptosis, with the latter being initiated by pro-apoptotic members of the Bcl-2 family but antagonized by anti-apoptotic members of this family [22,23]. Anti-apoptotic members of Bcl-2 can be inhibited by post-translational modification and/or by increased expression of PUMA, which is an essential regulator of p53-mediated cell apoptosis [24]. In addition, cytochrome c released from mitochondria can activate caspase 9, which then activates caspase 3 and caspase 7 [25]. In our study, we observed that vancomycin regulated expression level of a number of proteins, such as CAPN1, CDK1, and MAPK1. Furthermore, vancomycin regulated apoptotic signaling pathway via increasing the expression level of CAPN2, CAPNS1, LMNA, and SPTAN1 in HK-2 cells.
Furthermore, the proteomic results show that vancomycin exhibits a modulating effect on PI3K/Akt/mTOR signaling pathway. Under optimal growth conditions, activated mTORC1 inhibits autophagy by direct phosphorylation of Atg13 and ULK1 at Ser757 [26][27][28]. This phosphorylation inhibits ULK1 kinase activity and subsequent autophagosome formation. When the kinase activity of mTORC1 is suppressed, the autophagic machinery is initiated. In the present study, vancomycin regulated autophagy in HK-2 cells as indicated by the alteration in the expression of HSP90B1, ITGB1, MAPK1, PPP2R1A, SFN, YWHAE, YWHAG, YWHAH, and YWHAZ. Taken together, the autophagy-modulating effect of vancomycin may contribute to its nephrotoxic effect via the regulation of PI3k/Akt/mTOR signaling pathway.
EMT is characterized as epithelial cells to lose their polarization and specialized junction structures, undergoing cytoskeleton reorganization and acquiring morphological and functional features of mesenchymal-like cells [19,29]. In our proteomic study, we observed marked regulatory effects of vancomycin on the expression of a number of functional proteins that modulate epithelial adherent junction signaling pathway in HK-2 cells. Again, the SILAC-based quantitative proteomic analysis can discriminate the role of EMT modulation in vancomycin-associated nephrotoxicity.
Our data have provided new insights into the molecular mechanisms of vancomycin-induced nephrotoxicity that is often observed in clinical practice. Our data are consistent with previously observed biochemical changes at cellular levels induced by vancomycin in renal epithelial cells. Our data may help identify new targets that are useful for discovery of new therapeutic approaches for vancomycin-induced nephrotoxicity. Further functional studies are warranted to validate our proteomic data.

Chemicals and Reagents
Vancomycin, 13 C 6 -L-lysine, L-lysine, 13 C 6 15 N 4 -L-arginine, L-arginine, Dulbecco's phosphate buffered saline (PBS), heat inactivate fetal bovine serum (FBS), and dialyzed FBS were purchased from Sigma-Aldrich (St. Louis, MO, USA). DEME/F12 medium was bought from Invitrogen Inc. (Carlsbad, CA, USA). FASP™ protein digestion kit was purchased from Protein Discovery Inc. (Knoxville, TN, USA). The proteomic quantitation kits for acidification, desalting, and digestion, Ionic Detergent Compatibility Reagent (IDCR), DMEM/F12 medium for SILAC, Pierce bicinchoninic acid protein to determine geometric means and facilitate normalization. The average log 2 L/H ratios and standard deviation of the log 2 L/H ratios were determined for each data set, both before and after computational removal of the few significant outliers found in a few data sets. Every protein's log 2 L/H ratio was then converted into a z-score that is the measure of how many standard deviation units (expressed as "σ") that protein's log 2 L/H ratio is away from its population mean. Therefore, a protein with a z-score of >1.645σ indicates that that protein's differential expression lies outside the 90% confidence level, >1.960σ indicates that it is outside the 95% confidence level, 2.576σ indicates 99% confidence, and 3.291σ indicates 99.9% confidence. z-Scores of >1.960 were considered significant.

Pathway Analysis and Bioinformatics
The protein IDs were identified using Scaffold 4.3.2 from Proteome Software Inc. (Portland, OR, USA) and the pathway was analyzed using Ingenuity Pathway Analysis (IPA) from QIAGEN (Redwood City, CA, USA). Geometric means for total protein expression ratios across biological samples were calculated respective to intensity. Geometric means and Uniprot Protein identification numbers were uploaded to Ingenuity Pathway Analysis (IPA) to determine localization, molecular function, and protein interactions. Upstream regulator analysis was also performed within IPA where activity of potential upstream regulators is predicted based on the expression profile of known down-stream targets in relation to known upstream regulatormediated expression changes reported in the literature. This analysis determines the significance of overlap of the detected targets with the upstream regulator through a Fisher's exact test in addition to implementation of a z-score algorithm to make prediction of the direction of upstream regulator activity change. Description of the z-score algorithm is available on the IPA Web site (www.ingenuity.com).

Statistical Analysis
Data are expressed as the mean˘standard deviation (SD). Multiple comparisons were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison. A value of p < 0.05 was considered statistically significant.

Conclusions
In summary, the quantitative SILAC-based proteomic approach showed that vancomycin regulated cell proliferation, mitochondria-dependent apoptotic pathway and autophagy, and EMT in HK-2 cells, involving a number of key functional proteins and related molecular signaling pathways. This study may provide a clue to fully identify the molecular targets and elucidate the underlying mechanism of vancomycin-associated nephrotoxicity, resulting in an improved therapeutic effect and reduced side effect in clinical settings.