
 preeclampsia is a disorder affecting 5 to 10% of pregnancies and is clinically characterized by new - onset hypertension and proteinuria . despite significant progress in our understanding of preeclampsia , there is a need for a reliable diagnostic biomarker for use in clinical practice . in the search for a biologically plausible biomarker , there have been attempts to reconcile clinical findings with pathologic changes in renal biopsies of preeclampsia . for example , renal pathology of preeclampsia in the kidney is classically described as  endotheliosis  , or swelling of endothelial cells in the glomerulus .
the podocyte , a specialized visceral epithelial cell that lines and forms the slit diaphragm of the glomerular basement membrane , has traditionally been thought to be unaffected .
however , more recent microscopic studies demonstrate that podocytes are structurally changed and harbor protein resorption droplets .
furthermore , there is evidence that selected podocyte - specific proteins such as nephrin , synaptopodin , and glepp-1 are downregulated in preeclampsia , while vegf and flt-1 are increased [ 2 , 3 ] .
studied the use of podocytes in the urine ( podocyturia ) as diagnostic markers and found podocyturia to be highly sensitive and specific for preeclampsia .
we sought to study the use of podocyturia to diagnose preeclampsia and differentiate it from other conditions that may have a similar presentation in a high risk pregnancy population .
furthermore , podocyturia was found frequently in other high risk pregnancy states such as chronic hypertension and gestational diabetes .

 we recruited two groups of patients all 18 years of age : uncomplicated pregnant subjects ( control group ) and women at risk for pregnancy complications ( high - risk group ) as described below from the obstetric inpatient service at jacobi medical center , bronx , ny , usa . random urine samples
inclusion criteria for high - risk group were diagnosis of preeclampsia , chronic hypertension ( htn ) , gestational htn , type i and type ii diabetes mellitus ( dm ) , gestational dm , mixed connective tissue disease , and pregnancies with fetal chromosomal abnormalities .
exclusion criteria were patients under the age of 18 and absence of the above - mentioned diagnosis .
inclusion criteria for the control group were uncomplicated pregnancies and deliveries and absence of the above - mentioned high risk pregnancy states .
exclusion criteria for the control group were < 18 years of age , preexisting high risk pregnancy states or complicated deliveries .
diagnosis of preeclampsia fulfilled the criteria of new onset of htn with blood pressure of 140/90  mmhg after 20 weeks of gestation and proteinuria of > 300  mg of protein in a 24-hour urine specimen or 1 + protein on a urinalysis sample without evidence of another cause , such as urinary tract infection or inflammation .
chronic htn was defined as preexisting htn or blood pressure of 140/90  mmhg before 20 weeks of gestation .
gestational dm was defined as any degree of glucose intolerance with onset of first recognition during pregnancy .
this study was approved by the internal review board of albert einstein college of medicine .
urinary protein was quantified either from a 24-hour urine sample collection or extrapolated from a random urine dipstick .
for example , when only a dipstick urine protein was available , the value was extrapolated to a 24-hour value based on the following : negative protein corresponds to less than 150  mg/24 hours , trace protein corresponds to 150  mg/24 hours , 1 + corresponds to about 200500  mg/24 hours , 2 + to 0.51.5  g/24 hours , and 3 + to 25  g/24 hours .
twenty ml of freshly voided urine was centrifuged at 700  g for 5  min .
the sediment pellet was carefully recovered by aspirating the supernatant , washed twice with pbs and resuspended in 1  ml of pbs .
aliquots of 100  l of the resuspended sediment were centrifuged onto slides using the shandon cytospin 4 cytocentrifuge ( thermo electron corporation , asheville , nc ) , air - dried and fixed with 1  :  1 acetone / methanol for 10 minutes .
the slides were immersed with pbs/1% h2o2 for 15 minutes and washed with deionized water .
subsequently , antigen retrieval was achieved by steam - heating in a solution of citrate buffer , ph 6.0 , for 15 minutes and blocked with 10% horse serum in pbs and 2% bsa .
slides were incubated overnight with monoclonal mouse antihuman synaptopodin antibody at 1  :  1 dilution ( gift of dr .
peter mundel , massachusetts general hospital , boston , ma ) followed by horse anti - mouse igg at 1  :  1000 dilution ( dako inc .
sections were then incubated in avidin - biotin complex at 1  :  25 dilution ( vector labs , burlingame , ca ) and developed using diaminobenzidine ( dab ) as chromogen .
difference in clinical variables between more than two groups was determined by kruskal - wallis method , differences between two groups were determined by mann whitney method .
analyses were performed with stata version 8.2 and graphpad prism version 5.02 for windows software , and results were considered statistically significant if p < 0.05 . for test characteristics of podocyturia ,
sensitivity , specificity , positive predictive value , and negative predictive value were calculated for each high risk diagnosis .
the diagnoses at time of urine collection were as follows : preeclampsia ( n = 28 ) , eclampsia ( n = 1 ) , chronic hypertension ( n = 3 ) , gestational hypertension ( n = 6 ) , type i dm ( n = 1 ) , type ii dm ( n = 1 ) , gestational dm ( n = 4 ) , connective tissue disorder ( n = 1 ) , marginal previa ( n = 1 ) , chromosomal anomaly ( n = 1 ) , and uncomplicated pregnancy ( n = 9 ) . the clinical characteristics of all subjects are described in table 1 .
podocyturia ( figures 1(a ) and 1(b ) ) was present in 11 out of 29 ( 38% ) patients with preeclampsia / eclampsia , 3 out of 9 ( 33% ) with chronic and gestational htn , and 3 out of 6 ( 50% ) with gestational dm and type i / ii dm ( table 2 ) . among patients categorized as 
, 2 ( marginal previa and chromosomal anomaly ) out of 3 patients exhibited podocyturia ( 66% ) . in contrast , 0 out of 9 patients ( 0% ) with uncomplicated pregnancies demonstrated podocyturia . based on these findings
, we calculated the sensitivity and specificity of podocyturia for preeclampsia to be 38% and 70% , as compared to women of htn of any type , in whom it was 33% and 66% , respectively ( table 3 ) .
the sensitivity and specificity for dm of any type were 50% and 68% , respectively .
the positive predictive value was 57% for preeclampsia , as compared with 15% for both htn of any type and dm of any type .
the negative predictive value , however , was poor for preeclampsia at 51% , as compared with 83% and 91% for htn of any type and dm of any type , respectively .
podocyturia is well described in many glomerular diseases such as type i dm , iga nephropathy , lupus nephritis , and membranous nephropathy [ 5 , 6 ] .
though endothelial injury is thought to be the main lesion in preeclampsia , more recently , derangements of the podocyte with downregulation of selected podocyte - specific proteins in renal biopsies and presence of nephrin and podocalyxin ( podocyte specific proteins ) in the urine have been described [ 2 , 7 ] . in this study
, we sought to describe the presence of podocyturia in preeclampsia and other high risk pregnancy states using the podocyte marker synaptopodin for identification .
though the exact reason for this discrepancy is unclear , immunofluorescent staining of podocyte - specific proteins , in general , does not appear to be an accurate tool to identify podocytes , as these podocytes may be parietal in origin   and may also be apoptotic .
thus , the utility of urinary podocytes to detect ongoing glomerular damage in women with preeclampsia as previously suggested   is unclear .
furthermore , our study showed presence of podocyturia in other high - risk pregnancy states such as dm ( gestational or type
this finding is not unexpected since podocyturia and urinary podocyte mrna have been described in nonpregnant diabetic patients   and in nonpregnant hypertensive patients   respectively .
interestingly , the number of urinary podocytes has also shown a statistically significant correlation with blood pressure but not proteinuria in preeclampsia .
though podocyturia might help shed light on the pathophysiology of preeclampsia , we feel that its clinical utility is limited .
both cytospin methods ( as was performed in this study ) and cultivation of podocytes in culture are fraught with difficulties and are not cost - effective .
it may not be easy to eliminate podocyte cell debris when counting podocytes from cytospin specimen of fresh sediment .
growing urinary podocytes in cell culture , on the other hand , are frequently limited by bacterial or fungal contamination as the urine may not have been collected under sterile conditions .
furthermore , these cells may proliferate , undergo apoptosis , or not attach to the culture dish , thereby falsely representing the true podocyte count .
interobserver bias poses yet another obstacle as it requires a highly trained cytologist to correctly identify these cells .
since clinical guidelines are available to diagnose preeclampsia , some have questioned whether the addition of a urinary marker is necessary .
we feel that the utility of this marker becomes important when the diagnosis of preeclampsia is in question and when the clinical scenario is complicated by the presence of preexisting htn , dm , or other glomerular diseases such as lupus nephritis . in those cases ,
thus , a specific marker that is discovered through our understanding of the pathophysiology of preeclampsia remains crucial .
our goal was to confirm the previously shown high sensitivity and specificity of podocyturia in preeclampsia .
though a larger sample size would be ideal , we feel that of our total sample size of 56 with 29 patients diagnosed with preeclampsia / eclampsia , and 18 patients with other high - risk diagnoses , is an adequate sample size to demonstrate that podocyturia lacks sensitivity and specificity to be a diagnostic marker of preeclampsia .
we discovered that podocyte loss is present not only in preeclampsia but in other high risk pregnancy states .
in addition , podocyturia was not found in a majority of patients diagnosed with preeclampsia .
however , our findings raise an important note of caution of relying on the limited findings in the literature regarding the predictive value of podocyturia in preeclampsia and encourage larger studies .
given our current knowledge of the complex pathophysiology of the disease such as the significant role of vascular endothelial growth factor ( vegf ) signaling in maintaining a healthy endothelium and cross talk between the podocyte and vascular endothelial cell , it is unlikely that any single test or cell type will be able to predict preeclampsia .
 
 urinary podocyte ( podocyturia ) has been studied as a diagnostic marker for preeclampsia . 
 we sought to validate its use in preeclampsia and in differentiating it from other high risk pregnancy states . 
 we studied an obstetric population at high risk to develop preeclampsia ( study group ) and uncomplicated pregnancies ( control group ) by analyzing their urine sediment for podocytes within 24 hours of delivery . 
 podocytes were identified by immunohistochemistry using the podocyte - specific protein synaptopodin . 
 of the 56 patients who were enrolled , 29 patients were diagnosed with preeclampsia , 9 patients had hypertensive conditions such as chronic and gestational hypertension , 6 patients had type i / ii and gestational diabetes mellitus , 3 patients were classified as others , and 9 patients exhibited uncomplicated pregnancies . 
 podocyturia was identified in 11 out of 29 ( 38% ) of patients with preeclampsia / eclampsia , 3 out of 9 ( 33% ) with gestational and chronic hypertension , and 3 out of 6 ( 50% ) with type i / ii and gestational diabetes mellitus . 
 none of the 9 patients ( 0% ) with uncomplicated pregnancies demonstrated podocyturia . 
 the sensitivity and specificity of podocyturia for preeclampsia were found to be 38% and 70% . 
 our study showed that podocyturia does not appear to be a sensitive nor a specific marker to diagnose preeclampsia . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Limitations of the Study
6. Conclusions