Biomarkers and Outcome Predictors of Pediatric Nephrotic Syndrome: A Genetic, Transcriptomic, and Secretome Multiomics Study
Conditions: Nephrotic Syndrome Steroid-Dependent, Nephrotic Syndrome Steroid-Resistant, Nephrotic Syndrome in Children, Glomerulonephritis, Proteinuria, Hypoalbuminemia, Kidney Diseases, Chronic Kidney Disease
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

Cover Page 
 
Official Title of the Study: 
Biomarkers and outcome predictors of pediatric nephrotic 
syndrome: a genetic, transcriptomic and secretome 
multiomics study 
 
Acronym: 
PRECISE 
 
NCT Number: 
NCT06792448 
 
Date of the Document: 
31/07/2024 
 

 
 
 
 
 
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Biomarkers and outcome predictors of pediatric 
nephrotic syndrome: a genetic, transcriptomic and 
secretome multiomics study 
  
Acronimo: PRECISE 
 
Promoter: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,  
 Via Sforza 28, 20122 Milano, Italia 
 
Coordinating Centre: Struttura SC Nefrologia e Dialisi Pediatrica – Trapianti di 
Rene 
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,  
Via della Commenda 9, 20122 Milano, Italia 
 
Principal Investigator: 
 Prof. Giovanni Montini 
Firma ______________________________ 
 
Study design: biological non-pharmacological observational study. 
 
Numero della versione: v.1.0 
 
Data: 31/07/2024 
 
 
DECLARATION OF CONFIDENTIALITY 
All the information presented in this document must be considered confidential and will be property of Fondazione 
IRCCS Ca 'Granda Ospedale Maggiore Policlinico. The use of this confidential information is restricted to recipient for 
the aim agreed and must not be disclosed, published or communicated to unauthorised persons, for any reason, in any 
form, without the prior written consent of Fondazione IRCCS Ca 'Granda Ospedale Maggiore Policlinico. 

 
 
 
 
 
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Index 
 
 
1. Schedule of assessments 
2. Abbreviations 
3. Responsability 
4. Amendments 
5. Timelines  
6. Rationale and background  
7. Objectives  
8. Methods  
8.1 Study design 
8.2 Primary endpoints  
8.3 Secondary endpoints 
9. Enrollment and inclusion/exclusion criteria 
9.1 Study population (incident patients) 
9.2 Control group  
9.3 Validation cohort 
10. Study procedure 
10.1 Study visits 
10.2 Informed Consent and inclusion/exclusion criteria 
10.3 Demographic Data 
10.4 Medical History 
10.5 Physical Examination 
10.6 Assessment of Oedema 
10.7 Vital signs 
10.8 Routine laboratory tests  
10.9 Sample collection for molecular analysis 
11. Detailed technical methodologies  

 
 
 
 
 
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12. Source Document 
13. Sample Size 
14. Data management 
15. Statistical analysis  
   15.1 Primary endpoint: analysis 
   15.2 Secondary endpoints: analysis 
16. Quality control 
17. Study limitation 
18. Other aspects 
  18.1 Informative note to subjects and consensus module to personal data treatment 
18.2 Insurance  
19. Disclosure plan and communication of study results 
20. Publication and Intellectual property 
21. Financial support 
22. Bibliography 
Appendix 1. List of recruiting centers. 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
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1.  Schedule of assessments 
A. Schedule of assessments: main population (incident INS) 
 
 
Enrolment  
(Onset) 
6-weeks visit 
(End of metil-
prednisolone 
pulses*) 
6-months 
visit 
12-months 
visit 
First relapse 
Informed consent 
x 
 
 
 
 
Inclusion/exclusion 
criteria 
x 
 
 
 
 
Demographic data 
x 
 
 
 
 
Physiscal 
examination 
x 
x 
x 
x 
x 
Assessment of 
oedema 
x 
x 
 
x 
x 
Medical history 
x 
 
 
x 
 
Vitals sign 1 
x 
x 
x 
x 
x 
Clinical blood 
sample2 
x 
x 
x 
x 
x 
Clinical urine 
sample3 
x 
x 
x 
x 
x 
Blood for genetic 
analysis 
x 
 
 
 
 
Blood for 
mononuclear cells 
analysis 
x 
x 
 
x 
x 
Urine for 
molecular analysis 
x 
x 
x 
x 
x 
Serum for 
molecular analysis 
x 
x 
x 
x 
x 
 
1 Height, weight, blood pressure 
2 BUN, sCr, albumin, Na, K, total cholesterol, IgG, triglycerides, (local laboratory) serum 
complement C3 (local laboratory, only at enrolment) 

 
 
 
 
 
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3 Urine protein/creatinine ratio, urinalysis (local laboratory) 
* Only in patients not achieving remission at week 4 
 
B. Schedule of assessments: control group 
 
 
 
1 Height, weight, blood pressure  
2 BUN, sCr, albumin, Na, K, total cholesterol, triglycerides, IgG, IgM, IgA (at local site) 
3 urine protein/creatinine ratio, urinalysis (at local site) 
 
 
 
 
 
 
 
 
 
 
 
Enrolment  
 
Informed consent 
x 
Inclusion/exclusion 
criteria 
x 
Demographic data 
x 
Physiscal 
examination 
x 
Vitals sign 1 
x 
Clinical blood 
sample2 
x 
Clinical urine 
sample3 
x 
Blood for PBMC 
extraction 
x 
Urine 
x 
Serum 
x 

 
 
 
 
 
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C. Schedule of assessments: validation cohort (prevalent INS) 
 
 
 
 
Enrolment  
(Relapse) 
Remission 
 
Informed consent 
x 
 
Inclusion/exclusion 
criteria 
x 
 
Demographic data 
x 
 
Physiscal 
examination 
x 
x 
Assessment of 
oedema 
x 
x 
Medical history 
x 
 
Vitals sign 1 
x 
x 
Clinical blood 
sample2 
x 
x 
Clinical urine 
sample3 
x 
x 
Blood for PBMC 
extraction 
x 
x 
Urine 
x 
x 
Serum 
 
x 
x 
 
1 Height, weight, blood pressure 
2 BUN, sCr, albumin, Na, K, total cholesterol, triglycerides, IgG, IgM, IgA (at local site) 
3 urine protein/creatinine ratio, urinalysis (at local site) 
 
 
 
 
 

 
 
 
 
 
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2. Abbreviations  
 
INS 
Idiopathic nephrotic syndrome  
PBMC 
Peripheral blood mononuclear cells 
SSNS 
Steroid sensitive nephrotic syndrome 
SRNS 
Steroid resistant nephrotic syndrome 
SDNS 
Steroid-dependent nephrotic syndrome 
FRNS 
Frequent relapsing nephrotic syndrome 
IRNS 
Infrequent relapsing nephrotic syndrome  
BUN 
Blood Urea Nitrogen 
Ig 
Immunoglobulins 
Th17 
T helper 17 
Treg 
T regulatory 
UMI 
Unique Molecular Identifier counts 
uPr 
Proteinuria  
C3 
Serum complement 3  
PBS 
Phosphate Buffered Saline 
2D-DIGE 
two-dimensional difference gel electrophoresis 
TEM 
transmission Electron microscopy 
LC-MS 
Liquid chromatography-tandem mass spectrometry  
RedCap 
Research Electronic Data Capture 
ANOVA 
analysis of variance 
 
 

 
 
 
 
 
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3. Responsability 
 
The promoter of the study is the IRCCS Ca' Granda Ospedale Maggiore Policlinico Foundation. The 
coordinating center will be the SC Nephrology and Pediatric Dialysis – Kidney Transplants of the 
IRCCS Ca' Granda Ospedale Maggiore Policlinico Foundation. The role of Principal Investigator (PI) 
will be covered by Prof. Giovanni Montini, who will coordinate the clinical study and manage the 
consortium. Dr. William Morello will assume the role of Sub-investigator and will be directly 
responsible together with Prof. Giovanni Montini for patient enrollment. 
 
Internal collaborations 
 
Affiliation 
Name 
Study Role 
IRCCS Ca’ Granda Ospedale Maggiore Policlinico 
Dr. William Morello 
Sub-Investigator 
 
External collaborations 
 
Affiliation 
Department 
Name 
Study Role 
University of Milano 
Department of Clinical 
Sciences and Community 
Health 
Dr. Federica Collino 
Biological sample 
processing, 
biochemical and 
coordination of 
single cell RNAseq 
experiments and 
single vesicles 
studies. 
 
Heidelberg University 
Hospital 
Division of Pediatric 
Nephrology, Center for 
Pediatrics and Adolescent 
Medicine 
Prof. Franz Schaefer 
  
Patients 
recruitment, 
biological sample 
processing, 
management of the 
consortium. 
 

 
 
 
 
 
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Cologne University 
Hospital 
Pediatric Department of 
Pediatrics, Division of 
Pediatric Nephrology and 
Center for Molecular 
Medicine 
Dr. Max Liebau 
  
Patients 
recruitment, 
biological sample 
processing, 
proteomics and 
phospho-
proteomics and 
bioinformatic 
analysis.  
 
Medical University 
Gdansk 
Rare Diseases Centre and 
Department of Biology and 
Medical Genetics 
Prof. Beata S. Lipska-
Ziętkiewicz 
Biological sample 
processing, exome 
sequencing analysis 
University of Istanbul 
Division of Pediatric 
Nephrology. 
Prof. Alev Yilmaz 
Patients 
recruitment, 
biological sample 
processing, 
epigenetics analysis 
Vilnius University 
Faculty of Medicine 
Clinic of Children's Diseases 
Prof. Augustina 
Jankauskiene 
Patients recruitment 
and bioinformatic 
analysis 
 
 
 
 
External services 
 
Name 
Type of service 
University of Torino, Prof. B. Bussolati 
EV data analysis 

 
 
 
 
 
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Eurofins, Italy 
Novaseq 6000 
MacroGen UE 
Sample shipping and exome sequencing 
 
 
4. Amendments 
 
Not applicable. 
 
5. Timelines 
 
Stato dello studio 
Pianificazione 
Inizio della raccolta dati 
30/10/2024 
Fine della raccolta dati 
30/10/2027 
Report finale dello studio 30/03/2028 

 
 
 
 
 
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6. Rationale and background  
 
Idiopathic nephrotic syndrome (INS) is the most common glomerular disease in children, with an 
annual incidence of 2-7 cases per 100,000 children. INS is characterized by proteinuria, edema, 
hypoalbuminemia, hyperlipidemia, and thrombotic complications (1). The disease has an extremely 
variable clinical behavior, and the first-line treatment is based mainly on steroids and other 
immunosuppressive (IS) drugs. The response to steroids guides the actual classification. 
Approximately 80–90% of patients with nephrotic syndrome are initially steroid sensitive (SSNS), 
while 10–20% will not achieve remission despite second or third-line treatments and are defined as 
steroid resistant (SRNS) (2). The SSNS population exhibits an uncertain clinical course, 
characterized by various relapses and requiring immunosuppressive treatment, prolonged into 
adulthood in 1/3 of patients. These treatments have major side effects and come with an increased 
socio-economic burden as seen all over Europe. 
There is no biomarker able to predict the INS clinical course (response to treatment and occurrence 
of relapses) and to tailor the treatment for most patients. INS, at least in the steroid-sensitive forms, 
shows an immune pathogenesis. Potential causative circulating factors have been described, 
however none has been unequivocally shown to be pathogenic. The recent identification of genetic 
risk loci associated with HLA Class II region among the most represented in SSNS patients (3) 
emphasizes the central role of adaptive immunity in the NS pathogenesis. From the cellular side, 
various experimental evidence implies that dysfunction in some populations of T lymphocytes may 
be related to the pathogenesis of the disease (4). Among these, a Th17/Treg imbalance has been 
described in patients with minimal change glomerulopathy (5). Furthermore, the remission after the 
use of Rituximab, a monoclonal antibody against the CD20 surface antigen, suggests that B cells 
may also be involved in the pathophysiology of NS (6). The role of autoantibodies as circulating 
permeability factors able to directly damage the glomerular barrier, leading to proteinuria in INS, 
has been recently explored. Antibodies against Annexin A2, actin and ATP-synthase, ubiquitin-C-
terminal hydrolase L1, and CD40 antibodies were detected across both pediatric and adult INS 
patients in a variable percentage of INS patients highlighting the complexity of this pathology. 
Recently, the presence of anti-nephrin antibodies in post-transplant recurrent focal segmental 
glomerulosclerosis patients (7), as well as in the blood and kidney biopsies of patients with minimal 
change disease (8). 
As shown, INS displays a multifactorial and complex pathogenesis, where immunological, genetic, 
and epigenetic factors are involved. There is therefore an urgent medical need to find early 
biomarkers to allow for a precise selection and duration of the treatment, avoiding unnecessary 
severe side effects, and to characterize sub-cohorts for clinical studies. Biofluid secretome can 
harbor promising disease biomarkers (9). These biomarkers will also favor the understanding of 
the pathogenesis, allowing for an alternative classification and stratification of the patients. 
The PRECISE proposal aims to address unmet needs in INS children, searching for 
biomarkers/indicators/predictors of this rare kidney primary disease. In this context, we hypothesize 

 
 
 
 
 
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that biomarkers obtained from liquid biopsies (blood and urine) will improve the stratification and 
prognosis of INS patients, allowing for accurate tailoring of treatments. As the pathogenesis of the 
disease is multifactorial, an integrated cellular and secretome multi-omics approach will allow for 
the identification of biomarkers across multiple levels, to be then validated in the routine clinical 
setting. 
7. Objectives 
 
The PRIMARY OBJECTIVE of the PRECISE study is the generation of a predictive model to 
differentiate children with SRNS or SDNS from patients with IRNS at the onset through the 
integration of -omics, genetic, and clinical data (biomarker signature). 
 
The following 4 SECONDARY OBJECTIVES will be pursued in the study: 
 
1. Definition of the genetic/epigenetic modifications participating in the pathogenesis of INS. 
2. Evaluation of adaptive immune system alterations in INS children. 
3. Identification of specific protein patterns in serum and urine in different INS subtypes. 
4. Description of the molecular characteristics in healthy children. 
 
8. Methods 
 
8.1 Study design 
 
Open, multicenter, non-pharmacological observational study. 
 
 
 
 
 
 
 
 

 
 
 
 
 
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The PRECISE study is an open, multicenter, non-pharmacological biological study. The study will 
recruit a total of 310 INS patients and 40 controls. In details, the study will recruit 110 treatment-
naive childhood INS patients as a discover cohort, who will be followed for 12 months.  At 
enrolment urine and blood samples will be collected as for normal clinical practice for the planned 
analysis. At the 12-month follow-up, patients will be classified according to international guidelines 
as steroid-dependent (SDNS)/frequent relapsing (FRNS) and infrequent relapsing nephrotic 

 
 
 
 
 
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syndrome (IRNS) (10). Additional assessments and biological samples will be performed according 
to the study flowchart and as described in the study procedure chapter. A homogeneous control 
group (n=40) by age of children undergoing sampling for minor urological surgical correction 
procedures will be also enrolled. In the validation phase, biological samples from an independent 
group of prevalent INS (200 cases) cases will be collected. 
8.2. Primary endpoint 
 
To describe different molecular signatures (characteristics), predictive of the progression of INS, 
which would be able to discriminate among SRNS/SDNS and IRNS groups, based on 
genetic/epigenetic background, immune status, and vesicular proteome/transcriptomic profile. 
 
8.3. Secondary endpoints 
 
Related to secondary objective 1: 
1- Genetic kidney- and immune-related risk factors measured as number of variants in kidney- and 
immune-related genes, associated to INS incidence, progression, and poor response to therapy. 
2- A methylation profile of target genes measured as number of hypomethylated and 
hypermethylated immune-related gene regions, related to a faster progression to kidney failure and 
altered immune profile. 
Related to secondary objective 2: 
3- Difference in terms of % and absolute number of subpopulations of B cells and T cells among 
patients classified as SRNS/SDNS, IRNS, and healthy children. 
4- Changes in immune cell subsets genes, expressed as the number of expressed genes per cell (log-
transformed Unique Molecular Identifier counts, UMI), among patients classified as SRNS/SDNS, 
IRNS, and healthy children. 
5- Signaling dynamics changes, measured as number of phosphosites for each target protein 
expressed in the subpopulation of primary B and T cells, implicated in the development of kidney 
damage in INS. 
Related to secondary objective 3: 
6- Difference in the urine EV concentration expressed as total number/urine creatinine levels, and 
size as mean diameter, among patients classified as SRNS/SDNS, IRNS, and healthy children. 
7- Difference in the urine and serum EV surface proteome phenotype, in terms of the ratio of the 
average of label-free quantification intensities for each detected EV protein, among patients 
classified as SRNS/SDNS, IRNS, and healthy children. 

 
 
 
 
 
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8- Difference in the serum proteome, in terms of the ratio of average of label-free quantification 
intensities for each detected protein, among patients classified as SRNS/SDNS, IRNS, and healthy 
children. 
Related to secondary objective 4: 
9- To describe different molecular signatures (characteristics) for healthy children group in the same 
way done for the primary endpoint. 
9. Enrollment and inclusion/exclusion criteria  
The recruiting centers that will be involved in the study are listed in Appendix 1. 
9.1 STUDY POPULATION (incident patients) 
Inclusion criteria 
1. Clinical diagnosis of idiopathic nephrotic syndrome (nephrotic range proteinuria uPr/uCr 
ratio> 2mg/mg, hypoalbuminemia < 3.0 g/dl, oedema); 
2. No previous treatment for INS  
3. age between 1 and 18 years at onset 
4. signed informed consensus 
 
Exclusion criteria 
1. Congenital or infantile nephrotic syndrome (age < 1 year old); secondary nephrotic 
syndrome; glomerulonephritis; autoimmune diseases; vasculitis. 
2. Absence of signed informed consensus  
3. previous treatments with prednisone or prednisolone for INS  
 
9.2 CONTROL GROUP 
Inclusion criteria 
1. Clinical diagnosis of minor congenital abnormalities of kidney and urinary tract 
2. Absence of proteinuria (uPr/uCr < 0.2 mg/mg) 
3. age below 18 years 
4. signed informed consensus 
 
Exclusion criteria 
1. Immune-mediated diseases not associated with proteinuria (glomerulonephrities) 
2. age > 18 Years 
 
9.3 VALIDATION COHORT 

 
 
 
 
 
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Inclusion criteria 
1. Previous clinical diagnosis of idiopathic nephrotic syndrome (nephrotic range proteinuria 
uPr/uCr ratio> 2mg/mg, hypoalbuminemia < 3.0 g/dl, oedema); 
2. age between 1 and 18 years at onset 
3. signed informed consensus 
 
Exclusion criteria 
1. Congenital or infantile nephrotic syndrome (age < 1 year old); secondary nephrotic 
syndrome; glomerulonephritis; autoimmune diseases; vasculitis. 
2. Absence of signed informed consensus  
 
10. Study procedure 
 
10.1 Study visits 
Population 1 (main population_incident INS):  
At the enrollment visit, prior informed consent the following assessments will be performed: 
physical examination, assessment of oedema, measurement of vital signs, blood and urine samples 
collection (see A. Schedule of assessments for more details). Afterwards, patients will be assessed 
at 6 weeks, 6 months and 12 months according to A. Following the international guidelines, at the 
12 months visit patients will be clinically classified as SRNS, SDNS, or IRNS according to their 
medical history. Schedule of assessments: main population (incident INS). 
Patient will come back to the center in case of relapse (see definitions) to perform the ‘first relapse 
visit’ according to A. Schedule of assessments: main population (incident INS).  
Population 2 (control group): 
This population will perform after informed consent only the enrollment visit, according to B. 
Schedule of assessments: control group. 
Population 3 (validation cohort): 
Patient will be enrolled after informed consent at relapse (enrolment visit). During this visit, a 
physical examination, assessment of oedema, measurement of vital signs and collection of blood 
and urine samples will be performed according to C. Schedule of assessments: validation cohort. At 
the remission (see definitions), patients will come back to the center to perform the remission visit 
according to C. Schedule of assessments: validation cohort. 
10.2 Informed Consent and inclusion/exclusion criteria 
The Investigator or designee will ensure that each subject and/or subject’s parent/legal guardian is 
given full and adequate oral and written information about the nature, purpose, possible risks, and 

 
 
 
 
 
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benefits of the study. Subjects/parents/legal guardians will be notified that they are free to 
discontinue participation in the study at any time and will be given the opportunity to ask questions 
and allowed time to consider the information provided. After informed consent and assent (as 
appropriate) is obtained, subjects will undergo enrollment assessments. Once it is determined that 
they are eligible for the study, they will be enrolled in the study and baseline assessments will be 
completed. A copy of the signed ICFs will be provided to the subject and/or subject’s parent/legal 
guardian. 
10.3 Demographic data 
Demographic data (age, sex, and ethnicity, appearance age) and response to therapy will be 
collected for all participants during the enrollment visit. 
10.4 Medical history 
Medical history will be recorded at enrollment visit. Investigators should document the occurrence, 
signs, and symptoms of the participant’s preexisting conditions, including all prior significant 
illnesses. A detailed medical history about disease indication will be requested and will include 
prior surgeries, renal biopsies, hospitalizations, dialysis, transplantation, laboratory data, and 
extrarenal manifestations of the disease. Available disease medical history for the 3 years preceding 
screening will be requested. 
10.5 Physical examination 
A physical examination, performed by the investigator or designee, will include, at a minimum, 
assessment of the following: general, head, ears, eyes, nose, throat, heart, chest, lungs, abdomen, 
skin, extremities, back/neck, musculoskeletal, and lymph nodes. Additional symptom-driven 
physical examinations may be performed at any time, as deemed necessary by the investigator. See 
the schedule of assessments for the details regarding at which visit physical examination will be 
performed. 
10.6 Assessment of oedema 
Edema will be assessed at enrollment, 6-week visit, 12-month visit and at the first relapse. Assess 
for peripheral edema by pressing the area firmly with the thumb or index finger for 5 seconds and 
then release.  
10.7 Vital signs 
Vital signs including height, weight and systolic and diastolic blood pressure measurements will be 
evaluated at the visits indicated in the schedule of assessments.  
10.8 Routine laboratory tests (local laboratory) 
Blood samples. Blood samples for clinical assessments will be collected at the visits specified in the 

 
 
 
 
 
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schedule of assessments. The following analytes will be measured: BUN, sCr, albumin, Na, K, total 
cholesterol, triglycerides, IgG, IgM, IgA and serum complement 3 (C3). These exams will be 
conducted at each recruiting site.  
Urine samples. Urine for clinical assessments will be collected at the visits specified in the schedule 
of assessments. The following analytes will be measured: urine protein/creatinine ratio, and 
urinalysis. These exams will be conducted at each recruiting site.  
10.9 Sample collection for molecular analysis 
The following samples are needed:  
1. Blood sampling: for INS patients and controls, two additional aliquots of blood (a maximum of 
10-12 mL at a single time; body weight ＞12 kg) based on the patient's age and clinical 
condition during the sampling will be collected in the occasion of routine laboratory tests. 
2. Urine sampling: spot urines (20-30 mL) from residual urine from routine laboratory tests. 
Detailed sample processing: 
- Whole blood. Blood collection for genetic and epigenetic will be performed for all NS 
patients recruited into the study. Whole blood will be collected and stored at 4°C for 
analysis. Shipment of samples will be done at 4°C. 
- Mononuclear cells from peripheral blood. Whole blood will be isolated using the BD 
Vacutainer® CPT™ Mononuclear Cell Preparation Tube. Peripheral blood mononuclear 
cells (PBMCs) will be isolated by Ficoll procedure followed by washing step with PBS, 
and freezed in Cell Storage Media at density of 106 cells/vial and stored in liquid nitrogen 
or -80°C. Shipment of samples will be done by dry ice. 
- Urine. Urine samples will be centrifuged at 3,000 g for 15 minutes to remove large 
membrane fragments and other debris. A protease inhibitor cocktail will be added 
immediately (PI inhibitor). Urine aliquots will be stored at -80°C. Shipment of samples 
will be done by dry ice. 
- Serum. The serum will be obtained by direct cascade activation of coagulation from whole 
blood. The blood will then be centrifuged at 3,000g for 20 minutes to remove corpuscular 
components along with clots and debris. Serum aliquots will be stored at -80°C. Shipment 
of samples will be done by dry ice. 
Urine and serum will be centralised at Heidelberg University Hospital. The storage of peripheral 
blood mononuclear cells (PBMCs) will be centralized at the coordinating centre. 
Any residues of biological material that will not be used for the study will be eliminated at the end 

 
 
 
 
 
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of the study. 
11. Detailed technical methodologies 
Exome sequencing (collaborator involved: Medical University Gdansk). DNA samples extracted 
from blood leukocytes will be subject to sequencing using TruSeqExome Enrichment Kits 
according to the manufacturer’s instructions (Illumina). Data analysis will be carried out as 
previously described (11). 
Whole-Genome Bisulfite Sequencing Method (collaborator involved: University of Istanbul). For 
epigenetics analysis on CD45 lymphocytes, 5 μg of genomic DNA (OD260/280: 1.8–2.0) will be 
used as starting material. Bisulfite treatment of genomic DNA will be performed, and non-
methylated cytosine nucleotides will be converted to uracil and read as thymine (T) when 
sequenced. Methylated cytosines protected from conversion are still read as cytosine. The 
EpiGnome libraries will be diluted and loaded onto the cBot DNA Cluster Generation System. 
After cluster generation, the flow cell will be transferred to the HiSeq 2500 System for sequencing 
using 75 bp paired-end reads. The HiSeq 2500 generates approximately 500 Gb of sequence data 
per flow cell or about 62 Gb per lane allowing a coverage of approximately 120 Gb of data per 
sample. Additional sequencing can be completed for higher coverage. Data analysis of whole 
genome bisulfite sequencing will be conducted. FastQC tool will be used to check the raw data 
quality. Alignment will be done with the Bismark tool to identify the methylated regions 
(methylation calling). Statistical analyses will be performed with a methyl Kit.  
Cytofluorimetric analysis (collaborator involved: the University of Milano). Purified PBMCs will 
be discriminated by incubation with the dead cell Stain Buffer (BD) and surface staining will be 
then performed at 4°C for 20 min. Cells will be then fixed and permeabilized, stained at room 
temperature for 20 min with antibodies against intracellular antigens. For cell activation and 
intracellular cytokines staining, PBMCs will be stimulated with PMA/Ionomycin (1 μg/mL) for 2 
hours. Brefeldin A (10 ng/mL) will be added as a protein transport inhibitor. Cells will be stained 
with Fixable Viability Stain 780 (BD) followed by cell-surface marker (BD Pharmingen) and 
analyzed with the BD FACS Symphony flow cytometer. 
Single cell RNAseq experiments combined with CITE-seq (collaborators involved: IRCCS Ca’ 
Granda Ospedale Maggiore Policlinico and the University of Milano). CD3+ and CD19+ cells 
from peripheral blood will be sorted from purified PBMCs. CD3+ cells will be labeled using 
barcoded versions of anti-CD4 and anti-CD8 (TotalSeqC – _Biolegend). After cell number and 
viability determination by automated cell count, the appropriate volume of CD3+ and CD19+ cells 
will be loaded onto a 10x Genomics chip to recover 10.000 cells/sample. RNA-Seq and single-cell 
repertoire analyses will be performed according to 10x Genomics 5’ V(D)J protocol. Library 
sequencing will be performed using the Illumina NovaSeq 6.000 (Eurofins facilities, 
https://eurofinsgenomics.eu/en/custom-dna-sequencing/). 
scRNA-Seq data analyses (collaborators involved: IRCCS Ca’ Granda Ospedale Maggiore 

 
 
 
 
 
Dipartimento Area Materno - Infatile 
SC NEFROLOGIA E DIALISI PEDIATRICA – TRAPIANTI DI RENE–Direttore: Prof. Giovanni Montini 
Tel. 0255032883 
email: nefrologiapediatrica@policlinico.mi.it 
 
 
Policlinico and the University of Milano). Raw fastQ files will be aligned to the human reference 
genome GRCh38, subjected to quality control filtering, and data normalization high variable genes 
will be identified and used in a principal component analysis (PCA). Downstream analyses of 
scRNAseq data on different unsupervised algorithms (UMAP, tSNE) will allow the spatial 
visualization of the single-cell dataset and features. T- and B- -cell clusters will be defined using the 
Leiden algorithm, and differentially expressed genes (DEGs) between distinct cell clusters and 
experimental groups will be identified (P value ≤0.05, log2 fold change ≥1.5). The procedure will 
generate an enrichment in immune cell subsets with a specific molecular signature that will be 
analyzed bioinformatically. Gene Ontology (GO) analyses will also be performed. For scTCR-seq 
and scBCR-seq data analysis, CDR3 sequences and V(D)J gene rearrangement will be obtained 
using the Cell Ranger VDJ 10X pipeline. T/B cells showing consistent CDR3 amino acid sequence 
will be defined as clonotypes. 
EV isolation and characterization (collaborator involved: the University of Milano). Purification 
of EVs will be obtained by size exclusion chromatography using 70 nm qEV columns (IZON) after 
a 100 kDa centrifugal filter (Sartorius). The concentration and size distribution of EVs will be 
measured using NanoSight LS300 (NanoSight Ltd). EVs integrity will be characterized by 
transmission Electron microscopy (TEM) by adherence to glow-discharged formvar-coated copper 
grids 300 mesh. 
Single vesicles studies (collaborator involved: the University of Milano). EV evaluation at single 
levels will be performed following the pipeline described by (12). Super-resolution microscopy will 
be performed using the Nanoimager S Mark II microscope from ONI (Oxford Nanoimaging, 
Oxford, UK) equipped with a 100X. Tetraspanins and selective markers will be tested using the 
Apex Antibody Labelling Kit (Invitrogen) according to the manufacturer's protocol. Samples will 
be also analyzed with ExoView Plasma Tetraspanin kit on an ExoView R100 instrument 
(NanoView Biosciences) following the manufacturer’s instructions. 
EV protein corona and lipid evaluation (collaborator involved: the University of Milano). EVs 
surface phenotype will be analyzed using the MACSplex kit (Miltenyi Biotec) on a BD FACSCanto 
II (Becton Dickinson) as (12). Proteomic studies will be then performed by two-dimensional 
difference gel electrophoresis (2D-DIGE) followed by liquid chromatography-tandem mass 
spectrometry (LC-MS) as (13). Surface proteins will be separated from the ones from the lumen. 
Lipidomic profile will be also evaluated by gas chromatography (GC)-MS. 
Proteomics and phospho-proteomics (collaborator involved: Cologne University Hospital). For 
expression profiling and characterization of cellular phosphor-proteomes, a fully established 
standard pipeline at the Cellular Stress Responses in Aging-Associated Diseases and the Centre for 
Molecular Medicine Cologne will be used. Proteins are purified, alkylated, and digested. Peptides 
are purified after acidification by a Stage-Tip protocol using an SDB-RPS-Matrix (Styrene Divinyl 
Benzene reverse phase sulfonate-Matrix and measured in a Q Exactive Plus Tandem or a LTQ 
Orbitrap XL spectrometer. Raw data is processed using the MaxQuant software, is compared to 
reference datasets, and is analyzed and displayed using Perseus software. To obtain insights into the 

 
 
 
 
 
Dipartimento Area Materno - Infatile 
SC NEFROLOGIA E DIALISI PEDIATRICA – TRAPIANTI DI RENE–Direttore: Prof. Giovanni Montini 
Tel. 0255032883 
email: nefrologiapediatrica@policlinico.mi.it 
 
 
activation of specific signaling cascades by posttranslational modification (phosphorylation) we will 
also characterize the phospho-proteome. Phosphorylated peptides are enriched after standard 
isolation and purification with a special kit (High-Select Fe-NTA Phospho-peptide Enrichment Kit). 
For an unbiased serum proteomics approach, we can rely on the established fully automated clinical 
pipeline using liquid handling robots and state-of-the-art mass spectrometry platforms, including the 
latest generation Orbitrap Exploris 480. The approach enables the detection and quantification of on 
average 300 plasma/serum proteins per hour using a data-independent acquisition approach from a 
5 μl sample volume. 
12. Source Documents 
Medical history and all clinical data collected during the study will be collected from each visit’s 
medical records. 
Local laboratory records will be used to collect laboratory analysis results. 
 
13. Sample Size 
As this is a descriptive and explorative study without inference in the primary endpoint, and given 
the incidence of childhood idiopathic nephrotic syndrome, which is considered a rare disease, the 
number of recruiting centers, we foresee enrolling in this study 110 treatment-naive INS children. A 
homogeneous age control group of healthy children undergoing minor urological surgical correc-
tions will be enrolled as a control group (n=40 children in 3 years). These numbers are the maxi-
mum we can recruit, but at the same time, we think that this study could serve as an important basis 
for appropriate studies in the future. 
 
14.  Data Management 
A consortium was created under the direction of the Lead Unit (IRCCS Ca Granda of Milano, Prof. 
G. Montini, Proposal Coordinator) with the Partners involved in the study. At the enrollment visit, a 
unique code will be assigned to each participant. The de-identification of the data will take place in 
such a way that people who access the database will not be able to trace the identity of the subjects 
in any way. Only local investigators will be able to trace the identity of the enrolled subjects. 
The data necessary for the study will be recorded in a specific eCRF in a Data Management System 
validated according to national legislation, provided by the Scientific Management of the 
Foundation. The platform used will be RedCap (Research Electronic Data Capture). 
The REDCap Consortium is made up of >1000 institutional partners around the world (research 
institutions, universities, ministries, etc.). The consortium supports a secure web application 
(REDCap) designed exclusively to support data capture for research studies. The REDCap 
application allows users to create and manage online databases quickly and securely and is currently 
in use for more than 110,000 projects with approximately 150,000 users covering numerous areas of 
research interest across the consortium. 

 
 
 
 
 
Dipartimento Area Materno - Infatile 
SC NEFROLOGIA E DIALISI PEDIATRICA – TRAPIANTI DI RENE–Direttore: Prof. Giovanni Montini 
Tel. 0255032883 
email: nefrologiapediatrica@policlinico.mi.it 
 
 
Through REDCap, for this study the following will be implemented: a) identification at user level, 
with specific restrictions based on role in the study b) validation and control of data integrity in real 
time c) de-identification of patients before data export d) centralized data storage with daily backup, 
a secure server within the Foundation's IT structure. 
15. Statistical analysis 
 
15.1 Primary endpoints: analysis 
 
Statistical analysis using linear mixed effect models will ensure that data from the same individuals 
over time are properly considered. Network-based analysis using a linear mixed effect model 
LASSO implementation will allow us to identify key features associated with various clinical 
endpoints. To identify subsets of features, across all OMICs, associated with disease progression 
and treatment response, we will use a genetic algorithm-based feature selection approach. Unlike 
LASSO, this approach can identify non-linear relationships between features and can be linked to 
almost any predictive modeling approach. The feature subsets will further be evaluated in already 
available public domain data and then applied to a much larger cohort within the PRECISE 
consortium. 
 
15.2 Secondary endpoints: analysis 
Differences between two groups of patients for all the analysis conducted will be evaluated using 
Student's parametric T-test (in case of normality of variables distribution and homoscedasticity of 
variance) or Mann – Whitney's non-parametric test. The normality of the data will be evaluated by 
the Shapiro-Wilk test and the homoscedasticity assumption of the variance will be evaluated by the 
Hartley F-max test. In cases where more features are available than samples, regularized regression 
approaches, such as LASSO, or feature selection approaches will be used to identify potential 
combinations of features associated with a given outcome. Analyses of differences in continuous 
outcome measures between 3 or more levels of categorical features will take place through analysis 
of variance (ANOVA) using multiplicity-adjusted post-hoc tests by Dunnett or Tukey, as 
appropriate. Data analysis will be performed using GraphPad Prism software and P-values of 0.05 
will be accepted. 
16. Quality control 
Only after the approval from ethics committee and regulatory authorities, centers will enroll patients 
who meet inclusion and exclusion criteria and will propose them to participate to the study signing 
the informed consent. The coordinating center will have access to all registered data to verify any 
anomalies and report them to the participating center. All data will be handled, registered, and 
reported in accordance with the good clinical practice. As Promoter, Fondazione IRCCS Ca’ 
Granda Ospedale Maggiore Policlinico can carry out a quality check on the study at its discretion. 
In this case, the investigator should allow the monitor to directly access all relevant documentation 
and devote some of his or her staff time to the reviewer to discuss the results of the monitoring and 
any other aspects of the study. In addition, the regulatory authorities can carry out inspections. In 

 
 
 
 
 
Dipartimento Area Materno - Infatile 
SC NEFROLOGIA E DIALISI PEDIATRICA – TRAPIANTI DI RENE–Direttore: Prof. Giovanni Montini 
Tel. 0255032883 
email: nefrologiapediatrica@policlinico.mi.it 
 
 
this case, the investigator must authorize the inspector direct access to all relevant documentation 
and devote part of his time and his staff to the inspector himself to discuss the results of the 
monitoring and any other aspects of the study. 
 
17. Study limitation 
 
This study is a pilot study and shows some risks associated with it. The following limitations can be 
highlighted:  
  
1. Due to the nature of the pathology (rare disease), one of the risks could be not reaching the 
number of recruited patients (annual incidence of 2-7/100,000 children). Our contingency plan 
will be the integration of additional ERKnet and ESCAPE Network centers through an 
addendum to the protocol.  
2. The other limitation could be the absence of sample stability. Our contingency plan will be to 
define specific isolation workflow protocols, centralize the biobanking system, and perform 
sample stability tests for the different applications.  
3. Another problem could be a difficult blood draw based on the age of the new-onset children 
recruited in the study. We will exclude Infantile N from the study to overcome this problem.  
 
From the statistical side, all the data will be extensively scrutinized to ensure that misreporting is 
minimized. Potential confounders will be identified and assessed routinely throughout the analysis. 
Where possible, confounding factors will be adjusted for in the analysis. Where < 10% of missing 
data are present in a variable we will aim to impute missing data from other available data sources 
using a random Forest regression or classification approach. Variables with >20% missing values 
within a group will be kept aside and used only for descriptive quantitative evaluations. Statistical 
analysis using linear mixed effect models will ensure that data from the same individuals over time 
are properly considered.  
 
18. Other aspects 
The study will be conducted by the Standards of Good Clinical Practice, the ethical principles 
deriving from the Declaration of Helsinki.  
 
The study and the related documentation will be presented to the competent Ethics Committee of 
each center. The study will begin only after receiving the authorizations requested according to the 
institution's internal procedures. 
The Ethics Committee must also approve any changes to the protocol and advertising used to recruit 
subjects for the study, according to local regulations. 
18.1 Informative note to subjects and consensus module to personal data treatment. 
 
The investigators have the responsibility to obtain a signed informed consent by parents or legal 

 
 
 
 
 
Dipartimento Area Materno - Infatile 
SC NEFROLOGIA E DIALISI PEDIATRICA – TRAPIANTI DI RENE–Direttore: Prof. Giovanni Montini 
Tel. 0255032883 
email: nefrologiapediatrica@policlinico.mi.it 
 
 
guardians before collecting data. Signed documents must be stored by the investigators and a copy 
must be given to them. Parents, legal guardians or patients (if = 18 years old) will have all the time 
necessary for the evaluation of the information received before providing their informed consent to 
the use of sensitive data of minors. The investigator will have to obtain spontaneous informed 
consent from them before using it in any way for the study. The written consent to the handling of 
sensitive data must be subscribed by the date and signature of parents, legal guardians or patients (if 
= 18 years old) and by the investigator or his representative. The investigator has to give to them a 
signed copy of informed consent; the original form will be retained with the other documents of the 
study protocol; the module for the acquisition of informed consent to the treatment of sensitive data 
will be attached to the clinic folder.  
The Principal Investigator guarantees that the data of the patients enrolled during the study will be 
kept, archived, and processed in full compliance with the regulations on privacy under art. 13 of 
Regulation No. 2016/679/EU and the national privacy legislation in force, the Code of Ethics re-
garding data processing for statistical-scientific purposes, and the "Guidelines for the processing of 
personal data in the context of clinical trials of medical products" published in the Gazzetta 
Ufficiale n. 190 of 14 August 2008. 
Patient data will be accessible only to internal and external personnel specifically appointed and 
bound by an obligation of secrecy in relation to any information learned during the study. 
The Principal Investigator also guarantees that he has implemented the minimum security measures 
required by the aforementioned legislation for data processing using electronic and non-electronic 
tools, in order to avoid illicit data processing. 
18.2 Insurance 
Considering the observational nature of the study, extra insurance is not mandatory in addition to 
those already provided in normal good clinical practice. 
 
19. Disclosure plan and communication of study results 
The Principal Investigator of the study will undertake to draft a final report and make the results 
public at the end of the study. The data will be made public anonymously and presented as required 
in the aggregate model.  
20. Publication and Intellectual property 
Foundation as Promoter will guarantee the dissemination and publication of the study results, even 
in the event of negative results, without any constraints and guarantee the collaborating center 
visibility proportional to actual participation. Each scientific publication containing the results and 
data of the study must indicate the role and participation of the Centers and the Foundation, in a 

 
 
 
 
 
Dipartimento Area Materno - Infatile 
SC NEFROLOGIA E DIALISI PEDIATRICA – TRAPIANTI DI RENE–Direttore: Prof. Giovanni Montini 
Tel. 0255032883 
email: nefrologiapediatrica@policlinico.mi.it 
 
 
manner proportional to the actual contribution made in the study and the role played by each party. 
The data will be published in aggregate form or in any case anonymized, so as not to allow in any 
way the identification of the interested party to whom the data refers. 
The Parties recognize that for the conduct of the collaboration within the scope of the study, data, 
information, know-how, inventions (patentable or not) owned by each party may be used and 
shared, which remains the exclusive owner even if it grants them to the other a right of access and 
use, non-exclusive and free, for the sole purpose of carrying out the activities covered by the study 
and limited to its duration. It is understood that this right of use does not include the right to 
sublicense to third parties. In compliance with current legislation, the data and results generated 
within the Study will be the property of the Sponsor, unless specific agreements are made between 
the Sponsor and the Centres. 
 
21. Financial support 
The protocol study is financially supported by the European Joint call for Rare diseases (EJP RD 
JTC 2023).  
 
22. Bibliography 
1. Eddy AA, Symons JM. Nephrotic syndrome in childhood. Lancet. 2003. DOI: 
10.1016/S0140-6736(03)14184-0.  
2. Noone DG, Iijima K, Parekh R. Idiopathic nephrotic syndrome in children. Lancet. 2018 
Jul;392(10141):61–74. 
3. Barry A, McNulty MT, Jia X, Gupta Y, Debiec H, Luo Y, et al. Multi-population genome-
wide association study implicates immune and non-immune factors in paediatric steroid-
sensitive nephrotic syndrome. Nat Commun. 2023 Apr 29;14(1):2481. 
4. Kanasaki K, Kanda Y, Palmsten K, Tanjore H, Bong Lee S, Lebleu VS, et al. Integrin β1-
mediated matrix assembly and signaling are critical for the normal development and 
function of the kidney glomerulus. Dev Biol. 2008 Jan;313(2):584–93. 
5. li Liu L, Qin Y, fang Cai J, yun Wang H, ling Tao J, Li H, et al. Th17/Treg imbalance in 
adult patients with minimal change nephrotic syndrome. Clin Immunol. 2011 
Jun;139(3):314–20. 
6. Colucci M, Carsetti R, Rosado MM, Cascioli S, Bruschi M, Candiano G, et al. Atypical IgM 
on T cells predict relapse and steroid dependence in idiopathic nephrotic syndrome. Kidney 
Int. 2019 Oct;96(4):971–82. 
7. Shirai Y, Miura K, Ishizuka K, et al. A multi-institutional study found a possible role of 
anti-nephrin antibodies in post-transplant focal segmental glomerulosclerosis recurrence. 
Kidney Int. 2024;105(3):608-617. doi:10.1016/j.kint.2023.11.022 
8. Watts AJB, Keller KH, Lerner G, et al. Discovery of Autoantibodies Targeting Nephrin in 
Minimal Change Disease Supports a Novel Autoimmune Etiology. J Am Soc Nephrol. 
2022;33(1):238-252. doi:10.1681/ASN.2021060794. 
9. Cevenini A, Orrù S, Imperlini E. Secretome Proteomic Approaches for Biomarker 

 
 
 
 
 
Dipartimento Area Materno - Infatile 
SC NEFROLOGIA E DIALISI PEDIATRICA – TRAPIANTI DI RENE–Direttore: Prof. Giovanni Montini 
Tel. 0255032883 
email: nefrologiapediatrica@policlinico.mi.it 
 
 
Discovery: An Update on Colorectal Cancer. Medicina (Kaunas). 2020 Aug;56(9). 
10. Trautmann A, Boyer O, Hodson E, Bagga A, Gipson DS, Samuel S, et al. IPNA clinical 
practice recommendations for the diagnosis and management of children with steroid-
sensitive nephrotic syndrome. Paediatric Nephrology. 2023 Mar 21;38(3):877–919. 36.  
11. Koczkowska M, Jąkalski M, Birkholz-Walerzak D, Kostecka A, Iliszko M, Wójcik M, et al. 
Personalized health risk assessment based on single-cell RNA sequencing analysis of a male 
with 45, X/48, XYYY karyotype. Sci Rep. 2022 Dec 2;12(1):20854. 
12. Gebara N, Scheel J, Skovronova R, Grange C, Marozio L, Gupta S, et al. Single 
extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype 
alterations in preeclampsia. J Extracell Vesicles. 2022 May 18;11(5). 
13. Liangsupree T, Multia E, Saarinen J, Ruiz-Jimenez J, Kemell M, Riekkola ML. Raman 
spectroscopy 
combined with comprehensive gas 
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characterization of plasma-derived extracellular vesicle subpopulations. Anal Biochem. 
2022 Jun;647: 114672. 
Appendix 1. List of recruiting centers.