Research Article Podocyturia as a Diagnostic Marker for Preeclampsia amongst High-Risk Pregnant Patients

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1 Journl of Pregnncy Volume 2012, Article ID , 5 pges doi: /2012/ Reserch Article Podocyturi s Dignostic Mrker for Preeclmpsi mongst High-Risk Pregnnt Ptients Belind Jim, 1 Pscle Jen-Louis, 1, 2 Andi Qipo, 1 Dvid Grry, 2 Smi Min, 3 Tulio Mtos, 4 Christopher Provenzno, 1 nd Anjli Achry 1 1 Division of Nephrology, Deprtment of Medicine, Jcobi Medicl Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA 2 Deprtment of Obstetrics nd Gynecology, Montefiore Medicl Center, Bronx, NY, USA 3 Advocte Christ Medicl Center, Ok Lwn, IL, USA 4 Columbi University Medicl Center, New York, NY, USA Correspondence should be ddressed to Belind Jim, belind bun jim@yhoo.com Received 1 August 2011; Revised 1 November 2011; Accepted 18 November 2011 Acdemic Editor: Mri Kyrgiou Copyright 2012 Belind Jim et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Urinry podocyte (podocyturi) hs been studied s dignostic mrker for preeclmpsi. We sought to vlidte its use in preeclmpsi nd in differentiting it from other high risk pregnncy sttes. We studied n obstetric popultion t high risk to develop preeclmpsi (study group) nd uncomplicted pregnncies (control group) by nlyzing their urine sediment for podocytes within 24 hours of delivery. Podocytes were identified by immunohistochemistry using the podocyte-specific protein synptopodin. Of the 56 ptients who were enrolled, 29 ptients were dignosed with preeclmpsi, 9 ptients hd hypertensive conditions such s chronic nd gesttionl hypertension, 6 ptients hd Type I/II nd gesttionl dibetes mellitus, 3 ptients were clssified s others, nd 9 ptients exhibited uncomplicted pregnncies. Podocyturi ws identified in 11 out of 29 (38%) of ptients with preeclmpsi/eclmpsi, 3 out of 9 (33%) with gesttionl nd chronic hypertension, nd 3 out of 6 (50%) with Type I/II nd gesttionl dibetes mellitus. None of the 9 ptients (0%) with uncomplicted pregnncies demonstrted podocyturi. The sensitivity nd specificity of podocyturi for preeclmpsi were found to be 38% nd 70%. Our study showed tht podocyturi does not pper to be sensitive nor specific mrker to dignose preeclmpsi. 1. Introduction Preeclmpsi is disorder ffecting 5 to 10% of pregnncies nd is cliniclly chrcterized by new-onset hypertension nd proteinuri. Despite significnt progress in our understnding of preeclmpsi, there is need for relible dignostic biomrker for use in clinicl prctice. In the serch for biologiclly plusible biomrker, there hve been ttempts to reconcile clinicl findings with pthologic chnges in renl biopsies of preeclmpsi. For exmple, renl pthology of preeclmpsi in the kidney is clssiclly described s endotheliosis, or swelling of endothelil cells in the glomerulus. The podocyte, specilized viscerl epithelil cell tht lines nd forms the slit diphrgm of the glomerulr bsement membrne, hs trditionlly been thought to be unffected. However, more recent microscopic studies demonstrte tht podocytes re structurlly chnged nd hrbor protein resorption droplets [1]. Furthermore, there is evidence tht selected podocyte-specific proteins such s nephrin, synptopodin, nd GLEPP-1 re downregulted in preeclmpsi, while VEGF nd Flt-1 re incresed [2, 3]. Grovic et l. studied the use of podocytes in the urine (podocyturi) s dignostic mrkers nd found podocyturi to be highly sensitive nd specific for preeclmpsi [4]. We sought to study the use of podocyturi to dignose preeclmpsi nd differentite it from other conditions tht my hve similr presenttion in high risk pregnncy popultion. We discovered tht podocyturi ws not very sensitive nor specific in mking this dignosis. Furthermore, podocyturi ws found frequently in other high risk pregnncy sttes such s chronic hypertension nd gesttionl dibetes.

2 2 Journl of Pregnncy 2. Mterils nd Methods 2.1. Study nd Control Subjects. We recruited two groups of ptients ll 18 yers of ge: uncomplicted pregnnt subjects (control group) nd women t risk for pregnncy complictions (high-risk group) s described below from the obstetric inptient service t Jcobi Medicl Center, Bronx, NY, USA. Rndom urine smples were obtined from the subjects within 24 hours of delivery. Inclusion criteri for high-risk group were dignosis of preeclmpsi, chronic hypertension (HTN), gesttionl HTN, Type I nd Type II dibetes mellitus (DM), gesttionl DM, mixed connective tissue disese, nd pregnncies with fetl chromosoml bnormlities. Exclusion criteri were ptients under the ge of 18 nd bsence of the bove-mentioned dignosis. Inclusion criteri for the control group were uncomplicted pregnncies nd deliveries nd bsence of the bovementioned high risk pregnncy sttes. Exclusion criteri for the control group were <18 yers of ge, preexisting high risk pregnncy sttes or complicted deliveries. Dignosis of preeclmpsi fulfilled the criteri of new onset of HTN with blood pressure of 140/90 mmhg fter 20 weeks of gesttion nd proteinuri of >300 mg of protein in 24-hour urine specimen or 1+ protein on urinlysis smple without evidence of nother cuse, such s urinry trct infection or inflmmtion. Chronic HTN ws defined s preexisting HTN or blood pressure of 140/90 mmhg before 20 weeks of gesttion. Gesttionl DM ws defined s ny degree of glucose intolernce with onset of first recognition during pregnncy. This study ws pproved by the Internl Review Bord of Albert Einstein College of Medicine Urinry Protein Quntifiction. Urinry protein ws quntified either from 24-hour urine smple collection or extrpolted from rndom urine dipstick. For exmple, when only dipstick urine protein ws vilble, the vlue ws extrpolted to 24-hour vlue bsed on the following: negtive protein corresponds to less thn 150 mg/24 hours, trce protein corresponds to 150 mg/24 hours, 1+ corresponds to bout mg/24 hours, 2+ to g/24 hours, nd 3+ to 2 5 g/24 hours Podocyturi. Twenty ml of freshly voided urine ws centrifuged t 700 g for 5 min. The sediment pellet ws crefully recovered by spirting the superntnt, wshed twice with PBS nd resuspended in 1 ml of PBS. Aliquots of 100 μl of the resuspended sediment were centrifuged onto slides using the Shndon Cytospin 4 Cytocentrifuge (Thermo Electron Corportion, Asheville, NC), ir-dried nd fixed with 1 : 1 cetone/methnol for 10 minutes. The slides were immersed with PBS/1% H 2 O 2 for 15 minutes nd wshed with deionized wter. Subsequently, ntigen retrievl ws chieved by stem-heting in solution of citrte buffer, ph 6.0, for 15 minutes nd blocked with 10% horse serum in PBS nd 2% BSA. Slides were incubted overnight with monoclonl mouse ntihumn synptopodin ntibody t 1 : 1 dilution (gift of Dr. Peter Mundel, Msschusetts Generl Hospitl, Boston, MA) followed by horse ntimouse IgG t 1 : 1000 dilution (Dko Inc. Crpinteri, CA) s secondry ntibody for 30 minutes. Sections were then incubted in vidin-biotin complex t 1 : 25 dilution (Vector Lbs, Burlingme, CA) nd developed using diminobenzidine (DAB) s chromogen. After wshing, the sections were counter-stined with hemtoxylin nd coverslipped. Negtive controls were crried out by incubtion in the bsence of the primry ntibody. Podocytes were identified by positive DAB stining under light microscopy Sttisticl Anlysis. Difference in clinicl vribles between more thn two groups ws determined by Kruskl- Wllis method, differences between two groups were determined by Mnn Whitney method. Anlyses were performed with STATA Version 8.2 nd GrphPd Prism Version 5.02 for Windows softwre, nd results were considered sttisticlly significnt if P<0.05. For test chrcteristics of podocyturi, sensitivity, specificity, positive predictive vlue, nd negtive predictive vlue were clculted for ech high risk dignosis. 3. Results In totl, 56 ptients were recruited. The dignoses t time of urine collection were s follows: preeclmpsi (n = 28), eclmpsi (n = 1), chronic hypertension (n = 3), gesttionl hypertension (n = 6),Type I DM(n = 1), Type II DM (n = 1), gesttionl DM (n = 4), connective tissue disorder (n = 1), mrginl previ(n = 1), chromosoml nomly (n = 1), nd uncomplicted pregnncy (n = 9). The cliniclchrcteristicsofllsubjectsredescribedintble 1. Podocyturi (Figures 1() nd 1(b)) ws present in 11 out of 29 (38%) ptients with preeclmpsi/eclmpsi, 3 out of 9 (33%) with chronic nd gesttionl HTN, nd 3 out of 6 (50%) with gesttionl DM nd Type I/II DM (Tble 2). Among ptients ctegorized s other, 2 (mrginl previ nd chromosoml nomly) out of 3 ptients exhibited podocyturi (66%). In contrst, 0 out of 9 ptients (0%) with uncomplicted pregnncies demonstrted podocyturi. Bsed on these findings, we clculted the sensitivity nd specificity of podocyturi for preeclmpsi to be 38% nd 70%, s compred to women of HTN of ny type, in whom it ws 33% nd 66%, respectively (Tble 3).The sensitivity nd specificity for DM of ny type were 50% nd 68%, respectively. The positive predictive vlue ws 57% for preeclmpsi, s compred with 15% for both HTN of ny type nd DM of ny type. The negtive predictive vlue, however, ws poor for preeclmpsi t 51%, s compred with 83% nd 91% for HTN of ny type nd DM of ny type, respectively. 4. Discussion Podocyturi is well described in mny glomerulr diseses such s Type I DM, IgA nephropthy, lupus nephritis, nd membrnous nephropthy [5, 6]. Though endothelil injury is thought to be the min lesion in preeclmpsi, more recently, derngements of the podocyte with downregultion of selected podocyte-specific proteins in renl biopsies nd

3 Journl of Pregnncy 3 Preeclmpsi () Chronic HTN (b) Uncomplicted pregnncy (c) Uncomplicted pregnncy (d) Norml kidney tissue (e) Norml bldder tissue (f) Figure 1: Podocyturi in high risk nd uncomplicted pregnncies. () nd (b) representtive imges of podocyturi identified by positive synptopodin stining, (c) nd (d) Representtive imges of negtive stining in uncomplicted pregnncies, (e) synptopodin stining in norml kidney tissue s positive control, nd (f) synptopodin stining in norml bldder tissue s negtive control. presence of nephrin nd podoclyxin (podocyte specific proteins) in the urine hve been described [2, 7]. In this study, we sought to describe the presence of podocyturi in preeclmpsi nd other high risk pregnncy sttes using the podocyte mrker synptopodin for identifiction. We found tht podocyturi ws neither sensitive nor specific in mking this dignosis. These results re in contrst to n erlier studybygrovicetl.[2]. Though the exct reson for this discrepncy is uncler, immunofluorescent stining of podocyte-specific proteins, in generl, does not pper to be n ccurte tool to identify podocytes, s these podocytes my be prietl in origin [8] nd my lsobe poptotic [9]. Thus, the utility of urinry podocytes to detect ongoing glomerulr dmge in women with preeclmpsi s previously suggested [10] is uncler. Furthermore, our study showed presence of podocyturi in other high-risk pregnncy sttes such s DM (gesttionl or Type I/II), HTN (gesttionl or chronic). This finding is not unexpected since podocyturi nd urinry podocyte mrna hve been described in nonpregnnt dibetic ptients [6] nd in nonpregnnt hypertensive ptients [11] respectively. Interestingly, the number of urinry podocytes hs lso shown sttisticlly significnt correltion with blood pressure but not proteinuri in preeclmpsi [12].

4 4 Journl of Pregnncy Vrible Norml (9) Preeclmpsi/eclmpsi (29) Tble 1: Ptient Chrcteristics. HTN-chronic/gesttion (9) b DM-Type I/II/Gesttionl (6) c Other (3) P vlue Mternl ge (yr) 29.8 ± ± ± ± ± Gesttionl ge (wk) 37.4 ± ± ± ± ± 1.5 < Systolic blood pressure (mmhg) ± ± ± ± ± 6.6 < Distolic blood pressure (mmhg) 72.4 ± ± ± ± ± 9.8 < d Proteinuri (mg/24 hr) 149 ± ± ± ± ± 68 < HTN: hypertension. b DM: dibetes mellitus. c Other: dignoses mrginl previ (1), chromosoml nomly (1), connective tissue disorder (1). d 24-hour proteinuri extrpolted from urine dipstick vlue s described in methods section. Tble 2: Presence of podocyturi in vrious pregnncy ctegories. Podocyte positive Podocyte negtive % Positive Preeclmpsi/eclmpsi (29) % HTN-Gesttionl/chronic (9) % DM: ny type (6) % Others (3) % b Controls (9) 0 9 0% Other: dignoses of mrginl previ (1), chromosoml nomly (1), connective tissue disorder (1). b Controls: uncomplicted pregnncies. Tble 3: Test chrcteristics for podocyturi. Sensitivity Specificity Positive predictive vlue Negtive predictive vlue Preeclmpsi/eclmpsi 38% 70% 57% 51% HTN (ny type) 33% 66% 15% 83% DM (ny type) 50% 68% 15% 91% Though podocyturi might help shed light on the pthophysiology of preeclmpsi, we feel tht its clinicl utility is limited. There re severl limittions in relying on the podocytes s biomrkers. Identifying podocytes in the urine is highly lborious nd is techniclly chllenging. Both cytospin methods (s ws performed in this study) nd cultivtion of podocytes in culture re frught with difficulties nd re not cost-effective. It my not be esy to eliminte podocyte cell debris when counting podocytes from cytospin specimen of fresh sediment [9]. Growing urinry podocytes in cell culture, on the other hnd, re frequently limited by bcteril or fungl contmintion s the urine my not hve been collected under sterile conditions. Furthermore, these cells my proliferte, undergo poptosis, or not ttch to the culture dish, thereby flsely representing the true podocyte count [9]. Interobserver bis poses yet nother obstcle s it requires highly trined cytologist to correctly identify these cells. Since clinicl guidelines re vilble to dignose preeclmpsi, some hve questioned whether the ddition of urinry mrker is necessry. We feel tht the utility of this mrker becomes importnt when the dignosis of preeclmpsi is in question nd when the clinicl scenrio is complicted by the presence of preexisting HTN, DM, or other glomerulr diseses such s lupus nephritis. In those cses, the tretment would ger towrds the underlying condition, in ddition to supportive cre. Thus, specific mrker tht is discovered through our understnding of the pthophysiology of preeclmpsi remins crucil. Whether podocyturi is tht mrker remins unnswered. 5. Limittions of the Study The mjor limittion of our study is the smll smple size. Our gol ws to confirm the previously shown high sensitivity nd specificity of podocyturi in preeclmpsi. Though lrger smple size would be idel, we feel tht of our totl smple size of 56 with 29 ptients dignosed with preeclmpsi/eclmpsi, nd 18 ptients with other high-risk dignoses, is n dequte smple size to demonstrte tht podocyturi lcks sensitivity nd specificity to be dignostic mrker of preeclmpsi.

5 Journl of Pregnncy 5 6. Conclusions We discovered tht podocyte loss is present not only in preeclmpsi but in other high risk pregnncy sttes. In ddition, podocyturi ws not found in mjority of ptients dignosed with preeclmpsi. We relize our finding of reltively low sensitivity nd specificity is not conclusive. However, our findings rise n importnt note of cution of relying on the limited findings in the literture regrding the predictive vlue of podocyturi in preeclmpsi nd encourge lrger studies. Given our current knowledge of the complex pthophysiology of the disese such s the significnt role of vsculr endothelil growth fctor (VEGF) signling in mintining helthy endothelium nd cross tlk between the podocyte nd vsculr endothelil cell [13], it is unlikely tht ny single test or cell type will be ble to predict preeclmpsi. A pnel of biomrkers reflective of this complexity my be idel for dignosis. [10] J. Muller-Deile nd M. Schiffer, Renl involvement in preeclmpsi: similrities to VEGF bltion therpy, Journl of Pregnncy, vol. 2011, Article ID , 6 pges, [11]G.Wng,F.M.Li,B.C.Kwnetl., Podocytelossinhumn hypertensive nephrosclerosis, Americn Journl of Hypertension, vol. 22, no. 3, pp , [12] K. Ait, M. Etoh, H. Hmd et l., Acute nd trnsient podocyte loss nd proteinuri in preeclmpsi, Nephron Clinicl Prctice, vol. 112, no. 2, pp. c65 c70, [13] V. Eremin, H. J. Belde, nd S. E. Quggin, Role of the VEGF-A signling pthwy in the glomerulus: evidence for crosstlk between components of the glomerulr filtrtion brrier, Nephron Physiology, vol. 106, no. 2, pp. p32 p37, Acknowledgment The uthors thnk Dr. Hillel W. Cohen for his ssistnce in sttisticl nlysis nd editing of the pper. References [1] I. E. Stillmn nd S. A. Krumnchi, The glomerulr injury of preeclmpsi, Journl of the Americn Society of Nephrology, vol. 18, no. 8, pp , [2] V.D.Grovic,S.J.Wgner,L.M.Petrovicetl., Glomerulr expression of nephrin nd synptopodin, but not podocin, is decresed in kidney sections from women with preeclmpsi, Nephrology Dilysis Trnsplnttion, vol. 22, no. 4, pp , [3] S. Zho, X. Gu, L. J. Groome, nd Y. Wng, Decresed nephrin nd GLEPP-1, but incresed VEGF, Flt-1, nd nitrotyrosine, expressions in kidney tissue sections from women with preeclmpsi, Reproductive Sciences, vol. 16, no. 10, pp , [4] V. D. Grovic, S. J. Wgner, S. T. Turner et l., Urinry podocyte excretion s mrker for preeclmpsi, Americn Journl of Obstetrics nd Gynecology, vol. 196, no. 4, pp. 320.e1 320.e7, [5] S.U.Vogelmnn,W.J.Nelson,B.D.Myers,ndK.V.Lemley, Urinry excretion of vible podocytes in helth nd renl disese, Americn Journl of Physiology Renl Physiology, vol. 285, no. 1, pp. F40 F48, [6] T. Nkmur, C. Ushiym, S. Suzuki et l., Urinry excretion of podocytes in ptients with dibetic nephropthy, Nephrology Dilysis Trnsplnttion, vol. 15, no. 9, pp , [7] Y. Wng, S. Sho, S. Loyd, nd L. J. Groome, Incresed urinry excretion of nephrin, podoclyxin, nd βig-h3 in women with preeclmpsi, Americn Journl of Physiology Renl Physiology. In press. [8] J. Achenbch, M. Mengel, I. Tossidou et l., Prietl epitheli cells in the urine s mrker of disese ctivity in glomerulr diseses, Nephrology Dilysis Trnsplnttion, vol. 23, no. 10, pp , [9] M. Cmici, Urinry biomrkers of podocyte injury, Biomrkers in Medicine, vol. 2, no. 6, pp , 2008.

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