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Changes of Protein Levels in Human Urine Reflect the Dysregulation Of www.nature.com/scientificreports OPEN Changes of protein levels in human urine refect the dysregulation of signaling pathways of chronic kidney disease and its complications Yiming Hao1, Luis Tanon Reyes2, Robert Morris2, Yifeng Xu1, Yiqin Wang1* & Feng Cheng2* The increasing prevalence of chronic kidney disease (CKD) seriously is threatening human health and overall quality of life. The discovery of biomarkers of pathogenesis of CKD and the associated complications are very important for CDK diagnosis and treatment. In this paper, urine protein biomarkers were investigated because urine sample collection is convenient and non-invasive. We analyzed the protein concentrations in the urine of CKD patients and extracted abnormal protein signals comparing with the healthy control groups. The enriched signaling pathways that may characterize CKD pathology were identifed from these proteins. We applied surface-enhanced laser desorption and ionization time of fight mass spectrometry technology to detect diferent protein peaks in urine samples from patients with CKD and healthy controls. We searched the proteins corresponding to protein peaks through the UniProt database and identifed the signaling pathways of CKD and its complications by using the NIH DAVID database. 42 low abundance proteins and 46 high abundance proteins in the urine samples from CKD patients were found by comparing with healthy controls. Seven KEGG pathways related to CKD and its complications were identifed from the regulated proteins. These pathways included chemokine signaling pathway, cytokine–cytokine receptor interaction, oxidative phosphorylation, cardiac muscle contraction, Alzheimer’s disease, Parkinson’s disease, and salivary secretion. In CKD stages 2, 3, 4, and 5, fve proteins showed signifcantly diferential abundances. The diferential protein signals and regulated signaling pathways will provide new insight for the pathogenesis of CKD and its complications. These altered proteins may also be used as novel biomarkers for the noninvasive and convenient diagnosis methods of CKD and its complications through urine testing in the future. Chronic kidney disease (CKD) is a disease characterized by progressive loss of renal function over extended periods of time, of which spans from months to years. Its prevalence is increasing dramatically, and it threatens human health and quality of life. Currently, about 8–10% of the world’s population has a certain degree of CKD and is the 16th most frequent cause of mortality each year 1,2. Unfortunately, the increasing cost of treating CKD and its complications has placed a considerable economic burden on health care systems worldwide 3. However, in the early and middle stages of CKD, patients generally do not exhibit any noticeable signs or symptoms1. Te current diagnostic biomarkers (serum creatinine and urine albumin) are not enough to predict the develop- ment of CKD. Terefore, timely monitoring and diagnosis of CKD is important so that patients will fare better with treatment and better outcomes will be achieved. In recent years, using human urine samples to look for biomarkers have attracted more and more attention because of its convenient, non-invasive collection, and stable composition4. For example, some researchers indicated that some proteins in human urine may help to predict early-stage CKD patients 5. However, with the progression of CKD, some complications may appear gradually. Because of the complicated interplay between CKD and other comorbid conditions1, it is difcult to monitor the 1Shanghai Key Laboratory of Health Identifcation and Assessment/Laboratory of TCM Four Diagnostic Information, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL 33612, USA. *email: [email protected]; [email protected] Scientifc Reports | (2020) 10:20743 | https://doi.org/10.1038/s41598-020-77916-z 1 Vol.:(0123456789) www.nature.com/scientificreports/ Demographics and clinical information CKD group Healthy control group Samples number 147 24 Samples number (percentage) of stage 1 5 (3.40%) N/A Samples number (percentage) of stage 2 26 (17.69%) N/A Samples number (percentage) of stage 3 45 (30.61%) N/A Samples number (percentage) of stage 4 36 (24.49%) N/A Samples number (percentage) of stage 5 35 (23.81%) N/A Ratio of male to female 1:0.71 1:0.85 Average age (years) 53.64 ± 15.91 49.08 ± 11.60 eGFR (mL/min) 37.36 ± 28.27 116.67 ± 14.15 SCr (μmol/L) 264.24 ± 216.04 63.83 ± 11.24 Number (percentage) of samples with high blood pressure 62 (42.18%) N/A Number (percentage) of samples with heart disease 15 (10.20%) N/A Number (percentage) of samples with type 2 diabetes 3 (2.04%) N/A Number (percentage) of samples with Alzheimer’s disease 1 (0.68%) N/A Table 1. Summary of demographics and clinical information of the participants. N/A: not applicable. evolution of CKD by using protein concentration in human urine and thus, clinical timing of CDK diagnosis is crucial in reducing the risk of deterioration of the patient’s condition. Terefore, we hope to initially understand which proteins in the urine of CKD patients may have abnormal change as well as what kind of adverse events may result from these fuctuations. In this paper, we detected the diferent protein peaks existing in the samples of patients with CKD compared with healthy controls using surface enhanced laser desorption and ionization time of fight mass spectrometry (SELDI-TOF-MS) technology. Aferwards, we used proteomics databases to scan the proteins corresponding to the protein peaks observed. Finally, we explored the abnormal signaling pathways that may occur in CKD patients, as well as the related proteins and their functions that express changes in these pathways. Materials and methods Samples. CKD patients were selected from Longhua Hospital afliated with Shanghai University of Tradi- tional Chinese Medicine from 2008 to 2010. Te cause of CKD of patients was primary chronic glomerulone- phritis. One hundred and forty-seven urine samples were collected from these patients, of whom are receiving routine treatment of western medicine and traditional Chinese medicine. Te control group was selected from the healthy staf of Shanghai University of Traditional Chinese Medicine who had normal indicators of kidney function (including eGFR and SCr) and no other organic diseases. Twenty- four urine samples were collected from them. From Table 1, there was no signifcant diference (P > 0.05) between the CKD group and healthy control group with respect to gender and age, so the data from these groups were comparable. Te eGFR value of the CKD group was signifcantly lower than that of the healthy control group (P < 0.05) while SCr levels of the CKD patients were signifcantly higher than that of the healthy person (P < 0.05). In addition, among these CKD patients, there were 45 persons with stage 3, 36 individuals that had progressed to stage 4, 35 people diagnosed at stage 5, 26 with stage 2 CKD, and 5 patients with stage 1 CKD. Some of these CKD patients also had one or more secondary complications. Among them, sixty-two persons had high blood pressure, ffeen patients had heart disease, three individuals had type 2 diabetes, and one person had Alzheimer’s disease. Ethics approval. Te study was approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine in China in 2008 and performed in accordance with the Declaration of Helsinki. All the sub- jects got written informed consent. The criteria for inclusion, exclusion, and elimination. Te inclusion criteria of CKD patients include: 1. Patients accord with the diagnostic criteria of CKD which are referenced from “KDIGO 2012 clinical practice guideline for the evaluation and management of chronic kidney disease”6. 2. Patients’ age ranges from 20 to 85. Te exclusion criteria of CKD patients include: 1. Patients with primary heart, liver, lung, endocrine, blood, metabolism diseases, severe gastro-intestine pri- mary diseases or mental illness. 2. Patients with CKD caused by extra-renal diseases. 3. Patients with CKD and dialysis therapy is required. 4. Patients who are pregnant or breastfeeding. 5. Patients with allergic constitution or with drug allergy. Scientifc Reports | (2020) 10:20743 | https://doi.org/10.1038/s41598-020-77916-z 2 Vol:.(1234567890) www.nature.com/scientificreports/ Te elimination criteria include: Patients without complete clinical data; either due to incomplete collection or data missing. Experimental methods. 5 mL of morning urine from each subject was collected and placed into sterilized EP tubes. Te protein concentration of each urine sample was detected by ultraviolet spectrophotometry. Te sample with the lowest concentration was used as the reference, and the sample with higher concentrations were diluted with stroke-physiological saline solution to reach the minimum concentration. At 4 °C, we used a cen- trifuge (Allegra 21R, Beckman, Inc.) to remove the impurities in the urine samples at 3000 rpm/min for 10 min, and then took the supernatant. Te unpooled urine samples were analyzed independently. Tey were added to an H4 protein chip (Ciphergen Biosystems, Inc.). Te chip was placed in the SELDI-TOF mass spectrometer (PBSII, Ciphergen Biosystems, Inc.). Te instrument was calibrated to the range of 0.1% by H4 chip with all-in-one standard protein. Te parameters of mass spectrometer were as follows: laser intensity was 155, detector sensitivity was 7, the protein peaks with signal-to-noise ratio (S/N) over 5 and frequencies over 10% in all mass spectra were efective. Te optimum molecular weight (Mw) ranged from 1000 to 20,000. Te highest molecular weight was 40,000. Each point on the chip was collected 130 times. Data were collected with Ciphergen Protein Chip sofware version 3.1.1. Data processing, statistical analysis and substance identifcation. We used the total ion current to correct all the atlases to reduce the experimental errors caused by diferent chip and instrumental state fuc- tuations.
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