UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CURSO DE GRADUAÇÃO EM FARMÁCIA
INCREASED NEPHRIN IN URINARY EXTRACELLULAR VESICLES OF PREGNANT WOMEN WITH SEVERE PREECLAMPSIA
Adrielle Kétcia Alves de Lima
Natal – RN 2020
Adrielle Kétcia Alves de Lima
INCREASED NEPHRIN IN URINARY EXTRACELLULAR VESICLES OF PREGNANT WOMEN WITH SEVERE PREECLAMPSIA
Trabalho de Conclusão de Curso apresentado ao Curso de Graduação em Farmácia do Centro de Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito parcial para a obtenção do grau de bacharel em Farmácia.
Orientadora: Profa. Dra. Marcela Abbott Galvão Ururahy
Natal – RN 2020
Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI
Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde – CCS
Lima, Adrielle Kétcia Alves de.
Increased nephrin in urinary extracellular vesicles of pregnant women with severe preeclampsia / Adrielle Ketcia Alves de Lima. - 2020.
48f.: il.
Trabalho de Conclusão de Curso - TCC (Graduação em Farmácia) - Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Departamento de Farmácia. Natal, RN, 2020.
Orientadora: Marcela Abbott Galvão Ururahy.
1. Pré-Eclâmpsia - TCC. 2. Wilms tumor 1 - TCC. 3. Podocin - TCC. 4. Biomarker - TCC. 5. Pregnancy - TCC. I. Ururahy, Marcela Abbott Galvão. II. Título.
RN/UF/BS-CCS CDU 618.3
INCREASED NEPHRIN IN URINARY EXTRACELLULAR VESICLES OF PREGNANT WOMEN WITH SEVERE PREECLAMPSIA
Trabalho de Conclusão de Curso apresentado ao Curso de Graduação em Farmácia do Centro de Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito parcial para a obtenção do grau de bacharel em Farmácia.
Orientadora: Profa. Dra. Marcela Abbott Galvão Ururahy
________________________________________________________ Presidente: Profa. Marcela Abbott Galvão Ururahy, Dra., DACT/UFRN
________________________________________________________ Membro: Karla Simone Costa de Souza, Dra., PPgCF/UFRN ________________________________________________________
Membro: Prof. Igor de Farias Domingos, Dr., DACT/UFRN
A Deus por ser tão onipresente, me proporcionando toda força, ânimo e coragem para ter alcançado minha meta.
Aos meus pais, Fátima e Genildo, pelo apoio, cuidado е dedicação, que mesmo com alguns quilômetros de distância, conseguiam estar presentes e me dar esperança para seguir. O apoio deles significava segurança е o sentimento de nunca estar sozinha. Ao meu irmão amado, por seus ensinamentos e grande suporte. Você é o melhor.
Aos meus familiares que sempre me deram o suporte necessário para que meu objetivo fosse alcançado. À minha tia Eristejane Alves, por sempre estar presente e ser uma verdadeira segunda mãe. À minha avó Geraldina Alves, que apesar do Alzheimer fazer com que não me reconheça, nunca mediu esforços para que todos da família conseguissem sucesso. Ao meu avô Orlando, in memoriam, que estaria radiante se pudesse presenciar esse momento.
Ao meu namorado, Matheus Henrique, que está sempre ao meu lado com extrema dedicação e companheirismo.
A todos os meus amigos, em especial, Jean Aderaldo, Maria Daniele, Thalita Araújo, Luênia Dantas, Gabriel Camelo e Anna Júlia, por sempre estarem presentes nos momentos em que mais precisei e, também por toda força e apoio que vocês me proporcionam em toda minha trajetória.
Aos meus colegas de turma, por todos os momentos, que apesar das diferenças, conseguiram ser os melhores durante essa longa jornada, e com certeza não poderia pedir turma melhor.
À minha orientadora Marcela Ururahy, pelo empenho, respeito, cuidado e conhecimento. Por ser uma excelente professora e grande ser humano, agradeço por todos os momentos de descontração, aprendizado e principalmente pela confiança. Obrigada por ter se tornado uma verdadeira amiga, quase uma mãe em Natal.
À esta Universidade por acolher tão bem e dar oportunidades maravilhosas para todos os estudantes; e aos professores, que conseguiram transmitir todos os seus conhecimentos durante essa trajetória.
1. INTRODUCTION ... 13
2. MATERIALS AND METHODS ... 14
2.1. Ethics ... 14
2.2. Patients ... 14
2.3. Biochemical Analyses ... 15
2.4. uEV isolation ... 15
2.5. Quantification of Nephrin, Podocin and WT-1 ... 15
2.6. Statistical Analyses ... 16 3. RESULTS ... 17 4. COMMENT ... 18 REFERENCES ... 20 TABLES ... 24 FIGURES ... 25 ANEXO ... 27
Increased nephrin in urinary extracellular vesicles of pregnant women with severe preeclampsia
Adrielle K. A. de LIMA, Ms, Natal, Rio Grande do Norte, Brazil. Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte.
Magna M. F. de OLIVEIRA, MSc, Natal, Rio Grande do Norte, Brazil. Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte.
Natalee A. A. MENDONÇA, Ms, Natal, Rio Grande do Norte, Brazil. Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte.
Tatiana X. da COSTA, PhD, School Maternity Januário Cicco, Federal University of Rio Grande do Norte.
Adriana A. de REZENDE, PhD, Natal, Rio Grande do Norte, Brazil. Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte.
Ricardo N. O. COBUCCI, MD, Department of Obstetrics and Gynecology, Potiguar University
Rand R. MARTINS, PhD, Natal, Rio Grande do Norte, Brazil. Department of Pharmacy, Federal University of Rio Grande do Norte
Marcela A. G. URURAHY, PhD, Natal, Rio Grande do Norte, Brazil. Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte
Financial support: This study was supported by grants from CNPq (457656/2014-0 and 435645/2018-8) and from the Federal University of Rio Grande do Norte.
Corresponding Author:
Marcela Abbott Galvão Ururahy
Address: Rua General Gustavo Cordeiro de Farias, S/N. Centro de Ciências da Saúde, Faculdade de Farmácia, Laboratório de Bioquímica Clínica. Bairro: Petrópolis, Natal/RN, Brazil
Phone number: +55 84 3342-9807 Fax number: +55 84 3342-9833
E-mail: marcelaururahy@yahoo.com.br
Word count: 2026
Condensation: Quantify podocitary proteins in urinary extracellular vesicles of pregnant women with severe preeclampsia to investigate their potential use as biomarkers in early diagnosis.
Short title: Increased nephrin in severe preeclampsia
AJOG at a Glance
Preeclampsia is a major cause of mortality in pregnancy and due to the serious associated outcomes, it is necessary to obtain a reliable and predictive marker for the diagnosis that could lead to an early intervention and avoid/minimize complications.
B. What are the key findings?
The increase in the urinary nephrin/creatinine ratio in preeclampsia associated with the significant positive results of the Area Under the Receiver Operating Characteristics (AUROC) analysis, suggests its potential role in predicting the severe form of the disease.
C. What does this study add to what is already known?
To the best of our knowledge this is the first study that quantified the nephrin expression in urinary extracellular vesicles of women with preeclampsia.
Abstract
Background: Preeclampsia is a systemic syndrome that affects pregnancy, characterized by increased blood pressure and, frequently, proteinuria. It is considered one of the main causes of maternal mortality and due to its possible serious complications, a predictive diagnostic tool is required. There has been increasing evidence that podocytes are related to kidney damage associated to preeclampsia and that the presence of podocyturia was found to be highly sensitive and specific to this disease. Objective: To characterize the expression profile of the podocyte proteins nephrin, podocin and Wilms’ tumor 1 (WT-1) present in the urinary extracellular vesicles of pregnant women diagnosed with severe preeclampsia in order to investigate their potential use as biomarkers in the early diagnosis of the disease. Study Design: This is a cross-sectional study, including 20 pregnant women divided into two groups. The first group with 10 pregnant women diagnosed with severe preeclampsia (case group - PE) and the second, with 10 normotensive pregnant women (control group - CG). Fasting blood samples were used to evaluate the patients' overall metabolic status. First morning urine samples were collected to determinate the albumin/creatinine ratio and to isolate urinary extracellular vesicles by ultracentrifugation. Nephrin, podocin and WT-1 expressions were evaluated by Western-blot. Results: Increased serum creatinine (p = 0.015), uric acid (p <0.001) and urinary albumin/creatinine ratio (p <0.001), as well as decreased serum albumin (p <0.001), total protein (p = 0.037) and calcium (p = 0.003) were found in case group when compared control group. Regarding the podocyte proteins in the urinary extracellular vesicles, significant increased nephrin/creatinine ratio (p = 0.008) was observed in preeclampsia when compared to those from normotensive pregnant women. In addition, the area under de Receiver Operating Characteristic Curve (AUROC) analysis showed that nephrin/creatinine ratio is a good predictor of albuminuria (AUROC = 0.833; p = 0.018). Conclusion: Increase of the nephrin in the urinary extracellular vesicles in pregnant
women with preeclampsia proves to be a potential predictor for the early diagnosis of the disease.
1. INTRODUCTION
Preeclampsia (PE) is a systemic syndrome, specific of pregnancy, that occurs from the 20th week of gestation, characterized by hypertension and, frequently, proteinuria 1. PE has been considered one of the main causes of maternal and fetal / neonatal mortality, however, its pathophysiology is not yet fully elucidated. Due to the possible serious associated outcomes, such as renal damage / failure, pulmonary edema and even death 1,2, a predictive diagnostic tool to PE is needed.
In recent decades, there has been an increase in evidence that podocytes, epithelial cells differentiated from glomeruli, are related to renal damage in PE. It was demonstrated that the presence of podocyturia was highly sensitive and specific for PE 3,4. These cells form a filtration slit composed of several proteins, among them, nephrin and podocin, forming the main selective filtering barrier of the kidney. Therefore, the podocytes are the highly specialized functional units in selective permeability, not allowing the passage of proteins 5. Another important protein, expressed only in the podocytes, is the factor of Wilms’ tumor 1 (WT-1), a transcription factor that stimulates the expression of genes that encode podocyte proteins, being considered a podocyte marker 6–8. The loss of podocyte in urine has been shown in experimental membranous nephropathy and in the urinary sediment of patients with a variety of active glomerular diseases including PE, being considered a more specific marker for glomerular damage in progress 9.
In addition, there are various evidence to suggest that podocytes and their specific proteins are present in the urine of pregnant women in the diagnosis of PE, supporting the hypothesis that damage and loss of podocytes are involved in proteinuria present in PE 9,10.
that evaluation cellular components carried by extracellular urinary vesicles (uEVs), allowed to concentrate and quantify podocyte proteins in the urine 11. These vesicles that can originate in all segments of the nephron, including podocytes, and carry, in addition to constitutive proteins, specific cellular components, such as nephrin, podocin and WT-1 12–14.
Therefore, this study investigated expression profile of the nephrin, podocin and WT-1 released in uEVs of pregnant women with severe PE, as potential biomarkers in the early diagnosis of the disease.
2. MATERIALS AND METHODS
2.1.Ethics
The study was approved by the Research Ethics Committee of the University Hospital Onofre Lopes (HUOL) of the Federal University of Rio Grande do Norte (UFRN), under number 1,942,794. All the patients who agreed to participate in the study, after explaining the research, signed the Written Informed Consent Form.
2.2.Patients
This is a cross-sectional study, where 20 pregnant women were recruited and divided into two groups. The first group with 10 pregnant women diagnosed with preeclampsia (case group - PE), attended at the Intensive Care Unit of the Januário Cicco Maternity School (MEJC); and the second group with 10 normotensive pregnant women (control Group - CG), attended at Family Health Units in Natal/RN. As exclusion criterion for two groups were: chronic hypertension, autoimmune disease, nephropathies of other causes and Diabetes mellitus, whether previous or gestational. After filling out an individual data sheet, a blood sample (12-14 hours fasting) and the first morning urine were taken.
2.3.Biochemical Analyses
The general metabolic status of patients was evaluated, including glucose, total cholesterol, triglycerides, urea, creatinine, albumin, total proteins, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), calcium, phosphorus, uric acid and lactate dehydrogenase (LDH) determination, using Labtest kits (Lagoa Santa, MG, Brazil) and LABMAX PLENNO equipment (Labtest, Lagoa Santa, MG, Brazil). Urinary albumin and creatinine were quantified for later calculation of the albumin / creatinine ratio (ACR), using Wiener kits (Wiener Laboratories, Rosario, Argentina) and the CMD-800 equipment (Wiener, Laboratories, Rosario, Argentina).
2.4.uEV isolation
uEVs were isolated following the methodology already described 15,16. Briefly, first the urine was centrifuged at 1,500 rpm for 5 min to remove cellular debris, then the samples were submitted to differential centrifugation, first at 17,000 xg for 10 min, second at 200,000 xg for 1h (MTX 150 ultracentrifuge Sorvall by Thermo Scientific, Asheville, NC, USA). The uEVs were reconstituted in Laemmli buffer to perform the Western blot (Bio-Rad, Hercules, CA, USA).
2.5.Quantification of Nephrin, Podocin and WT-1
The proteins present in the uEVs were separated by NUPAGE® 4-12% Bis-Tris gel electrophoresis (Novex by Life Technologies, Carlsbad, CA, USA) and transferred, using a dry system (IBlot® Dry Blotting Transfer System, Invitrogen, Carlsbad, CA, USA), to polyvinylidene fluoride membranes. For the Western Blot, monoclonal mouse anti-nephrin and anti-WT-1 antibodies (Santa Cruz, Dallas, TX, USA) and a secondary antibody conjugated to mouse anti-Ig G peroxidase (Santa Cruz, Dallas, TX, USA) were used. As for podocin, rabbit polyclonal anti-podocin antibody (Boster Biological Technology, CA, USA)
and a secondary antibody conjugated to rabbit anti-Ig G peroxidase (Santa Cruz, Dallas, TX, USA) were used.
The antigen-antibody reaction was visualized after exposure to the chemiluminescent substrate ECL Prime® (GE Healthcare, Uppsala, Sweden) and the bands were detected using the ChemiDoc equipment (Bio-Rad, Hercules, CA, USA). The densitometric analysis of the bands was performed using the ImageJ program (NIH, Bethesda, MD, USA). Nephrin, podocin and WT-1 immunoreactivities were normalized with Ponceau S 17,18. Then, the nephrin/creatinine, podocin/creatinine and WT-1/creatinine ratios were calculated 19,20.
2.6.Statistical Analyses
Regarding statistical analysis, the Shapiro-Wilk test was initially performed to assess the normal distribution of continuous variables, with the variables classified as non-parametric and, consequently, analyzed by the Mann-Whitney test. Receiver Operating Characteristic Curve (ROC) was plotted to analyze the discriminative power of the prediction tools, as well as the Area Under the Receiver Operating Characteristic (AUROC), being classified as excellent (0.9 to 1), good (0.8 to 0.9), moderate (0.7 to 0.8), weak (0.6 to 0.7) and non-discriminatory (0.5 to 0.6). In addition, the corresponding 95% confidence intervals (95% CI) were calculated. The programs used were JASP version Q.9.1 (JASP Team, 2018) and SPSS version 15.0 (SPSS Inc., Chicago, IL, USA), with results whose descriptive levels (p values) were less than 0.05 considered statistically significant.
3. RESULTS
The gestational age, in weeks, of the patients included in the study was 32 in the control group and 35 in the case group (p = 0.139). Regarding blood pressure, the control group obtained a systolic pressure of 100 mmHg, while the case group had 135 mmHg. Regarding diastolic, the control group obtained 70 mmHg and the case group 83 mmHg. It was possible to observe that the pregnant women in the case group had high blood pressure values, both systolic (p <0.001) and diastolic (p = 0.036), characteristic of PE.
Regarding the biochemical profile of the studied group (Table 1), it was possible to observe metabolic alterations characteristics of pregnant women diagnosed with PE when compared to the CG. Increases in serum creatinine (p = 0.015), and uric acid (p < 0.001) concentrations, as well as, in the ACR (p < 0.001), and decreases in serum albumin (p < 0.001), total protein (p = 0.037) and calcium (p = 0.003) concentrations were found.
Figure 1 shows the results of nephrin, podocin and WT-1 expression in the uEVs of the patients. An increase in nephrin/creatinine ratio was observed (p = 0.008). No differences were found for podocin (p = 0.082) and WT-1 (p = 0.602) expression between the studied groups.
In order to verify whether podocyte proteins were predictors of albuminuria, the ROC curve was analyzed. In this evaluation, it was possible to observe that only nephrin showed significant high AUROC values, indicating that it is a good predictor of albuminuria (AUROC = 0.833; p = 0.018) (Figure 2).
4. COMMENT
The increase in nephrin expression may be due to podocyte damage, including the detachment of podocytes from the basement membrane, and is in agreement with studies carried out, in which an increase in nephrinuria has been demonstrated in pregnant women with PE compared to pregnant women normotensive 21,22. Thus, this result corroborates the
potential role of the nephrin as a marker for preeclampsia. Furthermore, the significant result of the AUROC analysis for nephrin/creatinine ratio, showing that this ratio is a good predictor of albuminuria, reinforce the potential role of nephrin in uEVs as a marker of early diagnosis of PE.
However, in relation to podocin, there were no differences due to the disease, as in a study already done 5. And with regard to WT-1, no study was found evaluating its relationship with PE, but in other associated glomerular diseases to podocyte damage an increase in expression was observed 23, however not presented in this study.
Regarding the overall metabolic status of the patient, the decreased values of proteins and albumin associated with the increase in the ACR and the concentration of serum creatinine occur as a result of kidney damage, since the entire protein filtration and reabsorption process will be affected. The reduction in calcium concentration is probably associated with a decrease in albumin, since calcium is transported in the bloodstream linked to this protein 24. And considering the increase in uric acid, it can be associated with the condition of glomeruloendotheliosis and spasms, observed in PE, which causes a reduction in the filtration rate and consequently resulting in an increase in the serum concentration of this metabolic, being hyperuricemia one of the first manifestations described in PE 25. Therefore, the biochemical profile, with metabolic changes, found in the patients in the case group is consistent with what is expected in this disease.
The limitations of the present study are: the small number of participants; it is transversal, not being possible to assess whether the increase in expression occurred in the early stages of disease; and, only patients with severe PE were included, not being possible to extrapolate our findings to those with the mild cases of the disease.
Finally, the significant increase in nephrin in uEVs of the women with preeclampsia, associated with the significant positive results of the AUROC analysis, suggests that this protein may be a potential predictor of severe preeclampsia. However, future research, with a larger number of patients and including those with mild PE, are necessary to better understand the potential of using these non-invasively collected and sensitive markers in preeclampsia.
Acknowledgments: We thank the support of the Federal University of Rio Grande do Norte, the Maternity School Januário Cicco and the students of the Research Group on Metabolic Diseases (GPDM) and the Multidisciplinary Laboratory (LabMult).
REFERENCES
1. Moghaddas Sani H, Zununi Vahed S, Ardalan M. Preeclampsia: A close look at renal dysfunction. Biomed Pharmacother. 2019;109(October 2018):408-416. doi:10.1016/j.biopha.2018.10.082
2. Belovic DK, Plešinac S, Dotlić J, et al. Biochemical markers for prediction of hypertensive disorders of pregnancy. J Med Biochem. 2019;38(1):71-82. doi:10.2478/jomb-2018-0001
3. Craici IM, Wagner SJ, Bailey KR, et al. Podocyturia predates proteinuria and clinical features of preeclampsia: Longitudinal prospective study. Hypertension. 2013;61(6):1289-1296. doi:10.1161/HYPERTENSIONAHA.113.01115
4. White WM, Garrett AT, Craici IM, et al. Persistent urinary podocyte loss following preeclampsia may reflect subclinical renal injury. PLoS One. 2014;9(3):1-5. doi:10.1371/journal.pone.0092693
5. Gilani SI, Anderson UD, Jayachandran M, et al. Urinary extracellular vesicles of podocyte origin and renal injury in preeclampsia. J Am Soc Nephrol. 2017;28(11):3363-3372. doi:10.1681/ASN.2016111202
6. Musante L, Tataruch DE, Holthofer H. Use and isolation of urinary exosomes as biomarkers for diabetic nephropathy. Front Endocrinol (Lausanne). 2014;5(SEP):1-13. doi:10.3389/fendo.2014.00149
7. Ranghino A, Segoloni GP, Lasaponara F, Biancone L. Lymphatic disorders after renal transplantation: New insights for an old complication. Clin Kidney J. 2015;8(5):615-622. doi:10.1093/ckj/sfv064
dictates podocyte health and disease. J Am Soc Nephrol. 2015;26(3):677-691. doi:10.1681/ASN.2013101067
9. Jim B, Mehta S, Qipo A, et al. A comparison of podocyturia, albuminuria and nephrinuria in predicting the development of preeclampsia: A prospective study. PLoS One. 2014;9(7):1-7. doi:10.1371/journal.pone.0101445
10. Garovic VD, Wagner SJ, Turner ST, et al. Urinary podocyte excretion as a marker for preeclampsia. Am J Obstet Gynecol. 2007;196(4):320.e1-320.e7. doi:10.1016/j.ajog.2007.02.007
11. Hoorn EJ, Pisitkun T, Zietse R, et al. Prospects for urinary proteomics: Exosomes as a source of urinary biomarkers. Nephrology. 2005;10(3):283-290. doi:10.1111/j.1440-1797.2005.00387.x
12. Karpman D, Ståhl AL, Arvidsson I. Extracellular vesicles in renal disease. Nat Rev Nephrol. 2017;13(9):545-562. doi:10.1038/nrneph.2017.98
13. He F-F, Chen S, Su H, Meng X-F, Zhang C. Actin-associated Proteins in the Pathogenesis of Podocyte Injury. Curr Genomics. 2013;14(7):477-484. doi:10.2174/13892029113146660014
14. Morrison EE, Bailey MA, Dear JW. Renal extracellular vesicles: from physiology to clinical application. J Physiol. 2016;594(20):5735-5748. doi:10.1113/JP272182
15. Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A. 2004;101(36):13368-13373. doi:10.1073/pnas.0403453101
16. Zhou H, Cheruvanky A, Hu X, et al. Urinary exosomal transcription factors, a new class of biomarkers for renal disease. Kidney Int. 2008;74(5):613-621.
doi:10.1038/ki.2008.206
17. Fosang AJ, Colbran RJ. Transparency is the key to quality. J Biol Chem. 2015;290(50):29692-29694. doi:10.1074/jbc.E115.000002
18. Romero-Calvo I, Ocón B, Martínez-Moya P, et al. Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem. 2010;401(2):318-320. doi:10.1016/j.ab.2010.02.036
19. Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW. Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells. J Physiol. 2011;589(24):6119-6127. doi:10.1113/jphysiol.2011.220277
20. Zhou H, Yuen PST, Pisitkun T, et al. Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int. 2006;69(8):1471-1476. doi:10.1038/sj.ki.5000273
21. Jung YJ, Cho HY, Cho SH, et al. The level of serum and urinary nephrin in normal pregnancy and pregnancy with subsequent preeclampsia. Yonsei Med J. 2017;58(2):401-406. doi:10.3349/ymj.2017.58.2.401
22. Zhai T, Furuta I, Akaishi R, et al. Feasibility of nephrinuria as a screening tool for the risk of pre-eclampsia: Prospective observational study. BMJ Open. 2016;6(8):1-9. doi:10.1136/bmjopen-2016-011229
23. Zhou H, Kajiyama H, Tsuji T, et al. Urinary exosomal wilms’ tumor-1 as a potential biomarker for podocyte injury. Am J Physiol - Ren Physiol. 2013;305(4):1-8. doi:10.1152/ajprenal.00056.2013
24. Baird GS. Ionized calcium. Clin Chim Acta. 2011;412(9-10):696-701. doi:10.1016/j.cca.2011.01.004
25. Martinez NF, de Oliveira Filgueira GC, de Souza Rangel Machado J, et al. Características clínicas e laboratoriais de gestantes com pré-eclâmpsia versus hipertensão gestacional. Rev Bras Ginecol e Obstet. 2014;36(10):461-466. doi:10.1590/SO100-720320140005029
TABLES
Table 1 – Biochemical parameters according to the groups studied
Variables CG n = 10 PE n = 10 p – value Glucose, mg/dL 73.3 ± 7.2 80.3 ± 8.7 0.067 Urea, mg/dL 15.8 ± 4.8 21.6 ± 10.7 0.153 Creatinine, mg/dL 0.51 (0.4 – 0.6) 0.75 (0.6 – 0.8) 0.015 Uric acid, mg/dL 3.1 ± 1.0 6.1 ± 1.2 <0.001 Total cholesterol, mg/dL 231.3 ± 29.5 195.1 ± 55.7 0.086 Triglycerides, mg/dL 212.0 ± 44.7 215.5 ± 80.3 0.914 AST, U/L 12 ± (8.5 – 15.5) 17.5 (10.7 – 63.5) 0.103 ALT, U/L 8.5 (7 – 13.2) 14 (7 – 78.7) 0.223 GGT, U/L 16.3 ± 5.2 38.6 ± 21.8 0.006
Alkaline phosphatase, U/L 121.6 ± 27.7 175.4 ± 84.9 0.073
LDH, U/L 335 (284 – 395) 381 (320 – 582) 0.218 Total proteins, g/dL 5.8 (5.3 – 6.6) 5.3 (4.5 – 5.7) 0.037 Albumin, g/dL 3.3 (3.0 – 3.5) 2.7 (2.3 – 3.0) <0.001 Calcium, mg/dL 9.0 ± 1.1 7.4 ± 0.8 0.003 Phosphorus, mg/dL 4.2 ± 0.5 4.6 ± 0.7 0.162 ACR, mg/g 6 (4 – 8) 117 (28 – 650) <0.001
Values are represented as mean ± standard deviation or median (interquartile range). AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma glutamyl transferase; LDH, lactate dehydrogenase; ACR, albumin / creatinine ratio.; LDH, lactate desidrogenase. n, absolute value. CG, control group; PE, preeclampsia group; n, number of individuals. Significant values were highlighted in bold.
FIGURES
Figure 1: Detection of nephrin, podocin and WT-1 in urinary extracellular vesicles of the studied patients. Densitometry analysis of urinary nephrin/creatinine ratio (A), urinary podocin/creatinine ratio (B) and urinary WT-1/creatinine ratio (C). Representative immunoblots for urinary nephrin (D), urinary podocin (E) and WT-1 proteins in uEVs (F). CG, Control group; PE, Preeclampsia group; WT-1, Wilms Tumor Factor – 1. Results are expressed as mean standard deviation.
Figure 2: Receiver operating characteristic curves (ROCs) for albumin to creatinine ratio (ACR).
AUROC, area under the receiver operating characteristic curve; 95% CI, 95% confidence interval.
