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

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.

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 Figure 1 and Figure 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 Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 , Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 , Figure 20 , Figure 21 , Figure 22 , Figure 23 , Figure 24 , Figure 25 and Figure 26 ). Since we have identified numerous molecular targets which were regulated by vancomycin in HK-2 cells, we predicted the modulating effects of vancomycin on cellular functions in HK-2 cells by functional analysis using IPA software. As shown in Table 5 , there were 500 categories of cellular functions which were altered by vancomycin in HK-2 cells. Treatment of vancomycin induced marked alterations in cell growth, cell proliferation, cell metabolism, cell morphology, gene expression, RNA post-translational modification, DNA replication and repair, cell-to-cell signaling and interaction, and cell death and survival. These cellular functional alterations are closely related to disease development. Notably, the functional analysis showed that vancomycin induced remarkable abnormalities in renal tissues and caused injury, probabaly resulting in renal tissue damage and cancer ( Table 6 ), with the involvement of molecular proteins of AHNAK, ANLN, BASP1, EDC4, NUMA1, PDXK, PNP, SDHAF2, SMARCA4, SNRNP200, and TUBA1C. Moreover, vancomycin induced the inflammation, degradation, and fibrosis of renal tubules involving PSAP and APRT. Taken together, the functional analysis of vancomycin suggests that vancomycin has a damaging effect on cellular functions and thus cause renal injuries. 492 molecular proteins regulated by vancomycin in HK-2 cells. 486 mapped molecular proteins by IPA. 290 canonical signaling pathways regulated by vancomycin in HK-2 cells. a The p value of the protein is computed by the permutation test. b The average ratio is for all peptides associated with the protein ( i.e. , log 2 L/H). c z -Score is the measure of how many standard deviation units that protein’s log 2 L/H ratio is away from its population mean. 24 networks of molecular signaling pathway regulated by vancomycin in HK-2 cells. 500 categories of predicted diseases and functions regulated by vancomycin in HK-2 cells. 187 categories of vancomycin-associated toxicity analyzed using IPA. Proteomic analysis revealed molecular interactome regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. There were 290 related pathways regulated by vancomycin in HK-2 cells. Red indicates an up-regulation; green indicates a down-regulation. Intergrated signaling pathways regulated by vancomycin in HK-2 cells. Vancomycin regulated RNA post-transcriptional modification, carbohydrate metabolism, and small molecule biochemistry. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated protein trafficking, cell death and survival, and nucleic acid metabolism. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated hematological disease, organismal injury and abnormalities, and RNA post-transcriptional modification. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated cellular assembly and organization, cell-to-cell signaling and interaction, and reproductive system development and function. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated infectious disease, small molecule biochemistry, and reproductive system disease. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated infectious disease, organismal injury and abnormalities, and immunological disease. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated protein synthesis, cellular movement, and gene expression. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated cellular growth and proliferation, infectious disease, and cellular development. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated molecular transport, protein trafficking, and RNA post-transcriptional modification. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated post-translational modification, protein folding, and drug metabolism. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated post-translational modification, protein folding, and cell morphology. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated cellular compromise, cellular assembly and organization, and cellular function and maintenance. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated cellular assembly and organization, cellular function and maintenance, and cell morphology. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated endocrine system development and function, lipid metabolism, and small molecule biochemistry. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated post-translational modification, protein folding, and dermatological diseases and conditions. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated DNA replication, recombination, and repair, cell morphology, and cellular function and maintenance. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated cellular movement, connective tissue disorders, and hematological disease. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and redcolors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated molecular transport, RNA trafficking, and cell death and survival. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated developmental disorder, hereditary disorder, and metabolic disease. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated cancer, endocrine system disorders, and organismal injury and abnormalities. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and redcolors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated RNA post-transcriptional modification, dermatological diseases and conditions, cell death and survival. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and redcolors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated carbohydrate metabolism, small molecule biochemistry, and cell morphology. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated cell-to-cell signaling and interaction, tissue development, and cardiovascular system development and function. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Vancomycin regulated connective tissue development and function, tissue morphology and behavior. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. 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. 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 reduced the expression level of CDK1, PRKDC, SFN, YWHAE, YWHAG, YWHAH, and YWHAZ by 1.1-, 1.3-, 1.6-, 1.2-, 1.1-, 1.6-, and 1.1-fold, respectively ( Figure 27 ). However, treatment of vancomycin induced the expression level of YWHAB and YWHAQ by 1.1- and 1.2-fold, respectively. Taken together, the results suggest that vancomycin affects the G 2 /M DNA damage check point signaling pathway in HK-2 cells. G 2 /M DNA damage check point signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Green indicates a down-regulation. The intensity of greencolor indicates the degree of down-regulation. Solid arrow indicates direct interaction. Apoptosis and autophagy are two predominant programmed cell death pathways, being promising targets for the treatment of cancer via regulating mitochondria-dependent or -independent or PI3K/Akt/mTOR-mediated signaling pathways. Furthermore, apoptosis and autophagy have been implicated in the pathogenesis of various diseases, including cancer [ 12 , 13 , 14 , 15 , 16 ]. In order to examine the effect of vancomycin on apoptosis and autophagy in HK-2 cells, the identified protein targets were subject to IPA pathway analysis. The results showed that vancomycin regulated apoptosis signaling pathway via the down-regulation of CAPN1, CDK1, and MAPK1 by 1.3-, 1.1-, and 1.7-fold, respectively ( Table 1 and Table 2 , and Figure 28 ). In addition, vancomycin regulated apoptotic signaling pathway via increasing the expression level of CAPN2, CAPNS1, LMNA, and SPTAN1 by 1.4-, 1.2-, 1.1-, and 1.21-fold, respectively. Moreover, Akt/mTOR signaling pathway plays a central role in the regulation of cell metabolism, growth, proliferation and survival through the integration of both intracellular and extracellular signals [ 17 ]. mTOR complexs 1 and 2 (mTORC1 and mTORC2) are two distinct complexes in mTOR signaling pathway transducting a variety of signals to downstream targets, including Akt, p70S6K, Atgs, eIF4G, PPAR-α, and PPAR-γ, to modulate cell growth, cell proliferation, energy metabolism, and autophagy [ 17 ]. Aberrant mTOR signaling pathway has been implicated in the pathogenesis of many diseases including cancer and targeting mTOR signaling pathway may be a promising strategy for cancer therapy [ 18 ]. As showed in Table 3 , vancomycin exhibited a capability of modulating PI3K/Akt/mTOR signaling pathway in HK-2 cells ( Figure 29 and Figure 30 ). The results showed that vancomycin decreased the expression level of a number of key proteins, such as HSP90B1, ITGB1, MAPK1, PPP2R1A, SFN, YWHAE, YWHAG, YWHAH, and YWHAZ by 1.1-, 1.2-, 1.7-, 1.2-, 1.6-, 1.2-, 1.1-, 1.6-, and 1.1-fod, respectively; but increased the expression level of EIF3C, EIF3M, EIF4A1, EIF4G1, FKBP1A, PPP2CA, RPS2, RPS3, RPS8, RPS11, RPS15, RPS24, RPS27A, RPS3A, RPS4X, and RPSA. Moreover, treatment of HK-2 cells with vancomycin induced mitochondrial dysfunction with alterations in the expression level of a number of proteins, including ATP5A1, ATP5B, ATP5H, CYB5R3, CYC1, HSD17B10, PARK7, PRDX5, SDHA, UQCRC1, VDAC1, and VDAC2 ( Figure 31 ). Taken together, the results suggest that the regulatory effects of vancomycin on apoptosis signaling pathway, PI3K/Akt/mTOR signaling pathway, and mitochondrial dysfunction contribute to the toxicity of vancomycin in HK-2 cells. Apoptosis signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and redcolors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. PI3K/Akt signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. mTOR signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Mitochondrial dysfunction was induced by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/ml vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. 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 ), tight junction signaling pathway ( Figure 33 ), and remodeling of epithelial adherent junction signaling pathway ( Figure 34 ) in HK-2 cells involving a number of functional proteins, including ACTA1, ACTG1, ACTN1, ACTN4, ACTR2, ACTR3, ARPC2, ARPC1B, CTNNA1, DNM1L, IQGAP1, MYH9, MAPRE1, MYH10, PPP2CA, PPP2R1A, RAB7A, RHOA, TUBA1B, TUBB, TUBB6, TUBB8, TUBB4B, VCL, ZYX, SPTAN1, VAPA, VCL, and YBX3. Taken together, the proteomic data show that vancomycin markedly induces EMT in HK-2. Epithelial adherent junction signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Tight junction signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction. Remodeling of epithelial adherent junction signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction. 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. 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. UPR signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Green indicates a down-regulation. The intensity of green color indicates the degree of down-regulation. Solid arrow indicates direct interaction. ER stress signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Green indicates a down-regulation. The intensity of green color indicates the degree of down-regulation. Solid arrow indicates direct interaction. ERK-MAPK signaling pathway was regulated by vancomycin in HK-2 cells. HK-2 cells were treated with 50 μg/mL vancomycin for 24 h and the protein samples were subject to quantitative proteomic analysis. Red indicates an up-regulation; green indicates a down-regulation. The intensity of green and red colors indicates the degree of down- or up-regulation. Solid arrow indicates direct interaction and dashed arrow indicates indirect interaction.

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.

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 assay kit, and Western blotting substrate were obtained from Thermo Fisher Scientific Inc. (Hudson, NH, USA). The human proximal tubule epithelial cell line HK-2 was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in regular DMEM/F12 (1:1) medium supplemented with 10% heat-inactivated FBS at 37 °C in a 5% CO 2 /95% air humidified incubator. Cells were seeded into the plates for 24 h to achieve a confluence of ~80% prior to vancomycin treatment. Vancomycin was dissolved in DMSO with a stock concentration of 50 mM, and was freshly diluted to the pre-determined concentrations with culture medium with 0.05% ( v / v ) final concentration of DMSO. For proteomic analysis, HK-2 cells were cultured in DMEM/F12 medium (for SILAC) with (heavy) or without (light) stable isotope labeled amino acids ( 13 C 6 - l -lysine and 13 C 6 15 N 4 - l -arginine) and 10% dialyzed FBS. HK-2 cells cultured in SILAC medium for six cell doubling times to achieve a high level (>98%) of labeled amino acid incorporation. Then the cells grown in “light” media were treated with 0.5% DMSO for 24 h to serve as the negative control; cells grown in “heavy” media were treated with pre-determined vancomycin for 24 h. All the experiments were performed three times independently. After vancomycin treatment, HK-2 cell samples were harvested, lysated with hot lysis buffer (100 mM Tris base, 4% SDS, and 100 mM dithiothreitol), and denatured for 5 min at 95 °C. Then, the samples were stored at −80 °C till further analysis. Prior to the quantitative proteomic analysis, the protein samples were subject to digestion and desalting which were performed using SILAC-based approach as described previously [ 8 , 10 , 30 ]. Briefly, the thermal denatured cell lysate was subject to a brief sonication at 20% AMPL for 3 s with 6 pulses. The sonicated samples were centrifuged at 15,000× g for 20 min at room temperature and the supernatant was collected. The protein concentration was determined using IDCR. Subsequently, equal amount of heavy and light protein sample was combined to reach a total volume of 30–60 μL containing 300–600 μg protein. The combined protein sample was digested using FASP™ protein digestion kit. A quota of 40 μL of combined lysate was added to a spin column prewetted with 8 M urea. The lysate buffer was exchanged to 8 M urea using centrifugation prior to iodoacetamide alkylation and then exchanged to 50 mM ammonium bicarbonate for trypsin digestion at a ratio of 1:100 ( w / w ). Digestion was carried out overnight in a humidified incubator at 37 °C and terminated with the addition of 5% formic acid. After the digestion, the resultant sample was acidified to pH of 3 and desalted using a C 18 solid-phase extraction column. Samples were loaded and washed in 0.1% formic acid and eluted using 90% acetonitrile/10% formic acid. Samples were concentrated in a vacuum concentrator at 45°C for 120 min and resuspended in 0.1% formic acid prior to LC-MS/MS analysis. The concentrated samples (5 μL) were subject to the hybrid linear ion trap-Orbitrap (LTQ Orbitrap XL, Thermo Scientific Inc.). LC-MS/MS was performed using a 10 cm-long 75 μm (inner diameter) reversed-phase column packed with 5 μm-diameter C 18 material with 300 Å pore size (New Objective, Woburn, MA, USA) with a gradient mobile phase of 2%–40% acetonitrile in 0.1% formic acid at 200 μL/min for 125 min. The Orbitrap full MS scanning was performed at a mass ( m / z ) resolving power of 60,000, with positive polarity in profile mode [M + H] + . Raw data files were submitted for simultaneous search using standard SILAC settings for LTQ Orbitrap XL instruments. Proteins for which at least two fully trypsin-digested L and H peptides were detected at >99% confidence were used for subsequent comparative quantitative analysis. Peptide SILAC ratio was calculated using MaxQuant version 1.2.0.13. The SILAC ratio was determined by averaging all peptide SILAC ratios from peptides identified of the same protein. Differential regulation within each experimental data set was determined by normalization of each data set. Briefly, the L/H ratio was converted into log 2 space 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. 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 ). 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.

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.