# Fetal biomarkers for lower urinary tract obstruction secondary to posterior urethral valves

Joost Schanstra, Stéphane Decramer, Bénédicte Buffin-Meyer, Julie Klein, Magdalena Fossum, Hsi-Yang Wu

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## To cite this version:

Joost Schanstra, Stéphane Decramer, Bénédicte Buffin-Meyer, Julie Klein, Magdalena Fossum, et al. Fetal biomarkers for lower urinary tract obstruction secondary to posterior urethral valves. Journal of Pediatric Urology, 2024, 20 (3), pp.492-496. 10.1016/j.jpurol.2024.01.011. hal-04920975

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## Review Article

# Fetal biomarkers for lower urinary tract obstruction secondary to posterior urethral valves

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updates

Joost P. Schanstra<sup>a,b</sup>, Stéphane Decramer<sup>a,b,c,d</sup>, Bénédicte Buffin-Meyer<sup>a,b</sup>, Julie Klein<sup>a,b</sup>, Magdalena Fossum<sup>e,f,g</sup>, Hsi-Yang Wu<sup>g,1</sup>

## Summary

Today, prenatal diagnosis of congenital urogenital malformations is mostly dependent on anatomical variations found on imaging. However, these findings can mislead us in telling us when to intervene, and about post-natal prognosis. Since many findings are dependent on multiple assessments, delayed diagnosis can occur, leading to less optimal outcomes compared to early intervention. Analyses of fetal urinary biomarkers have been proposed as a method of finding biological changes that are predictive for diagnosis and prognosis in fetuses at risk of kidney disease.

We interviewed a group of researchers that have demonstrated that by combining multiple omics traits extracted from fetal urine, the biological variability found in single omics data can be circumvented. By analyzing multiple fetal urine peptides and metabolites at single time point, the prognostic power of postnatal renal outcome in fetuses with lower urinary tract obstruction is significantly increased. In this interview, we inquired about the technical aspects of the tests, challenges, and limitations the research group have come across, and how they envision the future for multi-omics fetal analysis in the clinic.

## Bio-sketches

Drs Bénédicte Buffin-Meyer, Julie Klein and Joost P Schanstra are scientists at the French national institute of health and medical research (INSERM) in Toulouse, France. They have a longstanding track record and are passionate for research focusing on biomarkers of kidney disease. Prof Stéphane Decramer is a pediatric nephrologist from the Toulouse University Pediatric Hospital who specializes in developmental kidney disease with a practical experience of >1000 patients with these rare conditions.

Together they have developed state-of-the-art mass-spectrometry based prenatal body fluid marker signatures for the prediction of outcome of developmental kidney disease. A major focus of their work is on posterior urethral valves (PUV), and they are currently focusing on the validation of a fetal urinary peptide signature predictive of postnatal kidney function in the pan European ANTENATAL trial to come a step closer to implementation of such tests in clinical practice.

## Interview

**JPU: Moving from ureteropelvic junction obstruction (UPJO) to PUV: Initially the capillary electrophoresis – mass spectrometry (CE-MS) technique was used to study urinary peptides (proteomics) in newborns with unilateral UPJO, to predict which child with SFU grade 3–4 hydronephrosis with intermediate findings on MAG3 renography would eventually require surgery. Some of the differentially expressed peptides were different types of collagens [1]. This model was correct in 34/36 newborn patients (94 %). However, when the test was applied to both children <1 year or age and children >1 year of age in a different cohort, the area under the curve (AUC) dropped from 88 % to 57 % [2], showing that it was not applicable in the older age group.**

**Question:** With the benefit of hindsight, was this due to a normal maturational change in urinary peptide excretion in older children (implying that a new set of peptides needed to be studied), or do the patients who require

<footnote>ªNational Institute of Health and Medical Research (INSERM), UMR 1297, Institute of Metabolic and Cardiovascular Disease, Toulouse, France</footnote>

<footnote>ᵇUniversity Paul Sabatier, Toulouse III, Toulouse, France</footnote>

<footnote>ᶜDepartment of Pediatric Internal Medicine, Rheumatology and Nephrology, Toulouse University Hospital, Toulouse, France</footnote>

<footnote>ᵈCentre De Référence Des Maladies Rénales Rares du Sud-Ouest (SORARE), Toulouse University Hospital, Toulouse, France</footnote>

<footnote>ᵉDept. of Pediatric Surgery, Center or Organ Diseases and Transplantation, Rigshospitalet, Dept. of Clinical Medicine, Copenhagen University, Copenhagen, Denmark</footnote>

<footnote><fDepartment of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden</footnote>

<footnote n="8">⁸Division of Urology, Brown University, Providence, RI, USA</footnote>

<footnote>* Correspondence to: Magdalena Fossum, Department of Pediatric Surgery, Rigshospitalet, 2100 Copenhagen, Denmark maria.magdalena.fossum@regionh.dk (M. Fossum)</footnote>

<footnote>**Keywords** Biomarkers; Urinary Peptides; Fetal medicine; Pediatric urology; Obstructive kidney disease; Urethral Valves</footnote>

<footnote>Received 9 January 2024
Accepted 10 January 2024
Available online 14 January 2024</footnote>

<footnote n="1">¹ Shared last authorship.</footnote>
**surgery at >1 year of age have a kidney which compensates toward normal urinary peptide excretion since the obstruction was not as severe?**

**Answer:** A main concept in diagnostic or prognostic biomarker studies is the fact that biomarkers are only valid in the conditions where they are identified, unless proven otherwise. The Drube et al., 2010 study was a nice example of this. Although we cannot be certain, we believe that the drop in predictive value is due to the change in the urinary peptidome induced by kidney maturation in children >1 y. We already observed radical differences between the urinary peptidomes of young (2–10 y), adolescents (11–18 y) and adults (>19 y) that confirms this [3]. One can easily imagine that this is very similar during the first year of life where the kidney is still maturing. A new study would be necessary for older children with UPJO, although theoretically one should be able to mathematically "subtract" the maturation changes of the urinary peptidome. However, the latter would still need a novel study to determine the evolution of urinary peptides in healthy children from 0 to 2 y, currently not available.

**JPU:** The initial study using CE-MS in PUV patients compared the fetal urine of those with end stage renal disease (ESRD, n = 13) and without ESRD (n = 15) at age 2 years. 26 peptides which correlated to ESKD were identified out of 4199 candidates in the discovery cohort [4]. Of these, 12 peptides were chosen that most accurately selected out the patients with ESRD. This 12-peptide model was validated in subsequent population of 38 patients, with AUC 0.94, Sensitivity 88 %, Specificity 95 %. 11/12 peptides were collagen fragments, these were increased in ESRD. This is different than adults, where a decrease in collagen fragments in ESRD was seen.

**Question:** Why is there a difference in collagen expression in ESRD between children and adults? Is it specific to PUV vs other causes of ESRD in adults? Can we be sure that there is no collagen coming from the bladder?

**Answer:** Indeed, a striking difference is observed with progressive chronic kidney disease (CKD) in adults. In adults with CKD, the reduction in abundance of urinary collagen fragments is probably due to reduced in-situ

degradation of collagen that that could explain the development of fibrosis, a hallmark of CKD [5]. In contrast, we hypothesize that in PUV, the increased abundance of collagen fragments in fetal body fluids represents connective tissue remodeling (i.e. collagen degradation) due to kidneys with dys-/hypoplasia, cysts and hyperechogenicity (Fig. 1).

With respect to the origin of the (collagen) peptides, we fully agree. Collagens originating from the bladder will also be present in fetal urine. Our colleagues working on bladder cancer have identified urinary collagen peptides associated to the development of bladder cancer in adults [6]. However, in our case, since the selection of biomarkers was based on progression towards ESRD, i.e. impact on the kidney of PUV, we believe that collagen fragments included in the 12-peptide model mostly represent ongoing processes in the kidney, and not in the bladder. But, of course, we cannot completely exclude that some ESRD marker peptides originate from the bladder.

**JPU:** There were 2 reasons for studying urinary peptides: 1. They are filtered early in prenatal life. 2. They can be assayed from a small amount of urine. This is important as it is difficult to obtain large quantities of fetal urine. Inflammatory markers such as interleukins, TNF, TGF-β are also elevated at 22 weeks in fetuses with PUV [7].

**Question:** Are these inflammatory biomarkers much more difficult to assay using CE-MS? This study was done using a cytometric bead assay.

**Answer:** The aim of peptidome analysis is to cover an as large as possible number of peptides in one analysis. Hence the term "-ome", defined as "the complete whole of a class of substances for a species or an individual". However, this comes with a trade-off. Currently CE-MS can detect peptides down to the lower ng/mL range [8]. The collagen peptides, although very good markers of severe post-natal renal failure in fetuses with PUV, are thus relatively abundant. In contrast, small inflammatory and profibrotic cytokines are often present in the lower (pg/mL) range in body fluids including urine and

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Differences in (fetal) urinary collagen fragment content in adult CKD and in fetuses with PUV with early ESRD. We hypothesize that in adult CKD, characterized by kidney fibrosis, collagen degradation is reduced leading to a reduced abundance of collagen fragments in urine. In contrast, in fetuses with PUV that progress to early ESRD, kidney ultrasound often identifies dys-/hypoplasia, cysts, hyperechogenic kidneys etc., that could represent continuous connective tissue remodeling that necessitates collagen degradation leading to an increased abundance of collagen fragments in fetal urine.</figcaption></figure>
thus will not be readily detected by CE-MS [9,10]. In this
these cases ELISA-based approaches will be more
appropriate. However, the predictive value with respect
to postnatal kidney outcome of inflammatory markers in
PUV pregnancies remains to be determined.

**JPU:** The gray zone: One of the continuing problems that clinicians face is that sometimes a fetus that is predicted to develop ESRD develops normal renal function later in life. This falls into the diagnostic gray zone, as parents who are considering termination of pregnancy would like to avoid termination of a normal fetus. One approach is to add a complementary biomarker to increase the AUC.

Adding metabolomics to the peptide analysis was done in PUV patients with ESRD (n = 13) and without ESRD (n = 12) [11]. 24 metabolites were chosen and validated in a subsequent population of 35 patients. While the combination of peptide and metabolites (AUC 0.987) outperformed the metabolites alone (AUC 0.905), the peptide and metabolites did not significantly improve the AUC compared to peptide alone (AUC 0.967). Peptides and metabolites did improve the gray zone compared to peptides alone from 13% to 7%, but this was not statistically significant. To study the benefit of adding metabolomics and miRNAs to proteomics in a larger population, the international ANTENATAL study was organized, with a goal to study 400 fetuses [12]. It will study the use of amniotic fluid as a source of the peptides, assess if the peptides are changed by prenatal intervention, and look at the change in peptides over time if there are multiple specimens.

Adding metabolomics to the peptide analysis was done
in PUV patients with ESRD (n = 13) and without ESRD
(n = 12) [11]. 24 metabolites were chosen and validated in
a subsequent population of 35 patients. While the combi-
nation of peptide and metabolites (AUC 0.987) out-
performed the metabolites alone (AUC 0.905), the peptide
and metabolites did not significantly improve the AUC
compared to peptide alone (AUC 0.967). Peptides and
metabolites did improve the gray zone compared to pep-
tides alone from 13 % to 7 %, but this was not statistically
significant. To study the benefit of adding metabolomics
and miRNAs to proteomics in a larger population, the in-
ternational ANTENATAL study was organized, with a goal to
study 400 fetuses [12]. It will study the use of amniotic
fluid as a source of the peptides, assess if the peptides are
changed by prenatal intervention, and look at the change
in peptides over time if there are multiple specimens.

**Questions:** Do you think that the gray zone will be significantly improved with additional omics, given that this study will be appropriately powered? How rapidly would you expect improvement in the peptide profile after prenatal intervention?

**Answer:** As in one of the answers below, there is a lot of trial and error involved in these studies and it is really difficult to predict whether additional omics will reduce the gray zone and/or improve otherwise the prediction. Nevertheless, our results from 2018 suggest that this is the case at least in a context where the use of a single omics stratum (e.g. metabolites) is not completely satisfactory. We hope that for PUV a lot of the answers will come from the ANTENATAL study [12].

With respect to "normalization" in the peptide profile
after intervention, we believe that this is really depen-
dent on the severity of renal lesions already present at the
time of the intervention and the capacity of the devel-
oping kidney to reverse the impact of the obstruction.

**JPU:** Initial studies comparing amniotic fluid and fetal urine peptide levels have already been performed in 280 fetuses. 1831 peptides were found in both amniotic fluid (n = 2668) and fetal urine (n = 3257) [13]. Out of these, 67 peptides were found to have similar levels in amniotic fluid and fetal urine. Comparison of patients with ESRD (n = 6) and patients without ESRD (n = 8) found 7 peptides in amniotic fluid that discriminated between the groups. This was validated in another 16 patients. The amniotic fluid peptides performed similarly (AUC 0.97, Sensitivity 100 %,

Specificity 88 %) to fetal urine peptides (AUC 0.97, Sensi-
tivity 88 %, Specificity 100 %).

**Question:** Since there are 1426 peptides in fetal urine that are not found in amniotic fluid, where did they go? Are they degraded? Or are they diluted out so that they are not detectable?

**Answer:** We identified that some peptides exclusively found in fetal urine are still potential substrates for cleavage by proteases potentially present in amniotic fluid [13]. However, this is not necessarily true for all peptides. Furthermore, the amniotic fluid peptide content is also potentially modified by fetal deglutination and absorbance by the fetal skin and the amnion. Finally, the "dilution hypothesis" might also apply since the volume of the fetal bladder compared to the amniotic fluid volume is lower. Therefore, the difference between the fetal urine and amniotic fluid peptide content is probably due to a combination of these different factors.

**JPU:** The BIOMAN trial in France found 1008 identifiable peptides in amniotic fluid, 98 of which were associated with renal outcome [14]. Patients with ESRD (n = 18) were compared to patients without ESRD (n = 35), validation was done in another 51 patients. The amniotic fluid peptides had AUC 0.96, Sensitivity 88%, Specificity 97%. Adding amniotic fluid volume or gestation age did not significantly improve the AUC. More importantly, of the 8 fetuses that were terminated and subsequently found on pathology to have normal kidneys, the model correctly predicted that 6 of them would have normal renal function.

**Question:** Will adding additional omics treats increase the sensitivity of the peptide model so that healthy fetuses are not terminated?

**Answer:** This is a very tough one. Since we do not have a
crystal ball we really do not know. Especially here, where
the use of the peptidomic stratum alone already has a
very high predictive value. In theory adding different
biological levels should bring complementary informa-
tion. However, it can also introduce additional scatter
and errors. So, this is really a trial-and-error process.

**JPU:** Below we want to add some clarifications related to practical considerations for routine clinical use and end by speculating into the future. The current cost is 1200 euros for 5000 analytes.

**Questions:** Will specimens need to be sent to a central lab for processing due to quality control? How easy is it to set up the CE-MS in a new lab? What is the turn-around time? What other obstacles to routine use exist, aside from getting national approval for the technology? How often do the samples fail?

**Answer:** With respect to these analytical considerations:

Turn-around time and central laboratory: we are currently experimenting routine analysis of the amniotic fluid bCAKUTpep signature of samples coming from all-over France [14]. Mean turnaround time is about 2 weeks for around 50 samples analyzed to date in this context. This includes from shipping of frozen samples to a centralized laboratory until delivery of a prediction score.
In our view a central laboratory for peptidome analysis is nowadays still the best option. We are still far from a CE-MS setup around the corner of a clinic or hospital. Initial investment is still high and skilled labor is necessary, but if multiple tests, for different diseases, can be performed on the same equipment combined with standard operating protocols it will be feasible to implement such equipment locally. For drug detection, MS-based strategies in the analytical clinical laboratory have shown to be cost-effective already almost 2 decades ago [15]. Therefore, if such MS-based protein and peptide analyses become mainstream, local implementation will be feasible.

We have rare instances of failing samples. Bacterial contamination is a cause of failing samples. We have this now-and-then for adult urine which is not collected under sterile conditions. However, with respect to fetal urine or amniotic fluid, which are collected under very well controlled conditions, we to our knowledge never had issues with bacteria. Failing components in the pre-analytical pipeline (e.g. broken molecular weight cut-off filters) is another cause of failing samples, as it leads to high molecular weight compounds in the assay. But this is easily spotted during either the assay itself (on screen), or during the down-stream data analysis (number of detected peptides below the predefined threshold).

We feel that approval is still a long road mostly since these are rare-diseases and we are not focusing on gene modifications, that can use blood samples from any point in time allowing to include relatively easily several hundreds of patients. In contrast, (prenatal) body fluid-based omics require tedious planning and sample storage which is even more complicated in the case of a rare disease where patients must be recruited from geographically dispersed locations.

In addition, there is little interest in sponsoring of such studies by Pharma since the major goal of our studies is counselling and decision-making. Rare disease initiatives such as the International rare disease research consortium (IRDiRC) that created a specific Diagnostics Scientific Committee (DSC) might help here. However, the DSC of IRDiRC is currently mostly focusing on the development of gene-based diagnostics.

Fortunately, our team is strongly involved in the European rare kidney disease network ERKNet (https://www.erknet.org/) which helps us to promote our work and clinical implementation. Let's hope this will help to remove some of the obstacles!

**JPU:** In your previously published study, bladder puncture was performed at a mean of 27.8 gestational weeks (weeks of amenorrhea) [11].

**Questions:** Theoretically, how early after conception could bladder puncture be performed? Could amniotic fluid

proteomics be further developed for earlier analyses, to facilitate sampling and make it generally more available, or is it doomed to be of lower predictive quality?

Have you found any peptides that are completely lacking in patients with obstructive diseases, as an inverse marker of disease? Or peptides that imply a benign prognosis or marker of normality?

Could maternal blood sampling further be used for adding strength in the prediction of future disease?

What have been the biggest hurdles related to 1) consenting parents and patients to participate in these studies, 2) tissue/urine sampling and 3) performing the assays? Or any other hurdles?

**Answer:** Fetal urine puncture can be performed as early as 11-14 weeks of amenorrhea (wa). The earliest gestational age for fetal urine that we have analyzed was 14.6 wa [4]. Amniotic fluid punctures are generally performed a little later >16 wa, but we have analyzed amniotic fluid samples of a few CAKUT patients aged 13 wa [14]. There is a risk of club foot due to amniocentesis before 16 wa. So, we could perform the analysis earlier, but again, the added value of this will need to be demonstrated.

We have never found peptides completely lacking in one condition. We always observe either enrichment or decrease in abundance of a marker peptide in a specific condition, never a complete loss or a *de novo* appearance. It is, unfortunately, never black or white, and this is the reason why we need a combination of markers for the prediction algorithms.

With respect to maternal blood markers, although this clearly would reduce the invasiveness of the analysis, we believe it is clearly too "far" from the site of injury as we have described in the past [16].

The biggest hurdles:

* Parents: religion can be an obstacle in research and can influence the management of the disease.

* Sampling: sampling can be complicated due to the *in utero* position of the fetus and the presence of severe obesity in the mother.

* Assays: Since we are currently performing the analysis in an academic setting, the major hurdles were, *and are*, "logistics" including personnel and equipment availability, maintenance, etc ... The assay itself, including the preanalytical, analytical and data analysis pipeline is clearly defined, and is therefore not an issue.

**Questions:** How do you envision the future in the field of proteomics? Can you foresee other organ dysfunctions that could be prognosticated in *utero*? Do you think proteomics will be used within the field of precision medicine, in the sense that proteomics mapping on a particular patient, could be used for testing different pharmaceutical agents in vitro or in silico, as a personal preclinical trail before administering to the patient?
**Answer:** Our motto always has been to start from the clinical need. A prognostic need was clearly present in obstructive uropathies/CAKUT and we feel that the clinicians managing such pregnancies adhere to the prognostic methods based on fetal urinary and amniotic fluid peptides and are willing to push these towards clinic implementation. We are therefore confident on the future use of body fluid proteomics in this field. We have also been working on the prenatal identification of amniotic fluid peptide markers of symptomatic fetuses infected with cytomegalovirus that was identified as an important need in that field. In that context we identified a set of amniotic fluid peptide markers that yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90, to segregate 9 severely symptomatic from 12 asymptomatic CMV infected neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters [17]. However, despite these promising results there was no follow-up or additional interest from clinicians since this study from 2016. So, any potential prenatal application that has a strong prognostic need would fit, but ending up in routine clinical care is another issue.

In contrast, we reached in the field of adult medicine, an important milestone with respect to *in silico* prediction of treatment effects. Together with a large group of collaborators we have recently show-cased that based on urinary peptides we could predict *in-silico* whether a patient would respond to commonly used interventions such as antihypertensive drugs, or newer drugs like SGLT2i [18]. This opens the door to precision-based medicine approach in common and frequent diseases, which however still remains to be validated.

## Funding

None.

## Conflict of interest

Two last authors are Basic Science editors of the Journal.

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