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Klemens CA, Fedoriuk M, Semenikhina M, Stefanenko M, Zietara A, Levchenko V, Dissanayake LV, Palygin O, Staruschenko A. Electrolyte and metabolite composition of cystic fluid from a rat model of ARPKD. Commun Biol 2025; 8:230. [PMID: 39948436 PMCID: PMC11825955 DOI: 10.1038/s42003-025-07631-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 04/25/2024] [Accepted: 01/29/2025] [Indexed: 02/16/2025] Open
Abstract
Fluid-filled cysts are the key feature of polycystic kidney disease, which eventually leads to renal failure. We analyzed the composition of cyst fluid from a rat model of autosomal recessive polycystic kidney disease, the PCK rat, and identified sexual differences. Our results demonstrate that the ion composition of cyst fluid differs from that of urine or plasma. Untargeted metabolomics combined with transcriptomic data identified tryptophan metabolism, enzyme metabolism, steroid hormone biosynthesis, and fatty acid metabolism as pathways differing between male and female PCK rats. We quantified 42 amino acids in the cyst fluid (PCK only), plasma, and urine of male and female PCK rats and Sprague Dawley rats. Taurine was the most concentrated amino acid present in the cyst fluid, and PCK rat urinary taurine excretion was over 3-fold greater than Sprague Dawley rats. Understanding the composition of cyst fluid provides valuable insights into disease pathophysiology and may help identify potential dietary or pharmacological interventions to mitigate disease progression and improve patient outcomes.
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Affiliation(s)
- Christine A Klemens
- Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA.
- Hypertension and Kidney Research Center, University of South Florida, Tampa, FL, USA.
| | - Mykhailo Fedoriuk
- Department of Medicine, Medical University of South Carolina, Charleston, SC, USA
| | | | - Mariia Stefanenko
- Department of Medicine, Medical University of South Carolina, Charleston, SC, USA
| | - Adrian Zietara
- Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
| | - Vladislav Levchenko
- Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
| | - Lashodya V Dissanayake
- Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
| | - Oleg Palygin
- Department of Medicine, Medical University of South Carolina, Charleston, SC, USA
| | - Alexander Staruschenko
- Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA.
- Hypertension and Kidney Research Center, University of South Florida, Tampa, FL, USA.
- James A. Haley Veterans' Hospital, Tampa, FL, USA.
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Cheng T, Mariappan A, Langner E, Shim K, Gopalakrishnan J, Mahjoub MR. Inhibiting centrosome clustering reduces cystogenesis and improves kidney function in autosomal dominant polycystic kidney disease. JCI Insight 2024; 9:e172047. [PMID: 38385746 DOI: 10.1172/jci.insight.172047] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 05/08/2023] [Accepted: 01/17/2024] [Indexed: 02/23/2024] Open
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering," a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.
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Affiliation(s)
- Tao Cheng
- Department of Medicine, Nephrology Division, Washington University School of Medicine, St. Louis, Missouri, USA
| | - Aruljothi Mariappan
- Institute of Human Genetics, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany
| | - Ewa Langner
- Department of Medicine, Nephrology Division, Washington University School of Medicine, St. Louis, Missouri, USA
| | - Kyuhwan Shim
- Department of Medicine, Nephrology Division, Washington University School of Medicine, St. Louis, Missouri, USA
| | - Jay Gopalakrishnan
- Institute of Human Genetics, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany
- Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, Jena, Jena, Germany
| | - Moe R Mahjoub
- Department of Medicine, Nephrology Division, Washington University School of Medicine, St. Louis, Missouri, USA
- Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA
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Wang F, Lee SY, Adelnia F, Takahashi K, Harkins KD, He L, Zu Z, Ellinger P, Grundmann M, Harris RC, Takahashi T, Gore JC. Severity of polycystic kidney disease revealed by multiparametric MRI. Magn Reson Med 2023; 90:1151-1165. [PMID: 37093746 PMCID: PMC10805116 DOI: 10.1002/mrm.29679] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 12/22/2022] [Revised: 03/03/2023] [Accepted: 04/03/2023] [Indexed: 04/25/2023]
Abstract
PURPOSE We aimed to compare multiple MRI parameters, including relaxation rates (R 1 $$ {R}_1 $$ ,R 2 $$ {R}_2 $$ , andR 1 ρ $$ {R}_{1\rho } $$ ), ADC from diffusion weighted imaging, pool size ratio (PSR) from quantitative magnetization transfer, and measures of exchange from spin-lock imaging (S ρ $$ {S}_{\rho } $$ ), for assessing and predicting the severity of polycystic kidney disease (PKD) over time. METHODS Pcy/Pcy mice with CD1 strain, a mouse model of autosomal dominant PKD, were imaged at 5, 9, and 26 wk of age using a 7T MRI system. Twelve-week normal CD1 mice were used as controls. Post-mortem paraffin tissue sections were stained using hematoxylin and eosin and picrosirius red to identify histological changes. RESULTS Histology detected segmental cyst formation in the early stage (week 5) and progression of PKD over time in Pcy kidneys. InT 2 $$ {T}_2 $$ -weighted images, small cysts appeared locally in cystic kidneys in week 5 and gradually extended to the whole cortex and outer stripe of outer medulla region from week 5 to week 26. Regional PSR,R 1 $$ {R}_1 $$ ,R 2 $$ {R}_2 $$ , andR 1 ρ $$ {R}_{1\rho } $$ decreased consistently over time compared to normal kidneys, with significant changes detected in week 5. Among all the MRI measures,R 2 $$ {R}_2 $$ andR 1 ρ $$ {R}_{1\rho } $$ allow highest detectability to PKD, while PSR andR 1 $$ {R}_1 $$ have highest correlation with pathological indices of PKD. Using optimum MRI parameters as regressors, multiple linear regression provides reliable prediction of PKD progression. CONCLUSION R 2 $$ {R}_2 $$ ,R 1 $$ {R}_1 $$ , and PSR are sensitive indicators of the presence of PKD. Multiparametric MRI allows a comprehensive analysis of renal changes caused by cyst formation and expansion.
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Affiliation(s)
- Feng Wang
- Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center
- Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center
- Vanderbilt O’Brien Kidney Research Center, Vanderbilt University Medical Center
| | - Seo Yeon Lee
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center
| | - Fatemeh Adelnia
- Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center
| | - Keiko Takahashi
- Vanderbilt O’Brien Kidney Research Center, Vanderbilt University Medical Center
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center
| | - Kevin D. Harkins
- Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center
- Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center
- Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232
| | - Lilly He
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center
| | - Zhongliang Zu
- Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center
- Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center
| | - Philipp Ellinger
- Bayer AG Research & Development, Pharmaceuticals, 42113 Wuppertal, Germany
| | - Manuel Grundmann
- Bayer AG Research & Development, Pharmaceuticals, 42113 Wuppertal, Germany
| | - Raymond C. Harris
- Vanderbilt O’Brien Kidney Research Center, Vanderbilt University Medical Center
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center
| | - Takamune Takahashi
- Vanderbilt O’Brien Kidney Research Center, Vanderbilt University Medical Center
- Division of Nephrology and Hypertension, Vanderbilt University Medical Center
| | - John C. Gore
- Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center
- Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center
- Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232
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Liu A, Luo P, Huang H. New insight of complement system in the process of vascular calcification. J Cell Mol Med 2023; 27:1168-1178. [PMID: 37002701 PMCID: PMC10148053 DOI: 10.1111/jcmm.17732] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 12/22/2022] [Revised: 03/10/2023] [Accepted: 03/14/2023] [Indexed: 04/03/2023] Open
Abstract
The complement system defences against pathogenic microbes and modulates immune homeostasis by interacting with the innate and adaptive immune systems. Dysregulation, impairment or inadvertent activation of complement system contributes to the pathogenesis of some autoimmune diseases and cardiovascular diseases (CVD). Vascular calcification is the pivotal pathological basis of CVD, and contributes to the high morbidity and mortality of CVD. Increasing evidences indicate that the complement system plays a key role in chronic kidney diseases, atherosclerosis, diabetes mellitus and aging-related diseases, which are closely related with vascular calcification. However, the effect of complement system on vascular calcification is still unclear. In this review, we summarize current evidences about the activation of complement system in vascular calcification. We also describe the complex network of complement system and vascular smooth muscle cells osteogenic transdifferentiation, systemic inflammation, endoplasmic reticulum stress, extracellular matrix remodelling, oxidative stress, apoptosis in vascular calcification. Hence, providing a better understanding of the potential relationship between complement system and vascular calcification, so as to provide a direction for slowing the progression of this burgeoning health concern.
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Affiliation(s)
- Aiting Liu
- Department of Cardiology, The Eighth Affiliated Hospital, Joint Laboratory of Guangdong‐Hong Kong‐Macao Universities for Nutritional Metabolism and Precise Prevention and Control of Major Chronic Diseases Sun Yat‐sen University Shenzhen China
| | - Pei Luo
- State Key Laboratory for Quality Research in Chinese Medicines Macau University of Science and Technology Macau China
| | - Hui Huang
- Department of Cardiology, The Eighth Affiliated Hospital, Joint Laboratory of Guangdong‐Hong Kong‐Macao Universities for Nutritional Metabolism and Precise Prevention and Control of Major Chronic Diseases Sun Yat‐sen University Shenzhen China
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Liu J, Yin X, Dev H, Luo X, Blumenfeld JD, Rennert H, Prince MR. Pleural Effusions on MRI in Autosomal Dominant Polycystic Kidney Disease. J Clin Med 2023; 12:386. [PMID: 36615184 PMCID: PMC9820892 DOI: 10.3390/jcm12010386] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 11/24/2022] [Revised: 12/23/2022] [Accepted: 12/29/2022] [Indexed: 01/05/2023] Open
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) has cystic fluid accumulations in the kidneys, liver, pancreas, arachnoid spaces as well as non-cystic fluid accumulations including pericardial effusions, dural ectasia and free fluid in the male pelvis. Here, we investigate the possible association of ADPKD with pleural effusion. ADPKD subjects (n = 268) and age-gender matched controls without ADPKD (n = 268) undergoing body magnetic resonance imaging from mid-thorax down into the pelvis were independently evaluated for pleural effusion by 3 blinded expert observers. Subjects with conditions associated with pleural effusion were excluded from both populations. Clinical and laboratory data as well as kidney, liver and spleen volume, pleural fluid volume, free pelvic fluid and polycystic kidney disease genotype were evaluated. Pleural effusions were observed in 56 of 268 (21%) ADPKD subjects compared with 21 of 268 (8%) in controls (p < 0.0001). In a subpopulation controlling for renal function by matching estimated glomerular filtration rate (eGFR), 28 of 110 (25%) ADPKD subjects had pleural effusions compared to 5 of 110 (5%) controls (p < 0.001). Pleural effusions in ADPKD subjects were more prevalent in females (37/141; 26%) than males (19/127,15%; p = 0.02) and in males were weakly correlated with the presence of free pelvic fluid (r = 0.24, p = 0.02). ADPKD subjects with pleural effusions were younger (48 ± 14 years old vs. 43 ± 14 years old) and weighed less (77 vs. 70 kg; p ≤ 0.02) than those without pleural effusions. For ADPKD subjects with pleural effusions, the mean volume of fluid layering dependently in the posterior−inferior thorax was 19 mL and was not considered to be clinically significant. Pleural effusion is associated with ADPKD, but its role in the pathogenesis of ADPKD requires further evaluation.
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Affiliation(s)
- Jin Liu
- Department of Radiology, Weill Cornell Medicine, New York, NY 10065, USA
- Department of Cardiothoracic Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China
| | - Xiaorui Yin
- Department of Radiology, Weill Cornell Medicine, New York, NY 10065, USA
| | - Hreedi Dev
- Department of Radiology, Weill Cornell Medicine, New York, NY 10065, USA
| | - Xianfu Luo
- Department of Radiology, Weill Cornell Medicine, New York, NY 10065, USA
| | - Jon D. Blumenfeld
- Department of Medicine, Weill Cornell Medicine, New York, NY 10065, USA
- The Rogosin Institute, New York, NY 10065, USA
| | - Hanna Rennert
- Department of Pathology, Weill Cornell Medicine, New York, NY 10065, USA
| | - Martin R. Prince
- Department of Radiology, Weill Cornell Medicine, New York, NY 10065, USA
- Columbia College of Physicians and Surgeons, New York, NY 10027, USA
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JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging. Proc Natl Acad Sci U S A 2021; 118:2007194118. [PMID: 33798093 PMCID: PMC8000530 DOI: 10.1073/pnas.2007194118] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/18/2022] Open
Abstract
JNK signaling has been studied intensively in models of liver physiology and disease, but previous studies had focused on young mice. However, it had not been recognized that JNK plays a fundamental role in maintaining liver homeostasis and preventing the formation of biliary cysts in aging mice. These observations call for caution in all long-term pharmacological inhibition strategies targeting the JNK pathway. Finally, our results provide evidence of a molecular link between JNK and the cell-death mediator RIPK1. The specific overexpression of RIPK1 in cysts of a subset of patients with polycystic liver disease suggests that RIPK1 might be mechanistically involved in the pathogenesis of human biliary cysts. The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.
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The endothelin system as target for therapeutic interventions in cardiovascular and renal disease. Clin Chim Acta 2020; 506:92-106. [DOI: 10.1016/j.cca.2020.03.008] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 01/13/2020] [Revised: 03/05/2020] [Accepted: 03/05/2020] [Indexed: 12/12/2022]
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Proteomic Analysis of Urinary Extracellular Vesicles Reveals a Role for the Complement System in Medullary Sponge Kidney Disease. Int J Mol Sci 2019; 20:ijms20215517. [PMID: 31694344 PMCID: PMC6862015 DOI: 10.3390/ijms20215517] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 09/12/2019] [Revised: 10/15/2019] [Accepted: 11/04/2019] [Indexed: 12/15/2022] Open
Abstract
Medullary sponge kidney (MSK) disease is a rare and neglected kidney condition often associated with nephrocalcinosis/nephrolithiasis and cystic anomalies in the precalyceal ducts. Little is known about the pathogenesis of this disease, so we addressed the knowledge gap using a proteomics approach. The protein content of microvesicles/exosomes isolated from urine of 15 MSK and 15 idiopathic calcium nephrolithiasis (ICN) patients was investigated by mass spectrometry, followed by weighted gene co-expression network analysis, support vector machine (SVM) learning, and partial least squares discriminant analysis (PLS-DA) to select the most discriminative proteins. Proteomic data were verified by ELISA. We identified 2998 proteins in total, 1764 (58.9%) of which were present in both vesicle types in both diseases. Among the MSK samples, only 65 (2.2%) and 137 (4.6%) proteins were exclusively found in the microvesicles and exosomes, respectively. Similarly, among the ICN samples, only 75 (2.5%) and 94 (3.1%) proteins were exclusively found in the microvesicles and exosomes, respectively. SVM learning and PLS-DA revealed a core panel of 20 proteins that distinguished extracellular vesicles representing each clinical condition with an accuracy of 100%. Among them, three exosome proteins involved in the lectin complement pathway maximized the discrimination between MSK and ICN: Ficolin 1, Mannan-binding lectin serine protease 2, and Complement component 4-binding protein β. ELISA confirmed the proteomic results. Our data show that the complement pathway is involved in the MSK, revealing a new range of potential therapeutic targets and early diagnostic biomarkers.
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Kenter AT, van Rossum-Fikkert SE, Salih M, Verhagen PCMS, van Leenders GJLH, Demmers JAA, Jansen G, Gribnau J, Zietse R, Hoorn EJ. Identifying cystogenic paracrine signaling molecules in cyst fluid of patients with polycystic kidney disease. Am J Physiol Renal Physiol 2018; 316:F204-F213. [PMID: 30403162 DOI: 10.1152/ajprenal.00470.2018] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 12/16/2022] Open
Abstract
In autosomal dominant polycystic kidney disease (ADPKD) paracrine signaling molecules in cyst fluid can induce proliferation and cystogenesis of neighboring renal epithelial cells. However, the identity of this cyst-inducing factor is still unknown. The aim of this study was to identify paracrine signaling proteins in cyst fluid using a 3D in vitro cystogenesis assay. We collected cyst fluid from 15 ADPKD patients who underwent kidney or liver resection (55 cysts from 13 nephrectomies, 5 cysts from 2 liver resections). For each sample, the ability to induce proliferation and cyst formation was tested using the cystogenesis assay (RPTEC/TERT1 cells in Matrigel with cyst fluid added for 14 days). Kidney cyst fluid induced proliferation and cyst growth of renal epithelial cells in a dose-dependent fashion. Liver cyst fluid also induced cystogenesis. Using size exclusion chromatography, 56 cyst fluid fractions were obtained of which only the fractions between 30 and 100 kDa showed cystogenic potential. Mass spectrometry analysis of samples that tested positive or negative in the assay identified 43 candidate cystogenic proteins. Gene ontology analysis showed an enrichment for proteins classified as enzymes, immunity proteins, receptors, and signaling proteins. A number of these proteins have previously been implicated in ADPKD, including secreted frizzled-related protein 4, S100A8, osteopontin, and cysteine rich with EGF-like domains 1. In conclusion, both kidney and liver cyst fluids contain paracrine signaling molecules that drive cyst formation. Using size exclusion chromatography and mass spectrometry, we procured a candidate list for future studies. Ultimately, cystogenic paracrine signaling molecules may be targeted to abrogate cystogenesis in ADPKD.
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Affiliation(s)
- Annegien T Kenter
- Department of Internal Medicine, Division of Nephrology and Transplantation, Erasmus Medical Center, Rotterdam , The Netherlands.,Department of Cell Biology, Erasmus Medical Center, Rotterdam , The Netherlands.,Department of Developmental Biology, Erasmus Medical Center, Rotterdam , The Netherlands
| | | | - Mahdi Salih
- Department of Internal Medicine, Division of Nephrology and Transplantation, Erasmus Medical Center, Rotterdam , The Netherlands
| | | | | | | | - Gert Jansen
- Department of Cell Biology, Erasmus Medical Center, Rotterdam , The Netherlands
| | - Joost Gribnau
- Department of Developmental Biology, Erasmus Medical Center, Rotterdam , The Netherlands
| | - Robert Zietse
- Department of Internal Medicine, Division of Nephrology and Transplantation, Erasmus Medical Center, Rotterdam , The Netherlands
| | - Ewout J Hoorn
- Department of Internal Medicine, Division of Nephrology and Transplantation, Erasmus Medical Center, Rotterdam , The Netherlands
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Ai S, Zheng J, Qiu CX, Lu XL, Li XW. Urinary proteomics analysis based on mass spectrometry and identification of therapeutic targets of Shenkangling interventions in rats with adriamycin nephropathy using iTRAQ. Am J Transl Res 2018; 10:2115-2125. [PMID: 30093948 PMCID: PMC6079121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 06/02/2017] [Accepted: 06/01/2018] [Indexed: 06/08/2023]
Abstract
OBJECTIVE The isobaric tags for relative and absolute quantification (iTRAQ) technique for proteomic analysis was employed to identify diagnostic markers and therapeutic targets of Shenkangling intervention or prednisone tablets in rats with adriamycin nephropathy (AN). METHODS Fifty healthy, clean-grade Sprague-Dawley rats were selected, with 10 rats in the normal group and the remaining 40 rats receiving a tail vein injection of 5.5 mg/kg of adriamycin (ADR) to induce AN. Treatment began 1 week later. The normal group received gastric administration of normal saline. Forty rats with induced AN were further randomly divided into the AN modeling group (n = 10), AN modeling + prednisone treatment group (n = 10), AN modeling + Shenkangling intervention group (n = 10), and AN modeling + prednisone + Shenkangling intervention group (n = 10). iTRAQ was employed in combination with mass spectrometry to analyze the differentially expressed proteins in the urine after 3 weeks of treatment (in the fourth week of the experiment). RESULTS Compared with normal rats, AN rats had 6 down-regulated proteins and 1 upregulated protein. Compared with AN rats, prednisone rats had 2 down-regulated and 6 upregulated proteins. Compared with AN rats, combined treatment rats had 2 down-regulated and 8 upregulated proteins. Compared with the AN model group, the Shenkangling treatment group had 3 down-regulated and 9 upregulated proteins. Gro, Afamin, Cystatin-related protein 2, Afamin, and isoform CRA_a were considered diagnostic markers of primary nephrotic syndrome (PNS). Telomerase was considered the therapeutic target of prednisone. Urinary protein 2, Apolipoprotein A-II, 45 kDa calcium-binding protein, Vitronectin, and Osteopontin were the therapeutic targets of the Shenkangling intervention. Afamin, isoform CRA_a, Apolipoprotein A-IV, Coagulation factor XII, Prolactin-induced protein, and Coagulation factor XII were the therapeutic targets of the Shenkangling intervention combined with prednisone. CONCLUSION The feasibility of urinary proteomics analysis in rats using a large number of proteins with finite molecular weights is controversial. The markers screened in this study may be of clinical value for the diagnosis and treatment of nephropathy. However, these findings should be confirmed in future cohort studies.
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Affiliation(s)
- Si Ai
- The Affiliated People’s Hospital of Fujian University of Traditional Chinese MedicineFuzhou 350004, Fujian, China
- Key Laboratory of Integrative Kidney Disease in Fujian ProvinceFuzhou 350004, Fujian, China
| | - Jian Zheng
- The Affiliated People’s Hospital of Fujian University of Traditional Chinese MedicineFuzhou 350004, Fujian, China
- Fujian University of Traditional Chinese MedicineFuzhou 350122, Fujian, China
- Key Laboratory of Integrative Kidney Disease in Fujian ProvinceFuzhou 350004, Fujian, China
| | - Cai-Xia Qiu
- The Affiliated People’s Hospital of Fujian University of Traditional Chinese MedicineFuzhou 350004, Fujian, China
| | - Xiao-Lu Lu
- The Affiliated People’s Hospital of Fujian University of Traditional Chinese MedicineFuzhou 350004, Fujian, China
| | - Xu-Wei Li
- The Affiliated People’s Hospital of Fujian University of Traditional Chinese MedicineFuzhou 350004, Fujian, China
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Lai X, Umbricht CB, Fisher K, Bishop J, Shi Q, Chen S. Identification of novel biomarker and therapeutic target candidates for diagnosis and treatment of follicular carcinoma. J Proteomics 2017; 166:59-67. [DOI: 10.1016/j.jprot.2017.07.003] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 04/22/2017] [Revised: 06/23/2017] [Accepted: 07/04/2017] [Indexed: 12/19/2022]
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Caplan MJ. The tail of polycystin-1 pays the kidney a complement. Am J Physiol Renal Physiol 2016; 310:F1180-1. [DOI: 10.1152/ajprenal.00141.2016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/22/2022] Open
Affiliation(s)
- Michael J. Caplan
- Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
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13
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Salih M, Demmers JA, Bezstarosti K, Leonhard WN, Losekoot M, van Kooten C, Gansevoort RT, Peters DJM, Zietse R, Hoorn EJ. Proteomics of Urinary Vesicles Links Plakins and Complement to Polycystic Kidney Disease. J Am Soc Nephrol 2016; 27:3079-3092. [PMID: 26940098 DOI: 10.1681/asn.2015090994] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 09/08/2015] [Accepted: 01/19/2015] [Indexed: 12/21/2022] Open
Abstract
Novel therapies in autosomal dominant polycystic kidney disease (ADPKD) signal the need for markers of disease progression or response to therapy. This study aimed to identify disease-associated proteins in urinary extracellular vesicles (uEVs), which include exosomes, in patients with ADPKD. We performed quantitative proteomics on uEVs from healthy controls and patients with ADPKD using a labeled approach and then used a label-free approach with uEVs of different subjects (healthy controls versus patients with ADPKD versus patients with non-ADPKD CKD). In both experiments, 30 proteins were consistently more abundant (by two-fold or greater) in ADPKD-uEVs than in healthy- and CKD-uEVs. Of these proteins, we selected periplakin, envoplakin, villin-1, and complement C3 and C9 for confirmation because they were also significantly overrepresented in pathway analysis and were previously implicated in ADPKD pathogenesis. Immunoblotting confirmed higher abundances of the selected proteins in uEVs from three independent groups of patients with ADPKD. Whereas uEVs of young patients with ADPKD and preserved kidney function already had higher levels of complement, only uEVs of patients with advanced stages of ADPKD had increased levels of villin-1, periplakin, and envoplakin. Furthermore, all five proteins correlated positively with total kidney volume. Analysis in kidney tissue from mice with kidney-specific, tamoxifen-inducible Pkd1 deletion demonstrated higher expression in more severe stages of the disease and correlation with kidney weight for each protein of interest. In summary, proteomic analysis of uEVs identified plakins and complement as disease-associated proteins in ADPKD. These proteins are new candidates for evaluation as biomarkers or targets for therapy in ADPKD.
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Affiliation(s)
- Mahdi Salih
- Department of Internal Medicine, Division of Nephrology & Transplantation, and
| | - Jeroen A Demmers
- Proteomics Center, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Karel Bezstarosti
- Proteomics Center, Erasmus Medical Center, Rotterdam, The Netherlands
| | | | | | - Cees van Kooten
- Nephrology, Leiden University Medical Center, Leiden, The Netherlands; and
| | - Ron T Gansevoort
- Department of Nephrology, University Medical Center Groningen, Groningen, The Netherlands
| | | | - Robert Zietse
- Department of Internal Medicine, Division of Nephrology & Transplantation, and
| | - Ewout J Hoorn
- Department of Internal Medicine, Division of Nephrology & Transplantation, and
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14
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Waning DL, Mohammad KS, Reiken S, Xie W, Andersson DC, John S, Chiechi A, Wright LE, Umanskaya A, Niewolna M, Trivedi T, Charkhzarrin S, Khatiwada P, Wronska A, Haynes A, Benassi MS, Witzmann FA, Zhen G, Wang X, Cao X, Roodman GD, Marks AR, Guise TA. Excess TGF-β mediates muscle weakness associated with bone metastases in mice. Nat Med 2015; 21:1262-1271. [PMID: 26457758 PMCID: PMC4636436 DOI: 10.1038/nm.3961] [Citation(s) in RCA: 277] [Impact Index Per Article: 27.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 04/03/2015] [Accepted: 09/02/2015] [Indexed: 12/12/2022]
Abstract
Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases-representing breast, lung and prostate cancers, as well as multiple myeloma-exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-β, released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca(2+)) release channel (RyR1). The oxidized RyR1 channels leaked Ca(2+), resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-β signaling, TGF-β release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-β activity. Thus, pathological TGF-β release from bone contributes to muscle weakness by decreasing Ca(2+)-induced muscle force production.
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Affiliation(s)
- David L Waning
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Khalid S Mohammad
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Steven Reiken
- Department of Physiology and Cellular Biophysics, Helen and Clyde Wu Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Wenjun Xie
- Department of Physiology and Cellular Biophysics, Helen and Clyde Wu Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Daniel C Andersson
- Department of Physiology and Cellular Biophysics, Helen and Clyde Wu Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Sutha John
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Antonella Chiechi
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Laura E Wright
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Alisa Umanskaya
- Department of Physiology and Cellular Biophysics, Helen and Clyde Wu Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Maria Niewolna
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Trupti Trivedi
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Sahba Charkhzarrin
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Pooja Khatiwada
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Anetta Wronska
- Department of Physiology and Cellular Biophysics, Helen and Clyde Wu Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Ashley Haynes
- Department of Physiology and Cellular Biophysics, Helen and Clyde Wu Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Maria Serena Benassi
- Laboratory of Experimental Oncology, Instituto Ortopedico Rizzoli, Bologna, Italy
| | - Frank A Witzmann
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Gehua Zhen
- Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Xiao Wang
- Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Xu Cao
- Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - G David Roodman
- Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, Indiana, USA.,Richard L. Roudebush VA Medical Center, Indianapolis, Indiana, USA
| | - Andrew R Marks
- Department of Physiology and Cellular Biophysics, Helen and Clyde Wu Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Theresa A Guise
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA
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15
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Distinctive and pervasive alterations in aqueous humor protein composition following different types of glaucoma surgery. Mol Vis 2015; 21:911-8. [PMID: 26321865 PMCID: PMC4548793] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 04/27/2015] [Accepted: 08/22/2015] [Indexed: 11/29/2022] Open
Abstract
PURPOSE To investigate whether specific glaucoma surgeries are associated with differences in aqueous humor protein concentrations compared to eyes without filters. METHODS In this cross-sectional study, aqueous humor samples were prospectively collected from control subjects who underwent routine cataract surgery (n=14) and from patients who had different glaucoma filters: Baerveldt aqueous shunt (n=6), Ahmed aqueous shunt (n=6), trabeculectomy (n=5), and Ex-Press trabeculectomy (n=3). Total protein concentrations were determined with Bradford assay. Tryptic digests were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and were quantitatively compared with a label-free platform. Relative protein quantities were compared across groups with ANOVA. Post hoc pair-wise comparisons were adjusted for multiple comparisons. RESULTS Compared to the control eyes, the aqueous humor protein concentration was increased approximately tenfold in the Ahmed and Baerveldt eyes and fivefold in the trabeculectomy and Ex-Press eyes. Overall, 718 unique proteins, splice variants, or isoforms were identified. No differences in the protein concentrations were detected between the Baerveldt and Ahmed groups. Likewise, the trabeculectomy and Ex-Press groups were remarkably similar. Therefore, the aqueous shunt groups were pooled, and the trabeculectomy groups were pooled for a three-way comparison with the controls. More than 500 proteins differed significantly in relative abundance (ANOVA p<0.01) among the control, aqueous shunt, and trabeculectomy groups. Functional analyses suggested these alterations in relative protein abundance affected dozens of signaling pathways. CONCLUSIONS Different glaucoma surgical procedures were associated with marked increases in the aqueous humor protein concentration and distinctive changes in the relative abundance of numerous proteins involved in multiple signaling pathways.
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16
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Rithidech KN, Tungjai M, Jangiam W, Honikel L, Gordon C, Lai X, Witzmann F. Proteomic Profiling of Hematopoietic Stem/Progenitor Cells after a Whole Body Exposure of CBA/CaJ Mice to Titanium ( 48Ti) Ions. Proteomes 2015; 3:132-159. [PMID: 28248266 PMCID: PMC5217378 DOI: 10.3390/proteomes3030132] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 04/18/2015] [Revised: 07/06/2015] [Accepted: 07/09/2015] [Indexed: 12/31/2022] Open
Abstract
Myeloid leukemia (ML) is one of the major health concerns from exposure to radiation. However, the risk assessment for developing ML after exposure to space radiation remains uncertain. To reduce the uncertainty in risk prediction for ML, a much increased understanding of space radiation-induced changes in the target cells, i.e., hematopoietic stem/progenitor cells (HSPCs), is critically important. We used the label-free quantitative mass spectrometry (LFQMS) proteomic approach to determine the expression of protein in HSPC-derived myeloid colonies obtained at an early time-point (one week) and a late time-point (six months) after an acute whole body exposure of CBA/CaJ mice to a total dose of 0, 0.1, 0.25, or 0.5 Gy of heavy-ion titanium (48Ti ions), which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48Ti ions served as non-irradiated sham controls. There were five mice per treatment groups at each harvest time. The Trans-Proteomic Pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A proof-of-concept based Ingenuity Pathway Analysis (IPA) was used to characterize the functions, pathways, and networks of the identified proteins. Alterations of expression levels of proteins detected in samples collected at one week (wk) post-irradiation reflects acute effects of exposure to 48Ti ions, while those detected in samples collected at six months (mos) post-irradiation represent protein expression profiles involved in the induction of late-occurring damage (normally referred to as genomic instability). Our results obtained by using the IPA analyses indicate a wide array of signaling pathways involved in response to 1 GeV/n 48Ti ions at both harvest times. Our data also demonstrate that the patterns of protein expression profiles are dose and time dependent. The majority of proteins with altered expression levels are involved in cell cycle control, cellular growth and proliferation, cell death and survival, cell-to-cell signaling and interaction. The IPA analyses indicate several important processes involved in responses to exposure to 48Ti ions. These include the proteosme/ubiquination, protein synthesis, post-translation modification, and lipid metabolism. The IPA analyses also indicate that exposure to 1 GeV/n 48Ti ions affects the development and function of hematological system, immune cell trafficking, including the cytoskeleton. Further, the IPA analyses strongly demonstrate that the NF-κB and MAPKs (ERKs, JNKs, and p38MAPK) pathways play an essential role in signal transduction after exposure to 1 GeV/n 48Ti ions. At an early time-point (1 week), the top networks identified by the IPA analyses are related to metabolic disease, lipid metabolism, small molecule biochemistry, and development disorder. In contrast, the top networks identified in samples collected at a late time-point (6 mos post-irradiation) by the IPA analyses are related to cancer, hematological disorders, and immunological diseases. In summary, the proteomic findings from our study provide a foundation to uncover compounds potentially be highly effective in radiation countermeasures.
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Affiliation(s)
| | - Montree Tungjai
- Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA.
- Department of Radiologic Technology, Faculty of Associated Medical Sciences, Center of Excellence for Molecular Imaging, Chiang Mai University, Chiang Mai 50200, Thailand.
| | - Witawat Jangiam
- Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA.
- Department of Chemical Engineering, Burapha University, Chonburi 20131, Thailand.
| | - Louise Honikel
- Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA.
| | - Chris Gordon
- Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA.
| | - Xianyin Lai
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Room 0044, Indianapolis, IN 46202, USA.
| | - Frank Witzmann
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, Room 362A, Indianapolis, IN 46202, USA.
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17
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Lai X, Chen S. Identification of novel biomarker candidates for immunohistochemical diagnosis to distinguish low-grade chondrosarcoma from enchondroma. Proteomics 2015; 15:2358-68. [DOI: 10.1002/pmic.201400528] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 11/07/2014] [Revised: 02/04/2015] [Accepted: 03/03/2015] [Indexed: 01/14/2023]
Affiliation(s)
- Xianyin Lai
- Department of Biochemistry and Molecular Biology; Indiana University School of Medicine; Indianapolis IN USA
- Department of Cellular & Integrative Physiology; Indiana University School of Medicine; Indianapolis IN USA
| | - Shaoxiong Chen
- Department of Pathology and Laboratory Medicine; Indiana University School of Medicine; Indianapolis IN USA
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18
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Piazzon N, Bernet F, Guihard L, Leonhard WN, Urfer S, Firsov D, Chehade H, Vogt B, Piergiovanni S, Peters DJM, Bonny O, Constam DB. Urine Fetuin-A is a biomarker of autosomal dominant polycystic kidney disease progression. J Transl Med 2015; 13:103. [PMID: 25888842 PMCID: PMC4416261 DOI: 10.1186/s12967-015-0463-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 09/04/2014] [Accepted: 03/13/2015] [Indexed: 01/08/2023] Open
Abstract
Background Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by numerous fluid-filled cysts that frequently result in end-stage renal disease. While promising treatment options are in advanced clinical development, early diagnosis and follow-up remain a major challenge. We therefore evaluated the diagnostic value of Fetuin-A as a new biomarker of ADPKD in human urine. Results We found that renal Fetuin-A levels are upregulated in both Pkd1 and Bicc1 mouse models of ADPKD. Measurement by ELISA revealed that urinary Fetuin-A levels were significantly higher in 66 ADPKD patients (17.5 ± 12.5 μg/mmol creatinine) compared to 17 healthy volunteers (8.5 ± 3.8 μg/mmol creatinine) or 50 control patients with renal diseases of other causes (6.2 ± 2.9 μg/mmol creatinine). Receiver operating characteristics (ROC) analysis of urinary Fetuin-A levels for ADPKD rendered an optimum cut-off value of 12.2 μg/mmol creatinine, corresponding to 94% of sensitivity and 60% of specificity (area under the curve 0.74 ; p = 0.0019). Furthermore, urinary Fetuin-A levels in ADPKD patients correlated with the degree of renal insufficiency and showed a significant increase in patients with preserved renal function followed for two years. Conclusions Our findings establish urinary Fetuin-A as a sensitive biomarker of the progression of ADPKD. Further studies are required to examine the pathogenic mechanisms of elevated renal and urinary Fetuin-A in ADPKD. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0463-7) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Nathalie Piazzon
- Ecole Polytechnique Fédérale de Lausanne (EPFL), Bâtiment SV ISREC, Station 19, Lausanne, Switzerland. .,Department of Pharmacology and Toxicology, University of Lausanne (UNIL), Quartier UNIL-CHUV, Lausanne, Switzerland.
| | - Florian Bernet
- Ecole Polytechnique Fédérale de Lausanne (EPFL), Bâtiment SV ISREC, Station 19, Lausanne, Switzerland.
| | - Linda Guihard
- Service of Nephrology, Lausanne University Hospital (CHUV), Lausanne, Switzerland.
| | - Wouter N Leonhard
- Department of Human Genetics, Leiden Univ. Medical Center, Leiden, The Netherlands.
| | - Séverine Urfer
- Ecole Polytechnique Fédérale de Lausanne (EPFL), Bâtiment SV ISREC, Station 19, Lausanne, Switzerland.
| | - Dmitri Firsov
- Department of Pharmacology and Toxicology, University of Lausanne (UNIL), Quartier UNIL-CHUV, Lausanne, Switzerland.
| | - Hassib Chehade
- Department of Pediatrics, Division of Pediatric Nephrology, Lausanne University Hospital (CHUV), Lausanne, Switzerland.
| | - Bruno Vogt
- Department of Nephrology and Hypertension, Inselspital, Bern, Switzerland.
| | - Sophia Piergiovanni
- Department of Pharmacology and Toxicology, University of Lausanne (UNIL), Quartier UNIL-CHUV, Lausanne, Switzerland.
| | - Dorien J M Peters
- Department of Human Genetics, Leiden Univ. Medical Center, Leiden, The Netherlands.
| | - Olivier Bonny
- Department of Pharmacology and Toxicology, University of Lausanne (UNIL), Quartier UNIL-CHUV, Lausanne, Switzerland. .,Service of Nephrology, Lausanne University Hospital (CHUV), Lausanne, Switzerland.
| | - Daniel B Constam
- Ecole Polytechnique Fédérale de Lausanne (EPFL), Bâtiment SV ISREC, Station 19, Lausanne, Switzerland.
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19
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Fearn A, Sheerin NS. Complement activation in progressive renal disease. World J Nephrol 2015; 4:31-40. [PMID: 25664245 PMCID: PMC4317626 DOI: 10.5527/wjn.v4.i1.31] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Academic Contribution Register] [Received: 09/02/2014] [Revised: 11/14/2014] [Accepted: 12/10/2014] [Indexed: 02/06/2023] Open
Abstract
Chronic kidney disease (CKD) is common and the cause of significant morbidity and mortality. The replacement of functioning nephrons by fibrosis is characteristic of progressive disease. The pathways that lead to fibrosis are not fully understood, although chronic non-resolving inflammation in the kidney is likely to drive the fibrotic response that occurs. In patients with progressive CKD there is histological evidence of inflammation in the interstitium and strategies that reduce inflammation reduce renal injury in pre-clinical models of CKD. The complement system is an integral part of the innate immune system but also augments adaptive immune responses. Complement activation is known to occur in many diverse renal diseases, including glomerulonephritis, thrombotic microangiopathies and transplant rejection. In this review we discuss current evidence that complement activation contributes to progression of CKD, how complement could cause renal inflammation and whether complement inhibition would slow progression of renal disease.
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20
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Menezes LF, Germino GG. Systems biology of polycystic kidney disease: a critical review. WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE 2015; 7:39-52. [PMID: 25641951 DOI: 10.1002/wsbm.1289] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Academic Contribution Register] [Received: 10/02/2014] [Revised: 12/11/2014] [Accepted: 12/15/2014] [Indexed: 01/02/2023]
Abstract
The proliferation and diminishing costs of 'omics' approaches have finally opened the doors for small and medium laboratories to enter the 'systems biology era'. This is a welcome evolution that requires a new framework to design, interpret, and validate studies. Here, we highlight some of the challenges, contributions, and prospects of the 'cyst-ems biology' of polycystic kidney disease.
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Affiliation(s)
- Luis Fernando Menezes
- Kidney Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
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21
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Lai X, Liangpunsakul S, Li K, Witzmann FA. Proteomic profiling of human sera for discovery of potential biomarkers to monitor abstinence from alcohol abuse. Electrophoresis 2015; 36:556-63. [PMID: 25475211 DOI: 10.1002/elps.201400319] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 07/03/2014] [Revised: 08/19/2014] [Accepted: 11/20/2014] [Indexed: 11/09/2022]
Abstract
Although numerous biomarkers or biomarker candidates have been discovered to detect levels of drinking and intervals of time after last drinking episode, only a few biomarkers have been applied to monitor abstinence in a longer interval (≥6 wks) from alcohol abuse. Considering sample sources, sensitivity, and specificity, new biomarkers from blood with better accuracy are needed. To address this, serum proteomic profiles were compared between pre- and post- treatment samples from subjects seeking treatment for alcohol abuse and dependence in an intensive 6 wk daily outpatient program using high-abundance plasma protein immunodepletion and LC-MS/MS techniques. Protein identification, quantification, candidate biomarker selection, and prioritization analyses were carried out. Among the 246 quantified serum proteins, abundance of 13 and 45 proteins in female and male subjects were significantly changed (p ≤ 0.05), respectively. Of these biomarker candidate proteins, 2 (female) and 8 (male) proteins were listed in category 1, with high area under the receiver operating characteristic curve, sensitivity, specificity, and fold change. In summary, several new biomarker candidates have been identified to monitor abstinence from alcohol abuse.
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Affiliation(s)
- Xianyin Lai
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, IN, USA; Department of Cellular & Integrative Physiology, Indiana University School of Medicine, IN, USA
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22
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Su Z, Wüthrich RP, Mei C. Response to letter from the editor 'Complement C3 activation in cyst fluid and urine from autosomal dominant polycystic kidney disease patients'. J Intern Med 2014; 276:541-2. [PMID: 25205431 DOI: 10.1111/joim.12306] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Academic Contribution Register] [Indexed: 11/29/2022]
Affiliation(s)
- Z Su
- Department of Nephrology, Shanghai Changzheng Hospital, Kidney Institute, Second Military Medical University, Shanghai, China
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23
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Su Z, Wang X, Gao X, Liu Y, Pan C, Hu H, Beyer RP, Shi M, Zhou J, Zhang J, Serra AL, Wüthrich RP, Mei C. Excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease. J Intern Med 2014; 276:470-85. [PMID: 24494798 DOI: 10.1111/joim.12214] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Academic Contribution Register] [Indexed: 12/16/2022]
Abstract
OBJECTIVES The complement system is involved in many immune complex-mediated kidney diseases, yet its role in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) has not been examined in detail. METHODS AND RESULTS Screening of the glycoproteome of urine samples from ADPKD patients revealed that levels of complement factor B (CFB), serpin peptidase inhibitor, complement component 1 inhibitor (SERPING1) and complement component 9 (C9) increased, whereas complement component 1, r subcomponent-like (C1RL), CD55 and CD59 levels decreased with disease progression. Immunostaining and Western blot analysis confirmed the enhanced expression of CFB and C9 in cystic kidneys from ADPKD patients. Immunostaining also showed that the expressions of CFB and C9 in renal biopsy tissues from patients with other types of chronic kidney disease were lower than in tissues from ADPKD patients. The effect of the complement inhibitor rosmarinic acid (RMA) was evaluated in Pkd1(-/-) mice and Han:SPRD Cy/+ rats. Compared with vehicle-treated Pkd1(-/-) animals, RMA-treated mice had significantly lower serum creatinine (-50%) and blood urea nitrogen (-78%) levels, two kidneys/body weight ratio (-60%) and renal cystic index (-60%). Similar results were found in Cy/+ rats. Lower numbers of Ki67-positive nuclei and inflammatory cells and reduced fibrosis were observed in both animal models upon treatment with RMA. CONCLUSIONS These results suggest that excessive activation of the alternative complement pathway is associated with ADPKD progression, probably mediated by cyst-lining epithelial cell proliferation, tubulointerstitial inflammatory cell infiltration and fibrosis. Targeting the complement system might represent a new therapeutic strategy for ADPKD.
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Affiliation(s)
- Z Su
- Kidney Institute, Department of Nephrology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China
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Li P, Lai X, Witzmann FA, Blazer-Yost BL. Bioinformatic Analysis of Differential Protein Expression in Calu-3 Cells Exposed to Carbon Nanotubes. Proteomes 2013; 1:219-239. [PMID: 25177543 PMCID: PMC4148817 DOI: 10.3390/proteomes1030219] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/17/2022] Open
Abstract
Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 hexposure to 10 μg/mL and 100 ng/mLof two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformaticanalysis of proteins identified in LFQMS. Interestingly, after exposure to ahigh concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins in common to both nanotubes occurred within the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The majority of the protein changes represent a decrease in amount suggesting a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure.
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Affiliation(s)
- Pin Li
- Department of Biology, Indiana University Purdue University, 723 West Michigan Street, Indianapolis, IN 46202, USA; E-Mail:
| | - Xianyin Lai
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 1345 West 16th Street, Indianapolis, IN 46202, USA; E-Mails: (X.L.); (F.A.W.)
| | - Frank A. Witzmann
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 1345 West 16th Street, Indianapolis, IN 46202, USA; E-Mails: (X.L.); (F.A.W.)
| | - Bonnie L. Blazer-Yost
- Department of Biology, Indiana University Purdue University, 723 West Michigan Street, Indianapolis, IN 46202, USA; E-Mail:
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 1345 West 16th Street, Indianapolis, IN 46202, USA; E-Mails: (X.L.); (F.A.W.)
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +1-317-278-1145; Fax: +1-317-274-2846
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25
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Shannahan JH, Lai X, Ke PC, Podila R, Brown JM, Witzmann FA. Silver nanoparticle protein corona composition in cell culture media. PLoS One 2013; 8:e74001. [PMID: 24040142 PMCID: PMC3767594 DOI: 10.1371/journal.pone.0074001] [Citation(s) in RCA: 146] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 06/14/2013] [Accepted: 07/31/2013] [Indexed: 11/18/2022] Open
Abstract
The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index), the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic and hydrophobic interactions in the formation of the PC which may have broad biological and toxicological implications.
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Affiliation(s)
- Jonathan H. Shannahan
- Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America
| | - Xianyin Lai
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Pu Chun Ke
- Department of Physics and Astronomy, Clemson University, Clemson, South Carolina, United States of America
| | - Ramakrishna Podila
- Department of Physics and Astronomy, Clemson University, Clemson, South Carolina, United States of America
| | - Jared M. Brown
- Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America
| | - Frank A. Witzmann
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
- * E-mail:
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Domenighetti AA, Chu PH, Wu T, Sheikh F, Gokhin DS, Guo LT, Cui Z, Peter AK, Christodoulou DC, Parfenov MG, Gorham JM, Li DY, Banerjee I, Lai X, Witzmann FA, Seidman CE, Seidman JG, Gomes AV, Shelton GD, Lieber RL, Chen J. Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice. Hum Mol Genet 2013; 23:209-25. [PMID: 23975679 DOI: 10.1093/hmg/ddt412] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 01/15/2023] Open
Abstract
Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery-Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations.
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The KUPNetViz: a biological network viewer for multiple -omics datasets in kidney diseases. BMC Bioinformatics 2013; 14:235. [PMID: 23883183 PMCID: PMC3725151 DOI: 10.1186/1471-2105-14-235] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 07/12/2012] [Accepted: 07/21/2013] [Indexed: 02/08/2023] Open
Abstract
Background Constant technological advances have allowed scientists in biology to migrate from conventional single-omics to multi-omics experimental approaches, challenging bioinformatics to bridge this multi-tiered information. Ongoing research in renal biology is no exception. The results of large-scale and/or high throughput experiments, presenting a wealth of information on kidney disease are scattered across the web. To tackle this problem, we recently presented the KUPKB, a multi-omics data repository for renal diseases. Results In this article, we describe KUPNetViz, a biological graph exploration tool allowing the exploration of KUPKB data through the visualization of biomolecule interactions. KUPNetViz enables the integration of multi-layered experimental data over different species, renal locations and renal diseases to protein-protein interaction networks and allows association with biological functions, biochemical pathways and other functional elements such as miRNAs. KUPNetViz focuses on the simplicity of its usage and the clarity of resulting networks by reducing and/or automating advanced functionalities present in other biological network visualization packages. In addition, it allows the extrapolation of biomolecule interactions across different species, leading to the formulations of new plausible hypotheses, adequate experiment design and to the suggestion of novel biological mechanisms. We demonstrate the value of KUPNetViz by two usage examples: the integration of calreticulin as a key player in a larger interaction network in renal graft rejection and the novel observation of the strong association of interleukin-6 with polycystic kidney disease. Conclusions The KUPNetViz is an interactive and flexible biological network visualization and exploration tool. It provides renal biologists with biological network snapshots of the complex integrated data of the KUPKB allowing the formulation of new hypotheses in a user friendly manner.
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Shannahan JH, Brown JM, Chen R, Ke PC, Lai X, Mitra S, Witzmann FA. Comparison of nanotube-protein corona composition in cell culture media. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2013; 9:2171-81. [PMID: 23322550 PMCID: PMC3725593 DOI: 10.1002/smll.201202243] [Citation(s) in RCA: 96] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Academic Contribution Register] [Received: 09/12/2012] [Revised: 10/25/2012] [Indexed: 04/14/2023]
Abstract
In biological environments, nanomaterials associate with proteins forming a protein corona (PC). The PC may alter the nanomaterial's pharmacokinetics and pharmacodynamics, thereby influencing toxicity. Using a label-free mass spectrometry-based proteomics approach, the composition of the PC is examined for a set of nanotubes (NTs) including unmodified and carboxylated single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), polyvinylpyrrolidone (PVP)-coated MWCNT (MWCNT-PVP), and nanoclay. NTs are incubated for 1 h in simulated cell culture conditions, then washed, resuspended in PBS, and assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for their associated PC. To determine those attributes that influence PC formation, the NTs are extensively characterized. NTs had negative zeta potentials in water (SWCNT-COOH < MWCNT-COOH < unmodified NTs) while carboxylation increases their hydrodynamic sizes. All NTs are also found to associate a common subset of proteins including albumin, titin, and apolipoproteins. SWCNT-COOH and MWCNT-COOH are found to bind the greatest number of proteins (181 and 133 respectively) compared to unmodified NTs (<100), suggesting covalent binding to protein amines. Modified NTs bind a number of unique proteins compared to unmodified NTs, implying hydrogen bonding and electrostatic interactions are involved in PC formation. PVP-coating of MWCNT did not influence PC composition, further reinforcing the possibility of hydrogen bonding and electrostatic interactions. No relationships are found between PC composition and corresponding isoelectric point, hydropathy, or aliphatic index, implying minimal roles of hydrophobic interaction and pi-stacking.
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Affiliation(s)
- Jonathan H. Shannahan
- Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, 27834, USA
| | - Jared M. Brown
- Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, 27834, USA
| | - Ran Chen
- Department of Physics and Astronomy, Clemson University, Clemson, South Carolina, 29634, USA
| | - Pu Chun Ke
- Department of Physics and Astronomy, Clemson University, Clemson, South Carolina, 29634, USA
| | - Xianyin Lai
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, 46202, USA
| | - Somenath Mitra
- Department of Chemistry & Environmental Science, New Jersey Institute of Technology, Newark, New Jersey, 07102, USA
| | - Frank A. Witzmann
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, 46202, USA
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Owen MK, Witzmann FA, McKenney ML, Lai X, Berwick ZC, Moberly SP, Alloosh M, Sturek M, Tune JD. Perivascular adipose tissue potentiates contraction of coronary vascular smooth muscle: influence of obesity. Circulation 2013; 128:9-18. [PMID: 23685742 DOI: 10.1161/circulationaha.112.001238] [Citation(s) in RCA: 114] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Academic Contribution Register] [Indexed: 11/16/2022]
Abstract
BACKGROUND This investigation examined the mechanisms by which coronary perivascular adipose tissue (PVAT)-derived factors influence vasomotor tone and the PVAT proteome in lean versus obese swine. METHODS AND RESULTS Coronary arteries from Ossabaw swine were isolated for isometric tension studies. We found that coronary (P=0.03) and mesenteric (P=0.04) but not subcutaneous adipose tissue augmented coronary contractions to KCl (20 mmol/L). Inhibition of CaV1.2 channels with nifedipine (0.1 µmol/L) or diltiazem (10 µmol/L) abolished this effect. Coronary PVAT increased baseline tension and potentiated constriction of isolated arteries to prostaglandin F2α in proportion to the amount of PVAT present (0.1-1.0 g). These effects were elevated in tissues obtained from obese swine and were observed in intact and endothelium denuded arteries. Coronary PVAT also diminished H2O2-mediated vasodilation in lean and, to a lesser extent, in obese arteries. These effects were associated with alterations in the obese coronary PVAT proteome (detected 186 alterations) and elevated voltage-dependent increases in intracellular [Ca(2+)] in obese smooth muscle cells. Further studies revealed that the Rho-kinase inhibitor fasudil (1 µmol/L) significantly blunted artery contractions to KCl and PVAT in lean but not obese swine. Calpastatin (10 μmol/L) also augmented contractions to levels similar to that observed in the presence of PVAT. CONCLUSIONS Vascular effects of PVAT vary according to anatomic location and are influenced by an obese phenotype. Augmented contractile effects of obese coronary PVAT are related to alterations in the PVAT proteome (eg, calpastatin), Rho-dependent signaling, and the functional contribution of K(+) and CaV1.2 channels to smooth muscle tone.
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Affiliation(s)
- Meredith Kohr Owen
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr, Indianapolis, IN 46202, USA
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Lai X, Agarwal M, Lvov YM, Pachpande C, Varahramyan K, Witzmann FA. Proteomic profiling of halloysite clay nanotube exposure in intestinal cell co-culture. J Appl Toxicol 2013; 33:1316-29. [PMID: 23606564 DOI: 10.1002/jat.2858] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 11/16/2012] [Revised: 12/18/2012] [Accepted: 12/19/2012] [Indexed: 01/13/2023]
Abstract
Halloysite is aluminosilicate clay with a hollow tubular structure with nanoscale internal and external diameters. Assessment of halloysite biocompatibility has gained importance in view of its potential application in oral drug delivery. To investigate the effect of halloysite nanotubes on an in vitro model of the large intestine, Caco-2/HT29-MTX cells in monolayer co-culture were exposed to nanotubes for toxicity tests and proteomic analysis. Results indicate that halloysite exhibits a high degree of biocompatibility characterized by an absence of cytotoxicity, in spite of elevated pro-inflammatory cytokine release. Exposure-specific changes in expression were observed among 4081 proteins analyzed. Bioinformatic analysis of differentially expressed protein profiles suggest that halloysite stimulates processes related to cell growth and proliferation, subtle responses to cell infection, irritation and injury, enhanced antioxidant capability, and an overall adaptive response to exposure. These potentially relevant functional effects warrant further investigation in in vivo models and suggest that chronic or bolus occupational exposure to halloysite nanotubes may have unintended outcomes.
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Affiliation(s)
- Xianyin Lai
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA
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Happé H, van der Wal AM, Salvatori DCF, Leonhard WN, Breuning MH, de Heer E, Peters DJM. Cyst expansion and regression in a mouse model of polycystic kidney disease. Kidney Int 2013; 83:1099-108. [PMID: 23466997 DOI: 10.1038/ki.2013.13] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 12/23/2022]
Abstract
Autosomal-dominant polycystic kidney disease is characterized by progressive cyst formation and fibrosis in the kidneys. Here we describe an orthologous Pkd1(nl,nl) mouse model, with reduced expression of the normal Pkd1 transcript, on a fixed genetic background of equal parts C57Bl/6 and 129Ola/Hsd mice (B6Ola-Pkd1(nl,nl)). In these mice, the first cysts develop from mature proximal tubules around birth. Subsequently, larger cysts become visible at day 7, followed by distal tubule and collecting duct cyst formation, and progressive cystic enlargement to develop into large cystic kidneys within 4 weeks. Interestingly, cyst expansion was followed by renal volume regression due to cyst collapse. This was accompanied by focal formation of fibrotic areas, an increased expression of genes involved in matrix remodeling and subsequently an increase in infiltrating immune cells. After an initial increase in blood urea within the first 4 weeks, renal function remained stable over time and the mice were able to survive up to a year. Also, in kidneys of ADPKD patients collapsed cysts were observed, in addition to massive fibrosis and immune infiltrates. Thus, B6Ola-Pkd1(nl,nl) mice show regression of cysts and renal volume that is not accompanied by a reduction in blood urea levels.
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Affiliation(s)
- Hester Happé
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
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Lai X. Reproducible method to enrich membrane proteins with high purity and high yield for an LC-MS/MS approach in quantitative membrane proteomics. Electrophoresis 2013; 34:809-17. [PMID: 23334993 DOI: 10.1002/elps.201200503] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 07/05/2012] [Revised: 11/15/2012] [Accepted: 11/24/2012] [Indexed: 12/12/2022]
Abstract
The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC-MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC-MS/MS label-free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤30%. Western blot and LC-MS/MS results of markers for cytoplasm, nucleus, mitochondria, and their membranes indicated that the enriched membrane fraction was highly pure by the absence of, or presence of trace amounts of, nonmembrane marker proteins. The average yield of membrane proteins was 237 μg/10 million HT29-MTX cells. LC-MS/MS analysis of the membrane-enriched sample resulted in the identification of 2597 protein groups. In summary, the developed method is reproducible, produces a highly pure membrane fraction, and generates a high yield of membrane proteins.
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Affiliation(s)
- Xianyin Lai
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
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Prince KL, Colvin SC, Park S, Lai X, Witzmann FA, Rhodes SJ. Developmental analysis and influence of genetic background on the Lhx3 W227ter mouse model of combined pituitary hormone deficiency disease. Endocrinology 2013; 154:738-48. [PMID: 23288907 PMCID: PMC3548188 DOI: 10.1210/en.2012-1790] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Academic Contribution Register] [Indexed: 11/19/2022]
Abstract
Combined pituitary hormone deficiency (CPHD) diseases result in severe outcomes for patients including short stature, developmental delays, and reproductive deficiencies. Little is known about their etiology, especially the developmental profiles and the influences of genetic background on disease progression. Animal models for CPHD provide valuable tools to investigate disease mechanisms and inform diagnostic and treatment protocols. Here we examined hormone production during pituitary development and the influence of genetic background on phenotypic severity in the Lhx3(W227ter/W227ter) mouse model. Lhx3(W227ter/W227ter) embryos have deficiencies of ACTH, α-glycoprotein subunit, GH, PRL, TSHβ, and LHβ during prenatal development. Furthermore, mutant mice have significant reduction in the critical pituitary transcriptional activator-1 (PIT1). Through breeding, the Lhx3(W227ter/W227ter) genotype was placed onto the 129/Sv and C57BL/6 backgrounds. Intriguingly, the genetic background significantly affected viability: whereas Lhx3(W227ter/W227ter) animals were found in the expected frequencies in C57BL/6, homozygous animals were not viable in the 129/Sv genetic environment. The hormone marker and PIT1 reductions observed in Lhx3(W227ter/W227ter) mice on a mixed background were also seen in the separate strains but in some cases were more severe in 129/Sv. To further characterize the molecular changes in diseased mice, we conducted a quantitative proteomic analysis of pituitary proteins. This showed significantly lower levels of PRL, pro-opiomelanocortin (ACTH), and α-glycoprotein subunit proteins in Lhx3(W227ter/W227ter) mice. Together, these data show that hormone deficiency disease is apparent in early prenatal stages in this CPHD model system. Furthermore, as is noted in human disease, genetic background significantly impacts the phenotypic outcome of these monogenic endocrine diseases.
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Affiliation(s)
- Kelly L Prince
- Departments of Cellular and Integrative Physiology, Indiana University-Purdue University, Indianapolis, IN 46202, USA
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Lai X, Blazer-Yost BL, Clack JW, Fears SL, Mitra S, Ntim SA, Ringham HN, Witzmann FA. Protein expression profiles of intestinal epithelial co-cultures: effect of functionalised carbon nanotube exposure. ACTA ACUST UNITED AC 2013; 3. [PMID: 24228069 DOI: 10.1504/ijbnn.2013.054508] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/21/2022]
Abstract
To assess the biological effects of low level, water dispersible, functionalised carbon nanotube (f-CNT) exposure in an in vitro model simulating the digestive tract, cellular protein expression was quantified and compared using label-free quantitative mass spectrometry (LFQMS). Co-cultured cells were exposed to well-characterised SWCNT-COOH, MWCNT-COOH, and MWCNT-PVP. The relative expression of 2,282 unique proteins was compared across the dose groups. 428 proteins were found to be differentially expressed. At the high dose, the extent of differential protein expression was CNT-specific and directly related to CNT colloidal stability. Cells responded to low level MWCNT-PVP exposure with three-fold greater differential expression. Bioinformatic analysis indicated significant and f-CNT-specific effects on relevant molecular and cellular functions and canonical pathways, with little overlap across f-CNT type and in the absence of overt toxicity.
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Affiliation(s)
- Xianyin Lai
- Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 1345 West 16th Street, Indianapolis IN 46202, USA,
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Vidanapathirana AK, Lai X, Hilderbrand SC, Pitzer JE, Podila R, Sumner SJ, Fennell TR, Wingard CJ, Witzmann FA, Brown JM. Multi-walled carbon nanotube directed gene and protein expression in cultured human aortic endothelial cells is influenced by suspension medium. Toxicology 2012; 302:114-22. [PMID: 23026733 DOI: 10.1016/j.tox.2012.09.008] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 05/23/2012] [Revised: 08/08/2012] [Accepted: 09/20/2012] [Indexed: 11/17/2022]
Abstract
The use and production of multi-walled carbon nanotubes (MWCNTs) have significantly increased over the last decade due to their versatility in numerous applications. Their unique physical and chemical properties make them desirable for various biomedical applications, but the same properties also raise concerns about their safety to human health, particularly at the cellular level. The vascular endothelium could be exposed to nanomaterials either by direct intravenous administration in nanomedicine or by translocation following inhalational exposure in an occupational setting. We hypothesized that direct exposure to MWCNTs will increase the expression of inflammatory markers in human aortic endothelial cells (HAEC). We also investigated the effect of the route of exposure on activation by changing the suspension medium of the MWCNTs. HAEC were treated in vitro with MWCNTs (1 or 10 μg/cm(2)) suspended in either cell culture medium [(M)-MWCNTs] or 10% clinical grade pulmonary surfactant [(S)-MWCNTs]. The zeta potential of the (S)-MWCNTs was significantly more negative than the (M)-MWCNTs suggesting a more stable suspension. Treatment of HAEC with (S)-MWCNTs; as compared to (M)-MWCNTs resulted in a significantly higher up-regulation of mRNA transcripts for cell adhesion molecules VCAM1, SELE, ICAM1 and the chemokine CCL2. Time dependent changes in VCAM1 and CCL2 protein levels were confirmed by immunofluorescence, flow cytometry and ELISA. A label free quantitative mass spectrometry proteomic analysis was utilized to compare protein expression patterns between the two suspensions of MWCNTs. We identified significant expression changes in >200 unique proteins in MWCNT treated HAEC. However, the two suspensions of MWCNTs resulted in different protein expression patterns with the eIF2 pathway as the only common pathway identified between the two suspensions. These data suggest that direct exposure to MWCNTs induces acute inflammatory and protein expression changes in HAEC, which is influenced by the type of media used for suspension of MWCNTs and their resulting zeta potential.
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Affiliation(s)
- Achini K Vidanapathirana
- Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA
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Rithidech KN, Lai X, Honikel L, Reungpatthanaphong P, Witzmann FA. Identification of proteins secreted into the medium by human lymphocytes irradiated in vitro with or without adaptive environments. HEALTH PHYSICS 2012; 102:39-53. [PMID: 22134077 PMCID: PMC3744879 DOI: 10.1097/hp.0b013e31822833af] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Academic Contribution Register] [Indexed: 05/04/2023]
Abstract
There is increasing evidence to support the hypothesis of adaptive response, a phenomenon in which protection arises from a low-dose radiation (<0.1 Gy) against damage induced by subsequent exposure to high-dose radiation. The molecular mechanisms underlying such protection are poorly understood. The goal of this study was to fill this knowledge gap. Mass spectrometry-based proteomics was used to characterize global protein expression profiles in the medium collected from human lymphocyte cultures given sham irradiation (0 Gy) or a priming low dose of 0.03 Gy 137Cs γ rays 4 h prior to a challenging dose of 1 Gy 137Cs γ rays. Adaptive response was determined by decreased micronucleus frequencies in lymphocytes receiving low dose irradiation prior to high dose irradiation compared to those receiving only high dose irradiation. Adaptive response was found in these experiments. Proteomic analysis of media revealed: (a) 55 proteins with similar abundance in both groups; (b) 23 proteins in both groups, but 7 of them were high abundance in medium with adaptive environment, while 16 high abundance proteins were in medium without adaptive environment; (c) 17 proteins in medium with adaptive environment only; and (d) 8 proteins in medium without adaptive environment only. The results provide a foundation for improving understanding of the molecular mechanisms associated with the beneficial effects of low dose radiation that, in turn, will have an important impact on radiation risk estimation. Hence, these studies are highly relevant to radiation protection due to an increased use of low dose radiation in daily life (e.g., medical diagnosis or airport safety) or an unavoidable exposure to low level background radiation.
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Blazer-Yost BL, Blacklock BJ, Flaig S, Bacallao RL, Gattone VH. Lysophosphatidic acid is a modulator of cyst growth in autosomal dominant polycystic kidney disease. Cell Physiol Biochem 2011; 28:1255-64. [PMID: 22179013 DOI: 10.1159/000335857] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Accepted: 11/18/2011] [Indexed: 12/31/2022] Open
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of multiple fluid-filled cysts predominately in the kidney tubules and liver bile ducts. Elucidation of mechanisms that control cyst growth will provide the basis for rational therapeutic intervention. We used electrophysiological methods to identify lysophosphatidic acid (LPA) as a component of cyst fluid and serum that stimulates secretory Cl- transport in the epithelial cell type that lines renal cysts. LPA effects are manifested through receptors located on the basolateral membrane of the epithelial cells resulting in stimulation of channel activity in the apical membrane. Concentrations of LPA measured in human ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage and/or leakage of vascular LPA into the interstitial space are capable of stimulating epithelial cell secretion resulting in cyst enlargement. These observations are particularly relevant to the rapid decline in renal function in late-stage disease and to the "third hit" hypothesis that renal injury exacerbates cyst growth.
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Affiliation(s)
- Bonnie L Blazer-Yost
- Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202, USA.
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Lai X, Wang L, Tang H, Witzmann FA. A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS. J Proteome Res 2011; 10:4799-812. [PMID: 21888428 DOI: 10.1021/pr2005633] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 01/17/2023]
Abstract
Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by LC-MS/MS, two critical issues are generally ignored in this field: (i) peptides have multiple elution patterns across runs in an experiment, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a novel alignment method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. At least 90.8% of the proteins (up to 1,390) had CVs ≤ 30% across 10 technical replicates, and at least 93.6% (up to 2,013) had R(2) ≥ 0.9500 across 7 concentrations. Identical amounts of standard protein spiked in complex biological samples achieved a CV of 8.6% across eight injections of two groups. Further assessment was made by comparing mass spectrometric results to immunodetection, and consistent results were obtained. The new approach has novel and specific features enabling accurate label-free quantification.
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Affiliation(s)
- Xianyin Lai
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
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Abstract
Immunoaffinity chromatography (IAC) combines the use of LC with the specific binding of antibodies or related agents. The resulting method can be used in assays for a particular target or for purification and concentration of analytes prior to further examination by another technique. This review discusses the history and principles of IAC and the various formats that can be used with this method. An overview is given of the general properties of antibodies and of antibody-production methods. The supports and immobilization methods used with antibodies in IAC and the selection of application and elution conditions for IAC are also discussed. Several applications of IAC are considered, including its use in purification, immunodepletion, direct sample analysis, chromatographic immunoassays and combined analysis methods. Recent developments include the use of IAC with CE or MS, ultrafast immunoextraction methods and the use of immunoaffinity columns in microanalytical systems.
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Ly L, Wasinger VC. Protein and peptide fractionation, enrichment and depletion: Tools for the complex proteome. Proteomics 2011; 11:513-34. [DOI: 10.1002/pmic.201000394] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Received: 06/29/2010] [Revised: 10/03/2010] [Accepted: 10/18/2010] [Indexed: 12/28/2022]
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Lai X, Blazer-Yost BL, Gattone VH, Muchatuta MN, Witzmann FA. Protein composition of liver cyst fluid from the BALB/c-cpk/+ mouse model of autosomal recessive polycystic kidney disease. Proteomics 2009; 9:3775-82. [PMID: 19639592 DOI: 10.1002/pmic.200800379] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/12/2022]
Abstract
Cysts arising from hepatic bile ducts are a common extra-renal pathology associated with polycystic kidney disease in humans. As an initial step in identifying active components that could contribute to disease progression, we have investigated the protein composition of hepatic cyst fluid in an orthologous animal model of autosomal recessive polycystic kidney disease, heterozygous (BALB/c-cpk/+) mice. Proteomic analysis of cyst fluid tryptic digests using LC-MS/MS identified 303 proteins, many of which are consistent with enhanced inflammatory cell processes, cellular proliferation, and basal laminar fibrosis associated with the development of hepatic bile duct cysts. Protein identifications have been submitted to the PRIDE database (http://www.ebi.ac.uk/pride), accession number 9227.
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Affiliation(s)
- Xianyin Lai
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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Mason SB, Lai X, Bacallao RL, Blazer-Yost BL, Gattone VH, Wang KC, Witzmann FA. The biomarker enriched proteome of autosomal dominant polycystic kidney disease cyst fluid. Proteomics Clin Appl 2009; 3:1247-1250. [PMID: 20526430 DOI: 10.1002/prca.200800163] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/08/2022]
Abstract
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the development of numerous fluid-filled cysts in the kidneys of patients. We recently published our description of the proteome of renal cyst fluid in ADPKD. As a follow-up experiment, we hypothesized that the protein-bound subfraction consists of molecules of mechanistic or diagnostic interest in ADPKD. Using a manual biomarker enrichment kit, we have identified 44 distinct proteins in human cyst fluid.
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Affiliation(s)
- Stephen B Mason
- Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA
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Yoo KH, Kim YN, Lee MJ, Seong JK, Park JH. Identification of apolipoproteinA1 reduction in the polycystic kidney by proteomics analysis of the Mxi1-deficient mouse. Proteomics 2009; 9:3824-32. [DOI: 10.1002/pmic.200800753] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 01/06/2023]
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Lai X, Liangpunsakul S, Crabb DW, Ringham HN, Witzmann FA. A proteomic workflow for discovery of serum carrier protein-bound biomarker candidates of alcohol abuse using LC-MS/MS. Electrophoresis 2009; 30:2207-14. [PMID: 19544491 PMCID: PMC2756771 DOI: 10.1002/elps.200800775] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/10/2022]
Abstract
The diagnosis and care of patients with alcohol abuse and dependence is hampered by a lack of sensitive and specific screening and monitoring tests. Proteomics is a good approach to search for biomarkers of alcohol abuse. Serum carrier protein-bound proteins have attracted significant interest because they remain a relatively un-mined region of the proteome. In the present study, a proteomic workflow including LC-MS/MS with enrichment of serum carrier protein-bound biomarkers technique was applied to profile the changes in quality and quantity of serum carrier protein-bound proteins for the discovery of novel biomarker candidates of alcohol abuse. In total, 311 proteins identified with high confidence were discovered to be bound to serum carrier proteins. Complement isoforms, Ig fragments, and apolipoprotein family proteins are the main serum carrier-bound proteins. Protein quantification analysis with and without concern as to gender revealed that gender is a critical consideration for biomarker development in alcohol abuse. Identified proteins not previously associated with alcohol abuse include gelsolin, selenoprotein P, serotransferrin, tetranectin, hemopexin, histidine-rich glycoprotein, plasma kallikrein, and vitronectin. Altered abundance of these proteins suggests that they may be potential novel biomarkers for alcohol abuse.
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Affiliation(s)
- Xianyin Lai
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Suthat Liangpunsakul
- Department of Medicine – Division of Gastroenterology/Hepatology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - David W. Crabb
- Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Heather N. Ringham
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Frank A. Witzmann
- Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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From Our Sister Journal: Proteomics 17/2008. Proteomics 2008. [DOI: 10.1002/pmic.200890060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Academic Contribution Register] [Indexed: 11/10/2022]
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